Identification of Endophytic Bacillus BHL3501 from Wild Soybean and Its Inhibitory Effect against Soybean Cyst Nematode (Heterodera glycines Ichinohe)

[ Objective] The paper was to identify endophytic bacillus BHL3501 from wild soybean and its inhibitory effect against soybean cyst nematode. [ Meth- od] Through morphological characteristic, physiological and biochemical characteristics and 16S rDNA analysis, a strain of endophytic bacterium BHL3501 with ne- maticidal activity isolated and screened from the root of wild soybean was identified, the effects of BHL3501 metabolite solutions with different dilution multiples on egg hatching and activity of 2rd instar juvenile of soybean cyst nematode were also studied. [ Result ] BHL3501 strain was preliminarily identified to be Bacillus sp.. The relative inhibitory rate of its original fermentation broth on egg hatching of soybean cyst nematode after 24 h was 98.3% ; the relative inhibitory rate under 10- time diluted solution treatment was 79.5%, which had significant difference with sterile water control. The corrected mortality rate of 24 instar juvenile was 93.1% after treated by original fermentation broth for 24 h, the treatments of all diluted solutions had significant difference with sterile water control. [ Conclusion ] BHL3501 metabolites had strong inhibitory effect against egg hatching of soybean cyst nematode, which also had strong toxic effect on its 2nd instar juvenile.

Effects of Abiotic Environmental Factors on Soybean Cyst Nematode

As a pest, in order to complete its life history and reproduces effectively, soybean cyst nematode （SCN） （Heterodera glycines Ichinche 1952） must adapt to various environments and conditions for long periods of evolution. The nematode is widely dispersed year after year. Controlling this pest requires understanding characters and adaptability of SCN. Effects of abiotic factors, such as temperature, soil humidity, agrotype, pH value, ions, plant exudates, agricultural chemical and cultivation systems on SCN, are reviewed in this paper. The results show that SCN is able to endure various environmental stresses, especially low temperature. Because of its special life history, cyst stage help SCN over winter, resistance of SCN to environmental stress is strong. A few studies have reported the mechanism of SCN environmental adaptability. We emphasized the importance of studying environmental adaptability of SCN, which would benefit the control of SCN by ecological means.

Comparative Analysis of Gene Expression Profiling Between Resistant and Susceptible Varieties Infected With Soybean Cyst Nematode Race 4 in Glycine max

Soybean cyst nematode （SCN） is one of the most devastating pathogen for soybean. Therefore, identiifcation of resistant germplasm resources and resistant genes is needed to improve SCN resistance for soybean. Soybean varieties Huipizhiheidou and Wuzhaiheidou were distributed in China and exhibited broad spectrums of resistance to various SCN races. In this study, these two resistant varieties, combined with standard susceptible varieties （Lee and Essex）, were utilized to identify the differentially expressed transcripts after infection with SCN race 4 between resistant and susceptible reactions by using the Affymetrix Soybean Genome GeneChip. Comparative analyses indicated that 21 common genes changed signiifcantly in the resistant group, of which 16 increased and 5 decreased. However, 12 common genes changed signiifcantly in the susceptible group, of which 9 increased and 3 decreased. Additionally, 27 genes were found in common between resistant and susceptible reactions. The 21 signiifcantly changed genes in resistant reaction were associated with disease and defense, cell structure, transcription, metabolism, and signal transduction. The fold induction of 4 from the 21 genes was conifrmed by quantitative RT-PCR （qRT-PCR） analysis. Moreover, the gene ontology （GO） enrichment analyses demonstrated the serine family amino acid metabolic process and arginine metabolic process may play important roles in SCN resistance. This study provided a new insight on the genetic basis of soybean resistance to SCN race 4, and the identiifed resistant or resistant-related genes are potentially useful for SCN-resistance breeding in soybean.

Chemical mutagenesis and soybean mutants potential for identification of novel genes conferring resistance to soybean cyst nematode

