T6SS and T4SS redundantly secret effectors to govern the virulence and bacterial competition in Pectobacterium PccS1.

The 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 T6SS or T4SS. This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with stains defective in T6SS or/and T4SS confirm 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 T6SS and T4SS is believed to be the first of its kind in bacteria.

A Monoclonal Antibody-Based Immunochromatographic Test Strip and Its Application in the Rapid Detection of Cucumber Green Mottle Mosaic Virus.

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.

First Report of Colletotrichum siamense causing Anthracnose on Pecan in China.

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.

First Report of Bacterial Canker of Pecan Caused by Pseudomonas syringae pv. syringae in China.

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.

OxyR contributes to virulence of Acidovorax citrulli by regulating anti-oxidative stress and expression of flagellin FliC and type IV pili PilA.

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.

First Report of Scab Disease Caused by Venturia effusa on Pecan in Anhui Province of China.

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.

First Report of Dickeya fangzhongdai causing soft rot in Orchids in Canada.

Dickeya fangzhongdai was originally described as the causal agent of bleeding canker of pear tree in China. Recently, D. fangzhongdai was isolated and identified as the causal agent of soft rot in an orchid plant purchased in a local supermarket in Prince Edward Island, Canada. A water-soaked dark green spot on the leaf surface was observed and later became larger soft rot symptom. The origin of the orchid plants was traced back to a producer in Ontario, Canada who propagated them from with cuttings originally imported from the Netherlands and Taiwan. Bacterial isolations were made from a soft rot lesion on an orchid leaf by surface sterilization of small pieces of marginal tissue of the diseased leaf in 70% alcohol. The small pieces of leaf tissue were then washed three time using sterile water, and immersed in drops of sterile water. Bacterial streaming was observed under the microscope and non-fluorescing bacterial colonies were isolated on King's B and casamino acid-peptone-glucose agar plates and purified as isolates 908, 909, 910 and 911. The DNA samples were extracted from the four isolates, as well as the diseased leaf tissue, and tested by using a qPCR assay with the specific primer/probe set (DfF/DfR/DfP) for D. fangzhongdai (Tian et al. 2020). The assay yielded PCR amplicons of 135 bp with a melting temperature of 86.5±0.6 °C as did two control reactions using genomic DNA from D. fangzhongdai strains JS5T and QZH3 originally isolated in China, providing presumptive identification of the orchid isolates as D. fangzhongdai. To fulfill Koch's postulates, freshly purchased healthy orchid plants (n=4) were inoculated by leaf injection with the bacterial isolates obtained in this study and strains JS5 T and QZH3 at ~107 CFU/ml. Three leaves of the same side of the plants were inoculated with the same strains as triplicates. Sterile water was used as the negative control. Inoculated plants were incubated in a growth chamber with a 16 h photoperiod at 23 °C. Water soaked lesions developed in 3-5 days after inoculation followed by soft rotting in leaves inoculated with the new bacterial strains from orchid plants while strain QZH3 caused soft rot in 10 days after inoculation (Fig. S1). The non-fluorescing bacteria on King's B plates with colony morphology similar to those inoculated were re-isolated from the inoculated leaves and confirmed to be D. fangzhongdai by qPCR. Phylogenetic analysis of the assembled 16S rRNA sequence of isolate 908 (GenBank accession number: MT984340), together with GenBank data of all Dickeya spp. and some Pectobacterium spp, using neighbor-joining (NJ) method inferred with MEGA X software (Kumar et al. 2018) showed that isolate 908 clustered with strains JS5T and QZH3 at a phylogenetic distance of 0.0007. This clearly indicated that isolate 908 and JS5T and QZH3 belong to the same genus. Species-level identification of isolate 908 was achieved by genome sequencing and analysis based on average nucleotide identity (ANI). Genomic DNA of isolate 908 was sequenced with Illumina MiSeq to provide approximately 180X genome coverage. After quality checking using FastQC (Andrews 2010), de-novo assembly was performed with VelvetOptimiser v2.2.6 (Zerbino and Birney 2008). The draft genome size of strain 908 was 4,938,027 bp consisting of 76 contigs with 56.8% G+C content and 63,801 bp as N50. The draft genome was checked for misassembled fragments using QUAST v5.0.2 (Gurevich et al. 2013) and found to be of good quality. The draft genome sequence is deposited in GenBank under the accession number of JADCNJ000000000. The draft genome sequence of strain 908 was compared to that of D. fangzhongdai JS5T type strain genome using FastANI v1.2 (Jain et al. 2018) resulting in an ANI value of 98.9%, which is above the 95% cut-off for the same species. Previously, it was reported that D. fangzhongdai caused soft rot in orchid in Europe (Alic et al. 2018) and in onions in New York (Ma et al. 2020). The difference in virulence among D. fangzhongdai strains warrants further investigation and their pathogenicity on potato is being investigated to evaluate any threat to the potato industry. To our knowledge, this is the first report of D. fangzhongdai causing soft rot disease on orchids in Canada and North America.

