Chemical-Mediated Toxicity of N-Viro Soil to Heterodera glycines and Meloidogyne incognita

N-Viro Soil (NVS) is an alkaline-stabilized municipal biosolid that has been shown to lower population densities and reduce egg hatch of Heterodera glycines and other plant-parasitic nematodes; but the mechanism(s) of nematode suppression of this soil amendment are unknown. This study sought to identify NVS-mediated changes in soil chemical properties and their impact upon H. glycines and Meloidogyne incognita mortality. N-Viro Soil was applied to sand in laboratory assays at 0.5%, 1.0%, 2.0%, and 3.0% dry w/w with a nonamended treatment as a control. Nematode mortality and changes in sand-assay chemical properties were determined 24 hours after incubation. Calculated lethal concentration (LC₉₀) values were 1.4% w/w NVS for second-stage juveniles of both nematode species and 2.6 and >3.0% w/w NVS for eggs of M. incognita and H. glycines, respectively. Increasing rates of NVS were strongly correlated (r² = 0.84) with higher sand solution pH levels. Sand solution pH levels and, to a lesser extent, the production of ammonia appeared to be the inorganic chemical-mediated factors responsible for killing plant-parasitic nematodes following amendment with NVS.     

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.     

Pathogenicity of Pratylenchus penetrans,Heterodera glycines,and Meloidogyne incognita on Soybean Genotypes

The pathogenicity of Heterodera glycines, Meloidogyne incognita, and Pratylenchus penetrans on H. glycines-resistant ''Bryan,'' tolerant-susceptible ''G88-20092,'' and intolerant-susceptible ''Tracy M'' soybean cultivars was tested using plants grown in 800 cm³ of soil in 15-cm-diam. clay pots in three greenhouse experiments. Plants were inoculated with 0, 1,000, 3,000, or 9,000 H. glycines race 3 or M. incognita eggs, or vermiform stages of P. penetrans/pot. Forty days after inoculation, nmnbers of all three nematodes, except H. glycines on Bryan, generally increased with increasing inoculum levels in Experiment I. Heterodera glycines and M. incognita significantly decreased growth only of Tracy M. At 45 and 57 days after inoculation with 6,000 individuals/pot in experiments II and III, respectively, significantly more P. penetrans and M. incognita than H. glycines were found on Bryan. However, H. glycines and M. incognita population densities were greater than P. penetrans on G88-20092 and Tracy M. Growth of Tracy M infected by H. glycines and M. incognita and growth of G88-20092 infected by M. incognita decreased in Experiment III. Pratylenchus penetrans did not affect plant growth. Reduction in plant growth differed according to the particular nematode species and cultivar, indicating that nematodes other than the species for which resistance is targeted can have different effects on cultivars of the same crop species.     

Competition between Heterodera glycines and Meloidogyne incognita or Pratylenchus penetrans: Independent Infection Rate Measurements

Competition on soybean between Heterodera glycines (race 3) and Meloidogyne incognita or H. glycines and Pratylenchus penetrans were investigated in greenhouse experiments. Each pair of nematode species was mixed in 3-ml suspensions at ratios of 1,000:0, 750:250, 500:500, 250:750, and 0:1,000 second-stage juveniles or mixed stages for P. penetrans. Nematodes from a whole root system were counted and infection rates standardized per 1,000 nematodes (per replication) prior to testing the null hypothesis through a lack-of-fit F-test. Although the effect of increasing H. glycines proportions on the infection rate of M. incognita was generally adverse, the rate deviated significantly from a trend of linear decline at the 75% H. glycines level in one of two experiments. All lack-of-fit F-tests for the H. glycines and P. penetrans mix were significant, indicating that infection rates for both nematodes varied considerably across inocula. The infection rate of H. glycines decreased with increasing P. penetrans proportions. The rate of P. penetrans infection increased with increasing H. glycines proportions up to the 50% level, but declined at the 75% level. Competition had no effect on nematode development. The general adverse relationships between M. incognita and H. glycines and those between P. penetrans and H. glycines showed a linear trend. The relationship between H. glycines and P. penetrans indicates that the former may be competitive when present at higher proportions than the latter. In this study we have evaluated nematode competition under controlled conditions and provide results that can form a basis for understanding the physical and physiological trends of multiple nematode interactions. Methods critical to data analyses also are outlined.     

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.     