The resistance of soybean(Glycine max(L.) Merr.) to soybean cyst nematode(SCN, Heterodera glycines Ichinohe), which is a devastating pathogen in soybean production and causes a large quantity of annual yield loss worldwide, can shift during the long-term interaction and domestication. It is vital to identify more new resistance genetic sources for identification of novel genes underlying resistance to SCN for management of this pathogen. In the present study, first, two ethane methylsulfonate-mutagenesis soybean M2 populations of PI 437654, which shows a broad resistance to almost all of SCN races, and Zhonghuang 13, which is a soybean cultivar in China conferring strong resistance to lodging, were developed. Many types of morphological phenotypes such as four-and five-leaflet leaves were observed from these two soybean M2 populations. Second, 13 mutants were identified and confirmed to exhibit alteration of resistance to SCN race 4 through the forward genetic screening of 400 mutants of the PI 437654 M2 population, the rate of mutants with alteration of SCNinfection phenotype is 3.25%. Third, these identified mutants were further verified not to show any changes in the genomic sequences of the three known SCN-resistant genes, GmSHMT08, GmSNAP18 and GmSANP11, compared to the wildtype soybean; and all of them were still resistant to SCN race 3 similar to the wild-type soybean. Taken together, we can conclude that the 13 mutants identified in the present study carry the mutations of the new gene(s) which contribute(s) to the resistance to SCN race 4 in PI 437654 and can be potentially used as the genetic soybean sources to further identify the novel SCN-resistant gene(s).

Soybean cyst nematode-resistance: Gene identification and breeding strategies

Soybean cyst nematode(SCN,Heterodera glycines Ichinohe)is the most economically damaging disease of soybean worldwide,and breeding host plant resistance is the most feasible option for SCN management.In this review,we summarise the progress made so far in identifying nematode-resistance genes,the currently available sources of resistance,possible mechanisms of SCN resistance and strategies for soybean breeding.To date,only two sources of SCN resistance have been widely used,from the accessions PI 88788 and Peking,which has resulted in a shift in SCN resistance and created a narrow genetic base for SCN resistance.These resistant germplasms for SCN are classified into two types according to their copy number variation in a 31-kb genomic region:PI 88788-type resistance requires high copy numbers of a rhg1 resistance allele(rhg1-b)and Peking-type resistance requires both low copy numbers of a different rhg1 resistance allele(rhg1-a)and a resistant allele at another locus,Rhg4.Resistance related to rhg1 primarily involves impairment of vesicle trafficking through disruption of soluble NSF-attachment protein receptor(SNARE)complexes.By contrast,resistance via Rhg4 involves disturbance of folate homeostasis at SCN feeding sites due to alteration of the enzymatic activity of serine hydroxymethyltransferase(SHMT).Other potential mechanisms,including plant defences mediated by salicylic acid(SA)and jasmonic acid(JA)signalling modulation,have also been suggested for SCN resistance.Indeed,genome-wide association studies(GWAS)have identified other candidate SCN resistance genes,such as Gm SNAP11.Although gene functional analysis in a transient expression system could increase the efficiency of candidate gene identification,information on novel genes and mechanisms for SCN resistance remains limited.Any beneficial candidate genes identified might,when fully exploited,be valuable for improving the efficiency of marker-assisted breeding and dissecting the molecular mechanisms underlying SCN resistance.

A fragment of a 70-kDa Heterodera glycines heat shock protein(HgHSP70)interacts with soybean cyst nematode-resistant GmSHMT08

Soybean cyst nematode(SCN)Heterodera glycines is considered as the major constraint to soybean production.Gm SHMT08 at Rhg4 locus on chromosome 08,encoding a serine hydroxylmethyltransferase,is a major gene underlying resistance against H.glycines in Peking-type soybeans.However,the molecular mechanism underpinning this resistance is less well characterized,and whether Gm SHMT08 could interact with proteins in H.glycines remains unclear.In this study,yeast two-hybrid screening was conducted using Gm SHMT08 as a bait protein,and a fragment of a 70-kDa heat shock protein(Hg HSP70)was screened from H.glycines that exhibited interaction with Gm SHMT08.This interaction was verified by both GST pull-down and bimolecular fluorescence complementation assays.Our finding reveals Hg HSP70 could be applied as a potential candidate gene for further exploring the mechanism on Gm SHMT08-mediated resistance against SCN H.glycines.

Genetic Diversity of Soybean and the Establishment of a Core Collection Focused on Resistance to Soybean Cyst Nematode

Soybean cyst nematode （SCN; Heterodera glycines） Is one of the most Important pests affecting soybean production. The best method of control of SCN is through the development of resistant cultlvars. However, limited progress has been made in soybean breeding In China because most modern cultlvars have no resistance to SCN. The distribution and phenotype of 432 immune or highly resistant Chinese accessions were surveyed and a primary core collection was selected as a representative sample for further analyses. Using evenly distributed simple sequence repeat markers, five selection methods were applied to the primary core collection and the optimal method was chosen to establish a core collection, which consisted of 28 accessions. These encompassed 70.8% of the ailelic variation present in the overall resistant collection. The 28 accessions differed from the reference resistant accessions at the genomlc level, Indicating that Chinese resistant accessions are distinct from known resistant accessions. This applied core collection provides a rational framework for undertaking diversity surveys, using genetic variation for the investigation of complex traits and for the discovery of novel traits.

