Genomic characterization of Pseudomonas syringae pv. syringae from Callery pear and the efficiency of associated phages in disease protection.

The Pseudomonas syringae species complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize two P. syringae isolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified the P. syringae isolates as members of phylogroup 2, related to other strains previously isolated from Pyrus and Prunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related to Pseudomonas syringae pv. actinidiae phage PHB09 and the Flaumdravirus genus. Finally, using in planta infection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity of P. syringae and their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease.IMPORTANCEGlobal change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains of Pseudomonas syringae pv. syringae isolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novel P. syringae pv. syringae isolates that potentially pose a risk to local fruit production, or vice versa-but also provide us with novel associated phages, effective in disease mitigation.

Infection of postharvest pear by Penicillium expansum is facilitated by the glycoside hydrolase (eglB) gene.

The primary reason for postharvest loss is blue mold disease which is mainly caused by Penicillium expansum. Strategies for disease control greatly depend on the understanding of mechanisms of pathogen-fruit interaction. A member of the glycoside hydrolase family, ß-glucosidase 1b (eglB), in P. expansum was significantly upregulated during postharvest pear infection. Glycoside hydrolases are a large group of enzymes that can degrade plant cell wall polymers. High homology was found between the glycoside hydrolase superfamily in P. expansum. Functional characterization and analysis of eglB were performed via gene knockout and complementation analysis. Although eglB deletion had no notable effect on P. expansum colony shape or microscopic morphology, it did reduce the production of fungal hyphae, thereby reducing P. expansum's sporulation and patulin (PAT) accumulation. Moreover, the deletion of eglB (ΔeglB) reduced P. expansum pathogenicity in pears. The growth, conidia production, PAT accumulation, and pathogenicity abilities of ΔeglB were restored to that of wild-type P. expansum by complementation of eglB (ΔeglB-C). These findings indicate that eglB contributes to P. expansum's development and pathogenicity. This research is a contribution to the identification of key effectors of fungal pathogenicity for use as targets in fruit safety strategies.

New Insights and Approach Toward the Genetic Diversity and Strain Typing of Erwinia pyrifoliae Based on rsxC, an Electron Transport Gene.

Erwinia pyrifoliae, a causal agent of black shoot blight in apple and pear trees, is a plant pathogenic bacterium first reported in South Korea. The symptoms of black shoot blight are very similar to those of the fire blight disease in apple and pear trees caused by E. amylovora, as E. pyrifoliae has a genetically very close relationship with E. amylovora. Recently, there have been reports that E. pyrifoliae causes disease in European strawberries, resulting in severe fruit loss that aroused great concern about its spread, distribution, and host range. Therefore, it is essential to establish a trustworthy approach to understanding the distribution patterns of E. pyrifoliae based on the genetic background to strengthen the barrier of potential spreading risks, although advanced methods have been provided to accurately detect E. pyrifoliae and E. amylovora. Consequently, this study discovered a noble and noteworthy gene, rsxC, capable of providing the pathogen genotype by comparing E. pyrifoliae genomic sequences in the international representative genome archive. Different numbers of 40-unit amino acid repeats in this gene among the strains induced intraspecific traits in RsxC. By comparing their repeat pattern, E. pyrifoliae isolates were divided into two main groups, branching into several clades via sequence alignment of 35 E. pyrifoliae isolates from various apple orchards from 2020 to 2021 in South Korea. The newly discovered quadraginta amino acid repeat within this gene would be a valuable genetic touchstone for determining the genotype and distribution pattern of E. pyrifoliae strains, ultimately leading to exploring their evolution. The function of amino acid repeats and the biological significance of strains need to be elucidated further.

Biocontrol Potential of Streptomyces odonnellii SZF-179 toward Alternaria alternata to Control Pear Black Spot Disease.

Pear black spot disease, caused by Alternaria alternata, is a devastating disease in pears and leads to enormous economic losses worldwide. In this investigation, we isolated a Streptomyces odonnellii SZF-179 from the rhizosphere soil of pear plants in China. Indoor confrontation experiments results showed that both SZF-179 and its aseptic filtrate had excellent inhibitory effects against A. alternata. Afterwards, the main antifungal compound of SZF-179 was identified as polyene, with thermal and pH stability in the environment. A microscopic examination of A. alternata mycelium showed severe morphological abnormalities caused by SZF-179. Protective studies showed that SZF-179 fermentation broth could significantly reduce the diameter of the necrotic lesions on pear leaves by 42.25%. Furthermore, the potential of fermentation broth as a foliar treatment to control black leaf spot was also evaluated. Disease indexes of 'Hosui' and 'Wonwhang' pear plants treated with SZF-179 fermentation broth were lower than that of control plants. Overall, SZF-179 is expected to be developed into a safe and broad-spectrum biocontrol agent. No studies to date have evaluated the utility of S. odonnellii for the control of pear black spot disease; our study fills this research gap. Collectively, our findings provide new insights that will aid the control of pear black spot disease, as well as future studies of S. odonnellii strains.

