Streptomyces-triggered coordination between rhizosphere microbiomes and plant transcriptome enables watermelon Fusarium wilt resistance.

The use of microbial inoculant is a promising strategy to improve plant health, but their efficiency often faces challenges due to difficulties in successful microbial colonization in soil environments. To this end, the application of biostimulation products derived from microbes is expected to resolve these barriers via direct interactions with plants or soil pathogens. However, their effectiveness and mechanisms for promoting plant growth and disease resistance remain elusive. In this study, we showed that root irrigation with the extracts of Streptomyces ahygroscopicus strain 769 (S769) solid fermentation products significantly reduced watermelon Fusarium wilt disease incidence by 30% and increased the plant biomass by 150% at a fruiting stage in a continuous cropping field. S769 treatment led to substantial changes in both bacterial and fungal community compositions, and induced a highly interconnected microbial association network in the rhizosphere. The root transcriptome analysis further suggested that S769 treatment significantly improved the expression of the MAPK signalling pathway, plant hormone signal transduction and plant-pathogen interactions, particular those genes related to PR-1 and ethylene, as well as genes associated with auxin production and reception. Together, our study provides mechanistic and empirical evidences for the biostimulation products benefiting plant health through coordinating plant and rhizosphere microbiome interaction.

BCB1, a member of the acyl-coenzyme A synthetase family, regulates the morphogenesis and pathogenicity of Botrytis cinerea.

Botrytis cinerea is a non-host-specific phytopathogenic fungus capable of infecting numerous cash crops. Here, we analyzed the functions of the Bcb1 gene in B. cinerea, which encodes a membrane protein belonging to the acyl-coenzyme A synthase family. Compared to the wild type, Bcb1-deletion mutants exhibited obvious morphological abnormalities, including slower vegetative growth and reduced melanin production. The absence of Bcb1 causes B. cinerea to form only small and incompletely developed infection cushions and fail to produce spores. The Bcb1 mutants displayed hypersensitivity to the membrane stressor SDS, the cell wall stressor Congo red, and the oxidative stressor H2O2 and increased resistance to intracellular osmotic stress caused by KCl compared to the wild-type strain. However, there were no differences in tolerance to extracellular osmotic stress caused by NaCl. The deletion of Bcb1 also caused a reduction in pathogenicity. The qRT‒PCR results showed that the genes Bcpks12 and Bcpks13, which are related to melanin biosynthesis, and Bcpg2, BcBOT2, and cutA, which are related to virulence, were downregulated in ∆Bcb1. These data suggest that BCB1 is important for conidial morphogenesis, and pathogenesis in B. cinerea.

Transcriptomic and metabonomic insights into the biocontrol mechanism of Trichoderma asperellum M45a against watermelon Fusarium wilt.

Watermelon (Citrullus lanatus) is one of the most popular fruit crops. However, Fusarium wilt (FW) is a serious soil-borne disease caused by Fusarium oxysporum f. sp. niveum (FON) that severely limits the development of the watermelon industry. Trichoderma spp. is an important plant anti-pathogen biocontrol agent. The results of our previous study indicated that Trichoderma asperellum M45a (T. asperellum M45a) could control FW by enhancing the relative abundance of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of watermelon. However, there are few studies on its mechanism in the pathogen resistance of watermelon. Therefore, transcriptome sequencing of T. asperellum M45a-treated watermelon roots combined with metabolome sequencing of the rhizosphere soil was performed with greenhouse pot experiments. The results demonstrated that T. asperellum M45a could stably colonize roots and significantly increase the resistance-related enzymatic activities (e.g., lignin, cinnamic acid, peroxidase and peroxidase) of watermelon. Moreover, the expression of defense-related genes such as MYB and PAL in watermelon roots significantly improved with the inoculation of T. asperellum M45a. In addition, KEGG pathway analysis showed that a large number of differentially expressed genes were significantly enriched in phenylpropane metabolic pathways, which may be related to lignin and cinnamic acid synthesis, thus further inducing the immune response to resist FON. Furthermore, metabolic analysis indicated that four differential metabolic pathways were enriched in M45a-treated soil, including six upregulated compounds and one down-regulated compound. Among them, galactinol and urea were significantly positively correlated with Trichoderma. Hence, this study provides insight into the biocontrol mechanism of T. asperellum M45a to resist soil-borne diseases, which can guide its industrial application.

Protein kinase Ime2 is associated with mycelial growth, conidiation, osmoregulation, and pathogenicity in Fusarium oxysporum.

