Carot-4-en-9,10-Diol, a Conidiation-Inducing Sesquiterpene DiolProduced by Trichoderma virens PS1-7 upon Exposure to ChemicalStress from Highly Active Iron Chelators

Mengcen Wang, Makoto Hashimoto, Yasuyuki Hashidoko

Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan

To screen biocontrol agents against Burkholderia plantarii, the causative agent of rice seedling blight, we employed catechol, ananalog of the virulence factor tropolone, to obtain chemical stress-resistant microorganisms. The fungal isolate PS1-7, identifiedas a strain of Trichoderma virens, showed the highest resistance to catechol (20 mM) and exhibited efficacy as a biocontrol agentfor rice seedling blight. During investigation of metabolic traits of T. virens PS1-7 exposed to catechol, we found a secondarymetabolite that was released extracellularly and uniquely accumulated in the culture. The compound induced by chemical stressdue to catechol was subsequently isolated and identified as a sesquiterpene diol, carot-4-en-9,10-diol, based on spectroscopicanalyses. T. virens PS1-7 produced carot-4-en-9,10-diol as a metabolic response to tropolone at concentrations from 0.05 to 0.2mM, and the response was enhanced in a dose-dependent manner, similar to its response to catechol at concentrations from 0.1to 1 mM. Some iron chelators, such as pyrogallol, gallic acid, salicylic acid, and citric acid, at 0.5 mM also showed activation of T.virens PS1-7 production of carot-4-en-9,10-diol. This sesquiterpene diol, formed in response to chemical stress, promotedconidiation of T. virens PS1-7, suggesting that it is involved in an autoregulatory signaling system. In a bioassay of the metabolicand morphological responses of T. virens PS1-7, conidiation in hyphae grown on potato dextrose agar (PDA) plates was eitherpromoted or induced by carot-4-en-9,10-diol. Carot-4-en-9,10-diol can thus be regarded as an autoregulatory signal in T. virens,and our findings demonstrate that intrinsic intracellular signaling regulates conidiation of T. virens.

In 1985, it was first reported that an Omigawa isolate of Pseu-domonas sp. caused rice seedling blight in nursery boxes in

Chiba Prefecture, Japan (1). This isolate, initially named Pseu-domonas plantarii, was reclassified into the genus Burkholderia in1994 (2, 3), and tropolone produced by Burkholderia plantarii wascharacterized as the virulence factor responsible for rice seedlingblight (2).

Tropolone-type compounds possess a unique seven-memberaromatic ring system with 1-keto-2-hydroxy and other substitu-tions; several natural products, such as stipitatic acid, colchicine,and hinokitiol, contain this unique moiety (4, 5). As a nonben-zenoid aromatic compound, purified tropolone possesses uniqueproperties as a phenol and a highly active iron chelator (6, 7).Before discovery of its association with symptoms of B. plantarii-caused rice seedling blight, tropolone was defined as a potent an-tibiotic toward various bacteria and fungi (8, 9). Afterwards, it wasfound that tropolone exhibited cytotoxicity on plants— e.g., inhi-bition of ethylene production of excised peach seeds (10), as wellas inhibition of some enzymes, such as grape polyphenol oxidase(11), mushroom tyrosinase (12, 13), and metalloproteases ofTyrophagus putrescentiae and Dermatophagoides farinae (14). Inaddition, this compound and its derivatives reportedly inhibitgrowth of human and murine cell lines (5) and of methicillin-resistant Staphylococcus aureus (15). These reports indicate thatthe virulence of tropolone in rice seedlings can be attributed to itspotent cationic metal-chelating effect (15, 16).

As far as we know, a practical manner of controlling B. planta-rii-caused rice seedling blight using sterilization of rice seeds withchemical pesticides is sometimes ineffective and often environ-mentally unfriendly (17). B. plantarii infects rice either preemer-gence or postemergence as a seed-borne pathogen, and chemicalbactericides are rarely effective for controlling this disease (18).

Furthermore, almost all rice seedlings in Japan are grown for ma-chine transplanting in nursery boxes under well-controlled envi-ronments with relatively high temperature and humidity (2),which aggravates emergence of this disease and makes its controldifficult. It is thus desirable to identify biocontrol agents that willbe effective in preventing this disease. Tropolone is potently toxictoward a wide spectrum of bacteria and fungi (9, 10). Thus, mi-croorganisms suitable for biocontrol of tropolone-responsive riceseedling blight caused by B. plantarii should be capable of survivaland growth in the presence of tropolone.

To obtain candidate microorganisms for practical biocontrolof rice seedling blight, we first screened microorganisms from therhizosphere of paddy rice. Under selection for microorganismsresistant to chemical stress from catechol, which has iron-chelat-ing properties, a fungus resistant to high levels of catechol wasselected and its efficacy in biocontrol was tested. During furtherinvestigation of its metabolic traits, we found that its productionof a sesquiterpene was uniquely enhanced upon exposure to eithercatechol or tropolone at appropriate concentrations. In this arti-cle, we describe isolation and identification of the catechol-resis-tant fungus and of the sesquiterpene produced by this fungus and

Received 15 November 2012 Accepted 6 January 2013

Published ahead of print 11 January 2013

Address correspondence to Yasuyuki Hashidoko, yasu-h@abs.agr.hokudai.ac.jp.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03531-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03531-12

The authors have paid a fee to allow immediate free access to this article.

1906 aem.asm.org Applied and Environmental Microbiology p. 1906–1914 March 2013 Volume 79 Number 6

on August 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

further focus on its role as an autoinducer of morphodifferentia-tion induction in this fungus.

MATERIALS AND METHODSSampling sites and preparation of soil samples. Soil samples (5 to 10 geach) were collected from the rice rhizosphere in triplicate at six paddyfield sites (total of 18 samples) in Hokkaido, Japan, after the harvest pe-riod in late October 2010. A small portion of each soil (10 mg, two repli-cates for each soil sample) was suspended in a sterilized 18-cm test tubecontaining 10 ml of water sterilized on a Milli-Q Advantage-A10 system(Millipore, MA) and then vortexed for 1 min until the soil was evenlysuspended. The soil suspension was left to stand for 15 min, after which 50�l of the supernatant was used as an inoculant.

