Strains from the genus of Pectobacterium can cause soft rot in numerous important plants, leading to significant losses. 16S rRNA gene sequences reliably identify genera, but species identification is sometimes challenging due to indistinguishable species or strains and unrecognized new taxa. Therefore, a dependable and straightforward classification method is needed. In this study, we analyzed the complete genome sequence and predicted genome components of Pectobacterium strains. We also assessed their genetic relationships using average nucleotide identity and in silico DNA-DNA hybridization, alongside phylogenetic analysis. This was done by examining the whole-genome sequences of 14 new strains isolated in our laboratory, responsible for plant soft rot, and comparing them with 64 strains' genome sequences available in GenBank. The results reveal that three subspecies of P. carotovorum with genome sequences deposited in GenBank (PccS1, PCC21, and strain 67) need to be reclassified as separate species and also confirm that our new isolated strains are accurately categorized at the species level. Additionally, the virulence and adaptability of certain strains of Pectobacterium (PccS1, SCRI1043, and SCC3193) are influenced by horizontal acquisition genes. Furthermore, our findings suggest that the diversity in the car gene cluster among Pectobacterium strains likely stems from gene losing, as well as the auto-induced regulatory mechanisms underlying virulence determinant gene activation, and Car biosynthesis might act in different ways in PccS1, enhancing our knowledge of their genomic traits through comparative studies.

Bacterial pathogens secrete effector proteins that suppress plant immune responses and facilitate infection. This study focuses on Pectobacterium brasiliense NJAU180, a bacterial pathogen causing severe blackleg disease in potato plants in Inner Mongolia, China. Using exoproteomic analysis, plant-induced extracellular proteins were identified by comparing culture supernatants from P. brasiliense NJAU180 grown in minimal medium (MM) alone and in the presence of aseptically grown potato plantlets at an early growth stage (OD600 ≈ 0.5). The results reveal PurK as a novel plant-induced extracellular protein, and deletion of purK markedly reduces virulence. PurK, N5-carboxyaminoimidazole ribonucleotide synthetase, is a key enzyme in de novo purine biosynthesis. Its impact on virulence is distinct from the conventional production of plant cell wall-degrading enzymes: PurK promotes motility by modulating transcription of flagellar genes, acting through its three domains as an integrated unit to infect successfully. Extracellularly detected PurK suppresses callose deposition, a PAMP-triggered immunity (PTI)-like defense, while it also triggers a strong hypersensitive response and upregulates expression of PTI marker genes such as PR2 and WRKY7 when secreted into the host plant. Although PurK interacts specifically with PurE, our data indicate that PurK's pathogenic effects operate independently of purine biosynthesis. This study reveals a reliable experimental model for more accurate assessment of microbe-plant interactions and highlights new functional roles for PurK in P. brasiliense NJAU180 pathogenesis and identifies potential targets for disease control strategies.

Bacterial fruit blotch (BFB) caused by Paracidovorax citrulli is a severe threat to melon, watermelon, and other cucurbit crop production worldwide. The long-term adaptation of the pathogen to environmental conditions has resulted in substantial genetic diversity. In this study, we used P. citrulli strains from two groups as immunogens to obtain antibodies that were used to generate A.C1 and A.C2 colloidal gold immunochromatographic single test strips, which specifically identified group I and group II P. citrulli strains, respectively. We combined the A.C1 and A.C2 single test strips in a dual-channel plastic cartridge to construct a dual-channel colloidal gold immunochromatographic test strip able to distinguish between P. citrulli strains from two distinct groups. Test strip sensitivity reached 106 CFU/mL under ideal conditions. Moreover, it was relatively stable, with no cross-reactions with strains of closely related genera. The dual-channel test strip developed in this study may provide farmers with a useful tool for managing BFB through the prompt implementation of quarantine procedures in the field.

