Suppression of Heterodera glycines in soils from fields with long-term soybean monoculture

Experiments were conducted in a greenhouse to evaluate soils for their degree of suppressiveness against the soybean cyst nematode, Heterodera glycines. The soils were collected in 2003 and 2004 from two fields with long-term soybean monoculture and one field with corn/soybean annual rotation. The soils were autoclaved, treated with formalin, or untreated. Both formalin and autoclave treatments removed suppressive factors and increased the nematode egg population density as compared with untreated soil. The mixture of 10% untreated soil with 90% treated soil resulted in similar nematode population densities as in the untreated soils collected from the two monoculture fields, indicating the suppressive factors were biological and can be transferred. The soils from the monoculture fields appeared to be more suppressive than the soil from the corn/soybean rotation. Hirsutella rhossiliensis was observed in all three soils, and parasitised a large percentage of second-stage juveniles of H. glycines in the monoculture soils in 2004. The fungus may be a major factor for the suppression of the nematode population, but in this study other biological factors cannot be ruled out.     

大豆胞囊线虫的休眠

以前研究发现,辽宁地区大豆生长期间及收获期土壤中胞囊孵出的二龄幼虫量很少,推测线虫卵的休眠与大豆生长时期或季节相关。为明确该地区大豆胞囊线虫的休眠特点,2002-2003年采用田间随机多点取样、室内分离及模拟自然条件孵化等方法对大豆胞囊线虫的休眠进行深入研究。结果表明:在生长季节,感病品种辽豆10根围土壤中的白色雌虫、卵囊及褐色的胞囊均可孵出二龄幼虫,且孵化持续时间较长,第21d仍有幼虫孵出,白色雌虫及卵囊内的卵孵化率高于褐色胞囊;不同作物对其根围土壤中胞囊内卵的孵化影响不大,寄主作物大豆、非寄主作物玉米根围及休闲地土壤中的胞囊在条件适宜均可孵出二龄幼虫;季节对胞囊内卵的孵化有较大的影响,出苗期孵化率最高,收获期最低,2周时平均1个胞囊孵出幼虫分别为83.8和9.7条;胞囊皮对线虫卵的孵化有显著的影响。表明沈阳地区大豆胞囊线虫在正常和逆境条件下均有部分卵表现休眠。     

黑龙江大豆胞囊线虫胞囊可培养细菌多样性

【目的】了解黑龙江省大豆田大豆胞囊线虫胞囊可培养细菌的多样性。【方法】运用稀释平板法和16SrDNA基因序列的系统发育分析对胞囊可培养细菌多样性进行研究。【结果】用NA培养基从胞囊上分离90株具有不同菌落形态的细菌。16S rDNA序列分析结果表明:90株菌株分属于7个属22个种。46株属于变形菌门γ亚群(Gammaproteobacteria),32株属于厚壁菌门(Firmicutes),10株属于变形菌门β亚群(Betaproteobacteria),2株属于变形菌门ɑ亚群(Alphaproteobacteria)。假单胞菌属(Pseudomonas)和芽孢杆菌属(Bacillus)为优势菌属。【结果】黑龙江省大豆胞囊线虫胞囊中存在丰富的细菌物种多样性,这些细菌对大豆胞囊线虫可能具有一定的生理生态作用。     

