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 (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.

Bacterial fruit blotch (BFB) is a disease of cucurbit plants caused by Acidovorax citrulli. Although A. citrulli has great destructive potential, the molecular mechanisms of pathogenicity of A. citrulli are not clear, particularly with regard to its type III secreted effectors. In this study, we characterized the type III secreted effector protein, AopV, from A. citrulli strain Aac5. We show that AopV significantly inhibits reactive oxygen species and the expression of PTI marker genes, and helps the growth of Pseudomonas syringae D36E in Nicotiana benthamiana. In addition, we found that the aromatic dehydratase ADT6 from watermelon was a target of AopV. AopV interacts with ADT6 in vivo and in vitro. Subcellular localization indicated ADT6 and AopV were co-located at the cell membrane. Together, our results reveal that AopV suppresses plant immunity and targets ADT6 in the cell membrane. These findings provide an new characterization of the molecular interaction of A. citrulli effector protein AopV with host cells.

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.

Blueberry (Vaccinium spp.) has been cultivated in more than 20 provinces in China as reported in 2015. In July 2016, a root rot disease of blueberry with the whole plant died was found in Qujing (24°49′N; 103°59′E), Yunnan province. Approximately, 5% of the plants in the field showed symptoms characterized by leaf chlorosis, black rot on roots and stems, finally the whole plant wilted in about 20 days. In order to identify the causal agent of this disease, typical disease samples were collected. Twenty infected root tissues were sterilized and put on PDA at 25oC (Fang 1998) and 9 fungal isolates were obtained. The isolate YNQJ01Aa was chosen for further testing. Its colony on PDA was white to pale yellow. The conidia were collected from SNA colony after growing 10 d. Conidia presented with one to three septa (mostly one septa). Fifty conidia from one isolate were measured (59.37 to 71.33) × (5.93 to 7.91) μm, (mean = 62.25 µm× 6.22 µm). Ascospores were produced in perithecia on PDA and were (30.16 to 38.89) µm× (5.78 to 7.76) µm (mean = 34.97 µm× 6.52 µm). By DNA sequencing of the internal transcribed spacer (ITS) rDNA region (GenBank Accession No. MF785081), the identity of the isolate (YNQJ01Aa) was confirmed to be the genus Calonectria (99% homologous to several Calonectria spp. strains). Partial β-tubulin (TUB) gene sequence was also analyzed (GenBank Accession No. MF785082) and the result of BLAST search showed that it was 100% homologous to C. ilicicola strain (CBS 190.50). Further species identify was analyzed by phylogenic tree based on TUB (partial β-tubulin gene, T1/Bt2b ), tef1 (translation elongation factor 1 alpha gene, EF1-728F/EF1-986R) and HIS3(histone 3 gene, H3-1a/H3-1b) (Crous 2010 ; Chen et al 2011). Sequences obtained were deposited in GenBank (GenBank Accession No. TUB-MF785082, tef1-MF785083, HIS3-MF785084). Alignments were made using CLUSTAL X v.2.0, and a maximum likelihood phylogram was generated by MEGA v.7.0. Isolate YNQJ01Aa clustered together with C. ilicicola reference strain CBS190.50 (98% bootstrap). Three healthy 1-year-old ‘O’Neal’ blueberry plants were used to conduct pathogenicity tests by wound inoculation. Each plant was inoculated with 10 ml conidium suspension (1 × 105 conidia/ml) by root-irrigation method. Three plants were inoculated with the same amount of sterile water as control. All blueberry plants were incubated at 25 °C. After 20 days, the test plants showed typical symptom of black root rot and finally the whole plant wilted and died, while the control plants remained healthy. The same colonial fungus could be reisolated. It is the first report that C. ilicicola (anamorph Cylindrocladium parasiticum) can infect blueberry in China. Root rot disease can be difficult to detected early and therefore it is important for identification of etiology to develop appropriate disease management strategies. This fungus was previously cited on Medicago sativa in China (Pei et al. 2015). References: Z. D. Fang. Research Methods of Plant Disease. Agriculture Press, Beijing, China. 1998. P. W. Crous. In: Taxonomy and pathology of Cylindrocladium (Calonectria) and allied genera, APS Press, St. Paul, Minnesota, 2002. S. F. Chen et al. Persoonia Molecular Phylogeny & Evolution of Fungi, 26: 1, 2011. W. H. Pei et al. Plant disease, 99: 890, 2015.

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.