A type VI secretion system (T6SS) gene cluster has been reported in Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Δhcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Δtssm and double mutant ΔhcpΔtssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Δhcp and Δtssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Δhcp was delayed in KB medium. Biofilm formation ability of Δhcp, Δtssm and ΔhcpΔtssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe³⁺ medium. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Δhcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Nitrogen, one of the most important limiting elements in the environment, is necessary for the growth and reproduction of bacteria. As a nitrogen-regulating gene, ntrC plays an important role in maintaining bacterial nitrogen utilization and biological nitrogen fixation. However, the role of ntrC has not been determined for A. citrulli. In this study, we constructed a ntrC deletion mutant and a corresponding complementary strain in the background of the A. citrulli wild-type strain, Aac5. Through phenotype assays and qRT-PCR analysis, we investigated the role of ntrC in A. citrulli in nitrogen utilization, stress tolerance, and virulence against watermelon seedlings. Our results showed that the A. citrulli Aac5 ntrC deletion mutant lost the ability to utilize nitrate. The ntrC mutant strain also exhibited significantly decreased virulence, in vitro growth, in vivo colonization ability, swimming motility, and twitching motility. In contrast, it displayed significantly enhanced biofilm formation and tolerance to stress induced by oxygen, high salt, and copper ions. The qRT-PCR results showed that the nitrate utilization gene nasS; the Type III secretion system-related genes hrpE, hrpX, and hrcJ; and the pili-related gene pilA were significantly downregulated in the ntrC deletion mutant. The nitrate utilization gene nasT, and the flagellum-related genes flhD, flhC, fliA, and fliC were significantly upregulated in the ntrC deletion mutant. The expression levels of ntrC gene in the MMX-q and XVM2 media were significantly higher than in the KB medium. These results suggest that the ntrC gene plays a pivotal role in the nitrogen utilization, stress tolerance, and virulence of A. citrulli.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is a devastating watermelon disease that severely impacts the global watermelon industry. Like other Gram-negative bacteria, the type three secretion system (T3SS) is the main pathogenicity factor of A. citrulli. The T3SS apparatus gene hrpE codes for the Hrp pilus and serves as a conduit to secret effector proteins into host cells. In this study, we found that the deletion of hrpE in A. citrulli results in the loss of pathogenicity on hosts and the hypersensitive response on non-hosts. In addition, the A. citrulli hrpE mutant showed a reduction in in vitro growth, in planta colonization, swimming and twitching motility, and displayed increases in biofilm formation ability compared to the wild type. However, when HrpE was transiently expressed in hosts, the defense responses, including reactive oxygen species bursts, callose deposition, and expression of defense-related genes, were activated. Thus, the A. Citrulli growth in HrpE-pretreated hosts was suppressed. These results indicated that HrpE is essential for A. citrulli virulence but can also be used by hosts to help resist A. citrulli. Our findings provide a better understanding of the T3SS pathogenesis in A. citrulli, thus providing a molecular basis for biopesticide development, and facilitating the effective control of BFB.

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.

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.