The σ54 factor (RpoN), a significant transcriptional regulatory factor, plays crucial roles in regulating virulence, motility, biofilm formation, and the utilization of carbon and nitrogen sources in pathogenic bacteria. However, the function of RpoN has not been identified in Paracidovorax citrulli (formerly Acidovorax citrulli). To investigate this, we constructed a rpoN1 deletion mutant and a corresponding complement strain in the background of P. citrulli strain xjl12. The P. citrulli rpoN1 deletion mutant displayed attenuated virulence in melon. RNA-Seq analysis revealed that rpoN1 is involved in regulating the expression of certain pathogenicity-associated genes related to the secretion system, biofilm formation, and motility. Phenotypic analysis demonstrated that the rpoN1 deletion mutant of P. citrulli significantly attenuated biofilm formation, twitch motility, swarming motility, cotyledon colonization, and seed colonization. However, swimming motility was significantly enhanced in the rpoN1 mutant. As expected, qRT-PCR assays indicated that the type IV pili-related gene Aave_4679 (pilA) was barely expressed in the rpoN1 mutant, and western blot analysis revealed that RpoN1 positively regulated the expression of pilA. Additionally, bacterial one-hybrid assays and electrophoretic mobility shift assays indicated that RpoN1 directly binds to the promoter of pilA. Our investigation revealed that RpoN1 is essential for the virulence of P. citrulli and provides valuable insights into the physiology and pathogenic mechanisms of bacterial fruit blotch.

Fire blight, caused by Erwinia amylovora, is a devastating disease that affects apples and pears, leading to significant economic losses worldwide. The disease is typically managed through a combination of biological, cultural, and chemical strategies. Due to the emergence of antibiotic resistance and concerns regarding chemical residues, there is an urgent need for new bactericides that are both highly effective and low in toxicity. Recently, the Ministry of Agriculture and Rural Affairs of China approved the registration of a new bactericide, benziothiazolinone also known as 1,2-Benzisothiazol-3-one. The assessment of benziothiazolinone demonstrated an EC50 value of 0.48 µg/mL against E. amylovora BZ16 after a 10-hour inoculation period, and 0.67 µg/mL after 16 h. Furthermore, benziothiazolinone displays a broad antimicrobial spectrum against various microorganisms, including bacteria, fungi, and oomycetes, as evidenced by its EC50 values. Prolonged exposure to benziothiazolinone was found to enhance the production of amylovoran in the E. amylovora strain BZ16BITR; however, this treatment significantly diminished the pathogenicity of the strain. Resistance risk analysis indicates that E. amylovora has a low risk of developing resistance to benziothiazolinone. Results from two-year field trials demonstrated that benziothiazolinone could significantly reduce the disease incidence of fire blight. Compared to the streptomycin control, the application of benziothiazolinone exhibited excellent efficacy, achieving over 80

Acidovorax citrulli, the causal pathogen of bacterial fruit blotch of cucurbits, relies on a functional type III secretion system (T3SS) for pathogenicity. Two-component systems (TCSs) are primary signal transduction mechanisms for bacteria to detect and adapt to various environmental conditions. However, the role of TCS on regulating T3SS and other virulence factors in response to environmental stimuli is still poorly understood in A. citrulli. Here, we report the identification of a conserved TCS, OmpR/EnvZ, involved in hypersensitive response (HR) induction in Nicotiana benthamiana by screening a transposon-insertion library in the group II strain xjL12 of A. citrulli. Transcription analysis confirmed that OmpRAc/EnvZAc was upregulated in response to elevated osmotic pressure, low and high pH conditions, and host environment. Deletions of envZAc, ompRAc, or both envZAc and ompRAc in A. citrulli attenuated virulence to melon seedlings and mature leaf tissues, and delayed HR in N. benthamiana. OmpRAc was activated by EnvZAc and directly bound to the promoter region of hrpG, a major regulator of T3SS. This binding activated hrpG transcription and promoted T3SS assembly in T3SS-inducing medium, XVM2. Additionally, the OmpRAc/EnvZAc mutants of A. citrulli displayed reduced swimming motility due to impaired flagella formation, but also had enhanced biofilm formation and exopolysaccharide production. OmpRAc/EnvZAc regulation of these virulence factors in A. citrulli depended on its own conserved phosphorylation sites. This work illuminates a signalling pathway for regulating the T3SS and provides insights into the OmpR/EnvZ-mediated virulence regulatory network in A. citrulli.

