Antagonistic RALF peptides control an intergeneric hybridization barrier on Brassicaceae stigmas.

Pollen-pistil interactions establish interspecific/intergeneric pre-zygotic hybridization barriers in plants. The rejection of undesired pollen at the stigma is crucial to avoid outcrossing but can be overcome with the support of mentor pollen. The mechanisms underlying this hybridization barrier are largely unknown. Here, in Arabidopsis, we demonstrate that receptor-like kinases FERONIA/CURVY1/ANJEA/HERCULES RECEPTOR KINASE 1 and cell wall proteins LRX3/4/5 interact on papilla cell surfaces with autocrine stigmatic RALF1/22/23/33 peptide ligands (sRALFs) to establish a lock that blocks the penetration of undesired pollen tubes. Compatible pollen-derived RALF10/11/12/13/25/26/30 peptides (pRALFs) act as a key, outcompeting sRALFs and enabling pollen tube penetration. By treating Arabidopsis stigmas with synthetic pRALFs, we unlock the barrier, facilitating pollen tube penetration from distantly related Brassicaceae species and resulting in interspecific/intergeneric hybrid embryo formation. Therefore, we uncover a "lock-and-key" system governing the hybridization breadth of interspecific/intergeneric crosses in Brassicaceae. Manipulating this system holds promise for facilitating broad hybridization in crops.

The complete genome sequences of a negative single-stranded RNA virus and a double-stranded RNA virus coinfecting the entomopathogenic fungus Beauveria bassiana Vuillemin.

Beauveria bassiana Vuillemin is an entomopathogenic fungus that has been developed as a biological insecticide. B. bassiana can be infected by single or multiple mycoviruses, most of which are double-stranded RNA (dsRNA) viruses, while infections with single-stranded RNA (ssRNA) viruses, especially negative single-stranded RNA (-ssRNA) viruses, have been observed less frequently. In the present study, we sequenced and analyzed the complete genomes of two new different mycoviruses coinfecting a single B. bassiana strain: a -ssRNA virus which we have named "Beauveria bassiana negative-strand RNA virus 1" (BbNSRV1), and a dsRNA virus, which we have named "Beauveria bassiana orthocurvulavirus 1" (BbOCuV1). The genome of BbNSRV1 consists of a single segment of negative-sense, single-stranded RNA with a length of 6169 nt, containing a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) with 1949 aa (220.1 kDa). BLASTx analysis showed that the RdRp had the highest sequence similarity (59.79%) to that of Plasmopara viticola lesion associated mononegaambi virus 2, a member of the family Mymonaviridae. This is the first report of a -ssRNA mycovirus infecting B. bassiana. The genome of BbOCuV1 consists of two dsRNA segments, 2164 bp and 1765 bp in length, respectively, with dsRNA1 encoding a protein with conserved RdRp motifs and 70.75% sequence identity to the putative RdRp of the taxonomically unassigned mycovirus Fusarium graminearum virus 5 (FgV5), and the dsRNA2 encoding a putative coat protein with sequence identity 64.26% to the corresponding protein of the FgV5. Phylogenetic analysis indicated that BbOCuV1 belongs to a taxonomically unassigned group of dsRNA mycoviruses related to members of the families Curvulaviridae and Partitiviridae. Hence, it might be the member of a new family that remains to be named and formally recognized.

Hypovirulence-associated mycovirus epidemics cause pathogenicity degeneration of Beauveria bassiana in the field.

