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THE
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LINNEAN SOCIETY
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FOR THE YEAR
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VOL. LXXVI.
WITH FIFTEEN PLATES, 236 Text-figures.
SYDNEY: PRINTED AND PUBLISHED FOR THE SOCIETY BY
AUSTRALASIAN MEDICAL PUBLISHING CO. LTD. Seamer Street, Glebe, Sydney,
and SOLD BY THE SOCIETY. 1952.
CONTENTS OF PROCEEDINGS, 1951.
PARTS I-II (Nos. 353-354). (Issued 31st May, 1951.)
Presidential Address, delivered at the Seventy-sixth Annual General Meeting, 28th March, 1951, by D. J. Lee, B.Se. The Problems of Insect Quarantine ..
Elections Balance Sheet for the Year ending 28th February, 1951 ..
Serological Studies of the Root-nodule Bacteria. IV. Further Analysis of Isolates from Trifolium and Medicago. By Hilary F. Purchase, J. M. Vincent and Lawrie M. Ward ..
A Septoria Disease of Huphorbia peplus L. By Dorothy HE. Shaw. (Plate i and one Text-figure. )
Preservation Techniques for Scarabaeid and other Insect Larvae. By P. B. Carne
The Anatomy and Morphology of the Operculum in the Genus Eucalyptus.
Part I. The Occurrence of Petals in Hucalyptus gummifera (Gaertn.) Hochr. By J. L. Willis. (Plates ii-iii and two Text-figures.)
Some Notes on Athrotaxvis. By Charles G. Elliott. (Communicated by Dr. Patrick Brough.) (Sixteen Text-figures.)
The Paramphistomes (Trematoda) of Australian Ruminants. iPanvee Systematics. By P. H. Durie. (Plates iv—v.)
Pages.
i-xix
xix
xx
1-6
1-25
26-30
36-40
41-48
CONTENTS.
PARTS III-IV (Nos. 355-356). (Issued 17th September, 1951.)
A Review of the Australian Species of Sarcochilus (Orchidaceae). By H. M. R. Rupp. (One Text-figure.)
Australian Rust Studies. VIII. Puccinia graminis lolii, an Undescribed Rust of Lolium spp., and Other Grasses in Australia. By W. L. Waterhouse .
Celaenopsoides Gunther, 1942: A Synonym of Ophiomegistus Banks, 1914 (Acarina: Parasitidae). By Carl EH. M. Gunther
On Trombicula minor Berlese, 1905. By Carl E. M. Gunther. (Three Text- figures. )
The EHvolution of the Radio-medial Area in the Wings of the Muscoidea Acalyptrata (Diptera). By H. F. Lower. (Thirty Text-figures.)
Studies on Australian Marine Algae. VI. New Geographical Records of Certain Species. By Valerie May ..
The Marine Algae of Brampton Island, Great Barrier Reef, off Mackay, Queensland. By Valerie May ..
The Development of Bryozoan Faunas in the Upper Palaeozoic of Australia. By Joan Crockford (Mrs. Beattie.) (Four Text-figures.)
Suecinoxidase of Potato Tuber. By Adele Millerd. (Two Text-figures.)
The Nomenclature of Heteronychus sanctae-helenae Blanchard (Coleoptera: Scarabaeidae: Dynastinae). By EH. B. Britton. (Communicated by P. B. Carne.)
Controlled Pollination of Hucalyptus. By L. D. Pryor. (Plate vi.)
A Genetic Analysis of some Hucalyptus Species. By L. D. Pryor. (Plates vii—xi.)
Investigations on the Preference shown by Aédes (Stegomyia) aegypti Linn., and Culex (Culex) fatigans Wied., for Specific Types of Breeding Water. By Tom Manefield
A Mite from a Beehive on Singapore Island (Acarina: Laelaptidae). By Carl EH. M. Gunther. (Five Text-figures.)
ili
57-64
65
66-70
71-82
83-87
88-104
135-139
140-148
149-154
155-157
iv CONTENTS.
PARTS V-VI (Nos. 357-858). (Issued 15th January, 1952.)
A Critical Consideration of ec-mitosis with Reference to the Effects of Nitro- phenols. By Mary M. Hindmarsh, Linnean Macleay Fellow in Botany. (Plate xiii.)
The Life-history of a Penaeid Prawn (Metapenaeus) breeding in a Coastal Lake (Tuggerah, New South Wales). By Muriel C. Morris and Isobel Bennett. (Plate xii and ninety-six Text-figures. )
The Use of Excised Shoots in Linseed Investigations. By H. B. Kerr. (Plate xiv.>
Remarks on some Australian Laius Guér. (Col.: Malachiidae). By W. Wittmer. (Communicated by J. W. T. Armstrong.) (One Text-figure. )
A Revision of Australian Species previously referred to the Genus Himpousca (Cicadellidae, Homoptera). By Harold F. Lower. (Plate xv and seventy-
five Text-figures.)
Miscellaneous Notes on Australian Diptera. XV. Tabanus, Heteropsilopus. By G. H. Hardy
Balance Sheets
Pages.
158-163
164-182
183-186
187-189
190-221
222-225
XXi-Xxli
Abstract of Proceedings BPE ks MRO WTS tet, RNIB ot We Cree Sle saa XX11i-xxvii
ASEOERAMEMDET Sh sacar a etater: Oe toa ee) eee MMO Riot AM Nes) epee VAC Stee ee ae CONV T < ReN ERG NT
List of New Genera and Species
List of Plates
XXXiil
XXXiil
Index saat Sash oath as, exe allteae ae) Oe Ea RRO 4 ee Se eee Vee
ANNUAL GENERAL MEETING.
WEDNESDAY, 28TH MArcH, 1951.
The Seventy-sixth Annual General Meeting was held in the Society’s Rooms, Science House, Gloucester Street, Sydney, on Wednesday, 28th March, 1951.
Mr. D. J. Lee, B.Se., President, occupied the chair.
The Minutes of the Annual General Meeting held on 29th March, 1950, were read and confirmed.
i PRESIDENTIAL ADDRESS.
As is usual, the first part of my address is devoted to a brief review of the Society’s activities during the past year.
Volume 75 of the Society’s Proceedings was published during 1950. It consisted of 359 + xl pages, 12 plates and 442 text-figures, thus being a larger volume than the previous one. Financial assistance towards the cost of publication of three papers was received from Department of Anatomy, University of Sydney (£74 16s.), Commonwealth Scientific Publications Committee (£55) and University of Melbourne (£25). The annual grant from the State Government of £100 towards the cost of publication of the Proceedings was increased to £150 during the year.
Exchanges received from scientific societies and institutions totalled 1,414. Loans from the Library, particularly interstate inter-library loans, have been requested as in the previous year. Additional steel shelving has been installed in the Library and some duplicate books are still available for sale. New exchanges were commenced with: Academy of the Popular Republic of Roumania; California Zoological Club; Hlisha Mitchell Scientific Society; Estacao de Melhoramento de Plantas, Elvas, Portugal; Société des Sciences Naturelles de Tunisie; University of Upsala (““Symbolae Botanicae Upsalienses’”), and Victoria University College, Wellington, New Zealand. A number of books from the library of the late Dr. F. G. Hardwick were presented to the Library by his son, Mr. F. L. Hardwick.
Interesting programmes were given during the year at the following monthly meetings:
June: A talk on “Coral Genera of Heron Island (Barrier Reef)”, by Mr. K. HK. W. Salter and Miss M. A. Besley.
July: The following members contributed items of interest: Miss M. Hindmarsh— a note on “Mitotic Poisons’; Mr. A. Musgrave—exhibition of specimens of Stephanitis queenslandensis Hacker, 1927, a member of the family Tingidae (Lace Bugs); Mr. A. K. O’Gower—exhibition of the eggs, larvae and pupae of cat fleas. ©
August—A series of talks was given by members of the New South Wales Depart- ment of Agriculture, the speakers being: Mr. Grahame Edgar, Director of Veterinary Research (investigations on problems of animal industry in the State); Dr. S. L. Macindoe, Principal Research Agronomist (an account of the organization, progress and results of plant research conducted primarily on the ten Experiment Farms and two Agricultural Colleges of New South Wales); Dr. F. T. Bowman, Special Fruit Research Officer (outline of the research work of the Division of Horticulture); Mr. S. L. Allman, Senior Entomologist (description of the Entomological Branch and its work with insect pest problems); and Dr. C. J. Magee, Chief Biologist (research in the Biological Branch which is directed mainly at preventing losses from plant diseases).
September: Talks on “Recent Changes in the Vegetation of North-western New South Wales’, by Dr. N. C. W. Beadle and Professor N. A. Burges.
October: Short talks by Professor P. D. F. Murray on “The Fusion of Long Bones” and by Mr. B. R. A. O’Brien on “Some Aspects of Regeneration in Earthworms”’.
November: Talks on various aspects of Northern Australia were given by: Professor J. Macdonald Holmes—“The Geographical Background to the Problems of North
A
ii PRESIDENTIAL ADDRESS.
Australia’; Dr. W. Kirkland—‘‘Medical Problems of North Australia”; and Dr. N. W. G. Macintosh—“Comments on the Physical Types of the Aborigines of Arnhem Land”. A talk prepared by Mr. W. Poggendort on “Problems in Agriculture” was given by Mr. E. B. Furby.
We thank all who have contributed to these programmes.
Since the last Annual Meeting the names of fourteen membérs have been added to the list, three members have been lost by death, and eighteen have resigned. The number of members as at 1st March, 1951, is: Ordinary Members, 206; Life Members, 22: Honorary Member, 1; and Corresponding Members, 3—total 232.
As Dr. Dorothy Carroll tendered her resignation as Secretary to the Society as from 22nd January, 1951, it was found necessary to make interim arrangements for the successful prosecution of the business of the Society. To this end Council appointed Dr. W. R. Browne as Honorary Secretary and Dr. A. B. Walkom as Honorary Editor for the time being. It is hoped that Council will be able to decide on some more permanent arrangement in the near future. Council’s intention at present is to employ a part-time editor, if one can be found suitable and willing to undertake this responsi- bility, and carry on the secretarial work with an honorary secretary and have the assistant secretary in charge of the Society’s rooms and library. In view of a further 50% increase in the basic cost of publication of the Society’s Proceedings, Council has felt that some economy in the cost of the secretarial activities of the Society may be necessary in order to maintain the level of publication at one approximating that of previous years.
Two Special General Meetings were held on 25th October and 29th November, 1950, respectively, to make an addition to Rule VI as follows: “An Ordinary Member who has paid the Annual Subscription for forty years shall be exempt from payment of further subscriptions.” Council’s object was to relieve long-standing members of the burden of their subscriptions, particularly in their years of retirement.
Dr. Ida A. Browne resigned as a member of Council in April, 1950. Council expressed appreciation of her service on the Council during the past ten years.
Professor P. D. F. Murray was elected to. fill the vacancy on the Council caused by the resignation of Dr. Ida Browne.
Dr. G. D. Osborne was granted leave of absence from the Council for six months from ist January, 1951.
Dr. A. B. Walkom attended the Pan-Indian Ocean Science Congress at Bangalore in January, 1951, as one of the Australian delegates.
Miss Mary Tindale was appointed the Society’s representative at the VIIth Inter- national Botanical Congress in Stockholm, 1950, and accredited observer at the International Union for the Protection of Nature Conference. Miss Tindale has informed the Society of a number of points dealt with by the Nomenclature Committee and offered to answer questions concerning special points in changes of the Rules.
The total net return from Science House for the year was £597. In future halt- yearly meetings and half-yearly accounts and audits of the Science House Management Committee will be abandoned and only annual meetings will take place. A survey of the libraries of the owner-bodies of Science House has been made by the State Library Board of New South Wales at the request of the Societies’ Library Committee, and a report thereon was received.
Following a request from the Information Service, Commonwealth Scientific and_ Industrial Research Organization, which had resulted from one of the recommendations of the Royal Society Scientific Information Conference (July, 1948), the Council decided that a synopsis of each paper published in the Proceedings would appear below the title of the paper. Such a synopsis will not interfere in any way with the arrange- ment of the paper but will facilitate the reading of the papers in the Proceedings. These authors’ synopses will be introduced in Volume 76 for 1951.
Preservation of Natural Areas—The Joint Committee of the Royal Zoological Society and Linnean Society considered another survey, in this case at the Spencer’s Creek Dam site. The Snowy Mountains Hydro-electric Authority offered to supply
PRESIDENTIAL ADDRESS. : iii
transport and accommodation for a field party for the projected Natural History survey of this area. Under the leadership of Dr. W. R. Browne, and with the help of a grant of £75 from Mr. BH. J. Hallstrom, a party of eight geologists and biologists visited the Spencer’s Creek area during the period 17th to 30th January, 1951. A report on this survey is now in course of preparation. It is considered that an initial survey of this kind in any area shortly to be changed completely by the construction of a large dam will provide valuable basic data for continued observation of the changes in the natural history of the area which must inevitably take place during and after the construction of the dam. It is hoped that further observations will be possible every year until the new environment has reached a state of stability in relation to a changed fauna and flora.
The Society again supported a request to renew the proclamation protecting certain wild flowers and native plants for three years from ist July, 1950, and this has been done.
The Society has also supported appeals against the possible resumption of land within the Muogamarra Sanctuary. Similar action has been taken in a number of other cases wherein it appeared that natural areas within reserves were to be thrown open for development.
Commonwealth of Australia Jubilee Celebrations—The Society is co-operating with the Royal Society of New South Wales and other scientific bodies in the planning of a conversazione in the Great Hall of the University of Sydney on 18th April, 1951. An exhibit dealing with the early natural history in the State will be our contribution.
We offer congratulations to: Dr. I. M. Mackerras, on the award of the Clarke Memorial Medal of the Royal Society of New South Wales for 1950 for his work on Diptera; Dr. T. B. Kiely, on obtaining the degree of D.Sc.Agr., and the award of the Royal Society’s Medal for his work on the Black Spot of Citrus; Mr. Robert Endean, Miss Janet Harker and Miss Jean Liddell, on obtaining the M.Sc. degree of the University of Sydney; Miss Adele Millerd, on the award of a research fellowship which would enable her to study at the California Institute of Technology; and Miss Muriel Morris, on obtaining the degree of M.Sc. of the University of Sydney and receiving the award of an International Federation of University Women Fellowship to study at the University of Oxford.
Linnean Macleay Fellowships.
In 1949 the Council reappointed three Fellows for 1950, viz.: Miss M. Hindmarsh (Botany), Miss A. Millerd (Biochemistry) and Miss M. Morris (Zoology).
Miss Hindmarsh continued her investigation of mitotic poisons during 1950. In particular the effects of phosphates and nitrophenols were studied. Investigation of an unexpected result obtained when using phosphate buffers to control pH showed that phosphate inhibited mitosis in a manner similar to sulphanilamide inhibition. A similar result had been reported recently by I. Galinsky in the Journal of Heredity. Further work on this inhibition indicated that, although phosphate had a specific effect on cell division, other salts in an unbalanced solution may show a similar effect. 2,4-dinitro- phenol upsets cell division by delaying or inhibiting spindle formation and by inducing stickiness in the chromosomes. It has been found, however, that dinitrophenol acts as a slow fixative, gradually killing the cells, and not as a specific mitotic poison. The action of mononitrophenols which have been reported to have a “colchicine-like’ action on cell division is being investigated, since it is not known whether they too kill the cells.
Miss Millerd continued her investigation of the succinoxidase and carboxylase of the potato tuber. The investigation of the carboxylase activity of the potato tuber was carried along various lines: (i) the preparation of the enzyme, (ii) the splitting and reconstitution of the enzyme, and (iii) isolation of the product of reaction. Study of the succinoxidase system had almost reached finality when Miss Millerd was awarded a research fellowship which would enable her to study at the California Institute of Technology, and resigned her Fellowship as from 30th September, 1950.
iv x PRESIDENTIAL ADDRESS.
Miss Morris completed a study of the life-history of one of our commercial prawns, a new species of Metapenaeus. The interesting point about this work is the fact that it demonstrates conclusively that there is a commercial prawn species breeding in our coastal lakes. All other well-known species of commercial value definitely go to sea to breed. It is hoped that this work will be published shortly. Miss Morris was awarded an International Federation of University Women Fellowship and resigned her Fellowship as from 14th July, 1950, to continue her studies on plankton at the University of Oxford.
The Council reappointed Miss Mary Hindmarsh to a Fellowship in Botany for 1951 and appointed Mr. N. C. Stevens and Mr. T. G. Vallance to Fellowships in Geology for 1951.
Miss Hindmarsh proposes to continue with the investigation of mitotic poisons on plant cells. The work on the effects of phosphate and dinitrophenol on cell division will be completed. Levan’s results will be checked by testing the cytological action of nitrophenols and other related compounds, especially those which are not inhibitors of phosphate transfer. -A further study of mitotic poisons of the spindle inhibitor type will be made, to look for any similarity in action or true colchicine effects. Work on the inhibition of cell division by sulphanilamide and its reversal by p-aminobenzoic acid will be continued as suitable material becomes available.
The subject of Mr. Stevens’ studies will be the Geology and Petrology of Bathylithic Intrusions in the Cowra-Gunning Region.
Mr. Vallance proposes geological investigations in the Ordovician Metamorphic Belt of Central-western New South Wales.
We wish all three every success in their year’s work.
Macleay Bacteriologist.
Dr. Yao-tseng Tchan arrived from Paris and commenced his work as Macleay Bacteriologist on ist August, 1950. An opportunity to meet Dr. Tchan was given to members of Council and others at afternoon tea on 2nd August.
Dr. Tchan’s work on the Gilgai and red-brown earth soils has made some progress. Generally speaking there is no N-fixation, but the addition of wheat straw modifies the general microflora and as the result less N is lost as ammonia and the humus content is increased. Experiments with wheat are not yet finished because the vegetative cycle of the plants being used has not run its full course. Also, during the work, estimation techniques for the N-fixation bacteria and the total soil flora have had to be modified. Dr. Tchan gained some knowledge of Australian ecological conditions as the result of an excursion with members of the Faculty of Agriculture. Dr. Tchan proposes to continue work on the two soils mentioned above, as well as certain soils of the Sydney area.
Obituaries.
It is recorded with regret that the following members died during the year: Protessor W. J. Dakin, Dr. B. L. Middleton and Dr. G. A. Waterhouse.
William John Dakin, Emeritus Professor of Zoology in the University of Sydney, died on 2nd April, 1950. Before coming to Sydney as Professor of Zoology in 1929 Professor Dakin had wide experience of research and teaching in Liverpool, Kiel, Heligoland, Norway, Naples and Perth. Throughout his life he had a profound interest in the problems of marine biology, which he pursued with far more than normal vigour wherever he happened to be. He introduced this field of research to his department at Sydney University and from humble beginnings gradually developed a pattern of investigation which culminated in the foundation of the Commonwealth Scientific and Industrial Research Organization Fisheries Laboratory at Cronulla. During World War II Professor Dakin transferred his attention to problems of camouflage and was appointed Technical Director of Camouflage for the Commonwealth of Australia, an activity he continued until the end of the war. Throughout his life Professor Dakin not only produced a considerable volume of research papers, but also wrote several books, both text and documentary, and gained a widespread popularity
PRESIDENTIAL ADDRESS. Vv
as a radio speaker, expounding in understandable terms to a very wide audience the problems of science he loved so dearly. Professor Dakin joined the Society in 1929, was a member of Council from 1930 to 1943, and was our President for the 1934-35 session.
Bertram Lindsay Middleton died on 16th October, 1950. Dr. Middleton was a graduate in Arts and Medicine of Trinity College, Dublin, who came to Australia following a world tour many years ago. In 1912 he commenced practice in Murrurundi, New South Wales, which he continued until the time of his death. Dr. Middleton had a lifelong interest in Lepidoptera and amassed a most extensive collection of Australian moths and butterflies amounting to some 10,000 specimens, which he bequeathed to the Australian Museum. Dr. Middleton had been a member of this Society since 1937.
Gustavus Athol Waterhouse died on 29th July, 1950. Dr. Waterhouse was a graduate in both engineering and science from the University of Sydney. He was Assistant Assayer at the Sydney branch of the Royal Mint from 1900 until it closed in 1926. His scientific work is best known from his long study of the Lepidoptera, which culminated in the production of the books “The Butterflies of Australia” (in collaboration with Mr. G. Lyell) and “What Butterfly is That?”. Many other contribu- tions to our knowledge of Australasian Lepidoptera have been published in various journals, and the collection of Australian Hesperiidae he assembled in the Australian Museum is undoubtedly one of the world’s finest. Dr. Waterhouse was Keenly interested in the affairs and management of a number of scientific societies, and it was in this field that he made outstanding contributions to the organization of scientific work in Australia. He first joined the Society in 1897 and was our President for the period 1921-23. The Society is particularly indebted to him for his supervision of our finances during the period 1926-43, when he acted (except during 1928-29) as our Honorary Treasurer. In 1943 ill health forced him to resign from the Council and he was elected a Corresponding Member. The sound management of the Society’s finances carried out by Dr. Waterhouse has become increasingly obvious since his retirement from active participation in the Society’s affairs, and we are indeed grateful for the foresight he displayed in building our finances to a level which makes it still possible to ‘carry on without obvious retrenchment despite ever-rising costs for all our activities.
THE PROBLEMS OF INSECT QUARANTINE. 1. Definition and Introduction. - F 2. Insect Pests in Australia: (a) Exotic species now present in Australia; (b) Australian insects of importance in other countries. 38. Recent Instances of the Spread of Exotic Pests within Australia. 4. Evidence of the Introduction of InsectS into other Countries. 5. Exotic Insect Pests not yet introduced into Australia. 6. The Reality of the Economic Consequences of Pest Insect Introductions. 7. Reecapitulation of the General Problem. 8. Can Quarantine Procedures be Justified? 9. The Development of Insect Quarantine Legislation. 10. The Broader Aspects of Insect Quarantine.
1. Definition and Introduction. |
_The concept which lately has been described as insect quarantine simply implies considerations of the prevention of entry of noxious insects into areas where they are not known to occur, whether such areas be geographically or politically limited. Initially of course the enphasis is on the prevention of such entry into countries whether they be continents, major geographical units of continents, or islands large or small. Despite this initial emphasis, problems of prevention of spread of noxious insects within geographically or politically limited areas also arise and are generally considered within the field of insect quarantine. :
The basic problems are further complicated by the fact that certain of the harmless or relatively harmless insects of one country may prove to be pests or even serious hazards to the economy or health of another country, simply because a different set of environmental conditions may make an enormous difference in the level of population
B
vi PRESIDENTIAL ADDRESS.
attained and in the ecological relationships of the insect and the plant or animal life of the new environment. Furthermore, the insects themselves may not be intrinsically noxious but may act as vectors of human, animal or plant diseases and, for this reason, may be regarded as undesirable immigrants.
In actual fact there are natural ecological barriers of varying magnitude which, in themselves, offer considerable opposition to the spread of insects from one country to another, the most effective of these being the various oceans of the world. Deserts and mountain ranges operate in the same way, and tracts of country ecologically unsuitable to or otherwise uninhabited by the host plant or animal may also form barriers.
Such natural barriers have not proved adequate to prevent the spread of insect pests from country to country, even when separated by wide oceans, since the artificial methods of transfer by ships or aircraft are readily available. The tempo of such exchanges does seem to have increased very markedly with each increase in the speed of transport. For instance, Deputy (1948) claims that few field insects of any importance, other than the Hessian Fly and the Codling Moth, had established them- selves in the United States of America prior’ to 1850. In his opinion, few insects other than stored product pests were able to survive the lengthy journeys of the old sailing vessels, and it was not until the advent of steam vessels that the majority of exotic pest species now present in America were able to reach there. The story of recent introductions of insects into Hawaii, particularly by aircraft, is one to be told at a later stage, but, when unfolded, it does disclose an infinitely greater rate of exchange of insect species than obtained up till a few years ago, when extensive intercontinental air traffic was not in operation.
It is contended, then, that we are now, even more than previously, faced with the necessity of devising artificial protective measures, and some, but not all, of these must have a legislative content.
Most countries have varying enactments governing the entry of insect pests, and such legislation is usually embodied in Plant Protection Acts or Quarantine Regulations for Animal and Plant Diseases and Pests. Further, such legislation may be under the administrative control of various authorities such as departments of health or of agriculture.
As sometimes happens with a principle or, activity which has undergone gradual development over a period of years, the arguments for and against these become obscure and it may be found that the underlying reasons for certain actions appear to have been accepted without being adequately stated. Such a position may arise particularly when a system of legislation is built up item by item on specific issues, rather than being the result of a full investigation of a general problem at any particular time. Furthermore, when we are dealing with precautionary measures to prevent the entry of undesirable insects, or in the closely related field, plant diseases, it is particularly difficult, when such measures are successful, to establish that any real danger was imminent. Perhaps the most important evidence we can offer is the result of lapsed quarantine precautions due to the exigencies of World War II.
