Comparison of Populations of Pratylenchus Brachyurus Based on Isozyme Phenotypes 1

Journal of Nematology 22(4):538-545. 1990. © The Society of Nematologists 1990. Comparison of Populations of Pratylenchus brachyurus Based on Isozyme Phenotypes 1 L. A. PAYAN AND D. W. DICKSON~ Abstract: Enzymes from females of five Pratylenchus brachyurus populations and one P. scribneri population were analyzed by isoelectric focusing electrophoresis. Of the 18 enzyme systems inves- tigated, only malate dehydrogenase (MDH), phosphoglucomutase (PGM), and phosphoglucose isomerase (PGI) were detected from all five P. brachyurus populations and P. scribneri. Faint bands were detected for isocitrate dehydrogenase and phosphogluconate dehydrogenase from one P. brachyurus population. Three distinct phenotypic groups were found in the MDH and PGM systems for P. brachyurus populations, but only a single electromorph was detected for PGI. Multiple electromorphs for MDH, PGM, and PGI were detected for P. scribneri; there was no similarity among these patterns with those from P. brachywrus. No phenotypic differences in PGI were observed between females and mixed juveniles of one population of P. brachyurus. Key words: electrophoresis, interspecific variation, intraspecific variation, isocitrate dehydrogenase, isoelectric focusing, isozyme, lesion nematode, malate dehydrogenase, phosphoglucomutase, phosphogluconate dehydrogenase, phosphoglucose isomerase, Pratylenchus brachyurus, P. scribneri. The genus Pratylenchus is stenomorphic vermiform animals. Detection with elec- because of the small number of diagnostic trophoretic techniques of enzyme activity characters at the species level and the in- from a single vermiform nematode is not traspecific variability of some of these char- currently possible. Such analysis requires acters (11,13,18). Recent reappraisals or mass homogenates of hundreds or thou- compendia list the number of valid species sands of nematodes and generally includes in the genus as 49 (11), 63 (13), or 60 (18). mixed life stages (17). The principal prob- Modern biochemical systematic techniques lems in isolating proteins from single or a (16) are now available that may help our few vermiform nematodes are macerating understanding of the intraspecific and in- the nematodes and protecting the released terspecific variability within the genus. proteins from degradation. Techniques to Some enzyme phenotypes in some nema- macerate a single pyriform female have tode species appear to be descriptive been developed (8), but these techniques (6,9,10), although a range of genetic vari- will not work with vermiform nematodes. ability also occurs (7,19). Isoelectric focusing electrophoresis (IEF) Refinements and miniaturization of elec- is sensitive enough to detect enzyme activ- trophoretic techniques permit analysis of ity from a single Heterodera glycines Ichi- proteins from a single pyriform female in nohe female (14) and to detect intraspecific the Heteroderoidea (1,3). Most plant-para- variability among 12 H. glycines popula- sitic nematodes, however, are microscopic tions (20). This technique is a useful tool for studying protein polymorphism and genetic diversity among nematode populations (20). Received for publication 23 October 1989. Our objectives were 1) to compare five Florida Agricultural Experiment Station Journal Series No. R-00233. populations of Pratylenchus brachyurus A portion of a dissertation submitted by the first author in (Godfrey) Filipjev & Schuurmans-Stekho- partial fulfillment of the Ph.D. requirements, University of Florida. ven from various geographical regions and 2 Former Graduate Research Assistant and Professor, De- hosts and one P. scribneri Steiner popula- partment of Entomology and Nematology, University of Flor- ida, Gainesville, FL 32611-0611. Current address of first au- tion by analyzing 18 enzyme systems by IEF thor: Fruit Crops Department, University of Florida, and 2) to determine similarities between Gainesville. The authors thank K. R. Barker, N. A. Minton, and R. N. mixed juvenile stages and females of one Huettel for providing nematodes used in this study. population of P. brachyurus. 