BACKGROUND: The entomogenous fungus Beauveria bassiana is used as a biological insecticide worldwide, wild B. bassiana strains with high pathogenicity in the field play an important role in controlling insect pests via not only screening of highly virulent strains but also natural infection, but the pathogenicity degeneration of wild strains severely affected aforementioned effects. Previous studies have showed that multiple factors contributed to this phenomenon. It has been extensively proved that the mycovirus infection caused hypovirulence of phytopathogenic fungi, which has been used for plant disease biocontrol. However, it remains unknown whether the mycovirus epidemics is a key factor causing hypovirulence of B. bassiana naturally in the field. METHODS: Wild strains of B. bassiana were collected from different geographic locations in Jilin Province, China, to clarify the epidemic and diversity of the mycoviruses. A mycovirus Beauveria bassiana chrysovirus 2 (BbCV2) we have previously identified was employed to clarify its impact on the pathogenicity of host fungi B. bassiana against the larvae of insect pest Ostrinia furnacalis. The serological analysis was conducted by preparing polyclonal antibody against a BbCV2 coat protein, to determine whether it can dissociate outside the host fungal cells and subsequently infect new hosts. Transcriptome analysis was used to reveal the interactions between viruses and hosts. RESULTS: We surprisingly found that the mycovirus BbCV2 was prevalent in the field as a core virus in wild B. bassiana strains, without obvious genetic differentiation, this virus possessed efficient and stable horizontal and vertical transmission capabilities. The serological results showed that the virus could not only replicate within but also dissociate outside the host cells, and the purified virions could infect B. bassiana by co-incubation. The virus infection causes B. bassiana hypovirulence. Transcriptome analysis revealed decreased expression of genes related to insect epidermis penetration, hypha growth and toxin metabolism in B. bassiana caused by mycovirus infection. CONCLUSION: Beauveria bassiana infected by hypovirulence-associated mycovirus can spread the virus to new host strains after infecting insects, and cause the virus epidemics in the field. The findings confirmed that mycovirus infection may be an important factor affecting the pathogenicity degradation of B. bassiana in the field.

Cellular immune responses of the yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae), to the entomopathogenic fungus, Beauveria bassiana (Hypocreales: Cordycipitaceae).

The yellow peach moth (YPM), Conogethes punctiferalis, is a destructive insect pest of maize in eastern China and adapts to diverse environments, especially against pathogens. In insects, innate immunity comprising both humoral and cellular defense responses, is the primary defense against invading microbial pathogens. In this study, we identified five types of circulating hemocytes from the hemolymph of YPM larvae and analyzed their alterations and functions in immune responses to the infection of Beauveria bassiana, an entomopathogenic fungus infesting many lepidopteran species. The identified hemocytes included prohemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. Significant decreases of total and differential hemocyte counts were recorded over time in larvae, after they were injected with B. bassiana conidia. Additionally, hemocyte-mediated phagocytosis and nodulation were initiated in the hemolymph of larvae from the B. bassiana conidia challenge. The introduction of DEAE-Sepharose Fast Flow beads stained with Congo red also induced a strong encapsulation response in the larval hemolymph. Our observations unravel the occurrence of phagocytosis, nodulation and encapsulation in the hemocoel of YPM larvae to fight against the fungal infection, and offer the first insight into the YPM immune system.

Genome Sequence of Rhizoctonia solani Anastomosis Group 4 Strain Rhs4ca, a Widespread Pathomycete in Field Crops.

Rhizoctonia solani is an important soil-borne fungal pathogen that causes serious diseases on many agricultural crops and vegetables. Here, we report a complete genome assembly of R. solani AG4 (assembly: 45.47 Mb; contig N50: 1.56 Mb), using a combination of Illumina paired-end and PacBio long-read sequencing data. A total of 267 noncoding RNAs and 11,592 genes were predicted, including 109 genes associated with carbohydrate-active enzymes and 2,488 genes involved in host-pathogen interactions. The complete genome of R. solani AG4 represents a valuable base for studying interactions between host plants and pathogenic fungi and to search for potential antimicrobial targets.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Single-cell RNA-sequencing atlas reveals an MDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladder cancer.

BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.

The complete genome sequence of a novel chrysovirus from the entomopathogenic fungus Beauveria bassiana Vuillemin.