It is for these reasons that it has been felt worth while to review the activities of insect quarantine measures and precautions, particularly as they affect Australia, and endeavour to establish whether or not we have a great deal to gain by the continuance of such activities. ’
Australia, with its distinctive fauna and flora, and complete lack of agriculture and animal husbandry prior to the advent of the white man, and with her relatively brief period of colonization, should be a very favourable area for the investigation of the economic consequences of the introduction of noxious insects. Many of its insect pests are obvious introductions; some are indigenous and, moreover, the results of the emigration of some of its indigenous insects to other countries are well known. Its disadvantages are its size and the complexity of its insect fauna, which remains inadequately studied in most groups as compared with Hawaii, with a small island area and relatively small and better known insect fauna. However, the diversity of the
PRESIDENTIAL ADDRESS. vii
environments presented within Australia, as well as the diversity of its primary industries, may in some measure compensate for these disadvantages.
Finally, I should remark that in presenting this address I have in mind not the entomologists or even biologists who may happen to hear it, but rather those whose studies have followed different disciplines and yet who may feel that some elucidation of this problem is worthy of a few minutes of their attention.
2. Insect Pests in Australia. (a) Exotic species now present in Australia.
The noxious insects of Australia are partly indigenous species which have trans- ferred themselves from native hosts, either animal or plant, to introduced animals or to plants of agricultural or horticultural significance, and partly exotic species of insects which have entered the Commonwealth since 1788. There is no question concerning the relative importance of the two, the introduced pest species being far more numerous and damaging.
It is difficult to learn much about those pests which became established during the first hundred years or so of our history, and it is even difficult to pinpoint the entry of those which have appeared within the last twenty years. On the other hand, there is some information available concerning those pests which have been intercepted at the occasion of entry, but here we find a regrettable tendency to believe that such abortive introductions could not have established themselves in any case. That such a belief has no basis in fact is evidenced by a selection of cases cited at a later stage of this address.
However, let us consider those pests which may safely be considered to have entered Australia within the past 180 odd years. In the case of those which have been recorded from Australia over a long period, the evidence that they are exotic is simply that they have been known at least as long, if not longer, elsewhere and that they are associated with, particularly, plants which are themselves definite introductions to Australia.
One of the earliest Aphids to arrive in Australia was the Woolly Aphis of Apple, which was recorded as early as 1846 in Victoria. The Aphididae generally are of special interest since no native species are known and yet some 15 or more introduced species are established pests of a variety of plants. This is a remarkable record for one family, but when we consider that most Aphids over-winter in the egg stage one realizes how easily they might enter a new country on imported plants. Much the same applies to the scale insects, which have a long period of attachment to the plant host and hence considerable opportunity for transfer, with their hosts, from country to country. Among the more important scale pests are the Red Scale, the San José Scale, the Mussel Scale, and the White Wax Scale. All of these are definite introductions.
The Green Vegetable Bug has only been known in New South Wales since about ~ 1911, but has become a widespread pest in the intervening years. Similarly the Peach Tip Moth first appeared in the Sydney district about 1909 and has since spread through- out most coastal districts.
The Mediterranean Fruit Fly was first discovered in Western Australia in 1896 and the Brown Vegetable Weevil in Victoria in 1905. An earlier introduction was the Maize and Tomato Caterpillar, the first record of which dates back to 1858.
Our domestic fleas are introductions despite the fact that there are many native species. Most of our mosquitoes are indigenous, but the two most noteworthy domestic species, Culex fatigans and Aédes aegypti are undoubtedly introduced and have been here for many years.
It is safe to say that most, if not all, pests of stored products, grains and fabrics have been introduced with certain of the materials they commonly infest. The same applies in large measure to pests of stock and other domestic animals. Taken together with the pests associated with the introduced plants of agriculture and horticulture, the sum total of imsect pest species which have entered the Commonwealth from elsewhere is a very formidable one.
viii PRESIDENTIAL ADDRESS.
(b) Australian insects of importance in other countries.
The earliest and most often quoted case of an indigenous Australian insect becoming a pest elsewhere is that of the Cottony Cushion Scale. In Australia this scale appears to be restricted to the members of the large genera, Acacia, Pittosporum, Casuarina, Grevillea, Hakea and possibly other native Australian plants. When introduced into other parts of the world it almost immediately extended its host range to include Citrus, Prunus, Pyrus, Robinia, Vitis, Laurus, Magnolia, Quercus, Buxus and many other surprisingly different food plants (Hssig, 1948).
The introduction of the Cottony Cushion Scale to California took place about 1868, and it soon became one of the most serious pests of Citrus and later appeared on many other fruit crops and ornamentals. By 1890 it had killed hundreds of thousands of orange trees.*
Not so widely known are the cases cited by Miller (1948). ‘‘The Blue-gum Chalecid (Rhicnopeltella .eucalypti)” is “a Tasmanian insect established in New Zealand, where it is actually destroying, throughout the country, over a period of years, the Tasmanian blue gum (Hucalyptus globulus) but does not attack any other species of eucalypt. On the other hand the various species of Hucalyptus are attacked by the scale, Hriococcus coriaceus, the Chrysomelid beetle, Paropsis dilatata, and the weevil, Gonipterus scutellatus—all Australian species normal to the hosts. The scale actually kills the trees, especially the more susceptible Hucalyptus globulus, but little harm is caused by the beetles except that the weevil may produce a pronounced stunting of growth”. Certain Australian termites, Coptotermes acinaciformes and a species of Porotermes, have also been introduced to New Zealand within the last twenty years, and the eucalypt weevil, Gonipterus, has also caused severe damage to eucalypts in South Africa following its introduction there from Australia.
3. Recent Instances of the Spread of Exotic Pests within Australia.
The time of introduction of most exotic insect pests cannot be established for many of our major pests, either because their early establishment passed unnoticed some time during the last century, or because their importance as pests did not coincide with their early distribution in the Commonwealth. In regard to the latter it is possible that the Sheep Blowfly (Lucilia cuprina) did not achieve pest status until long after its arrival, such status being conditional on changes in the type of sheep developed in the country.
Recent introductions are few in number, and this may be a chance occurrence or a tribute to the quarantines which have operated for the past forty odd years. Evidence derived from elsewhere would almost conclusively establish the validity of the latter viewpoint.
We must look particularly to these more recent cases for evidence of what actually - happens following a new pest introduction, and one of the most illuminating is the case of the Buffalo Fly.
In one respect the Buffalo Fly is falsely entered in this class, since it seems most probable that it entered Australia as far back as 1825, when buffaloes came to the Northern Territory from Timor. However, the pest remained confined to the Darwin area for many years, and it was not until 1912 that Gilruth was the first to suggest that this fly might eventually prove a major one of cattle. Extensive movements of cattle in the north, which took place in succeeding years, distributed the fly over a much wider area. In 1927 it extended from Broome in Western Australia to the western border of Queensland, and in the following year it entered Queensland south of Camooweal. The range of distribution of Buffalo Fly in Queensland advanced and receded from time to time in the ensuing years in concert with cattle movements and seasonal conditions. A series of wet seasons from 1939 to 1941 resulted in the fly
* Fortunately for our international reputation this pest was eventually brought under complete control in California and in the other countries to which it had been introduced, by the introduction from Australia of a predatory ladybird beetle.
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crossing the low rainfall hinterland of the Gulf of Carpentaria, and once across this climatic, but intermittent, barrier its progress to the coast and southward down the eastern coast of Queensland, almost continuously populated with cattle, has been unimpeded (Belschner, 1946).
The history of Buffalo Fly in Australia provides a number of most interesting lessons, although of course we have only gained our wisdom after the event. The adult fly itself has an almost continuous association with cattle and only survives away from its host for a matter of 24 hours. Hence it is most unlikely that adults could be introduced unless cattle were being imported at the same time. The larval stages could survive and develop in cattle dung, but again it is most unlikely that such material would reach Australia without the importation of cattle. Given this knowledge, prevention of its entry would have been quite a simple matter.
Again, for almost 90 years the pest had a restricted distribution in the Northern Territory. At any time during this period an eradication programme might have been attempted, although it must be admitted that this would have had greater chances of success had modern insecticides then been available.
Further, the crossing of the intermittent climatic barrier south of the Gulf of Carpentaria took place in spite of fairly vigorous quarantine precautions in north- west Queensland, assisted, at least in part, by the lack of co-operation of those responsible for cattle movements.
Finally, it is interesting to note that once across this barrier the fly quickly extended its distribution to the hypothetical limits postulated for its distribution by Handschin (1932).
Compared with the Buffalo Fly, the spread of the Cabbage White Butterfly following its introduction, perhaps from New Zealand, was extremely rapid. It was first recorded in a restricted area of Victoria in 1939, following which it was found at Albury in 1940 and in Sydney in 1941, and thence spread rapidly to Queensland. A case of this nature emphasizes the urgent necessity for rapid decisions as to whether any attempts should be made at eradication following the first discovery of such a pest. Delay may mean the loss of all opportunity of eradication and leave us with just one more perennial pest species to be subjected to measures of control year after year. Further, the rapid spread through a number of States also emphasizes the tact that States distant from the site of introduction may really be equally concerned in the arrival, or in the eradication, of the pest within the area of early establishment in the country.
The Argentine Ant is again a recent introduction. It was first recognized in a Melbourne suburb in 1939 and in Perth in 1941. The first Sydney findings were in 1950, but it seems likely that it has been present here for at least three years. It is possible that three separate introductions were involved, but it is equally feasible that this pest has been unknowingly transferred from place to place, perhaps in potplants. There is no doubt, however, that this aggressive insect will continue to spread as opportunity offers (it rarely flies), unless eradicated or most carefully controlled.
In these cases at least we have very tangible evidence of introduction and subsequent spread within Australia. Similar events have taken place many times in the past; others are perhaps still going on. But it is:such cases of rapid dispersion that make it clear that widespread distribution following an introduction is possible and, indeed, is almost inevitable, although in many cases the time necessary for obvious dispersion may be a very variable factor.
' 4. Evidence of the Introduction of Insects into other Countries.
In the field of medical entomology clear evidence is available of the transfer of pest insects between the continents of Africa and America across the formidable barrier presented by the Atlantic Ocean.
The chigoe flea, Tunga penetrans, which occurs in the tropical and subtropical. areas of North and South America, including the West Indies, has been established in Africa for less than eighty years. It is not difficult to envisage how such an insect
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with a semi-permanent attachment to the body of man or pigs should effect its transfer from America to Africa.
On the other hand, it is a far more difficult matter for a mosquito. to traverse the same ocean, yet this has been accomplished by at least one non-domestic* species, Anopheles gambiae. This potent African vector of malaria reached Natal in Brazil about 1930 and in the ensuing years was responsible for epidemics of malaria of the greatest importance.
We may say that these cases all come from the past and that all that is likely to happen has already happened. A counter to this view comes from our knowledge of what has happened in Hawaii in very recent years.
Swezey (1948) discusses 31 species of insects which became established in Hawaii during and since World War II. Included are a variety of moths, wasps, beetles, bugs and flies, and a grasshopper and a cricket. Some of these have already been shown to be devastating pests, others are potentially dangerous, and some, on the other hand, are beneficial. It is revealed that some of these must have come from U.S.A. and others from various parts of the Western Pacific Zone.+ A significant number of these introduced insects were first found in the vicinity of Pearl Harbour, “which could be accounted for by reason of the excessive increase in shipping and airplane arrivals at this military centre, and the consequent great difficulty of carrying on quarantine inspections”. No attempt is made to include the large number of interceptions of migrant insects made by entomologists in their quarantine inspections. Another point of interest is the fact that-many of the first records of these introduced insects came from light traps which have been operated in the Pearl Harbour area with the express purpose of gaining prompt knowledge of the establishment of any anopheline mosquitoes, an event which has not so far taken place.
It is perhaps of interest to mention two of these introductions which are not known to be detrimental. One is the introduction of the wasp Humenes latreillei petiolaris (Schulz) from New Guinea. This wasp has effected a considerable measure of control on the moth Anacamptodes fragilaria (Grossbeck), which itself reached Pearl Harbour from California only two years prior to the wasp which has proved such a valuable addition to the fauna of Hawaii. Such a happy set of circumstances is unlikely to occur with any frequency, but it is interesting to record that a chance introduction from the east of a potentially destructive pest is brought under control, at least in part, by an equally chance introduction from the west.
The other of special interest is the introduction of an apparently harmless wingless grasshopper, Paraidemona mimica Scudder, from U.S.A., probably Texas. This is one of those cases where it is difficult to imagine how a wingless insect, in the absence of any host animal or plant or other material with which it might be closely associated, should manage to join a ship or an aircraft in order to accomplish such a journey.
In an address to the Entomological Society of Queensland in September, 1950, a guest speaker, Mr. C. Pemberton, stated that a total of 219 species of insects were introduced into Hawaii during the years of World War II. The most important among these was undoubtedly the Oriental Fruit Fly, Dacus dorsalis (Hendel), which appeared first in 1945 and rapidly became established throughout the entire group. Its attacks on avocardos and mangoes virtually ruined these industries until 1950, when the work of a team of 40 men on a half-million dollar budget started to show results, principally from the introduction of 15 different parasites gathered from various countries of the Western Pacific Zone. The Oriental Fruit Fly is one of those particularly devastating pests when divorced from its natural controls, and is capable of breeding in all sorts of different fruits and nuts (more than a hundred different host plants
* Certain domestic species, e.g. Aédes aegypti, have become tropicopolitan or, Culex fatigans, almost cosmopolitan. Such species which are likely to breed in drinking water might easily have been distributed in water barrels in the days of sailing vessels.
+ Of the 31 species discussed, 11 have come from the U.S.A., 6 from Guam, 5 from the Philippines, 5 from other Pacific islands or the Orient, while the origin of the remaining 4 species is uncertain.
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were recorded in Hawaii—Cooley, 1950) and should by no medns be considered as just another fruit fly. :
We have mentioned before the claim that the tempo of insect transfers from country to country underwent a marked increase with the advent of steam navigation. Then, subsequent to the more or less general introduction of quarantine precautions in the first decade of this century, there did appear to be a slowing up of such transfers. For instance, only four major insect pests have become established in the U.S.A. in the quarantine period 1912 to 1948 (Deputy, 1948). Of these, one, the Mediterranean Fruit Fly, has been eradicated and two entered from Mexico, the Mexican Fruit Fly and the Pink Bollworm. The fourth, the White Fringed Beetle, appears to have entered from South America.
In contrast to this, some 30 major pests entered and became established in the U.S.A. during the fifty years prior to the introduction of quarantine precautions.
How, then, do we reconcile the Hawaiian experience with such a trend? Admittedly, quarantine services were in part disrupted during the war, but beyond this we saw for the first time the real effect of extensive air transport. There seems no question that the increased speed of air transport over all previous methods has again brought in its train another and even greater increase in the tempo of insect transference from country to country and has presented us with quite a new set of problems.
At this stage I feel it would be pertinent to review the role of aircraft in insect pest introductions. To those who travel by aircraft, particularly in the northern half of Australia, the frequency with which mosquitoes and house flies are encountered inside passenger cabins is well known, but the extent to which other insects travel inside aircraft is not generally appreciated.
A number of detailed surveys of the insect passengers in aircraft have been made by various workers in the U.S.A. Hughes (1949) gives in considerable detail the entomological findings from a large number of aircraft entering various airports in the U.S.A. from other countries for the 10-year period 1937-47. Out of a total of 80,716 planes inspected during this period, 28,752, or 356% of the total, harboured arthropods. From the same total 3,873 or 4:8% harboured mosquitoes. The total number of arthropods found in this period was 106,106, of which 16,846 were alive, and this in spite of the increasing use of disinsectization measures.
The same author records that 12,825 mosquitoes were found during this period, representing 10 genera with a total of 73 species. Of these, 48 were indigenous to one or more of the areas at which they were intercepted, but 25, or 34:2%, of the total number of species, were truly exotic. Some of these were, of course, South American, but a number must have travelled considerably further and nine of them undoubtedly embarked on their flight at some point in the Western Pacific Zone. The distances involved would necessarily be from 4,000 to 6,000 miles.
For one year (1945) the total arthropod records comprised “taxonomically deter- mined categories consisting of 20 orders, 191 families, 680 genera and 524 species’, and within the class Insecta, exclusive of biting mosquitoes, 17 orders, 188 families, 670 genera and 491 species were determined, not all catches being identified as far as the species.
Admittedly many of these insects arrived at their destinations dead, but approxi- mately 15% were alive, despite, I must emphasize, the current disinsectization measures applied up till the time of disembarkation.
Another type of evidence of survival of insects in aircraft has been provided by Laird (1948), who transported mosquitoes in cages by aircraft from New Zealand to Japan and back. Of 75 female Aédes notoscriptus, 60% survived the 18-day journey of more than 12,000 miles over a wide range of conditions, and the average life span of those which survived was 61 days. Other details are given, but the above is surely sufficient to establish the ability of mosquitoes to withstand successfully long journeys by air.
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5. Exotic Insect Pests not yet Introduced into Australia.
A system of insect quarantine regulations and the precautions that go with these would not be justified if it could be shown that there were few, if any, major pests still occurring elsewhere which had not so far reached and successfully established themselves within the Commonwealth.
A little reflection on the part of entomologists will call to mind quite a formidable list of insect pests, well known elsewhere in the world, which have not yet appeared in Australia and yet would certainly occasion us serious trouble in the fields of agri- culture, animal husbandry or health.
Among the more obvious plant pests are the European Corn Borer, the Hessian Fly, the Oriental Fruit Fly, the Mexican Fruit Fly, the Boll Weevil, a number of Vine Moths, certain Sugar Cane Borers, the Colorado Potato Beetle, the Apple Capsid, the Pineapple Mealy Bug, Chinch Bugs, the Japanese Beetle, the Gypsy Moth, certain Rice Borers, the European Red Mite, Sirex Wood Boring Wasps and Dry Wood Termites.
In the veterinary field obvious exotic pests, the exclusion of which is most important, are the Warble Flies, the Screw Worm and certain species of ticks other than those already in Australia.
From the viewpoint of human disease we would be most concerned in excluding such potent vectors of malaria as Anopheles punctulatus punctulatus, Anopheles sundaicus and Anopheles gambiae, although quite a number of other species would be considered dangerous. Other arthropods to be excluded would be Tse-tse flies, certain ticks (e.g., Ornithodorus) and blood-sucking bugs (Reduviidae).
Particularly in the agricultural field, closer study would reveal an infinitely greater list of pest species one would wish to see prevented from entering the country. However, in each field we have mentioned only the most obvious of the recognized undesirables, and it must be emphasized that other problems exist of an equally serious nature. These are worthy of specific citation.
(i) Phytophagous insects of little or no status as pests in their native environment may be particularly devastating in a new environment or new host-relationship.
_ (ii) The danger of a plant insect pest does not always lie in the direct damage caused, but such insects may introduce with them a new plant disease. On the other hand, the disease may be present without an adequate vector species, which may find an opportunity of entering at some later stage. Our major concerns here are with plant-sucking bugs and virus diseases.
(iii) Again, in the case of human diseases particularly, we may find the vector well established without the disease, as in the classic case of Yellow Fever. One method of introducing the disease is in infected vectors, a possibility which increases with faster means of transport and the diversification of transport routes. On the other hand we may have the disease and certain of its vectors, but would still find a far more serious problem developing from the introduction of more potent vectors of the same disease.
(iv) Further, it is worth emphasizing that species differences are often of the greatest importance. The fact that we have present in the country certain ticks or certain mosquitoes is no reason for complacency concerning the possibility of the intro- duction of other species of these groups, and this applies time and time again in a wide variety of groups and genera.
(v) Finally, even though a pest is already present in the country, it may not necessarily be widely distributed and its reintroduction at other points may be just as damaging as a de novo introduction. Such circumstances can readily be visualized in the case of a pest of the nature of the Argentine Ant.
6. The Reality of the Consequences of Pest Insect Introductions. Following the introduction of Anopheles gambiae into Brazil in 1930 the loss in human lives directly attributable to this introduction was considerable, reaching an estimated total of 14,000 to 20,000 people. For the State of Rio Grande do Norte the estimated deaths exceeded 5,000 out of a population of 243,000 odd. (This State is only a portion of the total area invaded by A. gambiae and the estimates were made in
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1938, a year before the intensive anti-gambiae campaign commenced). In the same area more than 51,000 cases of malaria were estimated to occur and the economic losses occasioned by the debilitated population were considerable. To these consequences must be added the actual 3,000,000 dollar cost of the eradication of this species from the invaded area.*
Again in 1944 Anopheles gambiae caused more than 32,000 primary cases of malaria, of which almost 1,800 were fatal, after its introduction to Upper Egypt was first noticed in 1942. Here also a successful eradication campaign cost a total of £1,000,000 sterling.
Estimates of the economic losses due to insect pests have been made from time to time, but as they are usually based on reductions of yield for various crops they are not necessarily convincing. Estimates of the losses due to Sheep Blowfly were estimated to amount to £3,000,000 in this country during 1942, and this is rather more tangible than a loss of a percentage of a crop before its actual maturity. Even more convincing are figures given for control or eradication programmes. For instance, the Cattle Tick eradication programme on the North Coast of New South Wales had cost £3,000,000 up to the end of 1940.
Unequivocal evidence is also presented by those cases of almost complete annihilation of particular primary industries as was occasioned by the Cottony Cushion Scale on Citrus in California (see above) and more recently the ruin of the avocardo and mango industries in Hawaii by the Oriental Fruit Fly. In the latter case a sum of 500,000 dollars was allocated for the investigation of control measures.
Apart from such tangible cases there is no doubt that considerable losses are occasioned by most insect pests, even though they may be difficult to estimate con- vineingly. On the other hand, the actual cost of having insect pests, apart from such losses, is clearly indicated by the money spent on their control, and a partial estimate of this (exclusive of labour costs) is presented by the value of insecticides used. No accurate Australian estimates are available for insecticide consumption, but from the limited data of production of certain basic insecticides, and the market has by no means reached saturation for chlorine-containing insecticides at least, it would appear that, at a conservative estimate, two to three million pounds worth of insecticides are annually consumed in this country, and the real figure may be even higher.
Other figures on expenditure for individual pest control or eradication in American cases are quoted by Annand (1950). For example, one fairly recently established pest, the White Fringed Beetle, has occasioned the expenditure of about 1,200,000 dollars during 1950, to which we can add a total of some 9,000,000 dollars spent prior to 1949.
Generally, a perusal of appropriate sources of information does give more and more confirmation of the actual costs of insect pest introductions as being far-in excess of the costs of administering quarantine precautions. Up to a point increases in appropriations for such work do increase its efficiency, but beyond this it is a matter of training and knowledge.
7. Recapitulation of the General Problem. In reviewing the evidence presented above it is clear that the following points have been established: ' (i) That a very considerable transfer of insect pests has gone on in the past from country to country, even between those separated by wide oceans; and this has been largely accomplished through the agency of modern methods of transport.
* Soper and Wilson (1943) discount the theory that A. gambiae arrived by aircraft. One of the reasons (there are others) is that the first discovery, which had all the appearances of revealing a very recent introduction, was made at a distance of seven kilometres from the airfield with no foci of infestation closer to it. In this connection it is interesting to record that Mr. Hegener, a Dutch colleague, captured a live specimen of Anopheles bancroftii on the sixth floor of the Hotel Metropole, Sydney, in April, 1945. This species has not been found south of Queensland and is certainly not known to occur in the Sydney district. It is interesting to speculate just how this insect could have arrived at this location, a distance of more than three miles from the flying boat terminal and considerably more from the land plane airport.
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(ii) That, once introduced, insect pest species have proved themselves capable of rapid or delayed spread throughout large tracts of the invaded country.
(iii) That insects of little or no importance in one country may become major pests in another, or, in other words, it is not always possible to prejudge the importance of a particular species should it appear outside its native environment.
(iv) That the possibilities of insect transfers have increased with each marked improvement in methods of transportation, and that recent increases in the volume and diversity of routes of air transport have enormously increased the possibilities of further transfers.
(v) That serious economic losses are involved following the introduction of important pest species which may also be accompanied by serious losses of human life in the case of species of medical importance.
(vi) Finally, that even the reintroduction of existing species may have undesirable consequences either by extending the existing range of distribution of the pest or by the introduction of plant or animal diseases.
It seems obvious, then, that consideration should be given to the possibilities of preventing the entry of insect pests into countries new to them, and we do, indeed, find that insect pest quarantines have been in force in most countries for approximately the last forty years.
