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.     

Selection Against Heterodera glycines Males by Soybean Lines with Genes for Resistance

Soybeans with genes for resistance select against Heterodera glycines with the corresponding genes for avirulence. There may be a differential effect of sex with some specific gene interactions, which would influence the magnitude of gene frequency changes. No effect on H. glycines males was detected with one selected nematode population and the resistant soybean line PI88788. The selective effect of PI89772 against male nematodes was greater with two inbred nematode populations than with one selected (on PI88788) population, presumably due to differences in H. glycines gene frequencies. ''Peking'' also had few males with the one inbred nematode population, whereas Forrest and ''Pickett 71'' had intermediate numbers. Apparently Forrest and Pickett 71 did not get all the Peking genes for resistance that affect male as well as female nematode development. Other H. glycines-soybean genes stop only females, since there were few or no cysts, except on the susceptible soybean Williams. The number of males'' phenotype will help identify specific genes in both organisms.     

Nematode Population Attrition and Histopathology of Heterodera glycines-Soybean Associations

Selected populations of soybean cyst nematodes were inoculated to roots of compatible and incompatible soybeans. Rates of penetration of infective juveniles of nematode populations selected on PI 209332, PI 89772, and Pickett 71 were equivalent on compatible and incompatible soybean roots. The first two populations averaged about 10% and the last about 5% penetration in 24-hour inoculations of young seedlings. About 14% of those juveniles that entered roots in compatible combinations developed into maturing females, compared with only about 1% in incompatible combinations. Several aberrations from the pattern of syncytial development associated with mature females in compatible hosts were apparent. A rapid necrotic response occurred in both kinds of hosts but was more frequent in incompatible associations. Delayed necrosis and small syncytia were present in some combinations. Those few females that developed in incompatible soybeans were associated with a characteristic syncytium different from the kind seen in roots of compatible hosts.     

Characterization of the Virulence Phenotypes of Heterodera glycines in Minnesota

Knowledge of the virulence phenotypes of soybean cyst nematode, Heterodera glycines populations is important in choosing appropriate sources for breeding resistant cultivars and managing the nematode. We investigated races of 59 H. glycines populations collected from 1997 to 1998 and races and HG Types of 94 populations collected in 2002 from soybean fields across southern and central Minnesota. In the 1997 to 1998 samples, race 3 was predominant and represented 78% of the populations. The remaining populations were 11.9% race 1, 1.7% race 4, 6.8% race 6, and 1.7% race 14. In the 2002 samples, the populations were classified as 15.3% race 1, 77.6% race 3, 2.4% race 5, 3.5% race 6 and 1.2% race 9. Percentage of 1997 to 1998 populations with female indices (FI) higher than 10 were 10.2% on Pickett 71, 3.4% on Peking, 13.6% on PI 88788, 3.4% on PI 90763, 1.7% on PI 209332, and 1.7% on PI 437654. Percentage of 2002 populations with FI >10 was 1.1% on Peking, 17.0% on PI88788, 14.9% on PI 209332, 33.0% on PI 548316, 11.7% on Pickett 71, and 0% on the other three indicators, PI 90763, PI 437654, and PI 89772. The line PI 548316 was relatively susceptible to the Minnesota H. glycines populations and may not be recommended for breeding resistant cultivars in the state. There was no noticeable change of frequencies of virulence phenotypes in response to the use of resistant cultivars during 1997 to 2002 in Minnesota except that FI increased on the PI 209332.     

Correlation of Female Indices From Virulence Assays on Inbred Lines and Field Populations of Heterodera glycines

A lack of diversity and durability of resistant soybean varieties complicates management of the soybean cyst nematode (SCN), Heterodera glycines, exemplified by the current overdependence on the PI 88788 source of resistance. Of interest is the effect of adaptation of a SCN population to a source of resistance on its subsequent ability to develop on others. Female indices (FI) from virulence assays (race, HG Type and SCN Type tests) for SCN field populations and inbred lines were analyzed. Female indices on PI 88788, PI 209332 and PI 548316 were highly correlated, as were those of PI 548402, PI 90763, PI 89772 and PI 438489B. Previous studies on resistant SCN-infected soybean roots indicated that the cellular resistance response was similar within these two groups of soybean genotypes. In field populations, highly significant correlations were also found between FI on PI 88788 and PI 548402 and those on PI 89772 and PI 437654. In inbred lines, FI on PI 437654 were correlated with PI 90763 and PI 438489B. To avoid further adaptation, rotation of cultivars with resistance from these groups should be carefully monitored, including those from the most promising source of resistance, PI 437654, such as CystX. In a separate test, 10 soybean varieties developed from CystX were tested against HG Type 0, HG Type 2.5.7 and HG Type 1–7. Female development occurred in all tests but one. Although identification and deployment of unique resistance is needed, management strategies to prevent and detect adaptation should be emphasized.     