The RsmA RNA-Binding Proteins in Pseudomonas syringae Exhibit Distinct and Overlapping Roles in Modulating Virulence and Survival Under Different Nutritional Conditions.

The post-transcriptional regulator RsmA globally controls gene expression in bacteria. Previous studies showed that RsmA2 and RsmA3 played critical roles in regulating type III secretion system (T3SS), motility, syringafactin, and alginate productions in Pseudomonas syringae pv. tomato strain DC3000 (PstDC3000). In this study, we investigated global gene expression profiles of the wild-type PstDC3000, the rsmA3 mutant, and the rsmA2/A3 double mutant in the hrp-inducing minimum medium (HMM) and King's B (KB) medium. By comparing the rsmA2/A3 and rsmA3 mutants to PstDC3000, a total of 1358 and 1074 differentially expressed genes (DEGs) in HMM, and 870 and 1463 DEGs in KB were uncovered, respectively. When comparing the rsmA2/A3 mutant with the rsmA3 mutant, 277 and 741 DEGs in HMM and KB, respectively, were revealed. Transcriptomic analysis revealed that the rsmY, rsmZ, and rsmX1-5 non-coding small RNAs (ncsRNAs) were positively affected by RsmA2 and RsmA3, while RsmA3 positively regulates the expression of the rsmA2 gene and negatively regulates both rsmA1 and rsmA5 gene expression. Comparative transcriptomic analysis showed that RsmA2 and RsmA3 synergistically influenced the expression of genes involved in T3SS and alginate biosynthesis in HMM and chemotaxis in KB. RsmA2 and RsmA3 inversely affected genes involved in syringafactin production in HMM and ribosomal protein biosynthesis in KB. In addition, RsmA2 played a major role in influencing genes involved in sarcosine and thiamine biosynthesis in HMM and in mannitol and phosphate metabolism in KB. On the other hand, genes involved in fatty acid metabolism, cellulose biosynthesis, signal transduction, and stress responses were mainly impacted by RsmA3 in both HMM and KB; whereas RsmA3 played a major role in controlling genes involved in c-di-GMP, phosphate metabolism, chemotaxis, and capsular polysaccharide in HMM. Furthermore, regulation of syringafactin production and oxidative stress by RsmA2 and RsmA3 was experimentally verified. Our results suggested the potential interplay among the RsmA proteins, which exhibit distinct and overlapping roles in modulating virulence and survival in P. syringae under different nutritional conditions.

Fermentation: An Unreliable Seed Treatment for Bacterial Fruit Blotch of Watermelon.

Acidovorax citrulli is a seedborne pathogen that causes bacterial fruit blotch (BFB), a global threat to watermelon production. Treating watermelon seeds to eliminate A. citrulli is a critical component of BFB management, and several strategies have been evaluated to mitigate the impact of the disease. In China, watermelon seed producers routinely incubate seeds in watermelon juice (fermentation) to reduce the risk of seed infection by A. citrulli and seedling transmission of BFB. However, there has been limited effort to evaluate the efficacy of fermentation in mitigating A. citrulli seed infection. The current study showed that fermented watermelon fruit juice could inhibit A. citrulli population growth and demonstrated that the low pH conditions, not the temperature dynamic, generated during fermentation might play a major role in A. citrulli growth inhibition and could induce the viable but nonculturable (VBNC) state in A. citrulli. We developed an effective method that was based on propidium monoazide PCR to detect viable A. citrulli cells under low pH conditions or in fermented watermelon fruit juice. We also provided evidence that VBNC A. citrulli cells induced by fermented watermelon fruit juice could not be resuscitated and did not retain their virulence on watermelon seedlings. However, VBNC A. citrulli cells could be resuscitated in Luria-Bertani medium. Based on these observations, we conclude that fermentation in watermelon fruit juice may not be an effective seed treatment for BFB because it may increase the seed infection by A. citrulli.

