Association of the Plant-beneficial Fungus Verticillium lecanii with Soybean Roots and Rhizosphere

Colonization of soybean roots by the biocontrol fungus Verticillium lecanii was studied in vitro and in situ. For in vitro experiments, V. lecanii was applied to soybean root tip explant cultures. Four weeks after inoculation, the fungus grew externally on at least half of the roots (all treatments combined), colonizing 31% to 71% of root length (treatment means). However, when a potato dextrose agar plug was available as a nutrient source for the fungus, root tips inoculated soon after transfer were not colonized by V. lecanii unless Heterodera glycines was present. Scanning electron microscopy of colonized roots from in vitro cultures revealed a close fungus-root association, including fungal penetration of root cells in some specimens. In the greenhouse, soybeans in sandy soil and in loamy sand soil were treated with V. lecanii applied in alginate prills. The fungus was detected at greater depths from the sandy soil than from the loamy sand soil treatment, but fungus population numbers were small and variable in both soils. Root box studies coupled with image processing analysis of the spatial distribution of V. lecanii in sandy soil supported these findings. Verticillium lecanii was detected randomly in the rhizosphere and soil of root boxes, and was rarely extensively distributed. These in vitro and in situ experiments indicate that V. lecanii can grow in association with soybean roots but is a poor colonizer of soybean rhizosphere in the soil environment.     

Application of a Sex Pheromone,Pheromone Analogs,and Verticillium lecanii for Management of Heterodera glycines

A mutant strain of the fungus Verticillium lecanii and selected bioregulators of Heterodera glycines were evaluated for their potential to reduce population densities of the nematode on soybean under greenhouse conditions. The bioregulators tested were the H. glycines sex pheromone vanillic acid and the pheromone analogs syringic acid, isovanillic acid, ferulic acid, 4-hydroxy-3-methoxybenzonitrile, and methyl vanillate. A V. lecanii-vanillic acid combination and a V. lecanii-syringic acid combination were also applied as treatments. Syringic acid, 4-hydroxy-3-methoxybenzonitrile, V. lecanii, V. lecanii-vanillic acid, and V. lecanii-syringic acid significantly reduced nematode population densities in the greenhouse tests. Results with vanillic acid, isovanillic acid, and ferulic acid treatments were variable. Methyl vanillate did not significantly reduce cyst nematode population densities in the greenhouse tests.     

Interactions Between Calonectria crotalariae and Heterodera glycines on Soybean

The interactions of Heterodera glycines at four egg inoculum levels (0, 100, 1,000, and 10,000 per pot) and three cyst levels (0, 100, and 200 per pot) and Calonectria crotalariae at 500, 5,000, and 50,000 microsclerotia per pot were evaluated on soybean. At the two lowest nematode egg levels, the presence of C. crotalariae did not affect nematode reproduction. At 10,000 eggs per pot, however, nematode reproduction was increased significantly at each microsclerotial level. The increase in nematode reproduction was stepwise at 500 and 5,000 microsclerotia per pot but declined at 50,000 microsclerotia per pot. Similar results were obtained when cysts rather than eggs were used as nematode inoculum. The nematode x fungus interaction significantly affected 60-day plant growth parameters of both Lee 74 and Centennial soybean. The nematode x fungus interaction was antagonistic to plant roots and significantly influenced root injury ratings. The presence of C. crotalariae in tissues of stock plants or plants used as race differentials did not alter the analysis of this population as race 3.     

Dynamics of Belonolaimus longicaudatus Parasitism on a Susceptible St. Augustinegrass Host