538 Enzyme Phenotypes in Pratylenchus spp.: Payan, Dickson 539 mm drill bit. The dimensions of each well MATERIALS AND METHODS were ca. 2.0 mm wide at the bottom, 2.5 The designations and sources of the five mm wide at the surface, and 1.5 mm deep populations of P. brachyurus were as fol- (Fig. 1B). lows: 101 from corn (Zea mays L. cv. Pi- The surface of the slide, excluding the oneer 304C) and 102 from peanut (Arachis wells, was coated with Repel-Silane to ob- hypogaea L. cv. Florunner), Alachua Coun- tain a hydrophobic surface that prevented ty, Florida; 103 from Florunner peanut, the spreading of the droplet from the cap- Tiff County, Georgia; 105 from soybean illary tube and allowed the nematodes to (Glycine max L. cv. Forrest), Nash County, fall to the bottom of the well. Nematodes North Carolina; and 108 from citrus (Cit- that did not fall were pushed into the well rus sp.), Polk County, Florida. The iden- with a hair attached to the end of a needle. tification of each species was verified by The glass slide was then placed in a plastic morphometric studies using light micros- sandwich box (Fig. 1B) containing an ice copy. block, which cooled the nematodes during Each population was isolated from field maceration, and a mirror directly under soil and placed on root explant cultures of the glass slide, which enhanced visibility. corn (Zea mays L. cv. Iochie0 grown in plas- The entire process was done under the dis- tic petri dishes containing Gamborg's B-5 secting microscope at 30 x magnification. medium without auxins or cytokinins (Gib- After placing the nematodes in the well, co, Grand Island, New York) and main- most of the water was removed with a 1-ml tained in the dark at 29 C (15). The P. disposable syringe with the needle located scribneri population on corn root explants near the edge of the well. The remaining was obtained from USDA ARS, Beltsville, water was removed with filter paper (What- Maryland. Nematodes were extracted from man No. 2). The nematodes, which re- the culture dishes by removing a block of mained attached to the walls of the well by agar with roots and placing it in a modified a thin film of water, were easily macerated Baermann funnel containing water (5). for 30 seconds with a precooled ground- A controlled vacuum aspirator (12) was glass rod made to fit the well snugly. After used to pipet 250 females or 450 juveniles maceration, 2 gl solution containing 20% in deionized water from a dish into a sucrose (w/v) and 2% (v/v) Triton X-100 depression slide previously coated with Re- (8) was added to the well and the contents pel-Silane (LKB, Bromma, Sweden) to pro- were mixed thoroughly. Finally, the con- duce a hydrophobic surface. After the tents of the well were absorbed onto a 5- nematodes settled to the bottom, the vol- x 10-mm filter paper (LKB, Bromma, Swe- ume of water was reduced to about 20/A den) and applied directly to the surface of by pipetting out excess water. Nematodes the isoelectric focusing gel. and water were sucked into a 25-gl capil- Precast 24.5- x 10.5-cm polyacrylamide lary tube and used immediately or stored gels with a concentration that consisted of overnight at 5 C. The contents of each T = 5%, C = 3%, and 2.4% ampholytes capillary tube were placed into a small well with a pH range of 3.5-9.5 were used. The previously cut in the ground-glass surface LKB (Pharmacia LKB Biotechnology, Pis- of a Wheaton microculture slide (Thomas cataway, NJ) ultrophor system was used Scientific, Swedesboro, NJ) (Fig. 1A). Four with 1 M H~PO4 wicked across the anode wells with straight sides, flat bottoms, and and 1 M NaOH wicked across the cathode beveled lips were cut into the slide. To cut (21). During isoelectric focusing, the gels the wells, a starter hole was made with a were maintained at a constant temperature 1.6-mm drill bit then a 1.75-mm flat-head- of 5 C and run at a constant voltage. The ed miniscrewdriver, each attached to a 9.5- power was applied from an LKB macro- mm drill. Each well was drilled ca. 1.5 mm drive 5 constant power supply set at 1,500 deep, and the lips were beveled with a 2.8- volts, 50 milliamps, and 30 watts. The pre- 540 Journal of Nematology, Volume 22, No. 4, October 1990 A Well Slide Plastic sandwich box 1 Glass mirror B Ice block FIG. 1. Protein extraction equipment. A) Wheaton microculture slide showing the small wells and the glass rod constructed to macerate vermiform nematodes for protein extraction. B) Cross section of the microscope slide (not to scale) used for macerating vermiform nematodes. The dimensions of each well are ca, 2,0 mm wide at the bottom, 2.5 mm wide at the surface (beveled), and 1.5 mm deep. The slide was placed over a glass mirror that lay over an ice block contained in a plastic sandwich box. cast gels were cut generally into smaller The gels were immersed in buffer (the sections for runs with 5-10 nematode same buffer used in the stain mixture) for preparations. The settings of the power 3 minutes to remove excess electrolytes supply were reduced proportionally to the from the gel surface. The gels were then size of the gel. Each gel was prefocused for submerged into the enzyme reaction mix- 10 minutes, nematode samples were ap- tures in a plastic box lined previously with plied in duplicate, and the gel was run for plastic film and incubated in the dark at 25 1.5 hours. The pH gradient of each gel C until bands appeared. The enzymes used was determined immediately following a and the references to the stain and reaction run by measuring the pH at 0.5-cm inter- mixtures are in Table 1.

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