Cladobotryum protrusum is one of the mycoparasites that cause cobweb disease on cultivated edible mushrooms. However, the molecular mechanisms of evolution and pathogenesis of C. protrusum on mushrooms are largely unknown. Here, we report a high-quality genome sequence of C. protrusum using the single-molecule, real-time sequencing platform of PacBio and perform a comparative analysis with closely related fungi in the family Hypocreaceae. The C. protrusum genome, the first complete genome to be sequenced in the genus Cladobotryum, is 39.09 Mb long, with an N50 of 4.97 Mb, encoding 11,003 proteins. The phylogenomic analysis confirmed its inclusion in Hypocreaceae, with its evolutionary divergence time estimated to be ~170.1 million years ago. The genome encodes a large and diverse set of genes involved in secreted peptidases, carbohydrate-active enzymes, cytochrome P450 enzymes, pathogen–host interactions, mycotoxins, and pigments. Moreover, C. protrusum harbors arrays of genes with the potential to produce bioactive secondary metabolites and stress response-related proteins that are significant for adaptation to hostile environments. Knowledge of the genome will foster a better understanding of the biology of C. protrusum and mycoparasitism in general, as well as help with the development of effective disease control strategies to minimize economic losses from cobweb disease in cultivated edible mushrooms.

Cobweb disease is a fungal disease that can cause serious damage to edible mushrooms worldwide. To investigate cobweb disease in Morchella sextelata in Guizhou Province, China, we isolated and purified the pathogen responsible for the disease. Through morphological and molecular identification and pathogenicity testing on infected M. sextelata, we identified Cladobotryum mycophilum as the cause of cobweb disease in this region. This is the first known occurrence of this pathogen causing cobweb disease in M. sextelata anywhere in the world. We then obtained the genome of C. mycophilum BJWN07 using the HiFi sequencing platform, resulting in a high-quality genome assembly with a size of 38.56 Mb, 10 contigs, and a GC content of 47.84%. We annotated 8428 protein-coding genes in the genome, including many secreted proteins, host interaction-related genes, and carbohydrate-active enzymes (CAZymes) related to the pathogenesis of the disease. Our findings shed new light on the pathogenesis of C. mycophilum and provide a theoretical basis for developing potential prevention and control strategies for cobweb disease.

Since 2016, devastating bacterial blotch affecting the fruiting bodies of Agaricus bisporus, Cordyceps militaris, Flammulina filiformis, and Pleurotus ostreatus in China has caused severe economic losses. We isolated 102 bacterial strains and characterized them polyphasically. We identified the causal agent as Pseudomonas tolaasii and confirmed the pathogenicity of the strains. A host range test further confirmed the pathogen’s ability to infect multiple hosts. This is the first report in China of bacterial blotch in C. militaris caused by P. tolaasii. Whole-genome sequences were generated for three strains: Pt11 (6.48 Mb), Pt51 (6.63 Mb), and Pt53 (6.80 Mb), and pangenome analysis was performed with 13 other publicly accessible P. tolaasii genomes to determine their genetic diversity, virulence, antibiotic resistance, and mobile genetic elements. The pangenome of P. tolaasii is open, and many more gene families are likely to emerge with further genome sequencing. Multilocus sequence analysis using the sequences of four common housekeeping genes (glns, gyrB, rpoB, and rpoD) showed high genetic variability among the P. tolaasii strains, with 115 strains clustered into a monophyletic group. The P. tolaasii strains possess various genes for secretion systems, virulence factors, carbohydrate-active enzymes, toxins, secondary metabolites, and antimicrobial resistance genes that are associated with pathogenesis and adapted to different environments. The myriad of insertion sequences, integrons, prophages, and genome islands encoded in the strains may contribute to genome plasticity, virulence, and antibiotic resistance. These findings advance understanding of the determinants of virulence, which can be targeted for the effective control of bacterial blotch disease.

Aster tataricus is a perennial herb belonging to the family Asteraceae. It is a very important plant for traditional Chinese medicine. A decoction of the roots is used in treating chronic bronchitis, cough and phlegm. It also has anti-tumor, antibacterial, antiviral and antioxidant properties (Zhang et al. 2012). In 2014 and 2015, dark brown lesions were observed on leaves of A. tataricus plants in Changchun (125.39°E, 43.45°N), Jilin Province, China. The disease incidence reached approximately 30％. Symptoms on diseased leaves initially included small, light brown and circular spots. As the disease progressed, small circular spots expanded and gradually became round or irregular, and the spots turned gray in the center with dark brown margins, frequently surrounded by light yellow haloes. Eventually, the necrotic areas often coalesced and resulted in the appearance of larger spots with a diameter of 4.0-15.0 mm. Severely affected leaves often withered and died. Leaf tissues (3×5 mm2), cut from the margins of lesions, were surface disinfected (75％ ethanol for 30 s, 1％NaClO for 1 min, rinsed three times with sterile distilled water) and placed on potato dextrose agar (PDA). Single conidial isolate CDAT137107 was grown on PDA under a 12 h photoperiod at 25°C. The mycelia were initially pale white, the colony gradually expanded and turned into black, with some small black structures. On synthetic nutrient-poor agar (SNA) (Liu et al. 2016), the conidiophore of the fungi were cannular, hyaline and septate. Conidia (n = 150) were straight, cylindrical, both sides obtuse, with two oil droplets, and measured 8.9 to 37.5 (14.6 ± 3.8) μm in length, and 3.0 to 17.3 (4.9 ± 1.6) μm in width. Appressoria were brown sticks, and measured at 4.9 to 7.5 × 4.7 to 7.0 μm. Setae were slender, straight, subulate, dark brown, measured at 59.5 to 120.5 × 3.2 to 6.5 μm. Based on the morphological characteristics, the fungus was preliminarily identified as Colletotrichum destructivum (Damm et al. 2014). The internal transcribed spacer (ITS, KT355798) and chitin synthase (CHS-1, KT355799) sequence matched to C. destructivum (EU400156, JQ005785) with 100% identity, beta-tubulin (tub2, KT633397) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, KT751006) sequence matched to C. destructivum (JX625198, KC843523) with 99% identity. Pathogenicity tests were performed by spraying a 106 conidia/ml suspension of CDAT137107 on 30 healthy leaves of A. tataricus var. petersianus, and the controls were sprayed with sterilized water. All the inoculated leaves were covered with plastic bags and incubated in a growth chamber at 25°C for 7 days at 95% RH. Symptoms on the inoculated leaves were similar to those that naturally occurred in the field and there were no symptoms on the control leaves. The fungus was re-isolated from the infected leaves which was similar as the inoculated isolate. C. destructivum have been reported on curly dock in Korea and on common thyme in Florida (Lee et al. 2002 and Fu et al. 2015). To our knowledge, this is the first report of C. destructivum causing anthranose of A. tataricus. There is a need to develop disease management strategies for anthracnose on A. tataricus to prevent major economic constraints on production and quality of this medicinal plant in China. References: Damm, U., et al. 2014. Stud. Mycol. 79: 49. Fu Y., et al. 2015. Plant Dis. 99: 1184. Lee H. B., et al. 2002. Plant Dis. 86: 1271. Liu L. P., et al. 2016. Plant Dis. 100: 1240. Zhang Y. X., et al. 2012. Int. J. Biol. Macromol. 51: 509-513.