Abstract Arcopilus aureus is a plant-beneficial fungal species showing remarkable biocontrol capabilities. We investigated the antifungal activity of A. aureus YZXR strain and found that it strongly suppressed Polygonatum odoratum leaf spot disease caused by Fusarium fujikuroi. However, the antifungal substances and antifungal mechanisms remain unknown. To identify the antifungal substances, we extracted the fermentation liquid with ethyl acetate and found that the antifungal activity was mainly in the organic phase. Using column chromatography, semi-preparative HPLC, and GC–MS assays, we analyzed the compounds in the ethyl acetate phase and identified phenylethyl alcohol and 3,5-dihydroxytoluene as two compounds showing antifungal activity. The phenylethyl alcohol completely inhibited the growth of F. fujikuroi at a concentration of 0.25% (v/v), whereas the inhibition rate of 10.00 mg/mL 3,5-dihydroxytoluene on fungal growth was 80.60%. The fermentation broth of strain YZXR efficiently inhibited mycelial growth and conidiation of F. fujikuroi. Thus, this study uncovered the antifungal metabolites of A. aureus and the potential of A. aureus as a biological fungicide for managing P. odoratum leaf spot disease, providing a foundation for further research on its biocontrol mechanisms and field applications.

Previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single-copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either the T6SS or type IV secretion system (T4SS). This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with strains defective in the T6SS and/or T4SS confirmed that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose deposition in the course of infection. Notably, this redundant secretion mechanism between the T6SS and T4SS is believed to be the first of its kind in bacteria.

Bacterial-fungal interactions are widespread in nature.We observed that pear orchards affected by Cytospora pyri(formerly Valsa pyri)were often accompanied with Erwinia amylovora.However,the relationship of the two pathogens was unclear.The objective of this study was to determine whether the synergistic effect exists between E.amylovora and C.pyri.We first analyzed the coexistence frequencies of E.amylovora and C.pyri in pear trees.Virulence of the two pathogens,growth,physical interactions,amylovoran production,and expression of genes for amylovoran biosynthesis were conducted.Our results showed that E.amylovora and C.pyri could coexist on the same lesion and caused much more severe disease.We also found that E.amylovora could physically attach to C.pyri and the expression of amylovoran biosynthesis genes were up-regulated with fungal metabolite treatment.These results indicate that E.amylovora and C.pyri can cooperatively interact,which provides C.pyri with an opportunity to promote bacterial dispersal and production of virulence factor in E.amylovora.

ClpA is a widely conserved protease in bacteria that plays a key role in virulence. To investigate its specific mechanism of action in the pathogenicity of Paracidovorax citrulli (formerly Acidovorax citrulli), we constructed a ClpA deletion mutant, ΔClpA. The ΔClpA mutant of P. citrulli displayed reduced virulence on melon seedlings, and reduced motility, swarming ability, and antioxidant capacity. On the other hand, the ClpA deletion of P. citrulli mutant reduced the resistance to elevated temperature and enhanced biofilm formation ability. Using qRT-PCR, we observed that ClpA negatively regulates the expression of the virulence-related genes virB, pilR, pilA, and fliM, while positively regulating hrpG, hrcQ, and trbC. Bacterial double hybrid and Glutathione-S-transferase pulldown (GST-pulldown) results showed that ClpA interacts directly with RepA, and negatively regulates the expression of RepA. After deletion of the RepA gene, the pathogenicity of P. citrulli was lost, biofilm formation ability was enhanced, and the expression of hrpG, pilR, and trbC was positively regulated. These results indicate that ClpA plays a key role in the regulation of several virulence traits of P. citrulli, paving the way for future studies to better elucidate the virulence mechanisms of this bacterial plant pathogen.