Beauveria bassiana, an entomopathogenic fungus, is used for arthropod pest control worldwide. Here, we report the discovery and characterization of a novel double-stranded RNA (dsRNA) mycovirus, Beauveria bassiana chrysovirus 2 (BbCV-2), isolated from a Chinese B. bassiana strain. The genome sequence of the virus was determined by metagenomic sequencing, RT-PCR, and RACE cloning and was found to consist of four dsRNA segments that are 3441 bp, 2779 bp, 2925 bp, and 2688 bp long, respectively. Each dsRNA segment contains a single ORF. The ORF of dsRNA1 encodes a 1114-amino-acid (aa) protein (123.4 kDa) with a conserved RNA-dependent RNA polymerase (RdRp) motif, the sequence of which showed the highest identity of only 16.13% to that of Beauveria bassiana chrysovirus-1 (BbCV-1). The ORF of dsRNA2 encodes an 805-aa coat protein (CP) (84.7 kDa). The ORFs of dsRNAs 3 and 4 encodes proteins of undetermined function. The virus is a new member of the family Chrysoviridae from B. bassiana.

A novel polymycovirus with defective RNA isolated from the entomopathogenic fungus Beauveria bassiana Vuillemin.

A novel double-stranded RNA virus was isolated and identified from Beauveria bassiana Vuillemin, derived from the muscardine cadaver of an Ostrinia furnacalis larva in China. The virus contains six dsRNAs, and each viral dsRNA contains only one open reading frame (ORF). As in other polymycoviruses, dsRNA1 encodes an RNA-dependent RNA polymerase (RdRp), dsRNA3 encodes a methyltransferase (MTR), and dsRNA4 encodes a proline-alanine-serine-rich protein. A BLASTp search revealed that the viral RdRp domain showed 79.43%, 79.04%, and 59.05% sequence identity to Beauveria bassiana polymycovirus 2 and 3 (BbPmV-2, BbPmV-3) and Magnaporthe oryzae polymycovirus 1 (MoPmV-1), respectively. Phylogenetic analysis based on RdRp sequences showed that the phylogenetically closest relatives of this virus are BbPmV-2, BbPmV-3, and MoPmV-1. This virus, along with previously ill-defined polymycoviruses (BbPmV-2 and BbPmV-3), appears to belong to an as-yet-unestablished species. The findings further suggest that the virus is a new member of the genus Polymycovirus within the family Polymycoviridae, and we have named it "Beauveria bassiana polymycovirus 4" (BbPmV-4). However, the sixth dsRNA is a defective RNA with the same sequence as that of dsRNA4 except for a deletion of 312 bp from nt 185 to nt 496, but it still contains a complete ORF. To our knowledge, this is the first report of the existence of a defective RNA in a polymycovirus.

Endophytic colonization of entomopathogenic fungi increases plant disease resistance by changing the endophytic bacterial community.

Various mechanisms are involved in plant disease resistance mediated by entomopathogenic fungi; however, the role of plant endophytic microbes in disease resistance is unknown. In the present study, we showed that the disease incidence of northern corn leaf blight caused by Exserohilum turcicum (Et) on maize was reduced significantly by soil inoculation with Beauveria bassiana (Bb). Meanwhile, B. bassiana colonization and E. turcicum infection increased the diversity and abundance and diversity of endophytic bacteria and fungi, respectively, while the abundance of endophytic bacterial of the Bb + Et treatment decreased significantly compared with that of Et treatment alone. However, Bb + Et treatment increased the relative abundance of plant beneficial bacteria significantly, for example, Burkholderia and Pseudomonas. Network analyses showed that the microbiome complexity increased after soil inoculation with B. bassiana. Taken together, these results revealed the potential mechanism by which entomopathogenic fungi exert biological control of maize leaf spot disease.

Influence of genetic diversity of seventeen Beauveria bassiana isolates from different hosts on virulence by comparative genomics.

