Multiphasic investigations imply transfer of orange-/red-pigmented strains of the bean pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens to a new species as C. aurantiacum sp. nov., elevation of the poinsettia pathogen C. flaccumfaciens pv. poinsettiae to the species level as C. Poinsettiae sp. nov., and synonymy of C. albidum with C. citreum.

Curtobacterium flaccumfaciens (Microbacteriaceae), a plant-pathogenic coryneform species includes five pathovars with valid names and a number of proposed - but unvalidated - new members. In this study, phenotypic features and DNA similarity indexes were investigated among all C. flaccumfaciens members. Results showed that the C. flaccumfaciens pv. poinsettiae strains causing bacterial canker of Euphorbia pulcherrima in the USA as well as the orange-/red-pigmented strains of C. flaccumfaciens pv. flaccumfaciens pathogenic on dry beans in Iran are too distinct from each other and from the type strain of the species to be considered members of C. flaccumfaciens. Hence, the latter two groups were elevated at the species level as C. poinsettiae sp. nov. (ATCC 9682T = CFBP 2403T = ICMP 2566T = LMG 3715T = NCPPB 854T as type strain), and C. aurantiacum sp. nov. (50RT = CFBP 8819T = ICMP 22071T as type strain). Within the emended species C. flaccumfaciens comb. nov., yellow-pigmented strains causing bacterial wilt of dry beans and those causing bacterial canker of Euphorbia pulcherrima in Europe were retained as C. flaccumfaciens pv. flaccumfaciens and C. flaccumfaciens pv. poinsettiae, respectively; while taxonomic position of the sugar beet pathogen C. flaccumfaciens pv. beticola ATCC BAA144PT was confirmed. The newly described onion pathogen C. allii was also reclassified as C. flaccumfaciens pv. allii with the pathotype strain LMG 32517PT. Furthermore, C. flaccumfaciens pv. basellae causing bacterial leaf spot of malabar spinach (Basella rubra) was transferred to C. citreum pv. basellae with ATCC BAA143PT as pathotype.

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.

Clavibacter lycopersici sp. nov.: a peach-colored actinobacterium isolated from symptomless tomato plant.

In 2015, Gram-positive peach-coloured actinobacterial strains were isolated from symptomless tomato phyllosphere in Iran. Biochemical and physiological characteristics, as well as 16S rRNA phylogeny showed that the strains belong to Clavibacter sp., while they were non-pathogenic on the host of isolation, and morphologically distinct from the tomato pathogen C. michiganensis and other plant-associated bacteria. Multilocus sequence analysis of five housekeeping genes showed that the two peach-coloured strains CFBP 8615T (Tom532T) and CFBP 8616 (Tom495) were phylogenetically distinct from all validly described Clavibacter species. Whole genome sequence-based indices, i.e. average nucleotide identity (orthoANI) and digital DNA-DNA hybridization (dDDH), showed that the two peach-colored strains share nearly 100 % orthoANI value with one another, while they differ from all validly described Clavibacter species with the orthoANI/dDDH values <93 % and <50 %, respectively. Thus, based on both phenotypic features and orthoANI/dDDH indices the peach-coloured strains could belong to a new species within Clavibacter. In this study, we provide a formal species description for the peach-coloured tomato-associated Clavibacter strains. Clavibacter lycopersici sp. nov. is proposed for the new species with Tom532T = CFBP 8615T = ICMP 22100T as type strain.

Garlic Bulb Decay and Soft Rot Caused by the Cross-Kingdom Pathogen Burkholderia gladioli.

