Identiˆcation of Non-Pseudomonad Bacteria from Fruit Bodies of Wild Agaricales Fungi That Detoxify Tolaasin Produced by Pseudomonas Tolaasii

Biosci. Biotechnol. Biochem., 66 (10), 2201–2208, 2002

Identiˆcation of Non-Pseudomonad Bacteria from Fruit Bodies of Wild Agaricales Fungi That Detoxify Tolaasin Produced by Pseudomonas tolaasii

Takanori TSUKAMOTO,1,* Hitoshi MURATA,2,† and Akira SHIRATA1,**

1Department of Sericulture, National Institute of Sericultural and Entomological Sciences, Ohwashi 1-2, Tsukuba-City 305-8634, Japan 2Division of Bio-Resource Development, Forestry & Forest Products Research Institute, Kukizaki, Ibaraki 305-8687, Japan

Received April 17, 2002; Accepted June 24, 2002

Bacterial isolates from wild Agaricales fungi detoxi- the disease symptoms.4–8) We previously reported that êd tolaasin, the inducer of brown blotch disease of cul- a Gram-positive bacterium, which was then not as- tivated mushrooms produced by Pseudomonas tolaasii. signed to any deˆned bacterial genera, from rotted Mycetocola tolaasinivorans and Mycetocola lacteus fruit bodies of cultivated P. ostreatus detoxiêd were associated with fruit bodies of wild Pleurotus os- tolaasin and suppressed disease development on P. treatus and wild Lepista nuda, respectively. Tolaasin- ostreatus and A. bisporus under our laboratory assay detoxifying bacteria belonging to other genera were conditions.9) The following intensive systematic anal- found in various wild mushrooms. An Acinetobacter ysis showed that this bacterium was a new member of sp. was isolated from fruit bodies of Tricholoma the family Microbacteriaceae,andallowedustopro- matsutake, Bacillus pumilus was isolated from Copri- pose a new genus, Mycetocola, and to assign the bac- nus disseminatus,andSphingobacterium multivorum terium to Mycetocola tolaasinivorans.10) Later, we was isolated from Clitocybe clavipes.APedobacter sp., identiêd two other species in the genus, Mycetocola which seemed not be identiâble as any known bacterial lacteus and Mycetocola saprophilus, though a poten- species, was isolated from a Clitocybe sp. Tolaasin- tial for suppressing brown botch disease has not been detoxifying bacteria identiêd thus far were attached to evaluated, yet.10) Thus far, all three species of the surface of mycelia rather than residing within the Mycetocola were isolated only from cultivated fungal cells. M. tolaasinivorans, M. lacteus, B. pumilus, mushrooms.9,10) the Pedobacter sp., and S. multivorum e‹ciently detox- Since then, little progress has been made in the iêd tolaasin and strongly suppressed brown blotch de- elucidation of mechanisms involved in the detoxiˆca- velopment in cultivated P. ostreatus and Agaricus tion of tolaasin, because molecular genetic systems bisporus in vitro,buttheAcinetobacter sp. did so less applicable to this peptidoglycan type B bacterium are e‹ciently. These bacteria may be useful for the elucida- limited. As for the symbiotic habitat on mushrooms, tion of mechanisms involved in tolaasin-detoxiˆcation, it has not been clear whether Mycetocola species is and may become biological control agents of mushroom naturally parasitic to P. ostreatus or speciˆcally to disease. the cultivated mushrooms as an artifact brought through the substrate cultivation, and whether bac- Key words: Acinetobacter sp.; Bacillus pumilus; teria other than Mycetocola that detoxify tolaasin are Mycetocola spp.; Pedobacter sp.; Sphin- naturally associated with Agaricales fungi. gobacterium multivorum In this report, we describe various bacteria isolated from wild Agaricales fungi, which detoxify tolaasin Pseudomonas tolaasii causes brown blotch disease and have a potential to suppress the occurrence of the of the economically important cultivated mushrooms brown blotch disease. Some bacterial isolates were Pleurotus ostreatus and Agaricus bisporus.1–3) The found to belong to genera to which molecular genetic bacterium produces tolaasin, an extracellular approaches currently available may be applied. These lipodepsipeptide toxin needed for the elicitation of ˆndings may facilitate future studies of ecology and

