Napthoquinones from Neocosmospora sp.—Antibiotic Activity against Acidovorax citrulli, the Causative Agent of Bacterial Fruit Blotch in Watermelon and Melon

Bacterial fruit blotch (BFB) is a bacterial disease that devastates Cucurbitaceae crops worldwide, causing significant economic losses. Currently, there is no means to treat or control the disease. This study focused on exploring the antibacterial properties of endophytic fungi against Acidovorax citrulli (Aac), the causative agent of BFB. Based on disc diffusion, time kill and MIC microdilution broth assays, four endophytes showed promise in controlling Aac. Nonetheless, only one strain, Neocosmospora sp. MFLUCC 17-0253, reduced the severity of disease on watermelon and melon seedlings up to 80%. Structure analysis revealed production of several compounds by the fungus. Three of these secondary metabolites, including mixture of 2-methoxy-6-methyl-7-acetonyl-8-hydroxy-1,4-maphthalenedione and 5,8-dihydroxy-7-acetonyl-1,4-naphthalenedione, anhydrojavanicin, and fusarnaphthoquinones B exhibited antagonistic activity against Aac. The chemical profile data in planta experiment analyzed by LC-Q/TOF-MS suggested successful colonization of endophytic fungi in their host plant and different metabolic profiles between treated and untreated seedling. Biofilm assay also demonstrated that secondary metabolites of Neocosmospora sp. MFLUCC 17-0253 significantly inhibited biofilm development of Aac. To the best of our knowledge, secondary metabolites that provide significant growth inhibition of Aac are reported for the first time. Thus, Neocosmospora sp. MFLUCC 17-0253 possesses high potential as a biocontrol agent for BFB disease.

Etiology, diagnostic approaches and management strategies of Acidovorax citrulli, a bacterial fruit blotch pathogen of cucurbits

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli, represents one of the most destructing diseases of cucurbits, especially to watermelon- and melon producing-regions. This disease has been spread sporadically to many countries globally, due to the unintentionally dispersal of contaminated commercial seeds. The BFB causes massive yield losses up to 100% under conducive conditions. Once infected, all parts of the host plants are extremely susceptible to this bacterium, especially the seedlings and fruits parts. In recent years, various management approaches and detection tools have been employed to control A. citrulli. Genotypic characterization methods revealed two distinct groups of A. citrulli strains; (i) group I strains primarily isolated from non-watermelon cucurbits and consist of moderate to highly aggressive strains from wide range of cucurbit hosts, and (ii) group II strains isolated from watermelon which are highly aggressive on watermelon, but mildly aggressive on non-watermelon hosts. In this paper, an attempt has been made to review research findings where the impact of diverse methods and management approaches were applied in detection and controlling of A. citrulli infection. A better understanding of this devastating bacterium will serve as guidelines for agricultural practitioners in developing the most efficient and sustainable BFB control strategies.

Comprehensive genomic analysis of Bacillus subtilis 9407 reveals its biocontrol potential against bacterial fruit blotch

