First Report of Pseudomonas oryzihabitans Causing Pith Necrosis on Pepper in Hainan Provinces, China.

Abstract

Peppers (Capsicum annuum L.) are among the most widely consumed vegetable globally and cultivated in large areas in China (Zhou and Zhou 2021). In December 2023, pepper pith necrosis was discovered in a field located in Haiwan Town, Haitang District, Sanya City, Hainan, China (109.69°N, 18.35°E), with an incidence of 15%. The lower leaves of diseased plants exhibited yellowing, and the stem epidermis degenerated, revealing aberrantly thickened and browning vascular tissue. Additionally, a small number of adventitious roots were observed on the stem. When main stems were longitudinally cut, a brown and disintegrated, partially hollow pith was observed. To isolate and identify the causal agent of pith necrosis in peppers, the border tissue between diseased and healthy areas was excised, sterilized by immersion in 70% ethanol for 1 minute, and then washed three times with sterile water. After drying, the treated tissues were cut into small pieces measuring 5 x 5 mm and cultured on Luria-Bertani agar (LB) medium. Following incubation at 28°C for 48 hours, isolation and purification of the bacteria obtained from the cultures were continued. After several purification, the colonies appeared yellow, circular, and smooth-margined. The cells obtained from colonies were negative for Gram staining and rod-shaped under the optical microscope. They were positive for catalase and urease but negative for oxidase and amylase, and were able to utilize L-arabinose, D-glucose and D-mannitol. Morphological, physiological and biochemical characteristics of the isolates resembled that of Pseudomonas sp.. To verify the species identity of the bacteria, genomic DNA from the isolated strain HN6 was extracted, and the universal primers 27F/1492R (Lane 1991) were used to amplify the 16S rRNA gene, while specific primers LAPS27/VIC4 (Mulet et al. 2010) and gyrBPF/gyrBPR (Yamamoto et al. 2000) were used to amplify the rpoB and gyrB genes. The BLASTn analysis of 16S rDNA, rpoB, and gyrB sequences showed strain HN6 (PQ374171, PQ393140, and PQ393141) had 100.00, 98.94% and 98.97% identity with P. oryzihabitans(CP044074). The phylogenetic analysis ( Mega 11 ) indicates that strain HN6 is clustered with P. oryzihabitans into one branch with Bootstrap supported 100 using the neighbor-joining method with 5,000 bootstrap replicates, further confirms that strain HN6 is most closely related to P. oryzihabitans. Therefore, strain HN6 was identified as P. oryzihabitans based on morphology, metabolic profile, and sequence analysis. Peppers were planted in non-porous pots containing sterilized substrates for the pathogenicity test. Sterile syringes were used to prick the stems near the roots of the peppers. The wounds were wrapped with sterile defatted cotton dipped in bacterial suspension ( 108 CFU / mL ). As a control, sterile defatted cotton soaked in sterile water was applied. Subsequently, the pots were covered with a plastic film to prevent contamination and moisturize. The temperature was maintained above 28°C, and moisture levels were kept consistent to support plant growth. Each treatment had 6 replicates. The 10-15 days post-treatment group showed symptoms at the inoculation site, including epidermal degeneration, thickened and browned vascular tissue and a disintegrated, hollow pith in the stems, while no symptoms were observed in the controls. Koch's postulates were fulfilled through the reisolation of the pathogen from infected tissues and subsequently identified as the same pathogenic bacterium using the aforementioned methods. While Pseudomonas syringae pv. syringae has been reported as the causative agent of leaf spot disease in peppers (Miller et al. 2010), this study presents the first report of P. oryzihabitans causing pith necrosis on pepper worldwide. P. oryzihabitans can cause rice panicle blight and grain discoloration (Hou et al. 2020), and has also found to caused center blackening disease (Choi et al. 2019) as well as stem and leaf rot (Li et al. 2021 ) on muskmelon. Pseudomonas sp. can causing leaf spot disease in peppers (Miller et al. 2010). As far as we know, this is the first Report of P. oryzihabitans causing pith necrosis on Pepper in China.