Incubation and isolation of culturable rhizosphere microorgan-isms. Potato dextrose agar (PDA: 1� potato dextrose broth [PDB], pH6.2, solidified with 1.5% powdered agar) was used as a culture medium.The inoculant was spread evenly onto PDA plates with a glass spreaderand incubated for 14 days at 25°C in the dark. All distinguishable bacterialcolonies were isolated and were purified by streaking on fresh PDA plates.Fungi were isolated by cutting a hyphal plug from the margin of themycelium and subculturing it on fresh PDA plates. These isolated mi-crobes were used for screening chemical stress-resistant biocontrol agents.

Screening of microorganisms resistant to iron chelators. Potent re-sistance to tropolone, which is cytotoxic due to its iron-chelating proper-ties, was set as the screening criterion for identification of candidate bio-control agents for rice seedling blight. To screen for iron chelator-resistantmicroorganisms, we employed a simple analog of tropolone, catechol,which has relatively low cytotoxicity for bacteria and fungi (19–21). Cat-echol (Tokyo Chemical Industry, Tokyo, Japan) was added as a supple-ment to PDA at 0.2, 1, 5, 10, 15, and 20 mM. The initial screening was doneon PDA plates containing 0.2 mM catechol, from which isolates that ob-viously grew after a 14-day incubation were selected and further screenedon PDA plates containing a higher concentration of catechol (1, 5, 10, 15,and 20 mM). Catechol-resistant strains were preserved in 10% glycerolsolution at �80°C. PS1-7, the fungal isolate most resistant to catechol (20mM), was selected for identification and bioassay.

Identification of fungus PS1-7. PS1-7 was identified by two methods:by the morphology of its mycelia and conidia and through the sequencesof its internal transcribed spacer (ITS) and 5.8S rRNA gene. Characteris-tics of hyphae, phialide arrangement, and conidia were observed under anOlympus IX70 light microscope (Olympus, Tokyo, Japan) at magnifica-tions of 60 to 300�.

To obtain fresh mycelia for extraction of genomic DNA, PS1-7 wasshake-cultured in 50 ml of PDB medium for 2 days at 110 rpm at 25°C inthe dark. Mycelia collected from 1.5 ml of the culture by a brief centrifu-gation were washed with Milli-Q water several times and then transferredto 2-ml Eppendorf tubes filled with zirconia beads and then frozen inliquid nitrogen. The mycelia in the tube were disrupted in a Multi-beadsshocker (Yasui Kikai Co., Osaka, Japan) at 3,000 rpm for 2 min. DNA wasextracted from the disrupted mycelia using an Isoplant II DNA kit (Nip-pon Gene, Toyama, Japan). Using the resulting DNA as the template, theITS region was amplified by PCR using a pair of universal primers (for-ward ITS 1, 5=-TCCGTAGGTGAACCTGCGG-3=; and reverse ITS 4, 5=-TCCTCCGCTTATTGATATGC-3=) as reported before (22). The PCRamplicon (600 bp) was sequenced using a BigDye Terminator v 3.1 cyclesequencing kit (Applied Biosystems, Tokyo, Japan) with primer ITS 4according to the protocol recommended for an ABI Prism 310 geneticanalyzer (Applied Biosystems, CA).

Biocontrol assay. Burkholderia plantarii, originally isolated from arice seedling infested with blight, was a kind gift from Yuichi Takikawa(Faculty of Agriculture, Shizuoka University) and Kumiai Chemical In-dustry Co., Ltd. (Tokyo, Japan). We confirmed its production of tropo-lone and the sequence of its 16S rRNA gene. The culture was preserved in10% glycerol solution at �80°C and routinely grown on PDA plates. Totest the biocontrol efficacy of Trichoderma virens PS1-7 on rice seedlings

infested with B. plantarii, healthy rice seeds (Oryza sativa cv. Koshihikari)were placed in 1% NaCl solution and sterilized with 70% ethanol for 2min and then surface sterilized with 2% NaClO for 30 min and thoroughlywashed with sterile distilled water. The surface-sterilized seeds were inoc-ulated with B. plantarii by soaking them in a petri dish containing 10 ml ofbacterial cell suspension (106 CFU ml�1). The seeds were simultaneouslyinoculated with 100 �l of a conidial suspension of T. virens PS1-7 (106

conidia ml�1). Surface-sterilized rice seeds incubated with B. plantariionly (control), PS1-7 only (blank 1), or neither B. plantarii nor PS1-7(blank 2) were also prepared.

All seeds were incubated at 25°C for 2 days until germination. Weselected seeds at an early stage of germination and transplanted them intoa 6-cm-high glass dish (40 seeds per dish) containing 25 ml of Hoagland’sno. 2 solution solidified with 0.3% gellan gum (Wako, Osaka, Japan).After a 5-day incubation in a plant growth incubator (25°C, 12-h photo-period), we measured the lengths of the stem and root as parameters ofgrowth performance of the seedlings in each dish to assess the efficacy ofthe biocontrol treatment.

Semiquantitation and precise quantification of a secondary metab-olite produced by T. virens PS1-7 upon exposure to catechol. To mon-itor the metabolic traits of T. virens PS1-7 with exposure to 0.5 mM cate-chol, we inoculated 50 �l of PS1-7 conidial suspension (106 conidia ml�1)into 5 ml of PDB containing 0.5 mM catechol in an 18-cm test tube. For aseries of experiments, we prepared 18 or more of the culture tubes, and thetubes inoculated with T. virens PS1-7 were shake-cultured at 110 rpm at25°C in the dark. Three culture tubes were harvested at 24, 36, and 48 h orlater in some experiments. The entire culture medium harvested wastransferred into a 15-ml Falcon tube and centrifuged at 10,000 � g for 5min. The resulting supernatant (4 ml) was pipetted to an 18-cm test tube,and the pH was adjusted to 3.5 to 4.0 with HCl, at which point 1.5 ml ofethyl acetate (EtAOc) was added and the mixture was vigorously vortexedfor 1 min. For semiquantitation, a portion of the organic layer (5 �l) wasapplied using a volumetric glass capillary tube to a Kieselgel 60 GF254 silicagel thin-layer chromatography (TLC) plate (0.25 mm; Merck, Darmstadt,Germany) and developed in EtOAc-hexane (3:2 [vol/vol]). The TLC platewas sprayed with vanillin-H2SO4 reagent for detection.

