Phenotypic and Genomic Characterization of Pseudomonas wuhanensis sp. nov., a Novel Species with Promising Features as a Potential Plant Growth-Promoting and Biocontrol Agent.

Plant growth-promoting rhizobacterial strain FP607T was isolated from the rhizosphere of beets in Wuhan, China. Strain FP607T exhibited significant antagonism toward several phytopathogenic bacteria, indicating that FP607T may produce antimicrobial metabolites and has a stronger biocontrol efficacy against plant pathogens. Growth-promoting tests showed that FP607T produced indole-3-acetic acid (IAA), NH3, and ferritin. The genome sequence of strain FP607T was 6,590,972 bp long with 59.0% G + C content. The optimum temperature range was 25-30 °C, and the optimum pH was 7. The cells of strain FP607T were Gram-negative, short, and rod-shaped, with polar flagella. The colonies on the King's B (KB) agar plates were light yellow, smooth, and circular, with regular edges. A phylogenetic analysis of the 16S rRNA sequence and a multilocus sequence analysis (MLSA) showed that strain FP607T was most closely related to the type of strain Pseudomonas farris SWRI79T. Based on a polyphasic taxonomic approach, strain FP607T was identified as a novel species within the genus Pseudomonas, for which the name Pseudomonas wuhanensis sp. nov. was proposed. The type of strain used was FP607T (JCM 35688, CGMCC 27743, and ACCC 62446).

Pseudomonas hefeiensis sp. nov., isolated from the rhizosphere of multiple cash crops in China.

Three bacterial strains, FP250T, FP821, and FP53, were isolated from the rhizosphere soil of oilseed rape, licorice, and habanero pepper in Anhui Province, Xinjiang Uygur Autonomous Region, and Jiangsu Province, PR China, respectively. All strains were shown to grow at 4-37 °C and pH 6.0-9.0, and in the presence of 0-4.0 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences or housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) and phylogenomic analysis showed that strains FP250T, FP821, and FP53 belong to the genus Pseudomonas, and are closely related to Pseudomonas kilonensis DSM 13647T, Pseudomonas brassicacearum JCM 11938T, Pseudomonas viciae 11K1T, and Pseudomonas thivervalensis DSM 13194T. The DNA G+C content of strain FP205T was 59.8 mol%. The average nucleotide identity and digital DNA-DNA hybridization values of strain FP205T with the most closely related strain were 93.2 % and 51.4 %, respectively, which is well below the threshold for species differentiation. Strain FP205T contained summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) as major fatty acids, and diphosphatidylglycerol along with phosphatidylethanolamine and aminophospholipid as major polar lipids. The predominant isoprenoid quinone was ubiquinone-9. Based on these phenotypic, phylogenetic, and chemotaxonomic results, strain FP205T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas hefeiensis sp. nov. is proposed. The type strain is FP205T (=ACCC 62447T=JCM 35687T).

Effects of the invasion of Ralstonia solanacearum on soil microbial community structure in Wuhan, China.

This study investigated the change in the microbiome of tomato rhizosphere soils after the invasion of Ralstonia solanacearum and analyzed the correlation between microbes and soil physicochemical properties. Diversity analyses of the bacteria in healthy and diseased rhizosphere soil samples (HRS and DRS) revealed that HRS had a higher species diversity and were compositionally different from DRS (P ≤ 0.05). Substantial differences in the relative abundance of Actinobacteria (37.52% vs 28.96%, P ≤ 0.05) and Proteobacteria (29.20% vs 35.59%, P ≤ 0.05) were identified in HRS and DRS, respectively. Taxonomic composition analysis showed ten differentially abundant genera, and seven of them (Gaiella, Roseisolibacter, Solirubrobacter, Kribbella, Acidibacter, Actinomarinicola, and Marmoricola) are more abundant in HRS. Soil pH and enzyme activities were negatively correlated with the abundance of R. solanacearum. The contents of total nitrogen (TN), total phosphorus (TP), total potassium (TK), alkaline nitrogen (alkaline N), available phosphorus (AP), available potassium (AK), NO3-N(NN), NH4+-N (AN), and organic matter (OM) were all significantly increased in DRS. The composition and richness of protozoa in the samples show significant differences. Cephalobus, Acrobeles, Heteromita, norank_Tylenchida, and Rotylenchulus were enriched in DRS. Microbial interaction networks revealed that the HRS networks were more complex than the DRS networks. Overall, the results of this study demonstrate that healthy soil has a more complex microbial community structure and higher enzyme activity, and the invasion of R. solanacearum damages the soil microbial system.IMPORTANCEHow does the invasion of Ralstonia solanacearum affect tomato rhizosphere bacteria and protozoa? Which microbial changes can affect the growth of R. solanacearum? To date, most research studies focus on bacteria, with little research on protozoa, and even less on the synergistic effects between protozoa and bacteria. Here, we analyzed the correlation between tomato rhizosphere bacterial and protozoan communities and soil physicochemical properties during the invasion of R. solanacearum. We found that the diversity and abundance of rhizosphere microorganisms in healthy rhizosphere soil samples (HRS) were significantly higher than those in diseased rhizosphere soil samples (DRS), and there were significant changes in soil pH and enzyme activity. Overall, in this study, the analysis of microbial changes during the invasion of R. solanacearum provides a theoretical basis for the prevention and control of bacterial wilt.

