PLANT NEMATOLOGY NOTES ^.CA^-, PLANT NEMATOLOGY WORKSHOPS /4b > 7>i NORTH CAROLINA STATE COLLEGE RALEIGH, N. C. 1954 ALABAMA POLYTECHNIC INSTITUTE AUBURN, ALA. 1955 Sponsored By The SOUTHERN REGIONAL NEMATODE PROJECT (S-19) PLANT HEMATOLOGY NOTES PLANT NEMATOLOGY WORKSHOPS NORTH CAROLINA STATE COLLEGE RALEIGH, N. C. 1954 ALABAMA POLYTECHNIC INSTITUTE AUBURN, ALA. 1955 Sponsored By The SOUTHERN REGIONAL NEMATODE PROJECT (S-19) FOREWORD TO 1955 EDITION These lecture notes from the firct Plant Nematology Workshop, held at Raleigh, North Carolina, Sept'ember 7 to 18, were prepared for the use of the students at the Workshop and not for publication. Instructors at the V/orkshop were: E. J. Cairns, iMabama Polytechn.ic Institute^ J. N. Sasser, North Carolina State College; and A. L, Ta.^lor, Section of Nematology, Cf.S.D.A. This Workshop was a function of the Southern Regional Nematode Project and was held to provide training in nematology for professional plant pathologists. Facilities were furnished by the Division of Plant Pathology, North Carolina State College. The services of Mr. A. L. Taylor were made possible through the co- operation of Dr. G. Steiner of the Section of Nematology, U.S.D.A. Drawings of nematodes for these notes were made by Dr. Hedwig Hirsch"iann, North Carolina State College. Reproduction of the notes was made possible through a grant from the Rockefeller Foundation. J. N. Sasser, Chairman Technical Committee (S-19) FOREWORD TO 1958 EDITION Requests for copies of these Plant Nematology Notes soon depleted the first and second printings of it. Therefore, a final printing has been prepared and at the same time, extra sets of the new and revised portions have been made for distribution to owners of the earlier edition. This manual was intended as a stopgap measure due to lack of up to date and available texts j we hope it has served this purpose well. Gratifying as the demand for the manual has been it is even better to note that need for it should soon be over. A book dealing with the plant-parasitic nematodes, the diseases they cause, and their control is soon to be released by Dr. J. R. Christie. A text, which is to be more taxonomic in its approach, is being written by Mr. G, Thorne. The out of print book. Plant Parasitic Nematodes, by T. Goodey is to revised by J. B. Goodey. Soil and Freshwater Nematodes , also by T. Goodey and out of print, is to be reissued. In addition, the number of recent workshops in nematology thnt hnve been held snd the release of their notes have reduced the need for increasinf? the scope or making an extensive revision of Plant Nematology Notes. Appreciation is again acknowledged to Dr. Hedwig Hirschmann for her work in preparing the new plates for the section on morphology. Thanks are also due to the various authors from whose works we have collected information for these Notes. As before, reproduction of the Notes was made possible through a grant from the Rockefeller Fovindation. The services rendered by this Foundation to the southeastern regional plant-nematology program have set a pattern of activities for all the regions. The result is an arousal of interest and action in phytonematology, rewarding to all who are interested in this important subject and who have so generously supported it with money and active personal participation. E. J. Cairns, Chairman Technical Committee (S-19) First and second editions copyrighted 1955 aJ^d 1958. A TABLE OF CONTI'OTS Forwards to 1955 and 1958 Editions TECHNIQUF^ Tech. A: Equipment and materials B: Outline for processing samples C: Isolation of nematodes from soil D: Location and isolation of nematodes from plant tissue E: Preparation of nemrtode slide moiints F: Additional techniques and sources of information FX3RPH0L0GY Morph.A: General structure of nematodes B: External characters C: Digestive system D: Reproductive system E: Nervous system . F: Excretory system G: Plates I-IV SYSTEI-IATICS System. Nematode systematics B Key to the most common soil forms C Key to the females of the Tylenchoidea PLANT-F/JIASITIC NEMATODES Paras. A: Cyst-forming nematodes of the genus Heterodera B: Root-knot nematodes C: Meadow or root-lesion nematodes D: Bud and leaf nematodes E: Stem and bulb nematodes F: Spiral nematodes G: Sting nematodes H: Ring nematodes I: Stylet nematodes J: Stubby root nematodes K: Dagger nematodes FREE-LIVING MEMA.TODES Free-liv.A: Identification and biology CONTROL Control. A; Chemical B; Other means MISCELLANEOUS riisc.A: Useful references B: Scientific and common names . Tocl.aioue /. :1 :<^ EQUIPMENT AND MATERIALS >'^'^* '^*'.. /fe" 'i* I. Equipment and Materials for the Laboratory A. Optical Equipment: 1. Binocular sterescopic microscope of a type possessing a pedestal base or otherwise mounted so as to permit transmitted illumina- tion of the specimens. A range of magnifications from low to high powers is desirable. 