Conversion from mutualism to parasitism: a mutant of the ectomycorrhizal agaricomycete Tricholoma matsutake that induces stunting, wilting, and root degeneration in seedlings of its symbiotic partner, Pinus densiflora, in vitro

Abstract

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces matsutake mushrooms in association with conifers. Here, we isolated a pleiotropic mutant, designated as G1, following ␥-ray irradiation of T. matsutake NBRC 33136. In addition to exhibiting increased cellulose-and amylose-degrading activities and altered mycelial morphology, G1 degenerated lateral roots and caused stunting or fatal wilting of seedlings of its symbiotic partner, Pinus densiflora, in vitro. A mutant, designated Ar 59, previously isolated after argon-ion beam irradiation, exhibited the same phenotypes as G1, but without its detrimental effects. Ar 59, like NBRC 33136, developed a Hartig net around healthy cortical cells that was composed of uniseriate hyphae. In G1-inoculated seedlings, the Hartig net was composed of heavily bundled hyphae penetrating the intercellular space, and it was accompanied by somewhat unusual forms of plant cortical cells. Sequences of rRNA gene-related markers, including four single nucleotide polymorphisms present within each strain, were 100% identical among NBRC 33136, G1, and Ar 59, indicating that they are mutants of the wild-type. Thus, ␥-ray irradiation can convert the fungus from a beneficial to a harmful agent. These findings suggest the presence of an unknown mechanism in the fungal genome that can transform mutualism into parasitism.Résumé : Tricholoma matsutake est un agaromycète ectomycorhize qui produit les champignons matsutake en association avec les conifères. Les auteurs ont isolé ici un mutant pléiotropique qu'ils ont désigné G1, à la suite d'une irradiation ␥ de T. matsutake NBRC 33136. En plus de présenter une activité accrue de dégradation de la cellulose et de l'amylose et une morphologie mycéliale modifiée, G1 provoquait la dégénérescence des racines latérales et un retard de croissance ou une flétrissure fatale des jeunes plants de son partenaire symbiotique, Pinus densiflora, in vitro. Un mutant désigné Ar 59, isolé précédemment après une irradiation à un faisceau d'argon ionisé, présentait le même phénotype que G1, mais sans les effets néfastes. Ar 59, comme NBRC 33136, développait un réseau de Hartig autour des cellules corticales saines, composé d'hyphes unisériées. Chez les plantules inoculées avec G1, le réseau de Hartig était composé d'hyphes en faisceau pénétrant l'espace intercellulaire et était accompagné par des cellules corticales végétales de formes quelque peu inhabituelles. Les séquences de marqueurs reliés aux gènes de l'ARNr, y compris quatre polymorphismes de nucléotides uniques présents dans chaque souche, étaient 100% identiques entre NBRC 33136, G1 et Ar 59, indiquant qu'ils constituent des mutants de la souche sauvage. Ainsi, l'irradiation gamme peut convertir le champignon d'une forme bénéfique en un agent pathogène. Ces données suggèrent la présence d'un mécanisme inconnu dans le génome fongique qui peut transformer le mutualisme en parasitisme. [Traduit par la Rédaction] Mots-clés : enzymes de dégradation, mutagenèse par irradiation gamma, faisceau d'ion...

Argon-ion beam induced mutants of the ectomycorrhizal agaricomycete Tricholoma matsutake defective in β-1,4-endoglucanase activity promote the seedling growth of Pinus densiflora in vitro

“…Tricholoma matsutake NBRC 33136 significantly promoted P. densiflora growth in vitro ( Fig. 2a; Yamada et al 2006Yamada et al , 2010Murata et al 2013b, Murata et al 2019. Surprisingly, T.…”

“…The putative mutants Ar 5002 and Ar 5012, which resulted from 100-Gy argon-ion irradiation, which conferred a survival rate of 8%, showed no detectable -1,4 endoglucanase activity on PDA containing AZCL-HE cellulose. On AZCL-HE cellulose plates, the wild-type strain produced translucent blue halos, but not the clear ones that the over-producing mutants G1 and Ar 59 had previously produced (Murata et al 2019) at the margins of, as well as beneath, the mycelial colonies, resulting in enzymatic activities scored as + (Fig. 1a).…”

“…The dual cultivation experiment was carried out by following exactly the methods of our previous study (Murata et al 2019). The substrate was prepared by adding 160 mL of Hamada medium as modified by Yamanaka et al (2012Yamanaka et al ( , 2014 containing 1% glucose, 0.5% yeast extract and 0.25% FeCl 3 ⋅6H 2 O to 400 mL of dried pumiceous soil (particle diameter ~3 mm).…”

“…To carry out anatomical analyses of root tissues associated with fungi, semi-sections suitable for microscopic examination were prepared as described elsewhere (Shimomura et al 2008;Sawada et al 2014;Murata et al 2019). Briefly, root tips were fixed with 2.0% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h, washed with 0.1 M phosphate buffer (pH 7.2), further fixed with 1.0% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) for 2 h, washed with distilled water and then dehydrated in serially diluted (50%, 70%, 90%, 95% and 100%) ethanol solutions.…”

“…The rRNA intergenic spacer region 1 (rDNA IGS1) and the rRNA gene internal transcribed spacer (rDNA ITS) region were amplified by high-fidelity PCR (Tks Gflex DNA polymerase, Takara, Otsu, Japan) using primer pairs ITS1-F/ITS4-B (Gardes and Bruns, 1993) and IGS-1-P-1/5srRNA1_rev (Babasaki et al 2007;Murata et al 2013a), respectively. PCR and nucleotide sequencing of PCR products were carried out by following exactly the protocol described by Murata et al (2019). Using the software Geneious (Biomatters Limited, Auckland, NZ) and the reference sequences of NBRC 33136 [AB549435 (rDNA IGS1) and AB036890 (rDNA ITS)], the sequence data of DNA segments from putative mutants were subjected to multiple alignments.…”