Analysis of endophytic bacterial diversity in seeds of different genotypes of cotton and the suppression of Verticillium wilt pathogen infection by a synthetic microbial community.

BACKGROUND: In agricultural production, fungal diseases significantly impact the yield and quality of cotton (Gossypium spp.) with Verticillium wilt posing a particularly severe threat. RESULTS: This study is focused on investigating the effectiveness of endophytic microbial communities present in the seeds of disease-resistant cotton genotypes in the control of cotton Verticillium wilt. The technique of 16S ribosomal RNA (16S rRNA) amplicon sequencing identified a significant enrichment of the Bacillus genus in the resistant genotype Xinluzao 78, which differed from the endophytic bacterial community structure in the susceptible genotype Xinluzao 63. Specific enriched strains were isolated and screened from the seeds of Xinluzao 78 to further explore the biological functions of seed endophytes. A synthetic microbial community (SynCom) was constructed using the broken-rod model, and seeds of the susceptible genotype Xinluzao 63 in this community that had been soaked with the SynCom were found to significantly control the occurrence of Verticillium wilt and regulate the growth of cotton plants. Antibiotic screening techniques were used to preliminarily identify the colonization of strains in the community. These techniques revealed that the strains can colonize plant tissues and occupy ecological niches in cotton tissues through a priority effect, which prevents infection by pathogens. CONCLUSION: This study highlights the key role of seed endophytes in driving plant disease defense and provides a theoretical basis for the future application of SynComs in agriculture.

The effects and interrelationships of intercropping on Cotton Verticillium wilt and soil microbial communities.

BACKGROUND: Cotton Verticillium wilt, causing by Verticillium dahliae, has seriously affected the yield and quality of cotton. The incidence of Verticillium wilt in cotton fields has been on the rise for many years, especially after straw has been returned to the fields. Intercropping can reduce the incidence of soil borne diseases and is often used to control crop diseases, but the relationship between the effects of intercropping on microbial communities and the occurrence of plant diseases is unclear. This research explored the relationship between soil microbial community structure and Cotton Verticillium wilt in interplanting of cotton-onion, cotton-garlic, cotton-wheat and cotton monocultures. Amplicon sequencing applied to the profile of bacterial and fungal communities. RESULTS: The results showed that the disease index of Cotton Verticillium wilt was significantly reduced after intercropping with cotton-garlic and cotton-onion. Chao1 and Sobs indices were not significantly different in the rhizosphere soil and pre-plant soils of the four planting patterns, but the pre-plant fungal shannon index was significantly lower in the cotton-onion intercropping plot than in the other three plots. PCoA analysis showed that the soil microbial communities changed to a certain extent after intercropping, with large differences in the microbial communities under different cropping patterns. The abundance of Chaetomium was highest in the cotton-garlic intercropping before planting; the abundance of Penicillium was significantly higher in the cotton-wheat intercropping than in the other three systems. CONCLUSION: Cotton-garlic and cotton-onion interplanting can control Cotton Verticillium wilt by affecting the soil microbial community. Fungi of the genera Chaetomium and Penicillium may be associated with plant disease resistance.

Host-induced gene silencing of the acetolactate synthases VdILV2 and VdILV6 confers resistance to Verticillium wilt in cotton (Gossypium hirsutum L.).

Cotton Verticillium wilt caused by Verticillium dahliae (V. dahliae) is one of the most destructive fungal diseases and is difficult to control. However, resistant germplasm resources are scarce in cotton. Many studies have shown that host-induced gene silencing (HIGS) is a practical and effective technology in crop disease prevention by silencing virulence genes of pathogens. Acetolactate synthase (ALS) contains a catalytic subunit ILV2 and a regulatory subunit ILV6, which catalyzes the first common step reaction in branched-chain amino acid (BCAA) biosynthesis. We identified two acetolactate synthases, VdILV2 and VdILV6, which are homologs of ILV2 and ILV6, respectively, in Magnaporthe oryzae. To characterize the function of VdILV2 and VdILV6 in V. dahliae, we suppressed their expression in the strong pathogenic isolate Vd991 by using HIGS technology. VdILV2- or VdILV6-silenced V. dahliae had a dramatic reduction in pathogenicity. The results indicated that VdILV2 and VdILV6 are involved in the pathogenicity of V. dahliae. HIGS of VdILV2 or VdILV6 provides a novel fungicide target and an effective control to resist Verticillium wilt caused by V. dahliae.