Bacillus subtilis, a plant-beneficial bacterial species exhibiting good biocontrol capabilities, has been widely used in agricultural production. The endophytic strain 9407 can efficiently control bacterial fruit blotch (BFB) caused by the gram-negative bacterium Acidovorax citrulli. However, the mechanism underlying its biocontrol ability remains poorly understood. Given the genomic diversity of B. subtilis, strain 9407 was sequenced and assembled in this study to determine the genome information associated with its biocontrol traits. A combination of core genome phylogenetic analysis and average nucleotide identity (ANI) analysis demonstrated that the 9407 strain belonged to B. subtilis. Various functional genes related to biocontrol traits, i.e., biofilm formation, motility, pathogen inhibition, plant growth promotion, and induction of systemic resistance, were identified in B. subtilis 9407. Four secondary metabolite biosynthesis gene clusters with antibacterial ability were also found in the B. subtilis 9407 genome, including newly identified subtilosin A, bacilysin, and bacillaene, and the previously reported surfactin. Mutants lacking sboA or bacG, which are defective in synthesizing subtilosin A or bacilysin, showed decreased inhibitory activity against A. citrulli MH21, and the triple mutant with deleted sboA, bacG, and srfAB almost completely lost its inhibitory activity. The biofilm formation and swarming motility of the sboA and bacG mutants also decreased, in turn leading to decreased colonization on melon roots and leaves. Under greenhouse conditions, the biocontrol efficacy of the sboA and bacG mutants against BFB on melon leaves decreased by 21.4 and 32.3%, respectively. Here, we report a new biocontrol pathway of B. subtilis 9407 against BFB, in which subtilosin A and bacilysin contributed to the biocontrol efficacy by improving antibacterial activity and colonization ability of the strain. The comprehensive genomic analysis of B. subtilis 9407 improves our understanding of the biocontrol mechanisms of B. subtilis, providing support for further research of its biocontrol mechanisms and field applications.

Fermentation: an Unreliable Seed Treatment for Bacterial Fruit Blotch of Watermelon

Acidovorax citrulli is a seed-borne pathogen that causes bacterial fruit blotch (BFB), a global threat to watermelon production. Treating watermelon seeds to eliminate A. citrulli is a critical component of BFB management and several strategies have been evaluated to mitigate the impact of the disease. In China, watermelon seed producers routinely incubate seeds in watermelon juice (fermentation) to reduce the risk of seed infection by A. citrulli and seedling transmission of BFB. However, there has been limited effort to evaluate the efficacy of fermentation in mitigating A. citrulli seed infection. The current study first showed that fermented watermelon fruit juice could inhibit A. citrulli population growth, and further demonstrated that the low pH conditions, not the dynamic of temperature, generated during the fermentation process might play a major role in inhibition of growth of A. citrulli and could induce the viable but non-culturable (VBNC) state in A. citrulli. We developed an effective method that was based on PMA-PCR to detect viable A. citrulli cells under low pH conditions or in fermented watermelon fruit juice. We also provided evidence that VBNC A. citrulli cells induced by fermented watermelon fruit juice could not be resuscitated and did not retain their virulence on watermelon seedlings. However, VBNC A. citrulli cells could be resuscitated in LB medium. Based on these observations, we conclude that fermentation in watermelon fruit juice may not be an effective seed treatment for BFB as it may increase the seed infection by A. citrulli.

Acidovorax citrulli Type III Effector AopP Suppresses Plant Immunity by Targeting the Watermelon Transcription Factor WRKY6

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB), and BFB poses a threat to global watermelon production. Despite its economic importance, the molecular mechanisms underlying Ac pathogenicity and virulence are not well understood, particularly with regard to its type III secreted effectors. We identify a new effector, AopP, in Ac and confirm its secretion and translocation. AopP suppresses reactive oxygen species burst and salicylic acid (SA) content and significantly contributes to virulence. Interestingly, AopP interacts with a watermelon transcription factor, ClWRKY6, in vivo and in vitro. ClWRKY6 shows typical nuclear localization, and AopP and ClWRKY6 co-localize in the nucleus. Ac infection, SA, and the pathogen-associated molecular pattern flg22Ac promote ClWRKY6 production, suggesting that ClWRKY6 is involved in plant immunity and SA signaling. Furthermore, ClWRKY6 positively regulates PTI and SA production when expressed in Nicotiana benthamiana. Importantly, AopP reduces ClWRKY6 mRNA and ClWRKY6 protein levels, suggesting that AopP suppresses plant immunity by targeting ClWRKY6. In summary, we identify a novel effector associated with the virulence mechanism of Ac, which interacts with the transcription factor of the natural host, watermelon. The findings of this study provide insights into the mechanisms of watermelon immune responses and may facilitate molecular breeding for bacterial fruit blotch resistance.