St. Augustinegrass (Stenotaphrum secundatum) cv FX-313 was used as a model laboratory host for monitoring population growth of the sting nematode, Belonolaimus longicaudatus, and for quantifying the effects of sting nematode parasitism on host performance in two samples of autoclaved native Margate fine sand with contrasting amounts of organic matter (OM = 7.9% and 3.8%). Following inoculation with 50 Belonolaimus longicaudatus per pot, nematodes peaked at a mean of 2,139 nematodes per pot 84 days after inoculation, remained stable through 168 days at 2,064 nematodes per pot, and declined at 210 days. The relative numbers of juveniles and adults demonstrated senescence after 84 days. Root dry weight of nematode-inoculated plants increased briefly to an apparent equilibrium 84 days after inoculation, whereas root weights of uninoculated controls continued to increase, exceeding those of inoculated plants from 84 to 210 days (P < 0.01). At 210 days, uninoculated plants had 227% the root dry weight of inoculated plants. Transpiration of FX-313 was reduced by nematodes (P < 0.0001) at 84 and 126 days after inoculation; reduction was first observed at 42 days and last observed 168 days after inoculation (P < 0.05). OM content affected all plant performance variables at multiple dates, and generally there were no inoculation x OM content interactions. OM content had no effect on nematode numbers per pot, although there was a slight (P < 0.05) increase in the number of nematodes per gram root dry weight in the low-OM soil compared with the high-OM soil.     

Changes in Vertical Distribution of Pratylenchus scribneri under Potato and Corn

The vertical distribution of Pratylenchus scribneri populations was monitored under irrigated corn and potato grown in loamy sand soil. population estimates were based on the number of nematodes recovered from 100-cm³ soil samples and the roots contained therein. Reproduction was assessed by counting the number of second-stage juveniles. An index of population maturity was computed to evaluate the age structure of populations. At no time were nematodes distributed uniformly among five soil depths from 0 to 37.5 cm deep. During the summer (June-September), changes in the total number of P. scribneri and the number of second-stage juveniles recovered were not consistent among the depths sampled. Early (April-June) and late (September-November) in the season, changes in the abundance, reproduction, and maturity of populations were similar among depths. The timing and pattern of increases in numbers of nematodes suggests that variation in the abundance of P. scribneri in the soil profile beneath potato and corn was caused primarily by reproduction rather than the movement of nematodes.     

In-vitro Assays of Meloidogyne incognita and Heterodera glycines for Detection of Nematode-antagonistic Fungal Compounds

In-vitro methods were developed to test fungi for production of metabolites affecting nematode egg hatch and mobility of second-stage juveniles. Separate assays were developed for two nematodes: root-knot nematode (Meloidogyne incognita) and soybean cyst nematode (Heterodera glycines). For egg hatch to be successfully assayed, eggs must first be surface-disinfested to avoid the confounding effects of incidental microbial growth facilitated by the fungal culture medium. Sodium hypochlorite was more effective than chlorhexidine diacetate or formaldehyde solutions at surface-disinfesting soybean cyst nematode eggs from greenhouse cultures. Subsequent rinsing with sodium thiosulfate to remove residual chlorine from disinfested eggs did not improve either soybean cyst nematode hatch or juvenile mobility. Soybean cyst nematode hatch in all culture media was lower than in water. Sodium hypochlorite was also used to surface-disinfest root-knot nematode eggs. In contrast to soybean cyst nematode hatch, root-knot nematode hatch was higher in potato dextrose broth medium than in water. Broth of the fungus Fusarium equiseti inhibited root-knot nematode egg hatch and was investigated in more detail. Broth extract and its chemical fractions not only inhibited egg hatch but also immobilized second-stage juveniles that did hatch, confirming that the fungus secretes nematode-antagonistic metabolites.     

Effects of Heterodera glycines and Meloidogyne incognita on Early Growth of Soybean

Greenhouse and field microplot studies were conducted to compare soybean shoot and root growth responses to root penetration by Heterodera glycines (Hg) and Meloidogyne incognita (Mi) individually and in combination. Soybean cultivars Centennial (resistant to Hg and Mi), Braxton (resistant to Mi, susceptible to Hg), and Coker 237 (susceptible to Hg and Mi) were selected for study. In the greenhouse, pot size and number of plants per pot had no effect on Hg or Mi penetration of Coker 237 roots; root weight was higher in the presence of either nematode species compared with the noninoculated controls. In greenhouse studies using a sand or soil medium, and in field microplot studies, each cultivar was grown with increasing initial population densities (Pi) of Hg or Mi. Interactions between Hg and Mi did not affect early plant growth or number of nematodes penetrating roots. Root penetration was the only response related to Pi. Mi penetration was higher in sand than in soil, and higher in the greenhouse than in the field, whereas Hg penetration was similar under all conditions. At 14 days after planting, more second-stage juveniles were present in roots of susceptible than in roots of resistant plants. Roots continued to lengthen in the greenhouse in the presence of either Mi or Hg regardless of host genotype, but only in the presence of Mi in microplots; otherwise, responses in field and greenhouse studies were similar and differed only in magnitude and variability.     