Cotton polyamine oxidase is required for spermine and camalexin signalling in the defence response to Verticillium dahliae.

Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes vascular wilt disease in many economically important crops worldwide. A polyamine oxidase (PAO) gene was identified and cloned by screening suppression subtractive hybridisation and cDNA libraries of cotton genotypes tolerant to Verticillium wilt and was induced early and strongly by inoculation with V. dahliae and application of plant hormone. Recombinant cotton polyamine oxidase (GhPAO) was found to catalyse the conversion of spermine (Spm) to spermidine (Spd) in vitro. Constitutive expression of GhPAO in Arabidopsis thaliana produced improved resistance to V. dahliae and maintained putrescine, Spd and Spm at high levels. Hydrogen peroxide (H2 O2 ), salicylic acid and camalexin (a phytoalexin) levels were distinctly increased in GhPAO-overexpressing Arabidopsis plants during V. dahliae infection when compared with wild-type plants, and Spm and camalexin efficiently inhibited growth of V. dahliae in vitro. Spermine promoted the accumulation of camalexin by inducing the expression of mitogen-activated protein kinases and cytochrome P450 proteins in Arabidopsis and cotton plants. The three polyamines all showed higher accumulation in tolerant cotton cultivars than in susceptible cotton cultivars after inoculation with V. dahliae. GhPAO silencing in cotton significantly reduced the Spd level and increased the Spm level, leading to enhanced susceptibility to infection by V. dahliae, and the levels of H2 O2 and camalexin were distinctly lower in GhPAO-silenced cotton plants after V. dahliae infection. Together, these results suggest that GhPAO contributes to resistance of the plant against V. dahliae through the mediation of Spm and camalexin signalling.

Complete genome sequence data of Pseudomonas nitroreducens L4, an endophyte isolated from cotton plants.

Pseudomonas nitroreducens L4 was isolated from the interior of cotton plants, which showed strong biocontrol activity against Verticillium dahlia and other fungal pathogens. To elucidate the biocontrol mechanism, the genome sequence of L4 was sequenced using the Illumina and Nanopore sequencing platform. The assembled genome of L4 consisted of a single circular chromosome was 6,229,472 bp, with an average GC content of 64.95 %, 5,629 protein-coding genes, 72 tRNA, 16 rRNA and 1 tm RNA. Six secondary metabolite biosynthetic gene clusters are identified in the genome. The genome sequence provided a theoretical basis for analyzing the biocontrol mechanism of this strain.

Intelligent identification on cotton verticillium wilt based on spectral and image feature fusion.

BACKGROUND: Verticillium wilt is the major disease of cotton, which would cause serious yield reduction and economic losses, and the identification of cotton verticillium wilt is of great significance to cotton research. However, the traditional method is still manual, which is subjective, inefficient, and labor-intensive, and therefore, this study has proposed a novel method for cotton verticillium wilt identification based on spectral and image feature fusion. The cotton hyper-spectral images have been collected, while the regions of interest (ROI) have been extracted as samples including 499 healthy leaves and 498 diseased leaves, and the average spectral information and RGB image of each sample were obtained. In spectral feature processing, the preprocessing methods including Savitzky-Golay smoothing (SG), multiplicative scatter correction (MSC), de-trending (DT) and mean normalization (MN) algorithms have been adopted, while the feature band extraction methods have adopted principal component analysis (PCA) and successive projections algorithm (SPA). In RGB image feature processing, the EfficientNet was applied to build classification model and 16 image features have been extracted from the last convolutional layer. And then, the obtained spectral and image features were fused, while the classification model was established by support vector machine (SVM) and back propagation neural network (BPNN). Additionally, the spectral full bands and feature bands were used as comparison for SVM and BPNN classification respectively. RESULT: The results showed that the average accuracy of EfficientNet for cotton verticillium wilt identification was 93.00%. By spectral full bands, SG-MSC-BPNN model obtained the better performance with classification accuracy of 93.78%. By feature bands, SG-MN-SPA-BPNN model obtained the better performance with classification accuracy of 93.78%. By spectral and image fused features, SG-MN-SPA-FF-BPNN model obtained the best performance with classification accuracy of 98.99%. CONCLUSIONS: The study demonstrated that it was feasible and effective to use fused spectral and image features based on hyper-spectral imaging to improve identification accuracy of cotton verticillium wilt. The study provided theoretical basis and methods for non-destructive and accurate identification of cotton verticillium wilt.