For precise quantification of this chemical stress-responsive metabo-lite, a DB-1 capillary column (30 m by 0.25 mm; J&W Scientific, Folsom,CA) was installed on a GC-2025 gas chromatograph (Shimadzu, Kyoto,Japan) equipped with a flame ionization detector and using ultrapurehelium (99.999%) as the carrier gas. The oven temperature was initiallyheld at 120°C for 5 min and raised to 270°C at a rate of 10°C min�1. Thetemperatures of the injector and detector were set to 250°C and 280°C,respectively. The chemical stress-responsive metabolite was diluted inmethanol containing 100 �M m-tert-butylphenol as the internal standardinto a series of solutions of 1, 10, 50, 100, and 1,000 �M, from each ofwhich 2 �l was injected into the gas chromatograph. Peaks of the chemicalstress-responsive metabolite and m-tert-butylphenol were detected at tR

(times of retention) of 15.6 and 7.8 min, respectively. The peak intensity ofthe chemical stress-responsive metabolite relative to that of the internalstandard showed a linear relationship of y � 50.0x � 26.7 (r2 � 0.989),where y is the concentration of the metabolite (in �M) in culture fluid andx is the peak intensity ratio (ratio of the metabolite to internal standard).

Isolation and identification of the chemical stress-responsive me-tabolite. For isolation of the chemical stress-responsive metabolite, large-scale culturing of T. virens PS1-7 was done in 1,500 ml of PDB containing0.5 mM catechol. The PS1-7-inoculated medium was shake-cultured at110 rpm at 25°C in the dark. After a 3-day incubation, the culture mediumwas centrifuged at 10,000 � g for 10 min and then filtered through no. 101filter paper (Advantec, Tokyo, Japan). The culture filtrate was adjusted topH 3.5and then extracted exhaustively with EtOAc (500 ml � 3). Theorganic layer was combined and dried over anhydrous Na2SO4 and thenconcentrated under low pressure. The concentrates thus obtained (547mg) were resuspended in hexane-EtOAc (95:5 [vol/vol]) and then sepa-rated by chromatography on a GF60 silica gel column (50 g, 35-to-70

Conidiation Induction in Trichoderma by Sesquiterpene

March 2013 Volume 79 Number 6 aem.asm.org 1907

on August 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

mesh; Merck) with stepwise elution at 5 to 100% EtOAc in n-hexane.Fractions containing the chemical stress-responsive metabolite (thoseeluted with 25% and 30% EtOAc in n-hexane) were combined (113 mg),purified by preparative TLC, and recrystallized in chloroform to afford47.3 mg of colorless needles.

Field desorption mass spectroscopy (FD-MS) and electron ionizationMS (EI-MS) were conducted with a JEOL JMS-T100GCV and a JMS-SX-102, respectively. One-dimensional nuclear magnetic resonance (1D-NMR) and 2D-NMR were conducted using a JEOL JNM-EX270 and aBruker AM 500, respectively. The spectroscopic data of the isolated com-pound are as follows: FD-MS and FD-HR-MS, [M]� at m/z 238.1936(C15H26O2, calculated 238.1932) (see Fig. S2 in the supplemental mate-rial). EI-MS at m/z (relative intensity [%]), 238 (13%, [M]�), 220 (11%,[M-H2O]�), 202 (12%, [M-2H2O]�), 195 (98%, [M-Me2CH]�), 177(100%), 159 (69%), 123 (40%), 107 (42%), 93 (42%), and 43 (88%). Thecompound was acetylated with Ac2O-pyridine at 60°C and yielded amonoacetylated compound as a colorless syrup (24 mg, 68% yield). 1HNMR (nondecoupling [NON], H-H correlation spectroscopy [COSY],and nuclear Overhauser effect spectroscopy [NOESY]) and 13C-NMR (bi-level complete decoupling [BCM], distortionless enhancement by polar-ization transfer [DEPT], heteronuclear multiple-quantum correlation[HMQC], and heteronuclear multiple-bond correlation [HMBC]) spec-tra of the monoacetylated compound were taken in CDCl3 to confirm itschemical structure, including relative configuration.

Carot-4-en-9,10-diol production in T. virens PS1-7 exposed to cat-echol, tropolone, and other antifungal iron chelators. In a time courseexperiment, shake cultures of T. virens PS1-7 conidia in PDB containing0.5 mM catechol were sampled at 0, 12, 24, 48, 60, 72, 84, 96, 108, 120, 132,and 144 h after inoculation (2 ml each) and analyzed quantitatively bycapillary gas chromatography, as described above. T. virens PS1-7 wasinoculated into PDB containing different concentrations of catechol (0.1,0.5, 1, 2, or 5 mM) and cultured under the same incubation conditions toinvestigate the effect on carot-4-en-9,10-diol production. Samples of theculture medium (2 ml) were taken at 24, 48, 72, 96, 120, and 144 h forquantitative analyses.

Production of carot-4-en-9,10-diol by T. virens PS1-7 was examinedin the same manner. The metabolic effects of other iron chelators, includ-ing pyrogallol, gallic acid, citric acid, and salicylic acid, were also exam-ined in T. virens PS1-7. These chelators were tested at 0.5 mM; in addition,EDTA was tested, but at 0.2 mM, because it was toxic at 0.5 mM, as shownby prevention of hyphal growth. Cinnamic acid, which has neither anti-fungal nor iron-chelating properties, was used at 0.5 mM as a negativecontrol.