Nutritional Requirements of the Nematophagous Fungus Hirsutella rhossiliensis

Six natural media were examined for growth and sporulation of six isolates of the nematophagous fungus Hirsutella rhossiliensis , using solid and/or liquid culture. Twenty carbohydrates, 19 nitrogen (N) compounds, and nine vitamins were also tested for their effects on growth, sporulation, and spore germination of a further three isolates (ATCC46487, OWVT-1 and JA16-1). Variations in nutritional requirements existed among the fungal isolates. In general, V-8 juice agar (VA), cornmeal agar and potato dextrose agar were good media for growth, and malt extract agar, VA and yeast dextrose agar were good for sporulation of all six isolates. Glycogen was the best and sucrose, inulin, D- ( + ) - trehalose and soluble starch were also good carbon (C) sources for growth and spore germination of the three isolates ATCC46487, OWVT-1 and JA16-1 in both liquid and solid culture. None of the isolates utilized D- ( + )xylose as a C source. L- sorbose, D- ribose, citric acid and D- fructose were poor for growth of all isolates. The best C source for sporulation was D- ( + )-trehalose for ATCC46487, D- sorbitol for OWVT-1 and D- ( + )-cellobiose for JA16-1. Casein was the best N source for growth of ATCC46487 and OWVT-1, while peptone was best for JA16-1. L- asparagine, L- proline, and peptone were also good for growth of all three isolates. L - cystine was not utilized by H. rhossiliensis and DL- methionine inhibited growth of all isolates. Spore germination of all isolates was well supported by most N compounds examined but was inhibited by L- cystine. No significant difference in sporulation of ATCC46487 was observed among the N sources. DL- threonine was the best N source for spore production by OWVT-1 and L- phenylalanine was best for JA16-1. Vitamins generally enhanced fungal growth and sporulation, with thiamine having the greatest influence. Excluding some vitamins individually from the medium containing all other test vitamins sometimes increased growth and/or sporulation of certain isolates.     

Effect of starter nitrogen on soybeans under Heterodera glycines infestation

The soybean cyst nematode (SCN, Heterodera glycines) continues to spread in soils receiving many forms of soil nutrient amendments, including small amounts of N although applied with no particular reference to SCN and/or other stress inducing factors. The objective of this 2-year study was to test if standard at-planting application of 112.09 kg⁻¹ ha⁻¹ of 06–15–40 (N–P–K) containing urea (+N), or 0–15–40 (N–P–K) (−N) or a no fertiliser check (0) affect yield of SCN resistant (‘Jack’) and two susceptible (‘CX 252’ and ‘Kenwood-94’) soybean cultivars under high and low SCN and other soil abiotic stresses. Yield was higher under low compared to high stress during both years, with Jack producing the highest yield and Kenwood-94 the lowest. While not statistically significant, +N tended to decrease nodulation and increase yield under low stress in all cultivars. Nodulation was correlated positively with yield and negatively with daily nematode population density (DNPD), suggesting potential problems for N supply under nematode infestation. The susceptible cultivars accumulated significantly more leaf Ca and Mg than Jack, which was more pronounced in the presence of high rather than low stress, confirming known responses to stress. At high stress, only CX 252 showed a yield increase in response to N treatment, suggesting possible physiological adaptation mechanisms. Future research that account for the relationships amongst DNPD, frequency of sampling, and host response to better understand confounding factors and to conclusively prove or disprove any benefits from supplementary N under SCN infestation are discussed.     

Comparative Electrophoretic Analyses of Soluble Proteins from Heterodera glycines Races 1-4 and Three Other Heterodera Species

Modified polyacrylamide gel and SDS-polyacrylamide gel electrophoretic systems using a low molarity tris-HCl buffer and equal pH of homogenizing buffer and stacking gel provided improved stacking for separation of soluble proteins from Heterodera schachtii, H. trifolii, H. lespedezae, and H. glycines races 1, 2, 3, and 4, compared with previous studies with cyst nematodes, The four Heterodera species were easily distinguished using the polyacrylamide gel system, but H. trifolii and H. lespedezae had similar protein patterns. H. glycines races were not separable by that system. The SDS-polyacrylamide gel system produced different protein patterns for all four Heterodera species although H. trifolii and H. lespedezae differed by only a single band, suggesting that these two may be subspecifically related. A protein band unique to H. glycines races 3 and 4 was not detected in SDS-polyacrylamide gel profiles from races 1 and 2. Molecular weight determinations were 55,000 for distinctive proteins in profiles of H. trifolii and 75,000 for H. glycines races 3 and 4.     

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.     

Parasitism of the Nematode Heterodera glycines by the Fungus Hirsutella rhossiliensis as Influenced by Crop Sequence