8. Can Insect Quarantine Precautions be Justified?
Wardle (1929) is prepared to contend that “it is doubtful whether any pest which is at present to the fore would have been prevented by quarantine Acts from gaining admittance to the country” had U.S.A. instituted Plant Quarantine legislation fifty years earlier than it did. He considers that “the possibility of legislative barriers really fulfilling their intentions rests upon a perfection of circumstances which, in the present imperfect condition of civilization, simply does not exist’”’.
Wardle raises these additional objections to legislative barriers: (q@) that it is rarely possible to foresee that a potentially dangerous insect is likely to become introduced; (db) that the cost of maintaining the required inspection forces may be out of all proportion to the possible losses that insect introduction may bring about; and (c) that such legislation may act in restriction of international trade and may be used as a weapon by particular agricultural sections ‘that desire economic protection.
The statement by Strong (1923), ‘the fact that no pest of major importance has become established in the United States since the passage of the Plant Quarantine Act demonstrates in a graphic manner the value of plant quarantine to the United States as a nation”, is discounted by Wardle on the grounds that the period of time which had elapsed since the passing of the Act—ten years—was not long enough. We have already quoted the results of thirty-six years of Insect Quarantine operation for the. U.S.A. (Deputy, 1948), which is surely sufficient answer to this objection, and we have also given the evidence provided by the wartime breakdown of quarantine precautions in Hawaii. ‘
That the required perfection of circumstances does not exist is an indefinite criticism implying, in part, lack of sufficient knowledge and lack of efficiency on the part of the persons charged with the duties of administering the relevant quarantine procedures. Neither of these difficulties is insuperable, but it is important that quarantine staffs should be adequately trained and suited to their profession.
As experience is gained it becomes increasingly possible to foresee possibilities of pest insect introduction, and as insect and related quarantines become more and more international in their outlook the less credence can we give to this objection.
The critical objection surrounds the relative costs of the administration of such quarantines as compared with the subsequent costs of control or eradication following the establishment of a new pest. When we consider that quarantine is applied in a limited number of ports to ships and aircraft and their cargoes instead of control measures over wide areas of territory or large acreages of crops there seems little room for comparison. Then, when we realize that the quarantine procedures cope with
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far more than just individual pest species at the one time, instead of separate control measures for a number of different pests, there can be no doubt that precautionary measures will prove far less expensive. The fact that eradication programmes are seriously considered following the early discovery of a new insect pest is a further denial of Wardle’s contention.
Finally, to say that insect quarantine legislation may be used as an economic weapon is not a criticism of the principles of quarantine, but one of the integrity of governments.
If these are the most serious objections to the promulgation of insect quarantine precautions then it must be granted that we have strong reasons, along the lines which have been previously discussed, for the maintenance of such precautions.
9. The Development of Insect Quarantine Legislation.
A full discussion of the history of the development of insect and plant quarantine legislation, since the two usually go hand im hand, would be out of place in this address. However, we should realize that such legislation is of comparatively recent date. In California the first promulgations were issued in 1881, for the U.S.A. as a whole not until 1912. ‘
In Australia the Federal Constitution of 1901 empowered the Commonwealth to administer quarantine (Clause 9 of 51). Under the Constitution the first Quarantine Act, which included a Plants Division, which in its turn dealt with insect pests, was passed in 1908 and took effect on July 1, 1909. Until 1921 the Act was administered by the Department of Trade and Customs, but in this year the Department of Health was established by order of the Executive Council and the administration of quarantine became a function of this department, wherein it still remains. The year 1927 saw the foundation of the Divisions of Plant Quarantine and of Veterinary Hygiene as separate divisions of the Department of Health.
Hxisting procedures do, of course, come under critical review from time to time, and in the case of the Australian Act the most important contribution is found in the Report of the Central Committee appointed by the Australian Institute of Agri- cultural Science to deal with Overseas Plant Quarantine (Nicholson et al., 1941). Here criticism of existing practice is combined with a detailed series of recommendations concerning improvements in staff and regulations. These are too detailed to be quoted here, but it should be pointed out that at least some of these recommendations have been incorporated in the Regulations under the Quarantine Act, particularly by Statutory Rules No. 92 of 1948 which gives wide powers for the complete destruction of noxious animals or plants following their detection on entry, and by Statutory Rules No. 78 of 1950, which requires the registration of approved authorities who may desire to import nursery stock under permit. ;
What is of more immediate interest to us in the historical development of insect quarantine precautions is the gradual change in approach that is evident in the legisla- tion of most countries, including Australia. :
Harly quarantine legislation provided for embargoes against specific known pests, at least from the countries where these pests were known to occur, or restricted entry, subject to the fulfilment of particular provisions, of materials likely to harbour known pests. Inspection on arrival was provided for and any materials subject to restrictions would only be passed through quarantine if found free of the suspect insects, or following suitable treatments to destroy them. Providing the inspecting officer has been suitably trained and knows what to look for in any particular type of material, or has ready access to sources of appropriate information and identification, such a system works reasonably well. There are, of course, some cases wherein it is not possible to detect the pest insect by inspection, e.g., Narcissus Fly in various bulbs. To cope with such cases the material may be fumigated or subjected to heat treatment before shipment, this being done at a time when the bulbs will suffer less damage from such treatment. In other cases provision is now made for the, growing of suspect material under quarantine. This may be done by a Federal authority, as in the U.S.A.,
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or by the registered importers, as in Australia. A period of at least twelve months is then available for the detection of any ‘pests, disease or insect that may be incapable of detection on entry.
Various ways in which certain commodities, otherwise likely to be subject to embargoes, may receive treatment prior to their consignment or during their period of shipment are discussed by Annand (1950). Of particular interest is the shipment of pre-cooled and refrigerated apples in specially equipped ships from South Africa to the U.S.A., giving a complete mortality of fruit fly larvae, the most dangerous type of pest likely to be contained therein. f
Undoubtedly the various quarantine provisions increase in complexity as time goes on, but they do provide more and more protection against pest insects as they become revealed as potential dangers. The tendency is to channel all imports of suspect material through those agencies which would stand to lose most following the intro- duction of a pest of one of the materials in which they are most interested. And there can be little doubt that such restrictive precautions are ultimately understood to be protective to the importer as well as to the country generally.
There are still cases in which material is imported which is of no interest to anyone, and this applies to the various types of packing used in preparing goods for shipment. In such contingencies the onus is entirely on the quarantine service to see that such packing materials are free of pest insects.
Finally, following on the success of pre-flight treatment of aircraft leaving Hawaii for the U.S.A., particularly to cope with the danger of the introduction of Oriental Fruit Fly, similar arrangements are now the subject of negotiation between Australia, New Zealand and Hawaii. It is considered, on sound evidence (Cooley, 1950), that pre-flight inspection and treatment is a more effective way of dealing with possible insect passengers than similar operations carried out at the end of the journey and, moreover, they are more acceptable to airlines and passengers. There seems little doubt that extensions of this type of operation, will come about in the future, especially if reciprocal arrangements between countries can be agreed upon.
Except in the last case, we have dealt with problems usually associated with shipping. Insects harbouring in their host material is one problem, but insects purely as temporary passengers, .as they often are on aircraft, dissociated from any host materials, is an entirely different one. Consequently, in recent years, quarantine laws have had to cope with insects arriving in such a manner on aircraft. In the case of Yellow Fever the problem is so serious that International Air Navigation Agreements have been formulated.
Otherwise each country has added to its quarantine laws provisions regarding the treatment of aircraft to ensure that they arrive in an insect-free condition. Further precautions are often taken immediately after landing.
Annand (1950) foreshadows the wider use of point-of-origin inspection and clearance in the future, since it offers greater protection at less cost. He cites the proposal made in 1948 by the United States to the International Civil Aviation Organization. “Contracting States should make arrangements whereby one State will permit another State to station representatives of the public authorities concerned in its territory to examine aircraft, passengers, crew, baggage, cargo, and documentation for customs, immigration, public health and agricultural quarantine purposes, prior to departure for the other State concerned, when such action will facilitate clearance upon arrival in that State.”
Perhaps the most standard treatment in use at present for aircraft is the aerosol formulation G—382, containing 3% DDT, 5% of pyrethrum extract (20% pyrethrins), 5% cyclohexanone, 2% lubricating oil and 85% of Freon-12 at a dosage of 5 gm. per 1,000 cubic feet. Admittedly this dosage is not fully effective and pre-flight treatments, when carried out in the absence of passengers, are applied at four times this dosage (Cooley, 1950). Other formulations are discussed by McBride et al. (1950), who concluded that the best results were obtained by a combination of residual and aerosol treatments.
PRESIDENTIAL ADDRESS. Xvii
In the case of aircraft the problem is not a legislative one but one of technique. Considerable research has been undertaken to disclose a really effective insecticidal technique for application to this special problem, but even though routine aerosol treat- ments may cope with some of the more obvious Diptera in an aircraft, many other insects remain unaffected. This does not mean that the solution will not be found; eventually it probably will be. Automatic aerosol-dispensing systems for aircraft have been designed and are under trial, and there is little doubt that these would prove an improvement on hand application.
Another approach to the problem is contained in the Report on the Second Session of the Expert Committee on Malaria of the Interim Commission (W.H.O., 1948). This report includes the recommendation “that, whatever regulations be enforced regarding the disinsectization of sea- or aircraft, rigid anti-mosquito sanitation should, as far as practicable, be maintained within the mosquito flight-range of sea- or airports of the country to be protected, so that no imported mosquitoes will be able to survive”. The “reciprocal procedure, in the country of origin of such mosquitoes, should prove even _more effective.
The present importance of aircraft in the transfer of insect species: has already been emphasized. It is in this field particularly that uniform legislation and practice is desirable for all countries and varying procedures in different countries can only minimize the effectiveness of precautionary measures generally. The time available for such measures is so brief, and aircraft are liable to pass through many countries, so that changes in procedure will not only be confusing but are likely to evoke varying degrees of co-operation. ;
We hope, then, that uniform practices, based on critical research, will eventually be adopted on an international rather than a regional scale.
10. The Broader Aspects of Insect Quarantine.
We have discussed in some detail the overall background to the problem of insect quarantine, principally because it is a wide field about which relatively few have detailed .knowledge of all sections, even among entomologists, and also because its wide scope, covering pests of all horticultural and agricultural plants, stored products, the household, and pests of medical and veterinary significance is deserving of emphasis.
Some general discussion has been given to insect quarantine legislation, put a little more should be said about the implementation of such legislation.
The fact that a port-of-entry inspection service is required has been mentioned, but hand in hand with this must go an identification service for the less obvious of the suspect insect pests that may be intercepted,-and efficient treatment facilities for infested material. The identification service must be sufficiently skilled and knowledgeable to make rapid determinations, and the treatment facilities should be sufficiently flexible to cover a range of treatments for reasonable quantities of imported materials. Such facilities do vary from place to place, and it seems only reasonable that those of the Imajor ports of entry should be improved at least to the level of the most efficient.
Beyond this a quarantine service should also provide for prompt and effective action in the way of an eradication programme following the early detection of an insect importation which has escaped detection at entry. In the Australian Act this has been provided for in Statutory Rules No. 92 of 1948. It is not necessary that all the probable requirements for the implementation of such .a programme be held in waiting for such an event, but it is necessary that we should know where and how to marshal them when the occasion arises.
In the words of Annand (1947) “quarantines should have two functions, (i) to prevent the entrance of new pests or those not widely distributed, and (ii) to facilitate the movement of commerce by the application of treatments and other safeguards, when ‘ such’can be used, in lieu of embargoes. They are not intended to serve in place of tariffs, and they must be maintained on a biologically sound basis. To be fully effective they should be based on knowledge of the occurrence, distribution, abundance, hosts, and habits of harmful pests at home and abroad.”
xvili PRESIDENTIAL ADDRESS.
This introduces the general background to the operation of quarantine legislation, and Annand also stresses the need for exotic surveys of particularly important imports and the areas from which they come, linked with the findings of inspectors after the arrival of such imports.
In the detection of new pests Annand stresses the importance of initial surveys to establish what is present, and claims that these need to be exhaustive and co-operative and that greater allocations are required for the necessary taxonomic work.
Cooley (1947), writing particularly of the air traffic problem, defines three lines of defence: (i) pest surveys in foreign countries so that we may become familiar with species likely to be dangerous, (ii) inspections at airports, and (iii) continued surveys in the vicinity of ports of entry. The same author also recommends that all vegetation in the vicinity of international airports be continually. treated with DDT and other insecticides to prevent establishment of any pests which may escape from arriving planes and that various types of insect traps be maintained in the vicinity of all important air terminals. Such recommendations presuppose the maintenance of an entomological service at major air terminals.
In general, then, we are faced with the need for far more precise knowledge of insect pests on an international basis. For each country to gain its knowledge of exotic pests individually is more than a duplication of effort but, since few countries document their insect pest problems in a form suitable for quarantine purposes, such a procedure may be desirable, at least in special cases, for some time to come. Ultimately, when the type of information required by quarantine services is more widely realized, and there is co-operative effort on an international basis in the preparation of such data and the early circulation of information on new pests the need for exotic surveys will diminish.
But first of all we should know, and document, our own insect pest story, and this applies equally to those which are to us of major and of minor importance. Perhaps such information as we possess is available, but with six States in the Commonwealth and an equal number of journals recording agricultural pest information, the task of abstracting relevant details is a formidable one. The type of documentation envisaged is only accomplished when a specific allocation of staff and money is made for such a project, and it is one of our needs that the specific data on recorded pests, their distributions, habits, life histories, and animals, plants or other materials attacked should be gathered together in one series of publications.*
A prerequisite of such documentation is discussed by Hssig (1948), who stresses the need for insect surveys of literature, of collections, and of insect occurrence in the field on a scale sufficiently detailed to provide exact information for such areas with which any particular survey unit can cope. Here again we reveal the need of a wide basic taxonomic knowledge.
Finally, the efficient operation of quarantines depends not only on knowledge within the professional field but it also requires a general public realization of its purposes and a more specific knowledge of individual problems amongst those most concerned with goods likely to be subject to quarantine inspections.
Im other words, a public relations programme is also necessary, since lack of co-operation is usually based on ignorance.
In conclusion I should like to emphasize that the time has long since passed when we regarded insect, and also plant, quarantines as restrictive to the commercial exchange of various commodities. The basis on which they operate becomes biologically more sound as time goes on, and the needs of international commerce continually
* This implies a central information service as envisaged by Nicholson et al. (1941). It is pleasing to be able to record that for some years now the N.S.W. Department of Agriculture, through its Entomological Branch, has been bringing out an ,annual insect-pest survey which provides much useful information. For the past two years a section has also been devoted to pests intercepted in quarantine and this provides information of considerable significance.
PRESIDENTIAL ADDRESS. xix
demand that precautionary treatments and safeguards be developed. A. fuller realization of the problems involved is particularly desirable and there is further scope for the encouragement of co-operative effort amongst organizations not directly concerned in the implementation of quarantines.
References. ANNAND, P. N., 1947.—Preventive Entomology. J. Hcon. Ent., 40: 461-468. : , 1950.—Today in Foreign Plant Quarantine. J. Hoon. Ent., 43:139-145. BELSCHNER, H. G., 1946.—The Buffalo Fly—A Survey of the Position and Discussion of Control : Measures. Agric. Gaz. N.S.W., 57:149-152, 211-214, 218. Cooter, €. E., 1947.—International Air Commerce and Plant Quarantine. J. Hecon. Hnt.,
40: 129-132. —, 1940.—Plant Quarantine Activities in the Pacific, with Particular Reference _to the
7) Oriental Fruit Fly. Bull. State of Calif. Dept. Agric., 39:10-16. é Deputy, O. D., 1948.—Are Foreign Plant Quarantines Worth While? J. Econ. Ent., 41: 528-531. Essig, EH. O., 1948.—Insect Surveys in Relation to Quarantine and Control of Insect Pests.
J. Econ. Ent., 41:673-677. HANDSCHIN, H., 1932.—Investigations on the Buffalo Fly and its Parasites. O.S.I.R. Pamphlet
No. 31 (Australia). HuGHEs, J. H., 1949.—Aircraft and Public Health Service Foreign Quarantine Entomology.
U.S. Pub. Health Reps., Supplement 210:1-38. LairpD, M., 1948.—Reactions of Mosquitoes to the Aircraft Environment. Trans. Roy. Soe.
N.Z., 77: 93-114. McBribe. O. C., et al., 1950.—Treatment of Airplanes to Prevent the Transportation of Insects.
J. Hoon. Ent., 43: 66-70. Miturr, D., 1948.—In Report of the Fifth Commonwealth Hntomological Conference, p. 68.
(New Zealand Pests.) NicHotson, A. J., et al., 1941.—Plant Quarantine. J. Aust. Inst. Agric. Sci., 7: 143-146. Soper, F. L., and WILSON, D. B., 1943.—Anopheles Gambiae in Brazil. The Rockefeller
Foundation. New York. STRONG, L. A., 1923.—Western Views on Plant Quarantine. J. Econ. Ent., xvi: 266-270. Swezey, O. H., 1948.—Insect Invaders in Hawaii During and Since World War II. J. Econ.
Eint., 41: 669-672. WARDLE, R. A., 1929.—The Problems of Applied Entomology. Manchester University Press.
504-508. WHO ExPerT COMMITTEE ON MALARIA, 1948.—Report on the 2nd Session of the Expert Committee on Malaria of the Interim Commission. Bull. WHO, 1: 243.
s
The Honorary Treasurer, Dr. A. B. Walkom, presented the Balance Sheets for the year ended 28th February, 1951, duly signed by the Auditor, Mr. S. J. Rayment, F.C.A. (Aust.); and he moved that they be received and adopted, which was carried unanimously.
A vote of thanks to the Acting Honorary Secretary (Dr. W. R. Browne) and the Honorary Treasurer (Dr. A. B. Walkom) for their work for the Society was carried by acclamation.
No nominations of other candidates having been received, the Chairman declared the following elections for the ensuing year to be duly made:
President: Mr. A. N. Colefax, B.Sc.
Members of Council: Lilian Fraser, D.Sc.; Professor J. Macdonald Holmes, B.Sc., Ph.D., F.R.G.S., F.R.S.G.S.; Professor P. D. F. Murray, M.A., D.Se.; G. D. Osborne, D.Se., Ph.D.; T. C. Roughley, B.Sc., F.R.Z.S., and A. B. Walkom, D.Sc.
Auditor: S. J. Rayment, F.C.A. (Aust.).
A cordial vote of thanks to the retiring President was carried by acclamation.
xx
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SEROLOGICAL STUDIES OF THE ROOT-NODULE BACTERIA. Iv. FURTHER ANALYSIS OF ISOLATES FROM TRIFOLIUM AND MEDICAGO.
By Himary F. PURCHASE, J. M. VINCENT and LAWRIE M. WARD, School of Agriculture, University of Sydney.
[Read 28th March, 1951.]
Synopsis.
The present paper summarizes serological data accumulated over a period of about ten years in respect of reference strains of Rhizobium trifolii and Rh. meliloti. Over this time the antigenic properties of the organisms have shown a high degree of stability and the technique has proved useful for the typing of isolates in field and laboratory studies.
To obtain adequate typing it is necessary to distinguish flagellar and somatic reactions. The latter on its own permits more strains to be distinguished than the former, but maximum differentiation requires both to be taken into account.
Extending the method of analysis used in the earlier papers it has been found that the description of the 12 isolates of Rh. trifolii requires at least 2 flagellar and 9 somatic antigens. The corresponding figures for the 16 isolates of Rh. meliloti are 4 and 15.
INTRODUCTION.
Since the application of improved agglutination techniques to the study of root- nodule bacteria (Vincent, 1941, 1942) fair use has been made of these criteria for the classification and identification of serological strains in field and laboratory studies (Hughes and Vincent, 1942; Kleczkowski and Thornton, 1944; Vincent, 1944 and 1945; Purchase and Vincent, 1949; Read, 1950, private communication). Whilst there is no apparent relationship between serological constitution and the organism’s behaviour in association with the host, the method remains a useful technique for distinguishing and grouping strains, a means of studying field distribution, and for ‘labelling’ material for laboratory and field studies.
A comparison of our recent results with those reported in the earlier papers has shown a high degree of stability in antigenic properties; few cases have occurred where a culture has shown any significant change in this regard. The antigenic constitution appears in fact more stable than other characteristics (cf., for example, Kieczkowski and Thornton, 1944; Nutman, 1946).
The earlier papers from this laboratory included reasonably detailed analyses of several strains of both species. The number of fully studied strains has been added to in the intervening years so that a fair battery of testing sera is now available. It has been thought worthwhile, therefore, to record these further studies which provide the basis of our more recent investigations.
HE}XPERIMENTAL.
Organisms used for the development of antisera.—The isolates are identified by collection numbers and are mostly described in the earlier papers: Kh. meliloti (Vincent, 1941) and Rh. trifolii (Vincent, 1942). No, 204 has been added to the clover strains, having been obtained as “Clover F” strain from Dr. H. G. Thornton, Rothamsted Experiment Station, England. Rh. meliloti No. 52 originated from Medicago hispida var. denticulata growing at Warracknabeal, Victoria. Additionally it is worth noting that strains 7, 8, 10 and 12 came to us from the United States, the first three from. the collection of the University of Wisconsin, and the last via the N.S.W. State Depart- ment of Agriculture.
Methods—The methods of obtaining cultures and using them as antigens for the development and testing of antisera have been largely maintained as in the first paper (Vincent, 1941). It has been generally advantageous to obtain an earlier reading for ‘flagellar (H) agglutination at about one hour and advisable to check somatic (0) agglutination with heated antigen, particularly in the presence of flagellar agglutina-
Cc
2 SEROLOGICAL STUDIES OF THE ROOT-NODULE BACTERIA. IV,
tion. Occasional difficulties have been encountered with a less voluminous flagellar agglutination but, provided a two-day motile culture of sufficient density is used, the result has been almost always satisfactory. We have not found it necessary to distinguish H by the use of O-absorbed sera or by the removal of O antibody by heat, although some workers might well prefer this added check (Kleczkowski and Thornton, 1944). /
RESULTS. Strains of Rh. trifolii.—Tables 1 and 2 give the results for flagellar and somatic reactions respectively.
Two major groupings of flagellar antigens are revealed, one represented by strain 36, and the other by strain 46. Following the 1942 treatment these have been classed A and B respectively. The earlier paper also showed by absorption tests that the five members of the first group tested on that occasion were identical. Flagellar reactivity has now been found between 157, 161 and) 46—a result at variance with the earlier findings but supported by the agreement between reciprocal tests.
As has been found generally in these species, somatic antigens provide more groupings than do the flagellar. On the basis of what we now know of the somatic cross reactions of these strains the minimum number of somatic antigens would have to be extended from three to seven. Absorption tests have been applied in some detail to the 94-160 group of Table 2, and make it necessary to bring the number of antigens to nine. Some irregularities seem to be associated with antigen II of 94, 61, 111 and 161
TABLE 1. Flagellar Cross Reactions of Rhizobium trifolii.
Antigens. Sera. l 36 108 61 111 94 91 160 204 | 46 157 161 64 | | 36 58 63.8 28 PA 2 22 2 2 —_ = ae = 108 | 33 38 39 7, 2 33 2 2 ines als dea ae a 61 3 33 38 22 2 23 2 2 | —_ = = a 111 25 Be 33 23 2 22 2 2 — nna aG ae: 94 he 2 3 2 3 2 2 2 | —_ - Ree ee 91 33 33 3a 23 2 Be 2 2 | —_ = = Z 160 33 2 2 1 2 2 3 2 Pee ae eS = 204 3 2 2 2 2 2 1 3 ) = - aie eas ey el emee) een vee ete eur bath) APSO a og. a eel ee ee URE eb Th gen [SEE ES ES eee | | LGD roe = = = = = - - ! Pea Fe 3 0-1 ah Se Sa es Mea sey 2 p> |
Note-—Numbers indicate highest dilutions showing agglutination, viz. 1=1/25 to 1/50, 2=1/100 to 1/200, 3=1/400 to 1/800, 4=1/1600 to 1/3200 or greater. Top right-hand figure shows earlier result.
BY HILARY F. PURCHASE, J. M. VINCENT AND LAWRIE M. WARD. 3
and indicate the possibility that the antigen might be composite and its components variable in their relative proportions between strains and in the one strain at different times.
The findings can now be summarized:
Minimal Antigenic Composition.
Isolate. Flagellar. Somatic. 36, 108 ie ag Ae pd A I 94 ay es ah a A IN ay WARE 61, 111 Ha a we is A Oke WY
160 A IV, VII 91 A TII
204 A IX 64 B I
161 B LO TViveVali 46 B TIL
B VIII
157
Sera of the six strains underlined have been used for typing field isolates (Purchase and Vincent, 1949) and include representatives of both flagellar types and eight of the somatic antibodies.
t
TABLE 2. Somatic Cross Reactions of Rhizobium trifolii.
