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Here, we report the draft genome sequence of Xylella fastidiosa strain ATCC 35873, which was obtained from the American Type Culture Collection and was originally isolated from a symptomatic American elm tree grown in Washington, DC. The ATCC 35873 genome contains 2,454,216 bp and has a GC content of 51.68%.

Watermelon diseases caused by pathogenic bacteria were endemic in Liaoning and Jilin Provinces from 2019 to 2020 in China, resulting in serious economic losses to the watermelon industry. This study characterized 56 strains isolated from symptomatic watermelon leaves collected from Liaoning and Jilin Provinces. Through morphological observation, 16S rRNA and gyrB sequence analysis, and BIOLOG profiles, the pathogen was identified as Pseudomonas syringae. In China, the watermelon disease caused by P. syringae was reported for the first time. The multilocus sequence analysis showed that the isolated strains belonged to three different clades within P. syringae phylogroup 2. Interestingly, most of them (79%) belonged to clade 2a, 14% were clade 2b, and 7% were clade 2d. This indicates that bacterial leaf spot outbreaks of watermelon in China were caused by multiple sources and mainly by P. syringae clade 2a.

LuxR-type transcriptional regulators are essential for many physiological processes in bacteria, including pathogenesis. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. However, the LuxR-type transcriptional factors in A. citrulli have not been well studied, except for the previously reported LuxR-type regulatory protein, AcrR, involved in regulating virulence and motility. Here, we characterized a second LuxR-type regulator, AclR, in the group II strain Aac-5 of A. citrulli by mutagenesis, virulence and motility assays, and transcriptomic analysis. Deletion of aclR resulted in impaired twitching and swimming motility and flagellar formation and diminished virulence but increased biofilm formation. Transcriptomic analysis revealed that 1,379 genes were differentially expressed in the aclR mutant strain, including 29 genes involved in flagellar assembly and 3 involved in pili formation, suggesting a regulatory role for AclR in multiple important biological functions of A. citrulli. Together, our results not only indicate that AclR plays a global role in transcriptional regulation in A. citrulli influencing motility, biofilm formation, and virulence but also provide perspective regarding the regulatory network of biological functions in A. citrulli. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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

LuxR‐type regulators regulate many bacterial processes and play important roles in bacterial motility and virulence. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. We identified a LuxR‐type, nonquorum sensing‐related regulator, AcrR, in the group II strain Aac‐5 of A. citrulli. We found that the acrR mutant lost twitching and swimming motilities, and flagellar formation. It also showed reduced virulence, but increased biofilm formation and growth ability. Transcriptomic analysis revealed that 394 genes were differentially expressed in the acrR mutant of A. citrulli, including 33 genes involved in flagellar assembly. Our results suggest that AcrR may act as a global regulator affecting multiple important biological functions of A. citrulli., AcrR, a LuxR‐type regulator, acts as a global regulator affecting multiple important biological functions, especially swimming motility and flagellum assembly of Acidovorax citrulli.

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.

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 (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.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, seriously affects watermelon and other cucurbit crops, resulting in significant economic losses. However, the pathogenicity mechanism of A. citrulli is not well understood. Plant pathogenic bacteria often suppress the plant immune response by secreting effector proteins. Thus, identifying A. citrulli effector proteins and determining their functions may improve our understanding of the underlying pathogenetic mechanisms. In this study, a novel effector, AopN, which is localized on the cell membrane of Nicotiana benthamiana, was identified. The functional analysis revealed that AopN significantly inhibited the flg22-induced reactive oxygen species burst. AopN induced a programmed cell death (PCD) response. Unlike its homologous protein, the ability of AopN to induce PCD was dependent on two motifs of unknown functions (including DUP4129 and Cpta_toxin), but was not dependent on LXXLL domain. More importantly, the virulence of the aopN mutant of A. citrulli in N. benthamiana significantly decreased, indicating that it was a core effector. Further analysis revealed that AopN interacted with watermelon ClHIPP and ClLTP, which responds to A. citrulli strain Aac5 infection at the transcription level. Collectively, these findings indicate that AopN suppresses plant immunity and activates the effector-triggered immunity pathway.

Aconitum carmichaelii Debx. is a Chinese traditional medicine herb, and is widely planted in China. The processed lateral roots of A. carmichaelii is known as Fuzi, and is used for the treatment of...