Exploring the efficacy of carvacrol as a biocontrol agent against pear Valsa canker.

Valsa canker, a fungal disease caused by Valsa pyri, poses a significant threat to the pear industry. Currently, chemical control serves as the primary method to control valsa canker. However, the emergence of resistance can pose a challenge to its effectiveness. Biopesticides are a relatively new option for disease control, but there is limited research on their effects on pear Valsa canker. To determine the effectiveness of different biopesticides, we selected 10 common biopesticides to test their inhibition efficacy and impacts on mycelial growth rate and conidial germination. Results showed that carvacrol had very good antifungal activity; therefore its inhibition mechanisms were further investigated. Electron microscopy and transcriptome data analysis were utilized to examine how carvacrol impeded V. pyri by inducing mycelium deformation, wrinkling, and rupture. Carvacrol also affected plant hormones, thus improving plant resistance to the disease. This study lays the groundwork for the utilization of 10 distinct biopesticides to control V. pyri while elucidating how carvacrol harms the pathogen and prompts the plant defense control mechanism.

Exploration of the Biocontrol Activity of Bacillus atrophaeus Strain HF1 against Pear Valsa Canker Caused by Valsa pyri.

Valsa pyri-induced pear Valsa canker is among the most prevalent diseases to impact pear quality and yields. Biocontrol strategies to control plant disease represent an attractive alternative to the application of fungicides. In this study, the potential utility of Bacillus atrophaeus strain HF1 was assessed as a biocontrol agent against pear Valsa canker. Strain HF1 suppressed V. pyri mycelium growth by 61.20% and induced the development of malformed hyphae. Both culture filtrate and volatile organic compounds (VOCs) derived from strain HF1 were able to antagonize V. pyri growth. Treatment with strain HF1-derived culture filtrate or VOCs also induced the destruction of hyphal cell membranes. Headspace mixtures prepared from strain HF1 were analyzed, leading to the identification of 27 potential VOCs. Of the thirteen pure chemicals tested, iberverin, hexanoic acid, and 2-methylvaleraldehyde exhibited the strongest antifungal effects on V. pyri, with respective EC50 values of 0.30, 6.65, and 74.07 µL L-1. Fumigation treatment of pear twigs with each of these three compounds was also sufficient to prevent the development of pear Valsa canker. As such, these results demonstrate that B. atrophaeus strain HF1 and the volatile compounds iberverin, hexanoic acid, and 2-methylvaleraldehyde exhibit promise as novel candidate biocontrol agents against pear Valsa canker.

Current Situation of Fire Blight in China.

Fire blight, caused by the plant-pathogenic bacterium Erwinia amylovora, is a devastating disease that occurs on rosaceous plants, including pears and apples. E. amylovora is indigenous to North America and was spread to the Eurasian continent in the second half of the 20th century through contaminated plant materials. In 2016, fire blight was first observed in Yili, Xinjiang Province, in Northwestern China. Since then, it has spread to most pear-producing regions in Xinjiang Province and parts of Gansu Province. The disease has caused severe damage to China's pear and apple industries, including the 2017 disease epidemic in Korla, Xinjiang, which caused an overall yield reduction of 30 to about 50% in Korla and the destruction of over 1 million pear trees. Over the past few years, a combined effort of research, extension, and education by the Chinese government, scientists, and fruit growers has greatly alleviated outbreaks and epidemics in affected regions while successfully limiting the further spread of fire blight to new geographical regions. Here, we review the occurrence, spread, and damage of this disease to the Chinese fruit industry, as well as the management options used in China and their outcomes. We also discuss future perspectives for restraining the spread and alleviating the damage of fire blight in China.

Contribution of Native Plasmids of Pantoea vagans C9-1 to Epiphytic Fitness and Fire Blight Management on Apple and Pear Flowers and Fruits.

