First Report of Pantoea dispersa Causing Brown Blotch Disease in Flammulina filiformis in China

Flammulina filiformis (previously known as F. velutipes) is one of the most frequently cultivated and consumed edible mushrooms in China. In October 2020, brown blotch disease was observed on the pileus of F. filiformis at a mushroom factory in Ganzhou (25.74°N; 114.95°E), Jiangxi, China, with a disease incidence of approximately 6%. Symptoms initially appeared as small, irregular spots on the infected pileus, with color ranging from pale yellow to light brown. Such spots were enlarged and pitted at high relative humidity within several days, and finally caused malformation of the caps and yield reduction. To isolate the causal agent, the blotches on F. filiformis caps were homogenized and diluted with sterilized distilled water, and the resulting suspension (100 μl) was spread onto LB agar plates. After incubation at 28°C for 48 h, three colonial types were obtained: (i) yellow, convex, and smooth colonies, (ii) light yellowish, irregular, and rough colonies, and (iii) milky white, glistening, and smooth colonies. The first colonial type was predominant. A single colony of each type was randomly selected and streaked on fresh LB agar plates to obtain pure cultures namely PF1, PF2 and PF3 respectively. To test pathogenicity of the three isolates, young F. filiformis fruiting bodies (8- or 10-day-old primordium) grown in culture bottles were inoculated by spraying a bacterial suspension (108 CFU/ml, 3 ml per bottle on average), and cultivated in a mushroom house at 15±2°C and 95% relative humidity. The fruiting bodies sprayed with sterilized distilled water served as controls. The pathogenicity tests repeated three times, and at least five culture bottles were included in each experiment. Among the three types of bacteria, only strain PF1 induced symptoms similar to the original disease. The brown spots were observed on treated pileus 10 days after inoculation, and a fresh weight reduction of 30.9% per culture bottle was observed. In contrast, those fruiting bodies treated with water remained asymptomatic. Same yellow colonies were also re-isolated from the infected pileus, and identified by subsequent methods. The strain PF1 was gram negative, motile, and short rods. Biochemical analysis showed that the strains belonged to genus Pantoea (positive for citric acid, inositol, mannitol, methyl red test, and Voges-Proskauer test but negative for lysine, ornithine, phenylalanine, H2S, urease, D-melibiose, sorbitol, adonitol, and raffinose). Further PCR amplification and sequencing of four genes, 16S rRNA gene with primer 27F/1492R, fusA gene with primer fusA3/fusA4, gyrB gene with primer gyrBf1/gyrBr1, and rpoB genes with primer Vic3/Vic2 (Delétoile et al. 2009; Palemon et al. 2021), were performed to identify the species. A BLASTn showed 100%, 100%, 99.51%, and 99.26% homology, respectively, with those of P. dispersa (MT072166, CP045216, CP076369, MH015168). The four gene sequences were deposited in GenBank (accession numbers: MZ373179, MZ393661, MZ393662, MZ393663). A phylogenetic analysis based on the four concatenated genes also showed that the strain PF1 well clustered with the type strain of P. dispersa. This species has been reported to cause leaf blight in rice (Toh et al. 2019), soft rot in Agave angustifolia (Palemon et al. 2021), and bulb decay in onion (Chang et al. 2018). To the best of our knowledge, this is the first report of P. dispersa causing brown blotch diseases on cultivated F. filiformis, which was previously known to be caused by Pseudomonas tolaasii (Lee et al. 2002). Our results also indicate P. dispersa could induce malformation of pileus and lead to a severe yield loss if not controlled effectively. Therefore, it should be considered in future disease management of F. filiformis cultivation.

Confirmation de QTL et validation de marqueurs SNPs associés à la résistance du niébé à Colletotrichum capsici, agent responsable de la maladie des taches brunes