Pecan (Carya illinoinensis) is a world-famous nut tree which widely cultivated in China. Quanjiao County, located in Anhui province, is reputed to be the capital of pecan production in China. Since 2019, typical scab symptoms were observed on most pecan cultivars in orchards located in the regions of Quanjiao (32°5'7.08″ N, 118°16'2.91″ E). In April, dark brown to black lesions of scab could be observed on both the abaxial and adaxial surface of the lamina, and were often associated with the veins or midrib. In July, small, brownish, and circular lesions ranging from 1 to 2 mm in diameter were observed at the end of stems and shoulder of the fruit. In the surveyed orchards, disease incidence on the leaves reached more than 35%. While, according to the number of infected nut clusters, disease incidence ranged from 40 to 60% on the infected fruits. Using a sterilized scalpel, conidia were scraped from the surface of a single lesion from the infected leaves or fruits, and a dilute spore suspension was prepared in sterile distilled water, of which 100 microliters was spread on 1% water-agar plate (Bock et al. 2014). The conidia were incubated at 25°C for 48 h under fluorescent lights with a 12-hphotoperiod. Single germinated conidia were selected and transferred into potato dextrose agar (PDA) plate to obtain monospore isolates. From 2019 to 2020, more than 20 isolates were obtained from the infected leaves and fruits. Incubated at 24°C for 6 weeks in darkness on PDA, the colonies were gray-black with circular morphology and floccose texture, which were consistent with the characteristics of Venturia effusa described previously (Gottwald 1982). The conidia were pyriform to ellipsoid, zero to one septate, smooth, attenuated towards apex and base, base truncate, pale brown and 10.08 to 18.14 × 4.86 to 9.56 μm (n = 50) in size. To further identify the isolates, the regions of internal transcribed spacer (ITS), beta-tubulin 2 (TUB2) and translation elongation factor 1 alpha (EF1-a) were amplified and sequenced from genomic DNA for the three representative isolates (AH-81 and AH-82 from the infected leaves, and AH-41 from the infected fruits), respectively (White et al. 1990; Young et al. 2018; Bensch et al. 2006). Sequences of them were deposited in GenBank under nos. OP199056 to OP199058 (ITS), OP566581 to OP566583 (TUB2) and OP566578 to OP566580 (EF1-a). Multilocus phylogenetic analysis revealed that three isolates and V. effusa were clustered in the same clade, indicating high genetic similarity between these organisms. Their morphological and molecular characteristics were consistent with those for V. effusa. The pathogenicity of three isolates were tested on two-year-old container-grown pecan seedlings, which were grown in the nursery. The conidial suspension with a concentration of 5 × 105 conidia/ml was sprayed evenly on the surface of leaves of a healthy pecan seedling, and each isolate inoculated four pecan seedlings. The pathogenicity experiment was repeated three times. The plants inoculated with sterile water were used a negative control. The inoculated plants were enclosed in plastic bags for 2 days, and kept in the nursery greenhouse. Four weeks after inoculation, a similar symptom of scab was observed on leaves of cultivar Mahan, and V. effusa was isolated again from inoculated leaves with the frequency of 100% by the single-spore isolation, whereas no symptoms were observed on the control plants. To our knowledge, this is the first report of V. effusa as a scab pathogen on pecan in Anhui Province of China and underscores the need for monitoring this disease and developing disease control strategies to prevent severe reduction in the value of fruit. References: Bensch, K., et al. 2006. Studies in Mycology, 55(1): 299-305. Bock, C. H., et al. 2014. Forest Pathology, 44(4): 266-275. Gottwald, T. R. 1982. Taxonomy of the pecan scab fungus Cladosporium caryigenum. Mycologia. 74 (3), 382-390. White, T. T., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Application. Academic Press, San Diego, CA. Young, C. A., et al. 2018. Phytopathology, 108(7): 837-846. The author(s) declare no conflict of interest. Keywords: Venturia effusa, Scab, Pecan, Identification †Indicates the corresponding author.Y. Q. Zhao; zhaoyuqiang123@126.com.