Pantoea vagans C9-1 (C9-1) is a biological control bacterium that is applied to apple and pear trees during bloom for suppression of fire blight, caused by Erwinia amylovora. Strain C9-1 has three megaplasmids: pPag1, pPag2, and pPag3. Prior bioinformatic studies predicted these megaplasmids have a role in environmental fitness and/or biocontrol efficacy. Plasmid pPag3 is part of the large Pantoea plasmid (LPP-1) group that is present in all Pantoea spp. and has been hypothesized to contribute to environmental colonization and persistence, while pPag2 is less common. We assessed fitness of C9-1 derivatives cured of pPag2 and/or pPag3 on pear and apple flowers and fruit in experimental orchards. We also assessed the ability of a C9-1 derivative lacking pPag3 to reduce populations of E. amylovora on flowers and disease incidence. Previously, we determined that tolerance to stresses imposed in vitro was compromised in derivatives of C9-1 lacking pPag2 and/or pPag3; however, in this study, the loss of pPag2 and/or pPag3 did not consistently reduce the fitness of C9-1 on flowers in orchards. Over the summer, pPag3 contributed to survival of C9-1 on developing apple and pear fruit in two of five trials, whereas loss of pPag2 did not significantly affect survival of C9-1. We also found that loss of pPag3 did not affect C9-1's ability to reduce E. amylovora populations or fire blight incidence on apple flowers. Our findings partially support prior hypotheses that LPP-1 in Pantoea species contributes to persistence on plant surfaces but questions whether LPP-1 facilitates host colonization.

The PcMYB44-mediated miR397-PcLACs module regulates defence-induced lignification in pear resistance to fungal disease.

Diseases caused by Alternaria alternata and Botryosphaeria dothidea diminish pear yield and quality, and restrict the pear agricultural industry. Lignification is a conserved mechanism for plant resistance against pathogen invasion. The regulatory mechanisms underlying defence-induced lignification in pear in response to fungal pathogen infection remain unknown. In this study, analysis of lignification level and lignin content in pear revealed that A. alternata and B. dothidea induced lignification, and transcriptomics showed that lignin biosynthesis was affected. To explore whether laccases (LACs) mediated by miR397 regulate lignification in pear, we investigated the role of PcmiR397 in repressing the expression of PcLACs using 5'-RNA ligase-mediated-RACE and co-transformation in tobacco. Opposite expression patterns for PcmiR397 and PcLAC target genes were observed in pear in response to pathogens. Transient transformation in pear demonstrated that silencing PcmiR397 and overexpressing a single PcLAC enhanced resistance to pathogens via lignin synthesis. To further reveal the mechanism underpinning the PcMIR397 response of pear to pathogens, the PcMIR397 promoter was analysed, and pMIR397-1039 was found to be inhibited by pathogen infection. The transcription factor PcMYB44 was up-regulated, and it bound to the PcMIR397 promoter and inhibited transcription following pathogen infection. The results demonstrate the role of PcmiR397-PcLACs in broad-spectrum resistance to fungal disease, and the potential role of PcMYB44 involved in the miR397-PcLAC module in regulating defence-induced lignification. The findings provide valuable candidate gene resources and guidance for molecular breeding to improve resistance to fungal disease in pear.

Interspecificity of Scab Fungus Isolated from Wild Syrian Pear (Pyrus syriaca).

Scab on pear is caused by two pathogens, Venturia pyrina on European pear and V. nashicola on Asian pear. Five races of V. pyrina and seven races of V. nashicola have been reported thus far and pathological specialization occurs in both species. Among them, the five race isolates of V. pyrina were previously found from wild Syrian pear. In this study, mating and morphological characteristics of Venturia isolates from Syrian pear were compared with those of isolates from European and Japanese pear cultivated in Japan. The results from mating experiments showed that Syrian pear isolates were compatible with European pear isolates of V. pyrina to produce ascospores but were sterile with V. nashicola isolates in culture. Interestingly, however, the size and shape of conidia collected from naturally infected leaves of Syrian pear resembled those of V. nashicola. This finding may open the way to study coevolution between pear hosts and Venturia spp. in the future.

Exogenous dopamine improves resistance to Botryosphaeria dothidea by increasing autophagy activity in pear.