Pseudomonas cucumis sp. nov., isolated from the rhizosphere of crop plants.

Two bacterial strains, FP1935T and FP1962, were isolated from the rhizosphere soil of cucumber and Chieh-qua plants, respectively, in Jilin Province, PR China. These strains were Gram-stain-negative, aerobic, rod-shaped and motile with one or two polar flagella. Analysis of the 16S rRNA gene sequences revealed that they represented members of the genus Pseudomonas, with the highest similarity to Pseudomonas silesiensis A3T (99.45 %), Pseudomonas frederiksbergensis JAJ28T (99.45 %), Pseudomonas mandelii NBRC 103147T (99.38 %), Pseudomonas piscium P50T (99.27 %) and Pseudomonas meliae CFBP 3225T (99.18 %). The DNA G+C contents of FP1935T and FP1962 were 58.99 mol% and 58.98 mol%, respectively. The results of in silico genome-based analyses indicated that these strains were distinct from other species in the genus Pseudomonas, as the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were below the recommended thresholds of 95 % (ANI) and 70 % (dDDH) for prokaryotic species delineation, with no values exceeding 94.1 and 55.8 %, respectively, compared with any other related species. The results of phenotypic and chemotaxonomic tests confirmed their differentiation from their closest relatives. The fatty acid profiles of both strains mainly consisted of summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C12 : 0 and C16 : 0. The predominant respiratory quinone was Q-9. Polar lipids include phosphatidylethanolamine, unidentified aminophospholipids, unidentified lipids and an unidentified phospholipid. On the basis of these phenotypic and genotypic results, we propose the name Pseudomonas cucumis sp. nov. for these novel strains. The type strain is FP1935T (=ACCC 62445T=JCM 35690T).

Lysobacter changpingensis sp. nov., a novel species of the genus Lysobacter isolated from a rhizosphere soil of strawberry in China.

In the present work, we characterized in detail strain CM-3-T8T, which was isolated from the rhizosphere soil of strawberries in Beijing, China, in order to elucidate its taxonomic position. Cells of strain CM-3-T8T were Gram-negative, non-spore-forming, aerobic, short rod. Growth occurred at 25-37 °C, pH 5.0-10.0, and in the presence of 0-8% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CM-3-T8T formed a stable clade with Lysobacter soli DCY21T and Lysobacter panacisoli CJ29T, with the 16S rRNA gene sequence similarities of 98.91% and 98.50%. The average nucleotide identity and digital DNA-DNA hybridization values between strain SG-8 T and the two reference type strains listed above were 76.3%, 79.6%, and 34.3%, 27%, respectively. The DNA G + C content was 68.4% (mol/mol). The major cellular fatty acids were comprised of C15:0 iso (36.15%), C17:0 iso (8.40%), and C11:0 iso 3OH (8.28%). The major quinone system was ubiquinone Q-8. The major polar lipids were phosphatidylethanolamine (PE), phosphatidylethanolamine (PME), diphosphatidylglycerol (DPG), and aminophospholipid (APL). On the basis of phenotypic, genotypic, and phylogenetic evidence, strain CM-3-T8T (= ACCC 61714 T = JCM 34576 T) represents a new species within the genus Lysobacter, for which the name Lysobacter changpingensis sp. nov. is proposed.

Deciphering the rhizosphere bacteriome associated with biological control of tobacco black shank disease.