Gray mold caused by Botrytis cinerea is a critical disease that results in severe postharvest losses for the apple industry. In recent years, biological control has become an increasingly effective approach for controlling postharvest diseases in fruits. Brassica plants contain abundant natural compounds with known antimicrobial activity against numerous plant pathogens. In this study, a large-scale screening of 90 mustard cultivars was conducted to evaluate their biofumigation effects against B. cinerea. Among these, one mustard cultivar named Dilong-1, displayed the highest inhibitory effect against B. cinerea, and was able to completely inhibit mycelial growth. Further investigations showed that fumigation with Dilong-1 inhibited mycelial growth, sporulation, and spore germination of B. cinerea in vitro. In addition, fumigation using Dilong-1 showed a wide antifungal spectrum, including other fruit postharvest pathogens such as Phytophthora litchii. Furthermore, apple gray mold disease severity was significantly reduced by biofumigation using Dilong-1. Importantly, fumigation with Dilong-1 did not negatively impact final apple qualities, including weight loss, firmness, and total soluble solids. These results suggested that Dilong-1 significantly inhibited gray mold decay caused by B. cinerea without affecting the quality of apple fruits. In conclusion, biological fumigation of apple fruits with the mustard cultivar Dilong-1 is a promising eco-friendly approach for controlling apple gray mold during storage and shipment.

疮痂病是薄壳山核桃上最具毁灭性的病害,带菌植物材料是传播疮痂病的重要来源.准确、灵敏、快速的检测方法可为该病害流行规律调查和防控提供有力的依据.本文通过比较薄壳山核桃疮痂病菌Venturia effusa及其近似种之间的ITS序列差异,设计了特异性引物和TaqMan探针,建立了薄壳山核桃疮痂病菌的荧光定量PCR检测方法.特异性检测结果表明,该方法可以检测不同地区的薄壳山核桃疮痂病菌菌株,而对其近似种以及薄壳山核桃上的其他真菌均没有信号.本研究建立的检测方法对薄壳山核桃疮痂病菌DNA的最低检测限可达0.5 pg/μL.该方法用于田间样品检测时,检测时间仅需1 h,远快于常规的分离培养法.本研究建立的基于TaqMan探针的荧光定量PCR检测方法为薄壳山核桃疮痂病菌的快速检测和监测提供了有力工具.

Two specific monoclonal antibodies (mAbs) were screened, and an immunochromatographic strip (ICS) test for rapid and specific detection of cucumber green mottle mosaic virus (CGMMV) was developed. The coat protein of CGMMV was heterologously expressed as an immunogen, and specific capture mAb 2C9 and the detection mAb 4D4 were screened by an uncompetitive immunoassay. The test and control lines on the nitrocellulose membrane were coated with the purified 2C9 and a goat anti-mouse IgG, respectively, and a nanogold probe combined with 4D4 was applied to the conjugate pad. Using these mAbs, a rapid and sensitive ICS was developed. Within the sandwich mode of 2C9-CGMMV-4D4, the test line showed a corresponding positive relationship with CGMMV in infected samples. The ICS test had a detection limit of 1:5000 (w/v) for CGMMV in samples and was specific for CGMMV, with no observed cross-reaction with TMV or CMV.