The effect of crop sequence on parasitism of second-stage juveniles (J2) of Heterodera glycines by Hirsutella rhossiliensis was investigated. Data were collected from plots of a long-term crop rotation experiment established in 1982. Crop sequences included (i) continuous monoculture of corn and soybean; (ii) annual rotation of the two crops; and (iii) 1, 2, 3, 4, or 5 years of each crop following 5 years of the other crop. The nematode J2 density and percentage of J2 parasitized by the fungus were determined at planting, midseason, and end of season in 1997 and 1998. A significant effect of the crop sequence on parasitism of J2 was observed at midseason in both years and at end of season in 1998. In plots of first-year soybean following 5 years of corn, fungal parasitism increased from an undetectable level at planting to 2% and 4% of J2 parasitized by ends of season in 1997 and 1998, respectively. Fungal parasitism was similar in plots of second-through-fifth-year soybean after 5 years of corn and in plots of soybean monoculture. Parasitism of J2 in the soybean plots in annual rotation with corn increased from undetectable and 2% at planting to 6% and 23% at midseason in 1997 and 1998, respectively. The effect of crop sequence on the fungal parasitism of J2 may be attributed to a density-dependent relationship between the parasite and its host. Season also affected the fungal parasitism; percentage of J2 parasitized by the fungus was the highest at midseason and the lowest at planting.     

Molecular characterization of resistance to Heterodera glycines in soybean PI 438489B

Soybean (Glycine max L. Merr.) plant introduction (PI) 438489B is a newly found germplasm source that has resistance to multiple soybean cyst nematode (Heterodera glycines Ichinohe, SCN) races. We studied the inheritance of resistance to SCN races 1, 2, 3, 5 and 14 in PI 438489B using F₂ and F_2:3 families, which were generated by crossing to the susceptible cultivar ’Hamilton.’ The objectives of this study were to investigate the inheritance for resistance to SCN races in PI 438489B, to find molecular markers associated with resistances, and to study the allelic relationships among resistance loci for different SCN races. The results showed that the responses to SCN races were approximately normally distributed with large environmental effects, and were also highly correlated, which implied that genes giving resistance to different races were similar. The narrow-sense heritabilities of resistance to all five SCN races ranged from 0.55 to 0.88. Fifty one restriction fragment length polymorphism (RFLP) markers and 64 simple sequence repeat (SSR) markers were found to be polymorphic in the F₂ population. Quantitative trait loci (QTLs) associated with resistance to SCN races were anchored on soybean linkage groups (LGs) A1, A2, B1, B2, C1, C2, D1a, E and G. These QTLs explained 47.3%, 45.8%, 51.5%, 34.5% and 37.2% of the total phenotypic variances, respectively, for each race we investigated. Some QTLs for different races encompassed the same region of flanking markers; therefore, QTLs for multiple races may be linked or pleiotropic effects may be involved. Some loci provided resistance in a race-specific manner. Resistance to SCN race 14 had a different pattern compared to other races. Our results indicated that resistance to race 14 did not include loci on LGs A2 and G. These flanking markers associated with QTLs could be used to select for resistance to multiple SCN races in soybean breeding programs. Received: 25 March 2000 / Accepted: 4 August 2000     

Heterodera glycines in Indiana: II. Morphology of Geographical Isolates

Although much morphometric overlap occurs among five geographical isolates of Heterodera glycines in Indiana, significant differences in means exist among the isolates for various comparisons of second-stage juveniles. By using combinations of means, most of the isolates can be distinguished from the rest: e.g., the Vanderburgh County isolate (southern Indiana) has the longest esophagus, tail, and tail terminus; the Vigo County isolate (also from the south) has the shortest esophagus; the White County isolate (northern Indiana) has the shortest tail and tail terminus and the greatest total length; the Benton County isolate (north) is the shortest. Morphological similarities and differences do not appear to be coordinated with reproductive behavioral patterns we observed in the northern versus the southern isolates.     

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.     

Identification of a new major QTL associated with resistance to soybean cyst nematode (Heterodera glycines)

Resistance of soybean [Glycine max (L.) Merr.] to cyst nematode (SCN) (Heterodera glycines Ichinohe), one of the most destructive pathogens affecting soybean, involves a complex genetic system. The identification of QTLs associated with SCN resistance may contribute to the understanding of such system. The objective of this work was to identify and map QTLs for resistance to SCN Race 14 with the aid of molecular markers. BC₃F_2:3 and F_2:3 populations, both derived from an original cross between resistant cv. Hartwig and the susceptible line BR-92–31983 were screened for resistance to SCN Race 14. Four microsatellite (Satt082, Sat_001, Satt574 and Satt301) and four RAPD markers (OPAA-11₇₉₅, OPAE-08₈₃₇, OPR-07₅₄₈ and OPY-07₂₀₃₀) were identified in the BC₃F_2:3 population using the bulked segregant analysis (BSA) technique. These markers were amplified in 183 F_2:3 families and mapped to a locus that accounts for more than 40% of the resistance to SCN Race 14. Selection efficiency based on these markers was similar to that obtained with the conventional method. In the case of the microsalellite markers, which identify homozygous resistant genotypes, the efficiency was even higher. This new QTL has been mapped to the soybean linkage group D2 and, in conjunction with other QTLs already identified for SCN resistance, will certainly contribute to our understanding of the genetic basis of resistance of this important disease in soybean. Received: 12 October 1999 / Accepted: 14 April 2000     