Cleanup of culture medium for analysis of carot-4-en-9,10-diol bygas chromatography. To analyze the carot-4-en-9,10-diol in the culturefluid, the culture medium (2 ml) taken at each sampling was centrifuged at10,000 � g for 5 min and then the resulting supernatant (1.5 ml) under-went solid-phase extraction (SPE) using a Sep-Pak C18 (3-ml Vac car-tridge) containing 200 mg resin (Waters, MA). Methanol (3 ml) and water(3 ml) were added successively in a vacuum chamber to a cartridge pre-conditioned with acetone (3 ml). The supernatant was then gently loadedand passed through the column under low pressure. The column was thenwashed with water (1 ml) to remove the void culture fluid in the columnand then eluted with methanol (2 ml). The methanolic eluates thus ob-tained were concentrated and redissolved in 150 �l methanol containing100 �M m-tert-butylphenol as an internal standard; 2 �l of this mixturewas injected into a gas chromatograph for quantification of carot-4-en-9,10-diol.

Tropolone dynamics in cultures of T. virens PS1-7. We analyzedtropolone dynamics in medium inoculated with PS1-7 to investigate thecapacity of PS1-7 to degrade tropolone. Authentic tropolone (Wako) wasdissolved in sterilized Milli-Q water to a 10 mM stock solution, fromwhich 100 �l was added to 5 ml of PDB in an 18-cm test tube. PDBcontaining 0.2 mM tropolone was inoculated with 50 �l of a PS1-7 conid-ial suspension (106 conidia ml�1); water was used in place of tropolone as

the control. The culture was shaken at 110 rpm at 25°C in the dark andsampled at 0, 12, 24, 48, 72, 96, and 120 h. To analyze tropolone dynamicsin the culture fluid, the culture (2 ml) was centrifuged at 10,000 � g for 5min and the resulting supernatant (1.5 ml) was subjected to solid-phaseextraction, as mentioned above. The methanolic elutes from the cartridgewere concentrated and redissolved in a volumetric 150-�l methanol, fromwhich 10 �l was injected into a high-performance liquid chromatograph(HPLC) for quantitative analysis of remaining tropolone.

For the HPLC system, an L-column2 ODS column (250 mm by 4.6mm; inside diameter [i.d.], 5 �m) was installed on a Waters 600 HPLCsystem (Waters, MA) equipped with a photodiode array detector (wave-length, 270 nm) and using 5% water–CH3CN containing 1 mMEDTA·2Na as the mobile phase. Standard solutions of tropolone (0.01,0.1, 0.2, 2, and 20 mM) were prepared as a serial dilution series of the stocksolution; 10 �l of each concentration was separated by HPLC, and then astandard curve was obtained, which fit the equation y � 0.0004x � 0.0289(R2 � 0.998), where y is the concentration of tropolone (in mM) and x isthe absolute peak intensity of tropolone.

Autoregulatory function of carot-4-en-9,10-diol. The autoregula-tory function (23) of carot-4-en-9,10-diol on T. virens PS1-7 myceliumwas tested by two experiments. For macroscopic observation, a 5-mm-diameter PSF-7 mycelial plug placed on a PDA plate was allowed to de-velop hyphae on a fresh PDA plate (at full strength, 1/4�, or 1/10� PDB)containing 10 �M carot-4-en-9,10-diol; controls were without carot-4-en-9,10-diol. After a 4-day incubation, formation of green concentriccircles of conidiophores was recognized.

For microscopic observation, 10 ml of PDA (containing 1/10� PDB)was impregnated with 100 �l of a conidial suspension (106 conidia ml�1).After a 24-h incubation, an 8-mm-diameter, thick paper disc loaded with50 �l of 10 �M carot-4-en-9,10-diol solution (in acetone) was placed onthe resulting plates containing uniformly distributed PS1-7 hyphae. Ace-tone (50 �l) was used as the control. After a 4-day incubation, conidiationwas observed along the PS1-7 hyphae around the paper disc under a lightmicroscope.

Nucleotide sequence accession number. The DNA sequence of iso-late PS1-7 has been deposited in the DNA Data Bank of Japan (DDBJ)under accession no. AB744653.

RESULTSCatechol-resistant microorganisms isolated from the paddyrice rhizosphere. We obtained a total of 186 culturable and mor-phologically distinguishable microbial isolates from rhizospheresoil samples collected from six paddy fields (Table 1). Upon expo-sure to 0.2 mM catechol and later stepwise increases in concentra-tion up to 20 mM, the number of surviving microbial isolatesdecreased (Fig. 1). Upon exposure to catechol beyond 10 mM, thenumber of resistant microbial isolates decreased to 12 bacterialisolates and 4 fungal isolates. Considering that the majority of themicrobial isolates were capable of surviving exposure to less than 5mM catechol, 10 mM catechol was likely above the threshold levelof significant toxicity. As a result, 16 microbial isolates (Fig. 1) thatsurvived at 10 mM were stored for further investigation. The mostcatechol-resistant fungal isolate, PS1-7, the only one that survivedexposure to 20 mM catechol, was investigated for its biocontrolefficacy against rice seedling blight and for relevant metabolictraits upon exposure to chemical stress mediated by iron chela-tion.

Hyphae of PS1-7 developed a flat white mycelium on PDAplates that, after a 4-day incubation at 25°C, formed light greenconidia in a concentric circle within the mycelium. Conidio-phores were hyaline and smooth walled, and the phialide waslageniform or ampulliform, with a length of 8 to 10 �m and a baseof 2 to 3 �m. Conidia were ovate or obovate with a diameter of 3

Wang et al.

1908 aem.asm.org Applied and Environmental Microbiology

on August 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

to 4 �m, with a rough surface. The conidial zone turned darkgreen with an amber background on PDA plates after a 14-dayincubation. These morphological characteristics agreed withthose of Trichoderma sp., particularly T. virens. By combiningthese observations with the sequence of an amplicon derived fromits ITS (accession no. AB744653), this fungal isolate, PS1-7, wastentatively identified as a strain of T. virens.