薄壳山核桃（Carya illinoinensis （Wangenh.） K. Koch）又称美国山核桃，是绿化的优选树种，其果实又称碧根果是世界著名的干果之一。薄壳山核桃原产于美国和墨西哥北部，因其较高的经济价值已被引种到全球20多个国家和地区 [1-2] 。江苏是我国薄壳山核桃引种较早的省份之一 [3] ，江苏句容拥有万亩薄壳山核桃基地，发展势头良好。随着薄壳山核桃种植面积的扩大，病虫害问题突显，褐斑病在江苏句容相继发生，导致叶斑，果腐，落果，经济损失严重。从江苏句容两个薄壳山核桃种植区采集发病样品，进行病原菌的分离与鉴定，结果报道如下。

Pecan (Carya illinoinensis) is one of the important economic forest crops which has been widely cultivated in Anhui and Jiangsu Provinces, China. Since 2019, symptoms resembling anthracnose disease had been observed in 5-ha and 6.6-ha pecan orchards in Quanjiao ( 32°5'7.08″ N, 118°16'2.91″ E), Anhui Province, and Jintan (31°42'23.84″ N, 119°21'22.90″ E), Jiangsu Province. The disease severity was about 20 to 30% with 5 to 15% (about 500 trees) incidence. In May, symptoms of leaf initially appeared as small dark lesions, which gradually developed to irregular-shaped, sunken lesions (Figure S1, A). From August to October, similar symptoms were also observed on the fruits. Infected fruits appeared irregularly, dark and depressed necrotic lesions on which orange spore masses could be occasionally observed (Figure S1, B). As the disease progressed, the necrotic lesions gradually expanded and merged, resulting in abscission of the fruits. Small fragments (4 × 4 mm) from the necrotic borders of infected fruits or leaves were surfaced sterilized, plated on potato dextrose agar (PDA) and then incubated in darkness at 25°C for 3 days. Pure cultures were obtained from individual conidia by recovering single spores. On the PDA plate, the colonies surface was white and cottony. Observing from the back of the plate, the colonies were pale yellow at the centre and pale white at the margin (Figure S1, E). Spores were produced over PDA plates after 7 days growth. Conidia were hyaline, smooth walls, aseptate, guttulate, cylindrical with rounded ends with 14.8 to 17.5 × 3.3 to 4.7 μm (mean 16.5 × 4.1μm, n = 50) in size (Figure S1, F). These morphological characteristics were similar to those of the species of Colletotrichum siamense (Prihastuti et al. 2009; Weir et al. 2012; Fu et al. 2019). Thirty-two isolates Colletotrichum sp. were obtained from the infected leaves and fruits (isolation frequency about 80%). To further identify the isolates, the regions of internal transcribed spacer (ITS), calmodulin (CAL), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHSI), and beta-tubulin 2 (TUB2) were amplified and sequenced from genomic DNA for the four representative isolates (JS1 and AH1 from infected fruits; JS2 and AH2 from infected leaves), respectively (Weir et al. 2012). Sequences of them were deposited in GenBank under nos. OP389224 to OP389227 (ITS), OP413765 to OP413768 (CAL), OP413761 to OP413764 (ACT), OP413773 to OP413776 (GAPDH), OP413769 to OP413772 (CHSI), and OP413777 to OP413780 (TUB2). Blast analysis showed these sequences shared high identity with C. siamense (100% with ITS, CAL, CHSI, and TUB2; 98.94% with ACT; 98.19% with GAPDH). Multilocus phylogenetic analysis revealed that the four isolates and C. siamense were clustered in the same clade (Figure S2). Based on the results of morphological and molecular analysis, these isolates were identified as C. siamense. The pathogenicity of four isolates was tested on two-year-old container-grown pecan seedlings, which were grown in the nursery. The conidial suspension with a concentration of 5 × 106 conidia/ml was sprayed evenly on the surface of leaves of a healthy seedling, and each isolate inoculated three pecan seedlings. The pathogenicity experiment was repeated three times. For negative controls, pecan seedlings were sprayed with sterilized distilled water. Finally, all inoculated plants were kept in a greenhouse at 25°C under a 16 h/8 h photoperiod and 70% relative humidity. Three weeks after inoculation, the inoculated plants showed symptoms similar to those of the original diseased plants (Figure S1, C), while controls remained asymptomatic (Figure S1, D). Cultures were re-isolated from the infected leaves and were identified as C. siamense by both morphological characteristics and DNA sequence analysis. Previously, C. nymphaeae, C. siamense, C. fructicola and C. viniferum have been reported to cause anthracnose of Pecan worldwide (Zhang et al. 2019; Oh et al. 2021; Poletto et al. 2019; Zhao et al. 2022 ). To our knowledge, this is the first report of C. siamense causing anthracnose on pecan in China. The identification of this pathogen will facilitate the development of strategies for managing the disease in China. References: Oh, J. Y., et al. 2021. Plant disease. 105(10):3296. Poletto, T., et al. 2019. Plant disease. 103(12):3277. Prihastuti, H., et al. 2009. Fungal Divers. 39:89. Fu, M., et al. 2019. Persoonia-Molecular Phylogeny and Evolution of Fungi. 42(1):1-35. Weir, B. S., et al. 2012. Studies in Mycology. 73:115. Zhao, et al. 2022, Acta Phytopathologica Sinica, doi:10.13926/j.cnki.apps.000648 Zhang, Y. B., et al. 2019. Plant disease. 103(6):1432. The author(s) declare no conflict of interest. Keywords: Colletotrichum siamense, Anthracnose, Carya illinoinensis, Pathogenicity †Indicates the corresponding author. Y. Q. Zhao; zhaoyuqiang123@126.com.

