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.

In autumn 2018, cap discoloration and browning symptoms (up to 20% incidence) were observed on commercially grown white button mushrooms (Agaricus bisporus) in two distinct farms located in Shiraz and Marvdasht Counties in Southern Iran. Symptomatic caps (13 and six caps from Shiraz and Marvdasht Counties, respectively) were characterized by visible brown discoloration with no blotch symptoms, bacterial sticky mass and cap wilting. Isolation of bacterial strains from infected cap tissues was performed on yeast-extract peptone glucose agar (YPGA) according to Hamidizade et al. (2020). The resulted bacterial colonies were oyster-white in color, non-fluorescent, domed convex circular with smooth margins 1-2 mm in diameter. A total of six bacterial strains (Shiraz: Ir1002, Ir1003, Ir1004, Ir1005, Ir1007 and Marvdasht: Ir1027) were isolated from distinct mushroom samples. Standard biochemical and phenotypic analyses (Schaad et al. 2001) showed that the bacterial strains were Gram and oxidase negative, catalase positive and facultatively anaerobic, while no capsule or endospore was observed. All strains were positive in urease production, arginine dihydrolase, hydrolysis of tween 80, and utilization of sucrose and D-sorbitol, while they were negative in amylase, cellulose, lecithinase, pectinase, and protease production as well as casein hydrolysis. Based on these phenotypic characteristics, the strains were supposed to be members of Enterobacteriaceae. They also did not induce hypersensitive reaction (HR) on tobacco (Nicotiana tabacum cv. Turkish) leaves nor did they produce tolaasin when streaked side-by-side with “Pseudomonas reactans” strains on King B medium (Osdaghi et al. 2019). Pathogenicity of the strains was evaluated (repeated twice) on fresh caps of white button mushroom using cut-cap method (Hamidizade et al. 2020). Reference strains of Pseudomonas tolaasii (CFBP 8707) and Mycetocola spp. (CFBP 8708) were used as positive controls, while sterile distilled water was used as a negative control. Brown discoloration appeared 24-36 hours post inoculation on cap surfaces while control caps remained asymptomatic. Koch’s postulates were accomplished by re-isolation and identification of bacterial strains from the symptomatic caps using colony morphology and Gram staining. For molecular identification, all initial as well as re-isolated strains were subjected to amplification and sequencing of 16S rDNA and gyrB (Yamamoto and Harayama 1995; Hamidizade et al. 2020). Obtained nucleotide sequences were deposited into NCBI GenBank (16S: MZ298620 to MZ298625; gyrB: MZ313184 to MZ313189). BLAST search using the 16S rDNA and gyrB sequences showed that the strains isolated in this study had 97-99% sequence similarity to the reference strains of Cedecea neteri. Phylogenetic analyses also confirmed close relationship of bacterial strains from this study to C. neteri strains. Pure cultures of representative strains Ir1004 (CFBP 8900) and Ir1027 (CFBP 8896) are deposited in CIRM-CFBP culture collection. This is the first report of C. neteri causing brown spot disease on button mushroom in Iran, while the bacterium has previously been reported to cause soft rot on Pholiota nameko (Yan et al. 2018), and yellow sticky disease on Flammulina velutipes (Yan et al. 2019) in China. Further comprehensive investigations will shed a light on the economic impact of the brown spot disease on mushroom industry 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.

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 (2018-2021) 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 in 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 strains were identified as Ewingella americana based on biochemical assays and phylogeny of 16S rRNA and gyrB gene. Ewingella 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 non-pathogenic and environmental strains of the species. BOX-PCR-based fingerprinting revealed intraspecific diversity. Using the cutoff level of 73-76% 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. Taking together, due to the multifaceted nature of the pathogen, such a widespread occurrence of E. americana in mushroom farms in Iran could be an emerging threat for mushroom industry in the country.