We observed suppression of rice seedling blight symptom de-velopment in rice seedlings infested with B. plantarii when in-fested seedlings were inoculated at germination with a conidialsuspension of T. virens PS1-7. In control plants not inoculatedwith T. virens PS1-7 conidia, typical symptoms of the disease, suchas chlorosis, stunting, and wilting, were obvious. In contrast, thesymptoms of plants inoculated with the conidial suspension wereobviously remedied and were followed by significantly improvedgrowth (Fig. 2A and B). No difference in the growth of seedlingsinoculated only with PS1-7 (Fig. 2C, blank 1) or those inoculatedwith neither B. plantarii nor PS1-7 (Fig. 2D, blank 2) was observed(Fig. 2C and D).

Identification and quantification of carot-4-en-9,10-diol as achemical stress-responsive metabolite of T. virens PS1-7. A dis-tinctive metabolite was detected as a chemical stress-responsivemetabolite in the culture medium of T. virens PS1-7 grown in thepresence of 0.5 mM catechol (see Fig. S1 in the supplemental ma-terial). Its chemical structure and relative configuration (rel.) were

elucidated to be (rel. 1S,7R,9R,10S)-carot-4-en-9,10-diol (see Ta-ble S1 and Fig. S2 to S4 in the supplemental material), which isidentical to those of a carotane-type sesquiterpene, CAF-603, iso-lated from another strain of Trichoderma virens (24).

Upon exposure to 0.5 mM catechol, carot-4-en-9,10-diol pro-duction in PS1-7 was maintained from 12 h to 72 h and reached amaximum of 45 �� at 72 h, which was approximately 2.5-fold thelevel in the control (Fig. 3A and B). However, a culturing timelonger than 72 h led to a loss of carot-4-en-9,10-diol production inmycelial cultures of T. virens PS1-7. Upon exposure to 0.1, 0.5, and1 mM catechol, carot-4-en-9,10-diol production in T. virensPS1-7 was maintained up to 72 h, and its maximum level wasenhanced in a dose-dependent manner by catechol (Fig. 3A).Upon exposure to tropolone, the metabolic response of T. virensPS1-7 was a dose-dependent enhancement of carot-4-en-9,10-diol production (Fig. 3C), similar to what was observed whentreated with catechol.

The similar response of T. virens PS1-7 to chemical stress fromcatechol or tropolone (Fig. 3C) seemed to be attributable to theiriron-chelating properties. As for catechol (see Fig. S1 in the sup-plemental material), degradation of tropolone in T. virens PS1-7-cultured PDB was also observed by HPLC analysis (Fig. 3D). Thisunique capacity of T. virens PS1-7 toward tropolone seems to behighly related to the biocontrol efficacy of this fungus.

Induction of conidiation in PS1-7 with carot-4-en-9,10-diol.Enhanced conidiation and maturation of conidia by supplementedcatechol were clearly shown as increased numbers of conidial rings inT. virens PS1-7 mycelia grown on PDA plates (Fig. 4). Conidiation ofPS1-7 mycelia grown on nutrient-poor PDA plates (1/4� or 1/10�PDB), which is inhibited under these nutrient conditions (see Fig. S5in the supplemental material), was restored by supplementation with10 �M carot-4-en-9,10-diol (Fig. 5). In addition, formation of con-idiophores and maturation of conidia were accelerated by supple-mentation with 10 �M carot-4-en-9,10-diol. Based on microscopicobservation, conidiophores and conidia clearly formed in T. virensPS1-7 hyphae growing near paper discs loaded with 11.9 �g carot-4-en-9,10-diol under nutrient-poor conditions (e.g., 1/10� PDA). Dif-ferentiation from hyphae to conidiophores was inhibited in T. virensPS1-7 hyphae in the control area (see Fig. S6 in the supplementalmaterial).

DISCUSSION

The rhizosphere, a characteristic habitat for microorganisms, is anatural source of functional microorganisms that are highly asso-ciated with host plant defense or are competitive or antagonistic

FIG 1 Microbial isolates tolerant to iron chelator selected on PDA plate con-taining catechol. The value on top of each column is the number of microbesthat survived and developed a colony on the selection plate.

TABLE 1 Sampling sites and catechol-tolerant microbial isolates

Paddy field (latitude, longitude)

Isolate(s)a

Fungal Bacterial

Shimamatsu-Kitahiroshima Kyoei (43°00.147=N, 141°34.434=E) PS1-7 PS1-1, PS2-3, PS2-8Eniwa Kitajima (42°58.314=N, 141°36.647=E) PK1-3, PK2-1Chitose Komasato (42°56.627=N, 141°38.400=E) PC1-3, PC1-25, PC2-15

Atsuma Kamiatsuma (42°38.692=N, 141°50.674=E) PAK1-2, PAK2-13

Atsuma Shinmachi (42°43.126=N, 141°52.704=E) PAS1-1, PAS2-11

Hayakita Hokusin (42°46.558=N, 141°49.961=E) PH1-6 PH1-12, PH2-11a Both the fungal and bacterial isolates listed are tolerant to 10 mM catechol.

Conidiation Induction in Trichoderma by Sesquiterpene

March 2013 Volume 79 Number 6 aem.asm.org 1909

on August 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

against soilborne phytopathogenic microorganisms (25–27). Dueto long-term survival in the rhizosphere microenvironment, themicrobial community that is dominant and cooperative in thehost root system within the complex food web (28) is regarded as

an appropriate source of biopesticidal agents effective in host pro-tection. T. virens PS1-7 was isolated from the rice rhizosphere as acatechol-resistant fungus and showed great promise as a biocon-trol agent in controlling rice seedling blight.

FIG 2 Biocontrol efficacy of T. virens PS1-7 on growth of rice seedlings inoculated with B. plantarii. Shown are comparisons of stem and root growth amongcontrol rice seedlings inoculated with B. plantarii (A), treated rice seedlings inoculated with both B. plantarii and T. virens PS1-7 (B), rice seedlings inoculatedwith T. virens PS1-7 only (blank 1) (C), and rice seedlings without any inoculation (blank 2) (D). Rice seedlings grown for 5 days at 25°C under a 12-hphotoperiod (A to D) were harvested, the lengths aboveground (stem growth) and of the root (root growth) of each seedling were recorded (right panel). Valuesare means � standard deviations (SD [shown by error bars]) (n � 40). *, P � 0.001 by Student’s t test.