Introduction: The black shank disease seriously affects the health of tobacco plants. Conventional control methods have limitations in terms of effectiveness or economic aspects and cause public health concerns. Thus, biological control methods have come into the field, and microorganisms play a key role in suppressing tobacco black shank disease. Methods: In this study, we examined the impact of soil microbial community on black shank disease basing on the structural difference of bacterial communities in rhizosphere soils. We used Illumina sequencing to compare the bacterial community diversity and structure in different rhizosphere soil samples in terms of healthy tobacco, tobacco showing typical black shank symptoms, and tobacco treated with the biocontrol agent, Bacillus velezensis S719. Results: We found that Alphaproteobacteria in the biocontrol group, accounted for 27.2% of the ASVs, was the most abundant bacterial class among three groups. Heatmap and LEfSe analyses were done to determine the distinct bacterial genera in the three sample groups. For the healthy group, Pseudomonas was the most significant genus; for the diseased group, Stenotrophomonas exhibited the strongest enrichment trend, and Sphingomonas showed the highest linear discriminant analysis score, and was even more abundant than Bacillus; for the biocontrol group, Bacillus, and Gemmatimonas were the largely distributed genus. In addition, co-occurrence network analysis confirmed the abundance of taxa, and detected a recovery trend in the network topological parameters of the biocontrol group. Further functional prediction also provided a possible explanation for the bacterial community changes with related KEGG annotation terms. Discussion: These findings will improve our knowledge of plant-microbe interactions and the application of biocontrol agents to improve plant fitness, and may contribute to the selection of biocontrol strains.

Pseudomonas syringae Type III Secretion Protein HrpP Manipulates Plant Immunity To Promote Infection.

The bacterial plant pathogen Pseudomonas syringae deploys a type III secretion system (T3SS) to deliver effector proteins into plant cells to facilitate infection, for which many effectors have been characterized for their interactions. However, few T3SS Hrp (hypersensitive response and pathogenicity) proteins from the T3SS secretion apparatus have been studied for their direct interactions with plants. Here, we show that the P. syringae pv. tomato DC3000 T3SS protein HrpP induces host cell death, suppresses pattern-triggered immunity (PTI), and restores the effector translocation ability of the hrpP mutant. The hrpP-transgenic Arabidopsis lines exhibited decreased PTI responses to flg22 and elf18 and enhanced disease susceptibility to P. syringae pv. tomato DC3000. Transcriptome analysis reveals that HrpP sensing activates salicylic acid (SA) signaling while suppressing jasmonic acid (JA) signaling, which correlates with increased SA accumulation and decreased JA biosynthesis. Both yeast two-hybrid and bimolecular fluorescence complementation assays show that HrpP interacts with mitogen-activated protein kinase kinase 2 (MKK2) on the plant membrane and in the nucleus. The HrpP truncation HrpP1-119, rather than HrpP1-101, retains the ability to interact with MKK2 and suppress PTI in plants. In contrast, HrpP1-101 continues to cause cell death and electrolyte leakage. MKK2 silencing compromises SA signaling but has no effect on cell death caused by HrpP. Overall, our work highlights that the P. syringae T3SS protein HrpP facilitates effector translocation and manipulates plant immunity to facilitate bacterial infection. IMPORTANCE The T3SS is required for the virulence of many Gram-negative bacterial pathogens of plants and animals. This study focuses on the sensing and function of the T3SS protein HrpP during plant interactions. Our findings show that HrpP and its N-terminal truncation HrpP1-119 can interact with MKK2, promote effector translocation, and manipulate plant immunity to facilitate bacterial infection, highlighting the P. syringae T3SS component involved in the fine-tuning of plant immunity.

Duplicated Flagellins in Pseudomonas Divergently Contribute to Motility and Plant Immune Elicitation.