Suppression of Cyst Nematode by Natural Infestation of a Nematophagous Fungus

Penetration of cabbage roots by Heterodera schachtii was suppressed 50-77% in loamy sand naturally infested with the nematophagous fungus Hirsutella rhossiliensis. When Heterodera schachtii was incubated in the suppressive soil without plants for 2 days, 40-63% of the juveniles had Hirsutella rhossiliensis spores adhering to their cuticles. Of those with spores, 82-92% were infected. Infected nematodes were killed and filled with hyphae within 2-3 days. Addition of KCl to soil did not increase infection of Heterodera schachtii by Hirsutella rhossiliensis. The percentage of infection was lower when nematodes were touched to two spores and incubated in KCl solution than when nematodes naturally acquired two spores in soil.     

Entomopathogenic Nematodes and Bacteria Applications for Control of the Pecan Root-Knot Nematode, Meloidogyne partityla, in the Greenhouse

Meloidogyne partityla is a parasite of pecan and walnut. Our objective was to determine interactions between the entomopathogenic nematode-bacterium complex and M. partityla. Specifically, we investigated suppressive effects of Steinernema feltiae (strain SN) and S. riobrave (strain 7–12) applied as infective juveniles and in infected host insects, as well as application of S. feltiae''s bacterial symbiont Xenorhabdus bovienii on M. partityla. In two separate greenhouse trials, the treatments were applied to pecan seedlings that were simultaneously infested with M. partityla eggs; controls received only water and M. partityla eggs. Additionally, all treatment applications were re-applied (without M. partityla eggs) two months later. Four months after initial treatment, plants were assessed for number of galls per root system, number of egg masses per root system, number of eggs per root system, number of eggs per egg mass, number of eggs per gram dry root weight, dry shoot weight, and final population density of M. partityla second-stage juveniles (J2). In the first trial, the number of egg masses per plant was lower in the S. riobrave-infected host treatment than in the control (by approximately 18%). In the second trial, dry root weight was higher in the S. feltiae-infected host treatment than in the control (approximately 80% increase). No other treatment effects were detected. The marginal and inconsistent effects observed in our experiments indicate that the treatments we applied are not sufficient for controlling M. partityla.     

Induced Resistance to Meloidogyne hapla by other Meloidogyne species on Tomato and Pyrethrum Plants

Advance inoculation of the tomato cv. Celebrity or the pyrethrum clone 223 with host-incompatible Meloidogyne incognita or M. javanica elicited induced resistance to host-compatible M. hapla in pot and field experiments. Induced resistance increased with the length of the time between inoculations and with the population density of the induction inoculum. Optimum interval before challenge inoculation, or population density of inoculum for inducing resistance, was 10 days, or 5,000 infective nematodes per 500-cm³ pot. The induced resistance suppressed population increase of M. hapla by 84% on potted tomato, 72% on potted pyrethrum, and 55% on field-grown pyrethrum seedlings, relative to unprotected treatments. Pyrethrum seedlings inoculated with M. javanica 10 days before infection with M. hapla were not stunted, whereas those that did not receive the advance inoculum were stunted 33% in pots and 36% in field plots. The results indicated that advance infection of plants with incompatible or mildly virulent nematode species induced resistance to normally compatible nematodes and that the induced resistance response may have potential as a biological control method for plant nematodes.     

