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.

PacBio-Based Protocol for Bacterial Genome Assembly.

Acquisition of high-quality bacterial genomes is fundamental, while having in mind investigation of subtitle intraspecies variation in addition to development of sensitive species-specific tools for detection and identification of the pathogens. In this view, Pacific Biosciences technology seems highly tempting taking into consideration over 10,000 bp length of the generated reads. In this work, we describe a bacterial genome assembly pipeline based on open-source software that might be handled also by non-bioinformaticians interested in transformation of sequencing data into reliable biological information. With the use of this method, we successfully closed six Dickeya solani genomes, while the assembly process was run just on a slightly improved desktop computer.

First report of Pectobacterium aroidearum causing bacterial soft rot of carrot in Taiwan.

Carrot (Daucus carota) is an important root vegetable planted and consumed worldwide (Stein and Nothnagel 1995). In June 2020, carrots (cv. New Kuroda) showing soft rot symptoms were observed in a 600 sqft plot located in Pitou, Changhua, Taiwan (23°54'00.9"N, 120°28'37.3"E; with around 400 plants). About 10% of the plants on site had similar symptoms; infected taproot tissues were macerated (Figure S1) and emitted a foul odor. In most cases, the peels above the rotten tissues remain intact. Two infected plants were brought to the lab. Macerated tissues were suspended in water and examined under a microscope at 600X (without staining). Rod, motile bacteria were observed in all of the samples and the bacteria were isolated onto nutrient agar. Three bacterial strains were obtained from two taproots; strain Car1 was isolated from one plant, and strains Car2 and Car3 were isolated from the other. Their colonies were translucent, round and convex. All isolates could ferment glucose and induce soft rot symptoms on potato tuber slices (Schaad et al. 2001). They were not able to produce indigoidine on yeast dextrose calcium carbonate agar and were tested negative for phosphatase activity (Schaad et al. 2001). The 16S rDNA of Car1 to Car3 were amplified using primers 27F/1492R (Lane 1991). Cloning and sequencing of their 16S rDNA (GenBank accession no. MT889640) revealed that their sequences shared 99.9% identity (1,463/1,464 bp) with that of Pectobacterium aroidearum CFBP 8168T (SCRI 109T; GenBank accession no. NR_159926.1). Multilocus sequence analyses targeting the three isolates' dnaX, leuS and recA genes were conducted. The concatenated sequences (1,596 bp) of Car1 to Car3 and those included in a previous work (Portier et al. 2019) were subjected to phylogenetic analysis. The sequences of Car1 to Car3 were identical (GenBank accession nos. MT892671-MT892673). A maximum-likelihood tree showed that the three isolates belonged to the same clade as P. aroidearum CFBP 8168T (GenBank accession nos. MK516971, MK517115 and MK517259; Figure S2). For the concatenated sequences analyzed, the identity between P. aroidearum CFBP 8168T and our three isolates was 99.4% (1,587/1,596 bp). The pathogenicity of these isolates was determined by inoculating the bacteria into carrot (cv. Xiangyang No.2) taproots. Strains Car1 to Car3 were grown on NA for 48 h (28 °C) and cell suspensions with OD600 values of 0.3 (2.4 x 108 CFU/ml; in water) were prepared. The suspensions of each strain (100 µl) were loaded into 200 µl pipette tips. The tips were then pierced into intact carrot taproots (2.4 cm deep), ejected and left on the plants (one tip per plant). Three taproots were tested for each strain. Tips loaded with 100 µl of water were used for the controls (three replicates). The plants were incubated in a sealed plastic container kept in a growth chamber set at 28°C. After 48 h, all of the inoculated taproots produced soft rot symptoms resembling those observed in the field and plants in the control group did not. Bacteria were re-isolated from macerated tissues of the artificially infected plants and found to share the same leuS sequence with Car1 to Car3. Occurrences of carrot soft rot in Taiwan have only been attributed to Dickeya spp. (Erwinia chrysanthemi) in previous studies (Hsu and Tzeng 1981). The present study is the first report of P. aroidearum infecting carrots in Taiwan. The findings may add to our understanding of the diversity of soft rot pathogens affecting carrot production in Taiwan.

Capillary zone electrophoresis of bacterial extracellular vesicles: A proof of concept.

The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs.

A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac.