Flagellins are the main constituents of the flagellar filaments that provide bacterial motility, chemotactic ability, and host immune elicitation ability. Although the functions of flagellins have been extensively studied in bacteria with a single flagellin-encoding gene, the function of multiple flagellin-encoding genes in a single bacterial species is largely unknown. Here, the model plant-growth-promoting bacterium Pseudomonas kilonensis F113 was used to decipher the divergent functions of duplicated flagellins. We demonstrate that the two flagellins (FliC-1 and FliC-2) in 12 Pseudomonas strains, including F113, are evolutionarily distinct. Only the fliC-1 gene but not the fliC-2 gene in strain F113 is responsible for flagellar biogenesis, motility, and plant immune elicitation. The transcriptional expression of fliC-2 was significantly lower than that of fliC-1 in medium and in planta, most likely due to variations in promoter activity. In silico prediction revealed that all fliC-2 genes in the 12 Pseudomonas strains have a poorly conserved promoter motif. Compared to the Flg22-2 epitope (relative to FliC-2), Flg22-1 (relative to FliC-1) induced stronger FLAGELLIN SENSING 2 (FLS2)-mediated microbe-associated molecular pattern-triggered immunity and significantly inhibited plant root growth. A change in the 19th amino acid in Flg22-2 reduced its binding affinity to the FLS2/brassinosteroid insensitive 1-associated kinase 1 complex. Also, Flg22-2 epitopes in the other 11 Pseudomonas strains were presumed to have low binding affinity due to the same change in the 19th amino acid. These findings suggest that Pseudomonas has evolved duplicate flagellins, with only FliC-1 contributing to motility and plant immune elicitation. IMPORTANCE Flagellins have emerged as important microbial patterns. This work focuses on flagellin duplication in some plant-associated Pseudomonas. Our findings on the divergence of duplicated flagellins provide a conceptual framework for better understanding the functional determinant flagellin and its peptide in multiple-flagellin plant-growth-promoting rhizobacteria.

Diverse interactions of five core type III effectors from Ralstonia solanacearum with plants.

Ralstonia solanacearum is a widespread plant bacterial pathogen that can launch a range of type III effectors (T3Es) to cause disease. In this study, we isolate a pathogenic R. solanacearum strain named P380 from tomato rhizosphere. Five out of 12 core T3Es of strain P380 are introduced into Pseudomonas syringae DC3000D36E separately to determine their functions in interacting with plants. DC3000D36E that harbors each effector suppresses FliC-triggered Pti5 and ACRE31 expression, ROS burst, and callose deposition. RipAE, RipU, and RipW elicit cell death as well as upregulate the MAPK cascades in Nicotiana benthamiana. The derivatives RipC1ΔDXDX(T/V) and RipWΔDKXXQ but not RipAEK310R fail to suppress ROS burst. Moreover, RipAEK310R and RipWΔDKXXQ retain the cell death elicitation ability. RipAE and RipW are associated with salicylic acid and jasmonic acid pathways, respectively. RipAE and RipAQ significantly promote the propagation of DC3000D36E in plants. The five core T3Es localize in diverse subcellular organelles of nucleus, plasma membrane, endoplasmic reticulum, and Golgi network. The suppressor of G2 allele of Skp1 is required for RipAE but not RipU-triggered cell death in N. benthamiana. These results indicate that the core T3Es in R. solanacearum play diverse roles in plant-pathogen interactions.

Dynamics of rice microbiomes reveal core vertically transmitted seed endophytes.

BACKGROUND: Plants and their associated microbiota constitute an assemblage of species known as holobionts. The plant seed microbiome plays an important role in nutrient uptake and stress attenuation. However, the core vertically transmitted endophytes remain largely unexplored. RESULTS: To gain valuable insights into the vertical transmission of rice seed core endophytes, we conducted a large-scale analysis of the microbiomes of two generations of six different rice varieties from five microhabitats (bulk soil, rhizosphere, root, stem, and seed) from four geographic locations. We showed that the microhabitat rather than the geographic location and rice variety was the primary driver of the rice microbiome assemblage. The diversity and network complexity of the rice-associated microbiome decreased steadily from far to near the roots, rice exterior to interior, and from belowground to aboveground niches. Remarkably, the microbiomes of the roots, stems, and seeds of the rice interior compartments were not greatly influenced by the external environment. The core bacterial endophytes of rice were primarily comprised of 14 amplicon sequence variants (ASVs), 10 of which, especially ASV_2 (Pantoea) and ASV_48 (Xanthomonas), were identified as potentially vertically transmitted taxa because they existed across generations, were rarely present in exterior rice microhabitats, and were frequently isolated from rice seeds. The genome sequences of Pantoea and Xanthomonas isolated from the parental and offspring seeds showed a high degree of average nucleotide and core protein identity, indicating vertical transmission of seed endophytes across generations. In silico prediction indicated that the seed endophytes Pantoea and Xanthomonas possessed streamlined genomes with short lengths, low-complexity metabolism, and various plant growth-promoting traits. We also found that all strains of Pantoea and Xanthomonas exhibited cellulase activity and produced indole-3-acetic acid. However, most strains exhibited insignificant antagonism to the major pathogens of rice, such as Magnaporthe oryzae and X. oryzae pv. oryzae. CONCLUSION: Overall, our study revealed that microhabitats, rather than site-specific environmental factors or host varieties, shape the rice microbiome. We discovered the vertically transmitted profiles and keystone taxa of the rice microbiome, which led to the isolation of culturable seed endophytes and investigation of their potential roles in plant-microbiome interactions. Our results provide insights on vertically transmitted microbiota and suggest new avenues for improving plant fitness via the manipulation of seed-associated microbiomes.  Video Abstract.