The prevalence of deleterious mutations during the domestication and improvement of soybean

Soybean(Glycine max L.)is a protein and oil crop grown worldwide.Its fitness may be reduced by deleterious mutations,whose identification and purging is desirable for crop breeding.In the published whole-genome re-sequenced data of 2214 soybean accessions,including 221 wild soybean,1132 landrace cultivars and 861 improved soybean lines,we identified 115,275 deleterious single-nucleotide polymorphisms(SNPs).Numbers of deleterious alleles increased from wild soybeans to landraces and decreased from landraces to modern improved lines.Genes in selective-sweep regions showed fewer deleterious mutations than the remaining genes.Deleterious mutations explained 4.3%-48%more phenotypic variation than randomly selected SNPs for resistance to soybean cyst nematode race 2(SCN2),soybean cyst nematode race 3(SCN3)and soybean mosaic virus race 3(SMV3).These findings illustrate how mutation load has shifted during soybean domestication,expansion and improvement and provide candidate sites for breeding out deleterious mutations in soybean by genome editing and/or conventional breeding focused on the selection of progeny with fewer deleterious alleles.

Current Research Status of Heterodera glycines Resistance and Its Implication on Soybean Breeding

Heterodera glycines （i.e., soybean cyst nematode, SCN） is the most damaging nematode pest affecting soybean crop worldwide. This nematode is managed by means of crop rotation with selected resistant sources. With increasing reports of virulent SCN populations that are able to break the resistance within commonly used sources, there is an increasing need to find new sources of resistance or to broaden the resistance background. This review summarizes recent findings about the genes controlling SCN resis- tance in soybean, and about how these genes interact to confer resistance against SCN in soybean. It also provides an update on molecular mapping and molecular markers that can be used for the mass selection and differentiation of different resistance lines and cultivars in order to expedite conventional breeding programs. In-depth knowledge of SCN parasitism proteins and soybean resistance responses to the pathogen is critical for the diversification of resistant sources through gene modification, gene stacking, or incorporation of novel sources of resistance through backcrossing or genetic engineering.

Assaying the potential of twenty-one legume plants in Medicago truncatula and M. sativa for candidate model plants for investigation the interactions with Heterodera glycines

Soybean cyst nematode Heterodera glycines is one of the most serious soil-borne pathogens in soybean production. However, the researches were limited in China due to lack of an effective pathosystem. In this study, we screened 21 legume Medicago plants in both Medicago truncatula and Medicago sativa to obtain candidate model plants for establishing a new pathosystem for legume-H. glycines interactions. The nematode infection of tested plants was assayed with Race 3 and 4 respectively, which were two dominant H. glycines inbred races in China soybean producing areas. The results showed that the model legume plant M. truncatula A17 failed to allow Race 3 of H. glycines to complete its life cycle, in contrast, it provided the Race 4 population to form several cyst nematodes, however, the female index（FI） value was approximately 1.6. Three M. sativa cultivars, including Xunlu, Aergangjin and Junren, provided either Race 3 or 4 of H. glycines to develop into mature cysts with their FI value below 5 as well. Our results demonstrated that legume plants in both M. truncatula and M. sativa were not likely to be a model plant for H. glycines because of an extreme high resistance.

Functional response of the fungus Hirsutella rhossiliensis to the nematode, Heterodera glycines

Functional response is a key index in determining the population fluctuation in predation. However, the lack of operable research system limits the studies on functional response of fungal predators. Hirsutella rhossiliensis is a dominant parasite of the soybean cyst nematode, Heterodera glycines. In a soil microcosm bioassay, we determined fungal biomass at different days within 21 days after inoculation, and parasitism rate of H. glycines by the fungus was determined. The functional response of H. rhossiliensis to H. glycines was established and found to be Holling＇s type Ill, which was influenced by mycelial densities. Meanwhile, we conducted anti-fungal analysis of metabolic fractions extracted from H. rhossiliensis to explain the potential mechanism of the intraspecific competition illustrated by functional response. The result of anti-fungal experiments indicated that the fungal predators had more complicated interaction at population level than expected, which might be regulated by self-inhibition metabolite（s）. This study was the first functional response study of fungal predators in microcosm. With the in- creasing recognition of emerging fungal threats to animal, plant, and ecosystem health, the methodologies and hypotheses proposed in this study might inspire further research in fungal ecology.