FIG 3 Time course of carot-4-en-9,10-diol production and quenching in culture media of T. virens PS1-7. (A) Carot-4-en-9,10-diol was analyzed quantitativelyfor culture medium inoculated with T. virens PS1-7 containing catechol at 0, 0.1, 0.5, and 1 mM in triplicate at 24, 48, and 72 h. (B) Carot-4-en-9,10-diolproduction was quantified from cultures of T. virens PS1-7 in PDB containing 0.5 mM catechol (Œ) and in PDB without catechol (�). (C) Carot-4-en-9,10-diolwas analyzed quantitatively for culture medium inoculated with T. virens PS1-7 containing tropolone at 0, 0.05, 0.1, and 0.2 mM in triplicate at 24, 48, and 72 h.(D) Dynamics of supplemented tropolone (initial concentration 0.2 mM) in PDB inoculated with T. virens PS1-7 (Œ) or left uninoculated (�). Values aremeans � SD (shown by error bars) (n � 3).

Wang et al.

1910 aem.asm.org Applied and Environmental Microbiology

on August 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

During investigation of the metabolic traits of T. virens PS1-7exhibited in response to antifungal iron chelators in its culturemedium, we found that this fungus can degrade the virulencefactor tropolone as well as it can catechol (Fig. 3D). Some strainsof the genera Trichoderma and Penicillium resistant to tropoloneand catechol have shown potent degrading capacity toward trop-olone and catechol (29, 30). Decomposition of xenobiotics by thegenus Trichoderma was first reported in Trichoderma viride byBaarschers et al. in 1986 (31). A large portion of toxic xenobiotics,such as endosulfan (32), cyanide pollutants (33), dichlorvos (34),and even heavy metals (35), are removed from polluted environ-ments by the genus Trichoderma. The potent capacity of the genusto degrade various xenobiotics is due to both extracellular andintracellular metabolic pathways catalyzed by both specific and

nonspecific enzymatic systems. This cellular detoxification sus-tains the survival and growth of this fungus (36, 37).

In addition, we found that T. virens PS1-7 did not show theability to degrade either catechol or tropolone under carbon-poorconditions (unpublished data). Hence, it seems that T. virensPS1-7 metabolizes catechol and tropolone to detoxify them tofacilitate its survival and growth rather than to utilize such chem-icals as a carbon source. Whether such a tropolone-degradingability is necessary for a biocontrol agent to control rice seedlingblight is unclear. The use of T. virens in biocontrol of pathogenicfungal disease is mainly based on its antagonism against patho-gens via extracellular enzymes causative of mycoparasitism, secre-tion of antagonistic secondary metabolites, and cooperation withthe host plant by induction of systemic resistance and promotion

FIG 4 Conidiation of T. virens PS1-7 mycelia grown on PDA plates with exposure to catechol. Incubation was at 25°C for 5 days for all plates tested. Enhancedconidiation and maturation of conidia were observed as the morphological response of T. virens PS1-7 to exposure to 0.2 mM (center) and 1.0 mM (right)catechol added to a 1� PDA plate compared with the response of the control (without catechol addition).

FIG 5 Effect of carot-4-en-9,10-diol on conidiation of T. virens PS1-7 mycelia on PDA plates. (A, B, and C) PDA plates that contain 10 mM carot-4-en-9,10-diol;(D, E, and F) PDA plates without carot-4-en-9,10-diol. Conidiation of T. virens PS1-7 mycelia grown on PDA plates supplemented with 10 �M carot-4-en-9,10-diol (A, B, and C) was compared with conidiation on unsupplemented plates (D, E, and F). Plates A and D contained 10� diluted PD broth solidified with1.5% agar, while plates B and E contained the same mixture but 4� diluted. Red arrowheads indicate conidial rings on the mycelia. Incubation was at 25°C for5 days.

Conidiation Induction in Trichoderma by Sesquiterpene

March 2013 Volume 79 Number 6 aem.asm.org 1911

on August 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

of growth (38, 39). T. virens PS1-7 did not significantly affect thegrowth of rice seedlings (Fig. 2C) in our experiments. Hence, themechanism of efficacy of PS1-7 in biocontrol of rice seedlingblight is either antagonism against B. plantarii or degradation oftropolone.

Furthermore, we highlighted enhanced carot-4-en-9,10-diolproduction as a characteristic metabolic response of T. virensPS1-7 to catechol and tropolone; we regard this sesquiterpene as ametabolite responsive to chemical stress. Upregulation of sesquit-erpene biosynthesis has been reported for many sesquiterpenephytoalexins that are generally induced by chemical mediators,such as jasmonic acid, methyl jasmonate, and salicylic acid (40–42). We found that production of carot-4-en-9,10-diol in T. virensPS1-7 was enhanced by an array of compounds acting as ironchelators (Fig. 6; see Fig. S7 in the supplemental material),whereas it showed no response to cinnamic acid, which possessesneither antifungal nor iron-chelating properties. This on onehand indicates that carot-4-en-9,10-diol production in T. virensPS1-7 is an active metabolic response to chemical stress from anantifungal iron chelator and on the other hand indicates that T.virens PS1-7 can sense different antimicrobial iron chelatorsthrough a universal sensing system. G protein-coupled receptorsand/or nitrogen-sensing receptors spanning the cytoplasmicmembrane of hyphal cells of Trichoderma sp. specifically bind ex-tracellular chemical signals and stimuli, which is followed bytransmission of the chemical signal to intracellular signaling cas-cades (36). Hence, T. virens PS1-7 may sense structurally diverseiron chelators by a G protein-coupled receptor, leading to activa-tion of carot-4-en-9,10-diol biosynthesis.

T. virens PS1-7 also showed a morphological response to cate-chol, namely, promotion of conidiation and accelerated matura-

tion of the conidia, recognizable by their dark green pigmentation(Fig. 4). Conidiation leads to production and dispersal of prop-agules during an asexual stage in the life cycle of imperfect fungi(see Fig. S6 in the supplemental material), which are relativelyresistant to adverse environments (43), and hence, the promotionof conidiation in response to compounds like catechol likely facil-itates the survival of PS1-7. There seems to be a link between themetabolic and morphological responses, as shown by autoregula-tory signals acting in concert with environmental cues in regulat-ing a series of morphological events in filamentous fungi (21, 44).The discovery of this chemical stress response sheds light on thelink between carot-4-en-9,10-diol production and conidiation(Fig. 4 and 5).