First Report of Rhizopus arrhizus (syn. R. oryzae) Causing Garlic Bulb Soft Rot in Hebei Province, China.

Rhizopus soft rot occurs on the succulent tissues of vegetables, fruits, and ornamental plants throughout the world (Cui et al. 2019). When the garlic is in the seedling stage in the fields (Fig. S1) in November 2021, a disease outbreak on garlic bulbs suspected as Rhizopus soft rot occurred in Daming County, Handan City, Hebei Province of China (N 36°17', E 115° 13'). This disease symptom was first found in the garlic seedling stage in China. Disease incidence was 10% to 30% in cultivated garlic bulbs. There were soft water-soaked lesions on the surface of diseased garlic bulbs and the interiors were brown and soft. In the disease severe field, white to gray mycelia were observed on the diseased garlic bulbs. Infected garlic bulbs were sampled to isolate and determine the identity of the disease-causing organism. Symptomatic bulbs were surface sterilized with 1% NaClO for 2 min, dipped in 75% ethanol for 3 min and rinsed three times with autoclaved distilled water. Small pieces of the inner decayed tissue were removed and cultured on potato dextrose agar (PDA) at 28°C for 2 to 3 days. Five white colonies grew on PDA and then they became brownish gray to blackish-gray mycelium. The fungal strains were purified by hyphal-tip isolation method. To determine the identity of the five isolated fungi, we analyzed their internal transcribed spacer (ITS) region sequences (Jung et al. 2012). BLAST analysis of the ITS sequences from DSF-0-2 (accession no. ON706022), DSF-0-3 (accession no. ON706021), DSF-0-4 (accession no. ON706020), DSF-0-5 (accession no. ON706019) and DSF-0-6 (accession no. ON706018) were all 100% identical with Rhizopus arrhizus (syn. Rhizopus oryzae). Phylogenetic trees were constructed using the neighbor-joining method of MEGA11 based on the sequences of ITS rRNA gene (Walther et al. 2013). Phylogenetic trees indicated that isolates were most likely Rhizopus arrhizus (syn. Rhizopus oryzae) (Fig. S2). We selected one isolated strain, DSF-0-2, for characterize the morphology and test its ability to cause garlic bulb soft rot. Under the microscope, nonseptate rhizoids, sporangia, and sporangiospores were observed (Fig. S1). Sporangiospores were unequal, subglobose, numerous irregular, or oval, and 9.7 (6.2 - 12.5) × 6.5 (4.1 - 8.5) µm (n = 50) in diameter. The sporangia were globose, black, 121.5 (65 - 198) µm (n = 50) in diameter. Based on the rDNA-ITS sequencing and the morphological characteristics, the DSF-0-2 isolate was identified as Rhizopus arrhizus (syn. Rhizopus oryzae) (Zheng et al. 2007; Abeywickrama et al. 2020). To complete Koch's postulates, surface-sterilized healthy garlic bulbs were inoculated with R. arrhizus isolate DSF-0-2. A 1.0-ml sterile syringe was used to inject 50 µl of a 106 conidia/ml suspension into each of five healthy bulbs. As a control, garlic bulbs were treated with sterile distilled water. The inoculated and control bulbs were incubated at 28°C for 7 days. The bulbs inoculated with R. arrhizus DSF-0-2 showed symptoms of water soaking, and the tissues were brown and soft throughout the bulb at 7 days (Fig. S1). Results of the three trials were the same. No symptoms were observed in the control group. R. arrhizus was reisolated from the symptomatic garlic bulb and confirmed as such based-on colony and sporangia morphology and ITS sequence. There were some reports that R. arrhizus infects cassava tubers and potato tubers (Amadioha and Markson 2007; Cui et al. 2019). To our knowledge, this is the first report of R. arrhizus (syn. Rhizopus oryzae) associated with soft rot on garlic bulb in the seedling stage in China. This disease has posed a potential threat during garlic seedling stage in the field. Management measures should be considered before this disease outbreaks widely. Garlic bulbs died in the seedling stage, which caused production reduction, serious economic loss and soil pollution. This finding may help to take effective control measures for this disease.