yggS Encoding Pyridoxal 5'-Phosphate Binding Protein Is Required for Acidovorax citrulli Virulence.

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 ß-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.

Pectobacterium atrosepticum KDPG aldolase, Eda, participates in the Entner-Doudoroff pathway and independently inhibits expression of virulence determinants.

Pectobacterium carotovorum has an incomplete Entner-Doudoroff (ED) pathway, including enzyme 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda) but lacking phosphogluconate dehydratase (Edd), while P. atrosepticum (Pba) has a complete pathway. To understand the role of the ED pathway in Pectobacterium infection, mutants of these two key enzymes, Δeda and Δedd, were constructed in Pba SCRI1039. Δeda exhibited significant decreased virulence on potato tubers and colonization in planta and was greatly attenuated in pectinase activity and the ability to use pectin breakdown products, including polygalacturonic acid (PGA) and galacturonic acid. These reduced phenotypes were restored following complementation with an external vector expressing eda. Quantitative reverse transcription PCR analysis revealed that expression of the pectinase genes pelA, pelC, pehN, pelW, and pmeB in Δeda cultured in pyruvate, with or without PGA, was significantly reduced compared to the wild type, while genes for virulence regulators (kdgR, hexR, hexA, and rsmA) remained unchanged. However, Δedd showed similar phenotypes to the wild type. To our knowledge, this is the first demonstration that disruption of eda has a feedback effect on inhibiting pectin degradation and that Eda is involved in building the arsenal of pectinases needed during infection by Pectobacterium.

Transcriptome of Pectobacterium carotovorum subsp. carotovorum PccS1 infected in calla plants in vivo highlights a spatiotemporal expression pattern of genes related to virulence, adaptation, and host response.

Bacterial pathogens from the genus Pectobacterium cause soft rot in various plants, and result in important economic losses worldwide. We understand much about how these pathogens digest their hosts and protect themselves against plant defences, as well as some regulatory networks in these processes. However, the spatiotemporal expression of genome-wide infection of Pectobacterium remains unclear, although researchers analysed this in some phytopathogens. In the present work, comparing the transcriptome profiles from cellular infection with growth in minimal and rich media, RNA-Seq analyses revealed that the differentially expressed genes (log2 -fold ratio ≥ 1.0) in the cells of Pectobacterium carotovorum subsp. carotovorum PccS1 recovered at a series of time points after inoculation in the host in vivo covered approximately 50% of genes in the genome. Based on the dynamic expression changes in infection, the significantly differentially expressed genes (log2 -fold ratio ≥ 2.0) were classified into five types, and the main expression pattern of the genes for carbohydrate metabolism underlying the processes of infection was identified. The results are helpful to our understanding of the inducement of host plant and environmental adaption of Pectobacterium. In addition, our results demonstrate that maceration caused by PccS1 is due to the depression of callose deposition in the plant for resistance by the pathogenesis-related genes and the superlytic ability of pectinolytic enzymes produced in PccS1, rather than the promotion of plant cell death elicited by the T3SS of bacteria as described in previous work.

Comparative transcriptomic analysis of global gene expression mediated by (p) ppGpp reveals common regulatory networks in Pseudomonas syringae.