Inbreeding and Hybridizing Cyst Nematodes on Pruned Soybeans in Petri Plates

Inbred nematodes propagated on a selecting host are likely to have homozygous genes of interest for investigating the genetics of host-parasite associations. A technique is presented to inbreed soybean cyst nematodes, by sibling matings at each generation, and to cross inbred lines. Soybean seedlings with severely trimmed cotyledons survive well on 0.8% agar. Eggs from a single female are incubated in water in a microtiter well. Virgin as well as mated females result from inoculation of two juveniles per root. Sibling males from the same source are produced by mass inoculations of eggs. Males are added individually to unmated females. Overall success for fertile females was 14% in 1,368 isolations. Three generations of inbreeding by siblings were achieved using nematodes from two populations that differ in their ability to reproduce on differential soybeans. Hybrids from crosses of the two inbred lines tested on differential hosts showed that the influence of Population 1 (selected and inbred on PI 209332) is greater than that of Population 2 (selected and inbred on PI 89772). Reciprocal crosses suggest that the influence of males is stronger than that of females in determining host specificity of F₁ offspring in these crosses. Our technique is simple and effective for inbreeding and crossing soybean cyst nematodes.     

Effect of Soybean Tip Removal on Penetration and Development of Heterodera glycines

On a few occasions, soybeans with broken root tips were included in tests to evaluate resistance to Heterodera glycines. Although females developed on these plants, the numbers tended to be lower than on similarly treated intact roots. To test the possibility that removal of the root meristem affected nematode development, a culture system using pruned soybeans was devised that permitted access to the roots without disturbing the plants. Treatments included removal of 2 mm of root tip at various times ranging from 24 hours before to 10 days after inoculation, or roots left intact. In each experiment, all roots were inoculated at the same time with equal numbers of freshly hatched second-stage juveniles of Heterodera glycines. No differences in nematode development were detected in plants with root tips removed after inoculation compared to the control. When tips were removed at or before inoculation, fewer juveniles entered roots and relatively fewer nematodes developed. Penetration levels and development correlated with root tip removal such that progressively fewer nematodes entered roots and relatively greater numbers of nematodes remained undeveloped as the time interval between root tip removal and inoculation was increased.     

Selection and Inbreeding of Heterodera glycines on Glycine max

Few soybean cyst nematodes (SCN), Heterodera glycines, of a diverse gene pool developed into females on soybeans PI 89772 or PI 209332. Nematodes surviving the selection pressure were then inbred for nine generations by single cyst transfers on the same selecting soybean line. These nematodes appeared to tolerate concurrent selection and inbreeding. Effects of selection-inbreeding, selection only, and secondary selection were evaluated by relative ability to produce cysts on 11 soybean lines. The genetic differences of PI 89772 (also Peking and Pickett 71) and PI 209332 were reaffirmed. The random effects of inbreeding indicated that Ilsoy and Williams may have genes for resistance different from those in PI 89772 or PI 209332. Egg inoculum obtained from soil resulted in very few cysts in some tests. Fresh egg inoculum (from cysts on 27-30-day-old plants) generally resulted in more cysts and more consistent results. Concurrent with the change in inoculum, there was a large increase in relative numbers of cysts on several soybean lines but no change on other lines; the true cause of this large interaction is unknown. Secondary selection of two inbreds was effective and suppressed cyst numbers on the line on which one inbred was selected initially. These results are consistent with the allelism linkage of some SCN genes reported previously.     

Changes in the Heterodera glycines Female Index as Affected by Ten-year Cropping Sequences

The objective of this experiment was to measure the change in female index (FI) of Heterodera glycines from bioassays on Bedford, Peking, PI 89772, and PI 90763 soybean (Glycine max) for 12 cropping sequence treatments over a 10-year period. Cropping sequences included continuous plantings of Forrest, Peking, and D72-8927 soybean (all resistant to race 3); Bedford, Nathan, and D75-10710 soybean (all resistant to races 3 and 14); a Bedford-corn (Zea mays) rotation; a rotation of Bedford, Essex (susceptible), and Forrest; and a 70:30 blend of Bedford and Forrest. The FI from bioassays with PI 89772 and PI 90763 decreased over time from 24.3 to 1.6 with treatments involving continuous Bedford, Nathan, and D75-10710 and the Bedford-corn rotation. The FI increased in bioassays using Bedford with treatments involving Bedford, Nathan, D75-10710, the Bedford-Forrest blend, and the two rotations. Results of this field experiment confirm greenhouse experiments in which reciprocal changes occur in FI on PI 89772 and PI 90673 compared with FI on Bedford.     

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.     