Pear ring rot, a fungal disease caused by Botryosphaeria dothidea (B. dothidea), is one of the most damaging diseases in pear production, affecting fruit yield and causing economic losses. It is not clear whether dopamine, one of the catecholamines, has any role in pear ring rot resistance. In this study, we found that dopamine treatment of B. dothidea resulted in a significant upregulation of PbrTYDC expression compared to H2O treatment (control) and reduced the levels of Hydrogen Peroxide (H2O2) and Superoxide Anion (O2-), increased Peroxidase (POD), Catalase (CAT), Superoxide Dismutase (SOD) and Phenylalanine Ammonia-Lyase (PAL) activities, and induced a significant upregulation of related gene expression. Dopamine treatment promoted the oxidationreduction capacity of the AsA-GSH cycle to scavenge Reactive Oxygen Species (ROS), increased the expression of autophagy-related genes and the accumulation of autophagic structures, and enhanced autophagic activity. Silencing PbrTYDC and PbrATG8 in pear increased H2O2 and·O2-, decreased POD, CAT and SOD activities and reduced resistance to B. dothidea, which was restored by dopamine treatment. In conclusion, exogenous dopamine enhances resistance to B. dothidea by increasing the antioxidant capacity and autophagic activity of pears, and this study provides new insights for subsequent studies on B. dothidea as well as autophagy.

AaCaMKs Positively Regulate Development, Infection Structure Differentiation and Pathogenicity in Alternaria alternata, Causal Agent of Pear Black Spot.

Calcium/calmodulin-dependent protein kinase (CaMK), a key downstream target protein in the Ca2+ signaling pathway of eukaryotes, plays an important regulatory role in the growth, development and pathogenicity of plant fungi. Three AaCaMKs (AaCaMK1, AaCaMK2 and AaCaMK3) with conserved PKC_like superfamily domains, ATP binding sites and ACT sites have been cloned from Alternaria alternata, However, their regulatory mechanism in A. alternata remains unclear. In this study, the function of the AaCaMKs in the development, infection structure differentiation and pathogenicity of A. alternata was elucidated through targeted gene disruption. The single disruption of AaCaMKs had no impact on the vegetative growth and spore morphology but significantly influenced hyphae growth, sporulation, biomass accumulation and melanin biosynthesis. Further expression analysis revealed that the AaCaMKs were up-regulated during the infection structure differentiation of A. alternata on hydrophobic and pear wax substrates. In vitro and in vivo analysis further revealed that the deletion of a single AaCaMKs gene significantly reduced the A. alternata conidial germination, appressorium formation and infection hyphae formation. In addition, pharmacological analysis confirmed that the CaMK specific inhibitor, KN93, inhibited conidial germination and appressorium formation in A. alternata. Meanwhile, the AaCaMKs genes deficiency significantly reduced the A. alternata pathogenicity. These results demonstrate that AaCaMKs regulate the development, infection structure differentiation and pathogenicity of A. alternata and provide potential targets for new effective fungicides.

Novel Detection and Quantification Approach of Erwinia amylovora In Vitro and In Planta Using SYBR Green-Based Real-Time PCR Assay.

Fire blight, caused by the bacterial pathogen Erwinia amylovora, is a highly destructive disease of apple and pear. Because the apple tree gets systemically infected with E. amylovora and eventually dies, E. amylovora is a considerably important pathogen in the orchard that requires long-term management. In addition, it is crucial to prevent the spread of the pathogen by expeditious diagnosis. In this study, via comparative approaches to the genome sequences of the strains of various Erwinia spp., we designed specific primers targeting a hypothetical gene that is single copy and located in the chromosomal DNA of E. amylovora. This primer set specifically amplified the DNA of E. amylovora but no other bacteria, including E. pyrifoliae, Pectobacterium spp., Pantoea spp., and Dickeya chrysanthemi. Furthermore, the SYBR Green-based real-time PCR using the primer set allowed accurate estimation of the population of E. amylovora. Developing a rapid and accurate diagnostic method using the novel primer set enables effective defense against pathogen spread through continuous monitoring and quick response.

Isolation of Streptomycin-Resistant Erwinia pyrifoliae in Korea.

As a black shoot blight disease-causing agent, Erwinia pyrifoliae was first reported in 1995 in Korea. A total of 101 isolates of E. pyrifoliae were isolated from samples showing bacterial symptoms collected from apple and pear orchards between 2020 and 2021. These isolates were screened for streptomycin resistance, with one from an orchard in Gwangju showing resistance at 100 µg/ml streptomycin. This streptomycin-resistant E. pyrifoliae (EpSmR) isolate was identified via polymerase chain reaction amplification of the strA/strB gene and an internal region of the ribosomal rpsL gene containing codon 43. EpSmR has a point mutation that altered this codon from lysine (AAA) to threonine (ACA). The strA and strB genes were not identified in EpSmR. EpSmR showed a high resistance to streptomycin (>50,000 µg/ml). This is the first study reporting EpSmR, which emerged due to a mutation in codon 43 of the rpsL gene.