Fusarium oxysporum f.sp. niveum is one of the most serious diseases impairing watermelon yield and quality. Inducer of meiosis 2 (Ime2) is the founding member of a family of serine/threonine protein kinases and plays important roles in yeasts and other filamentous fungi. In this study, we analyzed the functions of FoIme2, the ortholog of Saccharomyces cerevisiae Ime2 in F. oxysporum f.sp. niveum. The FoIme2-deleted mutants exhibited obvious morphological abnormalities, including slower vegetative growth, more branches in the edge hyphae and a reduction in conidia production. Compared to the wild type, the mutants were hypersensitive to the osmotic stressor NaCl but were more insensitive to the membrane stressor SDS. The deletion of FoIme2 also caused a reduction in pathogenicity. Transcriptional analysis revealed that FoIme2 acts downstream of FoOpy2 which is an upstream sensor of the MAPK kinase cascade. These results indicate that FoIme2 is important in the development and pathogenicity of F. oxysporum, and provide new insight for the analysis of the pathogenic mechanism of F. oxysporum.

Fumigation practice combined with organic fertilizer increase antibiotic resistance in watermelon rhizosphere soil.

Chemical fumigants and organic fertilizer are commonly used in facility agriculture to control soil-borne diseases and promote soil health. However, there is a lack of evidence for the effect of non-antibiotic fumigants on the distribution of antibiotic resistance genes (ARGs) in plant rhizosphere soils. Here, the response of a wide spectrum of ARGs and mobile genetic elements (MGEs) to dazomet fumigation practice in the rhizosphere soil of watermelon was investigated along its branching, flowering and fruiting growth stages in plastic shelters using high-throughput quantitative PCR approach. Our results indicated that soil fumigation combined with organic fertilizer application significantly increased the relative abundance of ARGs and MGEs in the rhizosphere soil of watermelon plant. The positive correlations between the relative abundance of ARGs and MGEs suggested that soil fumigation might increase the horizontal gene transfer (HGT) potential of ARGs. This result was further confirmed by the enhanced associations between ARG and MGE subtypes in the networks of fumigation treatments. Moreover, bipartite associations between ARGs/MGEs and microbial communities (bacteria and fungi) revealed a higher percentage of linkage between MGEs and microbial taxa in the fumigated soils. Structural equation model analysis further suggested that the increases in antibiotic resistance after fumigation and organic fertilizer application were mainly driven by MGEs and fungal community. Together, our results provide vital evidence that dazomet fumigation process combined with organic fertilizer in plastic shelters has the great potential to promote ARGs' dissemination in the rhizosphere, and raise cautions of the acquired resistance by soil-borne fungal pathogen and the potential spreading of ARGs along soil-plant continuum.

Dazomet application suppressed watermelon wilt by the altered soil microbial community.

Fusarium wilt disease causes severe decline of watermelon yield and quality. Researches have been reported that soil fumigation with dazomet can help control crop disease. Firstly, we discovered that the dazomet application suppressed watermelon wilt in field experiment compared to the control group. While the importance of microbial community in regulating plant health has been rising up, we therefore focused on examining the soil microbial diversity at six different sampling times after dazomet application by using Illumina MiSeq platform. Remarkably, our research results showed that some beneficial microbial genera have been altered, and these beneficial microbial genera have dominated the entire community, such as Nitrolancea, Pseudomonas and Penicillium after dazomet application. Instead, the relative abundance of Fusarium genus and the pathogen FON (Fusarium oxysporum f. sp. niveum, FON) had the decreased. As there was a significant accumulation of AP (available soil phosphorus) after dazomet application, we noticed that the beneficial microbes as Bacillus, Nitrolancea, Paenibacillus and Penicillium have significant positive correlation with AP but negatively related to morbidity. Together, these results demonstrate that the altered soil microbial community structure by dazomet application is critical to suppress watermelon Fusarium wilt. Thus, our results will drive investigations aimed to deploy interaction of microbiota contribute and plant immunity.

Soil inoculation of Trichoderma asperellum M45a regulates rhizosphere microbes and triggers watermelon resistance to Fusarium wilt.

Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. niveum (FON) is a soil-borne disease that seriously limits watermelon production. In the present study, Trichoderma asperellum (T. asperellum) M45a was shown to be an effective biocontrol agent against FW. In a pot experiment, the application of 105 cfu/g of T. asperellum M45a granules had an improved control effect on FW during the blooming period (up to 67.44%) in soils subjected to five years of continuous cropping with watermelon, while the average length of watermelon vines was also significantly improved (P < 0.05). Additionally, the acid phosphatase (ACP), cellulase (CL), catalase (CAT), and sucrase (SC) activities in the M45a-inoculation group were significantly higher than those in the control (CK) group, and transformation of the soil nutrients (total N, NO3-N, and available P) was significantly increased. Moreover, T. asperellum M45a inoculation reduced fungal diversity, increased bacterial diversity and especially enhanced the relative abundance of plant growth-promoting rhizobacteria (PGPR), such as Trichoderma, Sphingomonas, Pseudomonas, Actinomadura, and Rhodanobacter. Through functional prediction, the relative abundance of ectomycorrhiza, endophytes, animal pathotrophs, and saprotrophs in the fungal community was determined to be significantly lower than that observed in the M45a-treated soil. Correlation analysis revealed that Sphingomonas, Pseudomonas, and Trichoderma had the most differences in terms of microorganism abundance, and these differences were positively correlated with ACP, CL, CAT, and SC. These findings provide guidance for the use of fungicides to achieve microecological control of FW in continuously cropped watermelon plots.

