Response of Resistant Soybean Plant Introductions to Meloidogyne arenaria Races 1 and 2

Resistant plant introductions, PI 230977 and PI 200538, and partially resistant Jackson and susceptible CNS were evaluated for seed yield in response to races 1 and 2 of Meloidogyne arenaria. Initial soil population densities (Pi) of the nematode were 0, 31, 125, and 500 eggs/100 cm³ soil. At the highest Pi, yield suppressions of CNS, Jackson, PI 230977, and PI 200538 were 55, 28, 31, and 29%, and 99, 86, 66, and 58% for races 1 and 2 compared with uninfested controls. Numbers of second-stage juveniles (J2) present in roots 14 days after planting increased as Pi increased, but did not differ between the two races. At the highest Pi, fewer race 1 (40-57%) and race 2 (53-68%) J2 were present in roots of the plant introductions than in roots of Jackson. Soil population densities of race 1 J2 at 135 days after planting were 83-89% lower on the resistant genotypes than on CNS. These numbers did not differ for race 2. Reproductive factors were considerably higher for race 2 compared to race 1 for all genotype by Pi combinations, except for CNS at the highest Pi.     

Cellular Responses of Resistant and Susceptible Soybean Genotypes Infected with Meloidogyne arenaria Races 1 and 2

The cellular responses induced by Meloidogyne arenaria races 1 and 2 in three soybean genotypes, susceptible CNS, resistant Jackson, and resistant PI 200538, were examined by light microscopy 20 days after inoculation. Differences in giant-cell development were greater between races than among the soybean genotypes. M. arenaria race 1 stimulated small, poorly formed giant-cells in contrast with M. arenaria race 2, which induced well-developed, thick-walled, multinucleate giant-cells. The number of nuclei per giant-celt was variable, but fewer nuclei were usually present in giant-cells induced by race 1 (mean 16 nuclei) than in giant-cells induced by race 2 (mean 41 nuclei). Differences observed in giant-cell development were related to differences in growth and maturation of M. arenaria races 1 and 2 and host suitability of the soybean genotypes.     

Penetration and Development of Meloidogyne incognita on Roots of Resistant Soybean Genotypes

Meloidogyne incognita penetration and development were studied in roots of highly resistant (PI 96354, PI 417444), resistant (Forrest), and susceptible (Bossier) soybean genotypes. Although more second-stage juveniles (J2) had penetrated roots of PI 96354 and PI 417444 than roots of Forrest and Bossier by 2 days after inoculation, fewer J2 were present in roots of PI 96354 at 4 days after inoculation. Juvenile development in all genotypes was evident by 6 days after inoculation, with the highest number of swollen J2 present in roots of Bossier. At 16 days after inoculation, roots of PI 96354 had 87%, 74%, and 53% fewer J2 than were present in roots of Bossier, Forrest, and PI 417444, respectively. Differential emigration of J2, not fewer invasion sites, was responsible for the low number of nematodes in roots of the highly resistant PI 96354. Some 72% of the J2 penetrating the roots of this genotype emerged within 5 days after inoculation, whereas 4%, 54%, and 83% emerged from roots of Bossier, Forrest, and PI 417444, respectively. Penetration of roots of PI 96354 decreased the ability of J2 emerging from these roots to infect other soybean roots.     

Effects of Resistance in White Clover (Trifolium repens) on Heterodera trifolii

Clones of two partially resistant and two susceptible white clover, Trifolium repens, genotypes were exposed to eggs of Heterodera trifolii and nematode development in stained roots measured at 2, 4, 7, 11, 18, 23, and 37 days after inoculation. The differences in development between nematode populations in resistant and susceptible genotypes showed that resistance operated after infection during feeding and development. At 7 days after inoculation, counts of second-stage juveniles did not differ between genotypes, whereas at 37 days more adults had developed in the susceptible than in the resistant genotypes. In a separate experiment, cysts hosted by susceptible genotypes were larger and contained more eggs than those on resistant genotypes so that the product of the values for cysts per plant and for eggs per cyst resulted in a more sensitive measure of resistance than from using cysts per plant alone.     

