First Report of Blight on Pinellia ternata (Banxia) Caused by Choanephora cucurbitarum in China.

Pinellia ternata (Thunb.) Makino ex Briet. (banxia, crow-dipper) is a perennial herbaceous plant native to China, Japan, and Korea. A member of the family Araceae, it is considered an invasive weed in parts of Europe and North America. In August 2020, P. ternata plants showing blight symptoms (8% incidence in a 30-ha field) were observed, near Qianjiang City (30°50′N, 112°92′E), Hubei Province, China. Brown water-soaked lesions first appeared on flowers followed by flower blight and leaf and stem rot during periods of more than 80% humidity (Supplementary figure 1). White, cottony mycelia grew from rotted tissues and produced sporangiophores with brown to black sporangiola. To identify the causal agent, 12 diseased samples were surface disinfested with 0.5% sodium hypochlorite and 75% ethyl alcohol, then plated on potato dextrose agar (PDA) maintained at 25°C. Ten fungal isolates were selected by hyphal tip isolation and placed on fresh PDA. White fungal colonies grew rapidly that later turned pale yellow and produced abundant sporangiola in 13 days. Sporangiophores were smooth, hyaline, aseptate, and produced monosporous sporangiola. Sporangiola were ellipsoid, indehiscent, pediculate, brown to dark brown, 8 to 16 × 14 to 21 μm (n = 50) in size, with visible longitudinal striations . Sporangia with a few or many sporangiospores were subglobose, pale brown to brown, and 55 to 165 μm (n = 40) in diameter. Sporangiospores were broadly ellipsoid, brown to pale brown, striate, 8 to 12 × 15 to 25 μm (n=30) in size, with hyaline polar appendages. Based on these morphological characteristics, the fungus was identified as Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. (Kirk 1984). To confirm the identification, the strain QJFY1 was chosen for DNA sequencing. The internal transcribed spacer (ITS) region of rDNA and large subunit (LSU) region of ribosomal RNA were amplified with primers ITS1/ITS4 and NL1/LR3 (Walther et al. 2013) and the amplicons were sequenced. BLAST analysis of the 593bp sequences (accession no. MW295532) and the 699bp sequence（accession no. MW341527）showed ≥99.5% identity with C. cucurbitarum strains CBS 674.93 (GenBank accession no. JN943006.1 and JN939195.1; Supplementary figure 2). Based on morphological and molecular characteristics, the fungus was identified as C. cucurbitarum. Koch’s postulates were fulfilled by inoculating flowers of three healthy 30-day-old P. ternata plants with 50 μL of inoculum suspension (1 x 104 conidia/ml) obtained from 13-day old cultures of C. cucurbitarum isolate QJFY1. Another three plants treated with sterile distilled water served as controls. All plants were placed in a greenhouse with relative humidity of 90% for 2 days and thereafter placed in the glasshouse at 25 ± 1°C. After three days, symptoms similar to those seen under field conditions, were observed on inoculated plants and non-inoculated plants remained healthy. C. cucurbitarum was reisolated and identified by molecular characteristics (ITS and LSU) from inoculated plants. The experiment was repeated thrice with similar results. To our knowledge, this is the first report of Choanephora blight caused by C. cucurbitarum on P. ternata in China and worldwide. Hubei Province is one of the most important banxia producing areas in China and C. cucurbitarum can pose a new threat to banxia production. Our results provide a basis to develop effective measures to manage this disease. References: Kirk, P. M. 1984. Mycol. Pap. 152:1. Walther, G., et al. 2013. Persoonia 30:11. http://dx.doi.org/10.3767/003158513X665070 Acknowledgements Science Funds for Young Scholar of Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences (grant no. 2019ZYCJJ01), Key Research and Development Program of Hubei Province (grant no. 2020BCA059), Support Plan of Hubei Academy of Agricultural Sciences (grant no. 2019fcxjh09), Key Laboratory of Integrated Management of Crops of Central China, Ministry of Agriculture, P. R.China / Hubei Key Laboratory of Crop Disease, Insect Pests and Weeds Control (grant no. 2019ZTSJJ6), Hubei Agricultural Science and Technology Innovation Center Project (grant no.2019-620-003-001)

First Report of Choanephora cucurbitarum Causing Leaf Wilt, Flower Rot, and Stem Necrosis on Crotalaria breviflora