Nucleotide sequences from tolaasin-detoxifying bacteria have been deposited in the DDBJ database under accession numbers AB012646- AB012648, AB020204-AB020208, and AB074152. † To whom correspondence should be addressed. Tel: +81-298-73-3211; Fax: +81-298-73-0507; E-mail: murmur＠Špri.aŠrc.go.jp * Present address: Yokohama Plant Protection Station, Shinyamashita 1-16-10, Naka-ku, Yokohama 231-0801, Japan ** Present address: Department of Research Planning and Coordination, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba 305-8602, Japan Abbreviations: PS, potato semi-synthetic broth; PSA, potato semi-synthetic agar 2202 T. TSUKAMOTO et al. physiology of tolaasin-detoxifying bacteria, molecular analysis of biological function involved in the detoxifying mechanisms, and the development of new means to control the devastating mushroom disease caused by P. tolaasii.

Materials and Methods

Growth conditions and media. Unless stated other- wise, bacteria were grown in PSB (potato semi-synthetic broth) or PSA (potato semi-synthetic agar) at 259C.9) PSB-Tol, a medium that contains tolaasin, was prepared by addition of the components of PSB to spent culture supernatants of P. tolaasii,which Fig. 1. EŠects of Talaasin-detoxifying Bacteria on Potato Slices. were obtained after culturing P. tolaasii in PSB at Potato slices treated with PSB-Tol in the presence of tolaasin- 259C for 48 h, harvested by centrifugation (49C, detoxifying bacteria remained colorless, but those treated 10,000×g) and sterilized by placing in boiling water together with non-detoxifying bacteria were blackened. for 10 min.9) Nutrient gelating agar plates were used to measure the extracellular proteolytic activity of bacteria.5) with API20NE, API50CH, API50CHB, APIZYM (bioMerieux s.a., Marcy-I'Etoile, France) or Biolog Isolation of Bacteria from Agaricales fruit bodies. GN2-MicroPlate (Biolog Life Science Institute, Altogether 43 isolates of the following 26 species of Hayward, CA), and standard protocols published wild Agaricales fungi were collected from a northern elsewhere.12–15) The 16S rDNA was ampliˆed from a part of Ibaraki Prefecture, Japan; Clitocybe sp., DNA sample from each bacterial isolate by PCR us- Clitocybe gibba, Clitocybe clavipes, Coprinus ing the 16S rDNA-universal primers 24f and 1492r.11) disseminatus, Coriolus versicolor, Helvella sp., Nucleotide sequences of the PCR products were ana- Kobayashia nipponica, Lactarius hatsudake, lyzed with BigDye terminator FS core kit and ABI Lenzites betulina, Lepiota praetervisa, Lepista nuda, Prism 377 (Applied Biosystems, Foster City, CA). Lycoperdon colossum, Lycoperdon perlatum, The phylogenetic analysis of the 16S rDNA was done Lyophyllum decastes, Marasmius androsaceus, with the CLUSTAL W program.16) Biological and Mycena pura, Mycena roseocandida, Phallus im- biochemical reactions for API- and Biolog-tests were pudicus, Pholiota malicola, Pleurotus cornucopiae, done at 309C for 48 and 24 h, respectively, and data Pleurotus ostreatus, Psathyrella piluliformis, Suillus were analyzed with the ATB-Expression system bovinus, Suillus laricinus, Tricholoma matsutake, (bioMerieux s.a.) and MicroLog System release and Tricholoma ustale. Bacteria associated with 4.01B (Biolog Life Science Institute), appropriately. fungi were collected by soaking fungal fruit bodies in 4 ml of sterile water for 30 min, followed by Assays for tolaasin-detoxiˆcation and suppression vigorously mixing with a Vortex mixer (Scientiˆc of the brown blotch. The bacteria were grown in Industries Inc., Bohemia, NY). The suspensions were PSB-Tol at 259C in a rotary shaker. Culture super- serially diluted and spread on PSA to allow bacteria natants were obtained by centrifugation, ˆlter-steri- associated with the mycelia to form colonies. lized, serially diluted, and applied onto potato tuber slices. The level of the tolaasin activity in culture su- Isolation of tolaasin-detoxifying bacteria. Each of pernatants were semiquantitatively measured by the bacterial isolates of 1032 colonies randomly selected extent of blackening of potato tuber slices (Fig. 1).9) was grown in 100 ml of PSB in wells of a 96-well To measure the suppression of the brown blotch, 2× microtiter plate at 259C for 48 h. Then, an equal 106 cells of each detoxifying bacterium were mixed volume of PSB-Tol was added and further incubated with equal cell numbers of P. tolaasii and used to at 259C for 72 h. The 50 ml of samples of the whole inoculate fruit bodies of P. ostreatus.9) The extent of cultures were then applied onto potato tuber slices, brown blotch development was scored after 48 h of wherein tuber slices normally become blackened due incubation.9) to the toxicity of tolaasin (Fig. 1).8,9) Bacterial strains that suppressed the occurrence of blackening were Results and Discussion scored as tolaasin-detoxifying agents (Fig. 1). Detoxifying bacteria from P. ostreatus and L. Taxonomic characterization of bacteria. Tolaasin- nuda detoxifying bacteria were genetically characterized by Tolaasin-detoxifying bacteria represented by the 16S rDNA analysis,11) and then biochemically strains OM-F11 and OM-A1 were isolated from P. Tolaasin-Detoxifying Bacteria from Wild Agaricales Fungi 2203

