First report of Pantoea dispersa causing strawberry root rot in China.

Abstract

China is the largest strawberry (Fragaria x ananassa) producer worldwide. In 2022 the area devoted to strawberry planting in China was 147.45 thousand hectares. Hebei province is the fourth-largest strawberry planting province in China, where strawberries play a crucial role in the agricultural economy. In November 2023, root rot was observed on strawberry plants (cultivar Benihoppe) in Xushui County (115°35'59″ E, 38°58'39″ N), Hebei province, affecting overall growth and reproduction. Disease incidence in two greenhouses (0.15 ha in size) was up to ~70%, with severe symptoms causing almost complete plant mortality. The initial symptoms included abnormally small new leaves, followed by wilting and collapse of the plants. As the disease progressed, the leaves curled and withered, ultimately leading to plant death. Under humid conditions, a sticky bacterial ooze was observed exuding from the stem base. Dissection of the stem base of diseased plants revealed reddish-brown discolored roots while the roots of healthy plants were white. To isolate the causal agent, 10 root samples from symptomatic plants were randomly selected from two greenhouses and surface sterilized sequentially in absolute ethanol (1 min), 3.125% NaOCl (6 min), and absolute ethanol (30 s), followed by rinsing in sterile water three times (Sahu et al. 2022). Root pieces were then placed on Luria-Bertani (LB) agar and incubated at 25℃ in darkness for 3 days. After isolation of bacteria from the symptomatic tissues, subculturing onto new media was performed to obtain pure cultures. The 10 bacterial isolates selected for further analysis exhibited a similar morphology of circular and smooth colonies that were initially milky white and later were yellow-pigmented. Three representative isolates (CM2402, CM2403, and CM2405) were selected for 16S rDNA sequencing using primers 27F/1492R (27F: 5'-AGAGTTTGATCMTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3') (Lane 1991). BLASTn analysis revealed that the three sequences were identical, with 99.86% (1,420/1,422 bp) identity to Pantoea sp. strain nts-7 (OR392975.1). Biochemical analysis confirmed that the isolates belong to the genus Pantoea, testing positive with the Voges-Proskauer test for production of acetylmethyl carbinol from glucose fermentation, utilization of citrate, D-mannose, inositol, and malonate, positive for β-galactosidase while negative for indole production, phenylalanine deaminase, lysine decarboxylase, urease, H₂S production, and acid production from raffinose, adonitol, and D-sorbitol (Gavini et al. 1989). Five housekeeping genes (gyrB, fusA, leuS, pyrG, rplB) were amplified and sequenced for further molecular identification (Delétoile et al. 2009). A phylogenetic tree based on the combined sequences of these genes was constructed using the neighbor-joining method, with the isolates clustering with P. dispersa. No sequence variation was detected among the isolates CM2402, CM2403, and CM2405 in the five genes. The sequences of the five housekeeping genes and the 16S rDNA from one representative isolate (CM2405) were deposited in GenBank under accession numbers: PQ100709 (gyrB), PQ100708 (fusA), PQ059271 (leuS), PQ100710 (pyrG), PQ100711 (rplB), and PQ095944 (16S rDNA). To fulfill Koch's postulates, pathogenicity tests were performed on 10 1-month-old strawberry plants (cultivar Benihoppe) with three independent replicates per treatment. The soil was shaken off of roots which were then soaked in bacterial suspensions of isolates CM2402, CM2403, and CM2405 (10⁷ cfu/mL) for 10 minutes. Strawberry plants were planted into sterilized soil and cultured at 25℃ under a 16 h light and 8 h dark photoperiod. Strawberry plants with roots soaked in sterilized water were used as the control. Four weeks after transplanting, the strawberry plants soaked with bacterial suspensions exhibited poor vigor and root necrosis, while the control plants remained symptomless. The pathogen reisolated from necrotic tissue was morphologically and molecularly identical to the original strain. These results confirmed P. dispersa as the causal agent of strawberry root rot in China. P. dispersa has been reported as a pathogen of rice in India and Malaysia (Jena et al. 2023; Toh et al. 2019), causing bulb decay in onion in Taiwan (Chang et al. 2018), and brown blotch disease in Flammulina filiformis in China (Hu et al. 2022). To our knowledge, this is the first report of P. dispersa causing strawberry root rot. This research is critical for effective management, facilitating early detection, informing targeted control measures, and preventing further disease spread, ultimately reducing crop losses and improving sustainability.