Antigens. * Sera. 36 108 64 | 94 61 161 111 160 91 46 204 157 l ! pavietolats ivy. 36 44 44 2 - — - ae. = = 25 S = 108 | 44 43 4 OR es Sg Sra = 2 ara ae | Py eel EER aM Se 64| 34 | 4 a= == = = - —\| - = a eS ae Baers ea. ooh eam ane bem omen Gy ne IE es 42 | 3 By We = = oe Ue RY; 161} — | - - 3 Ornate u 3 a ee eet ee Melee (eee, |= alls = 2 aoa Ne EO ce ne Fee seeen eee ame Or AIS cohuan Ion 7 gee ee wie a lea ———— ee 91 —_ — = = 44 AGS ak 3 = ig | | nee (gee bee Yee uel ellia rar oy eae ADs | se je = - => hie a een. Pca iat |
Note.—Numbers indicate highest dilutions showing agglutination, viz, 1 =1/25 to 1/50, 2=1/100 to 1/200, 3=1/400 to 1/800, 4=1/1600 to 1/3200 or greater. Top right-hand figure shows earlier result.
4 SEROLOGICAL STUDIES OF THE ROOT-NODULE BACTERIA. IV,
TABLE 3.
Antigens. Sera.
7 | 8 | 12 | 62 | 51 | 52 | .66 | 74 | 76 | 102 | 126 | 184! 10 | 47 | 101
~I ~ > 1 i
= rs 1 i cs w rs i
102 4 4 4 Cees 4 ARN Ni Aes eS 44) 4 4 ul ite = I
126 4 4 4 44 4 4 4 4 4 4 4 4 il =T || =
1349 | ae ape lira lwp Pal la la 0 liege on lane iy |e eee 0) 2 a el ee es ee |p | a | mf || | == AT Ape Dias oD, col Sian Cette th |e Sa hare = al 2 ea 54 oa gs AS er i (| atts EW fe ee eee | Pe ga ee at ee |
Note.— + =variable result at lowest titre ; code otherwise as for Table 1.
Strains of Rh. meliloti—Tables 3 and 4 give flagellar and somatic reactions of sixteen strains.
Most of the flagellar reactions are those previously seen in strain 27, but strain 10 has the major antigen of 47, and 101 is different from both ‘these groups. Using the notation of the first paper, 10 and 47 are described as Ab, the 27 group as bC and 101 is now characterized as D. The symbol Bb is used to describe a minor antigen that is commonly shared by members of the first two groups.
Somatic antigens provide a basis for further division. Approximate groups are represented by:
7, 27 and 62, 47 52, 102 126, 8, 12 134, 74 66 10, 76, 101 51 although there is a certain amount of cross reaction beyond these limits. Cross absorption tests have shown 7, 27 and 62 to be identical; they have, however, an antigen additional to those of 47 and the latter has an antigen not common to them. No. 76 also appears to have the same antigenic constitution as 101, otherwise there are differences even within the members of the groups set out. The group represented by 126, 8 and 12 shows a wide range of reactivity when they constitute the antigen, but outside the group itself reciprocal tests are mostly negative. It seems that the cells of
BY HILARY F. PURCHASE, J. M. VINCENT AND LAWRIE M. WARD.
or
TABLE 4. Somatic Cross Reactions of Rhizobium meliloti.
Antigens. = Sera. 27 7 62 47 52 | 102 | 126 8 12 | 134 | 74 66 10 76 | 101 | 51 oe ea aces eat 2) Stes Ee pe een bee he fire Nes (NS Pe a ae |e cae, || ee ea NDEs eS [a ev ree [est (5 Oi Eg as eo (ee A le ee Ree ea BB Bt ce dc Hee Maes aks ea aie (RR Ps a a ee 103 |) SEE SSS. S| ae Pe ee an ee 126 ea, Sipe pk eae 2 “9s! a oe ea 2 MS A 8} 2 es 3 8 = Lf Be a 4 d 4 3 PRE OX 2 3 3 12 ae SF Gh Oy 1°] 4 ho 5, & ae iss 4 2 3 3 reas ee ee ee Ee a gs! ai) = | sf 2) = ——— — —————| el — Ame el lear | oe |p |) ed 201 1a so joe fos Ps ; | [ees pees (| | pe | ea eee Po eo ge me —— a fa es eS eS SE 10 = = habs, ls vil aa (eee = = ae i | 4 3 4 1 «0 | 2 26) ee ee ae ee ae eee eee mms a lea | ie, sat ts I a aes ee ae ae ee
Note.— + =variable result at lowest titre, code otherwise as for Table 1.
this group are relatively easy to agglutinate, perhaps by a less specific antibody, and only reactions that have reciprocal tests in agreement have been used in stating somatic antigens. Cases 52 against serum 7, 66 vs. 134, 10 vs. 52, in which the reciprocal test is negative, have also been ignored. Antigen II, previously postulated as common to 66 and 74, would, on similar grounds, be regarded as a doubtful or minor antigen.
The data for cross reaction and absorption tests can be symbolized thus:
Minimal Antigenic Constitution.
Isolate. Flagellar. Somatic. 47 ms e 6 2 Ab I, Ill
10 ee = ie ve Ab IX, Si
Ys Ol, A> oe ae a oe be I, IV
52 25 ee = ae be III, V, XII 102 bc III, V, XIII 126 bC V, VIII, IX Pe be VIII, XIV
12 is an ae a bc VIII, XV 134 - By , ba be AW, IDs
74 be II, VI
66 bc II, VII
76 bc x
51 Le es a 2 be XI
101 Ae Sp ef. ah D XK
Sera of the strains underlined have been used in typing field isolates (Purchase, Vincent and Ward, 1950). These include the three flagellar groupings and all the somatic antigens so far revealed.
6 SEROLOGICAL STUDIES OF THE ROOT-NODULE BACTERIA. IV.
DISCUSSION.
These results emphasize the serological heterogeneity that exists within a ‘‘species” of Rhizobium. There are notably fewer differences in flagellar reaction than somatic, and distinction between the two types is obviously desirable for the more specific recognition of a strain. It is interesting to observe how, with both clover and medic isolates, the one somatic antigen can occur with any of the flagellar groupings.
Acknowledgements. The work reported in this paper has been supported in part by grants from the Commonwealth Research Committee and the Rural Bank of New South Wales.
References.
HuGHEs, D. Q., and VINCENT, J. M., 1942.—Serological Studies of the root-nodule bacteria. Ill. Tests of neighbouring strains of the same species. Proc. LINN. Soc. N.S.W., 67: 142-152.
KLECZKOWSKI, A., and THORNTON, H. G., 1944.—A serological study of root-nodule bacteria from pea and clover inoculation groups. J. Bact., 48: 661-672.
NutTMAN, P. S., 1946.—Variation within strains of clover nodule bacteria in the size of nodules produced and in the effectivity of the symbiosis. J. Bact., 51: 411-432.
PURCHASE, HILARY F., and VINCENT, J. M., 1949.—A detailed study of the field distribution of strains of clover nodule bacteria. Proc. LINN. Soc. N.S.W., 74: 227-236. = PURCHASE, HiLtary F., VINCENT, J. M., and WArp, LAWRIE M., 1950.—The field distribution of strains of nodule bacteria from species of Medicago. Aust. J. Agr. Res., 1951 (in press). VINCENT, J. M., 1941.—Serological studies of the root-nodule bacteria. I. Strains of Rhizobium
meliloti. Proc. LINN. Soc. N.S.W., 66: 145-154.
, 1942.—Serological studies of the root-nodule bacteria. II. Strains of Rhizobiwm trifolii. Idem., 67: 82-86. 3
, 1944.—Variation in the nitrogen fixing property of Rhizobium trifolii. Natwre, 153: 496-497. :
, 1945.—Host specificity amongst root-nodule bacteria isolated from several clover species. J. Aust. Inst. Agric. Sci., 11: 121-127.
sl
A SEPTORIA DISEASE OF HUPHORBIA PEPLUS L.
By DororHy EH. SHAw, Faculty of Agriculture, University of Sydney.
(Plate i; one Text-figure. ) [Read 28th March, 1951.]
Synopsis.
A Septoria disease of Petty Spurge is described, and the Australian distribution is given. The cultural characteristics and morphology of the causal organism are described. The results of investigations concerning the host-parasite relations, the longevity of the spores, the search for the perfect stage, and pathogenicity tests with other plants are given. The literature describing species of Septoria parasitizing species of Huphorbia is examined, and the name S. pepli, n. sp., is proposed for the causal organism.
INTRODUCTION.
A leaf and stem spot disease of Huphorbia peplus L., caused by a species of Septoria, was pointed out to the writer by Professor W. L. Waterhouse in 1947. He had had it under observation for some time, and noted that infection on Petty Spurge was quite widespread around Sydney. At his suggestion an investigation of the disease was carried out, with particular reference to the host range, as some serious diseases of economic plants are caused by species of Septoria.
EconomMic IMPORTANCE.
Huphorbia peplus L., Petty Spurge, native to Hurope and Asia, occurs on the New South Wales coast and tablelands, and in Queensland, Victoria, South Australia and Western Australia, as recorded by Hurst (1942). She listed its reputed medicinal and photographic properties, but to the writer’s knowledge it is not used commercially in Australia. Hurst also recorded it as containing a poisonous principle, euphorbin. It is a weed of gardens and waste places, but is easily eradicated. The disease is not of economic importance.
REVIEW OF LITERATURE. Australian Records.
No mention of a leaf and stem spot disease of H. peplus was made by Cooke (1892) or by McAlpine (1895). The disease was noted by Waterhouse (unpublished data) in June, 1921, in the Sydney area. Pieces of material collected by him and embedded in wax in 1921 are filed at Sydney University. Also filed are pieces of diseased material in wax from a collection made in July, 1932, again from the Sydney area. A record of a Septoria leaf spot disease of H. peplus was made by Noble et al. (1934), without either date or locality of occurrence. The herbarium specimen lodged at the Department of Agriculture, Sydney, is also without date of occurrence, locality or collector’s name. A Septoria species igs recorded occurring on ZH. peplus in Brittlebank’s catalogue of Australian Fungi (unpublished), compiled between 10th May, 1937, and 2nd March, 1940, but no indication of the date or locality is given, and no specimen is filed in the herbarium.*
\ Overseas Records.
Diseases caused by six species of Septoria and two species of Rhabdospora were recorded by Saccardo (1884, 1892, 19138)), on eight species of Huwphorbia, but none on #H. peplus. Oudemans (1921) listed species of Septoria, Phleospora and Rhabdospora on various Huphorbia species in Hurope, but none on H. peplus. No Septoria was recorded for any Huphorbia by Grove (1935), although a Rhabdospora was noted on one species of Huphorbia in Britain.
* Personal communication from Mr. S. Fish, Government Biologist, Department of Agri- culture, Victoria.
8 A SEPTORIA DISEASE OF EUPHORBIA PEPLUS L.,
In a personal communication, Dr. G. R. Bisby, of the Commonwealth Mycological Institute, reported that he could find only one record of a species of Septoria on E. peplus, and that was of Septoria euphorbiae Guep. in the Russian book “Key to Fungi, Vol. 2. Fungi Imperfecti’, by A. A. Jaczewski (1917). I am indebted to Dr. Bisby for his translation of the significant paragraph: “p. 102. S. euphorbiae Guep. on Euphorbia amygdaloides, E. peplus. Round, olive-coloured spots. Stylospores 40 to 454 by 2 to 2:54 with 3 to 4 indistinct septa.” Dr. Bisby also reported that no Septoria was recorded on Huphorbia spp. for North America by Seymour (Host Index of the Fungi of North America. Cambridge, Mass., 1929), this volume being unavailable to the writer.
No Septoria on any species of Huphorbia-was recorded for South Africa by Doidge and Bottomley (1931), by Brien (1939) for New Zealand, or by Shigekatsu Hirayama (1931) or Nakato Naito (1940) for Japan.
AUSTRALIAN OCCURRENCE.
The disease occurs on Petty Spurge over a wide area around Sydney, and collections were made throughout the area extending from Mangrove Mountain, near Gosford, in the north, Windsor in the west, and Mt. Keira, near Wollongong, in the south, in 1948 and 1949. In 1950, two further collections were made at Canberra, A.C.T., and Wagga, N.S.W.
The disease has not been recorded for Western Australia, South Australia, or for Queensland.* It is not known whether Brittlelank’s record was for Victorian occurrence. In Tasmania in 1948 the writer collected diseased Petty Spurge plants at Launceston, Hobart, and Port Arthur.
From the information available at present, the disease is known to occur around Sydney, at Wagga, Canberra and throughout Tasmania.
APPEARANCE OF THE DISEASE.
Lesions first appear on the leaves as small, mostly circular areas, pale green in colour, later spreading and turning pale yellow, then light brown, becoming papery and covered with scattered black pycnidia. Many lesions per leaf were recorded (in one case 14 apparently separate ones), but the majority of field specimens examined showed that infection was from one centre only, although sometimes two were observed. The pycnidia occur on both sides of the leaf, but mostly on the undersurface. A few lesions examined had a total lack of pycnidia on the upper surface, although numerous ones were found on the lower. In some cases a slight zonation of pycnidia occurs radially from the centre of infection. The pycnidia are produced singly, only rarely being grouped in twos or threes. As the necrotic tissue enlarges, slight puckering often occurs between this area and the rest of the leaf. The edge of the necrotic area is generally quite regular and definite, but without cicatrix formation. Pycnidial production is usually confined to the necrotic area, but occasionally lesions were found where pycnidia occurred at the edge of the lesions in advance of necrosis. Where several lesions occur per leaf, the diseased areas later coalesce. In the advanced stages of the disease premature leaf fall occurs.
Lesions on the stem occur less frequently than on the‘leaves. The leaf infection, in many cases, extends to the petiole and thence to the stem. Pycnidia occur on both petioles and stems. Stem infections were produced in the glasshouse, which were so severe as to cause death to the uninfected upper part of the plants.
Pycnidia were recorded on the seed capsules on material from the field, as well as on plants inoculated in the glasshouse. No pycnidia were noted on the seeds.
CULTURAL CHARACTERISTICS. Isolation Methods. Isolations of the causal organism were first made by the usual method of tissue transplants, pieces of diseased material of about 3” x 3” being cut from the edge of
* Personal communications from Mr. W. P. Cass Smith, Government Plant Pathologist, Western Australia, Mr. D. B. Adam, Department of Plant Pathology, University of Adelaide, and Professor D. A. Herbert, University of Queensland.
~
BY DOROTHY E. SHAW. 9
the lesions, or just in front, and taken through 95% alcohol for 5 seconds, mercuric chloride 1 in 1000 for 20 seconds, and three washings with sterile water, then transferred with sterile forceps to freshly poured and cooled plates of potato dextrose agar. It was found, however, that if a contaminant present had escaped the surface sterilization, it usually grew at a rate much in, excess of the Septoria mycelium, which was sub- sequently difficult to obtain in pure culture. The method later adopted was to allow the spores to exude from the pycnidia in the leaf, into a drop of distilled water, and to streak a loopful of the spore suspension over the surface of the P.D.A. plates. Bacterial and fungal contaminants were sometimes present, but because of the scattered nature of the Septoria spores isolations were not difficult. Transfers were then made to P.D.A. slopes, of single spores or hyphal tips, as well as mass transfer of mycelium. Nine isolations were made from collections, five from within the Sydney area, three from Tasmania, and one from Wagga. Nineteen collections were made from many diseased plants observed in the field, three of these being from Tasmania, one each from Wagga and Canberra, and the rest from a wide area around Sydney.
Media.
The fungus grew on P.D.A., water agar, standard agar, maize meal agar, maize husks and maize cobs, potato cubes, lucerne shoots, lima bean agar, dried pea agar, lentil agar, soaked rye and peanut husks, also on Czapeck-Dox with marmite, and wort agar. It also grew on a water extract of H. peplus leaves in agar, and on sterile shoots and stems of H. peplus. Growth on water agar was very slow and meagre. Pyenidia, mostly well formed, were produced on all the media, often with abundant pinkish exudate of spores, especially on the sterile stems of Petty Spurge, and on the rye-peanut husk media. P.D.A. proved the most satisfactory media for mycelium and spore production and for maintaining cultures.
Appearance of Cultures.
Spores germinate on P.D.A. usually within twenty-four hours. After three to four days the colonies are visible, being whitish in colour, and distinctly mucose, with a smooth surface and an entire edge to the naked eye. As the colonies develop, the edge becomes slightly irregular and the surface slightly ridged. At seven days most colonies have become black in the centre, and at 17 days are all black, sometimes with a tinge of greeny-grey, with a minute border of white at the edge.
On P.D.A., cultures later produce small greyish patches of very short, pile-like aerial mycelium. Black carbonaceous pycnidia are embedded over the surface, and these, in most cultures, give pink exudates of masses of spores. Where pycnidia occur near the side of a tube, cirri can be easily distinguished under the low power of the microscope. These spore masses are yeast-like in appearance to the naked eye. The mycelial mass later becomes carbonaceous, piled and convoluted in the centre, and with sub-surface hyphae at the edge. The hyphal mass remains very compact and difficult to separate, with growth upwards nearly as great as lateral growth. Seldom is the whole surface of the slope covered with mycelium, and often cracks occur in the agar. After about three months small tufts of white cottony mycelium appear in patches over the surface in some slopes. Colonies are usually non-sporulating about this time. In some cultures more than four months old, hard carbonaceous bodies appeared on the surface and along the cracks in the agar. The structures, when sectioned after fixing in chrom-acetic and stained with gentian violet-orange G, were roughly circular to oval, but measured anything from 200 to 5004 wide (measuring from the innermost walls). The interior had no definite structure, but consisted of wispy strands of fungal material, which could not be distinguished as hyphae. The walls consisted of 6-9 layers of very dark brown, thick-walled, pseudoparenchymatous cells, which pass into a region of brownish, twisted, strongly septate, clearly distinguished hyphae. No development further than this sclerotial-like stage was observed.
Optimum Temperature. Optimum temperature was determined by growing the fungus in small (24”) Petri dishes on P.D.A., and incubating over a range of temperatures. Three tests were
D
10 A SEPTORIA DISEASE OF EUPHORBIA PEPLUS L.,
carried out at different times. In the first two tests duplicate plates were used, in the third only one plate was inoculated for each temperature, except for 20°C., when dupli- eate plates were used. All the tests gave approximately the same results for the temperatures available. Inoculum was of the Pennant Hills isolate, approximately 2 mm. square, and readings were taken at four weeks. The readings for the third test are given, as the range available at that time was alittle more comprehensive.
TABLE 1. Growth of the Causal Organism at Various Temperatures.
Temperature. Colony Diameter. Spore Production and Character LOR mm. of Growth. 3 DX 92 No growth discernible. 5 | Dire vl Very slight growth. 10 7x 9 Colony black with small white margin; no spores. 15 13 x14 Colony similar to above ; no spores. 20 16x17 Colony black with greenish tinge; on the (mean of 2 reverse side, clumps of pyenidia in con- plates) centric circles; abundant spores. 25 10 x12 Colony black; no spores. 30 2x 2 No growth discernible. Room temp. (light) 13 x16 Colony as at 20° C.; abundant spores. Room temp. (dark) 12 x13 Colony as at 20° C.; very abundant spores. (June-July) |
Optimum temperature for mycelial growth and spore production is around 20°C. Colonies at the other temperatures might have produced spores when older. No measurement was made of the height of the colonies, but this increased with increase in diameter. :
Optimum pH.
Optimum pH was determined by growing the fungus on plates of P.D.A. adjusted with N/5 NaOH or N/5 HCI to give a series varying in hydrogen-ion concentration. Two tests were conducted, and the pH was read on a Vane Electronic pH Meter. Duplicate plates were used in the first test, triplicate plates, for the second. Readings of pH were taken before inoculation. Inoculum was of the Sandy Bay (Tasmania) isolate, approximately 2 mm. square. Plates were kept at room temperature during May and June, and measured after seven weeks.
TABLE 2. Growth of the Causal Organism at Various Hydrogen-ion Concentrations. Z
|
Colony Diameter. pH. mm. (Mean of 3.)
3°6 6-0x 9:3 4°8 23°6 x 25:3 5°6 26:0 x 29°5 6°8 24°3 x 26°3 (eT 22-0 x 24-0 8-6 16:0 18-6
Optimum pH is about 5-6 or a little more alkaline. Specificity is not very marked over a wide central range, the greatest effect being shown on the very acid side at the concentrations taken.
BY DOROTHY E. SHAW. 11
TEST FOR PATHOGENICITY.
Isolations of the organism were made as outlined previously, and the fungus maintained on P.D.A. slopes. Spores from pycnidia produced in culture were used to inoculate leaves of Petty Spurge seedlings in the glasshouse as outlined under ‘Host- parasite Relations, Method’. Typical symptoms of the disease appeared on the leaves, with production of pycnidia. Isolations made from these lesions yielded the organism which was similar in detail to that isolated initially.
MorPHOLOGY OF THE CAUSAL ORGANISM. Mycelium.
The mycelium in young colonies consists of stellately radiating hyphae, which are minutely guttulate, hyaline, septate, and branch profusely. Pigmentation occurs in hyphae about six days old. Old hyphae (i.e., after a period of months), become very nobbly in outline, olive in colour, often with large refractive globules, but usually without any apparent contents. Young hyphae at the edge of colonies are very fine. Mycelium in colonies 13 days old measured up to 3u in diameter, and at four months measured about 4u in diameter. Hyphae in the plant tissues measure 2-3 in diameter...
Pycnidia. .
Pycnidia occur on both sides of the leaf, but mostly on the under-surface, scattered over the centre and sometimes on the marginal green areas of the necrotic spots; singly, only rarely in twos or threes; visible to the naked eye, black in colour, but reddish-brown by transmitted light; globose, immersed but later erumpent; ostiole about one-quarter the diameter of the pycnidium; pycnidial wall smooth, composed of 2-3 layers of pseudoparenchymatous cells; 85-135u (65—-160u), mean 110-194 + 18-98y.
Pycnidiospores. Pyenidiospores hyaline, straight to very slightly curved, attenuated at one end, with a varying number of guttulae; usually three septa, often two, sometimes one, rarely aseptate or four-septate; 25—44u (17-51lu), mean 35-77u + 5-94u.
Cirrt.
Cirri were often found exuded from the ostioles in material examined straight from the field, and practically always from material in the glasshouse, owing to the high humidity. They are colourless under reflected light, and in strong sunlight can be seen with the naked eye as a glistening whitish spot on the pycnidium. The horns vary in length, nearly always curl over, and often adhere to the neighbouring horns. On diseased material from the glasshouse, cirri were noted from quite small pycnidia. Cirri were observed microscopically by placing diseased stems from the glasshouse (with cirri still attached) into lacto-phenol cotton-blue, and by allowing pycnidia produced in culture to exude spores into dilute lacto-phenol. The pycnidiospores are oriented with their long axes parallel to the horn. When material with cirri already exuded is placed into water the spores immediately separate from one another. When pycnidia with unreleased spores are placed in water, the spores are ejected separately, not exuded in cirri.
OBSERVATIONS ON PYCNIDIOSPORES. . Spore Structure.
Pyenidiospores of all isolates examined were hyaline, the septa being indistinguish- able unless stained. Stains used included cotton-blue lacto-phenol, aqueous gentian violet, nigrosin, aceto-carmine, and gentian violet, orcein and carbol fuchsin in lacto- phenol. Cotton-blue lacto-phenol proved -to be the best stain to show cell contents. This stain acts quickly, the protoplasm staining blue of varying intensity. Guttulae were of various sizes, sometimes two large ones appearing in each cell, one at each end near the septa, sometimes one large guttula only, as well as numerous small ones. The number of large guttulae was not constant per cell or per spore. Septa were unstained with gentian violet, the spore contents appearing granular with large refractive drops in most cells. In material fixed, sectioned and stained with gentian violet-orange G,
12 A SEPTORIA DISEASE OF EUPHORBIA PEPLUS L.,
nuclei were clearly distinguishable in the spores and conidiophores. Spores mounted in water and examined with dark field illumination, showed the cell contents as circular areas of light of varying size and intensity, as shown in Plate 1, B, by MacMillan and Plunkett (1942).
Septation of Spores.