A SIX1 Homolog in Fusarium oxysporum f. sp. conglutinans Is Required for Full Virulence on Cabbage.

Fusarium oxysporum is a soil-born fungus that induces wilt and root rot on a variety of plants. F. oxysporum f. sp. conglutinans (Foc) can cause wilt disease on cabbage. This study showed that a homolog of SIX1 protein in the Arabidopsis infecting isolate Fo5176 (Fo5176-SIX1) had four isoforms in the conidia of Foc by proteomic analysis. Thus, we analyzed the roles of protein Foc-SIX1. Gene expression analysis showed that, compared to the expression in mycelia, dramatically altered expression of Foc-SIX1 could be detected after infecting cabbages, and Foc-SIX1 was highly expressed in conidia under axenic culture condition. Furthermore, we knocked out the Foc-SIX1 gene and found that Foc-ΔSIX1 mutants had significantly reduced virulence compared with wild type isolate, and full virulence was restored by complementation of Foc-ΔSIX1 mutants with Foc-SIX1. Thus, we concluded that SIX1 in Foc was required for full virulence on cabbage. We also complemented Foc-ΔSIX1 with SIX1 gene in F. oxysporum f. sp. lycopersici (Fol) and found Foc-ΔSIX1::Fol-SIX1 mutants did not affect the virulence of Foc-ΔSIX1. The results confirmed that Fol-SIX1 was not capable of replacing the role of Foc-SIX1 in Foc on the disease symptom development of cabbage. The roles of Fol-SIX1 on virulence might rely on host specificity.

Comparative Proteomics Analyses of Two Races of Fusarium oxysporum f. sp. conglutinans that Differ in Pathogenicity.

Fusarium oxysporum is a soil-inhabiting fungus that induces vascular wilt and root rot in a variety of plants. F. oxysporum f. sp. conglutinans (Foc), which comprises two races, can cause wilt disease in cabbage. Compared with race 1 (52557(-TM), R1), race 2 (58385(-TM), R2) exhibits much stronger pathogenicity. Here, we provide the first proteome reference maps for Foc mycelium and conidia and identify 145 proteins with different abundances among the two races. Of these proteins, most of the high-abundance proteins in the R2 mycelium and conidia are involved in carbohydrate, amino acid and ion metabolism, which indicates that these proteins may play important roles in isolate R2's stronger pathogenicity. The expression levels of 20 typical genes demonstrate similarly altered patterns compared to the proteomic analysis. The protein glucanosyltransferase, which is involved in carbohydrate metabolism, was selected for research. We knocked out the corresponding gene (gas1) and found that Foc-∆gas1 significantly reduced growth rate and virulence compared with wild type isolates. These results deepened our understanding of the proteins related to F. oxysporum pathogenicity in cabbage Fusarium wilt and provided new opportunities to control this disease.

Development of a high-efficiency gene knockout system for Pochonia chlamydosporia.

The nematophagous fungus Pochonia chlamydosporia, which belongs to the family Clavicipitaceae (Ascomycota: Pezizomycotina: Sordariomycetes: Hypocreales), is a promising biological control agent for root-knot and cyst nematodes. Its biocontrol effect has been confirmed by pot and field trials. The genome sequence of the fungus was completed recently; therefore, genome-wide functional analyses will identify its infection-associated genes. Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150µgml(-1) G418 sulfate, representing a new selectable marker for P. chlamydosporia. The neomycin-resistance gene (neo), which was amplified from the plasmid pKOV21, conferred G418-resistance on the fungus; therefore, it was chosen as the marker gene. We subsequently developed a gene knockout system for P. chlamydosporia using split-marker homologous recombination cassettes with resistance selection and protoplast transformation. The split-marker cassettes were developed using fusion PCR, and involved only two rounds of PCR. The final products comprised two linear constructs. Each construct contained a flanking region of the target gene and two thirds of the neo gene. Alkaline serine protease and chitinase were confirmed to be produced by P. chlamydosporia during infection of nematode eggs and could participate in lysis of the eggshell of nematode eggs. Here, we knocked out one chitinase gene, VFPPC_01099, and two protease genes (VFPPC_10088, VFPPC_06535). We obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC_01099), 13% (VFPPC_10088) and 15% (VFPPC_06535). This efficient and convenient technique will accelerate functional genomic studies in P. chlamydosporia.