Joint metabolomic and transcriptomic analysis identify unique phenolic acid and flavonoid compounds associated with resistance to fusarium wilt in cucumber (Cucumis sativus L.).

Introduction: Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. cucumerinum (Foc) is a destructive soil-borne disease in cucumber (Cucumis sativus. L). However, there remains limited knowledge on the molecular mechanisms underlying FW resistance-mediated defense responses in cucumber. Methods: In this study, metabolome and transcriptome profiling were carried out for two FW resistant (NR) and susceptible (NS), near isogenic lines (NILs) before and after Foc inoculation. NILs have shown consistent and stable resistance in multiple resistance tests conducted in the greenhouse and in the laboratory. A widely targeted metabolomic analysis identified differentially accumulated metabolites (DAMs) with significantly greater NR accumulation in response to Foc infection, including many phenolic acid and flavonoid compounds from the flavonoid biosynthesis pathway. Results: Transcriptome analysis identified differentially expressed genes (DEGs) between the NILs upon Foc inoculation including genes for secondary metabolite biosynthesis and transcription factor genes regulating the flavonoid biosynthesis pathway. Joint analysis of the metabolomic and transcriptomic data identified DAMs and DEGs closely associated with the biosynthesis of phenolic acid and flavonoid DAMs. The association of these compounds with NR-conferred FW resistance was exemplified by in vivo assays. These assays found two phenolic acid compounds, bis (2-ethylhexyl) phthalate and diisooctyl phthalate, as well as the flavonoid compound gallocatechin 3-O-gallate to have significant inhibitory effects on Foc growth. The antifungal effects of these three compounds represent a novel finding. Discussion: Therefore, phenolic acids and flavonoids play important roles in NR mediated FW resistance breeding in cucumber.

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.