Because of the confusion in the literature regarding the number of septations in most species of Septoria, this aspect of the causal organism was particularly observed. MacMillan and Plunkett (1942), following on the work of Garman and Stevens* (cited by the afore-mentioned but unavailable to the writer), have shown that there is a great inadequacy on this point for most published descriptions of Septoria species. Sprague (1944) and MacMillan and Plunkett consider that the number of septations of the spores is usually 1, 3 or 7, depending on whether there are 1, 2, or 3 nuclear divisions in the spores, and that there is simultaneous division of the end cells, after formation of the primary septum. MacMillan and Plunkett concluded that the spores become mature on attaining the 3-septate condition, but not before, and that an even number of septa in the spores is anomalous. These authors found it difficult to account for the very large number of observations in the literature for even-numbered septate spores. Sprague (1944) found that some species infecting grasses are characterized by 2-septate spores, while five septa are common in others. He concluded that in some species the number. of nuclear divisions evidently is dependent on the available cell nutrients; that large spores produced in humid winter weather may have two or even three nuclear divisions, while later in the season, when the weather is warmer and drier, the same species may produce aseptate or 1-septate spores.
Septa are indistinguishable in unstained spores. Stained with cotton-blue lacto- phenol, the septations are visible, but the granular nature of the cells does not make for easy observation. The best method found was as follows: diseased material with pycnidia was placed in a drop of water on a slide, and the spores allowed to exude. After a minute or so, the tissue was lifted from the slide, and a drop of iodine in potassium iodide in 80% alcohol was added, this being a modification of the method used by Brodie and Neufeld (1942). The cover slip was lowered, and the septation counts made immediately. The spores, with this method, appear a homogeneous golden- brown, with the septa, under slightly reduced light, clearly distinguished.
TABLE 3. Septation of Spores from Different Collections, at Various Intervals from Time of Collection, and from Culture. | Percentages of Spores in the Various Septate No. of 1 } Classes. Collection. Spores Time from. | be ty $ Examined. | Collection. | | 0 1 2 3 4 | 5 weiss pian Ae | | | Mangrove Mtn. ue ie 200 2 weeks. — 10-0 28-5 60-5 1:0 —- Mt. Keira cas 4S 200 2 months. (Nal 7) “al7/9t) 37-0 44-5 0-5 — P. Arthur, Tas. .. ec 200 4 months. — | 19-0 46:5 33-0 1-0 0-5 Sandy Bay, Tas. .. “3 200 4 months. — 10°5 36-0 53°5 aS Sandy Bay, Tas. .. Be 100 6 months. ae 6-0 15-0 79-0 — _— *Sandy Bay, Tas. .. is 142 == [Pere Pea net) 46-0 52-1 say ih ce Penshurst .. WG AG 100 | 11 months. 1:0 | 21-0 38-0 40-0 | — —_ | } ' |
* Spores from culture.
The results shown in Table 3 indicate that the maximum number of septa in mature spores is three, practically no further division taking place until germination. The first cell-division in the spore would appear to take place early, because of the very small number of aseptate spores which appear in the counts. Spores in which
* Garman, P., and F. L. Stevens, 1920.—‘‘The genus Septoria.” Ill. State Acad. Sci. Trans., 13° 2176-219.
BY DOROTHY E. SHAW. 7 a2:
two cell-divisions have taken place (giving four-celled spores) would most probably be spores produced under optimum conditions. The large percentage of 2-septate spores could only be explained where only one cell divided after the formation of the primary septum. It is to be noted that a large number of 2-septate spores was also produced in culture. Counts from all the above collections are of the same general type, i.e., 3>>2>1- septate spores (except the Port Arthur isolate, where the 2-septate spores predominate), with 0-, 4-, and 5-septate spores occurring only very rarely. The relative percentage of 3-, 2-, and l-septate spores is most likely conditioned by nutrition. Age, under the conditions of storage of the material, did not appear to affect the relative percentages greatly. Germination of Spores.
1. Method and manner of germination.
Germinations were studied on plates of P.D.A., the media having been strained, and only that amount which would just cover the bottom poured into Petri dishes. Spores from a water suspension were streaked over the surface with a loop. With this method, each streak of spores could be followed under the microscope with ease. Germination conformed in general to that described by MacMillan and Plunkett (1942) for 20 representative spores of NS. apii-graveolentis. These workers noted that, at the end of four hours, the two end-cells had divided, and the tapered end of the spore had increased in length more than the other cells. At twelve hours the end-cells had divided, and at 24 hours the two original centre-cells had divided, and the outer cells of this division had sent out tubes. The spores were originally all 3-septate, and the germination was quite symmetrical.
In the present study, it was noted that germination was not always symmetrical, and counts were made of the positions of emergence of germ tubes. Readings were taken on 200 spores from the Penshurst collection, taken at random for both readings, on P.D.A.
TABLE 4. Germination of Spores at 24 and 50 Hours.
24 Hours. 50 Hours. Type of Germination. % % Not germinated .. Me aS ae Ee a Bs. 4 3 Germ tube at 1 end .. ais Ge Bs ss x 38 it 1 side ee is i ne ae 6-5 1 both ends sf <5 . ee 33 11 both sides Ne a =e a8 1:5 2 1 end, 1 side .. ae oe ff at 12-5 1 2 ends, 1 side .. 5 4 1V 1 end, 2 sides oe A oe 0-5 9 both ends and more than one side —- 55
. As indicated in Table 4, emergence of germ tubes was asymmetrical, although by the second reading at 50 hours, emergence had proceeded towards the “normal” type expected from a 3-septate spore, as shown by MacMillan and Plunkett. Spores from the Penshurst collection included many with one and two septations, and the asymmetrical emergences were most likely a reflection of this condition. The above workers considered that spores with less than three septations were immature, but it is to be noted that spores in this study which would be classed as “immature’’ were capable of germination, as shown in Table 4, where 96% had germinated in 24 hours.
Germinations on water agar were slower than on P.D.A. (e.g., in one test, 94% had germinated on P.D.A. in 48 hours, against 74-5% on water agar), and hyphae were shorter, with fewer branches. Fer this reason, strained P.D.A. was used in preference to water agar, although the latter is a little clearer. Germinations in water were comparable with those on P.D.A. Spores retained their identity in most cases, up to approximately 48 hours on P.D.A., but for a shorter time in water.
14 A SEPTORIA DISEASE OF EUPHORBIA PEPLUS L.,
No secondary spores borne directly on the mycelium were observed, as noted by Weber (1922) and Sprague (1944) for several species of Septoria. Anastomoses between germinating spores occurred in water, but none were observed on P.D.A. or water agar.
2. Effect of Temperature on Germination. ‘
Several loopfuls of a spore suspension in water were placed on coverslips, which were then inverted over van Tiegham cells containing several drops of water, and sealed with vaseline. After seven days cotton-blue lacto-phenol was run in, to fix and stain the hyphae, the coverslips were removed and placed on clean‘slides for observation.
TABLE 5.
Germination of Spores and Degree of Development at Different Temperatures.
} x ey | | Room
ASIC a eee Sag Ce: Weal olG-cem eeenem ps | ZONC: BOSC | PeesiiaGs ho) aiuikys | | | | | | | | | | |
He srcn Been ceearest ar ules Ear gamer a ok - ~ ;
| | |
The results of the temperature test for germination agree fairly well with that for vegetative growth, as in Table 1.
Spore Size. 1. Length.
Great discrepancy exists in the literature regarding methods employed for mounting and measuring, and methods of recording measurements, and the position has been reviewed by Bisby (1945) and Ramsbottom (1948). There are also wide differences in opinion as to the number of items, e.g., spores, which are required to be measured. Many workers have not recorded the number measured. Bisby (1945) suggested 20 or so, to include spores at both ends of the range. Cochrane (1932) measured 1,000 spores of each of the two species studied. Beach (1919) measured 200 spores for each test when checking the effect of various microclimates on the length of Septoria spores.
In this study, spores were allowed to exude from pycnidia into a drop of water on a slide, for not longer than two minutes, when lactophenol cotton-blue was added, to prevent swelling and to stain the hyaline spores. One thousand spores were measured, from different isolates and environments, and the results examined to see if there was any difference between the spores of seven collections, between the Tasmanian and Sydney area collections, and between the spores from diseased material and those from culture. In some of the collections, material was limited.
Examination of the results in Tables 6 and 7 shows that the means range from 32u to 41u, and variation between groups is no greater than variation within: groups. The greatest number of long spores was produced in culture. Other workers have found that even slightly different environments affect the length of Septoria spores, e.g., MacMillan and Plunkett (1942), found that spores of NS. apii-graveolentis from pycnidia measured 36-8u (average of 50 spores), while spores from cirri measured 44-5u (average of 20 spores). Moore, cited by Hughes (1949), found that the average length of spores of S. lactucae increased from 27-5u to 35u after a week of dull or wet weather. Beach (1919), measuring 200 spores for each test, with S. tritici and S. verbascicola, found that size was affected by environmental conditions such as bright and dull light, moist and dry culture conditions, and summer and winter development. While the readings shown in Table 6 are not sufficiently comprehensive for a detailed comparison, it is considered that the differences in length of spores of the various isolates can be accounted for by differences in environment at time of spore production.
BY DOROTHY E. SHAW. 15
TABLE 6. Length of Spores from Different Isolates. | | Standard Isolate. Month of Source.* No. } Mean. Deviation. Collection. | Measured. | UL. UL. | | | | | Sandy Bay, Tas. .. a iy .. | January. Ne 200 | 32-38 5-100 Sandy Bay, Tas. .. i ft .. | January. Cc. 100 | 33°46 4-676 Port Arthur, Tas. i, me .. | January. N. 200 35-60 5-821 Taunceston, Tas. .. as hs .. | January. N. 200 | 37-37 4-901 Pennant Hills (7) .. 3 Ka so |) IME C | 100 41-11 5-091 Rydalmere = nee sa ots .. | May. C. | 50 39-71 4-525 ‘Penshurst .. ae ae a Sanlediutliys N. 100 33.52 6-225 Pennant Hills (9) .. ee be .. | August. | N 50 38-08 3:°994
* N.=nature, C.=culture.
TABLE 7. Comparison between Groups. Bulked Tasmania Sydney Area | Mean Stan. Dev. UL. 5 U- | U- U- Nature .. ais 32°38 3 Snes 35-01 5-778 35-60 38-08 37-37 ; 41-11 | Culture .. Ae 33°46 39-71 37°77 5:775 | Bulked .. he 34-88 5-564 37°84 6-184 | 35-77 5-939 Mean Stan. Mean Stan. Mean Stan. Dev. - Dev. Dev.
2. Width.
Spores were prepared for measuring as described under the previous section. Three hundred spores were measured, at the widest part, using a 20x eyepiece with a calib- rated ocular micrometer, and fifty were measured using a filar micrometer.
TABLE 8. “ Width of Spores. Standard Method. Isolate. — Source. Number Mean. Deviation. ‘ Measured. UL. (2. Filar micrometer a .. | Penshurst. N. 50 1-8975 0-2204 Ocular micrometer .. .. | Sandy Bay. C. 100 Sandy Bay. N. 100 Launceston. N. 50 oe Ose Pennant Hills. C. 50
No difference between isolates was detected. The use of the calibrated ocular entailed making estimates for those spores whose widths fell between graduations. Although greater accuracy is obtained by using the filar micrometer, whose graduations are only 0-178u apart, the former method is quicker, and is sufficiently accurate to
16 A SEPTORIA DISEASE OF EUPHORBIA PEPLUS L.,
warrant its use under most circumstances, if a large number of spores are to be measured. SIze OF PYCNIDIA.
Diseased material was mounted in lactophenol to prevent shrinking and swelling, and a coverslip gently lowered on top to avoid squashing. Measurements were made from the outside of the pycnidial wall on those pycnidia whose ostiole was uppermost, i.e., if the adaxial surface of the leaf was uppermost, only adaxial pyenidia were measured. Readings for each collection, however, were made on pycnidia on both sides of the leaf. After a preliminary examination of the pycnidia, it was decided that their practically spherical formation required only one measurement. Where length and width differed, the diameter measured was the one which happened to lie parallel with the graduated scale of the eyepiece.
TABLE 9. Size of Pycnidia from Different Isolates. | iyi No. of Standard Tsolate. | Pycnidia Mean. | Deviation. Measured. {L. {L. | | Sandy Bay, Tas. Eyeva. won ye 200 117-55 | 18-694 Port Arthur, Tas. ee an oF el| 100 107-44 | 18-712 Pennant Hills (7) ae hd Be 50 102-34 15-760 Rydalmere ve Bs eck Ke 100 110-33 17-847 Penshurst ue th ae sh. 50 104-04 18-241 Pennant Hills (9), 56 as ast 100 105-06 | 18-093 Bulked .. *. a ah al 600 110-19 i 18-982 a \
As shown in Table 9, the mean of the bulked readings was 110-194 = 18-982u. Measurements of pycnidia grouped in twos and sometimes in threes fell within the range of solitary pycnidia, and are not included in the above figures.
The mean of 50 measurements of ostioles of pycnidia on diseased leaves was 29-24u, ranging from 17 to 47-6xu.
Host-PARASITE RELATIONS. Method.
Seedlings of HE. peplus, raised from seed, or transplanted from the field, were grown in pots in the glasshouse. Leaf surfaces were difficult to wet, and the best method proved to be moistening with the fingers, followed by spraying from an atomizer. Several loopfuls of inoculum were placed on both surfaces of the leaves, the inoculum consisting of a water suspension of spores, either from pycnidia on leaves, or from pycnidia in culture. Seedlings were incubated from 48 to 72 hours, then placed on benches in the glasshouse.
Leaves were picked and fixed after 24 and 48 hours, and thereafter every two days until pyenidia were abundant. Several methods of fixing and staining were tried, including staining with Pianese 111) as recommended by Weber (1922), but the method finally followed was as follows: leaves were fixed in Farmers (absolute alcohol and glacial acetic acid 3:1) for 20 minutes, washed several times in 95% alcohol, decolourized in 95% alcohol for 18—24 hours, and either mounted in lactophenol cotton- blue permanently, or taken from it to lactophenol after ten minutes. This method was found to be very satisfactory. The leaf tissue after treatment was quite colourless, and the spores and hyphae on the leaf surface stained. blue, standing out vividly. Spores in pycnidia stained in deepening amounts of blue, depending on the maturity. Hyphae in the tissues were quite colourless when first mounted, but after several months appeared faintly blue. Material mounted in this way and examined after fourteen months was in good condition. The hyphae were clearly visible below the epidermis,
BY DOROTHY E. SHAW. iW
and although the chloroplasts by this time were stained blue, this assisted observations by making cell boundaries easily discernible.
Stem tissue was stripped, fixed, stained and mounted as above.
Observations.
Spores had germinated on the leaf surface after 24 hours, usually from one end, sometimes from both, more rarely from a side branch. The infection hyphae were usually not much thinner than the width of the spore. Many germ tubes terminated in small, appressoria-like bodies, usually at the junction of two epidermal cells. In many cases hyphae were observed to pass alongside or over stomates. No entry was
Text-figure 1.
Camera-lucida drawings of spores germinating on the leaf surfaces. x 300. A, B and C, spores germinating on the upper surface. D, fusion of spores germinating on the under surface, with germ tube passing by stomate. BH, spore germinating om the under surface, with germ tube passing over stomate. F, multiple fusion of spores germinating on the uppper leaf surface.
observed through stomates, and there appeared to be no attraction for the hyphae to do so, One case only was observed where the hypha ended at a stomate, and in that ease there appeared to be further development.
Germination was similar on both leaf surfaces. Branching was observed only rarely, and spores retained their identity longer than on P.D.A. The number of septa
18 A SEPTORIA DISEASE OF EUPHORBIA PEPLUS L.,
did not increase in most spores during germination. Anastomoses were frequently observed between two spores, sometimes between three spores.
At a later stage, hyphae were observed in the leaf tissue, spreading out radially from the infection centre, branching and passing beneath the epidermis, and around the cells, mainly in the mesophyll region. Later, knots of hyphae were noted below the stomates, sometimes not directly beneath, but always in the sub-stomatal cavity. On no occasion did a knot develop on one hypha only: pyenidial formation therefore is probably symphogenous.
On a few leaves, notably in one collection kept wrapped in moist paper for 24 hours, hyphae were observed issuing from the stomates above immature pycnidia. These “aerial” hyphae measured approximately 3u wide, and were from several to 70u long. In many cases not just one hypha projected from the stomate, but two or three, and some cases were observed where bunches of five to eight short hyphal tips projected through the stomates.
Pyenidia were apparent to the naked eye after about 14 days. The most mature pycnidia occurred at the centre of the lesions, and radially from these were immature pycnidia, aggregations of hyphae under stomates, and then hyphae ramifying through the tissue. Hyphal tips were quite distinct, and measured about 3 across, being just slightly wider than the older hyphae. Tips were located up to 500u in advance of pycnidia formation. In the undiseased tissue, the chloroplasts appeared distributed evenly around the cell walls, and remained so even with the advance hyphae passing around them. In the region between the advance hyphae tips and the pyenidia, the chloroplasfs lose their discreteness, and the cells appear collapsed. The boundary between the normal-appearing cells and the collapsed cells was usually quite distinct. Not all the stomates had pycnidia forming under them, but where a stomate nad been missed in the first place, it often showed aggregations at a later stage, i.e., young pycnidia could form in the zone of older mature pycnidia.
Strippings from stems showed a similar condition, with hyphae mostly running up and down the stem, and pycnidia being more linearly placed, instead of zoned. Pyenidial formation was symphogenous.
Examination of healthy Petty Spurge leaves showed that stomates occur on the abaxial surface on the average of eight to a field (magnification x840), while very few occur on the adaxial surface, most fields having none, or two or three, at certain parts, e.g., along the sides of the main vein. This would account for the predominance of pycnidia on the lower surface of the leaves.
TABLE 10.
Viability of Spores at Various Periods After Collection.
|
No. of |
Collection. Age at Test. Spores | Germinated. Counted. | We = = aoe
| Mt. Keira Se 54 6 50 2 months. 170 94 Ban, 200 87 5 , 200 71 7 | 100 0 Mangrove Mtn. 7 ; | 200 | 12 Pennant Hills (9) 10 “ 200 0 Penshurst (ali is 300 0
Diseased tissue was also fixed in Flemming’s weaker solution and Farmers Fluid, the former being stained with gentian violet-orange G, the latter with carbol fuchsin- light green. Sections were cut at 8u and 10u. No cicatrix was ever observed at the edge of the lesion. Hyphae ramified throughout the tissue, and the stages in pycnidial formation were noted: hyphae loosely woven below the stomates, later developing into a knot, followed by the development of the pyenidial wall. The pycnidia were subepi-
BY DOROTHY E. SHAW. 19
dermal, and later erumpent with a widened ostiole. Spores were produced on one- celled pyenidiophores. Nuclei were clearly visible in sections stained with gentian violet-orange G.
LONGEVITY OF SPORES.
Germination tests were made with spores from diseased material which, after the initial test, was kept between paper at room temperature. Several loopfuls of the spore suspensions were streaked across strained P.D.A. plates, and germination counts were made at 48 hours. Spores from all isolates gave more than 95% germination at the initial tests.
Spores retained within the pyenidium under the above conditions of storage were viable for about six months.
SouRCE OF INOCULUM AND TRANSMISSION OF THE CAUSAL ORGANISM.
Diseased plants of Petty Spurge were found in the field’ throughout the year, with the exception of late spring, and further search might have revealed infected plants even during this period. These diseased plants, therefore, provide an immediate source of inoculum. Pycnidia on fallen leaf and stem fragments could also liberate spores, given favourable conditions of moisture, and provide an additional source of inoculum in the absence of growing infected plants.
To determine whether the disease is seed-borne or not, tests were carried out as follows:
A collection was made of the ripest capsules on heavily diseased plants at Penshurst. In the laboratory, seeds (most of which had left the capsule cases) were picked aut with forceps. The cases were floated and turned over in distilled water in watch glasses and examined under low power. Many pycnidia were noted on them. The cases were drawn up onto the sides of the watch glass with forceps, and the residue water examined with reduced light. Many spores were noted. The seed was turned over in distilled water in watch glasses and examined under low power. No pycnidia were noted on the seed, but spores were found in the water residue. The seed was then sown in pots containing sterilized soil, and the residue water with the spores sprayed over the seed. The seedlings were examined every day after emergence, but no sign of disease was apparent on the stems or leaves. Random seedlings were selected at four weeks, washed in fast running tap water, and several times in distilled water, and plated on P.D.A. No Septoria mycelium grew from the tissue. Capsules and seeds from diseased plants at Allawah were examined as above. Pycnidia were found on the cases, and spores in the washing, but no pycnidia were detected on the seeds.
Ripe seed capsules were collected from Petty Spurge plants in an isolated patch at the University, the plants being apparently quite free from disease. The capsules and seeds were examined as above, and as no pycnidia or spores were observed, the seed was presumed to be clean. The seed was divided into four groups of about 40 seeds each, and treated as follows: 5
(a) Seed sown in pots in sterilized soil (control).
(6) Seed sown in pots in sterilized soil, and the surface of the soil sprayed with a suspension of spores and mycelium of the Pennant Hills isolate.
(c) As (6), but from the Sandy Bay isolate.
(d@) Seed soaked for 24 hours in a spore and mycelium suspension, then sown in pots in sterilized soil.
The seedlings were examined after emergence, up to a period of eight weeks, but no sign of disease was observed on the stems or leaves. Random seedlings were washed as above, and plated on P.D.A., but no Septoria mycelium grew from the tissue.
Seeds from. heavily diseased plants were surface sterilized and plated on P.D.A., but no Septoria mycelium developed.
Although the above tests are not conclusive, it seems unlikely that mycelium is carried in the seed, and although spores are carried on the surface, no infection was established from these. It must not be overlooked that spores in pyenidia on shed
@
20 A SEPTORIA DISEASE OF EUPHORBIA PEPLUS I., ©
capsule cases could constitute a source of inoculum, for example, if splashed up by the rain on to the leaves of young seedlings.
SEARCH FOR THE PERFECT STAGE.
The perfect stage of some species of Septoria has been recorded, and the literature was examined to note the environment favourable for perithecial production. Stevens (1925) cited Klebahn, who recorded Mycosphaerella sentina as the perfect stage of .S. piricola, occurring on over-wintered leaves of apple and pear. Stevens also recorded Leptosphaeria phlogis as the perfect stage of S. phlogis. Roark (1921) showed that a Mycosphaerella, occurring on over-wintered leaves and in pure culture (media not given), was the perfect stage of S. rubri. Weber (1922) recorded Leptosphaeria avenaria as the perfect stage of Septoria avenae Frank, perithecia occurring in oatmeal agar and potato dextrose agar cultures. Stone (1916) showed that Mycosphaerella grossulariae, occurring on dead over-wintered leaves of currant and gooseberry, was the perfect stage of S. ribis, and that Mycosphaerella aurea, found on old leaves of Ribis aureum, was the perfect stage of S. aurea. Thompson (1941) recorded Mycosphaerella populorum as the perfect stage of S. musivad on poplars, and Mycosphaerella populicola as the perfect stage of S. populicola, the ascigerous stages occurring on over-wintered leaves. Ruggieri (1936) reported a Mycosphaerella as the perfect stage of S. aqurantiorum, having obtained perithecia in pure culture. Klebahn (1934) found that a Mycosphaerella on overwintered leaves of chestnut was the perfect stage of S. castanicola. Wollenweber (1938) found perithecia of Sphaerella linorum, the perfect stage of S. linicola, on flax straw in the Argentine. Johnson (1947) found a Leptosphaeria on wheat, and rarely, barley leaves in Canada, and also obtained perithecia on corn meal agar, this being the perfect stage of S. avenae Frank F. sp. triticea. Cochrane (1932) could not find the perfect stages of S. a@pii or S. apii-graveolentis, either in culture, on artificially wintered leaves, or in response to treatment with ultra-violet light. 4
During the present study, all cultures on P.D.A. were periodically examined for any evidence of the perfect stage, some slope cultures being kept for more than a year, Cultures on the various types of media used, on P.D.A. in various environments, and cultures used for temperature tests were examined, in case a necessary factor or combination of factors giving optimum conditions for perithecial growth had been supplied.
Cultures of the various isolates were opposed in all combinations on P.D.A., in nearly all combinations on corn meal agar and on sterile lucerne shoots. These cultures were kept for more than five months.
Heavily diseased leaves were placed on and just below the surface of soil sterilized for four hours, in Petri dishes, and were (a@) held at approximately 2°C. for four months, then at room temperature, or (0b) moistened periodically at room temperature.
Diseased leaves on plants infected in the glasshouse were allowed to fall onto the surface of the soil in the pots; some pots were kept in the glasshouse for six months, others were put into the open, and the decomposing leaves examined from time to time. A patch of heavily diseased plants was marked off in a garden at Hurstville, and allowed to remain undisturbed. The diseased leaves shed on to the soil surface, and later the stems, were examined. The Petty Spurge leaves are so delicate that they do not retain their identity for long on the soil surface.
No evidence of a perfect stage was found under any of the above conditions.
PATHOGENICITY TESTS WITH OTHER PLANTS.