Penetration and Development of Meloidogyne incognita in Roots of Resistant and Susceptible Corn Genotypes

Rates of penetration and development ofMeloidogyne incognita race 4 in roots of resistant (inbred Mp307, and S4 lines derived from the open-pollinated varieties Tebeau and Old Raccoon) and susceptible (Pioneer 3110) corn genotypes were determined. Seedlings grown in styrofoam containers were inoculated with 5,000 eggs of M. incognita. Roots were harvested at 3-day intervals starting at 3 days after inoculation (DAI) to 27 DAI and stained with acid fuchsin. Penetration of roots by second-stage juveniles (J2) at 3 DAI was similar for the four corn genotypes. Meloidogyne incognita numbers in Tebeau, Old Raccoon, Mp307, and Pioneer 3110 peaked at 12, 12, 15, and 27 DAI, respectively. Nematode development in the resistant genotypes was greatly suppressed compared to Pioneer 3110. Resistance to M. incognita in these genotypes appears to be expressed primarily as slower nematode development rather than differences in J2 penetration.     

Response of Resistant Soybean Plant Introductions to Meloidogyne incognita in Field Microplots

The response of two soybean plant introductions, PI 96354 and PI 417444, highly resistant to Meloidogyne incognita, to increasing initial soil population densities (Pi) (0, 31, 125, and 500 eggs/100 cm³ soil) of M. incognita was studied in field microplots for 2 years. The plant introductions were compared to the cultivars Forrest, moderately resistant, and Bossier, susceptible to M. incognita. Averaged across years, the yield suppressions of Bossier, Forrest, PI 417444, and PI 96354 were 97, 12, 18, and < 1%, respectively, at the highest Pi when compared with uninfested control plots. Penetration of roots by second-stage juveniles (J2) increased linearly with increasing Pi at 14 days after planting. At the highest Pi, 62% fewer J2 were present in roots of PI 96354 than in roots of the other resistant genotypes. Soil population densities of M. incognita were lower on both plant introductions than on Forrest. At 75 and 140 days after planting, PI 96354 had the lowest number of J2 in the soil, with 49% and 56% fewer than Forrest at the highest Pi. The resistance genes in PI 96354 should be useful in a breeding program to improve the level of resistance to M. incognita in soybean cultivars.     

Fitness Components and Selection of Biotypes of Heterodera glycines

Survival of biotypes of Heterodera glycines was studied in microplots and in the field. The field population was subjected to various cropping sequences. Viability of eggs overwintered in microplots was determined each spring by percentage hatch, percentage of hatched eggs penetrating roots, and numbers of females developing on Peking and PI 88788 soybeans. Eggs from the field were collected in the spring and fall and assayed for ability to develop on Peking and PI 88788. Hatch of isolates overwintered in the microplots averaged 13% in May 1989 and 19% in 1990. No differences in hatch were detected among the isolates in 1989. Numbers of juveniles penetrating susceptible roots averaged less than 20% of the hatched eggs each year. An isolate of a biotype parasitic on susceptible soybeans and the resistant soybean PI 88788 penetrated roots more successfully than other biotypes. A second isolate from North Carolina, parasitic on susceptible soybeans, PI 88788, and the resistant soybean Peking experienced selection against development on Peking during two winters. Only 17 % of the expected numbers of females developed on Peking from this isolate. In the microplot experiment, parasitism of PI 88788 and Peking had a selective disadvantage (selection coefficient) of s = 0.29 and 0.62 over all isolates, respectively. In the field experiment, the relative numbers of cysts on Peking and PI 88788 increased between the spring and fall on soybean, then decreased over the winter and under corn. Selection coefficients against parasitism of PI 88788 and Peking averaged 0,19 and 0.3 in the field population. In neither experiment did juveniles lose their ability to parasitize susceptible soybeans.     

