RESUMEN
Bacterial wilt (BW) is a devastating plant disease caused by the soil-borne bacterium Ralstonia solanacearum species complex (Rssc). Numerous efforts have been exerted to control BW, but effective, economical, and environmentally friendly approaches are still not available. Bacteriophages are a promising resource for the control of bacterial diseases, including BW. So, in this study, a crop BW pathogen of lytic bacteriophage was isolated and named PQ43W. Biological characterization revealed PQ43W had a short latent period of 15 min, 74 PFU/cell of brust sizes, and good stability at a wide range temperatures and pH but a weak resistance against UV radiation. Sequencing revealed phage PQ43W contained a circular double-stranded DNA genome of 47,156 bp with 65 predicted open reading frames (ORFs) and genome annotation showed good environmental security for the PQ43W that no tRNA, antibiotic resistance, or virulence genes contained. Taxonomic classification showed PQ43W belongs to a novel genus of subfamily Kantovirinae under Caudoviricetes. Subsequently, a dose of PQ43W for phage therapy in controlling crop BW was determined: 108 PFU*20 mL per plant with non-invasive irrigation root application twice by pot experiment. Finally, a field experiment of PQ43W showed a significantly better control effect in crop BW than the conventional bactericide Zhongshengmycin. Therefore, bacteriophage PQ43W is an effective bio-control resource for controlling BW diseases, especially for crop cultivation.
RESUMEN
Cowpea (Vigna unguiculata L.) is a legume consumed as a high-quality plant protein source in many parts of the world. In August 2023, it was observed that a plant disease affected cowpea growth in Yiyang (28.34°N, 112.55°E), China. The average disease incidence was 10%, resulting in 8.5% economic losses in approximately 3,000 m2. The symptoms initially appeared as brown lesions near the stem-soil interface and the lesions were colonized by white mycelia. As the disease progressed, the disease symptoms included constriction and brown staining at the base of the stem, covered with a small amount of white mycelia. Eventually, the entire plants withered and collapsed and many sclerotia were scattered on the ground around the diseased stem. Twenty samples (10 sclerotia and 10 diseased tissue fragments) were collected from symptomatic plants for causal agent isolation. Samples were disinfected with 70% ethanol for 30 s, 5% NaClO for 1 min, rinsed three times with sterile water, dried and placed on potato dextrose agar (PDA) plates at 28â in the dark. In total, 20 isolates were obtained by the hyphal tip method (Terrones et al. 2022) and showed a consistent phenotype of white cottony mycelia on PDA with an growth rate of 12.9 to 21.3 mm/day (n = 20). Sclerotia formed at five to eight days post inoculation, were initially whitish, turning beige and eventually dark brown. The diameter of mature sclerotia ranged from 0.89 to 2.13 mm (mean = 1.64±0.29 mm; n =50). For pathogen identification, ITS1/ITS4 (White et al. 1990) and EF1-983F/EF1-2218R (Rehner and Buckley 2005) primers were used to amplify the internal transcribed spacer regions (ITS) and translation elongation factor-1 alpha gene (TEF-1α), respectively. The sequences of all 20 isolates showed 99% to 100% similarity with Agroathelia rolfsii sequences from GenBank by BLAST analysis. The sequences of two representative strains, ID1 and ID4, were deposited in GenBank. The ITS sequences of ID1 (OR689482) and ID4 (OR689481) were ï¼99% similar to A. rolfsii strain QJ7 (593/596 bp; MZ750983) and A. rolfsii strain Kale078 (565/568 bp; MN872304), respectively. Also, TEF-1α sequences of ID1 (OR713735) and ID4 (OR713736) were ï¼99% similar to the sequences of A. rolfsii strain HS-Sr (1073/1073 bp; OL416131) and A. rolfsii strain MSB1-2 (1070/1075 bp; MN702790), respectively. Phylogenetic analysis based on ITS and TEF1-α sequences indicated that ID1 and ID4 clustered into the A. rolfsii clade. Based on morphology and sequence analyses, the isolates ID1 and ID4 were identified as A. rolfsii (anamorph Sclerotium rolfsii). Pathogenicity tests were conducted three times on healthy 30-day-old cowpea seedlings. Five plants were inoculated with 6-day-old mycelial discs (6 mm) of ID1 or ID4 at the base of the seedlings (n = 30) while four plants were inoculated with a sterile PDA disc as a control (n = 12). All seedlings were cultivated in a greenhouse with a temperature of 26°C to 28°C and relative humidity 60% to 80% with a 14/10 h light/dark photoperiod. Eight days later, all the fungal inoculated seedlings showed symptoms including brown necrosis and collapse of the stems, and eventual withering of the seedlings. Control plants remained asymptomatic. The causal pathogens were reisolated in PDA plates and identified by ITS sequence analysis, completing Koch's postulates. To our knowledge, this is the first report of A. rolfsii causing southern blight on cowpea in China. Early accurate diagnosis will help farmers to adopt suitable practices to control disease outbreaks and reduce losses.