Fig. 2. Phylogenetic Trees Based on the 16S rDNA Sequences of (A) Mycetocola Species and Some Close Relatives, (B) Sphingobacterium Species, Pedobacter Species and Some Close Relatives, (C) Acinetobacter Species and Some Close Relatives, and (D) Bacillus Species and Some Close Relatives. Phylogenetic trees were created by the neighbor-joining method. The numerals indicate the percentages of bootstrap samplings derived from 1000 replications.16) The 16S rDNA sequences were obtained from the National Center for Biotechnology Information (http:WWwww.ncbi.nlm.nih.govWirx WgenbankWqueryäform.html) with GenBankWEMBLWDDBJ accession numbers shown in paren- theses. ostreatus and L. nuda, respectively. Based on the strain OM-A1, which was positive in all, were identi- rDNA analysis, both strains were assigned to the ge- êd as M. tolaasinivorans and M. lacteus, respec- nus Mycetocola, a genus which has only been isolated tively. Thus far, we have not encountered M. from cultivated P. ostreatus (Fig. 2).9,10) These iso- saprophilus, which is positive in utilization of citrate lates had bacteriological features common to the and acid production from melezitose, and negative in genus Mycetocola: smooth yellow colonies, variable hydrolysis of Tween 80 and acid production from rod-shaped cells, aerobic growth, Gram-positive, no erythritol. spore formation, and no motility. All the isolates gave positive test results for hydrolysis of esculine, Detoxifying bacteria from C. clavipes b-galactosidase, and assimilation of D-glucose, D- From the fruit body of C. clavipes, tolaasin-detox- mannose, D-mannitol, and maltose. They gave nega- ifying bacteria represented by the strain OM-A8 were tive results in nitrate reduction to NO2 and N2, indol isolated. They had the sequence of 16S rDNA mar- production, arginine dihydrolase, urease, gelatinase, kedly similar to that of Sphingobacterium multivo- oxidase, and assimilation of L-arabinose, N-acetyl- rum (Fig. 2). The bacteriological properties of the glucosamine, gluconate, n-caprate, adipate, dl- isolate also corresponded to those of S. multivo- malate, and phenyl acetate. rum.14,15) For example, the strain OM-A8 had smooth Characteristics in hydrolysis of Tween 80, utiliza- yellow colonies, rod-shaped cells, and aerobic tion of citrate, and acid production from erythritol growth, and was Gram-negative, had no spore for- and melezitose allowed us to diŠerentiate these bac- mation and no motility. A positive reaction was not- terial isolates into deˆned species.10) Strain OM-F11, ed in the test of hydrolysis of esculine, b-galactosi- which was negative in of all the four phenotypes, and dase, and oxidase, and assimilation of D-glucose, L- 2204 T. TSUKAMOTO et al. arabinose, D-mannose, N-acetyl-glucosamine, and adipate, dl-malate, citrate, and phenyl acetate. Nega- maltose. Negative reaction was noted in nitrate tive reactions were noted in nitrate reduction to NO2 reductiontoNO2 and N2, indol production, arginine and N2, indol production, arginine dihydrolase, dihydrolase, gelatinase, and assimilation of D-man- urease, gelatinase, hydrolysis of esculine, b-galactosi- nitol, gluconate, n-caprate, adipate, dl-malate, dase, oxidase, and assimilation of D-glucose, L- citrate, and phenyl acetate. The feature of OM-A8 arabinose, D-mannose, D-mannitol, N-acetyl- based on the API20NE test scored 99.9z identity in glucosamine, maltose, and gluconate. These features the proˆle index of S. multivorum. This result was based on API20NE scored 91.9z probability in the consistent in the analysis based on Biolog (similarity proˆle index of Acinetobacter baumannii,thespecies index value＝0.842). Therefore, the strain OM-A8 that corresponds to genospecies 2.17) However, the was assigned to S. multivorum. result from Biolog strongly supported the idea that the strain OM-H10 is a member of the genospecies Detoxifying bacteria from Clitocybe sp. 11, a group of Acinetobacter spp. which has not been Tolaasin-detoxifying bacteria represented by strain clearly deˆned at the species level, by scoring a simi- OM-E81 were isolated from Clitocybe sp. The 