Pathogenicity, Molecular Characterization, and Mycotoxigenic Potential of Alternaria spp. Agents of Black Spots on Fruit and Leaves of Pyrus communis in Italy.

Brown and black spots, caused by Stemphylium and Alternaria species, are important fungal diseases affecting European pear (Pyrus communis) in orchards. Both fungal genera cause similar symptoms, which could favor misidentification, but Alternaria spp. are increasingly reported due to the changing climatic conditions. In this study, Alternaria spp. were isolated from symptomatic leaves and fruits of European pear, and their pathogenicity was evaluated on pear fruits from cultivar Abate Fétel, and molecular and chemical characterization were performed. Based on maximum likelihood phylogenetic analysis, 15 of 46 isolates were identified as A. arborescens species complex (AASC), 27 as A. alternata, and four as Alternaria sp. Both species were isolated from mature fruits and leaves. In pathogenicity assays on pear fruits, all isolates reproduced the symptoms observed in the field, by both wound inoculation and direct penetration. All but one isolate produced Alternaria toxins on European pears, including tenuazonic acid and alternariol (89.1% of the isolates), alternariol monomethyl ether (89.1%), altertoxin I (80.4%), altenuene (50.0%), and tentoxin (2.2%). These isolates also produced at least two mycotoxins, and 43.5% produced four mycotoxins, with an average total concentration of the Alternaria toxins exceeding 7.58 × 106 ng/kg. Our data underline the potential risks for human health related to the high mycotoxin content found on fruits affected by black spot. This study also represents the first report of AASC as an agent of black spot on European pear in Italy.

AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata.

AIMS: Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. METHODS AND RESULTS: A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6 h) and a 1.46-fold upregulation during the infection hyphae formation period (8 h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. CONCLUSIONS: AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. SIGNIFICANCE AND IMPACT OF STUDY: Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.

Biocontrol Efficacy of the Vishniacozyma Victoriae in Semi-Commercial Assays for the Control of Postharvest Fungal Diseases of Organic Pears.

Vishniacozyma victoriae NPCC 1263 was selected for this work because of the active antagonistic effect over several fungi in postharvest organic pears. Yeast biomass production was carried out in a 15 L stirred-tank bioreactor with 12 L of working volume at 20 °C and 300 rpm and 0.64 vvm of aeration. The selected production medium was based on cheese whey powder and salts. The present study aims to evaluate the possibility of using an inexpensive growth substrate for production of added value products (yeast biomass), this innovation also requires evaluate biocontrol efficacy of yeast against fungal diseases of pears in semi-commercial assays. The yeast biomass was collected, cold stored for 60 days (Treatment 1) and 15 days (Treatment 2) and sprayed on the pears in semi-commercial level testing assays. After 180 days of postharvest conservation, significant reduction of fungal infection by Penicillium expansum, Botrytis cinerea and Cladosporium sp. was observed. The Treatment 1 reduced total diseases incidence by 71%, instead Treatment 2 reduced it by 92%. The effect of spray application on the yeast viability with different cold storage was tested. These work provides information on the bench-scale bioreactor yeast production using a new low-cost medium, viability and biocontrol efficacy of Vishniacozyma victoriae in controlling common diseases affecting pears in semi-commercial assays.

Molecular Characterization of Phospholipase C in Infection Structure Differentiation Induced by Pear Fruit Surface Signals, Stress Responses, Secondary Metabolism, and Virulence of Alternaria alternata.

Fungal pathogens use plant surface physiochemical signals to trigger specific developmental processes. To assess the role of phospholipase C (PLC) in mediating plant stimuli sensing of Alternaria alternata, the function of three PLC genes was characterized by constructing ΔAaPLC mutants. Here we showed that fruit wax-coated surfaces significantly induced appressorium formation in A. alternata and mutants. Germination of ΔAaPLC mutants did not differ from the wild type. Deletion of AaPLC1 led to the decrease of appressorium formation and infected hyphae, but the degree of reduction varies between the different types of waxes, with the strongest response to pear wax. Appressorium formation and infected hyphae of the ΔAaPLC1 mutant on dewaxed onion epidermis mounted with pear wax (θ4) were reduced by 14.5 and 65.7% after 8 h incubation, while ΔAaPLC2 and ΔAaPLC3 formed the same infection hyphae as wild type. In addition, AaPLC1 mutation caused pleiotropic effects on fungal biological function, including growth deficiency, changes in stress tolerance, weakening of pathogenicity to the host, as well as destruction of mycotoxin synthesis. Both AaPLC2 and AaPLC3 genes were found to have some effects on stress response and mycotoxin production. Taken together, AaPLC genes differentially regulate the growth, stress response, pathogenicity, and secondary metabolism of A. alternata.

