Peroxidases h'om MeIoidogyne spp.: Starr 5 In: The Worthington Manual, Worthington 6. HUSSEY. R. S., and J. N. SASSER. 1973. Biochemical Corp., Freehold, New Jersey. Peroxidase from Meloidogyne incognita. 2. ttI3AN(;, C. W,, L. H. I,IN, and S. P. HUAN(;. Pltysiol, Plant I'athol. 3:223-229. 1971. Alteratimls in peroxidase activity in- 7. LOWRY, O. H., N. J. ROSENBROUGH, A. L. tliiced bv root-klu)t llelnatodes on tomato. FARR, and R. J. RANDALL. 1951. Protein Bot. Buli. Academia Sinica XII:74-83. measuremellt with the Folin phenol reagent. 3. HUAN(;, C. W., L. H. LIN, and S. P. HUANG. J. Biol, Chem. 193:265-275. 1971. Changes in peroxidase isoenzymes in 8. STARR, J. L. 1977. A micro-polyacrylamide gel tomato galls induced by Meloidogyne incog- electrophoresis system for analysis of nema- nita. Nematologica 17:460-466. tode enzymes. J. Nematol. 9:284-285 (Abstr.). 4. I-[tlSSEY, R. S. 1971. A technique for obtaining 9. STARR, J. L. 1977. Peroxidase activity in dif- quantities of living Meloidogyne females. J. ferent developmental stages of Meloidogyne Nematol. 3:99-100. spp. J. Nematol. 9:285 (Abslr.). 5. HUSSEY, R. S., J. N. SASSER, and 10, VRAIN, T. C, 1977. A technique for the collec- D. HU1SINGH. 1972. Disc-electrophoretic tion of larvae of Meloidogyne spp. and a studies of soluble proteins and enzymes of comparison of eggs and larvae as inocula. J. Meloidogyne incognita and M. arenaria. J. Nematol. 9:249-251. Nematol. 4:183-189. Morphological Comparison of Second-Stage Juveniles of Six Populations of Meloidogyne hapla by SEM~ J. D. EISENBACK and HEDWIG HIRSCHMANN ~ Abstract: External morphology of second-stage juveniles of six populations of Meloidogyne hapIa, hclonging to two cytological races (A and B), and one population each of M. arenaria, M, incog~ita, and M. ]ava~ica was compared by scanning electron microscopy (SEM). Race A of M. hapla included three facuhatively parthenogenetic populations with haploid chromosome numbers of 15. 16, and 17; race B consisted of three mitotically parthenogenetic populations with somalic chromosome numhers of 45, 45, and 48. The mitotically parthenogenetic popula- tions of M. arenaHa, hi. i~wogtzita, and M. ]avanica had 54, 41-43, and 44 chromosomes, respectively. Observations were made on head stntcturcs, lateral field, excretory pore, anal opening, and tail. Head morphology, including shape and proportion of labial disc and lips, expression of labial and cephalic sensilla, and markings on head region, was distinctly different h)r each species. AI, ha]da populations of race A were distinct from each other but showed much intrapopulatiou variation in head morphology. Populations of race B were difl'erent from those eft race A and were very stable and quite similar in head morphology. Considerable inter- and imrapopulatiou wn'iation lnade the structure of the lateral field, excretory pore, anal opening, and tail of little value in distinguishing species or popttlations. The results are discussed in relation to earlier SEM observations on the genus Helerodera. Key Words: M. arenaria, M. incognita, M. ]avanica. Species of the genus Meloidogyne are of value taxonomically, but many others Goeldi exltibit considerable variation in are variable and exltibit too much overlap their morpltology (10, l l, 23), physiology among species to be nseful in species differ- (12, 14, 15, 16, 17), and cytology (20, 21). entiation. MeIoidogyne species need to be In his review o[ the genus, Whitehead (23) examined more closely for distinctive mor- showed tltat many morphological characters phological characters. Cytological studies of in females, males, and second-stage juveniles several populations of M. hapla Chitwood emphasized the need for a detailed mor- Received for publication 13 March 1978. phological study, because this species 1 Paper No. 5570 of the Journal Series of tile North Carolina appears to be made up of two distinct Agricultural Experiment Station. Raleigh, North Carolina. A portion of the senior aulhor's Ph.D. dissertation. cytological races (21): One race includes "-Graduale Research Assistant and Professor, respectively populations that have a haploid chromo- l)eparonenl of Plant PathologT, North Carolina State University, Raleigh, North Carolina 27650. This study some number of 15, 16, or 17 and reproduce was supported, in part, by Nalional Science Foundation by [acultative meiotic parthenogenesis. The (;rant No. DEB 76-20968 A01 and A1D Contract No. ta-C- 1234. other race consists of populations with a 6 Journal of Nematology, Volume 11, No. 1, January 1979 somatic chromosome number of 45 or 48 (Lycopersicon esculentum Mill. 