Crotalaria breviflora (Fabaceae) is used as green manure crop because of its nitrogen fixation and nematode control (Nascimento et al. 2020). In April 2018, leaf wilting, flower rot, and stem necrosis symptoms were observed on C. breviflora with 100% incidence, in Sorriso (12° 33′ 31″ S, 55º 42′ 51″ W), Santa Carmem (11° 55′ 52″ S, 55º 16′ 47″ W), and Sapezal (12º 59′ 22″ S, 58º 45′ 52″ W) counties in the state of Mato Grosso, Brazil. Three monosporic isolates were isolated from symptomatic leaves, cultivated in potato dextrose agar (PDA) medium, and deposited at the Cultures Collection of the University of Brasilia (codes CCUB 1293, CCUB 1667, CCUB 1668). Colonies on PDA were white and cottony with presence of hyaline and coenocytic hyphae. The mycelia later became pale yellow with abundant reproductive structures. Sporangiophores were hyaline, aseptate, unbranched, and apically dilated to form a clavate vesicle, which produced secondary vesicles bearing sporangiola. Secondary vesicles were clavate, light brown, and 37 to 51 µm in diameter. Sporangia were brown to dark brown, globular to ellipsoid, 115 to 140 µm long, and 96 to 122 µm wide. Sporangiospores (n=30) were brown to reddish-brown, ellipsoid to ovoid, with longitudinal striae, 14 to 19 µm long, and 8 to 12 µm wide. Some with hyaline appendages at both ends. Their morphological characteristics were consistent with the descriptions of Choanephora cucurbitarum (Kirk 1984). To confirm the identity, the DNA of the three isolates was extracted and the sequences of Small Subunit (SSU), Large Subunit (LSU), and complete Internal Transcribed Spacer (ITS) of rDNA were amplified using V9G, ITS3, and LR5 primers (GenBank acc. no: MN897836, MN897837 and MN897838). The sequences were aligned with the MAFFT software. The alignment matrix was subjected to Maximum Likelihood (ML) analysis using RAxML v. 8 and Bayesian Inference performed in MrBayes v.3.1.2. The tree was edited in the FigTree software. The sequences showed 100% identity with the sequences from C. cucurbitarum found on the GenBank. To confirm pathogenicity, a suspension at 5.4 ×106 spores/ml was prepared from a 15-day-old culture grown at 25°C and sprayed on asymptomatic plants of C. breviflora. Sterilized water was sprayed as the control. Plants were kept in a humid chamber at 20°C for 48 h. Initial symptoms were visualized 16 days after inoculation. Complete necrosis of leaves and stems with spore mass on infected tissue was observed 19 days after inoculation. To satisfy the Koch’s postulates, the fungus was successfully reisolated from the infected tissues. No symptoms were observed on the control plants. In Brazil, this pathogen has been reported on Brassica oleracea var. capitata, Capsicum annuum, Crotalaria spectabilis, Cucurbita sp., and Vigna unguiculata (Alfenas et al. 2018; Mendes and Urben, 2019). C. cucurbitarum has been reported to have a wide range of hosts (Farr and Rossman, 2020). It can infect the crops grown in rotation or in succession, including common bean, corn, cotton, quinoa, soybean, and sunflower. Therefore, this pathogen is of epidemiological importance and poses a threat to the croplands where environmental conditions are conducive to the disease to develop and spread. To our knowledge, this is the first report of C. cucurbitarum causing leaf and flower wilt, and stem rot on C. breviflora in the world. Acknowledgment We thank the Environmental Sciences Graduate Program, Federal University of Mato Grosso, University of Brasilia, PROPeq/PROPG-UFMT, EMBRAPA, CODEX/UFMT, Institute of Agricultural and Environmental Sciences (ICAA)/UFMT and CAPES for providing the Master's scholarship. References Alfenas, R. F., et al. 2018. Plant Dis.102:1456. https://doi.org/10.1094/PDIS-10-17-1610-PDN, Google Scholar. Farr, D. F., and Rossman, A. Y. 2020. Fungal Databases, Syst. Mycol. Microbiol. Lab., ARS, USDA. Retrieved May 26, 2020 from https://nt.ars-grin.gov/fungaldatabases/, Google Scholar. Kirk, P. M. 1984. Mycol Paper. 152:1. Google Scholar. Mendes, M. A. S., and Urben, A. F. 2020. Fungos relatados em plantas no Brasil, Retrived May 26, 2020 from http://pragawall.cenargen.embrapa.br/aiqweb/michtml/fgbanco01.asp, Google Scholar. Nascimento, D. D. et al. 2020. Bioscience Journal. 36:713. https://doi.org/10.14393/BJ-v36n3a2020-42248, Google Scholar.

Novel Monomeric Fungal Subtilisin Inhibitor from a Plant-Pathogenic Fungus, Choanephora cucurbitarum: Isolation and Molecular Characterization

ABSTRACT The bacterial protease inhibitor domains known as Streptomyces subtilisin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are horizontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated “choanepins.” Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [Kd]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in inhibitory reactivity (reactive sites) and binding affinity (structural integrity). The function of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel serine protease inhibitors. IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not reactive sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional characterization of fungal SSI-like domains will be useful in understanding their biological role and future biotechnological applications.