16S larity index value of 0.869 with 96z probability.17) rDNA analysis identiêd the bacterial isolates as a Since both the systems suggested the same genus, the Pedobacter sp. (Fig. 2).14,15) These isolates also had isolate was assigned to Acinetobacter sp. OM-H10. bacteriological properties shared by the genus Pedobacter: smooth yellow colonies, rod-shaped Detoxifying bacteria from C. disseminatus cells, and aerobic growth, with no spore formation, Gram-positive spore-forming bacteria with rough Gram-negative reaction, and no motility.14,15) A posi- colony morphology represented by OM-F6 were iso- tive reaction was noted in the following features: lated from C. disseminatus. The sequence of 16S hydrolysis of esculine, oxidase, alkaline phos- rDNA of OM-F6 matched that of Bacillus pumillus phatases, esterase lipase (C8), leucine arylamidase, b- (Fig. 2). Biochemical analysis with API50CH showed galactosidase, a-glucosidase, N-acetyl-b-glucosami- the following features. Strain OM-F6 was aerobic dase, and assimilation of D-glucose, D-mannose, and motile, and positive for the acidiˆcation of amygdalin, and glucosamine.14,15) A negative reaction esculine, glucose, mannose, mannitol, N-acetyl- was noted in the following tests: urease, nitrate glucosamine, glycerol, L-arabinose, ribose, D-xylose, reduction, indol production, arginine dihydrolase, a- galactose, D-fructose, amygdaline, arbutine, salicine, fucosidase, and assimilation of n-caprate, adipate, cellobiose, saccharose, trehalose, b-gentiobiose, and phenyl acetate, erythritol, L-xylose, b-methyl-D-xylo- D-tagatose. side, L-sorbose, dulcitol, inositol, xylitol, D-tagatose, They were negative for the acidiˆcation of D-fucose, D-arabitol, D-melezitose, and gluco- maltose, gluconate, D-arabinose, erythritol, L-xylose, nate.14,15) adonitol, b-methy-xyloside, L-sorbose, rhamnose, However, the overall metabolic proˆle of OM-E81 dulcitol, inositol, sorbitol, a-methyl-D-mannoside, a- was unusual among those that are variable at the spe- methyl-D-glucoside, lactose, melibiose, inuline, cies level (Table 1).14,15) In addition, a negative reac- melezitose, D-ra‹nose, starch, glycogen, xylitol, D- tion for the following features excluded any possibili- turanose, D-lyxose, D-fucose, D-arabitol, D-arabitol, ties that the isolates represented by OM-E81 are 2-keto-gluconate, and 5-keto-gluconate. Having a Sphingobacterium spp., the genus the Pedobacter 99.9z match in the proˆle index, tolaasin-detox- spp. once had been classiêd in: urease, chymotryp- ifying bacteria represented by the strain OM-F6 were sin, a-mannosidase, and assimilation of a-methyl-D- identiêd as B. pumilus. mannoside, D-melezitose and, D-ra‹noase.14,15) The result was consistent with the analysis using Biolog, Detoxiˆcation and suppression of symptoms by in which again no deˆned bacterial species was speci- the bacterial isolates êd. Therefore, we named the isolate Pedobacter sp. The bacterial strains were compared for their strain OM-E81. e‹ciency in tolaasin detoxiˆcation. M. tolaasinivorans OM-F11 and M. lacteus OM-A1 isolated from Detoxifying bacteria from T. matsutake wild Agaricales fungi e‹ciently detoxiêd tolaasin as Tolaasin-detoxifying bacteria represented by strain we previously observed in M. tolaasinivorans CM-05 OM-H10 were isolated from the ectomycorrhizal from cultivated P. ostreatus (Fig. 3).9) In fact, M. fungus T. matsutake.The16SrDNAanalysis saprophilus CM-01 and M. lacteus CM-10 previously suggested that the isolate was an Acinetobacter sp. isolated from cultivated mushrooms for systematic (Fig. 2). Strain OM-H10 had the following bacterio- bacteriological analysis also detoxiêd tolaasin logical characteristics: creamy smooth colonies, rod- (Fig. 3). These observations suggested that the genus shaped cells, aerobic growth, no spore formation, Mycetocola is naturally parasitic to some species of Gram-negative reaction, and no motility. Positive Agaricales fungi and is capable of detoxifying tolaa- reactions were noted in assimilation of n-caprate, sin in general. Tolaasin-Detoxifying Bacteria from Wild Agaricales Fungi 2205