In 2021, two Gram-negative bacterial strains were isolated from garlic (Allium sativum) bulbs showing decay and soft rot symptoms in central Iran. The bacterial strains were aggressively pathogenic on cactus, garlic, gladiolus, onion, potato, and saffron plants, and induced soft rot symptoms on carrot, cucumber, potato and radish discs. Furthermore, they were pathogenic on sporophore of cultivated and wild mushrooms. Phylogenetic analyses revealed that the bacterial strains belong to Burkholderia gladioli species. Garlic bulb rot caused by B. gladioli has rarely been reported in the literature. Historically, B. gladioli strains had been assigned to four pathovars i.e. B. gladioli pv. alliicola, B. gladioli pv. gladioli, B. gladioli pv. agaricicola, and B. gladioli pv. cocovenenans infecting onion, Gladiolus sp., mushrooms, and poisoning foods, respectively. Multilocus (i.e., 16S rRNA, atpD, gyrB, and lepA genes) sequence-based phylogenetic investigations including reference strains of B. gladioli pathovars showed that the two garlic strains belong to phylogenomic clade 2 of the species which includes the pathotype strain of B. gladioli pv. alliicola. Although the garlic strains were phylogenetically closely related to the B. gladioli pv. alliicola reference strains, they possessed pathogenicity characteristics that overlapped with three of the four historical pathovars including the ability to rot onion (pv. alliicola), gladiolus (pv. gladioli) and mushrooms (pv. agaricicola). Further, pathotype of each pathovar could infect the hosts of other pathovars, undermining the utility of pathovar concept in this species. Overall, using phenotypic pathovar-oriented assays to classify B. gladioli strains should be replaced by phylogenetic or phylogenomic analysis.

Ewingella americana: An Emerging Multifaceted Pathogen of Edible Mushrooms.

Mycopathogenic bacteria play a pivotal role in the productivity of edible mushrooms grown under controlled conditions. In this study, we carried out a comprehensive farm survey and sampling (2018 to 2021) on button mushroom (Agaricus bisporus) farms in 15 provinces in Iran to monitor the status of bacterial pathogens infecting the crop. Mycopathogenic bacterial strains were isolated from pins, stems, and caps, as well as the casing layer on 38 mushroom farms. The bacterial strains incited symptoms on mushroom caps ranging from faint discoloration to dark brown and blotch of the inoculated surfaces. Among the bacterial strains inciting disease symptoms on bottom mushroom, 40 were identified as Ewingella americana based on biochemical assays and phylogeny of 16S rRNA and the gyrB gene. E. americana strains differed in their aggressiveness on mushroom caps and stipes, where the corresponding symptoms ranged from deep yellow to dark brown. In the phylogenetic analyses, all E. americana strains isolated in this study were clustered in a monophyletic clade closely related to the nonpathogenic and environmental strains of the species. BOX-PCR-based fingerprinting revealed intraspecific diversity. Using the cutoff level of 73 to 76% similarity, the strains formed six clusters. A chronological pattern was observed, where the strains isolated in 2018 were differentiated from those isolated in 2020 and 2021. Taken together, due to the multifaceted nature of the pathogen, such a widespread occurrence of E. americana on mushroom farms in Iran could be an emerging threat for the mushroom industry in the country.

Bacterial Brown Pit, a New Disease of Edible Mushrooms Caused by Mycetocola sp.

From September to December 2018, commercial button mushroom (Agaricus bisporus) farms in central Iran were surveyed to monitor the causal agent(s) of browning and blotch symptoms on mushroom caps. In addition to dozens of pseudomonads (i.e., Pseudomonas tolaasii and Pseudomonas reactans), six slow-growing gram-positive bacterial strains were isolated from blotched mushroom caps. These bacteria presented as creamy white, circular, smooth, nonfluorescent, and shiny colonies with whole margins resembling members of Microbacteriaceae (Actinobacteria). All of the actinobacterial strains were aggressively pathogenic on cut cap surface of two edible mushrooms (i.e., A. bisporus and Pleurotus eryngii), inducing brown pit symptoms 48 h postinoculation. The strains did not induce symptoms on the vegetables tested (i.e., carrot, cucumber, and potato), and they did not affect the growth of mycelium of tested plant-pathogenic fungi (i.e., Acremonium sp., Fusarium spp., and Phytopythium sp.). Phylogeny of 16S ribosomal RNA and multilocus sequence analysis of six housekeeping genes (i.e., atpD, dnaK, gyrB, ppK, recA, and rpoB) revealed that the bacterial strains belong to the actinobacterial genus Mycetocola spp., whereas the species status of the strains remains undetermined. Mushroom-associated Mycetocola species were previously reported to be capable of detoxifying tolaasin, a toxin produced by P. tolaasii, whereas the strains isolated in this study did not show tolaasin detoxification activities. Altogether, this is the first report of a mushroom disease caused by an actinobacterial species, and "bacterial brown pit" was assigned as the common name of the disease.

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.