Isolation and identification of the causal agent of gummy stem blight disease in Cucumis sativus caused by a bacterial pathogen in China

Abstract

Recently, a new bacterial disease was detected on cucumber stalks. In order to study the pathogenesis of this disease, the pathogenic bacteria were isolated and identified on the basis of morphological and molecular characteristics, and further analyzed for pathogenicity and antagonistic evaluation. Pathogenicity analysis showed that HlJ-3 caused melting decay and cracking in cucumber stems, and the strain reisolated from re-infected cucumber stalks was morphologically identical to HlJ-3 colonies, which is consistent with the Koch's postulates. The pathogenic strain HlJ-3 was identified as having similar morphological characteristics to Bacillus subtilis. Meanwhile, its internal transcribed spacer sequence (ITS) and DNA gyrase A subunit (gyrA) were both more than 99% homologous and clustered on the same branch with B. subtilis. Therefore, combined with morphological and molecular biological features, strain HlJ-3 was identified as B. subtilis. In addition, B. subtilis, which has a wide range of hosts, was able to infest other common crop species, including potato, tomato, pepper, melon, and radish. Furthermore, antagonistic evaluation confirmed that strain HlJ-3 strongly inhibited the mycelial growth of Colletotrichum coccodes and Alternaria tenuissima in vitro, with antagonistic effects of 69.92% and 68.08%, respectively. In conclusion, our results showed that strain HlJ-3 is B. subtilis, which is pathogenic to cucumber in vivo and can infect plants of Solanaceae, Cucurbitaceae and Brassicaceae with a wide range of hosts. In addition, this strain has good biocontrol effects against C. coccodes and A. tenuissima in vitro. The findings of this research will help to prevent and control the occurrence of this pathogen and regulate its use as a biocontrol agent.

Keywords: Bacillus subtilis; Cucumis sativus; Antagonistic function; Gummy stem blight disease; Identification; Isolation; Pathogenicity.

Fig. 1

In vitro antagonistic activity test method. Gray represents the pathogenic fungi tested for antagonistic activity

Fig. 2

A gelatinous secretion produced on the stalks and fruits of cucumber, found in the field. A–E symptoms on the surface of cucumber stems (the stems were wilted, yellowed and split). F, symptoms on the fruit of cucumber

Fig. 3

Pathogenicity of isolate HlJ-3 in cucumber stalks with surface wound inoculation (vivo inoculation tests). a, e, i, m and q represent in vivo controls with surface wounds that showed no disease symptoms; b, c, d, n, o and p represent yellowing and wilting of foliage, splitting of stalks, and yellowish brown of cucumber when cucumber stalks were inoculated with HlJ-3 by pinprick inoculation 10 days after inoculation; and j, k, l, r, s and t represent localised symptoms of cucumber stalks inoculated with HlJ-3 10 days after inoculation, w stand for the third pathogenicity, the leftmost stalk is CK.

Fig. 4

Pathogenicity of isolate HlJ-3 in cucumber stalks with surface non-wound inoculation (vivo inoculation tests). a and e, the control in vivo pathogenicity about surface non-wound inoculations, no disease symptoms appeared. b, c and d represent the disease status of the whole cucumber plant 15 days after inoculation with HlJ-3 under non-needling conditions; f, g and h represent the disease status of the inoculation site.

Fig. 5

Morphological characteristics of strain HlJ-3. a and g, represent morphological characteristics of the proto-bacteria isolated from field samples and inoculated into NA medium. b and h, Morphological characteristics of the pathogenic bacteria obtained after isolate HlJ-3 was inoculated onto Cucumber stalks and caused disease in Cucumber and then re-inoculated into NA medium. c and i, represent the morphological characteristics of the colonies obtained after inoculating the pathogen on LB plates. d and j represent the morphological characteristics of colonies inoculated on NB plates. e represents the characteristics of the pathogenic bacteria HlJ-3 after Gram staining. f represents the characteristics of the pathogenic bacteria HlJ-3 after Spore stain, and the direction of the arrow is the stained bacteriophage.

Fig. 6

Phylogenetic tree of B. subtilis strain HlJ-3 combined 16 S rRNA with gyrA. A amplifed by 16 S rRNA Primer; B amplifed by gyrA Primer.

Fig. 7

Lesions of infection by Bacillus subtilis HlJ-3 on melon, potato, tomato, corn, radish and chilli stems 14 days after surface wound and non-wound inoculation (a to f for needle inoculation, g to l for non-needle inoculation). m and n, models of different treatments for different crop species are in this figure. SW; Surface wound inoculation Bacillus subtilis HlJ-3. NW; Surface Non-wound inoculation Bacillus subtilis HlJ-3, CK (SW and NW) was inoculated with blank cultures.

Fig. 8

In vitro biocontrol of fungi by B. subtilis strain HlJ-3. The effects of HlJ-3 on C. coccodes (a1), A. tenuissima (a2), A. solani (a3), C. scovillei (a4), F. avenaceum (a5), F. verticillioides (a6), F. oxysporum (a7) for biological control. Pathogenic fungi and HlJ-3 were double-cultured on the same potato dextrose agar plate with a distance of 2.5 cm between them, and in the image, the pathogenic fungi are in the center, and HlJ-3 is around; a1 to a7 are negative controls, and the plates were cultured with a single pathogenic fungus. d1 to d2 are the effects of HlJ-3 on the mycelium of seven pathogenic fungi after standoff culture, respectively. c1 to c7 are mycelial controls, respectively.