Wild mushrooms as potential reservoirs of plant pathogenic bacteria: a case study on Burkholderia gladioli.

Fruit bodies (sporocarps) of wild mushrooms growing in natural environments play a substantial role in the preservation of microbial communities, for example, clinical and food-poisoning bacteria. However, the role of wild mushrooms as natural reservoirs of plant pathogenic bacteria remains almost entirely unknown. Furthermore, bacterial transmission from a mushroom species to agricultural plants has rarely been recorded in the literature. In September 2021, a creamy-white Gram-negative bacterial strain was isolated from the sporocarp of Suillus luteus (slippery jack) growing in Bermuda grass (Cynodon dactylon) lawn in Southern Iran. A similar strain was isolated from the same fungus in the same area in September 2022. Both strains were identified as Burkholderia gladioli based on phenotypic features as well as phylogeny of 16S rRNA and three housekeeping genes. The strains were not only pathogenic on white button mushrooms (Agaricus bisporus) but also induced hypersensitive reaction (HR) on tobacco and common bean leaves and caused soft rot on a set of diverse plant species, that is, chili pepper, common bean pod, cucumber, eggplant, garlic, gladiolus, narcissus, onion, potato, spring onion, okra, kohlrabi, mango, and watermelon. Isolation of plant pathogenic B. gladioli strains from sporocarp of S. luteus in two consecutive years in the same area could be indicative of the role of this fungus in the preservation of the bacterium in the natural environment. B. gladioli associated with naturally growing S. luteus could potentially invade neighboring agricultural crops, for example, vegetables and ornamentals. The potential role of wild mushrooms as natural reservoirs of phytopathogenic bacteria is further discussed.IMPORTANCEThe bacterial genus Burkholderia contains biologically heterogeneous strains that can be isolated from diverse habitats, that is, soil, water, diseased plant material, and clinical specimens. In this study, two Gram-negative pectinolytic bacterial strains were isolated from the sporocarps of Suillus luteus in September 2021 and 2022. Molecular phylogenetic analyses revealed that both strains belonged to the complex species Burkholderia gladioli, while the pathovar status of the strains remained undetermined. Biological investigations accomplished with pathogenicity and host range assays showed that B. gladioli strains isolated from S. luteus in two consecutive years were pathogenic on a set of diverse plant species ranging from ornamentals to both monocotyledonous and dicotyledonous vegetables. Thus, B. gladioli could be considered an infectious pathogen capable of being transmitted from wild mushrooms to annual crops. Our results raise a hypothesis that wild mushrooms could be considered as potential reservoirs for phytopathogenic B. gladioli.

Xanthomonas bonasiae sp. nov. and Xanthomonas youngii sp. nov., isolated from crown gall tissues.

The genus Xanthomonas contains a set of diverse bacterial strains, most of which are known for their pathogenicity on annual crops and fruit trees causing economically important plant diseases. Recently, five Xanthomonas strains were isolated from Agrobacterium-induced crown gall tissues of amaranth (Amaranthus sp.) and weeping fig (Ficus benjamina) plants in Iran. Phenotypic characteristics (i.e. biochemical tests and pathogenicity features) and whole genome sequence-based core-genome phylogeny followed by average nucleotide identity and digital DNA-DNA hybridization calculations suggested that these gall-associated strains belong to two new species within the genus Xanthomonas. In this study, we provide a formal species description for these new species where Xanthomonas bonasiae sp. nov. is proposed for the strains isolated from weeping fig with FX4T (=CFBP 8703T=DSM 112530T) as type strain. The name Xanthomonas youngii sp. nov. is proposed for the strains isolated from amaranth with AmX2T (=CFBP 8902T=DSM 112529T) as type strain.

Phenotypic and Molecular-Phylogenetic Analyses Reveal Distinct Features of Crown Gall-Associated Xanthomonas Strains.