A highly conserved core bacterial microbiota with nitrogen-fixation capacity inhabits the xylem sap in maize plants.

Microbiomes are important for crop performance. However, a deeper knowledge of crop-associated microbial communities is needed to harness beneficial host-microbe interactions. Here, by assessing the assembly and functions of maize microbiomes across soil types, climate zones, and genotypes, we found that the stem xylem selectively recruits highly conserved microbes dominated by Gammaproteobacteria. We showed that the proportion of bacterial taxa carrying the nitrogenase gene (nifH) was larger in stem xylem than in other organs such as root and leaf endosphere. Of the 25 core bacterial taxa identified in xylem sap, several isolated strains were confirmed to be active nitrogen-fixers or to assist with biological nitrogen fixation. On this basis, we established synthetic communities (SynComs) consisting of two core diazotrophs and two helpers. GFP-tagged strains and 15N isotopic dilution method demonstrated that these SynComs do thrive and contribute, through biological nitrogen fixation, 11.8% of the total N accumulated in maize stems. These core taxa in xylem sap represent an untapped resource that can be exploited to increase crop productivity.

Characterization of the SPI-1 Type III Secretion System in Pseudomonas fluorescens 2P24.

Pseudomonas fluorescens 2P24 is a plant growth-promoting rhizobacterium (PGPR) isolated from wheat take-all decline soil. Genomic analysis of strain 2P24 revealed the presence of a complete SPI-1 type III secretion system (T3SS) gene cluster on the chromosome with an organization and orientation similar to the SPI-1 T3SS gene clusters of Salmonella enterica and P. kilonensis F113. Phylogenetic analysis revealed that the SPI-1 T3SS gene cluster of strain 2P24 might be obtained from Salmonella and Shigella by horizontal gene transfer. Two transcriptional regulator homologs of HilA and InvF were found from the SPI-1 T3SS gene cluster of strain 2P24. HilA regulated the expression of the structural genes positively, such as invG, sipB, sipD, prgI, and prgK. Prediction of transcriptional binding sites and RNA-seq analysis revealed 14 genes were up-regulated by InvF in strain 2P24. Exploring potential roles of SPI-1 T3SS revealed that it was not associated with motility. However, 2P24ΔinvF reduced resistance against Fusarium graminearum significantly. 2P24ΔhilA enhanced formation of biofilm significantly at 48 h. All three mutants 2P24ΔhilA, 2P24ΔinvF, and 2P24ΔinvE-C reduced the chemotactic responses to glucose significantly. Finally, the determination of SPI-1 mutants to trigger innate immunity in Nicotiana benthamiana showed that 2P24ΔinvE-C reduced the ability to induce the production of reactive oxygen species compared with the wild type strain 2P24.

A Plant Lectin Receptor-like Kinase Phosphorylates the Bacterial Effector AvrPtoB to Dampen Its Virulence in Arabidopsis.

Plasma membrane-localized receptor-like kinases (RLKs) perceive conserved pathogen-associated molecular patterns (PAMPs) in plants, leading to PAMP-triggered immunity (PTI). The Arabidopsis thaliana lectin RLK LecRK-IX.2 has been shown to regulate the bacterial flagellin-derived peptide flg22-induced PTI. Here, we discover that Pseudomonas syringae effector AvrPtoB targets LecRK-IX.2 for degradation, which subsequently suppresses LecRK-IX.2-mediated PTI and disease resistance. However, LecRK-IX.2 can interact with and phosphorylate AvrPtoB at serine site 335 (S335). AvrPtoB self-associates in vitro and in vivo, and the association appears to be essential for its E3 ligase activity in ubiquitinating substrate in plants. Phosphorylation of S335 disrupts the self-association and as a result, phosphomimetic AvrPtoBS335D cannot ubiquitinate LecRK-IX.2 efficiently, leading to the compromised virulence of AvrPtoB in suppressing PTI responses. flg22 enhances AvrPtoB S335 phosphorylation by inducing the expression and activating of LecRK-IX.2. Our study demonstrates that host RLKs can modify pathogen effectors to dampen their virulence and undermine their ability in suppressing PTI.

New section and species in Talaromyces.