BACKGROUND: Beauveria bassiana (B. bassiana) is a famous entomopathogenic fungus that could parasitize on hundreds of insect species, which are being used as an environmentally friendly mycoinsecticide. Nevertheless, the possible effect of genetic diversity of these B. bassiana isolates from different hosts on virulence has not been explored before. In order to explore that issue, we compared the genome sequences among seventeen B. bassiana isolates from 17 different insects using whole genome re-sequencing, with B. bassiana strain ARSEF 2860 as the reference genome. RESULTS: There were a total of 10,098 missense mutated genes, 720 positively selected genes were identified in 17 strains of B. bassiana. Among these, two genes with high frequency mutations encode the toxin-producing non-ribosomal peptide synthase (NRPS) protein. Seven genes undergoing positive selection were enriched in the two-component signaling pathway that is known to regulate the fungal toxicity. In addition, the domain changes of three positively selected genes are also directly related to the virulence plasticity. Besides, the functional categorization of mutated genes showed that most of them involved in the biological functions of toxic proteins involved in. CONCLUSIONS: Based on our data, our results indicate that several mutated genes and positively selected genes may underpin virulence of B. bassiana towards hosts during infection process, which provide an insight into the potential effects of natural variation on the virulence of B. bassiana, which will be useful in screening out potential virulence factors in B. bassiana.

Overexpression of the chitinase gene CmCH1 from Coniothyrium minitans renders enhanced resistance to Sclerotinia sclerotiorum in soybean.

Pathogenic fungi represent one of the major biotic stresses for soybean production across the world. Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating fungal pathogen that is responsible for significant yield losses in soybean. In this study, the chitinase gene CmCH1, from the mycoparasitic fungus Coniothyrium minitans, which infects a range of ascomycetous sclerotia, including S. sclerotiorum and S. minor, was introduced into soybean. Transgenic plants expressing CmCH1 showed higher resistance to S. sclerotiorum infection, with significantly reduced lesion sizes in both detached stem and leaf assays, compared to the non-transformed control. Increased hydrogen peroxide content and activities of defense-responsive enzymes, such as peroxidase, superoxide dismutase, phenylalanine ammonia lyase, and polyphenoloxidase were also observed at the infection sites in the transgenic plants inoculated with S. sclerotiorum. Consistent with the role of chitinases in inducing downstream defense responses by the release of elicitors, several defense-related genes, such as GmNPR2, GmSGT-1, GmRAR1, GmPR1, GmPR3, GmPR12, GmPAL, GmAOS, GmPPO, were also significantly upregulated in the CmCH1-expressing soybean after inoculation. Collectively, our results demonstrate that overexpression of CmCH1 led to increased accumulation of H2O2 and up-regulation of defense-related genes and enzymes, and thus enhanced resistance to S. sclerotiorum infection while showing no detrimental effects on growth and development of soybean plants.

RNA sequencing analysis of Beauveria bassiana isolated from Ostrinia furnacalis identifies the pathogenic genes.

Beauveria bassiana (B. bassiana) is a broad-spectrum entomopathogenic species of fungi which is a natural enemy of Ostrinia furnacalis (O. furnacalis). Nevertheless, the precise mechanism of pathogenicity difference of B. bassiana strains on O. furnacalis has not been investigated before. In this study, two B. bassiana strains isolated from the infected O. furnacalis and exhibited different pathogenicity were chose to analyze the gene expression using RNA-sequencing analysis. To investigate the significantly differentially expressed genes (DEGs) of these two strains, total RNA was extracted and Cuffdiff software was applied to perform the significance analysis of the microarrays method. qRT-PCR was applied to verify the expression of DEGs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analyses were applied to evaluate the functions of DEGs. Data showed 72 up-regulated and 192 down-regulated genes in hyper-pathogenic strain ZK193 in comparison with hypo-pathogenic strain ZK203. Genes involved in fungal growth, sporulation and toxin production were up-regulated in hyper-pathogenic strain ZK193. GO enrichment analysis of DEGS showed that the most observably enriched biological processes of regulated genes were the single-organism process, the metabolic process, the cellular process and biological regulation. KEGG enrichment pathway demonstrated that the almost enriched groups were metabolic pathways, such as lipid metabolism, transport and catabolism, amino acid metabolism, and carbohydrate metabolism. In conclusion, these results will help us to further understand the reason why different B. bassiana strains exhibit different pathogenicity on the same host, even under the same conditions. In addition, transcriptome data will provide a theoretical basis for exploring latent virulence factors in the future.