Screening for Virulent Isolates of Entomopathogenic Fungi Against the Desert Locust, Schistocerca gregaria Forskal

The pathogenicity of 159 isolates of Metarhizium and Beauveria spp. was screened using Schistocerca gregaria adults. The median lethal time MLT , the average survival time and its standard deviation SD were used to describe the rate of kill from the cumulative observed response curve over time, following the application of conidia in bioassays. All screens included a standard strain of Metarhizium sp. from a single spore of IMI 330189ss , which gave an average MLT value of 4.4 days SD 0.4 in 46 assays. Approximately 50 isolates, all belonging to the genus Metarhizium, showed virulence that was comparable with this strain. Two previously unreported preliminary tests were carried out. The first test assessed the response to the standard isolate by an established laboratory stock of desert locusts compared with a recently introduced locust stock from Niger. There were insignificant differences in MLT and LD50 on day 5 between the different locust strains. The second test assessed the effects of inoculation at different times after the final ecdysis. This similarly had little effect, although applications before day 10 appeared to minimize the risk of control mortality. A computer program was developed to store bioassay results in a standardized format and to produce quick estimates of these statistics.     

Heterodera glycines in Indiana: I. Reproduction of Geographical Isolates on Soybean Differentials

Four of five geographical isolates of Heterodera glycines from Indiana classified as Race 3 using standard differentials showed many differences when classified using another group of differentials comprised of five soybean breeding lines and cultivars. Two isolates from northern Indiana produced cysts on more of the differentials tested than did three isolates from southern Indiana, suggesting that potential resistant lines should be tested on a range of H. glycines populations originating from the areas for which cultivars are being developed.     

Detection of the nematophagous fungus Hirsutella rhossiliensis in soil by real-time PCR and parasitism bioassay

Hirsutella rhossiliensis, a nematophagous fungus, has shown potential in biocontrol of plant-parasitic nematodes. Monitoring the population dynamics of a biocontrol agent in soil requires comprehensive techniques and is essential to understand how it works. Bioassay based on the fungal parasitism on the juveniles of soybean cyst nematode, Heterodera glycines, can be used to evaluate the activity of the fungus but fails to quantify fungal biomass in soil. A real-time polymerase chain reaction (PCR) assay was developed to quantify the fungal population density in soil. The assay detected as little as 100 fg of fungal genomic DNA and 40 conidia g⁻¹ soil, respectively. The parasitism bioassay and the real-time PCR assay were carried out to investigate the presence, abundance and activity of H. rhossiliensis in soil after application of different inoculum levels. Both of the percentage of assay nematodes parasitized by H. rhossiliensis based on the parasitism bioassay and the DNA yield of the fungus quantified by real-time PCR increased significantly with the increase of the inoculum levels. The DNA yield of the fungus was positively correlated with the percentage of assay nematodes parasitized by H. rhossiliensis. The combination of the two is useful for monitoring fungal biomass and activity in soil.     

A Technique for Determining Live Second-stage Juveniles of Heterodera glycines

We developed a quick and reliable technique to distinguish live and immobile (presumed dead) Heterodera glycines second-stage juveniles (J2) following their treatment with microbial culture filtrates. About 50 J2 in 1 ml of culture filtrate or water were placed in wells of a 24-well tissue-culture plate. After incubation, the nematodes in the wells were observed with the aid of an inverted microscope. The J2 lay straight and their viability could not be determined by direct microscopic observation. With the addition of one or two drops (50 to 100 µl) of 1 N NaOH into the well, the live nematodes changed their body shape from straight to curled or hook-shaped after about 30 seconds. The nematodes that responded to NaOH by changing their body shape within 3 minutes were considered a live, while those nematodes that failed to respond within 3 minutes and were immobile were presumed to be dead. The technique is simple, fast, and useful for the examination of a large number of samples in which one wants to determine the effects of microbial cultural filtrates on nematodes, or in similar tests.     