Differential Response of Thor Alfalfa to Meloidogyne chitwoodi Races and M. hapla

Second-stage juveniles (J2) of races 1 and 2 of Meloidogyne chiiwoodi and M. hapla readily penetrated roots of Thor alfalfa and Columbian tomato seedlings; however, few individuals of M. chitwoodi race 1 were able to establish feeding sites and mature on alfalfa. Histopathological studies indicate that J2 of race 1 either failed to initiate feeding sites or they caused cell enlargement without typical cell wall thickening. The protoplasm of these cells coagulated, and juveniles of race 1 did not develop beyond the swollen J2 stage. A few females of race 1 fed on small giant cells and deposited a few eggs at least 20 and 30 days later than M. chitwoodi race 2 and M. hapla, respectively. Failure of race 1 to establish feeding sites was related to egression of J2 from the roots. The M. chitwoodi race 1 J2 egression from alfalfa roots was higher than egression of race 2 and M. hapla. Egression of J2 of M. chitwoodi races 1 and 2 from tomato roots was similar and higher than that of M. hapla. Thus egression plays an important role in the host-parasite relationship of M. chitwoodi and alfalfa.     

Development of Meloidogyne arenaria on Peanut and Soybean under Two Temperature Cycles

Florunner peanut and three soybean cultivars, Centennial, Gasoy 17, and Wright, were inoculated with 48-hour age cohorts of Meloidogyne arenari race 1 second-stage juveniles and placed in a growth chamber set to simulate early season (low temperature) and midseason (high temperature) conditions. Percentages of the initial inoculum penetrating roots 4 and 8 days after inoculation were 2-3 times higher in soybean cultivars than in peanut; 25% on susceptible soybean and 9% on peanut. Penetration and early development of M. arenaria were greater in the higher temperature environment. Penetration percentages were expressed as a function of cumulative degree-days by regression models. Development of M. arenaria 10, 20, and 30 days after inoculation was more rapid on peanut than on soybean. The resistant soybean cultivar Wright had slower development rates than did the other two soybean cultivars. Nematode growth and development were dependent on temperature. In greenhouse experiments, production of eggs by M. arenaria was more than 10 times greater on peanut than on susceptible soybean. The reproductive factor for Wright soybean was less than one, but plant growth parameters indicated that this cultivar was intolerant of M. arenavia.     

Comparison of Methods for Assessing Resistance to Meloidogyne arenaria in Peanut

Use of resistant cultivars is a desirable approach to manage the peanut root-knot nematode (Meloidogyne arenaria). To incorporate resistance into commercially acceptable cultivars requires reliable, efficient screening methods. To optimize the resistance screening protocol, a series of greenhouse tests were done using seven genotypes with three levels of resistance to M. arenaria. The three resistance levels could be separated based on gall indices as early as two weeks after inoculation (WAI) using 8,000 eggs of M. arenaria per plant, while four or more weeks were needed when 1,000–6,000 eggs/plant were used. High inoculum densities (over 8,000 eggs/plant) were needed to separate the three resistance levels based on eggs per gram of root within eight WAI. A gall index based on percentage of galled roots could separate the three resistance levels at lower inoculum levels and earlier harvest dates than other assessment methods. The use of eggs vs. second-stage juveniles (J2) as inoculum provided similar results; however, it took three to five more days to collect J2 than to collect eggs from roots. Plant age affected gall index and nematode reproduction on peanut, especially on the susceptible genotypes AT201 and D098. The genotypes were separated into their correct resistance classes when inoculated 10 to 30 days after planting, but were not separated correctly when inoculated on day 40.     

Susceptibility of Soybean Introductions to Races 1, 2, 3, and 4 of Heterodera glycines

Thirteen soybean plant introduction (PI) lines, selected for their apparent susceptibility to Heterodera glycines, were compared with cultivar Lee 74 as hosts of H. glycines races 1, 2, 3, and 4. Race 3 produced the highest average number of females of the four races. Compared to Lee 74, more (P = 0.05) females of H. glycines race 1 were extracted from eI 274420, PI 274423, and PI 317333; PI 86457 had more females of H. glycines race 2; and PI 86443, PI 86457, PI 261467, PI 274420, PI 274421, and PI 274423 had more females of H. glycines race 3. Similar numbers of females of H. glycines race 4 developed on all of the soybean lines and Lee 74. PI 274421, PI 274420, or PI 196159 could provide a more or equally susceptible host for H. glycines races 1, 2, 3, and 4 than Lee 74. One of these three lines could be substituted for Lee as the standard susceptible cultivar in the race determination test.     