In order to determine the host range of the fungus, plants of the family Euphorbiaceae, being species of Huphorbia and Ricinus, were inoculated in the glass- house. The writer particularly desired to determine whether the species of Euphorbia reported as hosts of species of Septoria were susceptible or not to the fungus under study, but difficulty was experienced in obtaining seed, as most of the species mentioned are of European-Asian habitat, and are not present in Australia. Black
BY DOROTHY KE. SHAW. 21
(1922) recorded H. exigua as present in South Australia, and the C.S.I.R. Bulletin No. 156 (1942) listed H. exigua and H. Hsula as present in Australia, but seed of these species was unobtainable at Sydney Botanic Gardens, Adelaide University (Waite Institute), Melbourne Botanic Gardens and the Tasmanian University. Seed was also requested through -the medium of the Australian Plant Disease Recorder, circulating through all the States. Seed of H. Hsula, HE. exigua, EH. serrata, EH. silvatica, EH. palustris, EH. aspera, H. angulata and EH. amygdaloides was requested from overseas institutions by the Plant Introduction Office, C.S.I.R.O., and viable seed of H. Hsula and EH. exigua was eventually obtained.
Plants not of the family Huphorbiaceae, but known to be hosts of other species of Septoria, were also inoculated, together with other grasses, weeds and ornamentals.
The inoculum consisted of a suspension of spores, either from pycnidia on fresh leaves, or from cultures of the various isolates. The plants were either transplanted from the field, raised from seeds, or grown from cuttings. Seedlings of H. peplus
were inoculated at each test.
Plants inoculated were as follows: Huphorbia helioscopia L., “Sun Spurge’. E. Drummondii Boiss, “Caustic Weed’’. E. Lathyris L., “Caper Spurge’.
EH. terracina L., “False Caper’’.
EH. splendens Bojer (H. Milli Desm., Sterigmanthe splendens Kl. et Garcke), “Crown of Thorns”.
H. neriifolia L.
H. Bojeri Hook (Sterigmanthe Bojeri Kl. et Garcke).
H. enigua lL.
H, Hsula Ll.
HE pulcherrima Willd. ex Klotzsch, “Poinsettia”.
Ricinus communis L., “Castor Oil’.
Lycopersicon esculentum Mill., “Tomato”.
Linum usitatissimum L., “Flax”.
Pastinaca sativa L., “Parsnip’’.
Apium graveolens L., “Celery”.
Apium leptophyllum (DC) F. Muell.
Triticum vulgare Host. “Federation” wheat.
Triticum monococcum L., “Hinkorn” wheat.
Avena sativa L., “Richland” oats.
Hordeum vulgare L., “Kinver” barley. Secale cereale L., “Open-pollinated rye’’. Zea Mays L. (var. indentata).
Poa annua L., “Winter grass’.
Poa pratensis L., “Kentucky Blue grass’.
Bromus wunioloides H.B.K., “Prairie grass”.
Lolium multiflorum Lam., “Italian rye grass”.
Hordeum bulbosum lL.
Agrostis alba L.
Digitaria adscendens (H.B.K.) Henrard.
Dianthus spp., “Carnation”’’.
Malva_ parviflora L., “Small-flowered Mallow’”’.
Sonchus oleraceus L., “Sow Thistle’.
Geranium sp., “Geranium”.
Geranium dissectum L.
Hrodium cygnorum Nees, “Blue-flowered Crowsfoot”’.
Hrodium moschatum (l.) L’Hér., “Crows- foot”.
Oxalis corniculata L.
Stellaria media (L.) Vill.
No infection was obtained in any of the above species, while the H. peplus
controls gave lesions and pycnidia.
The plants were kept under observation for
weeks after inoculation, in case development of the fungus was slower than in “Petty
Spurge’”’.
No Septoria disease was detected in the field on plants of H. helioscopia at Hobart, Castle Hill and Hurstville, or on HE. Drummondii at Allawah and Sydney University, or on plants of the latter species sent from Toowoomba, Queensland. At Hurstville, several plants of H. helioscopia, growing in a patch of severely diseased H. peplus,
showed complete immunity.
Judged by the species tested, it appears as if the causal organism has a limited host range. Specificity by Septoria species for one or a few hosts has been noted by other workers, e.g., Beach (1919) working with 15 species, Weber (1922a, 1922b, 1923) with eight species, Cochrane (1932) with two species, Thompson (1941) with two species, and Sprague (1944) working with many species on grasses.
22 A SEPTORIA DISEASE OF KUPHORBIA PEPLUS L.,
NAME OF THE CAUSAL ORGANISM.
The history of the genus Septoria has been reviewed by Wakefield (1940) and Sprague (1944). The former’s recommendation that Septoria Sace. (1884) described from type species S. Cystisi Dem. be conserved against Septoria Fr. (1828), which was based on non-pycnidial species, was upheld by Sprague. Phleospora Wallr. (1833), based on non-pyenidial species, is an exact synonym of Septoria Fr. (1828). Grove (1935), Clements and Shear (1944) and Ainsworth and Bisby (1945) followed Saccardo’s use, employing Phleospora for the forms with incomplete pycnidia. The writer, on the above authorities, retains the genus as described by Sacecardo (1884) on page 474. 5
From examination of fresh diseased material and microtome sections, it is evident that the causal organism of the disease of “Petty Spurge” conforms to Septoria Sace. The filiform nature of the spores, their length in relation to their width (ratio approximately 15-20:1), the yeasty, later carbonaceous, scanty mycelium and distinct black pycnidia (to the naked eye), distinguish it from Stagnospora, whose spores are typically cylindrical (although grading into more filiform types), with a length: width ratio of less than 10:1, with a cottony appearance on P.D.A., and with pale brown, less prominently distinguished pycnidia to the naked eye (Sprague, 1944; Clements and Shear, 1941). The pycnidia are too well-formed for Phleospora, _ and lesions occur too often on the leaves to consider Rhabdospora.
The only species of Septoria recorded on Huphorbia spp. are as follows:
1. S. bractearum Mont., 1849. The spore length is given as 50u. It was described on H. serrata. (Saccardo, 1884.)
2. S. Kalchbrenneri Sacc. The type of this species is Rabenhorst Fungi europaei 1854, issued as S. euphorbiae Kalchbrenner in Hedwigia, 1865, p. 158, nec Guep. No spore measurements are given. It was described on E. silvatica, E. palustris and E. aspera. (Saccardo, 1884.)
3. S. Huphorbiae Guep., 1879. The spore measurements are given as “40—45u x 2-23u, with 3-4 indistinct septa. It was described on H. EHsula and E£. angulata. (Saceardo, 1884.)
The rest of the very brief descriptions of the above three species is more fe eR the same.
Oudemans (1902), after examining the Exsiccati of Desmazieres, recommended that S. bractearum Mont. should become S. Huphorbiae Desm., and that S. Huphorbiae Guep. should yield place to S. Guepini Oud. S. Kalechbrenneri Sace. remained unchanged.
4. §. media Sace. et Brun. A fairly full description is given. The spots are described as having a dark reddish margin, with spores 50-55u x In. It was described on EH. palustris. (Sacecardo, 1892.)
5. S. euphorbicola Hollos, 1910. The description of this species is fuller, and the spots are given as 1 mm. in diameter, pycnidia 140-160u in diameter, and spores 16-20u x 2-2-5u4. It was recorded on E. procera. (Saccardo, 1913.)
6. S. Hariotiana Sacc., 1906. A full description is given: the spots 1 mm. in diameter, with a dark purple margin, pycnidia 120-1254 in diameter, spores 3-4 septa, 30-324 x 34. It was recorded on EH. palustris. (Saccardo, 1913.)
Because of the inadequacy of the description of S. Kalchbrenneri Sace., a request was made to the Commonwealth Mycological Institute for further information, if it was available, and Dr. G. R. Bisby kindly supplied the following notes: “The Kew specimen of this No. 584 consists of three leaves bearing spots 1-3 mm. in diameter, roundish, visible on both sides of the leaf, at first brownish, then ashen, particularly on the upper surface of the leaf, at all times surrounded by a slightly raised, distinct, reddish brown margin; pyenidia circa 100—-200u wide, brown, with an ostiole which becomes 35u or more wide; spores (25) 30-35u x 2—2-5u, somewhat elevate and tapering
BY DOROTHY E. SHAW. 23
to 1-1-5u at one end, hyaline, 0-3 septate, straight or somewhat curved. Most of the pycnidia appear to open on the upper surface of the leaf.”
The only reference in the literature to a Septoria disease of H. peplus is in Jaczewski’s “Key to Fungi’, Vol. 2, p. 107, 1917: “S. euphorbiae Guep. on HL. amygda- loides, EH. peplus. Round, olive-coloured spots. Stylospores 40 to 454 by 2 to 2-5u with 3 to 4 indistinct septa.” (Jaczewski was apparently unaware of the entry in Revue Mycologique, 1902, whereby S. euphorbiae Guep. became S. Guepini Oud.). It is not known whether Jaczewski was referring to H. amygdaloides Lam. or E. amygda- loides L. Hooker and Jackson (1895) listed H. amygdaloides L. as being synonymous with #. sylvatica L., on which a Septoria disease had already been described, namely, S. Kalchbrenneri Sacc. Hooker and Jackson also listed H. amygdaloides Lam. as a synonym of H. nicaeensis All., and H. nicaeensis (St. Am. Fl. Agen., 192) as a synonym of H. Esula L. It was on H. Hsula L. that S. euphorbiae Guep. (now S. Guepini Oud.) was originally described. It is not known whether the Russian organism was identified on the grounds of morphological similarity. The spore sizes given in the Russian text are the same as those given by Saccardo (1884).
’ The fungus under study, with spores 35-84 + 5-9u long by 1-9 + 0-17u wide and pycnidia 110-2 + 19-0u and with unrestricted spots, differs in spore size from S. Huphorbiae Desm. (once S. bractearum Mont.) (spores 50”), and from S. media ‘Sace. et Brun (spores 50-554 by lu); and in pycnidia and spore size from S. ewphor- bicola Hollos (spores 16-204, pycnidia 140-1604). S. Huphorbiae Desm. was also recorded by Oudemans (1921) on H. exigua, and no infection was obtained on this plant with the organism from EH. peplus.
The fungus more closely resembles S. Hariotiana Sacc., S. Guepini Oud. and | S. Kalchbrenneri Sace. The spores of S. Hariotiana, however, are given as 3u wide, and the length of the spores of S. Guepini as 40—45u, and these are respectively wider and longer than the spores under study. The pycnidia of S. Kalchbrenneri (100-2004) are larger than those on Petty Spurge. S. Guwepini was recorded on H. Hsula, but no infection was obtained on this plant with the organism under study. The appearance of the lesions caused by S. Hariotiana and S. Kalchbrenneri on their hosts, differs markedly from those caused by the Septoria on EH. peplus.
It is realized that the same organism can sometimes produce quite dissimilar lesions on even closely related hosts, and that the difference in spore sizes of some of the above species could, perhaps, be accounted for by natural variation or environment. However, it is considered that the fungus is sufficiently different morphologically (as far as can be determined from the brief description of some of the other species), and with regard to host range, to be described as a new species. In correspondence with the C.M.I., Dr. Bisby was of the opinion that this would be the best course to follow. It is therefore proposed to name the fungus Septoria pepli, n. sp.
= SEPTORIA PEPLI, Nn. Sp.
Pycnidiis amphigenis, sed vulgo hypogenis, in orbicularibus non limitatis maculis; sparsis aut raro aggregatis, atris, globosis, innatis vel dein erumptibus, ostiolatis, 85-135 (65-165); sporulis hyalinis, rectus vel leniter curvatis, sursum attenuatis, guttulatis, 3-septatis, saepe 2-, raro 0- vel 4-septatis, 25-444 (17—-51lu) x 1-5-2-0u (1-3-2-5y).
Hab. in foliis et in cauli H. peplus L. in N.S.W., A.C.T. et Tasmania.
Pycnidia on both sides of the leaves, but mostly on the undersurface, in circular, unrestricted spots, singly or rarely aggregated in twos or threes, visible to the naked eye, black, but reddish-brown by transmitted light, globose or slightly elongate, immersed but later,erumpent; ostiole about one-quarter the diameter of the pycnidium; pycnidial wall smooth, composed of 2-3 layers of pseudoparenchymatous cells; 85-1354 (65-165,) ; spores hyaline, straight or slightly curved, tapering at one end, with a varying number of guttulae, 3 septate, often 2-, rarely 0- or 4-septate;, 25-44u (17-5luw) x 1-5-2-0u (1-3-2-5y).
Hab. on leaves and stems of E. peplus L., around Sydney, and at Wagga, N.S.W., -in the A.C.T. and Tasmania.
Type specimen collected at Pennant Hills, December, 1949.
24 A SEPTORIA DISEASE OF EUPHORBIA PEPLUS L.,
Acknowledgements.
I gratefully acknowledge the help of the following: Dr. G. R. Bisby of the C.M.I. for assistance as mentioned specifically in the text; Dr. D. B. Duncan for advice on the statistical analysis; Mr. 8. Fish, for two verbatim extracts from Revue Mycologique which is housed at Melbourne; Mr. J. Humpoletz and Mr. C. Warner for assistance in translating the extracts; Mr. J. Strang for the diseased material from Penshurst; the Sydney Botanic Gardens, Adelaide University, University of Tasmania and the Plant Introduction Office, C.S.I.R.O., Canberra, for cuttings and seed of the species of Euphorbia tested; Professor W. L. Waterhouse and the Department of Medical Illus- tration, Sydney University, for help with the photography. I am particularly grateful for the help given at all times by Professor Waterhouse during the course of the study.
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CLEMENTS, F. E., and C. L. SHEAR, 1941.—The Genera of Fungi. New York.
COCHRANE, L. C., 1932.—A Study of Two Septoria Leaf Spots of Celery. Phytopath., 22: 791-812.
CooKE, M. C., 1892.—Handbook of Australian Fungi. London.
C.S.1.R., 1942.—Standardised Plant Names. Bull. No. 156. Melbourne.
DoipGE, E. M., and A. M. BoTTOMLEY, 1931.—A Revised List of Plant Diseases Occurring in South Africa. Botanical Survey of South Africa, Mem. 11, Pretoria.
GROVE, W. B., 1935.—British Stem- and Leaf-fungi. Vol. 1. Cambridge.
Hooker, J. D., and B. D. JACKSON, 1895.—Index Kewensis Plantarum Phanerogamarum. Tomus 1.
HuGuHeEs, 8S. J., 1949.—Studies on Some Diseases of Sainfoin (Onobrychis sativa), ii. The Life History of Ramularia Onobrychidis Allescher. Trans. Br. Mycol. Soc., 32: 34-59.
Hurst., E., 1942.—The Poison Plants of N.S.W. Sydney.
JOHNSTON, T., 1947.—A Form of Leptosphaeria avenaria on Wheat in Canada. Can. Jour. IMG (Ca 268 2470)
IKLEBAHN, H., 1934.—EHine Blattkrankheit und einige sie begleitende Pilze. JZeitschr. fur Pflanzenkrankh. wu. Pflanzenschutz, 33, i, 1-23. Abstr. R.A.M., 13:407, 1934.
MACMILLAN, H. G., and O. A. PLUNKETT, 1942.—Structure and Germination of Septoria Spores. Jour. Agr. Res., 64: 547-559.
MCALPINE, D., 1895.—Systematic Arrangement of Australian Fungi. Melbourne.
NAKATO NAITO, 1940.—Studies on Septorioses in Plants. New or Noteworthy Species of Septoria Found in Japan. Mem. Coll. Agric. Kyoto, 47: 31-43.
NOBLE, R. J.. H. J. Hynes, F. C. McCurrery, and W. A. BIRMINGHAM, 1934.—Plant Diseases recorded in N.S.W. Sci. Buwill., 46, Sydney.
OUDEMANS, C. A. J. A., 1921.—Enumeratio systematica fungorum. Vol. 3, p. 1071 et seq.
1902. Rectifications Systematiques, rédigées en ordre Alphabétique. Rev. Mycol., p: LZ: :
RAMsBOTTOM, J., 1948.—Presidential Address. Trans. Brit. Mycol. Soc., 30: 22-39 (pp. 33-34).
ROARK, E. W., 1921.—The Septoria Leaf Spot of Rubus. Phytopath., 11: 328-333.
RUGGIERI, G., 1933.—Lesions on Citrus sinensis Obeck caused by Mycosphaerella aurantiorum. Boll. Staz. Pat. veg. Roma, N.S., 15, 2, 338-346, 1935. Abstr. R.A.M., 15:85, 1936.
SACCARDO, P. A., 1884.—Sylloge fungorum, 3: 474 and, 515.
—-— ——, 1892.—Sylloge fungorum, 10: 379.
, 1913.—Sylloge fungorum, 222: 1092.
SHIGEKATSU HIRAYAMA, 1931.—Studies of Septorioses of Plants. IV. New or Noteworthy Species of Septoria Found in Japan.- Mem. Coll. Agric. Kyoto, 13: 33-39.
SPRAGUE, R., 1944.—Septoria Disease of Gramineae in Western United States. Oregon State College, Oregon.
STEVENS, F. L., 1925.—The Fungi which Cause Plant Disease. New York.
STONE, R. E., 1916.—Studies in the Life Histories of Some Species of Septoria occurring on Ribes. Phytopath., 6: 419-427.
THOMPSON, G. E., 1941.—Leaf Spot Diseases of Poplars caused by Septoria musiva and S. populicola. Phytopath., 31: 241-254.
WAKEFIELD, E. M., 1940.—Nomina generica conservanda. Contributions from the Nomenclature Committee of the British Mycological Society. 3. Trans. Brit. Mycol. Soc., 24: 245-293.
BY DOROTHY E. SHAW. 25
WEBER, G. F., 1922a@.—Septoria Diseases of Cereals. Phytopath., 12: 449-470. , 19226.—Septoria Diseases of Cereals. Phytopath., 12: 5387-585. , 1923.—Septoria Diseases of Cereals. 3. Phytopath., 13: 1-23. WOLLENWEBER, H. W., 1938.—Sphaerella linicola n. sp., the Agent of the American Flax Epidemic (‘“‘Spasm” or Septoria Disease). Rev. Bot. Inst. ‘Miguel Tillo’, ii, 2a, pp. 483-492, 1938. Abstr. R.A.M., 18: 111-112, 1939.
DESCRIPTION OF PLATE I.
Fig. 1.—Leaves of Huphorbia peplus L. showing lesions caused by Septoria pepli n. sp. x 2.
Fig. 2.—Six months old culture of the fungus on P.D.A., showing ‘‘staling’’. Note the convoluted centre and the sub-surface hyphae at the edge. x 2.
Fig. 3.—Pycnidium produced in culture, on rye and peanut husks, with spores exuded in a cirrus. Mounted in cotton-blue lactophenol. x 100.
Fig. 4.—Section through pycnidium showing filiform spores. Cut at 84% and stained with gentian violet-orange G. x 400. :
26
PRESERVATION TECHNIQUES FOR SCARABAEID AND OTHER INSECT LARVAE. By P. B. Carne, B.Agr.Se., Division of Entomology, C.S.I.R.0. [Read 28th March, 1951.]
Synopsis. 4
Methods commonly employed in preserving insect larvae are compared and the use, especially for scarabaeid larvae, of Peterson’s “KAAD” fixative is recommended. The various fixatives in use are compared in regard to price; a modified cheaper form of Peterson’s fixative, giving equally good results, is described.
The probable function of the fixative components is discussed and evidence recorded which suggests that the discoloration of preserved larvae is due to tyrosinase activity; in vivo, the tyrosinase is inhibited by a dehydrogenase system. The very rapid distension of larvae killed in KAAD or its modification is discussed in the light of recent work on insect cuticle.
Detailed recommendations are given for the cleaning, fixing and storage of larval Scarabaeidae.
INTRODUCTION.
The writer has experimented with a variety of methods of preserving insect larvae. Much of his earlier collected material, and of that obtained from the-collections of earlier workers, shows marked faults, resulting from the use of unsatisfactory methods of preservation. While ethyl alcohol is used almost universally as a storage fluid, previous fixation is essential with most species; larvae killed and stored directly in alcohol become blackened and distorted and almost valueless for scientific study.
The purpose of this paper is to compare preservation techniques in common use, to bring to notice a valuable new fixative devised by Alvah Peterson (Peterson, 1943, 1948), and to discuss the functions of the constituents of the latter.
DEFINITION OF A SATISFACTORY FIXATIVE.
An ideal fixative would have the following characteristics:
(a) Larvae fixed in it should be distended and turgid, neither soft and flaccid, nor hardened and shrivelled. (0b) The larvae so fixed should retain normal, or close to normal, coloration (Lepidopterous larvae present much greater difficulties in this regard than do Scarabaeid larvae), with no darkening of the softer parts, nor bleaching of the head capsule. An increase in body opacity is desirable for morphological work where setal patterns are to be studied. (c) The fixative fluid should be reasonably inexpensive, as large quantities are used in the field collecting of larvae; and (d) the fixative should be one which may be used cold, and in which the larvae may be held for prolonged periods without deterioration. This is particularly desirable on collecting expeditions, when regular transference of larvae from fixative to storage fluid is some- times inconvenient. 2
PRESERVATION TECHNIQUES COMPARED.
The bulk of the larval collections seen by the writer have been treated by one or other of the following methods. The following comments are based upon a critical comparison of these methods, using series of larvae of Adoryphorus couloni Blackb., Semanopterus sp. and Sericesthis sp., and on the writer’s general observations on the preservation of a wide range of species of soil-inhabiting larvae. The compositions of the fixatives are given below.
a. By direct killing in a storage fluid of 70-95 per cent. ethyl alcohol or in methylated spirits.
This technique, which appears to be frequently used, is most unsatisfactory for scarab larvae. Darkening of the cuticle begins within 24 hours of killing, and older specimens are completely blackened, and may become either very soft or hardened, according to the strength of alcohol used.
BY P. B. CARNE. 27
b. By direct killing in a storage fluid of 4 per cent. formalin.
This technique is equally unsatisfactory, severe discoloration occurring in time. Formaldehyde vapour from the specimens is a continual source of irritation to the eyes during their examination.
c. By killing and fixing in boiling water, and subsequent storage in 70-95 per cent. ethyl alcohol, or in 4 per cent. formalin.
Quite good results can be obtained by this method, and darkening on storage is prevented to a great extent. Small larvae appear to respond better than large larvae, which may fail to become, or to remain, turgid. Collapse is frequent if the larvae are held in actively boiling water; much better results are obtained if the larvae are placed in a beaker of boiling water, which is then allowed to cool. If the high temperature is maintained, the fat lining the body wall is melted, and is deposited about the gut. Any heat treatment is undesirable when larvae are to be dissected.
d. Killing and fixing in one of the acetic-formalin fixatives, e.g., Carls, Blés, or Bouin, and transfer to 70 per cent. ethyl alcohol.
Carls’ or Blés’ fixatives can give good results in the laboratory where careful attention can be given to time and temperature of fixation. Overfixation, resulting in hardening and buckling of the body wall, is the common error. Such overfixation makes examination of setal patterns extremely difficult, and special “renovation” tech- niques may be necessary before determination of the larva is possible. These fixatives can be used cold for longer periods, but larvae should be sorted into size groups, because in the period required to ensure adequate fixation of large larvae, small larvae in the same series becomes grossly overfixed. However, where collections are being made at a number of localities, this introduces difficulties in that the number of containers to be carried is multiplied.
Bouin easily results in overfixation, and imparts an undesirable picric staining to the larvae, which is difficult to remove.
e. Killing and fixing in Carnoy’s fixative and storage in 90 per cent. ethyl alcohol.
Killing and fixing in cold Carnoy gives excellent results, although larvae become somewhat soft and transparent if allowed to remain in the fixative for more than a few days.
Larvae fixed by methods a—d invariably die with their mandibles closely opposed or overlapping, and these cannot be moved apart, the articulations becoming rigid, so that dissection of the mouthparts is difficult. On the other hand larvae fixed in Carnoy die with the mandibles opposed, but the articulations remain quite flexible and are easily dissected.
f. Killing and fixing in Peterson’s KAAD fixative, or derivatives thereof, and storing in 95 per cent. ethyl alcohol.
The use of Peterson’s KAAD results in larvae in an ideal state of preservation. The larvae are well distended, firm and completely free of any discoloration. There is an increase in opacity of the body wall.
Although Peterson states that best results are obtained by fixing for periods not longer than four hours, the writer has not observed any deterioration when larvae are left in the fixative for periods of up to three weeks. Best results for scarab larvae are given by fixation for not less than 2-3 hours.
As the writer’s use of KAAD began only two months prior to the drafting of this paper, he has not seen larvae so preserved for periods longer than this. It is the writer’s observation that, with all other preservation techniques, any tendency towards deterioration becomes evident within a week of treatment. Peterson states that the larvae retain their good condition for “a prolonged period”.