In many bacteria, OxyR acts as a transcriptional regulator that facilitates infection via degrading hydrogen peroxide (H2O2) generated by the host defense response. Previous studies showed that OxyR also plays an important role in regulating biofilm formation, cell motility, pili relate-genes expression, and surface polysaccharide production. However, the role of OxyR has not been determined in Acidovorax citrulli strain xjl12. In the current study, the qRT-PCR and western blot assays revealed that the expression level of oxyR was significantly induced by H2O2. The oxyR deletion mutant of A. citrulli was significantly impaired bacterial tolerance to oxidative stress and reduced catalase (CAT) activity. In addition, oxyR mutant resulted in reduced swimming motility, twitching motility, biofilm formation, virulence, and bacterial growth in planta by significantly affecting flagellin and type IV pili-related gene (fliC and pilA) expression. The qRT-PCR assays and western blot revealed that OxyR positively regulated the expression of fliC and pilA. Furthermore, bacterial one-hybrid assay demonstrated that OxyR directly affected pilA and fliC promoter. Through bacterial two-hybrid assay, it was found that OxyR can directly interact with PilA and FliC. These results suggest that OxyR plays a major role in the regulating of a variety of virulence traits, and provide a foundation for future research on the global effects of OxyR in A. citrulli.

Pecan (Carya illinoinensis) is a world-famous nut tree that is widely cultivated in China, especially in Jiangsu Province (Zhang et al. 2015). In April 2022, cankers on trunks were recorded in pecan (cv. Pawnee) fields located in Taizhou (32°27'58″ N, 120°0'49″ E), Jiangsu. Cankers on the trunks resulted in wilt of the plants. Usually, the color of infected bark on the trunk became darker than the healty bark. When the outer bark was peeled away, the inner tissues were water-soaked, often with reddish streaks. In the surveyed orchards, disease incidence ranged from 10 to 20% among young saplings (about 200 three-year-old trees). While no fungal mycelium or spores were found in the diseased areas by microscope, bacterial colonies were isolated by surface-sterilizing small fragments (25 mm2) of symptomatic tissue in 0.5% NaOCl, rinsing the sections twice in sterilized water, and then streaking them on Luria-Bertani (LB) plates. More than 20 bacterial isolates were obtained and all isolates induced a hypersensitive response on Nicotiana tabacum. All isolates were fluorescent on King's medium B, and were gram-negative based on lysis by KOH. Isolates were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices. Based on above information, the isolates thus belonged to Lelliot's LOPAT group 1, P. syringae (Lelliott and Stead 1988). The 16S rRNA sequences of five representative isolates (accession numbers OP175939-OP175943) were amplified by PCR, sequenced, and compared with the NCBI GenBank database (Weisburg et al. 1991; Sarkar and Guttman 2004), finding a 99.92% genetic similarity with a previously reported 16S rRNA sequence of a Pseudomonas syringae pv. syringae (Pss) isolate (accession numbers NW389777). Additional housekeeping genes gap1(accession numbers OP186937-OP186941), rpoD (accession numbers OP186952-OP186956), gyrB (accession numbers OP186947-OP186951), and gltA (accession numbers OP186942-OP186946) were PCR-amplified and sequenced as reported by Hwang et al. (2005), followed by