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase.

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71-82.73% reduction of lesion length in a detached stem assay (T3 and T4 generations) and 76.67-82.0% reduction of lesion area in a detached leaf assay (T4 generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T3 and T4 transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H2O2) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H2O2 at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H2O2 levels, and eliciting defense responses mediated by multiple signaling pathways.

Increased multiple virus resistance in transgenic soybean overexpressing the double-strand RNA-specific ribonuclease gene PAC1.

Viruses constitute a major constraint to soybean production worldwide and are responsible for significant yield losses every year. Although varying degrees of resistance to specific viral strains has been identified in some soybean genetic sources, the high rate of mutation in viral genomes and mixed infections of different viruses or strains under field conditions usually hinder the effective control of viral diseases. In the present study, we generated transgenic soybean lines constitutively expressing the double-strand RNA specific ribonuclease gene PAC1 from Schizosaccharomyces pombe to evaluate their resistance responses to multiple soybean-infecting virus strains and isolates. Resistance evaluation over three consecutive years showed that the transgenic lines displayed significantly lower levels of disease severity in field conditions when challenged with soybean mosaic virus (SMV) SC3, a prevalent SMV strain in soybean-growing regions of China, compared to the non-transformed (NT) plants. After inoculation with four additional SMV strains (SC7, SC15, SC18, and SMV-R), and three isolates of bean common mosaic virus (BCMV), watermelon mosaic virus (WMV), and bean pod mottle virus (BPMV), the transgenic plants exhibited less severe symptoms and enhanced resistance to virus infections relative to NT plants. Consistent with these results, the accumulation of each virus isolate was significantly inhibited in transgenic plants as confirmed by quantitative real-time PCR and double antibody sandwich enzyme-linked immunosorbent assays. Collectively, our results showed that overexpression of PAC1 can increase multiple virus resistance in transgenic soybean, and thus provide an efficient control strategy against RNA viruses such as SMV, BCMV, WMV, and BPMV.

Over-expression of the Pseudomonas syringae harpin-encoding gene hrpZm confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean.

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T2-T4 generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.

RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean.

KEY MESSAGE: Robust RNAi-mediated resistance to multiple Potyvirus strains and isolates, but not to Secovirus BPMV, was conferred by expressing a short SMV P3 hairpin in soybean plants. Engineering resistance to multiple Potyvirus strains is of great interest because of a wide variability of the virus strains, and mixed infections of multiple viruses or strains commonly associated with field grown soybean. In this study, RNAi-mediated silencing of the soybean mosaic virus (SMV) P3 cistron, which is reported to participate in virus movements and pathogenesis and to be the putative determinant of SMV virulence, was used to induce resistance to multiple Potyvirus strains and isolates in soybean. A 302 bp inverted repeat (IR) of the P3 cistron, isolated from the SMV strain SC3, was introduced into soybean. The transgenic lines exhibited stable and enhanced resistance to SMV SC3 under field conditions over 3 consecutive years. The transgenic lines also showed significantly enhanced resistance to four other SMV strains (SC7, SC15, SC18, and SMV-R, a novel recombinant found in China), the soybean-infecting bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV). Nevertheless, no significant differences were found between transgenic plants and their non-transformed (NT) counterparts in terms of resistance to bean pod mottle virus (BPMV, Secoviridae). Consistent with the results of resistance evaluations, the expression of the respective viral CP cistrons and virus accumulation were significantly lower in seven Potyvirus strains and isolates than in the NT plants, but not in BCMV-inoculated transgenic lines. The results demonstrate the effectiveness of engineering resistance to multiple Potyvirus strains and isolates via RNAi-mediated SMV P3 cistron silencing, and thus provide an effective control strategy against Potyvirus infections in soybean and other crops.

Introgression between divergent corn borer species in a region of sympatry: Implications on the evolution and adaptation of pest arthropods.