Viability of Heterodera glycines Exposed to Fungal Filtrates

Filtrates from nematode-parasitic fungi have been reported to be toxic to plant-parasitic nematodes. Our objective was to determine the effects of fungal filtrates on second-stage juveniles and eggs of Heterodera glycines. Eleven fungal species that were isolated from cysts extracted from a soybean field in Florida were tested on J2, and five species were tested on eggs in vitro. Each fungal species was grown in Czapek-Dox broth and malt extract broth. No toxic activity was observed for fungi grown in Czapek-Dox broth. Filtrates from Paecilomyces lilacinus, Stagonospora heteroderae, Neocosmospora vasinfecta, and Fusarium solani grown in malt extract broth were toxic to J2, whereas filtrates from Exophiala pisciphila, Fusarium oxysporum, Gliocladium catenulatum, Pyrenochaeta terrestris, Verticillium chlamydosporium, and sterile fungi 1 and 2 were not toxic to J2. Filtrates of P. lilacinus, S. heteroderae, and N. vasinfecta grown in malt extract broth reduced egg viability, whereas F. oxysporum and P. terrestris filtrates had no effect on egg viability.     

Pathogenicity of Fungi to Eggs of Heterodera glycines

Twenty-one isolates of 18 fungal species were tested on water agar for their pathogenicity to eggs of Heterodera glycines. An egg-parasitic index (EPI) for each of these fungi was recorded on a scale from 0 to 10, and hatch of nematode eggs was determined after exposure to the fungi on water agar for 3 weeks at 24 C. The EPI for Verticillium chlamydosporium was 7.6, and the fungus reduced hatch 74%. Pyrenochaeta terrestris and two sterile fungi also showed a high EPI and reduced hatch 42-73%. Arthrobotrys dactyloides, Fusarium oxysporum, Paecilomyces lilacinus, Stagonospora heteroderae, Neocosmospora vasinfecta, Fusarium solani, and Exophiala pisciphila were moderately pathogenic to eggs (EPI was 2.0-4.5, and hatch was reduced 21-56%). Beauveria bassiana, Hirsutella rhossiliensis, Hirsutella thompsonii, Dictyochaeta heteroderae, Dictyochaeta coffeae, Gliocladium catenulatum, and Cladosporium sp. showed little parasitism of nematode eggs but reduced hatch. A negative correlation was observed between hatch and fungal parasitism of eggs. Fusarium oxysporum, H. rhossiliensis, P. lilacinus, S. heteroderae, V. chlamydosporium, and sterile fungus 1 also were tested in soil in a greenhouse test. After 3 months, the nematode densities were lower in soil treated with H. rhossiliensis and V. chlamydosporium than in untreated soil. The nematode population densities were correlated negatively with the EPI, but not with the percentage of cysts colonized by the fungi. Plant weights and heights generally increased in the soil treated with the fungi.     

Factors Affecting the Suppression of Heterodera glycines by N-Viro Soil

Previous laboratory research demonstrated that N-Viro Soil (NVS), an alkaline-stabilized municipal biosolid, suppressed plant-parasitic nematodes. This study continued to explore the use of NVS as a nematode management tool specifically addressing factors that could influence its use. N-Viro Soil from different locations, the components of NVS (de-watered biosolids and fly ash admixtures), and sterilized NVS were applied to sand microcosms to determine effects on nematode survival sand solution pH and ammonia concentrations. This study confirmed the previous finding that an important mechanism of Heterodera glycines suppression by NVS was the generation of alkaline soil conditions. Only the fly ash admixture that resulted in an increase in pH to 10.0 suppressed H. glycines to the same level as NVS. Alkaline-stabilization of biosolids was necessary to achieve nematode suppression. Biosolids applied at rates <3% dry w/w did not suppress H. glycines to the same level as equivalent amounts of NVS. Sand solution pH levels after biosolid application, regardless of rate, were approximately 8.5 whereas 1% and 4% w/w NVS amendment resulted in pH levels of 10.3 and 11.6, respectively. NVS from different processing facilities were all effective in suppressing H. glycines. The NVS source that produced the highest concentration of ammonia did not reduce H. glycines survival to the same level as those sources generating pH levels above 10.1. Microbes associated with NVS appeared not to be responsible for the nematode suppressiveness of the amendment; there was no difference in nematode suppression between autoclaved and nonautoclaved NVS. The role that ammonia plays in the suppression of H. glycines by NVS is still unclear.     

Characteristics and Efficacy of a Sterile Hyphomycete (ARF18), a New Biocontrol Agent for Heterodera glycines and Other Nematodes

A filamentous, nonsporulating fungus, designated Arkansas Fungus 18 (ARF18), was isolated from 9 of 95 populations of Heterodera glycines, the soybean cyst nematode, in Arkansas. In petri dishes, ARF18 parasitized 89% of H. glycines eggs in cysts. The fungus also infected eggs of Meloidogyne incognita and eggs in cysts of Cactodera betulae, H. graminophila, H. lespedezae, H. leuceilyma, H. schachtii, and H. trifolii. In pot tests, reproduction of SCN was 70% less in untreated field soil that was naturally infested by ARF18 than in autoclaved field soil. Although ARF18 grew well at 25 C on cornmeal agar over a wide pH range, it did not sporulate on 28 media and thus could not be identified to genus or species.     

