First Report of Pectobacterium brasiliense Causing Soft Rot of Melon in China.

Abstract

In May of 2024, a stem soft rot disease in melon (Cucumis melo L.) was observed in Ningbo (29.52°N, 121.31°E), Zhejiang Province of China. The disease incidence was approximately 30% in two greenhouses (ca. 8 m × 50 m each). The soft rot yellowish-brown lesions developed on infected stems, soft rotted with a bad foul odor. To identify the pathogen, the diseased stems from four plants were surface sterilized by soaking in 75 % ethanol for 30 s, followed by rinsing three times in sterile distilled water (SDW). The stems were cut in 5 mm fragments and let stand in SDW. After 5 minutes 100 µL of the water was spread on nutrient agar (NA). After incubation at 28°C for 24 h, seven bacterial colonies were selected and individually transferred to new NA plates. Three strains (PB052001, PB052002 and PB052003) were randomly selected as representatives for further study. They were streak-inoculated on NA for formation of single colonies (28°C, 3 d). The colonies were white and round in shape with neat, slightly raised margin. Total genomic DNA was extracted from the three bacterial strains using EasyPure Bacterial Genomic DNA Kit (TransGen, Beijing). It was used as template for PCR amplification of 16S rDNA as well as the five house-keeping genes (pgi, proA, mtlD, mdh and icdA) using specific primers (Lane 1991; Ma et al. 2007; Waleron et al. 2008). The resulting DNA fragments were cloned and sequenced. The three bacterial strains had 100% identical DNA sequences at each of the six loci, therefore, only the DNA sequences from strain PB052001 were deposited in GenBank. Sequence analysis showed that the 16S rDNA in PB052001 (GenBank Acc No. PQ146559) was 99.67% (1501/1506 nt) and 99.60% (1500/1506 nt) identical to those in Pectobacterium brasiliense SR10 (CP084655) and 21PCA AGRO2 (CP113504), respectively. A multilocus phylogenetic analysis was done based on pgi, proA, mtlD, mdh and icdA from PB052001 (GenBank Acc Nos. PQ145967, PQ145968, PQ145969, PQ145970 and PQ145971, respectively). The result showed that the three strains clustered together with P. brasiliense. To confirm pathogenicity, a bacterial suspension of PB052001 (108 cfu/mL) was injected into stems (10 per treatment) of melon seedlings (cultivar Fengsu No.10) at 1 cm below the cotyledons, 10 µL for each plant. Ten seedlings was injected SDW as negative control. The treated seedlings were grown at 28°C under 80% relative humidity. After 24 h, while the control plants remained healthy, the plants inoculated with the bacteria showed soft rot symptoms. Additionally, the bacterial suspension was sprayed on melon leaves (10 per treatment), and SDW was sprayed on melon leaves as a control. The plants were incubated under the same conditions described above. After 3 d, the control leaves remained healthy, the leaves treated with the bacteria exhibited soft-rot symptoms. Bacteria were re-isolated from the diseased tissues, and morphological and molecular identification showed that it was identical to PB052001. P. brasiliense has a broad host range, especially in Cucurbitaceae such as cucumber and watermelon (Ma et al. 2007; Waleron et al. 2008). To our knowledge, this is the first report of P. brasiliense causing soft rot disease on melon in China. The finding expands our knowledge about the host range of P. brasiliense. This disease poses a threat to the local melon industry.