First Report of Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum on Pepper Fruits (Capsicum annuum) in Malaysia

Symptoms of water-soaked lesions and soft rot were first observed in June 2011 on bell pepper fruits (Capsicum annuum cv. Annuum) in the two main regions of pepper production in Malaysia (Cameron Highlands and Johor State). Economic losses exceeded 40% in severely infected fields and greenhouses with the estimated disease incidence of 70%. In pepper fruits damaged by insects, sunscald, or other factors, symptoms initially appeared in the peduncle and calyx tissues and entire fruits were turned into watery masses within 2 to 6 days. Fruits infected in the field tended to collapse and hang on the plant. When the contents leaked out, the outer skin of the fruit dried and remained attached to the plant. Field-grown transplants and infected soil were identified as probable sources of inocula. A total of 50 attached fruits were collected from 10 pepper fields and greenhouses located in the two growing regions. Tissue from the margins of water-soaked lesions was surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto nutrient agar (NA) and eosin methylene blue agar (EMB) media (3). A similar bacterium was isolated from all samples. After 2 days, white to creamy bacterial colonies on NA and emerald green colonies on EMB developed. Five independent strains were subjected to further biochemical, molecular, and pathogenicity tests. Bacterial strains were gram-negative, motile rods, grew at 37°C, were facultatively anaerobic, oxidase-negative, phosphatase-negative, and catalase-positive. They degraded pectate, were sensitive to erythromycin, did not utilize Keto-methyl glucoside, were indole production-negative, and reduced sugars from sucrose (3). Acid production was negative from sorbitol and arabitol, but positive from melibiose and citrate. PCR amplification of the pel gene by Y and Y primers produced a 434-bp fragment (2). Amplification of the intergenic transcribed spacer (ITS) region by G and L primers (4) gave two amplicons ca. 550 and 580 bp long. The expected amplicon was not produced with any of the strains using primers Br1f/L1r and Eca1f/Eca2r (1), whereas a 550-bp PCR product, typical of Pectobacterium carotovorum subsp. carotovorum, was obtained with primers EXPCCF and EXPCCR (1). Based on biochemical and molecular characteristics, and analysis of PCR-RFLP of 16S-ITS-23R rRNA genes using Rsa I enzyme (4), all five bacterial strains were identified as P. carotovorum subsp. carotovorum. BLAST analysis of the 16S rRNA sequ