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.     

Flow Cytometric Analysis and Sorting of Heterodera glycines Eggs

A nondestructive technique was developed to characterize and separate eggs of soybean cyst nematode, Heterodera glycines, by developmental stage using flow cytometry. Eggs from cysts cultured on susceptible soybean roots were suspended in 0.1% xanthan gum or 59% sucrose and loaded into either a Coulter EPICS 752 or EPICS 753 flow cytometer. Eggs were analyzed and sorted according to forward angle and 90° light scatter, flow cytometric parameters that are relative measures of object size and granularity, respectively. Mature eggs containing vermiform juveniles were less granular and slightly larger than eggs in earlier stages of embryogeny, allowing for separation of mature eggs from immature eggs. The effectiveness of flow cytometric sorting was evaluated by comparing the developmental stages of subpopulations of unsorted and sorted eggs. Of a subpopulation of unsorted eggs, 62% contained vermiform juveniles, whereas 85 to 95% of sorted subpopulations of larger, less granular eggs contained vermiform juveniles. Suspending H. glycines eggs in 0.1% xanthan gum or 59% sucrose for flow cytometric analysis had no effect on subsequent egg hatch in vitro. This technique is an efficient and effective means to collect large, relatively homogeneous quantities of H. glycines eggs in early or late embryogeny, and would likely be useful for analyzing and sorting eggs of other nematode species for use in developmental, genetic, or physiological research, or for identification and collection of parasitized eggs.     

Relationship Between Egg Viability and Population Densities of Meloidogyne incognita on Cotton

Cotton seedlings were inoculated with a range of initial populations (Pi) of Meloidogyne incognita in greenhouse experiments to test the relationship between nematode population densities and egg viability. In two of three experiments, a significant (P < 0.05) negative linear relationship was detected between percentage of hatch of first generation eggs and log Pi. A similar relationship between hatch and root-gall index was observed. In two experiments numbers of eggs judged to be nonviable based on appearance were significantly greater (P < 0.05) in the highest Pi (60,000 eggs/seedling) treatments than in treatments with lower Pi (600-6,000 eggs/seedling). It was concluded that Pi affects egg viability measured as percentage of hatch and that this relationship may play a role in the density-dependent winter survival rates of Meloidogyne species.     

Separation and Characterization of Heterodera glycines Acetylcholinesterase Molecular Forms

The composition and biochemical properties of acetylcholinesterases isolated from Heterodera glycines were determined. Heterodera glycines contains three separable AChE molecular forms that can be grouped into two classes corresponding to classes A and C found in some other nematode species. The apparent lack of class B AChE is unusual and may have significant behavioral ramifications. The class C enzyme isolated from H. glycines is similar to that from Meloidogyne arenaria and M. incognita but is somewhat more sensitive to AChE inhibitors such as eserine. Heterodera glycines possesses a larger percentage of its total acetylcholinesterase as class C than other nematodes thus far examined.     

Overwinter Population Dynamics of Heterodera glycines

The purpose of this research was to compare the overwinter survival of populations of Heterodera glycines at different latitudes in the United States and the effect of changing latitudes before and after the initiation of dormancy. Soil samples infested with H. glycines were collected in August or October in 1992 to 1994 from soybean fields in two to four states (combinations of Arkansas, Florida, Minnesota, Missouri, and Wisconsin). The samples were mixed thoroughly, divided into subsamples, shipped to an overwinter location, and buried until time for processing. To determine survival, cysts, eggs, and second-stage juveniles were extracted from replicated subsamples and counted each month from December to May. Survival generally was between 50% and 100%, and often was best in the state of origin. In Florida, survival was at the 50 to 100% level in soil from most locations, and in Wisconsin was near 100%. Survival of H. glycines in Arkansas and Missouri varied more than at the other locations. In a separate test, survival in microplots in Arkansas, in a more natural environment than that of buried samples, was 70 to 94% for field isolates from Arkansas, Minnesota, and Missouri and 100% for isolates of races 1, 3, and 14 that had been maintained in a greenhouse for several years. Survival appears to be better than previous tests had indicated. High survival rates require cultivars with high levels of resistance and long-term rotations for management.     

Detection and Partial Characterization of Egg Polypeptides from Heterodera glycines

The presence of two major egg polypeptides was demonstrated in the plant-parasitic nematode Heterodera glycines. The polypeptides were present in equal amounts in, and were most abundant in, eggs from yellow females. They were also present in brown females but were not detected in second-stage juveniles (J2). The two major egg polypeptides, MEP-I and MEP-II, accounted for more than 50% of the total protein in egg extracts evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. During development of females from the yellow stage to the brown stage, the levels of MEP-I and MEP-II declined at twice the rate as total protein. MEP-I and MEP-II had estimated molecular masses of 190 kD and 180 kD, respectively, similar to those reported for female-specific proteins, vitellins, from free-living nematodes.     