Talaromyces is a monophyletic genus containing seven sections. The number of species in Talaromyces grows rapidly due to reliable and complete sequence data contributed from all over the world. In this study agricultural soil samples from Fujiang, Guangdong, Jiangxi, Shandong, Tibet and Zhejiang provinces of China were collected and analyzed for fungal diversity. Based on a polyphasic approach including phylogenetic analysis of partial ITS, BenA, CaM and RPB2 gene sequences, macro- and micro-morphological analyses, six of them could not be assigned to any described species, and one cannot be assigned to any known sections. Morphological characters as well as their phylogenetic relationship with other Talaromyces species are presented for these putative new species. Penicillium resedanum is combined in Talaromyces section Subinflati as T. resedanus.

Genomic insights into a plant growth-promoting Pseudomonas koreensis strain with cyclic lipopeptide-mediated antifungal activity.

Strain S150 was isolated from the tobacco rhizosphere as a plant growth-promoting rhizobacterium. It increased plant fresh weight significantly and lateral root development, and it antagonized plant pathogenic fungi but not phytobacteria. Further tests showed that strain S150 solubilized organic phosphate and produced ammonia, siderophore, protease, amylase, and cellulase, but it did not produce indole-3-acetic acid. Using morphology, physiological characteristics, and multi-locus sequence analysis, strain S150 was identified as Pseudomonas koreensis. The complete genome of strain S150 was sequenced, and it showed a single circular chromosome of 6,304,843 bp with a 61.09% G + C content. The bacterial genome contained 5,454 predicted genes that occupied 87.7% of the genome. Venn diagrams of the identified orthologous clusters of P. koreensis S150 with the other three sequenced P. koreensis strains revealed up to 4,167 homologous gene clusters that were shared among them, and 21 orthologous clusters were only present in the genome of strain S150. Genome mining of the bacterium P. koreensis S150 showed that the strain possessed 10 biosynthetic gene clusters for secondary metabolites, which included four clusters of non-ribosomal peptide synthetases (NRPSs) involved in the biosynthesis of cyclic lipopeptides (CLPs). One of the NRPSs possibly encoded lokisin, a cyclic lipopeptide produced by fluorescent Pseudomonas. Genomic mutation of the lokA gene, which is one of the three structural NRPS genes for lokisin in strain S150, led to a deficiency in fungal antagonism that could be restored fully by gene complementation. The results suggested that P. koreensis S150 is a novel plant growth-promoting agent with specific cyclic lipopeptides and contains a lokisin-encoding gene cluster that is dominant against plant fungal pathogens.

Bacillus telluris sp. nov. Isolated from Greenhouse Soil in Beijing, China.

A novel Gram-stain-positive, rod-shaped, endospore-forming bacterium, which we designated as strain 03113T, was isolated from greenhouse soil in Beijing, China. Phylogenetic analysis based on 16S rRNA gene sequences showed strain 03113T is in the genus Bacillus and had the highest similarity to Bacillus solani CCTCC AB 2014277T (98.14%). The strain grew at 4 °C-50 °C (optimum 37 °C), with 0-10% (w/v) NaCl (optimum 5%), and in the range of pH 3.0-12.0 (optimum pH 8.0). Menaquinone was identified as MK-7, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The main major cellular fatty acids detected were anteiso-C15:0 (51.35%) and iso-C15:0 (11.06%), which are the predominant cellular fatty acids found in all recognized members of the genus Bacillus. The 16S rRNA gene sequence and core-genome analysis, the average nucleotide identity (ANI), and in silico DNA-DNA hybridization (DDH) value between strain 03113T and the most closely related species were 70.5% and 22.6%, respectively, which supported our conclusion that 03113T represented a novel species in the genus Bacillus. We demonstrated that type strain 03113T (=ACCC 03113T=JCM 33017T) was a novel species in the genus Bacillus, and the name Bacillus telluris sp. nov. was proposed. Strain 03113T secreted auxin IAA and carried the nitrogenase iron protein (nifH) gene, which indicated that strain 03113T has the potential to fix nitrogen and promote plant growth. Bacillus telluris sp. nov. 03113T is a potential candidate for the biofertilizers of organic agriculture areas.

Characterization of a Versatile Plant Growth-Promoting Rhizobacterium Pseudomonas mediterranea Strain S58.