BACKGROUND: Pseudomonas syringae is an important plant pathogen, which could adapt many different environmental conditions. Under the nutrient-limited and other stress conditions, P. syringae produces nucleotide signal molecules, i.e., guanosine tetra/pentaphosphate ((p)ppGpp), to globally regulate gene expression. Previous studies showed that (p) ppGpp played an important role in regulating virulence factors in P. syringae pv. tomato DC3000 (PstDC3000) and P. syringae pv. syringae B728a (PssB728a). Here we present a comparative transcriptomic analysis to uncover the overall effects of (p)ppGpp-mediated stringent response in P. syringae. RESULTS: In this study, we investigated global gene expression profiles of PstDC3000 and PssB728a and their corresponding (p)ppGpp0 mutants in hrp-inducing minimal medium (HMM) using RNA-seq. A total of 1886 and 1562 differentially expressed genes (DEGs) were uncovered between the (p)ppGpp0 mutants and the wild-type in PstDC3000 and PssB728a, respectively. Comparative transcriptomics identified 1613 common DEGs, as well as 444 and 293 unique DEGs in PstDC3000 and PssB728a, respectively. Functional cluster analysis revealed that (p) ppGpp positively regulated a variety of virulence-associated genes, including type III secretion system (T3SS), type VI secretion system (T6SS), cell motility, cell division, and alginate biosynthesis, while negatively regulated multiple basic physiological processes, including DNA replication, RNA processes, nucleotide biosynthesis, fatty acid metabolism, ribosome protein biosynthesis, and amino acid metabolism in both PstDC3000 and PssB728a. Furthermore, (p) ppGpp had divergent effects on other processes in PstDC3000 and PssB728a, including phytotoxin, nitrogen regulation and general secretion pathway (GSP). CONCLUSION: In this study, comparative transcriptomic analysis reveals common regulatory networks in both PstDC3000 and PssB728a mediated by (p) ppGpp in HMM. In both P. syringae systems, (p) ppGpp re-allocate cellular resources by suppressing multiple basic physiological activities and enhancing virulence gene expression, suggesting a balance between growth, survival and virulence. Our research is important in that due to similar global gene expression mediated by (p) ppGpp in both PstDC3000 and PssB728a, it is reasonable to propose that (p) ppGpp could be used as a target to develop novel control measures to fight against important plant bacterial diseases.

Genome Sequence and Comparative Analysis of Colletotrichum gloeosporioides Isolated from Liriodendron Leaves.

Colletotrichum gloeosporioides is a hemibiotrophic pathogen causing significant losses to economically important crops and forest trees, including Liriodendron. To explore the interaction between C. gloeosporioides and Liriodendron and to identify the candidate genes determining the pathogenesis, we sequenced and assembled the whole genome of C. gloeosporioides Lc1 (CgLc1) using PacBio and Illumina next generation sequencing and performed a comparative genomic analysis between CgLc1 and Cg01, the latter being a described endophytic species of the C. gloeosporioides complex. Gene structure prediction identified 15,744 protein-coding genes and 837 noncoding RNAs. Species-specific genes were characterized using an ortholog analysis followed by a pathway enrichment analysis, which showed that genes specific to CgLc1 were enriched for the arginine biosynthetic process. Furthermore, genome synteny analysis revealed that most of the protein-coding genes fell into collinear blocks. However, two clusters of polyketide synthase genes were identified to be specific for CgLc1, suggesting that they might have an important role in virulence control. Transcriptional regulators coexpressed with polyketide synthase genes were detected through a Weighted Correlation Network Analysis. Taken together, this work provides new insight into the virulence- and pathogenesis-associated genes present in C. gloeosporioides and its possible lifestyle.

Prevalence of Acidovorax citrulli in Commercial Cucurbit Seedlots During 2010-2018 in China.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit fruit and seed production worldwide. In recent years, the BFB has spread to many areas of China, mainly via the inadvertent distribution of contaminated commercial seeds. To assess the prevalence of seedborne A. citrulli in commercial watermelon and other cucurbitaceous seedlots in China, a 9-year survey was conducted between 2010 and 2018. A total of 4,839 seedlots of watermelon and other cucurbitaceous species were collected from 13 major seed production areas of China and tested by a semiselective media-based colony PCR technique for A. citrulli. Overall, A. citrulli was detected in 18.00% (871/4,839) of all cucurbitaceous seedlots. The bacterium was detected in 21.59% (38/176), 19.19% (33/172), 23.44% (214/913), 40.76% (247/606), 13.28% (85/640), 15.40% (95/617), 13.25% (73/551), 8.03% (48/598), and 6.71% (38/566) of all commercial seedlots tested from the 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, and 2018 growing seasons, respectively. Additionally, the prevalence of A. citrulli in cucurbit seedlots was determined for different seed production areas. The prevalence of A. citrulli in cucurbitaceous seedlots produced in Xinjiang, Gansu, Ningxia, Inner Mongolia, and 9 other provinces was 18.76% (582/3103), 26.34% (103/391), 21.47% (82/382), 11.11% (14/126), and 10.75% (90/837), respectively. This is the first survey for A. citrulli in commercial cucurbit seeds in China, and the relatively high prevalence suggests that commercial seeds represent a substantial source of primary inoculum that can threaten cucurbit seed and fruit production in China.