Complete Characterization of the Race Scheme for Heterodera glycines

One hundred thirty-eight isolates of Heterodera glycines from nine states in the United States, People''s Republic of China, and Indonesia were tested on the four standard soybean race differentials. A total of 12 variants were found, including the five races described previously. The seven variants that did not correspond to one of the described races and reports from other areas of populations that could not be classified are evidence that the present race classification system needs to be fully characterized. Eleven additional races are described; this expands the total to 16 races, the maximum possible using the four prescribed differentials and a + or - rating for each. The seven new races are designated as 6, 9, 10, 13, 14, 15, and 16. This complete characterization of the race scheme will allow for immediate communication of the discovery of the remaining four races plus the identification of previous undescribed races.     

A Revised Classification Scheme for Genetically Diverse Populations of Heterodera glycines

Heterodera glycines, the soybean cyst nematode, is a major yield-limiting pathogen in most soybean production areas worldwide. Field populations of H. glycines exhibit diversity in their ability to develop on resistant soybean cultivars. Since 1970, this diversity has been characterized by a bioassay used to assign a race classification to a population. The value of the race scheme is reflected in the number and quality of resistant soybean cultivars that have been developed and released by soybean breeders and nematologists working in concert. However, the race scheme also has been misapplied as a means of studying H. glycines genotypes, in part due to the use of the term "race." For fungal and bacterial pathogen species, "race" can theoretically be applied to individuals of a population, thus allowing inference of individual genotypes. Application of a race designation to an individual egg or second-stage juvenile (J2) of H. glycines is not possible because a single J2 cannot be tested on multiple hosts. For other nematode species, "race" is defined by host ranges involving different plant species, whereas the H. glycines race test involves a set of lines of the same plant species. Nonetheless, because H. glycines populations vary in genetic diversity, and this variation has implications for management strategies, a mechanism is needed for documenting and discussing population differences. The HG Type scheme described herein avoids the implication of genetic uniformity or predictability in contrast to the way the race scheme has been used.     

Coating Soybean Seed with Oxamyl for Control of Heterodera glycines

Oxamyl coated on soybean (Glycine max (L.) Merr. cv. Elgin) seeds in solutions of 20, 40, 80, and 160 mg/ml had no serious deleterious effects on seedling emergence and growth when planted in sterile soil. Seedling emergence on day 3 was less than that of the uncoated control, but by day 7 emergence was equal to, or greater than, the control. Shoot and root growth from seed coated with oxamyl in 40 and 80 mg/ml solutions was greater than that of the control. In soil infested with soybean cyst nematode, Heterodera glycines, shoot weight of soybean plants from seeds coated with oxamyl in 80 mg/ml solution was 11 and 9% greater at weeks 3 and 7, respectively, than from uncoated seeds. Numbers of juveniles (J3 and J4) and adults of H. glycines observed on the roots of plants from oxamyl-coated seeds were 83, 42, and 49% less at weeks 3, 5, and 7, respectively, than numbers on the roots of the untreated control. Numbers of J2 extracted from the roots of plants from oxamyl-coated seeds were 75% less at weeks 5 and 7 than those extracted from roots of uncoated seeds. The numbers of J2 extracted from the soil planted to oxamyl-coated seeds were 51 and 33% less at weeks 5 and 7, respectively, than from soil planted to uncoated seed.     

Influence of Selected Plant Species on Hatching of Eggs and Development of Juveniles of Heterodera glycines

The influence of selected plant species on egg hatch and subsequent development of Heterodera glycines race 3 was investigated. Plants tested included four soybean cultivars, red clover, alfalfa, hairy vetch, field corn, sweet corn, cabbage, tobacco, cotton, and wheat. Soybean stimulated egg hatching more than any of the other plant species, with H. glycines-resistant cultivars being more stimulating than susceptible ones. Hairy vetch also increased hatch. Roots of cabbage, red clover, alfalfa, and hairy vetch were readily penetrated by juveniles of H. glycines. Maturation to adult occurred only on soybean and hairy vetch.