Plant growth-promoting rhizobacterial strain S58 was isolated from the tobacco rhizosphere. It showed strong antagonism against a battery of plant pathogenic fungi and bacteria, and controlled wheat sharp eyespot and tobacco wildfire diseases efficiently. Further tests showed that strain S58 solubilized organic phosphate and produced siderophore, protease, ammonia, and indole-3-acetic acid. In Arabidopsis thaliana, it promoted plant growth and changed root system architecture by restricting the growth of primary roots and increasing lateral root numbers. We relied on morphological, biochemical, physiological characteristics, and molecular phylogenic analysis to identify strain S58 as Pseudomonas mediterranea. The complete genome of strain S58 has a single circular chromosome of 6,150,838 bp with a 61.06% G+C content. The bacterial genome contained 5,312 predicted genes with an average length of 992.90 bp. A genome analysis suggested that P. mediterranea S58 was a rich cyclic lipopeptide (CLP)-producing strain that possessed seven non-ribosomal peptide gene clusters for CLP synthesis. Leaf inoculation of the bacterial culture and supernatants triggered cell death-like immunity in tobacco. Quantitative real-time PCR assays showed that the strain S58 induced the expression of pattern-triggered immunity and cell death marker genes, but not jasmonic acid marker genes. The results suggested that P. mediterranea S58 is a novel, versatile plant growth-promoting agent with multiple beneficial traits for plants.

Pseudomonas syringae AlgU Downregulates Flagellin Gene Expression, Helping Evade Plant Immunity.

Flagella power bacterial movement through liquids and over surfaces to access or avoid certain environmental conditions, ultimately increasing a cell's probability of survival and reproduction. In some cases, flagella and chemotaxis are key virulence factors enabling pathogens to gain entry and attach to suitable host tissues. However, flagella are not always beneficial; both plant and animal immune systems have evolved receptors to sense the proteins that make up flagellar filaments as signatures of bacterial infection. Microbes poorly adapted to avoid or counteract these immune functions are unlikely to be successful in host environments, and this selective pressure has driven the evolution of diverse and often redundant pathogen compensatory mechanisms. We tested the role of AlgU, the Pseudomonas extracytoplasmic function sigma factor σE/σ22 ortholog, in regulating flagellar expression in the context of Pseudomonas syringae-plant interactions. We found that AlgU is necessary for downregulating bacterial flagellin expression in planta and that this results in a corresponding reduction in plant immune elicitation. This AlgU-dependent regulation of flagellin gene expression is beneficial to bacterial growth in the course of plant infection, and eliminating the plant's ability to detect flagellin makes this AlgU-dependent function irrelevant for bacteria growing in the apoplast. Together, these results add support to an emerging model in which P. syringae AlgU functions at a key control point that serves to optimize the expression of bacterial functions during host interactions, including minimizing the expression of immune elicitors and concomitantly upregulating beneficial virulence functions.IMPORTANCE Foliar plant pathogens, like Pseudomonas syringae, adjust their physiology and behavior to facilitate host colonization and disease, but the full extent of these adaptations is not known. Plant immune systems are triggered by bacterial molecules, such as the proteins that make up flagellar filaments. In this study, we found that during plant infection, AlgU, a gene expression regulator that is responsive to external stimuli, downregulates expression of fliC, which encodes the flagellin protein, a strong elicitor of plant immune systems. This change in gene expression and resultant change in behavior correlate with reduced plant immune activation and improved P. syringae plant colonization. The results of this study demonstrate the proximate and ultimate causes of flagellar regulation in a plant-pathogen interaction.

Modular Study of the Type III Effector Repertoire in Pseudomonas syringae pv. tomato DC3000 Reveals a Matrix of Effector Interplay in Pathogenesis.

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of Nicotiana benthamiana and other plants by injecting a complex repertoire of type III secretion effector (T3E) proteins. Effectorless polymutant DC3000D36E was used with a modularized system for native delivery of the 29 DC3000 T3Es singly and in pairs. Assays of the performance of this T3E library in N. benthamiana leaves revealed a matrix of T3E interplay, with six T3Es eliciting death and eight others variously suppressing the death activity of the six. The T3E library was also interrogated for effects on DC3000D36E elicitation of a reactive oxygen species burst, for growth in planta, and for T3Es that reversed these effects. Pseudomonas fluorescens and Agrobacterium tumefaciens heterologous delivery systems yielded notably different sets of death-T3Es. The DC3000D36E T3E library system highlights the importance of 13 T3Es and their interplay in interactions with N. benthamiana.