The Asian corn borer, Ostrinia furnacalis, and European corn borer, O. nubilalis (Lepidoptera: Crambidae), cause damage to cultivated maize in spatially distinct geographies and have evolved divergent hydrocarbons as the basis of sexual communication. The Yili area of Xinjiang Uyghur Autonomous Region in China represents the only known region where O. furnacalis has invaded a native O. nubilalis range, and these two corn borer species have made secondary contact. Genetic differentiation was estimated between Ostrinia larvae collected from maize plants at 11 locations in Xinjiang and genotyped using high-throughput SNP and microsatellite markers. Maternal lineages were assessed by direct sequencing of mitochondrial cytochrome c oxidase subunit I and II haplotypes, and a high degree of genotypic diversity was demonstrated between lineages based on SNP genotypes. Furthermore, historical introgression was predicted among SNP genotypes only at sympatric locations in the Yili area, whereas in Xinjiang populations only O. furnacalis haplotypes were detected and no analogous introgressed genotypes were predicted. Our detection of putative hybrids and historical evidence of introgression defines Yili area as a hybrid zone between the species in normal ecological interactions and furthermore, might indicate that adaptive traits could spread even between seemingly divergent species through horizontal transmission. Results of this study indicate there may be a continuum in the degree of reproductive isolation between Ostrinia species and that the elegance of distinct and complete speciation based on modifications to the pheromone communication might need to be reconsidered.

Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA.

Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.

Jilinibacillus soli gen. nov., sp. nov., a novel member of the family Bacillaceae.

A Gram-positive, aerobic, rod-shaped, motile, endospore-forming bacterium, designated strain A12(T), was isolated from a saline and alkali soil samples in Baicheng City, western of Jilin Province, China. Growth occurred in 15-45 °C (optimum, 30 °C) and at pH 7.0-11.5 (optimum, pH 9.0) and in the presence of 0-10 % (w/v) NaCl [optimum, 1-3 % (w/v) NaCl]. Meso-DAP was present in the peptidoglycan. The predominant menaquinone was MK-7. The major polar lipid profile was phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl inositol-methyl and phosphotidylinositol dimannosid. The major fatty acid (>10 % of total fatty acids) was anteiso-C15:0. DNA G + C content was 36.2 mol %. The level of 16S rRNA gene sequence similarity between strain A12(T) and other recognized species of the family was below 95.6 %. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the strain A12(T) fell with the family Bacillaceae and formed a distinct taxon. Based on physiological, chemotaxonomic and phylogenetic analyses, strain A12(T) was considered to represent a novel species of a new genus, for which the name Jilinibacillus soli gen. nov., sp. nov. was proposed. The type strain of Jilinibacillus soli was A12(T) (=GIMN1.014(T) = CCTCC M2011164(T) = KCTC 33417(T)).

Mating Disruption or Mass Trapping, Compared With Chemical Insecticides, for Suppression of Chilo suppressalis (Lepidoptera: Crambidae) in Northeastern China.

Asiatic rice borer, Chilo suppressalis (Walker), larvae cause extensive crop losses worldwide. Because chemical control is problematic, and sex pheromone applications are a valuable management tactic in China, judicious timing of a minimal density of pheromone dispensers is important in developing a cost-effective C. suppressalis IPM program. During June-October in 2011, 20, 30, 40, and 50 dispensers per hectare for mass trapping, and 200, 300, 400, and 500 dispensers per hectare for mating disruption were placed in northeastern China rice fields. Based on those results, only the two highest mass trapping densities were used in 2012-2013. The 40, 50, and 500 dispenser densities reduced egg masses to <2.0 per 100 tillers, compared with >9.5 in the insecticide-treated plots in 2011-2013. The reduced oviposition resulted in >85% reduction of larval damage, which was comparable with the currently used insecticides, dimethoate and deltamethrin (0.35 kg/ha), which gave no egg reduction, but ≍80 and 89% reduction in larval damage. The 40 and 500 densities are recommended to Chinese rice farmers for mass trapping and mating disruption programs, respectively.