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Bacterial fruit blotch (BFB) is a devastating disease caused by Acidovorax citrulli, severely impacting the watermelon and melon industries and leading to significant economic losses. Currently, researchers have not identified any commercial melon varieties with high resistance to BFB, and farmers primarily rely on chemical agents for prevention and control. However, the extensive use of these agents contributes to increased drug resistance among pathogenic bacteria, making it essential to develop environmentally friendly control methods. To explore the feasibility of combining the Bacillus velezensis ZY1 strain with chemical agents for BFB management, we assessed the efficacy of 10 bactericides and the ZY1 strain against Acidovorax citrulli. The results show that Prothioconazole, Zhongsheng Tetramycin Solution, Streptomycin Sesquisulfate, Tetramycin, Zhongshengmycin, and ZY1 exhibited significant inhibitory effects on the growth of Acidovorax citrulli. We determined the biocompatibility of the bactericides with the ZY1 strain using the plate confrontation method and the flat counting method. Zhongsheng Tetramycin Solution, Zhongshengmycin, and Kasugamycin exhibited good compatibility with the ZY1 strain. Additionally, we established the optimal compounding ratio of the bactericide and ZY1 using the Horsfall method. Among these, Zhongshengmycin demonstrated the best performance when combining its efficacy against Acidovorax citrulli with its biocompatibility with the ZY1 strain. The combination of Zhongshengmycin and ZY1 at a volume ratio of 5:5 significantly inhibited Acidovorax citrulli, exhibiting a clear synergistic effect with a synergy index (IR) value of 1.542. Field tests conducted over 21 days in Beijing greenhouses, Hainan Field facility greenhouses, and Xinjiang showed that the control efficacy of the Zhongshengmycin and ZY1 combination (89.23%) significantly surpassed that of the single agent Zhongshengmycin (80.35%) and the biocontrol bacterium ZY1 (72.12%). Notably, the application rate of Zhongshengmycin in the mixture was only half that of the single agent, resulting in a significant reduction in chemical usage. The combination of Bacillus velezensis ZY1 and Zhongshengmycin not only decreases chemical usage but also significantly enhances control efficacy compared to using Zhongshengmycin alone.

Acidovorax citrulli populations exhibit genetic and phenotypic variations, particularly in terms of copper tolerance. Group I strains of A. citrulli generally exhibit higher copper tolerance compared to group II strains. This study aims to identify genes involved in copper tolerance to better understand the differences in copper tolerance between group I and group II strains. Representative strains pslb65 (group I) and pslbtw14 (group II) were selected for comparison. Deletion mutants of putative copper-tolerance genes and their corresponding complementary strains were constructed. The copper tolerance of each strain was evaluated using the minimum inhibitory concentration method. The results showed that the copA, copZ, cueR, and cueO genes played major roles in copper tolerance in A. citrulli, while cusC-like, cusA-like, and cusB-like genes had minor effects. The different expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 under copper stress indicated that they had different mechanisms for coping with copper stress. Overall, this study provides insights into the mechanisms of copper tolerance in A. citrulli and highlights the importance of specific genes in copper tolerance.

Acidovorax citrulli can cause bacterial fruit blotch of watermelon, melon, and other cucurbits, and has the potential to cause severe economic losses to growers throughout the world. This article investigated the functions and interactions of the pilR and pilS genes, two important genes in bacterial type IV pili systems, in A. citrulli. For each gene, deletion mutants and complementary strains were constructed via homologous recombination, and their phenotypes were determined. The results showed that the absence of pilR and pilS could significantly reduce the pathogenicity and twitching motility of A. citrulli while increasing the swimming motility, biofilm formation, and in vitro growth. Conversely, complementary strains were no different than the wild-type strain. Using quantitative reverse transcription PCR and promoter activity assays, we confirmed that the deletion of pilR and pilS genes leads to a significant decrease in the transcription level of pilA. Meanwhile, three methods including yeast two-hybrid, glutathione S-transferase pull-down, and luciferase complementation imaging assays were used to verify the direct interaction between the PilR and PilS proteins. These findings revealed the biological function of the pilR and pilS and confirms their regulatory role on pilA.