Background: Acidovorax citrulli causes bacterial fruit blotch (BFB), a disease that poses a global threat to watermelon and melon production. Despite the economic importance of BFB, relatively little is known about the regulation of A. citrulli pathogenesis. Light has a role in regulating the virulence factors of many plant pathogenic bacteria; however, the role of light in A. citrulli is unknown. This study aimed to investigate the roles of light in A. citrulli pathogenicity. Results: We found that light suppressed swimming motility andbiofilm formation in A. citrulli Aac5 strain. Furthermore, we found that A. citrulli Aac5 strain exhibited significant differences in colony morphology under light and dark conditions. In fact, the twitching motility of A. citrulli Aac5 strain also was supressed cultured by light conditions compared to dark conditions. In addition, the expression of hrpG, hrpX, and hrcC genes, which are important for type 3 secretion system (T3SS), was inhibited under light compared to dark conditions. However, in A. citrulli host interactions, light suppressed ROS production and enhanced callose deposition by watermelon when induced by A. citrulli; in addition, light enhanced the ability of A, citrulli to infect watermelon. These results suggest that light affected the pathogenicity of A. citrulli Aac5 strain by regulating its virulence factors and watermelon immune response. Conclusions: Light as an important environmental factor plays an important role in regulating the occurrence of plant diseases. BFB as an important watermelon disease seriously threatens the development of the industry. However, there are few reports on the impact of environmental factors on the disease. In our studying, we report for the first time that light can inhibit the expression of major virulence factors, such as swimming motility, biofilm formation, twitching motility, and even key gene of T3SS in A. citrulli Aac5 strain. But surprisingly, the light enhances the pathogenicity of A. citrulli Aac5 strain for natural host watermelons. The important phenotypes of host immune response are also different between ROS and callose deposition. These phenomena reflect the close relationship between BFB occurrence and light factor, which is of great significance for the study of BFB.

In 2015, red or black-brown necrotic lesions on main or lateral veins associated with the wilting of leaves and dry rot caused by plant pathogenic bacteria were found in tobacco plants in different fields in Shaowu city, Fujian province, China. Two bacteria (FM3 and FM4) were isolated and selected for further tests. Pathogenicity test results showed that all the tested isolates developed typical vein rot symptoms in tobacco 4 days after inoculation and soft rot symptoms in potato tuber, okra, pepper, capsicum, celery, and Chinese cabbage 2 days after inoculation, with the bacterium re-isolated from all the inoculated plants. On the basis of phenotypic properties, maceration characteristics, biochemical tests, Biolog system, and 16S rRNA sequences analysis, isolates FM3 and FM4 were identified as Pectobacterium carotovorum subsp. carotovorum . To our knowledge, this is the first report that the new bacterial vein rot disease in tobacco in Shaowu city, China, was caused by P. carotovorum subsp. carotovorum .

Acidovorax citrulli is a seed-borne pathogen causing bacterial fruit blotch of cucurbits including melon and watermelon. We investigated the roles of quorum sensing in the wild-type group II strain Aac-5 of A. citrulli by generating aacR and aacI knockout mutants and their complementation strains. We found that twitching motility and virulence were reduced, but biofilm formation and seed attachment were increased significantly in the two mutants as compared to the wild type strain. Deletion of aacR and aacI, however, had no effect on swimming motility and polar flagella formation of Aac-5. Furthermore, deletion of aacR resulted in reduced gene expression of hrpE, hrcN and pilT, while deletion of aacI affected only the expression of hrpE and pilT, not hrcN.

Acidovorax citrulli can be divided into two genetic groups: group I and group II based primarily on pulsed-field gel electrophoresis (PFGE) and multilocus sequence classification (MLST). To distinguish more rapidly between strains of the two groups, a pair of specific primer for specific polymerase chain reaction (PCR) that can identify group II strains was designed based on the pilL gene of a group II strain, AAC00-1. PCR results showed that a 332-bp band was generated for 51 of 52 group II strains whereas only three of 93 group I strains were positive, largely consisting with previous studies of A. citrulli classification. Results of PCR showed the primers were able to detect group II strains of A. citrulli and distinguish between strains of groups I and II rapidly and accurately.

In August 2012, a new tobacco root disease was observed in a number of tobacco fields in Shi-Ka Village, Pianbai Town, Youyang County of Chongqing City, China. The disease incidence varied from 10% to 40%, and the seedling mortality ranged from 50% to 80%. Affected tobacco plants developed symptoms characterized by a progressive yellowing and wilting of leaves, brown or black streaks in the stems, and root necrosis that eventually lead to the death of the plant. In this study, we were able to confirm field observations in the greenhouse using fungal isolates. The seedling mortality rate under artificial inoculation was 100%. The results of a pathogenicity test and morphological studies as well as rDNA internal transcribed spacer (ITS) and β-tubulin sequence analyses indicated that the disease was caused by Gelasinospora reticulata. This is a novel root rot disease on tobacco caused by this pathogen.