Conidiation, as an outcome of normal cellular differentiationin fungi, is also repressed under nutrient-poor conditions due tothe reduction of cell competence to form conidia (45) (see Fig.S5A in the supplemental material). Similarly, radial growth of T.virens PS1-7 mycelium grown on nutrient-poor PDA plates wasunaffected (see Fig. S5B), but cellular differentiation recognizableas formation of vertical hyphae and subsequent conidiophore for-mation was drastically inhibited (Fig. 5; see Fig. S5B). This inhi-bition of conidiation was restored by supplementation with 10�M carot-4-en-9,10-diol, and under nutrient-rich conditions,carot-4-en-9,10-diol accelerated conidiophore formation andconidial maturation of T. virens PS1-7. This finding indicates thatcarot-4-en-9,10-diol is an autoregulatory signal molecule. Similarto our findings, sporogen AO1, an eremophilane-type sesquiter-pene produced in Aspergillus oryzae and a Penicillium sp., also hasan autoregulatory activity capable of exerting a sporogenic effect(46, 47).

Although the mechanism of autoregulatory signal regulationof this morphological event is not well understood (48), our find-ing that exogenously added carot-4-en-9,10-diol induces conidi-ation in T. virens PS1-7 is consistent with a hypothesis proposedby Nemcovic et al. for conidiation of Trichoderma atroviride (49).In their estimation, specific receptors on the plasma membranefor C8 volatile organic compounds that induce conidiation in T.atroviride transduce the signal from these compounds into conidi-ation regulation via the mitogen-activated protein and/or G pro-tein signaling pathways (49, 50). A complex autoregulatory systemrecently discovered in Aspergillus nidulans indicated the impor-tance of log P values (P is partition coefficient) of the signal com-pounds (51) and suggested that carot-4-en-9,10-diol of T. virensPS1-7 is thus capable of solely exerting autoregulatory functiondue to its high log P value (3.152; calculated with ACD/Labs soft-ware V11.02 in the SciFinder program). Hence, it is possible thatcarot-4-en-9,10-diol binds to a specific receptor on the hyphal cellmembrane that subsequently upregulates genes in the down-stream signaling cascade involved in conidiation of T. virens (42).

In conclusion, tropolone-resistant T. virens PS1-7 isolatedfrom the rice rhizosphere is a candidate biocontrol agent for riceseedling blight caused by B. plantarii. In combination with detox-ification of antifungal iron chelators, production of carot-4-en-9,10-diol as a metabolic response that mediates conidiation facil-itates the adaptation of T. virens PS1-7 to hostile environments.The autoregulatory signal molecule carot-4-en-9,10-diol inducedconidiation in PS1-7 mycelia, revealing that this compound trig-gers chemical stress-mediated conidiation in T. virens.

FIG 6 Production of carot-4-en-9,10-diol in T. virens PS1-7 upon expo-sure to several types of iron chelators. Production of carot-4-en-9,10-diolin the presence of 0.5 mM an iron chelator was compared with that in thecontrol after a 72-h incubation, except for with EDTA-Na� at used at aconcentration of 0.2 mM because the fungal cells did not grow well in itspresence at 0.5 mM. (E)-Cinnamic acid was used as a negative control at 0.5mM. Values are means � SD (shown by error bars) (n � 6). *, P � 0.0001by Student’s t test.

Wang et al.

1912 aem.asm.org Applied and Environmental Microbiology

on August 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

ACKNOWLEDGMENTS

We gratefully acknowledge Eri Fukushi (GC-MS and NMR Laboratory, Re-search Faculty of Agriculture, Hokkaido University) for helpful assistance inMS and NMR analyses. We thank Y. Takikawa (Shizuoka University) andKumiai Chemical Industry Co. Ltd. for providing us B. plantarii.

We are also grateful to the Chinese Scholarship Council for a scholar-ship (CSC 2010632028 to M.W.). This research work was financially sup-ported by a Grant-in-Aid for Scientific Research A (no. 20248033 to Y.H.)from the Japan Society for the Promotion of Science and by the METIProject, Japan (highly efficient gene design for microbial production ofinnovative biomaterials).

REFERENCES1. Azegami K, Nishiyama K, Watanabe Y, Suzuki T, Yoshida M, Nose K,

Toda S. 1985. Tropolone as a root growth-inhibitor produced by a plantpathogenic Pseudomonas sp. causing seedling blight of rice. Ann. Phyto-pathol. Soc. Jpn. 51:315–317.

2. Azegami K, Nishiyama K, Watanabe Y, Kadota I, Ohuchi A, FukazawaC. 1987. Pseudomonas plantarii sp. nov, the causal agent of rice seedlingblight. Int. J. Syst. Bacteriol. 37:144 –152.

3. Urakami T, Itoyoshida C, Araki H, Kijima T, Suzuki KI, Komagata K.1994. Transfer of Pseudomonas plantarii and Pseudomonas glumae toBurkholderia as Burkholderia spp. and description of Burkholderia vandiisp. nov. Int. J. Syst. Bacteriol. 44:235–245.

4. Dewar MJS. 1950. Tropolone. Nature 166:790 –791.5. Inamori Y, Tsujibo H, Ohishi H, Ishii F, Mizugaki M, Aso H, Ishida N.

1993. Cytotoxic effect of hinokitiol and tropolone on the growth of mam-malian-cells and on blastogenesis of mouse splenic T-cells. Biol. Pharm.Bull. 16:521–523.