Le niébé (Vigna unguiculata (L.) Walp.) est une légumineuse à graine très importante et constitue la principale source de protéines végétales pour l’alimentation des populations d’Afrique Subsaharienne. Sa production au Burkina Faso est entravée par la maladie des taches brunes provoquée par un champignon, Colletotrichum capsici (Syd.) Butler et Bisby. C’est dans la perspective d’accroître la productivité du niébé que nous avons entrepris de renforcer la lutte variétale contre cet agent pathogène. L’identification de marqueurs SNPs (Single Nucleotide Polymorphism) et QTL liés à la résistance à la maladie des taches brunes a été entrepris à partir d’une population biparentale F2 issus du croisement entre la variété sensible Tiligré et celle résistante KN-1. L’analyse QTL de la résistance du niébé à C. capsici à partir de la méthode ICIM add. a permis de confirmer et de valider respectivement un QTL majeur dénommé qBBDR2.1 et 9 marqueurs SNPs convertis, lesquels ont été cartographiés sur le chromosome Vu02 du niébé. Ce QTL dominant a présenté des effets additifs élevés liés aux allèles favorables de KN-1 et des valeurs de PVE de l’ordre de 51,50% et 55,33%, respectivement aux 21ème et 28ème JAI. English title: Confirmation of QTL mapping and validation of SNPs markers associated to cowpea resistance to Colletotrichum capsici, causal agent of brown blotch disease Cowpea (Vigna unguiculata (L.)Walp.) is one of the most important grain legume crops and constitutes the main source of plant protein for people food in sub-Saharan Africa. Cowpea production in Burkina Faso is constrained by brown blotch disease caused by a fungal, Colletotrichum capsici (Syd.) Butler and Bisby. In order to increase cowpea productivity we initiated a project to enhance host plant resistance to control the pathogen. The identification of SNP (Single Nucleotide Polymorphism) markers and QTL associated with brown blotch disease resistance was undertaken from a bi-parental F2 population resulting from a cross between the sensitive variety Tiligre and the resistant KN-1 to the disease. QTL analysis of cowpea resistance to C. capsici using the ICIM add method. Allowed to confirm and validate respectively a major QTL named qBBDR2.1 and 9 converted SNP markers, which were mapped on cowpea chromosome Vu02. This dominant QTL showed higher additive effects associated to alleles from KN-1 and PVE values of 51.50% and 55.33% respectively at 21 and 28 days after inoculation

Insights into detoxification of tolaasins, the toxins behind mushroom bacterial blotch, by Microbacterium foliorum NBRC 103072T

Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of brown blotch disease of mushrooms, and are the toxins that cause the brown spots. We previously reported that Microbacterium foliorum NBRC 103072T is an effective tolaasin-detoxifying bacterium. In this study, we aimed to characterize the tolaasin-detoxification process of M. foliorum NBRC 103072T. The tolaasin-detoxification by M. foliorum NBRC 103072T was carried out by hydrolyzation of tolaasins at two specific sites in the peptide moiety of tolaasins by its cells, and the resulting fragments were released from bacterial cells. The tolaasin-hydrolyzing activity can be extracted by neutral detergent solution from M. foliorum NBRC 103072T cells. Moreover, tolaasin-adsorption to the bacterial cells occurred prior to hydrolyzation of tolaasins, which might contribute to the effective tolaasin-detoxification by M. foliorum NBRC 103072T. It is notable that the tolaasin-degradation process by M. foliorum NBRC 103072T is carried out by hydrolyzation at specific sites in the peptide moiety of lipopeptide by bacterial cells as a novel biological degradation process of cyclic lipopeptides.

Helper bacteria halt and disarm mushroom pathogens by linearizing structurally diverse cyclolipopeptides

The bacterial pathogenPseudomonas tolaasiiseverely damages white button mushrooms by secretion of the pore-forming toxin tolaasin, the main virulence factor of brown blotch disease. Yet, fungus-associated helper bacteria of the genusMycetocola(Mycetocola tolaasinivoransandMycetocola lacteus) may protect their host by an unknown detoxification mechanism. By a combination of metabolic profiling, imaging mass spectrometry, structure elucidation, and bioassays, we found that the helper bacteria inactivate tolaasin by linearizing the lipocyclopeptide. Furthermore, we found thatMycetocolaspp. impair the dissemination of the pathogen by cleavage of the lactone ring of pseudodesmin. The role of pseudodesmin as a major swarming factor was corroborated by identification and inactivation of the corresponding biosynthetic gene cluster. Activity-guided fractionation of theMycetocolaproteome, matrix-assisted laser desorption/ionization (MALDI) analyses, and heterologous enzyme production identified the lactonase responsible for toxin cleavage. We revealed an antivirulence strategy in the context of a tripartite interaction that has high ecological and agricultural relevance.

Resistance Sources to Brown Blotch Disease (Pseudomonas tolaasii) in a Diverse Collection of Pleurotus Mushroom Strains

Brown blotch disease (BBD) caused by Pseudomonas tolaasii is one of the most devastating diseases of Pleurotus spp. worldwide. Breeding for resistant strains is the most effective method for controlling BBD. To identify resistant germplasm for BBD management, 97 strains comprising 21 P. cf. floridanus, 20 P. ostreatus, and 56 P. pulmonarius were screened by two different methods; namely, inoculation of the pathogen on the mushroom pileus (IMP) and on the spawned substrate (IMSS) under controlled conditions. Out of the 97 strains screened, 22 P. pulmonarius, and four P. cf. floridanus were moderately resistant to BBD using the IMP method. Eleven P. pulmonarius, six P. cf. florida, and one P. ostreatus strains were highly resistant to BBD using the IMSS method. All of the 97 strains showed varying degrees of susceptibility using the IMP method, but eight strains were completely resistant using the IMSS method. Combining these two methods, five strains were highly resistant (four P. pulmonarius and one P. cf. floridanus) and 11 were moderately resistant (eight P. pulmonarius and three P. cf. floridanus). The resistance sources to P. tolaasii identified in P. pulmonarius and P. cf. floridanus could be used for further breeding of Pleurotus spp.