Bacterial soft rot caused by Pectobacterium species is a serious disease in konjac (Amorphophallus konjac), a healthy source of starch particularly in East Asia. An effective diagnostic method is crucial to control the disease and reduce losses in konjac production. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay with a specific primer set for the rapid and accurate detection of P. aroidearum. A comparative genomics approach was used to identify the specific genes suitable for the design of LAMP primers. The candidate target genes were determined through a first-round comparison with a 50-genome nucleotide database, and subjected to a second-round screening with the GenBank NR database. As a result, nine specific genes of P. aroidearum were selected for LAMP primer design. After screening of the primers, the primer set 1675-1 was chosen for LAMP detection owing to its high specificity and sensitivity. The LAMP assay could detect the presence of P. aroidearum genomic DNA at a concentration as low as 50 fg and 1.2 × 104 CFU/g artificially infected soil within 40 min at 65 °C. Subsequently, this primer set was successfully used to specifically detect P. aroidearum in naturally infected and non-symptomatic plant samples or soil samples from the field. This study indicates that a comparative genomic approach may facilitate the development of highly specific primers for LAMP assays, and a LAMP diagnostic assay with the specific primer set 1675-1 should contribute to the rapid and accurate detection of soft-rot disease in konjac at an early stage.

Chemotactic response of Ginseng bacterial soft-rot to Ginseng root exudates.

Our purpose was to evaluate chemotactic response of Ginseng bacterial soft-rot to ginseng root exudates. The exudates of plant roots has a significant influence on the population changes of rhizosphere microorganisms and chemotaxis is an important way in which many pathogens sense the signals of host plants and invade the host plants. In this study, with the capillary method, we tested the chemotactic responses of Ginseng bacterial soft-rot for three ginseng roots exudates under four chemotactic parameters (concentration, temperature, pH and time). The results showed that the chemotatic response of the Ginseng bacterial soft-rot for the ginseng roots exudates at the water layer where pH = 7 and the concentration was 0.0125 mg/L reached its peak value under the circumstance that the exudates was cultivated for 60 min at 25 °C. The chemotatic ratios were respectively 124.89% and 89.44%. For the butanol extract layer and the petroleum ether faction at the concentration of 0.125 mg/L and the pH value at 7, the ginseng roots exudatess reached peak values at 25 °C and 30 °C and 60 min and 75 min respectively, and the chemotatic ratios were respectively 139.64% and101.87%, and 115.29% and 81.36%. The three ginseng roots exudates had positive effects for the chemotaxis of the Ginseng soft-rot bacteria, but the effect declined as the concentration increased.

Genome-wide identification of potato long intergenic noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection.

BACKGROUND: Long noncoding RNAs (lncRNAs) represent a class of RNA molecules that are implicated in regulation of gene expression in both mammals and plants. While much progress has been made in determining the biological functions of lncRNAs in mammals, the functional roles of lncRNAs in plants are still poorly understood. Specifically, the roles of long intergenic nocoding RNAs (lincRNAs) in plant defence responses are yet to be fully explored. RESULTS: In this study, we used strand-specific RNA sequencing to identify 1113 lincRNAs in potato (Solanum tuberosum) from stem tissues. The lincRNAs are expressed from all 12 potato chromosomes and generally smaller in size compared to protein-coding genes. Like in other plants, most potato lincRNAs possess single exons. A time-course RNA-seq analysis between a tolerant and a susceptible potato cultivar showed that 559 lincRNAs are responsive to Pectobacterium carotovorum subsp. brasiliense challenge compared to mock-inoculated controls. Moreover, coexpression analysis revealed that 17 of these lincRNAs are highly associated with 12 potato defence-related genes. CONCLUSIONS: Together, these results suggest that lincRNAs have potential functional roles in potato defence responses. Furthermore, this work provides the first library of potato lincRNAs and a set of novel lincRNAs implicated in potato defences against P. carotovorum subsp. brasiliense, a member of the soft rot Enterobacteriaceae phytopathogens.

N-acyl homoserine lactones in diverse Pectobacterium and Dickeya plant pathogens: diversity, abundance, and involvement in virulence.

Soft-rot bacteria Pectobacterium and Dickeya use N-acyl homoserine lactones (NAHSLs) as diffusible signals for coordinating quorum sensing communication. The production of NAHSLs was investigated in a set of reference strains and recently-collected isolates, which belong to six species and share the ability to infect the potato host plant. All the pathogens produced different NAHSLs, among which the 3-oxo-hexanoyl- and the 3-oxo-octanoyl-L-homoserine lactones represent at least 90% of total produced NAHSL-amounts. The level of NAHSLs varied from 0.6 to 2 pg/cfu. The involvement of NAHSLs in tuber maceration was investigated by electroporating a quorum quenching vector in each of the bacterial pathogen strains. All the NAHSL-lactonase expressing strains produced a lower amount of NAHSLs as compared to those harboring the empty vector. Moreover, all except Dickeya dadantii 3937 induced a lower level of symptoms in potato tuber assay. Noticeably, aggressiveness appeared to be independent of both nature and amount of produced signals. This work highlights that quorum sensing similarly contributed to virulence in most of the tested Pectobacterium and Dickeya, even the strains had been isolated recently or during the past decades. Thus, these key regulatory-molecules appear as credible targets for developing anti-virulence strategies against these plant pathogens.