FMRFamide-like Immunoactivity in Heterodera glycines (Nemata: Tylenchida)

Material antigenically related to the neuromodulatory peptide FMRFamide was detected and examined in preparations of the soybean cyst nematode, Heterodera glycines, and in the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. FMRFamide-related peptides were quantified by an enzyme-linked immunosorbent assay. Specific activities were remarkably similar among all of the vermiform members of the three species. FMRFamide-related peptide immunoactivity was present in both sexes and all stages of H. glycines examined. The highest specific activity was present in second-stage juveniles and in males, and the lowest in white and yellow females. Total FMRFamide-related peptide level per individual was highest in brown females, with 90% of the activity associated with the eggs. Peptide levels in these eggs and in second-stage juveniles were comparable and increased in adults, especially in females. Chromatographic analysis of FMRFamide-related peptide preparations from H. glycines juveniles, C. elegans, and P. redivivus revealed distinct qualitative differences between the infective plant parasite and the free-living nematodes.     

Penetration and Post-infectional Development and Reproduction of Meloidogyne arenaria Races 1 and 2 on Susceptible and Resistant Soybean Genotypes

Penetration, post-infectional development, reproduction, and fecundity of Meloidogyne arenaria races 1 and 2 were studied on susceptible (CNS), partially resistant (Jackson), and highly resistant (PI 200538 and PI 230977) soybean genotypes in the greenhouse. The ability to locate and invade roots was similar between races, but more juveniles penetrated roots of susceptible CNS than the resistant genotypes. At 10 days after inoculation, 56% and 99% to 100% of race 1 second-stage juveniles were vermiform or sexually undifferentiated in CNS and the resistant genotypes, respectively. In contrast, only 2%, 42%, 44%, and 62% of race 2 juveniles had not initiated development in CNS, Jackson, PI 200538, and PI 230977, respectively. By 20 days after inoculation, 88% to 100% of race 2 nematodes in roots of all genotypes were females, whereas only 25% and 1% of race 1 were females in CNS and the resistant genotypes, respectively. For all four genotypes, race 1 produce 85% to 96% fewer eggs per root system 45 days after inoculation than race 2. At 45 days after inoculation race 2 produced more eggs on CNS than the other genotypes.     

Early Growth of Soybean as Altered by Heterodera glycines,Phenamiphos and/or Alachlor

Greenhouse and field experiments were conducted to determine the effects of phenamiphos and/or alachlor on early growth of soybean, root morphology, and infection and resurgence of Heterodera glycines (race 1). All tests were planted to ''Ransom'' soybeans. In greenhouse experiments without nematodes, root growth was inhibited at 5 days by alachlor treatments and at 10 days by phenamiphos treatments; with nematodes, phenamiphos treatments enhanced root growth. Phenamiphos also suppressed early penetration of soybean roots by H. glycines in the greenhouse. Early soybean growth parameters among treatments were generally similar in the field. Nematode penetration was limited with treatments containing phenamiphos at one location. Plants treated with only alachlor had less nematode infection than did the control; however, plants treated with herbicide/nematicide combinations had more nematode penetration than did plants treated with phenamiphos alone. Alterations of root growth and interference with the efficacy of phenamiphos are two processes by which alachlor may enhance soybean susceptibility or suitability to H. glycines.     

Resistance Genes in a 'Williams 82' x 'Hartwig' Soybean Cross to an Inbred Line of Heterodera glycines

The number of resistance genes in soybean to soybean cyst nematode (SCN) Heterodera glycines was estimated using progeny from a cross of ''Williams 82'' x ''Hartwig'' (derived from ''Forrest''³ x PI 437.654) screened with a fourth-generation inbred nematode line derived from a race 3 field population of SCN. Numbers of females developing on roots of inoculated seedlings were assigned to phenotype cells (resistant, susceptible, or segregating) using Ward''s minimum variance cluster analysis. The ratio obtained from screening 220 F₃ soybean families was not significantly different from a 1:8:7 (resistant:segregating:susceptible) ratio, suggesting a two-gene system for resistance. The ratio obtained from screening 183 F₂ plants was not significantly different from a 3:13 (resistant:susceptible) ratio, indicating both a dominant (Rhg) and a recessive (rhg) resistance gene.     

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.     

Development of Heterodera glycines on Soybean Damaged by Soybean Looper and Stem Canker

Short-term greenhouse studies with soybean (Glycine max cv. Bragg) were used to examine interactions between the soybean cyst nematode (Heterodera glycines) and two other common pests of soybean, the stem canker fungus (Diaporthe phaseolorum var. caulivora) and the soybean looper (Pseudoplusia includens), a lepidopterous defoliator. Numbers of cyst nematode juveniles in roots and numbers of cysts in soil and roots were reduced on plants with stem cankers. Defoliation by soybean looper larvae had the opposite effect; defoliation levels of 22 and 64% caused stepwise increases in numbers of juveniles and cysts in both roots and soil, whereas numbers of females in roots decreased. In two experiments, stem canker length was reduced 40 and 45% when root systems were colonized by the soybean cyst nematode. The absence of significant interactions among these pests indicates that the effects of soybean cyst nematode, stem canker, and soybean looper on plant growth and each other primarily were additive.     