A TaqMan-based real-time PCR assay was developed for specific detection of strains of X. fastidiosa causing oleander leaf scorch. The assay uses primers WG-OLS-F1 and WG-OLS-R1 and the fluorescent probe WG-OLS-P1, designed based on unique sequences found only in the genome of oleander strain Ann1. The assay is specific, allowing detection of only oleander-infecting strains, not other strains of X. fastidiosa nor other plant-associated bacteria tested. The assay is also sensitive, with a detection limit of 10.4fg DNA of X. fastidiosa per reaction in vitro and in planta. The assay can also be applied to detect low numbers of X. fastidiosa in insect samples, or further developed into a multiplex real-time PCR assay to simultaneously detect and distinguish diverse strains of X. fastidiosa that may occupy the same hosts or insect vectors. Specific and sensitive detection and quantification of oleander strains of X. fastidiosa should be useful for disease diagnosis, epidemiological studies, management of oleander leaf scorch disease, and resistance screening for oleander shrubs.

Acidovorax citrulli has become quite common in China. A collection of 118 strains of A. citrulli was made from throughout China and other countries to determine their genetic relatedness. Strains were identified as A. citrulli by pathogenicity, phenotypic characterization, and PCR. Genetic diversity was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PFGE electrophoresis resulted in nine genotypes, which could be typed into two groups based on host; group 1 included strains mainly from melon and group 2 included strains mainly from watermelon. MLST analysis resulted in 73 sequence types (ST) among the 118 A. citrulli strains. All A. citrulli strains were typed into three groups: group 1 with 82 strains (including type strain Fc247), group 2 with 35 strains and group 3 a singleton (Fc380). Similar to PFGE results, group 1 included strains mainly from melon and group 2 included strains mainly from watermelon. The difference was the 10 watermelon strains (pslbtw1-3, 5–11) from Beijing grouped with melon strains of group 1 based on MLST, suggesting that these 10 watermelon strains had a close relationship with melon. Our study indicated that there was genetic differentiation among A. citrulli strains between watermelon and melon. Also, our study was the first attempt to compare PFGE and MLST on analyzing genetic diversity of A. citrulli strains and proved MLST could better distinguish A. citrulli strains.

Iron is essential for the growth and survival of many organisms. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the high-affinity ferric uptake system in many bacteria. To investigate the function of the fur gene in Xanthomonas vesicatoria (Xv), we generated a fur mutant strain, fur-m, by site-directed mutagenesis. Whereas siderophore production increased in the Xv fur mutant, extracellular polysaccharide production, biofilm formation, swimming ability and quorum sensing signals were all significantly decreased. The fur mutant also had significantly reduced virulence in tomato leaves. The above-mentioned phenotypes significantly recovered when the Xv fur mutation allele was complemented with a wild-type fur gene. Thus, Fur either negatively or positively regulates multiple important physiological functions in Xv.

We determined the draft genome sequence of the Pseudomonas syringae pv. tabaci strain yuexi-1. It was isolated from tobacco sample of yuexi-1, Sichuan province, China, by our laboratory. The genome contains 6,232,497 bp and has a G+C content of 58.2 mol%.

Bacterial fruit blotch (BFB) of melons is a seed-borne disease caused by Acidovorax citrulli . We determined the draft genome of A. citrulli Tw6. The strain was isolated from a watermelon collected from Beijing, China. The A. citrulli Tw6 genome contains 5,080,614 bp and has a G+C content of 68.7 mol%.

Acidovorax citrulli is typed into two groups, mainly based on the host. We determined the draft genome of A. citrulli group 1 strain pslb65. The strain was isolated from melon collected from Xinjiang province, China. The A. citrulli pslb65 genome contains 4,903,443 bp and has a G+C content of 68.8 mol%.

The ice nucleation (IN) gene iceA of Erwinia ananas 110 was integrated into the chromosomes of two Enterobacter cloacae strains (Enc1.2022 and Enc1.181). These two newly derived transgenic strains, designated Enc2022-I and Enc181-I, respectively, possessed ice nucleation activity at -2.5 degrees C, significantly higher than their parent strains (active at approx -10 degrees C or lower). After ingesting these transgenic bacteria, the mean supercooling points (SCPs) of corn borer and cotton bollworm larvae were -3 to -4 degrees C, significantly higher than those of untreated controls. The SCPs remained significantly elevated over the 9-day period after ingestion, which matched well with the efficient gut colonization of the bacteria during this period. All treated larvae froze and eventually died after exposure for 6 h to a temperature of -7 degrees C, and more than 95% died after 12 h at -5 degrees C. In contrast, few or none of the untreated control larvae froze and died under the same conditions. Furthermore, the growth ability of these transgenic ice nucleation-active (INA) En. cloacae strains on corn leaves was reduced, compared to that of wild-type epiphytic E. ananas, as revealed by pot tests conducted in both greenhouse and outdoor conditions. The stable colonization in insect guts and their lower affinity to plants would make these transgenic INA bacteria useful as a novel tool for biological control of insect pests in agricultural fields.