Table 1. Biochemical Characteristics of Pedobacter sp. Strain OM-E81

Bacterial strains

Characteristics Pedobacter P. P. P. P. Pedobacter Pedobacter Pedobacter Pedobacter sp. strain heparinus africanus piscium saltans sp. strain sp. strain sp. strain sp. strain OM-E81 Ia1 Ia3 III Ia2 Ib1 Ib2 II

Enzymatic activity (APIZYM): Esterase + „, + „ + „ „ „ „ „ Lipase „ „,„ „ v,„ „ „ „ „ Valine acrylamidase + „, „ v + v v + + + Cystine acrylamidase + „, + „ + „ „ „ „ „ Trypsin+„,„„+„„„„„ Chymotrypsin „ „, + „ + „ „ „ „ „ a-Galactosidase „ „, + „ + „ „ „ „ „ b-Glucuronidase + „, „ „ v v „ „ „ „ b-Glucosidase „ „, + v + + v + + „ a-Mannosidase „ „, + v + „ „ „ + „ Assimilation of (API50CH): Glycerol „ „ „ „ + „ v + „ D-Arabinose „ „ v „ v v + + „ L-Arabinose „ + v + + v + + „ Ribose „ „ v „ „ + „ „ „ D-Xylose„+v+++++„ Adonitol„+„„+++„„ D-Galactose „ + + v + + v + „ D-fructose++v+v++„+ Rhamnose „ + + v + + + + „ Mannitol + + „ „ „ + + „ „ Sorbitol „ + „ „ „ + v „ „ Arbutin + v v + + v + + + Salicin++v+++++„ D-Cellobiose + + + + „ + + + „ Maltose++v+++++„ Lactose++++++++„ D-Melibiose + + + + v + + + „ Sucrose + + + + v + + + „ Trehalose++++v+++„ Inulin „ „ „ „ „ „ „ „ „ D-Melezitose „ „ „ „ „ „ „ „ „ D-Ra‹nose „ „ „ + v „ + „ „ Starch+„v+„„+++ Glycogen „ „ „ „ „ „ v „ „ b-Gentobiose + + v + + + + + „ D-Turanose + + + + v + + + „ D-Lyxose„„„„„„„„„ L-Fucose „ + v „ „ v v „ „ D-Arabitol „ „ „ „ „ v „ „ „ 2-Keto-gluconate „ „ „ „ „ „ v „ „ 5-Keto-gluconate „ „ „ „ „ „ „ + „ Methyl-D-mannoside „ + + „ „ „ + + „ Methyl-D-glucoside++++v++++