Larvae killed in KAAD almost invariably die with the mandibles apart, the articulations remaining flexible. Important taxonomic characters occur on the mandibles, and dissection may often be avoided when the mandibles remain apart.
The fixative is expensive, containing approximately 8 per cent. dioxane. For this reason the writer has tried omitting this component, with results equal to those
28 PRESERVATION TECHNIQUES FOR SCARABAEID AND OTHER INSECT LARVAE,
obtained with the complete fixative. The acetic acid content may be reduced to 25 per cent. of that recommended by Peterson, or may be omitted altogether if the larvae are previously killed in hot water.
For purposes of gross dissection, larvae fixed in KAAD are considerably superior to those prepared by any of the other methods described. Dr. M. F. Day, of the Division of Entomology, C.S.I.R.O., has kindly examined mid-gut tissues of cetoniid larvae so treated. He found that histologically the mid-gut was fairly well preserved, considering the gross method of fixation employed, and that staining of the tissues was satisfactory.
DISCUSSION.
The writer has rarely seen any discussion of the possible function of components of fixative mixtures, and has attempted to gain some understanding of these functions.
Firstly, all fixatives known to the writer contain acetic acid and alcohol. It is generally stated that acetic acid, together with the alcohol, is responsible for precipita- tion (“fixation”) of the body protein, and that for most larvae its presence is necessary to prevent subsequent blackening.
The fact that hot water treatment prevents discoloration suggests that the blacken- ing is an enzymatic process. That the enzyme is probably tyrosinase is supported by the following observations. Blackening is prevented by acids, suggesting that inactiva- tion of the enzyme results from denaturation of the protein portion of the enzyme. Tyrosinase is known to contain copper in its prosthetic group and to be inactivated by cyanide. Larvae killed in cyanide do not blacken when stored in alcohol but will do so if first placed in an alcoholic solution of cupric chloride. The period of immersion in hot water necessary to destroy the enzyme varies from one minute at 100°C. to 30 minutes at 55°C. with Semanopterus sp.
The rapidity with which larvae blacken depends upon the treatment, which suggests that there is some system present which prevents blackening. Dennell (1949) considers that the tyrosinase in the larvae of Calliphora erythrocephala is prevented from acting upon its substrate by the presence of a dehydrogenase system which maintains a low redox potential in the insect tissues. Destruction of this system allows the redox potential to rise and the tyrosinase to act upon its substrate. Chloroform inhibits dehydrogenase systems and Adoryphorus and Sericesthis larvae placed in chloroform vapour are completely blackened an hour after anesthesia. When killed in ethyl acetate vapour these larvae show no trace of blackening in the same period of time, which suggests the presence in these larvae of a tyrosinase-inhibiting dehydrogenase system.
Hurst (1940) has observed that a polar substance of low dielectric constant such as alcohol, is greatly assisted in its passage through the lipoid layer of insect cuticle by the presence of a non-polar compound of high dielectric constant, such as kerosene. The very rapid distension of larvae killed in Peterson’s “KAAD” appears to be due to this phenomenon. Scarab larvae placed in either ethyl alcohol or kerosene die very slowly, and distension takes place very slowly. Death occurs very rapidly in a mixture of these two substances, and larvae are fully distended in less than an hour. The rate of entry of alcohol may be reduced by lowering the proportion of kerosene in the mixture, and Peterson found this necessary with some soft-bodied larvae, which otherwise burst before equilibrium was established.
Some kerosenes are not completely miscible with alcohol-acetic acid mixtures, and Peterson finds that adding dioxane results in complete miscibility. While Peterson considers that the dioxane itself may improve the quality of some larvae, the writer has found larvae preserved in KAA equally good. The KAA becomes cloudy during fixation to a much greater extent than does KAAD, although the cloudiness may be greatly reduced by the use of absolute rather than 95 per cent. alcohol in preparing the fixative. The fixative may be filtered and used again a number of times,
BY P. B. CARNE. 29
COMPOSITION AND ESTIMATED COSTS OF FIXATIVES.*
Bouin.—Picrie sat. aqueous soln. 71 per cent., formalin 24 per cent., glacial acetic 3 per cent., approx. 7s. per gallon.
Carls.—Water 57 per cent., absolute alcohol 28 per cent., formalin 11 per cent., glacial acetic 4 per cent., approx. 4s. per gallon.
Blés.—70 per cent. alcohol 90 per cent., formalin 7 per cent., glacial acetic 3 per cent., approx. 3s. 6d. per gallon.
Carnoy.—Absolute alcohol 60 per cent.,’ chloroform 30 per cent., glacial acetic 10 per cent., approx. £1 1s. per gallon.
‘KAAD.—Kerosene 8 per cent., 95 per cent. alcohol 70 per cent., glacial acetic 14 per cent., dioxane 8 per cent., approx. £1 per gallon.
KAA (1).—Kerosene 8 per cent., 95 per cent. alcohol 77 per cent., glacial acetic acid 15 per cent., approx. 9s. per gallon.
KAA(2) (with acetic acid content reduced).—Kerosene 8 per cent., 95 per cent. alcohol 87 per cent., glacial acetic 5 per cent., approx. 5s. per gallon.
While there is probably little to choose between Carnoy and KAA(2), it will be seen that the price of the former is approximately four times that of the latter.
OTHER F‘AULTS IN PRESERVED LARVAE.
When larvae are immersed in any fluid other than actively boiling water, regurgita- tion of part of the gut contents occurs. The black fluid coagulates on the mouthparts. The latter possess taxonomically important structures, which must be examined in detail for specific determination; the coagulated material must therefore first be removed. This is a tedious operation, and one likely to damage delicate structures: it may be made unnecessary either by starving the larvae for several days before killing, so that the regurgitated fluid is colourless and leaves no deposit, or the larvae may be anesthetized before placing in the fixative, when no regurgitation occurs. Many scarab larvae are remarkably slow to succumb to cyanide, and the most rapid anesthesia is brought about by carbon dioxide or chloroform. Deep anesthesia is necessary, or the larvae recover sufficiently in cold fixative to regurgitate.
Tubes containing preserved larvae must be sealed to prevent evaporation, other- wise the percentage alcohol content of the fluid decreases rapidly, due to the higher volatility of aleohol over water in the mixture. Browning and changes of texture of the larvae.then occur which are highly undesirable. If some evaporation does occur, the tube should be topped up with an alcohol of higher strength than that originally used. a :
RECOMMENDED PROCEDURE FOR PRESERVATION OF SCARABAEID LARVAE.f
(a) Handle larvae with blunt forceps, and remove superficial dirt by blowing, or by gentle washing in cold water. With some cetoniid larvae, a soft brush may be necessary to dislodge adhering soil. Such cleaning movements should be made in a caudal direction to avoid damage to the setae clothing the body.
(bo) Anaesthetize the larvae deeply in carbon dioxide, or chloroform or ethyl acetate vapours.
(c) Kill and fix in Peterson’s KAAD or KAA for not less than two hours.
(d@) Wash larvae in alcohol briefly and store in 95 per cent. alcohol. Not more than two-thirds of the effective length of the tube should contain larvae, which should always be well covered by alcohol.
LARVAE OTHER THAN SCARABABIDS.
Peterson’s KAAD or KAA has been tried with a number of larval types. Excellent fixation was given with larvae of Carabidae, Elateridae, Cerambycidae, Chrysomelidae, Asilidae, and Lepidoptera. The fixative appears to be unsatisfactory for some Calli- phorid larvae.
* Based. on Australian prices as at July, 1950.
+ This refers to larvae killed in the laboratory. In the field, anesthesia may have to be omitted, and cleaning postponed until transfer to storage fluid in the laboratory.
30 PRESERVATION TECHNIQUES FOR SCARABAEID AND OTHER INSECT LARVAE. Acknowledgements. i The writer is indebted to Dr. R. H. Hackman, Division of Industrial Chemistry, and Drs. M. F. Day and K. H. L. Key of the Division of Entomology for advice and comment.
References.
DENNELL, R., 1949.—Weismann’s ring and the control of tyrosinase activity in the larva of Calliphora erythrocephala. Proc. Roy. Soc. L., (B), 136: 94.
Hurst, H., 1940.—Permeability of insect cuticle. Nature, 145: 462. PETERSON, ALVAH, 1943.—Some new killing fluids for larvae of insects. J. Hcon. Ent., 36: 115. , 1948.—Larvae of insects, Lepidoptera and Hymenoptera. Pt. 1. Columbus, Ohio.
31
THE ANATOMY AND MORPHOLOGY OF THE OPERCULUM IN THE GENUS HUCALYPTUS.
PART I. THE OCCURRENCE OF PETALS IN EUCALYPTUS GUMMIFERA (GAERTN.) HOCHR.
By J. L. WILLIS, Museum of Applied Arts and Sciences, Sydney.
[Read 26th April, 1951.]
Synopsis. The nature of the operculum present in the genus Hucalyptus has been for many years the subject of a considerable amount of conjecture, and a number of conflicting interpretations as to its morphological nature have been proposed.
In the young buds of H. gummifera, four minute imbricate petals have been found, which gradually fuse together to form an inner corolline operculum. This inner operculum remains quite distinct from the outer operculum which is probably calycine in origin.
There is, therefore, a close morphological relationship between the flowers of this species and the two New Caledonian genera Piliocalyx and Acicalyptus, which may also indicate a close phyletie relationship.
INTRODUCTION. Although a considerable volume of literature dealing with the anatomy of the genus Hucalyptus has accumulated, no investigations have yet been carried out on that unique organ, the operculum, most attention having been focussed upon the wood anatomy, leaf structure, etc. This is somewhat surprising, as the exact nature of the operculum has been the subject of a considerable amount of conjecture since 1788 when the genus was first described by L’Héritier.
HISTORICAL.
In his original description, and after consideration of the one species only (#. obliqua), L’Héritier held the operculum to be corolline in nature. On the other hand, Jussieu (1812) considered that it was formed by the fusion of two bracts. Robert Brown (1814), however, came to the conclusion that the operculum had different origins in different groups of species. He thought that in most species it represented a fusion of the calyx and corolla; in those species with double opercula, the inner structure represented the corolla, and the outer one the calyx; and in the genus Hudesmia R.Br., now Eucalyptus L’Hér., Series Hudesmiae (Blakely, 1934), the operculum was formed from the corolla alone. Hooker (1860) concluded that the operculum was a combined calyx and corolla, but Bentham (1866) was uncertain as to the correct interpretation of the organ, although he considered it to be most likely corolline in nature. He noted the presence of an additional outer operculum in some species, but regarded the nature of this outer organ as doubtful. Bentham thought that this outer operculum would eventually be found to be present in nearly all species but that it was deciduous so early that it was not noticeable in most buds. Maiden (1923) regarded the operculum as corolline in origin except in those species with double opercula where he considered the inner one to be corolline and the outer one calycine. He predicted that eventually all species would be found to have double opercula, the outer calycine one being deciduous very early in most species. Naudin (1883), Deane (1900), Andrews (19138), Hutchinson (1926), Blakely (1934), Rendle (1938), and Osborne (1947) all considered the operculum to be formed from the fused petals, whereas von Mueller (1879-84) concluded that the organ was nearly always calycine in nature. Hardy (1935, 1939), however, thought the operculum was either a modified corolla or a fusion of both calyx and corolla, but he considered the evidence insufficient to determine definitely which interpretation was the correct one.
32 ANATOMY AND MORPHOLOGY OF THE OPERCULUM IN EUCALYPTUS. I,
In view of these conflicting opinions, it was thought that a study of the organogeny of the operculum would definitely decide which of these interpretations should be | adopted.
THE PRESENT STATUS OF THE PROBLEM.
It is apparent from the descriptions of the various species that there can be no general interpretation of the morphological nature of the operculum, and that the species fall roughly into three groups: (1) the Hudesmiae with four minute calyx teeth surmounted by a single operculum; (2) those species with distinct double opercula throughout most or all of the stages of bud development, notably the Corymbosae- peltatae; and (3) those species with a single operculum only throughout most or all of the bud’s development. This last group includes the majority of Eucalypt species. These three groups coincide with those of Robert Brown (1814).
There are still good grounds for considering group (1) (Eudesmiae) to be a separate genus closely related to Hucalyptus. Also, group (3) is probably derived from group (2) by suppression of one of the opercula or by the fusion of both structures. Consequently, an examination of the opercula in group (2) is the most likely to provide information regarding the fundamental nature of the organ. It may also assist -in interpreting the morphological nature of the single operculum in group (3).
MATERIALS AND METHODS.
The Bloodwood EL. gummifera was the first species to be examined as it is a common tree on the Hawkesbury sandstone in the vicinity of Sydney, and so little difficulty was experienced in collecting adequate material. The buds used in this study were collected from three different localities—Avalon, Roseville, and National Park.
The buds at different stages were fixed under reduced pressure in F.A.A. or F.P.A. and embedded in paraffin in the usual way. Staining was carried out with Safranin and Delafield’s Haematoxylin using the method of Boke (1939).
The photographs were taken on Kodak Process Pan plates at a magnification of x 30, and have been reproduced at the same magnification.
THE ANATOMY OF THE OPERCULUM.
An examination of the very young buds of H. gummifera revealed the presence of four minute, imbricate petals, inserted between the staminal ring and outer operculum (Plate ii, figs. 1 and 2). The petals are simple in construction with a uniform epidermis and no cuticle, whilst a constant feature is the presence of many large, well-defined oil glands (Fig. 2). The petals are attached by a broad base, and have a peculiar arrange-
| Text-figures 1 and 2.
1. The petal arrangement in Hucalyptus gummifera. 2. The expected petal arrangement when the phyllotaxis is opposite and decussate.
ment in that they are not symmetrically imbricate. For example, in the case of three of the petals, the right (or left) edge overlaps the left (or right) edge of the petal next to it, whilst the fourth petal has both right and left edges enclosed by the petals on either side (Text-fig. 1 and Plate iii, fig. 8). As Huealypts have basically a phyllotaxis of one-half (Jacobs, 1986), one would expect a simple dimerous arrangement as shown in Text-figure 2.
BY J. L. WILLIS. 33
This arrangement described above leads to one petal being folded and enclosed by the other three, so that when the bud is cut transversely, the upper part of the folded petal is sectioned somewhat longitudinally (Plate iii, fig. 7).
As the bud develops, the petals gradually fuse together to form a solid inner operculum, which remains quite separate from the outer conspicuous operculum (Plate iii, figs. 10 and 11). The calyx tube grows faster than both the operculum and the fused petals, and as the proportional volume occupied by the fused petals becomes less and less, they spread out to form an almost flat cap just under the operculum proper (Plate iii, fig. 11). There is always a small space between the two structures and they fall together when the stamens unfold. A comparison of the ratio opercular volume to bud volume in Plate ii, fig. 1 and Plate iii, fig. 11, shows the much more rapid growth of the calyx tube.
Hardy (1935, 1939) has pointed out that the operculum is not a solid mass of tissue as most authors have assumed. Instead, a distinct lobing at or near the apex of the operculum marks the:presence of a minute pore, which may either run right through the organ, or only part of the way, according to the species under consideration. Hardy found this pore or traces of it in 55 species, and there seems little doubt that it will be found to be of general occurrence in the genus.
In H. gummifera this channel is lined with cuticularized elongated cells, and runs completely through the organ (Plate iii, fig. 9). It nearly always emerges at one side of the operculum near the apex, and rarely through the apex itself (Plate ii, fig. 3; Plate iii, fig. 9). At its upper end the channel is usually three lobed (Plate ii, fig. 4) but lower down it becomes a single elongated slit (Plate ii, fig. 5). It is eventually obliterated as the bud matures.
DISCUSSION.
Angophora is generally considered to be the genus with the closest affinity to Eucalyptus, the two genera being the only members of the Subtribe Eucalypteae (Bentham and Hooker, 1862-67). However, in view of the occurrenee of four minute petals in H. gummifera as described above, it now seems more likely that the genera most closely related to Hucalyptus will be found to be the two genera Piliocalyx Brong. and Gris., and Acicalyptus A. Gray. (Bentham and Hooker (1862-67) placed Piliocalyx in the genera anomala, and Acicalyptus in the Subtribe Metrosidereae. )
Acicalyptus is confined to the Fiji Islands and New Caledonia. Its buds are Eucalypt-like and four-angled, with a beaked operculum which falls off, revealing four minute imbricate petals inserted on the margin of the calyx tube by broad bases. These petals lightly cohere, thus forming a lid which falls in one piece when the operculum is detached. There are numerous inflexed stamens but the ovary has only two loculi. The fruit is unknown (Gray, 1854).
Piliocalyx also possesses an operculum covering four small, unequal imbricate petals which cohere to form an inner operculum. The stamens are indefinite and the fruit is unknown (Brongniart and Gris, 1865). One species is found on Lord Howe Island (von Mueller, 1873), but otherwise the genus is known only from New Caledonia. Both these genera, therefore, bear flowers having a very close morphological relation- ship to those of H. gummifera, and a closer examination of them should prove of great interest, as this similarity in floral morphology may well denote a close phyletic kinship.
It seems likely also that on investigation a situation similar to that described for E. gummifera will be found to hold for all other species of Eucalyptus with double opercula (mainly the Corymbosae-peltatae), and that by fusion or loss, the single operculum of the majority of Eucalypts has been formed. However, the position of -such species as H. camaldulensis Dehnh. (Series Exsertae) and #. microtheca F. Muell. (Series Buxeales) described by Maiden (1923) as having double opercula, is still obscure.
The presence of the opercular channel described above, may well indicate that the operculum is calycine in nature, especially in view of the lobed nature of the channel near the apex. In this connection it is significant that Hardy (1935, 1939) recorded this channel in two other members of the Corymbosae-peltatae, H. calophylla R.Br. and
Fr
34 ANATOMY AND MORPHOLOGY OF THE OPERCULUM IN EUCALYPTUS. I,
E. ficifolia F. Muell., the former having four symmetrical lobes at the apex of the operculum, and the latter either four, three or two lobes irregularly arranged, ‘but approximating to a symmetrical arrangement of four about the axial point”. Although Hardy considered the operculum, in the latter species at least, to be a combined calyx and corolla, for simplicity he termed the lobes ‘“‘petaline vestiges’’.
However, it has been found (Willis, unpub.) that in #. ficifolia (and almost certainly in #. calophylla) there are four imbricate petals similar to those reported above for H#. gummifera, but larger and not completely concrescent. They are pressed closely to the inside surface of the operculum but not fused to it, indicating that the operculum is most likely calycine in nature and is not a composite structure.
The indistinct lobing or lack of lobing, and the incomplete nature of the opercular channel found in such series as the Pachyphloiae and the Piperitales indicates that fusion takes place earlier and earlier in the ontogeny as the species becomes more advanced.
Jacobs (1936) has shown that the phyllotaxis of Hucalyptus is basically opposite and decussate. The alignment of juvenile leaves in two rows and the varying arrange- ments of mature leaves are caused by the growth in length of the stem between each pair of leaves, the twisting of the petioles, and the twisting of the internodes between each leaf pair. Consequently, the petals of a Hucalyptus flower would be expected to show two dimerous whorls instead of the arrangement described. The arrangement of the petals of Piliocalyx and Acicalyptus is at present unknown, but if they show an arrangement similar to that shown by LH. gummifera, it will be additional confirmation for the close morphological relationship to Hucalyptus postulated above for these two genera.
SUMMARY.
1. The presence of four small imbricate petals in H. gummifera is demonstrated.
2. The petals have an unusual unsymmetrical arrangement instead of the expected two dimerous whorls.
3. These petals fuse during the development of the bud forming an inner operculum.
4. The outer operculum has a distinct channel or slit running through it which is obliterated before the bud reaches maturity.
5. The relationship between H. gummifera and the two genera Piliocalyx and Acicalyptus is discussed.
6. There is some evidence indicating that the outer operculum represents a fusion of the sepals.
ACKNOWLEDGEMENTS. Thanks are due to the Trustees and the Director of the Museum of Applied Arts and Sciences, Sydney, for permission to publish this work, and to Dr. P. Brough for much encouragement and helpful criticism.
References. ,
ANDREWS, E. C., 1913.—The Development of the Natural Order Myrtaceae. Proc. LINN. Soc. N.S.W., 38: 558.
BENTHAM, G., 1866.—Flora Australiensis, Vol. 3. Lovell Reeve and Co., London.
, and Hooxrmr, J. D., 1862-67.—Genera Plantarum, Vol. I. Reeve and Co., London.
BLAKELY, W. F., 1934.—A Key to the Eucalypts. The Worker Trustees, Sydney.
Boker, N. H., 1939.—Delafield’s Haematoxylin and Safranin for Staining Meristematic Tissues. Stain Techn., 14: 129-131. ‘
BRONGNIART, AD., and Gris, A., 1865.—Observations sur Diverses Plantes Nouvelles ou peu connues de la Nouvelle-Calédonie. Ann. Sci. Nat., Sér. V, 3: 225-227.
Brown, Ropert, 1814.—Appendix to Matthew Flinders’ Voyage to Terra Australia, Vol. 2. Lovell Reeve and Co., London.
DBANB, H., 1900.—Observations on the Tertiary Flora of Australia, etc. Proc. LINN. Soc. N.S.W., 25:471. Incorrectly cited by Maiden (1923) as 22:471 (1897).
Gray, ASA, 1854.—Wilkes’ U.S. Expl. Exped. Botany Phanerogamia. Vol. I and Atlas. George Putnam and Co., New York.
Harpy, A. D., 1935.—Petaline Vestiges in Eucalyptus. Rept. Aust. N.Z. Assoc. Adv. Sci., 22: 372-4.
, 1939.—Additional Notes on Petaline Vestiges in Eucalyptus. Proc. Roy. Soc. Vic., 51
(N.S.) : 215-18.
BY J. L. WILLIS. 35
Hooker, J. D., 1860.—Flora Tasmaniae. Vol. I. Dicotyledons. Lovell Reeve and Co., London.
HUTCHINSON, J., 1926.—The Families of Flowering Plants. Vol. I. Dicotyledons. Macmillan and Co., London.
JACOBS, M. R., 1936.—The Primary and Secondary Leaf Bearing Systems of the Eucalypts and their Sylvicultural Significance. Commonwealth Forestry Bureau. Bulletin No. 18. Com- monwealth Government Printer, Canberra.
Jussieu, A. L. DE, 1812.—Note sur le Calice et la Corolle. Ann. Mus. Hist. Nat., 19: 431-2.
MaIpEN, J. H., 1923.—A Critical Revision of the Genus Hucalyptus. Vol. 6. Govt. Printer, Sydney.
MUELLER, F. von, 1873.—Fragmenta Phytographiae Australiae. Vol. 8. Govt. Printer, Melbourne. Fj
————,, 1879-84.—Hucalyptographia. Government Printer, Melbourne.
NAvDIN, C., 1883.—Mémoire sur les Hucalyptus Introduits dans la Région Méditerranéenne. Ann. Sci. Nat., Sér. VI, 16: 344. :
OsBorN, T. G. B., 1947.—Book Review. New Phytol., 46: 289.
“RENDLE, A. B., 1938.—The Classification of Flowering Plants. Vol. 2. Dicotyledons. Cambridge Univ. Press.
EXPLANATION OF PLATES II-III. (All photographs x 30.) Plate ii.
Fig. 1.—Longitudinal section through young bud showing insertion of petals.
Fig. 2.—Transverse section of young bud just below the level of the stigma showing the four imbricate petals. Two of the petals are beginning to fuse.
Fig. 3.—Transverse section of operculum of young bud showing lateral position of the entrance to the opercular channel.
Fig. 4.—Transverse section slightly lower than Fig. 3 showing triple nature of the channel near the apex of the operculum.
Fig. 5.—Transverse section slightly lower than Fig. 4 showing the opercular channel.
Fig. 6.—Transverse section slightly lower~ than Fig. 5 showing three imbricate petal segments.
?
Plate iii.
Fig. 7.—Transverse section slightly lower than Fig. 6 showing the four imbricate petals. The characteristic infolding of the fourth petal beneath the other three is clearly shown.
Fig. 8.—Slightly oblique transverse section at the level of the stigma showing the four imbricate petals. i
Fig. 9.—Longitudinal section of bud showing petals and opercular channel.
Fig. 10.—Longitudinal section showing gradual fusion of petal segments.
Fig. 11.—Longitudinal section of mature bud showing the outer calycine operculum, and the inner operculum formed from the fusion of the petals. The irregularly pentagonal objects just below the inner operculum are transverse sections through the filaments of inflexed stamens.
36
SOME NOTES ON ATHROTAXIS. By CHARLES G. ELLIOTT. (Communicated by Dr. Patrick Brough.) (Sixteen Text-figures. )
[Read 26th April, 1951.]