Relationship Between Time of Infection with Heterodera glycines and Soybean Yield

Experiments were conducted to determine the relationship between time of infection by Heterodera glycines and soybean growth in the greenhouse and yield of plants grown in the field. Soybean cultivar Essex seedlings growing in the greenhouse were inoculated with H. glycines at 2, 4, or 6 weeks after planting. Seedling growth was inhibited by H. glycines infection at 2 or 4 weeks after planting but not at 6 weeks. Infection of Essex by H. glycines in the field was delayed 2-6 weeks by nematicides. Yields were significantly increased when H. glycines infection was delayed 2 weeks by nematicide treatment. Essex yields were highest when infection was delayed 6 weeks, equalling the yield of the H. glycines-resistant cultivar Asgrow 5474. The effect of H. glycines on soybean growth in the greenhouse and yields in the field declined when infection was delayed 6 weeks. Thus, soybean sensitivity to H. glycines seemed to diminish with age of the soybean plants.     

Heterodera glycines Cyst Components and Surface Disinfestants Affect H. glycines Hatching

We investigated the effects of Heterodera glycines cyst components and surface disinfestants on hatching of H. glycines eggs in vitro. Eggs were incubated in either H. glycines cyst wall fragments, cyst wall and egg rinsate, egg homogenate, or control solutions of soybean root diffusate, sterile distilled water, or zinc sulfate. Hatch in cyst wall and egg rinsate, and egg homogenate, was greater (α = 0.05) than hatch in sterile distilled water; however, it was not different from hatch in zinc sulfate according to Dunnett''s test. Hatch in cyst wall fragments was similar to hatch in sterile distilled water. To determine whether surface disinfestants affected hatch, eggs were treated first with chlorhexidine diacetate, mercuric chloride, sodium hypochlorite, or streptomycin sulfate and then incubated in H. glycines egg homogenate, soybean root diffusate, sterile distilled water, or zinc sulfate. Hatch of eggs treated with chlorhexidine diacetate, mercuric chloride, and streptomycin sulfate was reduced (α = 0.05), and hatch of eggs treated with sodium hypochlorite was increased (α = 0.05) relative to hatch of nontreated eggs in all incubation solutions except zinc sulfate according to Dunnett''s Test. Hatch in zinc sulfate was similar among all surface disinfestants except mercuric chloride, where hatch was reduced relative to hatch of nontreated and other surface disinfestant-treated eggs.     

Effects of a Mutant Strain and a Wild Type Strain of Verticillium lecanii on Heterodera glycines Populations in the Greenhouse

A wild type strain ofVerticillium lecanii and a mutant strain with increased tolerance to the fungicide benomyl were evaluated in greenhouse experiments for effects on Heterodera glycines populations. Nematodes were applied at 300 eggs and juveniles per 4,550-cm³ pot (two soybean plants in 4,990 g loamy sand per pot) and at both 300 and 10,000 eggs and juveniles per 1,720-cm³ pot (one soybean plant in 2,060 g sand per pot). With 300 nematodes added per pot, both V. lecanii strains significantly reduced nematode populations in loamy sand (fungus applied at 0.02% dry weight per dry weight loamy sand) and sand (0.006% and 0.06% fungus application rates). The mutant strain applied at 0.002% to sand also significantly reduced cyst numbers. When 10,000 nematodes were added per pot, only the mutant strain at 0.06% significantly decreased population. Various media were tested for isolation of the fungus strains from prills, loamy sand, and sand, but the fungi were recovered from few of the greenhouse pots.     

Monoclonal Antibodies to the Esophageal Glands and Stylet Secretions of Heterodera glycines

Three monodonal antibodies (MAbs) that bound to secretory granules within the subventral esophageal glands of second-stage juveniles (J2) of the soybean cyst nematode (SCN), Heterodera glycines, were developed from intrasplenic immunizations of a mouse with homogenates of SCN J2. Two MAbs to the secretory granules within subventral glands and one MAb to granules within the dorsal esophageal gland of SCN J2 were developed by intrasplenic immunizations with J2 stylet secretions. Stylet secretions, produced in vitro by incubating SCN J2 in 5-methoxy DMT oxalate, were solubilized with a high pH buffer and concentrated for use as antigen. Three of the five MAbs specific to the subventral esophageal glands bound to stylet secretions from SCN J2 in immunofluorescence and ELISA assays. Two of these three MAbs also bound to secretory granules within both the dorsal and subventral esophageal glands of young SCN females. All five of the subventral gland MAbs bound to the subventral glands of Heterodera schachtii and one bound to the subventral glands of Globodera tabacum, but none bound to any structures in Meloidogyne incognita or Caenorhabditis elegans.