Xylella fastidiosa causes bacterial leaf scorch in many landscape trees including elm, oak, sycamore and mulberry, but methods for specific identification of a particular tree host species-limited strain or differentiation of tree-specific strains are lacking. It is also unknown whether a particular landscape tree-infecting X. fastidiosa strain is capable of infecting multiple landscape tree species in an urban environment. We developed two PCR primers specific for mulberry-infecting strains of X. fastidiosa based on the nucleotide sequence of a unique open reading frame identified only in mulberry-infecting strains among all the North and South American strains of X. fastidiosa sequenced to date. PCR using the primers allowed for detection and identification of mulberry-infecting X. fastidiosa strains in cultures and in samples collected from naturally infected mulberry trees. In addition, no mixed infections with or non-specific detections of the mulberry-infecting strains of X. fastidiosa were found in naturally X. fastidiosa-infected oak, elm and sycamore trees growing in the same region where naturally infected mulberry trees were grown. This genotype-specific PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. Unexpectedly but interestingly, the unique open reading frame conserved in the mulberry-infecting strains in the U. S. was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. When the primer set was tested against naturally infected olive plant samples collected in Italy, it allowed for detection of olive-associated strains of X. fastidiosa in Italy. This PCR assay, therefore, will also be useful for detection and identification of the Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution and biology of this new group of X. fastidiosa strains.

Xylella fastidiosa causes bacterial leaf scorch in landscape trees including sycamore. We determined the draft genome of X. fastidiosa strain Sy-Va, isolated in Virginia from a sycamore tree displaying leaf scorch symptoms. The Sy-VA genome contains 2,477,829 bp, and has a G+C content of 51.64 mol%.

Xylella fastidiosa causes bacterial leaf scorch in landscape trees, including mulberry. We determined the draft genome of the mulberry strain Mul-MD in order to gain a better understanding of the molecular basis of strain divergence, host specificity, nutrient requirements, and pathogenicity, as well as to develop genome-based specific detection methods.

One hundred and thirty-three bacterial strains were isolated from inner tissue of potato tubers collected from Datong, Taiyuan and Inner Mongolia Autonomous regions. On the basis of antagonistic examination in vitro, greenhouse and field tests, five strains named as 069, 085, 110, 116 and 118 were chosen for their suppression of bacterial ring rot or their growth promotion. Strain 118 is an endophytic bacterium with three effects of colonization, growth promotion and suppression of the pathogenic bacteria, showing good prospects for commercial use.

Fibroblast is a crucial kind of cell in the construction of the tissue engineered dermal equivalent. In order to optimize the cryopreservation protocols of the tissue-engineered dermis, the characteristics of dermal fibroblast in subzero temperatures are required, which include the water permeability of the cell membrane and the apparent activation energy. Using the differential scanning calorimeter (DSC), the volumetric shrinkage during freezing of human dermal fibroblast suspensions was obtained at the cooling rate of 5°C·min−1 in the presence of extracellular ice. To ensure the presence of extracellular ice, a small quantity of ice nucleation bacteria (INA bacteria), pseudomonas syringae was added in the samples. And based on the Karlsson’s model, a nonlinear-least-squares curve fitting technique was implemented to calculate the cryogenic parameters. At the reference temperature TR(=0°C), the water permeability of membrane Lpg=0.578 μm·min−1·atm−1 and the apparent activation energy, ELP=308.8 kJ·mol−1. These parameters were then used to simulate water transport of fibroblast during constant cooling at rates between 0.01–50°C · min−1. The simulation results were analyzed to predict the amount of water left in the cell after dehydration and the “optimal cooling rate” for fibroblast cryopreservation. For the dermal fibroblast with DMEM solution, a cooling rate of 4.6°C · min−1 was optimal.

Ice nucleation active (INA) bacteria are the most potent heterogeneous ice nuclei in nature, which have become an important biological resource for diverse applications. Many researches have proved that INA bacteria can raise the supercooling points (SCPs) of insect pests, then reduce their cold hardiness. However, INA bacteria’s inefficient colonization on the surface or in the guts of insects, and the high incidence of frost injury induced by their release hampered the application of INA bacteria in controlling insect pests in agricultural fields. In this study, we constructed a recombinant plasmid mob-Tn5 -iceA with the ability of broad-host-range conjugal mobilization and integration of the ina gene of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition. In addition, Ent. cloacae strains stably carrying iceA and expressing high ice nucleation activity (INA), even in the absence of antibiotic pressure, were constructed through conjugal mobilization and Tn5 transposition. Ent. cloacae strains have been reported to be able to efficiently colonize in the guts of insects, but have weak plant epiphytic ability. Therefore, these transgenic Ent. cloacae may be promising candidates for control of insect pests in agricultural fields.