6. Doering WVE, Knox LH. 1951. Tropolone. J. Am. Chem. Soc. 73:828–838.7. Zhao J. 2007. Plant troponoids: chemistry, biological activity, and biosyn-

thesis. Curr. Med. Chem. 14:2597–2621.8. Lindberg GD. 1981. An antibiotic lethal to fungi. Plant Dis. 65:680–683.9. Lindberg GD, Larkin JM, Whaley HA. 1980. Production of tropolone by

a Pseudomonas. J. Nat. Prod. 43:592–594.10. Mizutani F, Rabbany ABMG, Akiyoshi H. 1998. Inhibition of ethylene

production by tropolone compounds in young excised peach seeds. J. Jpn.Soc. Hortic. Sci. 67:166 –169.

11. Valero E, Garciamoreno M, Varon R, Garciacarmona F. 1991. Time-dependent inhibition of grape polyphenol oxidase by tropolone. J. Agric.Food Chem. 39:1043–1046.

12. Espin JC, Wichers HJ. 1999. Slow-binding inhibition of mushroom (Aga-ricus bisporus) tyrosinase isoforms by tropolone. J. Agric. Food Chem.47:2638 –2644.

13. Kahn V, Andrawis A. 1985. Inhibition of mushroom tyrosinase by trop-olone. Phytochemistry 24:905–908.

14. Morita Y, Matsumura E, Okabe T, Shibata M, Sugiura M, Ohe T,Tsujibo H, Ishida N, Inamori Y. 2003. Biological activity of tropolone.Biol. Pharm. Bull. 26:1487–1490.

15. Arima Y, Nakai Y, Hayakawa R, Nishino T. 2003. Antibacterial effect of-thujaplicin on staphylococci isolated from atopic dermatitis: relation-ship between changes in the number of viable bacterial cells and clinicalimprovement in an eczematous lesion of atopic dermatitis. J. Antimicrob.Chemother. 51:113–122.

16. Hendershott L, Gentilcore R, Ordway F, Fletcher J, Donati R. 1982.Tropolone: a lipid solubilizing agent for cationic metals. Eur. J. Nucl. Med.7:234 –236.

17. Adachi N, Tsukamoto S, Inoue Y, Azegami K. 2012. Control of bacterialseedling rot and seedling blight of rice by bacteriophage. Plant Dis. 96:1033–1036.

18. Hashidoko Y. 2011. Burkholderia plantarii, causative of rice bacterialseedling blight disease, is not killed but repressed its tropolone biosynthe-sis by avirulent B. mimosarum strain 901-5B. J. Pestic. Sci. 36:157.

19. Andjelkovic M, Van Camp J, De Meulenaer B, Depaemelaere G, So-caciu C, Verloo M, Verhe R. 2006. Iron-chelation properties of phenolicacids bearing catechol and galloyl groups. Food Chem. 98:23–31.

20. Azegami K, Nishiyama K, Kato H. 1988. Effect of iron limitation on“Pseudomonas plantarii” growth and tropolone and protein production.Appl. Environ. Microbiol. 54:844 – 847.

21. Kocacaliskan I, Talan I, Terzi I. 2006. Antimicrobial activity of catecholand pyrogallol as allelochemicals. Z. Naturforsch. C. 61:639 – 642.

22. Tamura R, Hashidoko Y, Ogita N, Limin SH, Tahara S. 2008. Require-ment for particular seed-borne fungi for seed germination and seedlinggrowth of Xyris complanata, a pioneer monocot in topsoil-lost tropicalpeatland in Central Kalimantan, Indonesia. Ecol. Res. 23:573–579.

23. Ugalde U. 2006. Autoregulatory signals in mycelial fungi, p 203–213. InKües U, Fischer R (ed), The Mycota, vol 1. Growth, differentiation andsexuality. Springer-Verlag, Berlin, Germany.

24. Watanabe N, Yamagishi M, Mizutani T, Kondoh H, Omura S, HanadaK, Kushida K. 1990. CAF-603: a new antifungal carotane sesquiterpene-isolation and structure elucidation. J. Nat. Prod. 53:1176 –1181.

25. Barea JM, Pozo MJ, Azcon R, Azcon-Aguilar C. 2005. Microbial co-operation in the rhizosphere. J. Exp. Bot. 56:1761–1778.

26. Compant S, Duffy B, Nowak J, Clement C, Barka EA. 2005. Use of plantgrowth-promoting bacteria for biocontrol of plant diseases: principles,mechanisms of action, and future prospects. Appl. Environ. Microbiol.71:4951– 4959.

27. Hashidoko Y. 2005. Ecochemical studies of interrelationships betweenepiphytic bacteria and host plants via secondary metabolites. Biosci. Bio-technol. Biochem. 69:1427–1441.

28. Denison RF, Bledsoe C, Kahn M, O’Gara F, Simms EL, Thomashow LS.2003. Cooperation in the rhizosphere and the “free rider” problem. Ecol-ogy 84:838 – 845.

29. Karetnikova EA, Zhirkova AD. 2005. Degradation of phenols formedduring lignin pyrolysis by microfungi of genera Trichoderma and Penicil-lium. Biol. Bull. 32:445– 449.

30. Smith RS, Cserjesi AJ. 1970. Degradation of nootkatin by fungi causingblack heartwood stain in yellow cedar. Can. J. Bot. 48:1727–1729.

31. Baarschers WH, Heitland HS. 1986. Biodegradation of fenitrothion andfenitrooxon by the fungus Trichoderma viride. J. Agric. Food Chem. 34:707–709.

32. Katayama A, Matsumura F. 1993. Degradation of organochlorine pesti-cides, particularly endosulfan, by Trichoderma harzianum. Environ. Toxi-col. Chem. 12:1059 –1065.

33. Ezzi MI, Lynch JM. 2005. Biodegradation of cyanide by Trichoderma spp.and Fusarium spp. Enzyme Microb. Technol. 36:849 – 854.

34. Tang J, Liu L, Hu S, Chen Y, Chen J. 2009. Improved degradation oforganophosphate dichlorvos by Trichoderma atroviride transformantsgenerated by restriction enzyme-mediated integration (REMI). Biores.Technol. 100:480 – 483.

35. Massaccesi G, Romero MC, Cazau MC, Bucsinszky AM. 2002. Cad-mium removal capacities of filamentous soil fungi isolated from industri-ally polluted sediments, in La Plata (Argentina). World J. