Decomposition of Plant Debris by the Nematophagous Fungus ARF

In the study of the biological control of plant-parasitic nematodes, knowledge of the saprophytic ability of a nematophagous fungus is necessary to understand its establishment and survival in the soil. The objectives of this study were (i) to determine if the nematophagous fungus ARF (Arkansas Fungus) shows differential use of plant residues; and (ii) to determine if ARF still existed in the soil of a field in which ARF was found originally and in which the population level of Heterodera glycines had remained very low, despite 15 years of continuous, susceptible soybean. Laboratory studies of the decomposition of wheat straw or soybean root by ARF were conducted in two separate experiments, using a CO₂ collection apparatus, where CO₂-free air was passed through sterilized cotton to remove the microorganisms in the air and then was passed over the samples, and evolved CO₂ was trapped by KOH. Milligrams of C as CO₂ was used to calculate the percentage decomposition of the plant debris by ARF. Data indicated ARF decomposed 11.7% of total organic carbon of the wheat straw and 20.1% of the soybean roots in 6 weeks. In the field soil study, 21 soil samples were taken randomly from the field. Only 3 months after the infestation of the soil with H. glycines, the percentage of parasitized eggs of H. glycines reached 64 ± 19%, and ARF was isolated from most parasitized eggs of H. glycines. Research results indicated ARF could use plant residues to survive.     

Fungal Parasitism of Heterodera glycines Eggs as Influenced by Egg Age and Pre-colonization of Cysts by Other Fungi

The objective of this study was to determine the effect of egg age and pre-colonization of cysts by a saprophytic or parasitic fungus on parasitism of Heterodera glycines eggs by other parasitic fungi. In agar and in soil tests, fungi generally parasitized more eggs in early developmental stages than eggs containing a juvenile. The effect of pre-colonization of cysts by a fungus on parasitism of eggs by other fungi depended on the fungi involved. In most cases, pre-colonization of cysts by an unidentified, saprophytic fungal isolate (A-1-24) did not affect parasitism of eggs in the cysts subsequently treated with other fungi. However, pre-colonization of cysts by A-1-24 reduced fungal parasitism of eggs in cysts subsequently treated with Cylindrocarpon destructans isolate 3. In agar tests, pre-colonization of cysts by Chaetomium cochliodes, a saprophytic or weakly parasitic fungus, reduced parasitism of eggs in cysts subsequently treated with Verticillium chlamydosporium Florida isolate, Fusarium oxysporum, Fusarium solani, ARF18, and another sterile fungus. However, in soil tests, pre-colonization of cysts by C. cochliodes had no effect on parasitism of eggs by subsequent fungal parasites. In another test, parasitism of eggs by V. chlamydosporium in cysts was not affected by pre-colonizing fungi C. destructans, F. oxysporum, and F. solani but was reduced by Mortierella sp., Pyrenochaeta terrestris, and C. cochliodes. Parasitism of eggs in cysts by ARF18 was reduced by pre-colonizing fungi C. destructans, F. oxysporum, F. solani, P. terrestris, and C. cochliodes but not Mortierella sp.     

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.     

Glomus fasciculatum,a Weak Pathogen of Heterodera glycines

The occurrence ofchlamydospores of Glomus fasciculatum (Gf) within cysts of the soybean cyst nematode, Heterodera glycines, and the effects of vesicular-arbuscular mycorrhizae on nematode population dynamics and soybean (Glycine max) plant growth were investigated. Chlamydospores occupied 1-24% of cysts recovered from field soil samples. Hyphae of Missouri isolate Gfl penetrated the female nematode cuticle shortly after she ruptured the root epidermis. Convoluted hyphae filled infected eggs, and sporogenesis occurred within infected eggs. G. microcarpum, G. mosseae, and two isolates of Gf were inoculated with H. glycines on plants of ''Essex'' soybeans. Each of the two Gf isolates infected about 1% of the nematode eggs in experimental pot cuhures. The Gfl isolate decreased the number of first-generation adult females 26%, compared with the nonmycorrhizal control. The total numbers of first-generation plus second-generation adult females were similar for both Gf isolates and 29-41% greater than the nonmycorrhizal control. Soybean plants with Gf and H. glycines produced more biomass than did nonmycorrhizal plants with nematodes, but only Gfl delayed leaf senescence.     

Field Interrelationships among Heterodera glycines,Pratylenchus scribneri,and Three Other Nematode Species Associated with Soybean

Field experiments were conducted in 1982 and 1983 to assess interactions between Heterodera glycines and Pratylenchus scribneri on soybean in southern Illinois. Soybean cyst nematode susceptible cultivar Williams 79 and resistant cultivar Fayette were treated or not treated with aldicarb 15G. Initial population densities were 35 H. glycines cysts containing eggs, 100 P. scribneri, 30 Helicotylenchus pseudorobustus, 225 Paratylenchus projectus, and 85 Tylenchorhynchus martini per 250 cm³ soil in 1982, whereas in 1983 populations were 11 H. glycines cysts, 330 P. scribneri, and 620 H. pseudorobustus. In both years H. glycines populations increased on nontreated Williams 79, decreased on both treated and nontreated Fayette, and remained at initial levels on treated Williams 79. Recovery of P. scribneri per gram dry root was different between nontreated cultivars in 1982 but not in 1983. Aldicarb treatment suppressed soil and root populations of P. scribneri on both cultivars in both years. Populations of H. pseudorobustus, P. projectus, and T. martini at harvest indicated little population increase on either nontreated cultivar. In 1982 H. glycines caused yield suppression but P. scribneri did not, as differences in yield occurred between cultivars but not between aldicarb treatments. In 1983, however, there were no yield differences between cultivars, but aldicarb application resulted in yield increase in both cultivars. In 1983 the yield increase resulting from P. scribneri control was approximately 25%. No synergistic effect on yield was observed between H. glycines and P. scribneri.     

Effects of Some Pesticides on the Growth of ARF18 and Its Pathogenicity to Heterodera glycines

The effects of 22 pesticides on the mycelial growth and pathogenicity of the biocontrol fungus ARFI8 to Heterodera glycines were tested in vitro. The chemicals were added to agar at 10, 100, and 1,000 ppm a.i.; a block of agar containing the fungus was added to each test concentration; and fungal growth was measured. Subsequently, a block of the fungus on the pesticide-containing agar was used to determine the ability of the fungus to parasitize eggs of H. glycines. Aldicarb, bentazone, and chlorothalonil had little or no effect on fungal growth, whereas benomyl and thiophanate methyl completely inhibited growth of the fungus at 10 ppm. The relative insensitivity of ARF18 to certain pesticides would permit selected use of those pesticides with ARF18 in an integrated control program if the effects on the fungus in the field are similar to results from petri dish studies.     

Multivariate Analysis of Selected Edaphic Factors and Their Relationship to Heterodera glycines Population Density

The influence of selected soil physical and chemical factors on population density of Heterodera glycines was investigated in 1988 and 1989 in two different locations of a soybean (Glycine max) field. Soil variables of a Norfolk loamy sand were measured after planting soybeans susceptible to H. glycines. Cyst and egg populations were determined after harvest. Nematode population density was found to be clustered. Up to 91% of the eggs were parasitized by a sterile fungus. Principal component analysis with orthogonal VARIMAX rotation grouped 12 variables into five uncorrelated factors in 1988 and three in 1989. In 1988, the factor "pH and Mg" was positively correlated (P < 0.001) with cyst and egg population density. Also, the factor "fine texture and Cu" was negatively correlated (P < 0.05) with egg population density. In 1989, the factor "pH, Mg and Cu" was positively correlated (P < 0.05) with levels of cysts and percentage of parasitized eggs, but not with total egg number. Across 2 years, factors containing soil pH and Mg were positively associated with cyst nematode population density. Copper appeared to be negatively associated with populations of H. glycines.     

Variation in Efficacy of Isolates of the Fungus ARF Against the Soybean Cyst Nematode Heterodera glycines

An unnamed fungus, designated ARF, that parasitizes eggs and sedentary stages of cyst nematodes is a potential biological control agent of Heterodera glycines. The objectives of this study were to determine whether ARF isolates differ in their ability to suppress nematode numbers in soil and to compare the efficacy of ARF in heat-treated and native soil. The effectiveness of 11 ARF isolates was compared by introducing homogenized mycelium into heat-treated soil. Soybean seedlings were transplanted into pots containing fungus-infested soil and inoculated with H. glycines. After 30 or 60 days, the number of nematodes and the percentage of parasitized eggs were determined. Three isolates (907, 908, and TN14), which were previously reported to be weak egg parasites in vitro, consistently suppressed nematode numbers by 50% to 100%. Of the isolates previously reported to be aggressive egg parasites, four (903, BG2, MS3, and TN12) reduced nematode numbers by 56% to 69% in at least one experimental trial, but the other four had no effect on nematode numbers. When the efficacy of isolate TN14 was tested in heat-treated and native soil, nematode suppression was greater in the heat-treated soil in only one of two trials. In both soil treatments, nematode numbers were reduced by more than 60%. We conclude that virulence toward nematode eggs in vitro is a poor indicator of effectiveness of an ARF isolate in soil, and that the presence of soil microbes may reduce, but does not completely inhibit, activity of isolate TN14.     

Fungi Associated with Females and Cysts of Heterodera glycines in a Florida Soybean Field

Fungal colonization was determined for females and cysts of Heterodera glycines on soybean roots or in rhizosphere soil from a Florida soybean field. A total of 1,620 females and cysts were examined in 1991, and 1,303 were examined in 1992. More than 35 species of fungi were isolated from females and cysts. The frequency of fungi colonizing white and yellow females was low, but a high frequency of fungi was encountered in brown cysts, which increased with time of exposure of the cysts to the soil. No single fungal species predominated in the nematode females or cysts in this field. Rarely was a female or cyst colonized by more than one fungus. The common fungi isolated from the females and cysts were Neocosmospora vasinfecta, Fusarium solani, Fusarium oxysporum, Dictyochaeta coffeae, Dictyochaeta heteroderae, Pyrenochaeta terrestris, Exophiala pisciphila, Gliocladium catenulatum, Stagonospora heteroderae, and a black yeast-like fungus. The communities of common fungal species isolated from cysts in several regions in the southeastern United States appear to be similar.     

Multiple Pest Interactions in Soybean: Effects on Heterodera glycines Egg Populations and Crop Yield

Population changes of Heterodera glycines eggs on soybean in small field plots were influenced by the lepidopterous insect pest, Helicoverpa zea; however, few effects on eggs due to the presence of annual weeds were detected. Soybeans defoliated 15-35% by H. zea during August remained green and continued to produce new flowers and pods later into the season than soybeans without H. zea, resulting in higher numbers of H. glycines eggs at harvest on insect-defoliated soybeans. Final H. glycines populations also were influenced by soil population density (Pi) of the nematode at planting. Fecundity of H. glycines was generally greater at the undetected and low Pi than at high Pi levels. Soybean yields were suppressed 12, 22, and 30% by low, moderate, and high H. glycines Pi, respectively. When weed competition and H. zea feeding damage effects were added, yields were suppressed 34, 40, and 57% by the three respective nematode Pi levels. Effects among the three pests on soybean yield were primarily additive.     

Dormancy of Heterodera glycines in Missouri

A 2-year study was conducted in field microplots to determine the relative importance of soybean phenology and soil temperature on induction of dormancy in Heterodera glycines in Missouri. Four near-isogenic soybean lines differing for maturity date were planted in microplots infested with a race 5 isolate of H. glycines. Soil temperature was monitored at a depth of 15 cm. Eggs of H. glycines, extracted from cysts collected monthly from each microplot, were used in hatching tests and bioassays to determine dormancy. Egg hatching and second-stage juvenile (J2) infectivity rates decreased sharply from their highest levels in midsummer (July-August) to a low level by October of each year and remained low (< 10% hatching and < 0.2 J2/cm root) until May or June of the following year. The patterns of numbers of females and eggs in the bioassays were similar. The decreases were not related to soil temperature and did not differ consistently among soybean isolines. The monophasic changes in all nematode responses with peak midsummer rates suggest that H. glycines produces one primary generation per year in central Missouri. Changes in hatching rates and the timing of minimum and maximum rates suggested that H. glycines eggs exhibit more than one type of dormancy.     

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.     

Influence of Temperature and Soybean Phenology on Dormancy Induction of Heterodera glycines

Growth room and field experiments were conducted to determine the influence of soil temperature and soybean phenology on dormancy induction of a North Carolina population of Heterodera glycines race 1. Three temperature regimes and two photoperiods to regulate plant phenology were investigated in growth rooms. H. glycines hatch was greatest from the 26 and 22 C (day and night) temperature treatment, intermediate at 22 and 18 C, and least from the decreasing regime (26 and 22 C, 22 and 18 C, and 18 and 14 C). More eggs hatched and greater nematode reproduction occurred on pod-producing soybeans than on those that remained vegetative. In the field study, hatching patterns were not different between depodded and naturally senescing soybeans nor between the different maturity groups of soybean cultivars (groups V through VIII). Egg hatch (9-16%) was greatest in August and September when mean soil temperatures were between 25 and 29 C. Hatch declined to 1% in vitro and was not detectable in the bioassay in November. Greatest nematode numbers were observed on the latest maturing cultivar (group VIII) and fewest on the cultivar which matured earliest (group V). Decreasing temperature appears to be more important than soybean phenology in dormancy induction of H. glycines.     

Effect of Rotation Crops on Heterodera glycines Population Density in a Greenhouse Screening Study

Crop rotation is a common means of reducing pathogen populations in soil. Several rotation crops have been shown to reduce soybean cyst nematode (Heterodera glycines) populations, but a comprehensive study of the optimal crops is needed. A greenhouse study was conducted to determine the effect of growth and decomposition of 46 crops on population density of H. glycines. Crops were sown in soil infested with H. glycines. Plants were maintained until 75 days after planting, when the soil was mixed, a sample of the soil removed to determine egg density, and shoots and roots chopped and mixed into the soil. After 56 days, soil samples were again taken for egg counts, and a susceptible soybean (‘Sturdy’) was planted in the soil as a bioassay to determine egg viability. Sunn hemp (Crotalaria juncea), forage pea (Pisum sativum), lab-lab bean (Lablab purpureus), Illinois bundleflower (Desman-thus illinoensis), and alfalfa (Medicago sativa) generally resulted in smaller egg population density in soil or number of cysts formed on soybean in the bioassay than the fallow control. Sunn hemp most consistently showed the lowest numbers of eggs and cysts. As a group, legumes resulted in lower egg population densities than monocots, Brassica species, and other dicots.     

Interrelationship of Heterodera glycines and Fusarium solani in Sudden Death Syndrome of Soybean

Experiments were established in field microplots to examine the association between Heterodera glycines and the blue form of Fusarium solani in sudden death syndrome of soybean (SDS). Foliar disease symptoms occurred on more plants per plot, appeared 3 to 7 days earlier, and were more severe on plants grown in plots infested with F. solani + H. glycines than on those inoculated with F. solani only. Yields were suppressed only in treatments that included the nematode. Numbers of H. glycines cysts and second-stage juveniles were significantly lower in plots infested with F. solani + H. glycines than with the nematode alone. Fusarium solani was able to infect cysts and eggs.     

Isolation of Fungi from Heterodera glycines and in vitro Bioassays for Their Antagonism to Eggs

Twenty fungi were assayed in vitro for antagonism to eggs of Heterodera glycines. Eight of the fungi were isolated from cysts or eggs of H. glycines during the current study, one was isolated from Panagrellus redivivus, and eleven were obtained from other researchers or collections. The bioassays were conducted on eggs from nematodes that had been grown monoxenically on excised root tips. Phoma chrysanthemicola, one strain of Verticillium chlamydosporium, and one strain of V. lecanii caused a decrease (P < 0.01, P < 0.05, P < 0.05, respectively) in the number of viable eggs, although no hyphae were observed colonizing live eggs. Trichoderma polysporum infected live eggs but enhanced (P < 0.05) egg survival. Acremonium bacillisporum, Chaetomium sp., Drechmeria coniospora (two strains), Epicoccum sp., Exophiala jeanselmei, Fusarium sp., Neocosmospora vasinfecta, Scytalidium fulvum, Trichoderma harzianum (two strains), V. chlamydosporium (one strain), V. lecanii (three strains), and an unidentified fungus did not measurably affect egg viability, even though hyphae of five of these fungi were seen in live eggs. The bioassay provides a useful step in the selection of a biological control agent for this major nematode pest.     

Population Development of Heterodera glycines and Soybean Yield in Soybean-maize Rotations Following Introduction into a Noninfested Field

An 11-year field study was initiated in 1979 to monitor population development of Heterodera glycines. Fifty cysts of a race 5 population were introduced into plots in a field with no history of soybean production and that had been in sod for 20 years. Soybean cultivars either susceptible or resistant to H. glycines were grown either in monoculture or rotated with maize in a 2-year rotation. During the first 5 years, resistant cultivars with the Peking source of resistance were planted. After year 5, monocuhure of Peking resistance resulted in 18 cysts/250 cm³ of soil, whereas populations resulting from the continuous cropping of susceptible soybean resulted in 45 cysts/250 cm³. Some plots in all treatments, including control plots, were contaminated at the end of year 5. Crop rotation delayed population development of H. glycines. During years 6 through 11 cv. Fayette (PI88.788 source of resistance) was planted. In year 6 numbers of cysts declined to 1/250 cm³ of soil in the treatment consisting of monocultured Fayette. At the end of year 10, cysts were below the detection level in all treatments in which Fayette was planted. Yield of susceptible soybean in monoculture with or without H. glycines infestation was lower beginning in year 6 when compared to yield of soybean grown in rotation and remained lower throughout the duration of the experiment except for 1987 (year 9). Yields of susceptible and resistant soybean were different each year except for drought years in 1980 and 1988. From 1979 to 1982 differences in yield were due to lower yield potential of resistant cultivars. Except for the drought year, yield of cv. Fayette was greater than susceptible Williams 82 during years 6 through 11.     

Development of Heterodera glycines as Affected by Fusarium solani,the Causal Agent of Sudden Death Syndrome of Soybean

The effects of the blue form of Fusarium solani, the causal agent of sudden death syndrome (SDS), on Heterodera glycines were examined in the greenhouse. Roots of soybean cv. Coker 156 were inoculated with either H. glycines alone or F. solani + H. glycines in combination. Population levels of H. glycines were reduced 47% in the presence of F. solani. Life-stage development of H. glycines increased 3% in 30 days in the presence of F. solani. Fusarium solani colonized epidermal and cortical cells adjacent to developing juveniles of H. glycines and the nematode-induced syncytia within the soybean root tissue. At 40 days after inoculation, F. solani was isolated from 37% of the cysts in soil recovered from the F. solani + H. glycines combination treatment. Fusarium solani significantly affected H. glycines population density, life-stage development, and succeeding populations.     

Seasonal Biochemical Changes in Eggs of Heterodera glycines in Missouri

Changes in the carbohydrate (glucose, trehalose, and glycogen) and total protein contents of eggs retained within Heterodera glycines cysts were monitored monthly in a field microplot experiment conducted from March 1993 to March 1995. Treatments included two near-isogenic lines of soybean cv. Clark differing for date of maturity, and one corn hybrid. The soybean lines were planted in microplots infested with H. glycines at a high average initial population density (Pi) (23,810 eggs/100 cm³ soil), and the corn was planted in microplots infested at high (24,640) and low (5,485) Pi. Soil temperatures at 15 cm depth and rainfall were monitored. Carbohydrate contents varied in the same pattern, with the highest levels measured before planting (May) and after harvest (October) in both years. Neither Pi nor soybean isoline had an effect on any measured response, but the carbohydrate contents of eggs from corn and soybean microplots differed during the overwinter (October-May) periods (P < 0.0001). Trehalose accumulation was negatively correlated with soil temperature (r = -0.78 and r = -0.84, P = 0.0001, July through November 1993 and 1994, respectively), which reflects its role as a cryoprotectant. In contrast to the pattern for carbohydrates, total protein was lowest before planting and after harvest, and highest (>20 μg/1,000 eggs) June through October. Protein content was unaffected by plant cultivar or species. Protein and carbohydrate levels in H. glycines eggs showed seasonal changes that appeared to be primarily temperature-dependent.     

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.