Life Cycle and Mating Behavior of Belonolaimus longicaudatus in Gnotobiotic Culture

The life cycle of Belonolaimus longicaudatus was observed in vitro on excised roots of Zea mays. Roots were cultured on Gamborg''s B5 medium in petri dishes with 1.5% agar adjusted to pH 5.8 and incubated at 28 °C in darkness. Second-stage juveniles (J2) fed on the roots and started the second molt (M2) to the third-stage juveniles 2 days after inoculation (DAI). The third molt (M3) to the fourth-stage juveniles occurred 7 DAI, followed by the fourth molt (M4) to males 13 DAI or to females 14 DAI. Nematode gender differences were observed by the end of the fourth molt. The first male appeared 15 DAI and the first female 17 DAI, after which mating occurred. Males were attracted to females, and mating was observed. Mating was required for reproduction. Fertilized females began to lay eggs 19 DAI and continued egg laying without the further presence of males during a 90-day observation. All of the eggs hatched. Unfertilized females rarely laid eggs, and none of the eggs were able to hatch. Feeding took place between each molt and before egg deposition occurred. The first-stage juveniles molted in the eggs 4 days after deposition, and J2 hatched from eggs 5 days after egg deposition. The life cycle from J2 to J2 was completed in 24 days.     

Temperature Effects on Race Determination in Heterodera glycines

Currently there are 16 possible races for Heterodera glycines, and these are differentiated based on ability of a nematode population to develop on a set of four differential soybean genotypes. Because results are based on numbers of nematode females that develop to a specific stage rather than on the reproductive capability of these females, race determinations based on female indices may not represent results obtained after several reproductive cycles of H. glycines. Counting numbers of eggs and juveniles, and then developing corresponding indices, would allow reproduction to be considered in making race determinations. Our objectives were to compare the present race identification scheme for H. glycines based on female indices with those using egg and juvenile indices and to examine the effect of temperature on race designations using female, egg, and juvenile indices. Race designations for H. glycines populations from two locations in Illinois were determined at 20, 27, and 30 °C in a water bath. The numbers of females, eggs, and juveniles (at 19 days) were recorded, and an index based on each life stage was calculated. Race determinations based on female, egg, or juvenile indices were inconsistent when conducted at 20 °C, which demonstrates that this temperature is not suitable for identifying races of H. glycines. However race designations at 27 and 30 °C were consistent for all three indices. This indicates that counting females, eggs, or juveniles should be equally reliable when race determinations are conducted at these two temperatures, and choice of method would depend on investigator preference or research objective.     

Differentiation of Two New York Isolates of Pratylenchus penetrans Based on Their Reaction on Potato

The behavior of two isolates of Pratylenchus penetrans on six potato clones was assessed to test the hypothesis that these nematode isolates from New York were different. Four potato cultivars (Superior, Russet Burbank, Butte, and Hudson) and two breeding lines (NY85 and L118-2) were inoculated with nematode isolates designated Cornell (CR) and Long Island (LI). Population increase and egression of nematodes from roots were used to distinguish resistance and susceptibility of the potato clones. Based on numbers of eggs, juveniles, and adults in their roots 30 days after inoculation, potato clones Butte, Hudson, and L118-2 were designated resistant to the CR isolate and susceptible to the LI isolate. More eggs were found in the roots of all plants inoculated with the LI isolate than with the CR isolate. The clones NY85 and L118-2 were inoculated with the CR and LI isolates in a 2 x 2 factorial experiment to assess differences in nematode egression. Egression was measured, beginning 3 days after inoculation, for 12 days. The rates of egression were similar for the four treatments and fit linear regression models, but differences were detected in numbers of egressed nematodes. More nematodes of the CR isolate than the LI isolate egressed from L118-2. Differences in egression of females was particularly significant and can be used as an alternative or supplement to reproduction tests to assess resistance in potato to P. penetrans and to distinguish variation in virulence.     