The RNA-Binding Protein ProQ Impacts Exopolysaccharide Biosynthesis and Second Messenger Cyclic di-GMP Signaling in the Fire Blight Pathogen Erwinia amylovora.

Erwinia amylovora is a plant-pathogenic bacterium that causes fire blight disease in many economically important plants, including apples and pears. This bacterium produces three exopolysaccharides (EPSs), amylovoran, levan, and cellulose, and forms biofilms in host plant vascular tissues, which are crucial for pathogenesis. Here, we demonstrate that ProQ, a conserved bacterial RNA chaperone, was required for the virulence of E. amylovora in apple shoots and for biofilm formation in planta. In vitro experiments revealed that the deletion of proQ increased the production of amylovoran and cellulose. Prc is a putative periplasmic protease, and the prc gene is located adjacent to proQ. We found that Prc and the associated lipoprotein NlpI negatively affected amylovoran production, whereas Spr, a peptidoglycan hydrolase degraded by Prc, positively regulated amylovoran. Since the prc promoter is likely located within proQ, our data showed that proQ deletion significantly reduced the prc mRNA levels. We used a genome-wide transposon mutagenesis experiment to uncover the involvement of the bacterial second messenger c-di-GMP in ProQ-mediated cellulose production. The deletion of proQ resulted in elevated intracellular c-di-GMP levels and cellulose production, which were restored to wild-type levels by deleting genes encoding c-di-GMP biosynthesis enzymes. Moreover, ProQ positively affected the mRNA levels of genes encoding c-di-GMP-degrading phosphodiesterase enzymes via a mechanism independent of mRNA decay. In summary, our study revealed a detailed function of E. amylovora ProQ in coordinating cellulose biosynthesis and, for the first time, linked ProQ with c-di-GMP metabolism and also uncovered a role of Prc in the regulation of amylovoran production. IMPORTANCE Fire blight, caused by the bacterium Erwinia amylovora, is an important disease affecting many rosaceous plants, including apple and pear, that can lead to devastating economic losses worldwide. Similar to many xylem-invading pathogens, E. amylovora forms biofilms that rely on the production of exopolysaccharides (EPSs). In this paper, we identified the RNA-binding protein ProQ as an important virulence regulator. ProQ played a central role in controlling the production of EPSs and participated in the regulation of several conserved bacterial signal transduction pathways, including the second messenger c-di-GMP and the periplasmic protease Prc-mediated systems. Since ProQ has recently been recognized as a global posttranscriptional regulator in many bacteria, these findings provide new insights into multitiered regulatory mechanisms for the precise control of virulence factor production in bacterial pathogens.

The transmembrane protein AaSho1 is essential for appressorium formation and secondary metabolism but dispensable for vegetative growth in pear fungal Alternaria alternata.

The high-osmolarity glycerol response (HOG) pathway is pivotal in environmental stress response, differentiation and virulence of Alternaria alternata. The synthetic high osmolarity sensitive sensor Sho1 has been postulated to regulate the HOG pathway. To determine the regulatory role of transmembrane protein Sho1 on vegetative growth, secondary metabolism and infection structure formation, a gene (AaSho1) encoding Sho1 was cloned and characterized from A. alternata (JT-03). Sequence analysis showed that AaSho1 has all four characteristic transmembrane domains and the SH3 domain present in another Sho1 gene from several filamentous fungal. The quantitative RT-PCR analysis showed that fruit wax extract significantly up-regulated AaSho1 gene expression in vitro. Pharmacological experiments showed that A. alternata treated with nystatin, a specific AaSho1 inhibitor, had no significant effect on the morphology of A. alternata and the invasive growth in pear fruit. However, nystatin treatment significantly reduced spore germination rates on different wax-coated hydrophobic surfaces, with 58.00, 46.70 and 83.72% reduced for fruit wax, beeswax and paraffin coated. Meanwhile, the secondary metabolism altenuene (ALT), tentoxin (TEN) toxin, and melanin content were also affected by nystatin treatment. These findings suggest that AaSho1 is required for the infection structure differentiation and secondary metabolism of A. alternata in response to physiochemical signals on the host surfaces.