In summer 2019, widespread occurrence of crown gall disease caused by Agrobacterium spp. was observed on commercially grown ornamental plants in southern Iran. Beside agrobacteria, pale yellow-pigmented Gram-negative strains resembling the members of Xanthomonas were also associated with crown gall tissues on weeping fig (Ficus benjamina) and Amaranthus sp. plants. The purpose of the present study was to characterize the crown gall-associated Xanthomonas strains using plant inoculation assays, molecular-phylogenetic analyses, and comparative genomics approaches. Pathogenicity tests showed that the Xanthomonas strains did not induce disease symptoms on their host of isolation. However, the strains induced hypersensitive reaction on tobacco, geranium, melon, squash, and tomato leaves via leaf infiltration. Multilocus sequence analysis suggested that the strains belong to clade IA of Xanthomonas, phylogenetically close to Xanthomonas translucens, X. theicola, and X. hyacinthi. Average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of the strains isolated in this study and reference Xanthomonas strains are far below the accepted thresholds for the definition of prokaryotic species, signifying that these strains could be defined as two new species within clade IA of Xanthomonas. Comparative genomics showed that the strains isolated from crown gall tissues are genetically distinct from X. translucens, as almost all the type III secretion system genes and type III effectors are lacking in the former group. The data obtained in this study provide novel insight into the breadth of genetic diversity of crown gall-associated bacteria and pave the way for research on gall-associated Xanthomonas-plant interactions. IMPORTANCE Tumorigenic agrobacteria-members of the bacterial family Rhizobiaceae-cause crown gall and hairy root diseases on a broad range of plant species. These bacteria are responsible for economic losses in nurseries of important fruit trees and ornamental plants. The microclimate of crown gall and their accompanying microorganisms has rarely been studied for the microbial diversity and population dynamics of gall-associated bacteria. Here, we employed a series of biochemical tests, pathogenicity assays, and molecular-phylogenetic analyses, supplemented with comparative genomics, to elucidate the biological features, taxonomic position, and genomic repertories of five crown gall-associated Xanthomonas strains isolated from weeping fig and Amaranthus sp. plants in Iran. The strains investigated in this study induced hypersensitive reactions (HR) on geranium, melon, squash, tobacco, and tomato leaves, while they were nonpathogenic on their host of isolation. Phylogenetic analyses and whole-genome-sequence-based average nucleotide identity (ANI)/digital DNA-DNA hybridization (dDDH) calculations suggested that the Xanthomonas strains isolated from crown gall tissues belong to two taxonomically unique clades closely related to the clade IA species of the genus, i.e., X. translucens, X. hyacinthi, and X. theicola.

Genomic Analyses of Rose Crown Gall-Associated Bacteria Revealed Two New Agrobacterium Species: Agrobacterium burrii sp. nov. and Agrobacterium shirazense sp. nov.

Agrobacterium tumefaciens species complex contains a set of diverse bacterial strains, most of which are well known for their pathogenicity on agricultural plants causing crown gall diseases. Members of A. tumefaciens species complex are classified into several taxonomically distinct lineages called "genomospecies" (13 genomospecies until early 2021). Recently, two genomospecies, G19 (strains RnrT, Rew, and Rnw) and G20 (strains OT33T and R13) infecting Rosa sp. plants in Iran, were described based on biochemical and molecular-phylogenetic data. Whole genome sequence-based core-genome phylogeny followed by average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) calculations performed in this study suggested that genomospecies G19 and G20 could be described as two novel and standalone species. In the phylogenetic tree, these two new genomospecies were clustered separately from other genomospecies/species of A. tumefaciens species complex. Moreover, both ANI and dDDH indices between the G19/G20 strains and other Rhizobiaceae members are clearly below the accepted thresholds for prokaryotic species description. Hence, Agrobacterium burrii sp. nov. is proposed to encompass the G19 strains, with RnrT = CFBP 8705T = DSM 112541T as type strain. Agrobacterium shirazense sp. nov. is also proposed to include G20 strains, with OT33T = CFBP 8901T = DSM 112540T as type strain.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Phenotypically and Genotypically Heterogeneous Strains of Pseudomonas syringae Associated With Alfalfa Leaf Spot Disease in Iran.