a Pedobacter sp. strain OM-E81 was characterized with APIZYM and API50CH as described by Steyn et al.14) Data from Steyn et al. and Takeuchi and Yokota were used to describe characteristics of P. heparinus Ia1, P. africanus Ia3, P. piscium, P. saltans III, and Pedobacter sp. strains Ia2, Ib1, Ib2, and II.14,15) Only characteristics that allowed us to diŠerentiate species in the genus of Pedobacters are given. See the text for features shared by the genus. +, Positive; „, negative; v, variable.

Similarly, B. pumilus OM-F6, S. multivorum OM- it is plausible that the detoxiˆcation of tolaasin in- A8, and Pedobacter sp. strain OM-E81 detoxiˆed volves speciˆc mechanisms operating exclusively in a tolaasin as e‹ciently as strains of Mycetocola group of bacteria rather than a generalized pro- (Fig. 3). In contrast, Acinetobacter sp. strain OM- teolytic processes. H10 was the least e‹cient (Fig. 3). With the excep- Suppression of the occurrence of brown blotch tion of B. pumilus OM-F6, tolaasin-detoxifying symptom on cultivated P. ostreatus by tolaasin- bacteria did not produce extracellular protease, but detoxifying bacteria was semiquantitively evaluated. about 20z of the bacteria that did not detoxify Suppression of the symptom was well correlated with tolaasin showed some proteolytic activity. Therefore, their ability to detoxify tolaasin (Fig. 3). For exam- 2206 T. TSUKAMOTO et al.