Acidovorax citrulli, the causative agent of bacterial fruit blotch, can be divided into two main groups based on factors such as pathogenicity and host species preference. PilA is an important structural and functional component of type IV pili (T4P). Previous studies have found significant differences in pilA DNA sequences between group I and group II strains of A. citrulli. In this study, we characterized pilA in the group I strain pslb65 and the group II strain Aac5. pilA mutants, complementation strains, and cross-complementation strains were generated, and their biological phenotypes were analyzed to identify functional differences between pilA in the two groups. pilA deletion mutants (pslb65-ΔpilA and Aac5-ΔpilA) showed significantly reduced pathogenicity compared with the wild-type (WT) strains; pslb65-ΔpilA also completely lost twitching motility, whereas Aac5-ΔpilA only partially lost motility. In King’s B medium, there were no significant differences in biofilm formation between pslb65-ΔpilA and WT pslb65, but Aac5-ΔpilA showed significantly reduced biofilm formation compared to WT Aac5. In M9 minimal medium, both mutants showed significantly lower biofilm formation compared to the corresponding WT strains, although biofilm formation was recovered in the complementation strains. The biofilm formation capacity was somewhat recovered in the cross-complementation strains but remained significantly lower than in the WT strains. The interspecies competitive abilities of pslb65-ΔpilA and Aac5-ΔpilA were significantly lower than in the WT strains; Aac5-ΔpilA was more strongly competitive than pslb65-ΔpilA, and the complementation strains recovered competitiveness to WT levels. Furthermore, the cross-complementation strains showed stronger competitive abilities than the corresponding WT strains. The relative expression levels of genes related to T4P and the type VI secretion system were then assessed in the pilA mutants via quantitative PCR. The results showed significant differences in the relative expression levels of multiple genes in pslb65-ΔpilA and Aac5-ΔpilA compared to the corresponding WT stains. This indicated the presence of specific differences in pilA function between the two A. citrulli groups, but the regulatory mechanisms involved require further study.

Acidovorax citrulli, the causal agent of bacterial fruit blotch, can be divided into two groups. Group I is mainly isolated from melon, and group II is mainly isolated from watermelon. In this study, group I and II strains were used to assess competition in vivo and in vitro by evaluating inhibition activity assays and by measuring population growth dynamics. Our results indicated that there is no mutual inhibition of growth between the two groups of strains in King’s B medium. The bacterial populations of A. citrulli strains were counted within 7 days after inoculation in melon and watermelon cotyledons and plotted against time to generate population growth curves. Area under the population growth curves was calculated. When the two groups of strains were inoculated separately into melon and watermelon cotyledons, the results of analysis of variance show that there was no significant difference. In this study, for the first time in an experimental setting, we inoculated two A. citrulli groups into melon and watermelon cotyledons at the same time and determined the population dynamics of each strain. The results showed that there was no significant difference between two group strains in melon cotyledons. However, in watermelon cotyledons, the area under population growth curves of group II strain were significantly higher than those of group I strain. Three-way analysis of variance results showed that there was interaction between host and grouping on the influence of strain population level (p < 0.0001). These data provide additional information on the host preference of different groups of A. citrulli.

Bacterial fruit blotch caused by Acidovoraxcitrulli is a serious disease of cucurbit crops. Here we report characterization of a mutant strain of A. citrulli M6 defective in lip1, a gene encoding a lipolytic enzyme. The M6-lip1- mutant was detected in a mutant library screen aimed at identifying M6 mutants with altered levels of twitching motility. In this screen M6-lip1- was the only mutant that showed significantly larger twitching motility haloes around colonies than wild-type M6. Sequence analyses indicated that lip1 encodes a member of the GDSL family of secreted lipolytic enzymes. In line with this finding, lipolytic assays showed that the supernatants of M6-lip1- had lower lipolytic activity as compared with those of wild-type M6 and a lip1-complemented strain. The mutant was also affected in swimming motility and had compromised virulence on melon seedlings and on Nicotiana benthamiana leaves relative to wild-type and complemented strains. Lip1 contains a predicted N-terminal signal sequence for type II secretion. Evidence from our study confirms Lip1 is indeed secreted in a type II secretion-dependent manner, and this is required for full virulence of A. citrulli. To the best of our knowledge this is the first study reporting contribution of lipolytic activity to virulence of a plant-pathogenic Acidovorax species.

Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB), is an economically important pathogen of watermelon and related plant species worldwide. In the period 2014–2018, several outbreaks of BFB were observed in major watermelon production regions in Serbia. A total of 43 strains, isolated from symptomatic watermelon tissue, were analyzed by biochemical, pathogenic and molecular tests. Based on the phenotypic characteristics, PCR assay, and 16S rRNA gene sequence analysis, all strains were identified as A. citrulli. A multilocus sequence analysis of the four housekeeping (adk, gyrB, pilT and gltA) and three virulence genes (Aave_1548, avrRx01 and luxR) revealed that studied A. citrulli strains represent the homogeneous population and they clustered together with group II reference strain AAC00-1. Strain affiliation to group II was confirmed by PCR based on the putative type III secretion effector gene and by duplex PCR test. High homogeneity of studied strains was also confirmed by BOX-PCR. Differences were observed for two strains in their pathogenicity as well as susceptibility to copper compounds. Moreover, six major watermelon varieties grown in Serbia showed high sensitivity to the pathogen, while cross inoculation assay revealed that the strains were able to infect other species within the Cucurbitaceae family.

Bacterial fruit blotch caused by Acidovorax citrulli is known to be the major threat to cucurbit crop production worldwide. The pathogen can penetrate into seed coat and cause disease symptoms at any stage of plant growth, which results in fruit loss. Two main genotypes (genotype I and II) are reported in A. citrulli, in which genotype II is the main cause of Bacterial Fruit Blotch (BFB) in watermelon and group I is known to be a causal agent of BFB in melon. To date, there are no commercially available cultivars resistant to BFB, and available strategies are not able to completely manage the disease. In this study, we aim to isolate bacteriophages to control BFB. Samples collected from watermelon, melon, and pumpkin were used to isolate bacteriophages. All isolated bacteriophages were tested against 42 strains of A. citrulli, among which two phages with the ability to lyse a greater number of hosts were selected and characterized. Bacteriophage ACP17 from the Myoviridae family, with a head size of 100 ± 5 nm and tail of 150 ± 5 nm, infected 29 strains of A. citrulli mostly belonging to genotype group I, whereas the second isolated bacteriophage, ACPWH from Siphoviridae, with a head size of 60 ± 5 nm and tail of 180 ± 5 nm, infected 39 A. citrulli strains. Genome analysis of both bacteriophages using Next generation Sequencing (NGS) showed that ACP17 and ACPWH have double-stranded DNA with sizes of 156,972 kb and 424,299 kb, respectively. Watermelon seeds coated with ACPWH showed a germination rate of up to 90% in the presence of A. citrulli in contrast to untreated seed, which showed no germination or germinated juveniles with BFB symptoms in the presence of A. citrulli. The results of this study show that the use of bacteriophages of A. citrulli represents a potential biocontrol method for controlling BFB.

Bacterial fruit blotch (BFB) is an economically important disease in melons and watermelons for which no effective control method is available. Application of phytobacterium-infecting phage has been evaluated as an alternative means of preventing bacterial diseases in plants. Coating of seeds with bacteriophages infecting Acidovorax citrulli, the causal agent of BFB, is effective for controlling the disease, as shown in our previous study. We evaluated the transport of bacteriophage ACPWH from soil to the leaves of melon plants, and we also evaluated its effect on BFB. Leaves of melon plants were spray-inoculated with A. citrulli, and bacteriophage ACPWH was added to soil after symptoms had developed. ACPWH was detected by PCR in foliar tissue 8 h after addition to soil. DAPI-stained ACPWH accumulated at the leaf tip after 24 h. Melon treated with ACPWH showed 27% disease severity, compared to 80% for the non-treated control, indicating that ACPWH can be used to control BFB.