Ferric Uptake Regulator (FurA) is Required for Acidovorax citrulli Virulence on Watermelon.

Acidovorax citrulli is the causal agent of bacterial fruit blotch, a serious threat to commercial watermelon and melon crop production worldwide. Ferric uptake regulator (Fur) is a global transcription factor that affects a number of virulence-related functions in phytopathogenic bacteria; however, the role of furA has not been determined for A. citrulli. Hence, we constructed an furA deletion mutant and a corresponding complement in the background of A. citrulli strain xlj12 to investigate the role of the gene in siderophore production, concentration of intracellular Fe2+, bacterial sensitivity to hydrogen peroxide, biofilm formation, swimming motility, hypersensitive response induction, and virulence on melon seedlings. The A. citrulli furA deletion mutant displayed increased siderophore production, intracellular Fe2+ concentration, and increased sensitivity to hydrogen peroxide. In contrast, biofilm formation, swimming motility, and virulence on melon seedlings were significantly reduced in the furA mutant. As expected, complementation of the furA deletion mutant restored all phenotypes to wild-type levels. In accordance with the phenotypic results, the expression levels of bfrA and bfrB that encode bacterioferritin, sodB that encodes iron/manganese superoxide dismutase, fliS that encodes a flagellar protein, hrcN that encodes the type III secretion system (T3SS) ATPase, and hrcC that encodes the T3SS outer membrane ring protein were significantly downregulated in the A. citrulli furA deletion mutant. In addition, the expression of feo-related genes and feoA and feoB was significantly upregulated in the furA mutant. Overall, these results indicated that, in A. citrulli, FurA contributes to the regulation of the iron balance system, and affects a variety of virulence-related traits.

Homologues of the RNA binding protein RsmA in Pseudomonas syringae pv. tomato DC3000 exhibit distinct binding affinities with non-coding small RNAs and have distinct roles in virulence.

Pseudomonas syringae pv. tomato DC3000 (PstDC3000) contains five RsmA protein homologues. In this study, four were functionally characterized, with a focus on RsmA2, RsmA3 and RsmA4. RNA electrophoretic mobility shift assays demonstrated that RsmA1 and RsmA4 exhibited similar low binding affinities to non-coding small RNAs (ncsRNAs), whereas RsmA2 and RsmA3 exhibited similar, but much higher, binding affinities to ncsRNAs. Our results showed that both RsmA2 and RsmA3 were required for disease symptom development and bacterial growth in planta by significantly affecting virulence gene expression. All four RsmA proteins, especially RsmA2 and RsmA3, influenced γ-amino butyric acid utilization and pyoverdine production to some degree, whereas RsmA2, RsmA3 and RsmA4 influenced protease activities. A single RsmA, RsmA3, played a dominant role in regulating motility. Furthermore, reverse transcription quantitative real-time PCR and western blot results showed that RsmA proteins, especially RsmA2 and RsmA3, regulated target genes and possibly other RsmA proteins at both transcriptional and translational levels. These results indicate that RsmA proteins in PstDC3000 exhibit distinct binding affinities to ncsRNAs and have distinct roles in virulence. Our results also suggest that RsmA proteins in PstDC3000 interact with each other, where RsmA2 and RsmA3 play a major role in regulating various functions in a complex manner.

Optimization of culture conditions for promoting heat-stable antifungal factor production level in Lysobacter enzymogenes.

