First Report of Brown Rot Disease on Cultivated Auricularia cornea Caused by Trichoderma pleuroticola in Sichuan Province, China.

Auricularia cornea Ehrenb. is a well-known, rare, and valuable edible mushroom, with considerable culinary and medicinal value. It is distributed worldwide and especially common in Asia (Khatua et al. 2022). In China, more than 1.89 million tons of A. cornea are artificially cultivated annually, particularly in Sichuan Province, which produces 0.97 million tons, accounting for about 51% of the nation's total (Ye et al. 2022). However, farmers in Shifang, a county-level city in Sichuan Province, who practiced traditional greenhouse cultivation on a large scale, reported that brown rot disease affected up to 20% of their A. cornea crop and caused severe failures each year between 2016 and 2022, resulting in devastating economic losses. Worse, when the diseased fruit bodies were not removed promptly by farmers, the disease could spread to 100% of crop in a given greenhouse in 10 to 15 days. The symptoms mainly occur in fresh unfolded fruiting bodies. Lesions were brown, putrefied, and foul smelling, and eventually the fruiting bodies wilted. Naturally air-dried fruiting bodies were dark brown with yellow deposits at the edges (Fig. 1A). In this backdrop, we collected more than 60 diseased samples from Shifang (104°1'15''E, 31°12' 30'' N) and isolated pathogens from May 2021 through May 2022. On the clean bench, the surfaces of the lesions were disinfected with cotton balls soaked in 75% ethanol and rinsed three times with sterile water, and then the internal tissue block was picked with a sterile scalpel and cultured in potato dextrose agar (PDA) medium at 25°C. The pathogen was repeatedly isolated and purified, and we conducted pathogenicity tests. Colonies of the pathogen on PDA medium were white and cottony, with a mycelial growth rate of 13.92 ± 1.24 mm/day at 25°C. Then the spores began to turn yellow-green and soon turquoise, converging into a wide concentric wheel. The spores were elliptical with dimensions of 4.0 to 8.0 µm × 3.0 to 4.5 µm (N=50). Pathogenicity tests were conducted in an incubator. Ninety pure white, healthy A. cornea fruiting bodies were randomly picked and placed in groups of 10. On the clean bench, the bodies surface were sterilized with 75% alcohol cotton balls, then washed three times with sterile water, dried with sterile absorbent paper, and placed in sterile petri dishes for subsequent tests. The first control group (CK1) did not receive additional treatment other than the surface disinfection mentioned above. These bodies were immediately sealed with parafilm. The second control group (CK2) was not inoculated with the pathogen, and a blank sterile PDA plug (diameter: 0.8 cm) was placed on the surface of the fruiting body and sealed with parafilm. All seven treatment groups (TS) were inoculated with single-pathogen mycelium plugs (diameter: 0.8 cm) in the center surface of each fruiting body. They were then sealed with parafilm. All total of nine experimental treatment groups were cultured in a light incubator at 25°C. The test results showed that the pathogen could visibly infect the A. cornea within 24 hours. At 48 hours after inoculation, the lesions were round and brown, radiating outward along the inoculum, consistent with the symptoms of the original sample. As culture time continued, the extent of each lesion gradually expanded (Fig. 1B). After 120 hours, the fruiting bodies showed rot, stench, and loss of other traits relevant to commercial value. In contrast, the CK1 and CK2 groups had no lesions. Furthermore, the fungal cultures with the same phenotypic characteristics could be continuously isolated from the lesions of TS, and the pathogenic factors were verified by Koch's postulates. Similarly, uninoculated and inoculated tests were conducted in a greenhouse, and the results were consistent with those of incubator testing. The pathogen was designated MMEBYJ202206. The DNA of the pathogen was extracted using CTAB, and the rDNA internal transcribed spacer (ITS) of the isolates was amplified using ITS1/ITS4 primers. PCR was conducted in a 25 μL reaction mixture, and a 596 bp sequence was obtained by sequencing. The ITS sequence has been submitted in GenBank with accession number ON974844.1. BLAST database in NCBI was used to compare ITS sequences and phylogenetic tree was constructed based on the neighbor-joining algorithm from MEGA (Fig. 2). The results indicate that the MMEBYJ202206 was located on a common clade of the phylogenetic tree with KX343129.1, KX343130.1, KX343131.1, and MF871554.1 of T. pleuroticola, and it showed 99% support in bootstrap (500 replicates), but it was not in the same clade as other Trichoderma spp., suggesting that the pathogen was T. pleuroticola. To our knowledge, this is the first report to show that T. pleuroticola can cause brown rot disease on artificially cultivated A. cornea. However, a previous study showed T. pleuroticola to be the causal agent of considerable decline in the yield of Pleurotus ostreatus and P. florida (Siwulski et al. 2011; Blaszczyk et al. 2013) and capable of infecting A. heimuer raised on artificial bed-log (a rod used for growing mycelium of edible mushrooms) (Liu et al. 2019). Brown rot disease is important in China because it has caused considerable damage to yield in artificially cultivated A. cornea, a decline in the external and internal qualities of the product, and a reduction in the enthusiasm of farmers for this crop. Consequently, this study provides a foundation for further research and prevention of this pathogen in China.