In this study, we provide a polyphasic characterization of 18 Pseudomonas spp. strains associated with alfalfa leaf spot symptoms in Iran. All of the strains were pathogenic on alfalfa, although the aggressiveness and symptomology varied among the strains. All strains but one were pathogenic on broad bean, cucumber, honeydew, and zucchini, whereas only a fraction of the strains were pathogenic on sugar beet, tomato, and wheat. Syringomycin biosynthesis genes (syrB1 and syrP) were detected using the corresponding PCR primers in all of the strains isolated from alfalfa. Phylogenetic analyses using the sequences of four housekeeping genes (gapA, gltA, gyrB, and rpoD) revealed that all of the strains except one (Als34) belong to phylogroup 2b of P. syringae sensu lato, whereas strain Als34 placed within phylogroup 1 close to the type strain of P. syringae pv. apii. Among the phylogroup 2b strains, nine strains were phylogenetically close to the P. syringae pv. aptata clade, whereas the remainder were scattered among P. syringae pv. atrofaciens and P. syringae pv. syringae strains. Pathogenicity and host range assays of the bacterial strains evaluated in this study on a set of taxonomically diverse plant species did not allow us to assign a "pathovar" status to the alfalfa strains. However, these results provide novel insight into the host range and phylogenetic position of the alfalfa-pathogenic members of P. syringae sensu lato, and they reveal that phenotypically and genotypically heterogeneous strains of the pathogen cause bacterial leaf spot of alfalfa.

Two Novel Genomospecies in the Agrobacterium tumefaciens Species Complex Associated with Rose Crown Gall.

In this study, we explored the pathogenicity and phylogenetic position of Agrobacterium spp. strains isolated from crown gall tissues on annual, perennial, and ornamental plants in Iran. Of the 43 strains studied, 10 strains were identified as Allorhizobium vitis (formerly Agrobacterium vitis) using the species-specific primer pair PGF/PGR. Thirty-three remaining strains were studied using multilocus sequence analysis of four housekeeping genes (i.e., atpD, gyrB, recA, and rpoB), from which seven strains were identified as A. larrymoorei and one strain was identified as A. rubi (Rer); the remaining 25 strains were scattered within the A. tumefaciens species complex. Two strains were identified as genomospecies 1 (G1), seven strains were identified as A. radiobacter (G4), seven strains were identified as A. deltaense (G7), two strains were identified as A. nepotum (G14), and one strain was identified as "A. viscosum" (G15). The strains Rnr, Rnw, and Rew as well as the two strains OT33 and R13 all isolated from rose and the strain Ap1 isolated from apple were clustered in three atypical clades within the A. tumefaciens species complex. All but eight strains (i.e., Nec10, Ph38, Ph49, fic9, Fic72, R13, OT33, and Ap1) were pathogenic on tomato and sunflower seedlings in greenhouse conditions, whereas all but three strains (i.e., fic9, Fic72, and OT33) showed tumorigenicity on carrot root discs. The phylogenetic analysis and nucleotide diversity statistics suggested the existence of two novel genomospecies within the A. tumefaciens species complex, which we named "G19" and "G20." Hence, we propose the strains Rew, Rnw, and Rnr as the members of "G19" and the strains R13 and OT33 as the members of G20, whereas the phylogenetic status of the atypical strain Ap1 remains undetermined.

Phenotypic and Molecular-Phylogenetic Analysis Provide Novel Insights into the Diversity of Curtobacterium flaccumfaciens.

A multiphasic approach was used to decipher the phenotypic features, genetic diversity, and phylogenetic position of 46 Curtobacterium spp. strains isolated from dry beans and other annual crops in Iran and Spain. Pathogenicity tests, resistance to arsenic compounds, plasmid profiling and BOX-PCR were performed on the strains. Multilocus sequence analysis (MLSA) was also performed on five housekeeping genes (i.e., atpD, gyrB, ppk, recA, and rpoB) of all the strains, as well as five pathotype strains of the species. Pathogenicity test showed that six out of 42 strains isolated in Iran were nonpathogenic on common bean. Despite no differences found between pathogenic and nonpathogenic strains in their plasmid profiling, the former were resistant to different concentrations of arsenic, while the latter were sensitive to the same concentrations. Strains pathogenic on common bean were polyphyletic with at least two evolutionary lineages (i.e., yellow-pigmented strains versus red/orange-pigmented strains). Nonpathogenic strains isolated from solanaceous vegetables were clustered within either the strains of C. flaccumfaciens pv. flaccumfaciens or different pathovars of the species. The results of MLSA and BOX-PCR analysis were similar to each other and both methods were able to discriminate the yellow-pigmented strains from the red/orange-pigmented strains. A comprehensive study of a worldwide collection representing all five pathovars as well as nonpathogenic strains of C. flaccumfaciens is warranted for a better understanding of the diversity within this phytopathogenic bacterium.