Fig. 4. Suppression of the Brown Blotch Disease by Tolaasin- detoxifying Bacteria. Fig. 3. Tolaasin Detoxiˆcation and the Suppression of the Panel A＝P. tolaasii,PanelB:P. tolaasii+Acinetobacter sp. BrownBlotchbyM. saprophilus CM-01 (01), M. tolaasinivo- strain OM-H10, Panel C＝P. tolaasii+S. multivorum OM-A8, rans CM-05 (05) and M. lacteus CM-10 (10) Isolated from Culti- Panel D＝a buŠer control. Lane 1–3: potato tuber slice, A. vated P. ostreatus,andM. lacteus OM-A1 (A1) and M. bisporus fruit body, and P. ostreatus fruit body, respectively. tolaasinivorans OM-F11 (F1), S. multivorum OM-A8 (A8), M. tolaasinivorans OM-F11, M. lacteus OM-A1, Pedobacer sp. Pedobacter sp. Strain OM-E81 (E8), Acinetobacter sp. Strain strain OM-E81 and B. pumilus OM-F6 also suppressed the dis- OM-H10 (H1) and B. pumilus OM-F6 (F6) Isolated from Wild ease development to the same extent as S. multivorum OM-A8. Agaricales. See the text and the legend of Fig. 3 for the assay conditions. PSB-Tol without detoxifying bacteria was also applied as a control (Cn) (see below). The bar diagram denotes the level of relative tolaasin activity, the degree of blackening in a potato ‰uorescens, have been recognized as potential biolog- tuber slice scored from 0 (no blackening) to 8 (complete blacken- ical control agents antagonistically suppressing the ing) as described elsewhere.9) Solid bar＝6 h incubation of bac- 29–31) teria in PS-Tol, shaded bar＝12 h incubation of bacteria in PSB- incoming population of P. tolaasii. This ap- Tol. The extent of brown blotch development on P. ostreatus proach, however, may call for precautions, because fruit bodies was examined visually after 48 h of incubation and P. ‰uorescens produces a number of antifungal scored from—(no symptoms) to +++ (completely blotched) as 9) agents and may become an opportunistic pathogen to described previously. cultivated mushrooms.32) This study showed that various non-pseudomonad bacteria capable of detoxifying tolaasin are also asso- ple, inoculation of either M. tolaasinivorans OM- ciated with fruit bodies of wild Agaricales fungi, F11, M. lacteus OM-A1 or CM-10, M. saprophilus waiting for further studies on their symbiotic or para- CM-01, S. multivorum OM-A8, Pedobacer sp. strain sitic roles. The fact that tolaasin-detoxifying bacteria OM-E81, or B. pumilus OM-F6 reduced the occur- were released into water from fungal fruit bodies rence of brown blotch induced by P. tolaasii to the without disrupting the fungal cells indicates that same extent as M. tolaasinivorans CM-05, unlike these bacteria are attached to the surface of fungal Acinetobacter sp. strain OM-H10 (Fig. 3). Similarly, mycelia like P. tolaasii rather than residing in the tolaasin-detoxifying bacteria suppressed the develop- fungal protoplasm.33) Burkholderia, an rRNA group ment of disease symptoms in cultivated A. bisporus, II pseudomonad, was recently recognized inside of as well (Fig. 4). spores of arbascular-mycorrhizal fungi, the biological contribution of which to host fungi has been yet Conclusions unknown.34) It may be interesting for further research Studies done to date on the interaction between development to explore such endosymbiotic bacteria Agaricales fungi and bacteria have been focused on that could detoxify tolaasin in Agaricales fungi and the species of one bacterial genus, i.e., Pseudomo- confer immunity to the bacterial disease. nas, as pathogens of cultivated mushrooms, as sym- Previously, we reported that M. tolaasinivorans bionts mediating a mycorrhizal association between isolated from cultivated P. ostreatus detoxiêd tolaa- fungi and plants, as agents inducing basidiomes, and sin and prevented brown blotch disease under our as parasites of yet unknown functions.1–3,18–28) Be- laboratory assay conditions.9) Consistent isolation of cause of their parasitism on mushrooms, some various species of Mycetocola from both wild and species of pseudomonads, such as Pseudomonas cultivated P. ostreatus suggests that the genus Tolaasin-Detoxifying Bacteria from Wild Agaricales Fungi 2207 Mycetocola has a niche for association with this mushroom pathogen Pseudomonas tolaasii Paine. J. Agaricales fungus and could have a great potential Am. Chem. Soc., 113, 2621–2627 (1991). for a biocontrol agent against the bacterial disease of 7) Rainey,P.B.,Brodey,C.L.,andJohnstone,K., not only cultivated P. ostreatus but also A. bispo- Identiˆcation of a gene cluster encoding three high- rus.9,10) We have, however, had di‹culty in analyzing molecular-weight proteins, which is required for synthesis of tolaasin by the mushroom pathogen Pseudo- the mechanisms of tolaasin detoxiˆcation in Myceto- monas tolaasii. Mol. Microbiol., 8, 643–652 (1993). due to the lack of molecular genetic techniques cola 8) Shirata, A., Sugaya, K., Takasugi, M., and Monde, for use in this group of Gram-positive bacteria. In K., Isolation and biological activity of toxins pro- view of this situation, it is meaningful for the duced by a Japanese strain of Pseudomonas tolaasii, research development in future that we have identi- the pathogen of bacterial rot of cultivated oyster êd species of Acinetobacter, Bacillus, Pedobacter, mushroom. Ann. Phytopathol. Soc. Japan, 61, and Sphingobacterium as tolaasin-detoxifying 493–502 (1995). agents, for some molecular genetic tools are currently 9) Tsukamoto, T., Shirata, A., and Murata, H., Isola- available for these bacteria. Information about tion of a Gram-positive bacterium eŠective in sup- tolaasin-detoxifying bacteria may also be useful in pression of brown blotch disease of cultivated the aspect of plant disease control since some mushrooms, Pleurotus ostreatus and Agaricus bisporus,causedbyPseudomonas tolaasii. 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