Trichoderma spp. are widely used beneficial microbes in agricultural production; however, the improper carrier choice for Trichoderma agent preparation can alter the effectiveness of Trichoderma fungicides. In this study, the co-culture of four Trichoderma strains produced a large amount of free amino acids, with a content of 392.8414 ug/mL, and significantly improved the production level of γ-aminobutyric acid. A greenhouse experiment further showed that the co-culture of Trichoderma synergistically improved the female flower development and bacterial angular leaf spot resistance. The effects of ten kinds of carriers were compared in terms of water absorption and heat generation, as well as their effects on the seedling emergence rate and the plant growth promotion of maize, cucumber, and pakchoi cabbage. Each carrier was screened to mix with four strains of co-culture metabolites to prepare highly soluble and quality powders. The results showed that there were different effects of the carriers themselves and Trichoderma strain co-culture metabolite powder prepared with the carriers on seedling emergence rate and seedling growth. Β-cyclodextrin performed best in high solubility and low heat generation upon absorbing water and in easy drying in processing operations. Trichoderma strains co-culture metabolite powder with β-cyclodextrin as a carrier provided the most obvious promotion effects on seedling emergence rate and seedling growth. Therefore, β-cyclodextrin was determined to be an ideal carrier to prepare a highly water-soluble Trichoderma agent. Taken together, the study successfully developed a new type of highly soluble powder containing Trichoderma co-culture metabolites that is expected to benefit farming drip irrigation and spraying systems for the promotion of crop growth and disease control.

In September 2022, rice spikelets rot disease (RSRD) was investigated in Songjiang District (30.94132N, 121.18393E), China, leading to a 26.77% yield loss. At the heading stage, infected spikelets exhibited small, yellowish-brown dots with water-stained husks, subsequently coalescing to form irregular brown to black lesions. Later, the lesions were enlarged and rotted, which eventually caused blighted grains. About 10% of husked grains showed black spots. 30 infected grains and 30 husked grains with black spots were surface sterilized in 75% ethanol for 2 min, then rinsed with ddH₂O and plated on PDA medium at 28°C in darkness for 4 d. 22 and 13 fungal isolates with similar morphology were obtained in shriveled and husked grains, respectively. Three isolates (SJTU1, SJTU2 and SJTU3) were selected by the single-spore isolation method. The colonies were brown to blackish green, smooth, and contained a large number of stolons with a few aerial mycelia in the center. Hyphae and conidiophores were blackish green, thick-walled, branched with septa. Conidia were 14.77 to 26.82×4.74 to 11.36 μm (average 20.42×8.58 μm, n= 100) in size, lightly curved with blackish green. Conidia with three septa and four cells, apical and basal cells transparent, middle cell unequal in size. Based on morphological characteristics, the isolates were preliminarily identified as Curvularia plantarum (Raza et al. 2019). The genomic DNA of the three isolates (SJTU1 to 3) was extracted for molecular identification. 3 pairs of primers ITS1/TTS4 (Peever et al. 2004), gpd1/gpd2 (Berbee et al. 1999), and EF-983F/EF-2218R (Rehner and Buckley 2005) were used to amplify ITS, GAPDH, and EF1-α genes, respectively. These sequences were all uploaded in GenBank (ITS: OR726053 to 55; EF1-α: OR732471 to 73; GAPDH: OR732474 to 76). According to data in GenBank, the ITS, EF1-α, and GAPDH genes of 3 isolates (SJTU1 to 3) showed 99-100% identity (573/575 bp, 542/543 bp, and 531/531 bp) to the ITS (MW581905, MN044755, and MN215690), 99-100% identity (869/869 bp, 868/869 bp, and 855/856 bp) to the EF1-α (MN263982 to 83, and MT628901), and 99-100% identity (543/544 bp, 528/528 bp, and 540/540 bp) to the GAPDH (MT628902, MN264120, and MT432926) gene of C. plantarum, respectively. The phylogenetic analysis by Maximum Likelihood (ML) method based on the concatenated sequences of ITS, EF1-α, and GAPDH genes showed that the three isolates (SJTU1 to 3) clustered with C. plantarum. According to morphology and molecular identification, these fungal isolates were identified as C. plantarum. Pathogenicity tests were conducted in the field used only for inoculation with pathogens by spraying 30 spikelets of rice cultivar 'Song1013' at the heading stage with conidial suspension (5 × 10⁵ conidia/mL). 30 spikelets sprayed with ddH₂O were designated as control. The test was conducted 3 times at 22 to 31°C with 78 to 89% RH. All the inoculated spikelets exhibited similar symptoms to those of the infected spikelets in paddy at 10 d after spraying, while the control spikelets remained healthy. All reisolated strains from infected spikelets were identified the same as the original inoculated strains by morphology and ITS sequences, fulfilling Koch's postulates. To our knowledge, this is the first report of C. plantarum causing RSRD in China. The discovery of this new disease and its pathogens will facilitate the provision of pathogenically relevant information vital for management strategies to RSRD caused by C. plantarum in the future.

Trichoderma spp., Metarhizium spp., and Bacillus spp. are commonly used as biocontrol microorganisms domestically and internationally. However, microbial pesticides currently prepared from single living microorganisms have problems such as a short shelf life, particularly under stressful environment conditions. Secondary metabolites produced from biocontrol microorganisms are comparatively stable when used under field conditions. This study screened the optimal combination of biocontrol metabolites, referred to as TMB, composed of culture filtrates from certain isolates of Trichoderma asperellum 10264, Bacillus subtilis S4-4-10, and Metarhizium anisopliae 3.11962 (1:4:1 (v/v)). RNA-seq analysis and transmission electron microscope observations were carried out to identify the major functions of the most effective culture filtrates against Magnaporthe oryzae (the pathogen causing rice blast disease) and Chilo suppressalis (an insect pest in rice cultivation). TMB was found to disrupt the midgut subcellular structure of C. suppressalis larvae and inhibit the expression of genes related to immunity, membrane components, protein synthesis, and other functions in C. suppressalis larvae and M. oryzae, thereby interfering with their normal growth, reproduction, and infection potential in rice. In addition, TMB was also able to promote rice growth and trigger host defense responses against infections by the target pests and pathogens. In summary, TMB generated different inhibitory activities against multiple targets in C. suppressalis and M. oryzae and induced plant immunity in rice. Therefore, it can be used as a new environmentally friendly agent or alternative to control rice pests and diseases.

As critical biocontrol agents, Trichoderma and Metarhizium have demonstrated vast potential in disease prevention and pest control respectively. Current applications, however, are predominantly confined to singular use or simplistic combinations of the two fungi. This study innovatively utilizes Metarhizium and Trichoderma as experimental subjects, employing non-targeted metabolomics to compare the differences in metabolites between co-culture and separate fermentation. The metabolite distinctions in co-culture are meticulously analyzed using sample correlation, least squares discriminant analysis, cluster analysis, and KEGG enrichment analysis. Metabolome sequencing analysis is then applied to compare the co-culture fermentation filtrate with Trichoderma atroviride and Metarhizium anisopliae. Differential metabolite analysis found that co-culture had significantly up-regulated or down-regulated metabolites compared to singular cultures.However,significant metabolite discrepancies are discovered in singular cultures, notably the up-regulated metabolites such as osthole and spirocyclic amine, which have documented antibacterial functions that may prove effective against Fusarium graminearum. Ultimately, the findings from this research contribute to the exploration of synergistic applications of biocontrol fungi and serve as a valuable reference for future development of bio-pesticides for disease and pest control.