The Effect of Resistant Soybean on Male and Female Development and Adult Sex Ratios of Heterodera glycines

To determine whether currently used sources of resistance (soybean Plant Introductions [PI] 548402, 88788, 90763, 437654, 209332, 89772, and 548316) influence sex ratios in H. glycines, four inbred lines of the nematode characterized by zero or high numbers of females on resistant soybean were used to observe the number of adult males produced. Nematodes were allowed to infect soybean roots for 5 days in pasteurized sand. Infected plants were washed and transferred to hydroponic culture tubes. Males were collected every 2 to 3 days up to 30 days after infestation (DAI), and females were collected at 30 DAI. Resistance that suppressed adult females also altered adult male numbers. On PI 548402, 90763, and 437654, male numbers were low and close to zero, whereas on PI 88788, male numbers were higher (α = 0.05). In a separate experiment, the same PIs were infected by an inbred line that tested as an HG Type 0 (i.e., the numbers of females that developed on each PI were less than 10% of the number that developed on the standard susceptible soybean cultivar Lee). In this experiment, male numbers were similar to female numbers on PI 548402, 90763, 437654, and 89772, whereas male numbers on PI 88788, 209332, and 548316 were higher than those of females (α = 0.05). In all experiments, the total number of adults that developed to maturity relative to the number of second-stage juveniles that initially penetrated the root was less on resistant than on susceptible soybean (P ≤ 0.05), indicating that resistance influenced H. glycines survival and not sexual development.     

Effects of Resistance in Phaseolus vulgaris on Development of Meloidogyne Species

Use of resistant Phaseolus vulgaris germplasm has a potential role in limiting damaging effects of Meloidogyne spp. on bean production. Effects of two genetic resistance systems in common bean germptasm on penetration and development of Meloidogyne spp. were studied under growth room conditions at 22°C to 25°C. Nemasnap (gene system 1) and G1805 (gene system 2) were inoculated with second-stage juveniles (J2) of M. incognita race 2 and M. arenaria race 1, respectively; Black Valentine was used as the susceptible control. Up to 7 days after inoculation, there were no differences in numbers of M. incognita J2 penetrating roots of Black Valentine and Nemasnap; subsequently, more nematodes were present in Black Valentine roots (P < 0.05). More nematodes reached advanced stages of development in Black Valentine than in Nemasnap roots (P < 0.05). Total numbers of M. arenaria were greater in Black Valentine than in G 1805 roots from 14 days after inoculation (P < 0.05). Advanced stages of development occurred earlier and in greater numbers in Black Valentine plants than in G1805 plants. In these studies, resistance to M. incognita race 2 and M. arenaria race 1 in bean germplasm, which contain gene system 1 and gene system 2, respectively, was expressed by delayed nematode development rather than by differential penetration compared with susceptible plants.     

Identification of Sources of Resistance to Four Species of Root-knot Nematodes in Tobacco

Resistance to the southern root-knot nematode, Meloidogyne incognita races 1 and 3, has been identified, incorporated, and deployed into commercial cultivars of tobacco, Nicotiana tabacum. Cultivars with resistance to other economically important root-knot nematode species attacking tobacco, M. arenaria, M. hapla, M. javanica, and other host-specific races of M. incognita, are not available in the United States. Twenty-eight tobacco genotypes of diverse origin and two standard cultivars, NC 2326 (susceptible) and Speight G 28 (resistant to M. incognita races 1 and 3), were screened for resistance to eight root-knot nematode populations of North Carolina origin. Based on root gall indices at 8 to 12 weeks after inoculation, all genotypes except NC 2326 and Okinawa were resistant to M. arenaria race 1, and races 1 and 3 of M. incognita. Except for slight root galling, genotypes resistant to M. arenaria race 1 responded similarly to races 1 and 3 of M. incognita. All genotypes except NC 2326, Okinawa, and Speight G 28 showed resistance to M. javanica. Okinawa, while supporting lower reproduction of M. javanica than NC 2326, was rated as moderately susceptible. Tobacco breeding lines 81-R-617A, 81-RL- 2K, SA 1213, SA 1214, SA 1223, and SA 1224 were resistant to M. arenaria race 2, and thus may be used as sources of resistance to this pathogen. No resistance to M. hapla and only moderate resistance to races 2 and 4 of M. incognita were found in any of the tobacco genotypes. Under natural field infestations of M. arenaria race 2, nematode development on resistant tobacco breeding lines 81-RL-2K, SA 1214, and SA 1215 was similar to a susceptible cultivar with some nematicide treatments; however, quantity and quality of yield were inferior compared to K 326 plus nematicides.     

