Pseudomonas cichorii causing leaf spot disease on Banxia (Pinellia ternata) in China.

Abstract

Banxia (Pinellia ternata) is an important Chinese medicinal material in the family Araceae and is a widely grown herb in China. In September 2021, a leaf spot disease was observed on Banxia field, with an incidence rate of 35 to 40 % in a 4-ha field, in Zhongxiang City, Hubei Province of China. Symptoms were observed as yellow-white centers, water-soaked edges, irregular lesions, and gradually developed into a yellowish-brown center and a dark-brown edge. Necrotic spots gradually increased, leading to leaf chlorosis and plant death. Margins of leaf lesions were excised form diseased tissue and were plated on nutrient agar (NA) using serial dilution. Growth on NA was predominantly cream-colored circular bacterial colonies with undulated margins. Characterization of three randomly chosen bacterial isolates (JYB1, JYB7 and JYB8) suggests they are Gram-negative, levan negative, arginine dihydrolase negative, oxidase positive, potato soft rot positive, and tobacco hypersensitive positive. Isolates were identified as Pseudomonas cichorii based on the LOPAT scheme (Cottyn et al. 2009). Taxonomic positioning was confirmed genetically by PCR analysis using primers set: 16S rRNA gene universal primers 27F/1492R (Weisburg et al. 1991) and hrcRST gene specific primers Hcr1/Hcr2 (Cottyn et al. 2011). Homology search of 16S rRNA gene sequences (GenBank accessions: JYB1, MZ749668; JYB7, MZ823822; and JYB8, MZ823823) indicated 99.93 % (1396 bp) identity with P. cichorii strains (GenBank accessions: MK356431, JX913785, MZ723344). Similarly, comparison of the hrcRST locus (GenBank accessions: JYB1, MZ977010; JYB7, MZ977011; and JYB8, MZ977012) shared 99.38% (812 bp) with P. cichorii strains (GenBank accessions: CP007039, CP074349, GU324131). Koch's postulate was performed on healthy 30-day-old Banxia plants to confirm pathogenicity of the isolated strains. Leaves were injected with 50 μL bacterial suspensions (1x108 cfu/ml) by sterile syringe. The negative control was inoculated with sterile water. The inoculated Banxia plants were incubated at 28 °C, 70 to 80 % relative humidity, and exhibited water-soaked lesions on the leaf surface within two days around the inoculation sites. Within seven days, all leaves withered and plants died. In contrast, control plants remained healthy and symptomless. The pathogen was consistently reisolated from diseased plants and morphologically and molecularly identified as P. cichorii, while no bacterial colonies were isolated from the control plants, fulfilling Koch's postulates. To our knowledge, this is the first report of bacterial leaf spot caused by P. cichorii on Banxia in China. As one of the main producing areas of Banxia in China, Jianghan Plain of Hubei Province has a planting area of nearly 20 square kilometers. The occurrence of this bacterial disease has the potential threat to the Banxia industry, more research is needed for breeding disease resistance and for developing chemical control.