Rhizopus oryzae causes the leaf rot disease of Epimedium sagittatum in Guizhou, China.

Epimedium sagittatum is a collective term for herbaceous plants belonging to the family Berberidaceae. Their dried leaves and stems have significant therapeutic effects on tumor inhibition, hypertension control, and coronary heart disease (Ke et al. 2023; Zhao et al. 2019). In 2021 and 2022, plants with similar leaf rot symptoms ranging from 30% to 55% was observed on E. sagittatum in Congjiang County, Guizhou province. The initial symptoms of the disease manifest locally on the leaf, with yellowing on the surface edge of the affected tissue, browning in the middle part, and brown-white discoloration in the innermost part (Supplementary Figure S1B). As the disease progresses, the entire infected leaf gradually softens, while the veins remain intact (Supplementary Figure S1C). Ultimately, the leaf withers and dehisces. The nine samples with typical symptoms were collected from Congjiang County, Guizhou province (26.598°N, 106.707°E). Twenty-seven fungi were isolated, including ten isolates of Rhizopus and seventeen isolates of seven other genera. On isolate YYH-CJ-17 many sporangia were formed and turned to a brown-gray to black color on potato dextrose agar medium (PDA) after culturing 5 days under dark at 25 ℃ (Supplementary Figure S2A and S2B). The branches of mycelium were finger-shaped or root-shaped. The sporangium was spherical or nearly spherical, 60-250 µm in diameter, and sporangiospores were elliptical or spherical and 4-8 µm in diameter. The obtained 547 bp ITS fragment (accession OR225970) and 1231 bp EF-1α region (accession OR242258) from isolate YYH-CJ-17 were compared with NR database using the BLAST tool provided by NCBI, which revealed more than 99.5% identity (query cover more than 98%) with the sequences of ITS (accessions MF522822.1) and EF-1α (accession AB281541.1) of Rhizopus oryzae Went & H.C. Prinsen Geerlings (Gao et al. 2022; Zhang et al. 2022). The phylogenetic tree constructed with the ITS and EF-1α gene sequences demonstrates that strain YYH-CJ-17 clusters with R. oryzae in the same branch and the bootstrap value was greater than 99% (Supplementary Figure S3). Based on the morphological characteristics and ITS and EF-1a sequences, the isolate YYH-CJ-17 is identified as R. oryzae. Pathogenicity tests were performed on detached healthy leaves and living plants of E. sagittatum. Healthy leaves of E. sagittatum were subjected to inoculation with isolate YYH-CJ-17 with 5 × 105 CFU mL-1 concentration in sterile culture dishes. The progression of the disease was marked by the gradual softening of the infected leaves and the expansion of the lesions, which ultimately produced black-brown sporangium (Supplementary Figure S4A). Furthermore, the E. sagittatum living plants were sprayed with 5 × 105 CFU mL-1 conidial suspension of isolate YYH-CJ-17, with ddH2O as a negative control, and then were cultivated at 25℃ and 90% humidity for 21 days in the greenhouse. This assay found that the E. sagittatum leaves treated with isolate YYH-CJ-17 exhibited the same symptoms observed on plants in fields (Supplementary Figure S4B). The fungus re-isolated from the inoculated leaves were identified as R. oryzae by ITS sequencing and were blasted with NR database, which highest matched with the sequence of ITS (accessions MF522822.1) mentioned above, thus fulfilling Koch's postulates. R. oryzae has been identified as a causative agent of a diverse array of host diseases, including leaf mildew of tobacco, fruit rot of yellow oleander and pears, and soft rot of bananas (Farooq et al. 2017; Khokhar et al. 2019; Kwon et al. 2012; Pan et al. 2021). To the best of our knowledge, this is the first report of leaf rot on E. sagittatum caused by R. oryzae in China, which will provide clear prevention and management target for the leaf rot disease of E. sagittatum.

A Probiotic Bacillus amyloliquefaciens D-1 Strain Is Responsible for Zearalenone Detoxifying in Coix Semen.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium spp., which commonly and severely contaminate food/feed. ZEN severely affects food/feed safety and reduces economic losses owing to its carcinogenicity, genotoxicity, reproductive toxicity, endocrine effects, and immunotoxicity. To explore efficient methods to detoxify ZEN, we identified and characterized an efficient ZEN-detoxifying microbiota from the culturable microbiome of Pseudostellaria heterophylla rhizosphere soil, designated Bacillus amyloliquefaciens D-1. Its highest ZEN degradation rate reached 96.13% under the optimal condition. And, D-1 can almost completely remove ZEN (90 µg·g-1) from coix semen in 24 h. Then, the D-1 strain can detoxify ZEN to ZEM, which is a new structural metabolite, through hydrolyzation and decarboxylation at the ester group in the lactone ring and amino acid esterification at C2 and C4 hydroxy. Notably, ZEM has reduced the impact on viability, and the damage of cell membrane and nucleus DNA and can significantly decrease the cell apoptosis in the HepG2 cell and TM4 cell. In addition, it was found that the D-1 strain has no adverse effect on the HepG2 and TM4 cells. Our findings can provide an efficient microbial resource and a reliable reference strategy for the biological detoxification of ZEN.