A Technique for Evaluating Heterodera glycines Development in Susceptible and Resistant Soybean

A technique was developed to evaluate Heterodera glycines development in susceptible and resistant soybean. Roots of 3-day-old soybean were exposed to infective juveniles of H. glyci.nes in sand for 8 hours followed by washing and transfer to hydroponic culture. The cotyledons and apical meristem were removed and plants were maintained under constant light, which resulted in a dwarfed plant system. After 15 or 20 days at 27 C, nematodes were rated for development. Emerged males were sieved from the culture water and females were counted directly from the roots. Nematodes remaining in the roots were rated for development after staining and clearing the tissues. The proportion of nematodes at each stage of development and the frequency of completed molts for each stage were calculated from these data. This technique showed that resistance to H. glycines was stage related and did not affect males and females equally in all resistant hosts. The resistance of plant introduction PI 209332 primarily affected development of third and fourth-stage juveniles; ''Pickett'' mainly affected second and third-stage juveniles, whereas PI 89772 affected all stages. Male development was markedly affected in PI 89772 and ''Pickett'' but not in PI 209332.     

Characterization of Resistance in a Somatic Hybrid of Solanum bulbocastanum and S. tuberosum,to Meloidogyne chitwoodi

A somatic hybrid, CBP-233, between resistant Solanum bulbocastanum (SB-22) and susceptible S. tuberosum (R4) was tested for resistance to Meloidogyne chitwoodi race 1. One week after inoculation, only 0.04-0.4% of the initial inoculum (Pi, 5,000 eggs) as second stage-juveniles infected SB-22 and CBP-233 root systems, compared to 2% in R4. After 8 weeks, the number of M. chitwoodi in SB-22 and CBP-233 roots remained lower (0.3-1.5% of Pi) compared to R4, which increased from 2% to ca. 27%. Development of M. chitwoodi was delayed on SB-22 and CBP-233 by at least 2 weeks, and only half of the infective nematodes established feeding sites and matured in resistant clones compared to 99% in susceptible R4. Necrotic tissue surrounded nematodes that failed to develop in SB-22 and CBP-233. The reproductive factor (ratio of final number of eggs recovered from roots to Pi) was <0.01 for both SB-22 and CBP-233 and 46.8 for R4. Delaying inoculation of CBP-233 from 1 to 3 months after planting did not increase the chance or rate of tuber infection. Only a few M. chitwoodi developed to maturity on CBP-233 tubers and deposited a small number of eggs. SB-22 rarely produced tubers in these experiments, and like CBP-233 were resistant to M. chitwoodi. It appeared that the mechanisms of resistance to M. chitwoodi in roots and tubers of CBP-233 are similar.     

Variability in Reproduction of Four Races of Meloidogyne incognita on Two Cultivars of Soybean

Variability in the reproduction of the four races ofMeloidogyne incognita on the soybean cuhivars Pickett 71 and Centennial was studied in growth chamber experiments. Analysis of variance in the number of eggs produced by the races 6 weeks after the plants had been inoculated with 5,000 eggs of each race revealed that the nematode race by soybean cultivar interaction was highly significant (P = 0.001). Races 1, 3, and 4 produced from about 5,000 to 15,000 eggs per root system on Pickett 71 and only from about 300 to 600 eggs per root system on Centennial. In contrast, race 2 produced about 8,000 eggs per root system on Centennial and about 1,200 eggs per root system on Pickett 71. In a second experiment, in which the plants were inoculated with 2,000 second-stage juveniles, race 1 and race 2 produced about 13,000 and 3,000 eggs per root system, respectively, on Pickett 71 and about 600 and 10,000 eggs per root system, respectively, on Centennial. The results suggest that M. incognita resistance in soybean is race-specific.