Heat-stable antifungal factor (HSAF), which was first isolated from Lysobacter enzymogenes, exhibits inhibitory activities against a wide range of pathogens; however, a low level of HSAF was obtained from L. enzymogenes cultured in 0.1 × tryptic soy broth (TSB), an amount that does not satisfy HSAF application in disease control. In this study, the optimization of media components and environmental conditions were examined for improving the production of HSAF from L. enzymogenes OH11. The one factor at a time method was used to screen optimal nitrogen and carbon sources and inorganic salt. Then the orthogonal matrix method was used to determine the optimal concentration of the media components and environmental factors. The results showed that the maximum level of HSAF (23361 mAU·s) was achieved when OH11 cultured in the media of 0.7% (w/v) soybean powder, 0.5% (w/v) glucose and 0.08% CaCl2 at 200 rpm at 30°C for 60 h, which is much higher than that cultured in 0.1 × TSB. This opens up the possibility of HSAF or L. enzymogenes utilization for biological control of plant disease.

Identification and Characterization of Phomopsis amygdali and Botryosphaeria dothidea Associated with Peach Shoot Blight in Yangshan, China.

Peach (Prunus persica (L.) Batsch) is produced locally in Yangshan, Wuxi City, China. In recent years, a widespread shoot blight has been observed in many peach orchards of Yangshan that kills the twigs and results in high losses in fruit production. Disease incidences ranged from 10 to 20% in the affected orchards and, in extreme cases, 40% of the trees were affected. Shoot blight of peach is caused by a fungus, previously identified as Phomopsis amygdali. Between 2014 and 2015, samples were collected four times from three peach orchards located in Yangshan to understand the etiology of shoot blight. Interestingly, two types of shoot blight symptoms were observed: one characterized by necrotic lesions with rings and one without rings. Based on conidial morphology, cultural characteristics, and analysis of nucleotide sequences of three genomic regions (the internal transcribed spacer region, a partial sequence of the ß-tubulin gene, and the translation elongation factor 1-α), isolates were identified as P. amygdali and Botryosphaeria dothidea. Remarkably, most of the P. amygdali isolates were recovered from twigs showing necrotic lesions without rings. In contrast, most of the B. dothidea isolates were recovered from twigs with rings in the necrotic lesions. Correlations among pathogens, sampling regions, and disease symptoms were noted, and growth rates of these pathogens were characterized. Pathogenicity tests showed that B. dothidea isolates could induce necrotic lesions with rings but P. amygdali isolates could only induce necrotic lesions. Moreover, the B. dothidea isolates exhibited higher levels of virulence than P. amygdali isolates on the peach twig. Additionally, high frequencies of detection of both P. amygdali and B. dothidea from buds indicated that buds may be the primary site of fungal invasion. Cankers and necrotic twigs may also serve as infection courts. Our results suggest that B. dothidea and P. amygdali are the common causal agents of peach shoot blight in Yangshan, China. This finding provides a basis for the development of effective management strategies.

Hfq, a RNA Chaperone, Contributes to Virulence by Regulating Plant Cell Wall-Degrading Enzyme Production, Type VI Secretion System Expression, Bacterial Competition, and Suppressing Host Defense Response in Pectobacterium carotovorum.

Hfq is a RNA chaperone and participates in a wide range of cellular processes and pathways. In this study, mutation of hfq gene from Pectobacterium carotovorum subsp. carotovorum PccS1 led to significantly reduced virulence and plant cell wall-degrading enzyme (PCWDE) activities. In addition, the mutant exhibited decreased biofilm formation and motility and greatly attenuated carbapenem production as well as secretion of hemolysin coregulated protein (Hcp) as compared with wild-type strain PccS1. Moreover, a higher level of callose deposition was induced in Nicotiana benthamiana leaves when infiltrated with the mutant. A total of 26 small (s)RNA deletion mutants were obtained among a predicted 27 sRNAs, and three mutants exhibited reduced virulence in the host plant. These results suggest that hfq plays a key role in Pectobacterium virulence by positively impacting PCWDE production, secretion of the type VI secretion system, bacterial competition, and suppression of host plant responses.