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.

Clarifying the taxonomy of the causal agent of bacterial leaf spot of lettuce through a polyphasic approach reveals that Xanthomonas cynarae Trébaol et al. 2000 emend. Timilsina et al. 2019 is a later heterotypic synonym of Xanthomonas hortorum Vauterin et al. 1995.

Assessment of the taxonomy and diversity of Xanthomonas strains causing bacterial leaf spot of lettuce (BLSL), commonly referred to as Xanthomonas campestris pv. vitians, has been a long-lasting issue which held back the global efforts made to understand this pathogen. In order to provide a sound basis essential to its study, we conducted a polyphasic approach on strains obtained through sampling campaigns or acquired from collections. Results of a multilocus sequence analysis crossed with phenotypic assays revealed that the pathotype strain does not match the description of the nomenspecies provided by Brown in 1918. However, strain LMG 938=CFBP 8686 does fit this description. Therefore, we propose that it replaces LMG 937=CFBP 2538 as pathotype strain of X. campestris pv. vitians. Then, whole-genome based phylogenies and overall genome relatedness indices calculated on taxonomically relevant strains exhibited the intermediate position of X. campestris pv. vitians between closely related species Xanthomonas hortorum and Xanthomonas cynarae. Phenotypic profiles characterized using Biolog microplates did not reveal stable diagnostic traits legitimizing their distinction. Therefore, we propose that X. cynarae Trébaol et al. 2000 emend. Timilsina et al. 2019 is a later heterotypic synonym of X. hortorum, to reclassify X. campestris pv. vitians as X. hortorum pv. vitians comb. nov. and to transfer X. cynarae pathovars in X. hortorum as X. hortorum pv. cynarae comb. nov. and X. hortorum pv. gardneri comb. nov. An emended description of X. hortorum is provided, making this extended species a promising model for the study of Xanthomonas quick adaptation to different hosts.

Role of the acquisition of a type 3 secretion system in the emergence of novel pathogenic strains of Xanthomonas.

Cases of emergence of novel plant-pathogenic strains are regularly reported that reduce the yields of crops and trees. However, the molecular mechanisms underlying such emergence are still poorly understood. The acquisition by environmental non-pathogenic strains of novel virulence genes by horizontal gene transfer has been suggested as a driver for the emergence of novel pathogenic strains. In this study, we tested such an hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four associated type 3 secreted proteins (T3SPs) to the non-pathogenic strains of Xanthomonas CFBP 7698 and CFBP 7700, which lack genes encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped on Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. Wild-type, non-pathogenic strains induced a hypersensitive response (HR)-like necrosis, whereas strains complemented with T3SS and T3SPs suppressed this response. Such suppression depends on a functional T3SS. Amongst the T3SPs encoded on the plasmid, Hpa2, Hpa1 and, to a lesser extent, XopF1 collectively participate in suppression. Monitoring of the population sizes in planta showed that the sole acquisition of a functional T3SS by non-pathogenic strains impairs growth inside leaf tissues. These results provide functional evidence that the acquisition via horizontal gene transfer of a T3SS and four T3SPs by environmental non-pathogenic strains is not sufficient to make strains pathogenic. In the absence of a canonical effector, the sole acquisition of a T3SS seems to be counter-selective, and further acquisition of type 3 effectors is probably needed to allow the emergence of novel pathogenic strains.

Xanthomonas prunicola sp. nov., a novel pathogen that affects nectarine (Prunus persica var. nectarina) trees.

Three isolates obtained from symptomatic nectarine trees (Prunus persica var. nectarina) cultivated in Murcia, Spain, which showed yellow and mucoid colonies similar to Xanthomonas arboricola pv. pruni, were negative after serological and real-time PCR analyses for this pathogen. For that reason, these isolates were characterized following a polyphasic approach that included both phenotypic and genomic methods. By sequence analysis of the 16S rRNA gene, these novel strains were identified as members of the genus Xanthomonas, and by multilocus sequence analysis (MLSA) they were clustered together in a distinct group that showed similarity values below 95 % with the rest of the species of this genus. Whole-genome comparisons of the average nucleotide identity (ANI) of genomes of the strains showed less than 91 % average nucleotide identity with all other species of the genus Xanthomonas. Additionally, phenotypic characterization based on API 20 NE, API 50 CH and BIOLOG tests differentiated the strains from the species of the genus Xanthomonas described previously. Moreover, the three strains were confirmed to be pathogenic on peach (Prunus persica), causing necrotic lesions on leaves. On the basis of these results, the novel strains represent a novel species of the genus Xanthomonas, for which the name Xanthomonas prunicola is proposed. The type strain is CFBP 8353 (=CECT 9404=IVIA 3287.1).