Ochratoxins (OTs) are worldwide regulated mycotoxins contaminating a variety of food-environment and agro-environment. Several Aspergillus and Pencillium species synthesize OTs from a six-gene biosynthetic gene cluster (BGC) to produce the highly toxic final product OTA. Although many studies on OTA-degrading enzymes were performed, high efficiency enzymes with strong stability are extremely needed, and the OTA degrading mechanism is poorly understood. The study aimed to explore the OT-degradation enzyme and investigate its degradation mechanisms in Metarhizium, which contain an OT biosynthetic gene cluster. Phylogenomic relationship combined with RNA expression analysis were used to explore the distribution of OT BGC in fungi. Bioactivity-guided isolation and protein mass spectrometry were conducted to trace the degrading enzymes in Metarhizium spp., and the enzymes were heterologously expressed in E. coli and verified by in vitro assays. Structure prediction and point mutation were performed to reveal the catalytic mechanism of MbAmh1. Beyond Aspergillus and Pencillium species, three species of the distant phylogenetic taxon Metarhizium contain an expressed OT-like BGC but lack an otaD gene. Unexpectedly, no OT BGC products were found in some Metarhizium species. Instead, Metarhizium metabolized both OTA and OTB to their non-toxic degradation products. This activity of M. brunneum was attributed to an intracellular hydrolase MbAmh1, which was tracked by bioactivity-guided proteomic analysis combined with in vitro reaction. Recombinant MbAmh1 (5 μg/mL) completely degraded 1 μg/mL OTA within 3 min, demonstrating a strong degrading ability towards OTA. Additionally, MbAmh1 showed considerable temperature adaptability ranging from 30 to 70 °C and acidic pH stability ranging from 4.0 to 7.0. Identification of active sites supported the crucial role of metal iron for this enzymatic reaction. These findings reveal different patterns of OT synthesis in fungi and provide a potential OTA degrading enzyme for industrial applications.

Cabbage Fusarium Wilt (CFW) is a serious disease caused by Fusarium oxysporum f. sp. conglutinans in many parts of the world. The use of chemical fungicides has placed a heavy burden on the environment and is prone to drug resistance in plant pathogens. As a method with great potential, biological control has attracted the attention of many academics both at home and abroad. In this study, we have found that strains B5 and B6 had a strong inhibitory effect on various pathogens and significantly inhibited mycelium growth. They were both identified as Bacillus velezensis by morphological features, biochemical determinations, 16S rRNA gene and gyrA gene sequence analysis. When different concentrations of bacterial suspension were applied to cabbage seeds, hypocotyl and taproot length increased to varying degrees. The in vivo results showed that B5 and B6 decreased the incidence of cabbage seedling wilt disease, with B6 performing significantly better. Furthermore, B. velezensis B6 had the ability to colonize cabbage plants and rhizosphere soil. Thus, strain B6 has great potential for biocontrol development and this research could lead to the development of a promising biological agent for CFW.

Colletotrichum capsici is one of the most important pathogens on chili peppers. Unreasonable application of chemical fungicides will lead to threats to human and animal health, environmental damage, and increased fungicide resistance to pathogens. As an alternative strategy, biological control has been paid more and more attention by academics. In this study, the Yb-1 strain was isolated from healthy cucumber leaves, which could significantly restrict the mycelium growth of C. capsici and Botrytis cinerea, with inhibition rates of 93.49% and 74.03%, respectively. Strain Yb-1 was identified as Bacillus velezensis by morphological features and 16S rRNA gene, gyrA gene, and gyrB gene sequence analysis. When pepper seeds were treated with different concentrations of bacterial suspension, we found that a medium-concentration treatment (10⁴ CFU/mL and 10⁵ CFU/mL) could promote seed germination and growth, while high-concentration treatments (10⁸ CFU/mL) limited seed germination and growth. In addition, the root-irrigation method, acupuncture-leaf method, and leaf-cutting method were used to evaluate the safety of strain Yb-1 on peppers. The results indicated that Yb-1 did not affect the normal growth of peppers. The results of further field trials showed that the control effect of strain Yb-1 on pepper anthracnose was 59.45%. Thus, the strain B. velezensis Yb-1 has great biocontrol potential for pepper anthracnose and promotes plant growth.

The Gleditsia sinensis Lam. widely grown in China is a perennial plant with medicinal properties (Zhang et al. 2016). Since 2019, the leaves of G. sinensis have exhibited yellowing and wilting, and...

Rab GTPases play a crucial role in facilitating the transportation of vesicles during the process of fungal biogenesis. Currently, there is limited understanding regarding the specific biological functions of Rab small GTPase elements within Fusarium oxysporum. In this study, we examined the three proteins FoRab5, FoRab7, and FoRab8 of Foc, which exhibit homology to the Rab protein family found in Saccharomyces cerevisiae. In addition, we also employed a PEG-mediated homologous recombination approach to create deletion mutants and complementary strains for the FoRab5, FoRab7, and FoRab8 genes, thereby facilitating a comprehensive investigation into the functional roles of these genes. FoRab5 was localized on vesicles of various sizes within the cell. Compared to the wild-type strain, the growth rate of the mutant ΔFoRab5 strain decreased, the aerial hyphae decreased, the sporulation decreased, and the spore germination rate decreased. The sensitivity to cell membrane stress, cell wall stress, and endoplasmic reticulum stress increased, the activity of laccase and glucoamylase decreased significantly, and the pathogenicity to cabbage seedlings decreased. FoRab7 was localized on the vacuolar membrane. Compared to the wild type, the growth rate of the mutant ΔFoRab7 strain decreased, the bacteria produced a large amount of pigment deposition, the aerial hyphae decreased significantly, the hyphal branches increased, and the mutant almost lost the ability to produce spores. The sensitivity to osmotic stress, cell membrane stress, cell wall stress, metal ion stress, and endoplasmic reticulum stress was enhanced, and the vacuole was fragmented. Laccase and glucoamylase activities decreased in a significant manner. Moreover, there was a decrease in the pathogenicity of cabbage seedlings. FoRab8 was localized at the tip of the mycelium. Compared to the wild type, the growth rate of the mutant ΔFoRab8 strain decreased, the sporulation decreased, and the sensitivity of the mutant to osmotic stress and endoplasmic reticulum pressure increased. There was a significant decrease in the activity of laccase, glucoamylase, and cellulase. A reduction in the pathogenicity to cabbage seedlings occurred. In summary, these results indicate that members of the Rab family proteins FoRab5, FoRab7, and FoRab8 regulate a series of processes such as growth, sporulation, pathogenicity, and ectoenzyme secretion in Foc.