multilocus sequence typing analysis (MLSA). Molecular phylogenetic trees (MEGA vesion 6.0, maximum likelihood with Jukes-Cantor model, 1,000 bootstraps) were generated based on each of these five DNA regions and revealed that all five isolates were clustered together with the strains in P. syringae genomospecies 2, and grouped these isolates with Pss in the PAMDB database (Hwang et al. 2005). As a result, these isolates were identified as Pss. Pathogenicity on pecan (cv. Pawnee) was confirmed by cutting the trunks of two-year-old pecan trees with sterilized blades dipped in cell suspensions containing 107 CFU/ml of each isolate. Plants inoculated in a similar manner with sterile water served as negative controls. The inoculated plants were incubated in a greenhouse maintained at 25°C and 80% relative humidity. After 7 to 8 days, all inoculated plants showed the symptoms of necrosis previously described for the original field plants, while the control plants did not show symptoms. The bacteria reisolated from the inoculated plants were identified as Pss using the LOPAT tests. These results and the sequence analysis of the 16S rRNA and four housekeeping genes described above, fulfilled Koch's postulates. No target bacteria were isolated from the control plants. To our knowledge, this is the first report of Pseudomonas syringae pv. syringaecausing bacterial canker of pecan worldwide. The identification of this pathogen will allow the study of strategies for managing the disease. References: Hwang, M. S., et al. 2005. Applied and Environmental Microbiology, 71:5182-5191. Lelliott, R. A., and Stead, D. E. 1988. Blackwell Scientific, Sussex, UK. Sarkar, S. F., and Guttman, D. S. 2004. Applied and Environmental Microbiology, 70:1999. Weisburg, W. G., et al. 1991. Journal of Bacteriology, 173: 697. Zhang, R., et al. 2015. Scientia Horticulturae, 197: 719-727. The author(s) declare no conflict of interest. Keywords: Carya illinoinensis, Pseudomonas syringae, Canker, Identification †Indicates the corresponding author.Y. Q. Zhao; zhaoyuqiang123@126.com.

Bacterial fruit blotch, caused by seed-borne pathogen Acidovorax citrulli, poses a serious threat to the production of cucurbits globally. Although the disease can cause substantial economic losses, limited information is available about the molecular mechanisms of virulence. This study identified that, a random transposon insertion mutant impaired in the ability to elicit a hypersensitive response on tobacco. The disrupted gene in this mutant was determined to be Aave_0638, which is predicted to encode a YggS family pyridoxal phosphate-dependent enzyme. YggS is a highly conserved protein among multiple organisms, and is responsible for maintaining the homeostasis of pyridoxal 5′-phosphate and amino acids in cells. yggS deletion mutant of A. citrulli strain XjL12 displayed attenuated virulence, delayed hypersensitive response, less tolerance to H2O2 and pyridoxine, increased sensitivity to antibiotic β-chloro-D-alanine, and reduced swimming. In addition, RNA-Seq analysis demonstrated that yggS was involved in regulating the expression of certain pathogenicity-associated genes related to secretion, motility, quorum sensing and oxidative stress response. Importantly, YggS significantly affected type III secretion system and its effectors in vitro. Collectively, our results suggest that YggS is indispensable for A.citrulli virulence and expands the role of YggS in the biological processes.