Investigating Verticillium wilt occurrence in cotton and its risk management by the direct return of cotton plants infected with Verticillium dahliae to the field.

Verticillium wilt is one of the most crucial diseases caused by Verticillium dahliae that threatens the cotton industry. Statistical results showed that the return of cotton plants infected with V. dahliae to the field might be an essential cause of the continuous aggravation of cotton Verticillium wilt. The correlation among the cotton plants infected with V. dahliae returning to the field, the occurrence of Verticillium wilt, and the number of microsclerotia in rhizosphere soil need further investigation. A potted experiment was carried out to explore the effects of the direct return of cotton plants infected with Verticillium dahliae to the field on the subsequent growth and Verticillium wilt occurrence in cotton. As a risk response plan, we investigated the feasibility of returning dung-sand (i.e., insect excreta) to the field, the dung-sand was from the larvae of Protaetia brevitarsis (Coleoptera: Cetoniidea) that were fed with the V. dahliae-infected cotton plants. The results demonstrated that the return of the entire cotton plants to the field presented a promotional effect on the growth and development of cotton, whereas the return of a single root stubble or cotton stalks had an inhibitive effect. The return of cotton stalks and root stubble infected with V. dahliae increased the risk and degree of Verticillium wilt occurrence. The disease index of Verticillium wilt occurrence in cotton was positively correlated with the number of microsclerotia in the rhizosphere soil. The disease index increased by 20.00%, and the number of soil microsclerotia increased by 8.37 fold in the treatment of returning root stubble infected with V. dahliae to the field. No Verticillium wilt microsclerotia were detected in the feed prepared from cotton stalks and root stubble fermented for more than 5 days or in the transformed dung-sand. There was no risk of inoculation with Verticillium wilt microsclerotia when the dung-sand was returned to the field. The indirect return of cotton plants infected with V. dahliae to the field by microorganism-insect systems is worthy of further exploration plan of the green prevention and control for Verticillium wilt and the sustainable development of the cotton industry.

Pseudomonas spp. Enriched in Endophytic Community of Healthy Cotton Plants Inhibit Cotton Verticillium Wilt.

The plant microbiome plays a fundamental role in plant growth and health. However, detailed information regarding the plant endophytic microbiome during the infection period of a pathogen is largely unknown. Here, we investigated the microbial community of healthy and diseased cotton plants and the root exudate profiles of susceptible and resistant cultivars utilizing high-throughput sequencing and metabolomics. The results showed that the pathogen infection reduced bacterial diversity and significantly affected the bacterial community composition. The microbiome assembly is shaped predominantly by cultivars. The endophytic microbiome of the infected plants showed greater complexity than the healthy plants in network analysis. The results displayed that a total of 76 compounds were significantly different in the two groups, with 18 compounds showing a higher relative abundance in the resistant cultivars and 58 compounds in the susceptible cultivars. Pathway enrichment analysis showed that pathways related to plant hormone signal transduction, biosynthesis of various secondary metabolites, and biosynthesis and metabolism of amino acids were prominently altered. We also demonstrate that plants inoculated with Pseudomonas sp. strains showed increased resistance to the cotton Verticillium wilt compared with the control plants in pot experiments. Overall, it showed that the pathogen infection affected the community composition, and healthy plants displayed an enriched beneficial microbiome to combat the plant disease. These findings significantly advance our understanding of the endophytic microbiome assembly under the pathogen infection and develop microbiome-based solutions for sustainable crop production systems.

Rhizosphere fungal community structure succession of Xinjiang continuously cropped cotton.

The large-scale long-term plantation of cotton in the Xinjiang region has been accompanied by a regular and wide outbreak of soil-borne fungal diseases such as verticillium wilt, which significantly damaged the local cotton industry. High-throughput sequencing data showed that the cotton field cultivation management measures pose a significant influence upon the original ecological soil fungal community structure. During long-term continuous cropping of cotton, a new soil fungal community structure emerges after several repeated adjustments over five years. The number of verticillium wilt pathogens in the soil increased rapidly with prolonged continuous cropping time, reaching a maximum at around the 10th y; moreover, the abundance of the verticillium wilt pathogen only serves as one of numerous essential factors for disease occurrence. The fungal community structure and the abundance of verticillium wilt pathogens in local cotton fields are gradually formed under joint effects of year-long continuous cropping and supporting cultivation management measures.