Hybridization of Races of Heterodera glycines

Progeny from single females of four known races of Heterodera glycines Ichinohe were used to establish relatively uniform populations. Single females from these populations were mated with males of other races in all possible combinations to study compatibility and inheritance patterns. When race 1 or 3 was crossed with either race 2 or 4, there was a significant reduction in number of females and a greater number of eggless females than in crosses of races 1 × 3 and 2 × 4. More females matured and fewer were eggless when matings were of the same race. Parasitic capabilities of races 2 and 4 were dominant or partially dominant over those of races 1 and 3, based on parasitism of F₁ hybrids. Segregation patterns were generally similar for reciprocal crosses between races. There appeared to be either one or two major genes segregating for parasitism of ''Pickett'' soybean in the different crosses. A hybrid isolate (race 3 × 4) that differed in parasitic capability from the four known races produced as many females on the resistant soybean genotype, PI 90,763, as on the susceptible Lee cultivar. Those data indicate that isolates of H. glycines with a different parasitic capability may develop from gene recombination.     

Changes in the Reproduction of Heterodera glycines on Different Lines of Glycine max

Selection for ability of soybean cyst nematode (SCN), Heterodera glycines, to reproduce on soybeans with different sources of resistance divides some SCN race 4 field populations into two distinct subpopulations. These subpopulations reproduce well on ''Bedford'' and plant introduction (PI) 88788 or PI 89772 and PI 90763 but not on both pairs of soybean lines. The ability of these subpopulations to reproduce on the four soybean lines was reversed by changing the soybean line used as a host during a second cycle of selection. When SCN populations previously selected for reproduction on Bedford and PI 88788 were selected for their ability to reproduce on D72-8927 and J74-88, the ability of these populations to reproduce on Bedford and PI 88788 decreased significantly and their ability to reproduce on PI 89772 and PI 90763 increased significantly. Conversely, when SCN populations, previously selected for reproduction on P189772 and P190763, were selected for their ability to reproduce on Bedford, the reproduction of these populations on Bedford increased significantly and reproduction on PI 89772 and PI 90763 decreased significantly. Selection for ability of a SCN race 4 field population to reproduce on soybean lines derived from SCN race 4 resistant PIs resulted in the same division of the field population into two distinct subpopulations. These data substantiate earlier proposals to rotate cultivars with different genes for SCN resistance as a means of managing SCN populations.     

Differences in Hatching of Heterodera glycines Egg-mass and Encysted Eggs in vitro

Hatching studies with Heterodera glycines typically have been conducted with a mixture of egg-mass and encysted eggs. Laboratory research was conducted to compare hatching of H. glycines eggs from external egg masses with that of eggs extracted from within females and cysts (encysted eggs). Egg-mass eggs were collected by soaking infected soybean roots in 0.5% sodium hypochlorite, and encysted eggs were collected from females and cysts dislodged from the same roots with a stream of water. Eggs were incubated at 25 °C in deionized water, 3.0 mM ZnSO₄solution, or one of three synthetic H. glycines hatch inhibitors, mad hatched juveniles were counted every other day for 22 days. Samples of eggs collected at the beginning and end of all experiments were analyzed to determine extent of embryo development. Egg-mass eggs hatched more rapidly than encysted eggs during the first 16 days, but not thereafter. Throughout the experiments, hatch of egg-mass eggs in deionized water was greater than that of encysted eggs. From day 8 to day 22, egg-mass eggs were less sensitive than encysted eggs to the hatch inhibitor 2-(2''-carboxyethyl)-5-[carboxy(hydroxy)methylidenyl]cyclopentanone. A greater proportion of egg-mass eggs contained vermiform juveniles than did encysted eggs at the beginning of the experiments, but not at the end. Results indicated that H. glycines egg-mass and encysted eggs have different hatching behaviors that cannot be explained entirely by differences in embryological development.     

Penetration Rates by Second-stage Juveniles of Meloidogyne spp. and Heterodera glycines into Soybean Roots

The rates of soybean root penetration by freshly hatched second-stage juveniles (J2) of Meloidogyne arenaria, M. hapla, M. incognita, M. javanica, and Heterodera glycines races 1 and 5 were examined over a period of 1 to 240 hours. Heterodera glycines entered roots more quickly than Meloidogyne spp. Penetration by most nematodes was accomplished within 48 hours. The increases in penetration after 48 hours were insufficient to warrant further assessments. Penetration of J2 into roots of soybean seedfings in a styrofoam container was as good or better than in a clay pot. Thus, rapid and accurate root-penetration assessments can be made at 48 hours after inoculation.