Synopsis.
This paper records some observations on Athrotaxvis made in Tasmania in 1946-47. The relationships between the three species of the genus are briefly discussed. The pollen of Athrotaxis differs from other Taxodiaceae in the absence of a germ pore or germinal papilla. The intine is 2-layered. Female cones often contain larvae of a fly. Some features of the gametophytes described by earlier workers are commented on in the light of more recent work on Sequoia and Sequoiadendron. Athrotaxis differs from all other Taxodiaceae in the absence of cleavage polyembryony. All the embryo initials contribute to a single dicotyledonous embryo. The relationships between Athrotaxvis and Sequoia and Sequoiadendron are briefly discussed, but no conclusions reached.
INTRODUCTION.
It is now more than 20 years since Saxton and Doyle (1929) published their fragmentary account of the life history of Athrotaxis selaginoides. Since then a description of the stem apex (Cross, 1943) is all that has been published on the morphology of the genus. On the other hand, the life histories of Sequoia sempervirens and Sequoia gigantea have been worked out (Looby and Doyle, 1937, 1942; Buchholz, 1939a, 1939b). Buchholz considers the two species sufficiently distinct to be placed in different genera, and instead of Lindley’s invalid Wellingtonia has proposed the genus Sequoiadendron for 8S. gigantea (Buchholz, 1939c). While some still consider the two to belong to one genus (Doyle, 1945), many have accepted Sequoiadendron (see especially Stebbins, 1948). This, together with the discovery in China of Metasequoia glyptostroboides, the morphology and ecology of which are being investigated by a number of botanists,* makes it highly desirable to know something more about Athrotaxis. During 1946-47, at the University of Tasmania, I carried out some observa- tions mainly on A. cupressoides Don, and though results were far from complete, the following points seem worthy of record.
RELATIONSHIPS BETWEEN THE THREE SPECIES.
No one in Tasmania could agree with Dallimore and Jackson’s (1948, p. 207) remark that the three forms ‘might well be regarded as gradations of one species”. The two common species, A. cupressoides and A. selaginoides, are quite distinct, both in the morphological characters used in taxonomy, such as the shape of the leaves and cone-seales, and ecologically. A. cupressoides forms stands or occurs as single trees by the side of tarns and along streams, while A. selaginoides is a taller forest tree generally growing on sloping ground. It does sometimes grow beside tarns, but IL have not observed it in such habitats when A. cuwpressoides is found there. Where the two species occur near one another, their habitats are distinct, as has been noted by Sutton (1928). <A. lawvifolia is rare, and does not form stands. Many people entertain the possibility that it is a hybrid between the other two species, although there is little substantial evidence for the view at present. However, many more trees than exist in any one locality would be needed to show much segregation of characters.
MALE CONES AND POLLEN. The male cones of Athrotaxis cupressoides can first be distinguished from vegetative tips about mid-February, and their development proceeds until May, when spore mother cells are found. Cones collected at Lake Dobson (Mt. Field National Park) on 25th
* A bibliography is given by Chu and Cooper (1950).
Led
BY CHARLES G. ELLIOTT. 37
August, 1946, and fixed in the field, contained microspores, but in some, the uppermost sporangia had quadrinucleate protoplasts with the spindles of the second meiotic division still present. Meiosis thus occurs about the second or third week of August. The pollen is mature about a month later. I have not observed the shedding of pollen in the field, but twigs with male cones collected at Lake Dobson on 9th September, 1946, and placed in jars of water in the laboratory at Hobart shed pollen on 17th September, and cones eollected at the Hugel Lakes (Lake St. Clair National Park) on 14th September, 1947, shed pollen in Hobart on 18th September.
The pollen of Athrotaxis, in common with that of other Taxodiaceae and Cupres- saceae, lacks air bladders, nor are there any male prothallial cells. As in other members of these families, the exine is thin and is thrown off by the swelling of the intine when the mature pollen grain comes into contact with water. The pollen of A. cupressoides is spherical or subspherical, and has no trace of any germ pore or germinal papilla. The diameter of acetolysed grains is 27-2 + 0-274. Three of the layers of the exine recognized by Erdtman (1948) can be observed in Athrotazis. The nexine, about 2u thick, is made up of an endonexine and an ectonexine of approximately equal thickness. The sexine is represented by minute granulae, irregularly scattered, and less than ly in diameter (Text-fig. 1).
Text-figures 1-6.—Athrotaxis cuwpressoides.
1. Acetolysed pollen grain. 2. Pollen, mounted in water, from which the exine has come off, showing the protoplast surrounded by a gelatinous “halo’’. 38, 4. Pollen after four days in water, showing enlargement of the protoplast within the ‘“‘halo’’. 5. Pollen after eight days in M/4 sucrose. Protoplast with its wall (ii) emerging from the ‘‘halo” (oi). 6. Pollen mounted in potash. es, exine; # and oi, inner and outer layers of the intine. Text-figure 1, x 640; all others, x 400.
Text-figures 7-8.—Cones of Athrotaxis cupressoides. x 2.
7, about the time of fertilization, 14th December, 1946. 8, with nearly mature seed, 24th February, 1947.
The intine itself consists of two layers. When fresh pollen grains are mounted in water, the grains first become turgid, and the average diameter is 30u. After the exine is thrown off, the contents of the grain are seen to be surrounded by a spherical gelatinous “halo”, the average diameter of the latter being 38u (Text-fig. 2). The pear-shaped “cell” within the halo is closely surrounded by a wall of its own, especially well seen in dry pollen mounted in potash (Text-fig. 6), which we may call the “endintine”’ (ii), and the halo then is the “exintine”’ (oi). After four days in water or sugar solutions the protoplast contained within the endintine has elongated consider- ably, and although the shape of the protoplast is irregular, the exintine still preserves a spherical form except whére it is actually forced out of position (Text-figs. 3, 4). When the cell surrounded by the endintine emerges (Text-fig. 5), the exintine is seen to have a definite thickness. A two-layered intine does not appear to have been
38 SOME NOTES ON ATHROTAXIS,
described before in a conifer, but since only the exine is preserved in fossils, it has monopolized the attention of pollen morphologists to the exclusion of the intine.
The pollen of Athrotaxis differs markedly from that of Sequoia, Sequoiadendron, and Metasequoia, in all of which there is a prominent germinal papilla, and it likewise differs from Cryptomeria, Taxodium, and Glyptostrobus, which also have a germinal papilla of some sort. It resembles more closely Cunninghamia and the Cupressaceae (Wodehouse, 1935; Erdtman, 1943; Sterling, 1949).
FEMALE CONES.
Young female cones have not been distinguished from vegetative tips before mid- September. In some localities production of cones varies from season to season. Cones of A. cupressoides were found near Lake St. Clair both in 1946-47 and 1947-48. But in the Mt. Field National Park, near the easterly limit of its distribution, A. cupressoides formed no cones in 1946-47, although A. selaginoides several miles away did form cones with fertile seeds. The same trees of A. cupressoides at Lake Dobson, however, bore cones in 1947-48. Text-figure 7 is a sketch of a cone about the time of fertilization. A fully grown cone is shown in Text-figure 8. Hames (1913, pp. 32, 33) has referred to the arrangement of the vascular bundles in the cone scales. The ovules are in a single row, and have their micropyles directed towards the cone axis. Cones of A. cupressoides have frequently been found to contain larvae of a fly. Heavily infested cones were deformed. The larvae appeared to be eating the ovules.
FEMALE GAMETOPHYTE.
Observations on the development of the female gametophyte are very incomplete. As Saxton and Doyle (1929) reported, during the enlargement of the megaspore the nucellus is soon consumed up to a thickness of one cell. In this respect Athrotazis seems to be more advanced than Sequoia and Sequoiadendron. An important feature is that in the free nuclear stages the nuclei are not evenly distributed round the embryo sac but are congregated at the end away from the micropyle, where they are two deep (Text-fig. 9). In this respect A. cupressoides resembles Sequoia sempervirens and differs from Sequoiadendron giganteum (Looby and Doyle, 1942). Saxton and Doyle’s (1929) Figure 8 of A. selaginoides shows nuclei distributed round the embryo sac in a single layer, though more densely packed lower down. Looby and Doyle (1942) have shown that in Sequoiadendron alveolation proceeds evenly all round the embryo sac. In Sequoia, however, while alveoli are formed against the central vacuole, in the lower part where nuclei are not in a single layer, walls form cutting out cells of irregular arrangement. In A. selaginoides, Saxton and Doyle’s Figure 18 shows that wall forma- tion takes place by the method in Sequoia, with alveoli being formed at the vacuolar | edge of the basal portion.
Another feature reported by Saxton and Doyle in which Athrotaxis resembles Sequoia and not Sequoiadendron is that the pollen tube grows over the surface of the nucellus. This is apparently also the case in Metasequoia (Sterling, 1949).
Y EMBRYO.
Proembryo stages were not found. The earliest stage observed showed eight cells in two tiers of four cells below the four-celled prosuspensor (Text-figs. 10, 13). The early growth of these embryo cells (Text-figs. 11, 12) gives rise to a single embryonic mass which may show some lobing (Text-fig. 14) and whose origin from four primary cells is generally apparent for some time. However, cleavage of this embryo, such as occurs in Sequoiadendron and Taxodium, was found never to take place.
There is no. primary suspensor. A massive secondary suspensor is produced by the single embryo. An early stage of development of embryonal tubes is seen in Text- figure 15. As the suspensor elongates and its upper part becomes folded, the nucellus and rather indefinite prothallial tissue are included in the folds, and all together form a compact mass at the micropylar end of the mature seed (Text-fig. 16). The embryo has two cotyledons. Rosette cells are present in the early stages (Text-figs. 13, 14). Nuclear divisions are numerous in the chalazal part of the gametophyte, but unfor- tunately in no case could the chromosome number be counted; however, it does not
BY CHARLES G. ELLIOTT. 39
appear to be large. Embryo systems are frequently found in pairs, the result presumably of the fertilization of two adjacent archegonia by the two male cells (Text-fig. 15). Thus Athrotaxis exhibits only Simple Polyembryony as defined by Buchholz. In this respect it is unique in the Taxodiaceae.
Text-figures 9-16.—Athrotaxis cwpressoides.
9. Longitudinal section of ovule at free nuclear stage, showing micropyle, integument, etc. Embryo sac badly shrunken, but it shows nuclei congregated at lower end in two layers, i4th December, 1946. x 60. 10. 8-celled embryo, 27th January, 1947. x 168. 11. Embryo with +wo tiers of cells each of more than four cells, 27th January, 1947. x 168. 12. Three-tiered embryo, 27th January, 1947. x 168. 13. Embryo system with two rosette cells, 4-celled prosuspensor, and 8-celled embryo, similar to Text-figure 10. 27th January, 1947. x 72. 14. Embryo system with rosette cells and lobed multicellular embryo, 19th January, 1947. x 72. 15. Embryos of two different systems showing early stage in development of embryonal tubes, 26th January, 1947, x 72. (Material from which Text-figures 10-13 is taken is from a more exposed locality at a higher altitude than those which gave Text-figures 14-15, hence the earlier dates of the latter. Text-figures 10-15 from dissected embryos.) 16. Longitudinal section of nearly mature seed, 24th February, 1947. x 24.
Free nuclear stages in the gametophyte were found in mid-December, and nearly mature embryos late in February.
DISCUSSION. Athrotaxis shows some significant differences from both Sequoia and Sequoiadendron in its morphological features—in its pollen grain, simple polyembryony; also in its one-cell-thick nucellus. It agrees with Sequoia in the method of wall formation in the
40 SOME NOTES ON ATHROTAXIS,
embryo sac, thin megaspore membrane, and two cotyledons. It resembles Sequoiadendron in its mature leaves being of one type only, in the presence of a prosuspensor and rosette cells, and one might expect in general features of proembryo development. It is clearly impossible to derive the Athrotaxis type of embryogeny with its prosuspensor from the Sequoia type which has none; but it may have been derived from that in Sequoiadendron. Buchholz (1940, 1948) has rightly suggested that Athrotaxis, Sequoia- dendron and Sequoia constitute a distinct subfamily, the Sequoidae. But it must be borne in mind that Florin (1940) has shown that while Athrotaxvis was widely distributed in the Southern Hemisphere in Tertiary times, “no representatives of this genus are known with certainty from the Northern Hemisphere” (p. 90). Reports of the southern occurrence of Sequoia, Florin shows, were the results of misdeterminations. On the other hand, Sequoia was abundant in Europe and Metasequoia in North America (Chaney, 1948). It is clear that the position of Athrotaxvis is not merely intermediate between Sequoia and Sequoiadendron, but we are not yet able to define their relationships more precisely.
Acknowledgements.
I am greatly indebted to Professor Joseph Doyle, of University College, Dublin, who agreed that some notes should be written on this work before his own observations from trees growing in Ireland are published.
My thanks are also due to Drs. E. I. McLennan and I. C. Cookson, of the University of Melbourne, and to Dr. P. Brough, of the University of Sydney, for their interest in this work.
References.
BucHHo.uz, J. T., 1939a.—The Morphology and Embryogeny of Sequoia gigantea. Amer. J. Bot., 26: 93-101.
, 1939b.—The Embryogeny of Sequoia sempervirens with a comparison of the Sequoias. Amer. J. Bot., 26; 248-257.
, 1939ce.—The Generic Segregation of the Sequoias. Amer. J. Bot., 26: 535-8. , 1940.—The Embryogeny of Cunninghamia. Amer. J. Bot., 27: 877-883. , 1948.—Generic and Subgeneric Distribution of the Coniferales. Bot. Gaz., 110: 80-91.
CHANEY, R. W., 1948.—The bearing of the living Metasequoia on problems of Tertiary Palaeo- botany. Proc. Nat. Acad. Sci. Wash., 34: 503-515.
CHu, K., and Cooper, W. S., 1950.—An ecological reconnaissance in the native home of Metasequoia glyptostroboides. EHcology, 31: 260-279.
Cross, G. L., 1943.—The Shoot Apex of Athrotaxis and Taiwania. Bull. Torrey Bot. Cl., 70: 335-348.
DALLIMORE, W., and JAcKSON, A. B., 1948.—A Handbook of Coniferae. 3rd Edition. Arnold, London.
Doy.LeE, J., 1945.—Naming of the Redwoods. Natwre, 155: 254-7.
EAMES, A. J., 1913.—The Morphology of Agathis australis. Ann. Bot., Lond., 27; 1-38.
ERDTMAN, G., 1943.—Introduction to Pollen Analysis. Chronica Botanica Co., Waltham.
, 1948.—Did Dicotyledonous Plants exist in early Jurassic Times? Geol. Foren. Stockh. Forh., 70: 265-271.
FLORIN, R., 1940.—The Tertiary Fossil Conifers of South Chile and their phytogeographical significance, with a Review of the fossil Conifers of Southern Lands.’ K. Svenska Vetensk. Akad. Handl., 3rd Ser., 19 (2).
Loopy, W. J., and Doyue, J., 1937.—Fertilization and Proembryo Formation in Sequoia. Sci. Proc. R. Dublin Soc., 21: 457-476.
———.,, 1942.—Formation of Gynospore, Female Gametophyte, and Archegonia in Sequoia. Sci. Proc. R. Dublin Soc., 23: 35-54.
SAxTON, W. T., and Doytg, J., 1929.—The Ovule and Gametophytes of -Athrotaxis selaginoides. Ann. Bot., Lond., 43: 833-840.
STEBBINS, G. L., 1948.—The Chromosomes and Relationships of Metasequoia and Sequoia. Science, 108: 95-8.
STERLING, C., 1949.—Some Features in the Morphology of Metasequoia. Amer. J. Bot., 36: 461-471.
Sutton, C. S., 1928.—A Sketch of the Vegetation of the Cradle Mountain, Tasmania, and a Census of its Plants. Pap. Roy. Soc. Tasm., 1928: 132-159.
WODEHOUSE, R. P., 1935.—Pollen Grains. McGraw Hill, New York.
41
THE PARAMPHISTOMES (TREMATODA) OF AUSTRALIAN RUMINANTS. PART I. SYSTEMATICS. By P. H. Durig£, B.Sc., Division of Animal Health and Production, C.S.I.R.O., Veterinary Parasitology Laboratory, Yeerongpilly. (Plates iv—v.) [Read 26th April, 1951.]
Synopsis.
A study has been made of the species of Paramphistomidae occurring in the rumen and reticulum of Australian cattle.
Identifications were based on the system devised by Nasmark 1937, involving an examination of the acetabulum, genital atrium and pharynx, as seen in median sagittal sections.
The species previously known as Paramphistomum cervi, was found to consist of Ceylonocotyle streptocoelium (Fischoeder, 1901) Nasmark, 1937, and Calicophoron calicophorum (Fischoeder, 1901) N&smark, 1937, whilst the species previously known as P. cotylophorwm was determined as P. ichikawat Fukui, 1922. A fourth species was present among the material examined but as yet remains undetermined.
In view of the importance given by Edgar (1938), Roberts (1934), Ross and Gordon (1936) to Paramphistomes as causal agents of parasitic gastro-enteritis in sheep and cattle in Australia, investigations were commenced into their bionomics, pathogenicity, and control. Three species of Amphistome flukes have been recorded from Australian eattle (Seddon, 1947), namely, Paramphistomum cervi (Zeder, 1790), P. explanatum (Creplin, 1847), and P. cotylophorum (¥ischoeder, 1901). P. cervi is recorded from Queensland, New South Wales, Victoria, Western Australia and Tasmania; P. explanatum from Queensland; and P. cotylophorum from Queensland and New South Wales. P. cervi and P. cotylophorum are noted as occurring also in sheep.
The author (Durie, 1949) published a preliminary note implicating the Planorbid snails, Glyptanisus gilberti and Segnitilia alphena as the intermediate hosts of P. cervi and P. cotylophorum respectively. These observations were based on the recovery of flukes, identified as those two species, from lambs fed cysts obtained from naturally infested snails. Subsequent attempts to infest snails experimentally with miracidia, considered to be those of P. cervi, were unsuccessful, and it seemed evident that the flukes that had been identified as P. cervi probably consisted of more than one species. A careful, taxonomic study of the Australian species was therefore considered essential before further work on the bionomics of these parasites could be attempted.
MATERIALS AND METHODS.
The bulk of the material examined has been collected from the rumen and reticulum of cattle slaughtered at abattoirs near Brisbane, Queensland, and drawn from the coastal and sub-coastal areas of the State. Additional material has been obtained from other States through the courtesy of slaughtering inspectors and veterinary research laboratories.
It is evident from the literature, Fischoeder (1901), Stiles and Goldberger (1910), Maplestone (1923), Travassos (1934), Dawes (1936, 1946) and Nasmark (1937), that the family Paramphistomidae is a very difficult one from the systematic point of view. This has been discussed fully by Nasmark (1937), who attempted to obtain a clearer conception of the family’s classification by careful studies on serial sections, particularly median sagittal sections, in which attention was largely directed to the muscle structures of the acetabulum and pharynx.
Nasmark’s system has been used by the author in the studies reported here and is considered a satisfactory one, at least, in so far as the Australian species are concerned.
q
42 THE PARAMPHISTOMES OF AUSTRALIAN RUMINANTS. I,
The measurements of body length and breadth were made on entire specimens fixed in Carnoy’s fluid, whereas all other measurements were made on median sagittal sections. Ova dimensions were obtained from eggs deposited in physiological saline.
It was noted very early in these studies that Gigantocotyle (Paramphistomum) explanatum (Creplin, 1847) Nasmark, 1937, was not represented in the Australian material under examination. This species has a readily recognized and distinct musculature of the acetabulum and pharynx (Nasmark, 1937), and specimens received from Ceylon were easily identified on this character. G. explanatum occurs in the gall bladder and bile ducts of bovines (Dawes, 1946; Nasmark, 1937). In view, therefore, of the absence of reports from Australia of amphistomes occurring in these parts of the body, together with the fact that the species was not represented among the hundreds of amphistomes obtained for examination from various parts of Australia, it is highly probable that G. explanatum is not present in this country and that records of its occurrence by Roberts (1984) and Ross and Gordon (1936) are erroneous.
Two and possibly three species have been recognized among the flukes previously identified as P. cervi. Calicophoron calicophorum (Fischoeder, 1901) Nasmark, 1937, is very common and widespread and is probably the most prevalent amphistome in Australian cattle. Ceylonocotyle streptocoelium (Fischoeder, 1901) Nasmark, 1937, is the second species, whereas the third species as yet remains undetermined, but appears to be closely related to C. calicophorum.
A re-examination of the species previously recorded in Queensland and New South Wales as Paramphistomum cotylophorum has shown it to be Paramphistomum ichikawai Fukui, 1922. A detailed description of each of these species is given below.
CEYLONOCOTYLE STREPTOCOELIUM (Fischoeder, 1901) Nasmark, 1937.
Host: Bos taurus—rumen and reticulum.
Distribution: Beaudesert, Queensland; Gympie, Queensland.
Length 4:55 mm. (3:0-5:0 mm.), breadth 2-4 mm. (2-3 mm.); D.V. measurement 1-4 mm. (1:1-1:8 mm.); dorsal line curved, curvature greater in the posterior third of the body; ventral line plain to slightly concave. Acetabulum conforms to Nasmark’s Streptocoelium type opening postero-ventrally, its maximum diameter 1:0 mm. (0-7-1-2 mm.), the ratio of its diameter to body length being 1:4:°5 (1:3:5-1:5:0). Pharynx conforms to Nasmark’s Paramphistomum type, 0:40 mm. (0:30-0:46 mm.) in length; ratio of pharynx length to body length 1:10:0 (1:9-1:12). Oesophagus 0:22 mm. (0-17- 0-25 mm.) in length; oesophageal sphincter present. Genital atrium with genital sphincter, conforms to Nasmark’s Streptocoelium type; ratio of genital atrium to acetabulum diameter 1:3:9 (1:3:5-1:4:5). Testes moderately lobed, oval to rectangular in shape, situated tandem and measuring 0°-5 mm. in length and 1:0 mm. in D.V. direction. Egg 0-148 x 0:074 mm. (0:154 x 0-070 — 0-145 x 0:078 mm.).
Acetabulum (Pl. iv, fig. 1).—The acetabulum conforms to Nasmark’s Streptocoelium type. The circular muscle series are characterized by their relatively few muscle units, with the interior series presenting a greater number than the exterior series. There is no division into de, and de, circular. De and di circular correspond closely in structure and in the number of units to ve and vi circular. - The de series shows a slight variation in the size of the units, these being smaller externally and increasing in size internally. The number of units present in 10 specimens is shown in Table 1.
Pharynz.—The pharynx conforms to Nasmark’s Paramphistomum type. The internal circular muscle layer consists of small closely packed units. The internal surface of the pharynx is smooth or with very small papillae. The interior longitudinal layer appears as a clearly developed band and extends inwards for about one-quarter the width of the pharynx. The middle circular layer is absent. The radial muscles are not strongly developed but are clearly visible. The exterior circular layer is rather indistinct with small units. The exterior longitudinal layer is indistinct and narrow. The basally circular layer is strongly developed and the series appear to be arranged in two rows. A slight trace of the anterior sphincter is present, but both the lip sphincter and posterior sphincter are absent.
BY P. H. DURIE. 43
Genital Atrium (PI. iv, fig. 2)—The genital atrium of this species is much smaller than that of P. ichikawai, and possesses both a genital sphincter and sphincter papillae.
The genital papilla is rather thick and variable in shape. Sphincter papillae, although present, are not as strongly developed as described by Nasmark (1937). The genital sphincter consists of several bundles of closely packed fibres. It is quite conspicuous, but again not as strongly developed as described by Naésmark (1937). The ventral atrium is only slightly developed.
TABLE 1. C. streptocoelium: Unit Series of Circular Musculature of the Acetabulum from a Median Sagittal Section.
de. di ; | vi ve : < 12 | 32 37 15 14 | 30 25 15 17 32 | 29 15 15 30 27 13 15 30 30 14 14 31 22 12 12 28 25 13 14 29 26 14 13 31 28 14 ne 32 26 13 Discussion.
The specimens differ from Nasmark’s description mainly in body length (7:6 mm.), but the measurements of the pharynx and other structures, except the oesophagus, agree closely with his description. Consequently any ratios given by Nasmark involving body length are greater than those given by the author.
Body length is regarded as a useful character to define the approximate size of a species, and to differentiate species which vary greatly in size. However, the length ~ of the body in living specimens is an extremely variable one, as flukes actively elongate and contract continually. In fixed specimens the body length may vary according to the type of fixative and method of fixing employed and is therefore considered unsuitable for determining critical ratios.
In specimens belonging to the genus Ceylonocotyle Laurer’s canal and the excretory canal (PI. iv, fig. 3)