'Rutgers') (personal communication, A. C. Trianta- in a gTeenhouse; the M. hapla populations phyllou) and reproduces exclusively by were kept at 21 C, and the other species at mitotic parthenogenesis. Little effort has 28 C. Newly hatched second-stage juveniles been made so far to make a detailed mor- were obtained by incubating egg masses in phological comparision of populations of a moist c/lamber at room temperature. The Meloidogyne of different chromosome num- specimens were transferred to 0.5 ml of ber and mode of reproduction. Previous filtered tap water in a BPI watch glass, and morphological studies of this genus have were chilled and killed by adding 3-4 drops been limited almost exclusively to the light of cold (8 C) 4% gultaraldehyde solution microscope; only recently has the electron (buffered with 0.1M sodium-cacodylate at microscope become a useful tool in clarify- pH 7.4) every half an hour until the final ing the morphology of Meloidogyne species. concentration of the fixative reached 2%. A few studies on the fine structm'e by Fixation of the material continued for an transmission electron microscopy have con- additional 72 h in tile cold. The specimens tributed greatly to our understanding of were then transferred to a processing the internal morphology of these nematodes chamber modified from DeGrisse (7), and (1, 2, 3, 8, 9, 22). Scanning electron micro- the remaining steps, except critical point scope (SEM) observations of the external drying, were taken without the top of the morphology of Meloidogyne spp. are lim- chamber in place. Fixation was followed by ited (13). rinsing with 0.1M sodium-cacodylate plus The present study was done to com- 7.5% sucrose solution at pH 7.4 for 12 h in pare external morphological characters in the cold. Postfixation with 2% osmium second-stage juveniles of the chromosomal tetroxide, buffered with 0.1M sodium- populations that now form the species M. cacodylate at pH 7.4, for 12 h was followed hapla by SEM and to relate morphological hy rinsing with tile buffer-sucrose solution findings with cytological data. In addition, for 12 h in the cold. Specimens were de- one population each of 3I. arenaria (Neal) hydrated with an 8-step graded series of Chitwood, M. incognita (Kofoid and ethanol during a 3-h period at room tem- V~qfite) Chitwood, and M. javanica (Treub) perature, and Freon 13 was used as an Chitwood was observed with the SEM as a intermediate fluid in a 5-step graded series guide to the differences that may occur of fifteen minutes for each step. The nema- between species. todes were then critical-point dried with Freon II3 by standard procedures (5), and dried specimens were transferred indi- MATERIALS AND METHODS vidually with a dental root-canal file onto Six populations of M. hapla, differing a stub covered with double-coated tape. in mode of reproduction and chromosonm Tile specimens were propped up against a number, and one population each of 3I. hair to ensure an unobstructed view of the arenaria, M. incognita, and M. javanica entire head region; they were coated with were selected from the Meloidogyne collcc- approximately 400 A of gold and viewed tion at N.C. State University. Populations and photographed with an ETEC scanning of M. hapla were designated by a number, electron microscope operated at 20 Kv. At an abbreviated word indicating their ori- least 100 second-stage juveniles from each gin, and their chromosome number in population were examined. parentheses, as follows: 42-Can (15) from Canada; 6-NC (16), 86-NC (17), and 48-NC OBSERVATIONS (45) from North Carolina; 66-Md (45) from Maryland; and 230-Chile (48) from Chile. Head morphology: Fig. 1 shows the Populations of the other three species were basic plan of the cephalic characters for the designated similarly, as follows: M. arenaria genus Meloidogyne, based on SEM obser- 351-Fla (54) from Florida; M. incognita vations of second-stage juveniles of M. 68-NC (41-4~) from North Carolina; and arenaria, M. hapla, M. incognita, and M. M. javanica 78-Ga (44) from Georgia. All iavanica. Centrally located on the labial populations were propagated on tomato disc is the ovoid opening of the prestoma, SEM of Meloidogyne hapla Juveniles: Eisenback, Hirschmann 7 A PS B FIG. l-A, B. Diagrams illustrating the head morphology of a second-stage juvenile of the genus Meloidogyne. A) Face view, B) View from the lateral side. AA, amphidial aperture; BA, body almule; CS, cephalic sensillnm; HR, head region; ILS, inner labial sensillum; LD, labial disc; LL, lateral lip; ML, medial lip; PS, prestoma. which leads to the slitlike stomatal opening edge of tile labial disc. Cephalic sensilla are medially below it. The prestoma is en- expressed externally, and tile head region is circled by tire small pitlike openings of the not ammlated. six inner labial sensilla. Posterior to the M. incognita, population 68-NC (41-43) lahial disc, tile subdorsal and subventral (Fig. 4, 5), possesses a small round labial lips are fused medially, forming one dorsal disc that is slightly raised above the medial and one ventral lip, respectively.