Infection of Pratylenchus penetrans by Nematode-pathogenic fungi

Eleven fungal isolates were tested in agar dishes for pathogenicity to Pratylenchus penetrans. Of the fungi that produce adhesive conidia, Hirsutella rhossiliensis was a virulent pathogen; Verticillium balanoides, Drechmeria coniospora, and Nematoctonus sp. were weak or nonpathogens. The trapping fungi, Arthrobotrys dactyloides, A. oligospora, Monacrosporium dlipsosporum, and M. cionopagum, killed most of the P. penetrans adults and juveniles added to the fungus cultures. An isolate of Nematoctonus that forms adhesive knobs trapped only a small proportion of the nematodes. In 17-cm³ vials, soil moisture influenced survival of P. penetrans in the presence of H. rhossiliensis; nematode survival decreased with diminishing soil moisture. Hirsutella rhossiliensis and M. ellipsosporum were equally effective in reducing numbers of P. penetrans by 24-25% after 4 days in sand. After 25 days in soil artificially infested with H. rhossiliensis, numbers of P. penetrans were reduced by 28-53%.     

Parasitism of Nematodes by the Fungus Hirsutella rhossiliensis as Affected by Certain Organic Amendments

Experiments were conducted to determine whether the addition of organic matter to soil increased numbers of bacterivorous nematodes and parasitic activity of the nematophagous fungus Hirsutella rhossiliensis. In a peach orchard on loamy sand, parasitism of the plant-parasitic nematode Criconemella xenoplax by H. rhossiliensis was slightly suppressed and numbers of C. xenoplax were not affected by addition of 73 metric tons of composted chicken manure/ha. In the laboratory, numbers of bacterivorous nematodes (especially Acrobeloides spp.) and fungivorous nematodes increased but parasitism of nematodes by H. rhossiliensis usually decreased with addition of wheat straw or composted cow manure to a loamy sand naturally infested with H. rhossiliensis. These results do not support the hypothesis that organic amendments will enhance parasitism of nematodes by H. rhossiliensis.     

Effects of Interactions among Heterodera glycines,Meloidogyne incognita,and Host Genotype on Soybean Yield and Nematode Population Densities

The effects of host genotype and initial nematode population densities (Pi) on yield of soybean and soil population densities of Heterodera glycines (Hg) race 3 and Meloidogyne incognita (Mi) race 3 were studied in a greenhouse and field microplots in 1983 and 1984. Centennial (resistant to Hg and Mi), Braxton (resistant to Mi, susceptible to Hg), and Coker 237 (susceptible to Hg and Mi) were planted in soil infested with 0, 31, or 124 eggs of Hg and Mi, individually and in all combinations, per 100 cm³ soil. Yield responses of the soybean cultivars to individual and combined infestations of Hg and Mi were primarily dependent on soybean resistance or susceptibility to each species separately. Yield of Centennial was stimulated or unaffected by nematode treatments, yield of Braxton was suppressed by Hg only, and yield suppressions caused by Hg and Mi were additive and dependent on Pi for Coker 237. Other plant responses to nematodes were also dependent on host resistance or susceptibility. Population densities of Mi second-stage juveniles (J2) in soil were related to Mi Pi and remained constant in the presence of Hg for all three cultivars. Population densities of Hg J2 on the two Hg-susceptible Cultivars, Braxton and Coker 237, were suppressed in the presence of Mi at low Hg Pi.     

Reproduction and Development of Meloidogyne incognita and M. javanica on Guardian Peach Rootstock

Guardian peach rootstock was evaluated for susceptibility to Meloidogyne incognita race 3 (Georgia-peach isolate) and M. javanica in the greenhouse. Both commercial Guardian seed sources produced plants that were poor hosts of M. incognita and M. javanica. Reproduction as measured by number of egg masses and eggs per plant, eggs per egg mass, and eggs per gram of root were a better measure of host resistance than number of root galls per plant. Penetration, development, and reproduction of M. incognita in Guardian (resistant) and Lovell (susceptible) peach were also studied in the greenhouse. Differences in susceptibility were not attributed to differential penetration by the infectivestage juveniles (J2) or the number of root galls per plant. Results indicated that M. incognita J2 penetrated Guardian roots and formed galls, but that the majority of the nematodes failed to mature and reproduce.     

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.     