2. Honobjective microscope equiped with a mechanical stage, sub- stage condenser, and objectives ranging from low to high magni- fications (oil immersion). A set of oculars with powers from about $1 to l^X and interchangeable binocular body tube are useful additions. Achromatic lenses are satisfactory for most work, but an apochromatic oil immersion objective with compensat- ing eyepieces is much to be preferred for critical examinations requiring maximum resolution at high magnification. 3. Illumination froia an electric illuminator of a condenser type capable of providing the Koehler system of illumination is desirable, if detailed microscooic examinations of the nematodes are to be" made U. Measurements of nematodes and their parts v/ill require an ocular micrometer disc. A filiar micrometer eyeoiece is a useful acces- sory for very accurate measurements, but it is not a necessity. Calibration of the ocular disc or the filiar micrometer will require access to a stage micrometer, 5. Camera lucida apparatus is also useful, if not necessary. Most nematoiogists dispense with the small mirror provided and instead use a large-sized, front- surfaced mirror. A source of these mirrors, which are made to size and stocked just for this purpose, is the Pancro Mirrors Co. Inc., 2958 Los Feliz Blvd., Los Angeles 39, California. The item is listed as camera lucida mirror, 6X9 inches, 1/8 inch thick mirror- quality glass, front-surfaced Pancro coating. The mirror is mounted on a piece of wood, and a very satisfactory assembly for the mirror can be made from standard Flexiframe supports obtainable from scientific supply houses. B. Slide Making Materials: 1. Microscope slides of the 3X1 inch size are most used. The best quality, non-corrosive slides should be used for permanent slides. Clinical grade or other lees highly finished or selected grades are satisfactory for routine work. A special metal slide which holds the nematode specimens moiinted between two coverglasses is obtainable. This device permits study of both sides of the 2 Tech. A: mounted specimen. Equipment for making these slides is available on loan from various laboratories. Contact one of the authors for more information. 2, Covfar-gLasses of best quality are to be preferred. They should be of #0 thickness for routine work. Circles of 3/U inch diameter are generally used. Square cover-slips may be satisfactory and are less expensive, 3. Slide sealing materials are of various kinds. An ideal sealing cement ZUT was devised by G, Thome (1935) and is widely used by phytonematologists. It is used to seal water, formalin, T A F, lactophenol, and glycerine m.ounts. It dries quickly and resists action of solvents used for removing immersion oil from the cover-slips. ZUT is obtainable in pint and quart amounts from; Bennett's, 65 'iest First South Street, Salt Lake City 10, Utah. The recommended thinner for ZUT is butyl acetate. Ethyl acetate has been found to be a satisfactory suiDstitute, and acetone may also be used, but the latter may produce small bubbles in the applied ZUT seal. Lactophenol gum is a cement used to seal lactophenol mounts. The directions for its preparation (Davis, 192ii) are as follows: Dissolve 38 grams of pure gum arable in 50 ml, of distilled water, add 5 grams of glucose and 6 grams of lactophenol. The solution is then filtered through glass-wool. Lactophenol consists of a solution made by mixing 3 parts melted phenol, 1 part lactic acid, 2 parts glycerine, and 1 part water. Other slide sealing cements include: Cleared , used to seal lactophenol mounts, obtainable from H. W. Clark, 5Ul9 - 32nd Street, N. W., Washington, D. C; paraffin-vaseline mixtiire) 50-50, for temporary water and formalin mounts; gold-size varnish and bakelite resin varnish and other materials can be used, pro- vided that they are not effected by the mounting medium or by the solvents used to remove immersion oil. A slide-ringing turntable is not necessary. When used for making neat seals of round cover-glasses, extra precaution should be taken to provide a seal of sufficient thickness, particularly if the ringing compound is thinned for easier application. Seals applied freehand are likely to be thicker, and round as well as square cover-glasses can be safely sealed in this manner, even if somewhat less neatly. Sources of turntables are the Will Corp., Ho Chester 3, N. Y.j and the Southern Scientific Co., Inc., Atlanta 3, Georgia. U. Cover-glass supports are necessary to prevent distortion of the nematode specimens by the pressure of the cover-glass, liJhatever the kind of support used, it is imperative that it be only slightly thicker than the cross-sectional diameter of the nematodes. Tech. A:3 This is necessary in orJor that tlie oil immersion objective, which has a veiy limited working distance, may be focused on and within the nematode without contacting the cover-glass.