Influence of Lysobacter enzymogenes Strain C3 on Nematodes

Chitinolytic microflora may contribute to biological control of plant-parasitic nematodes by causing decreased egg viability through degradation of egg shells. Here, the influence of Lysobacter enzymogenes strain C3 on Caenorhabditis elegans, Heterodera schachtii, Meloidogyne javanica, Pratylenchus penetrans, and Aphelenchoides fragariae is described. Exposure of C. elegans to L. enzymogenes strain C3 on agar resulted in almost complete elimination of egg production and death of 94% of hatched juveniles after 2 d. Hatch of H. schachtii eggs was about 50% on a lawn of L. enzymogenes strain C3 on agar as compared to 80% on a lawn of E. coli. Juveniles that hatched on a lawn of L. enzymogenes strain C3 on agar died due to disintegration of the cuticle and body contents. Meloidogyne javanica juveniles died after 4 d exposure to a 7-d-old chitin broth culture of L. enzymogenes strain C3. Immersion of A. fragariae, M. javanica, and P. penetrans juveniles and adults in a nutrient broth culture of L. enzymogenes strain C3 led to rapid death and disintegration of the nematodes. Upon exposure to L. enzymogenes strain C3 cultures in nutrient broth, H. schachtii juveniles were rapidly immobilized and then lysed after three days. The death and disintegration of the tested nematodes suggests that toxins and enzymes produced by this strain are active against a range of nematode species.     

Hirsutella rhossiliensisand Verticillium chlamydosporium as Biocontrol Agents of the Root-knot Nematode Meloidogyne hapla on Lettuce

Hirsutella rhossiliensis and Verticillium chlamydosporium infected second-stage juveniles (J2) and eggs of Meloidogyne hapla, respectively, in petri dishes and in organic soil in pots planted to lettuce in the greenhouse. In vitro, H. rhossiliensis produced 78 to 124 spores/infected J2 of M. hapla. The number of J2 in roots of lettuce seedlings decreased exponentially with increasing numbers of vegetative colonies of H. rhossiliensis in the soil. At an infestation of 8 M. hapla eggs/cm³ soil, 1.9 colonies of H. rhossiliensis/cm³ soil were needed for a 50% decrease in J2 penetration of lettuce roots. Egg-mass colonization with V. chlamydosporium varied from 16% to 43% when soil was infested with 8 M. hapla eggs and treated with 5,000 or 10,000 chlamydospores of V. chlamydosporium/cm³ soil. This treatment resulted in fewer J2 entering roots of bioassay lettuce seedlings planted in the infested soils after harvesting the first lettuce plants 7 weeks after infestation with M. hapla. Hirsutella rhossiliensis (0 to 4.3 colonies/cm3 soil), V. chlamydosporium (500 to 10,000 chlamydospores/cm3 soil), or their combination, added to organic soils with 8 M. hapla eggs/cm³ soil, generally did not affect lettuce weight, root galling, or egg production of M. hapla. However, when lettuce was replanted in a mix of infested and uninfested soil (1:3 and 1:7, v:v), egg production was lower in soils with V. chlamydosporium than in soils without the fungus. Both fungi have potential to reduce the M. hapla population, but at densities below 8 eggs/cm³ soil.     

Field Efficacy of Verticillium lecanii,Sex Pheromone,and Pheromone Analogs as Potential Management Agents for Soybean Cyst Nematode

A soybean cyst nematode sex pheromone (vanillic acid), chemical analogs of the pheromone, and the fungus Verticillium lecanii were applied in alginate prills (340 kg/ha) to microplots and small-scale field plots as potential management agents for Heterodera glycines on soybean. In 1991 microplot tests, treatment with V. lecanii, vanillic acid, syringic acid plus V. lecanii, or vanillic acid plus V. lecanii lowered midseason cyst numbers compared with the untreated susceptible cultivar control, autoclaved V. lecanii treatment, or aldicarb treatment, At-harvest cyst numbers were lowest with V. lecanii and with vanillic acid treatments. Aldicarb treatment reduced midseason cyst numbers in 1992. There were no differences among seed yields either year. In the field trials, numbers of cysts were reduced one or both years with aldicarb, ferulic acid, syringic acid, vanillic acid, or 4-hydroxy-3-methoxybenzonitfile treatments, or with a resistant cultivar, compared to an untreated susceptible cultivar. Highest yields were recorded after treatment with 4-hydroxy-3-methoxybenzonitrile (1991), methyl vanillate (1992), and aldicarb (1992). These studies indicate that some chemical analogs of vanillic acid have potential for use in soybean cyst nematode management schemes.     

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.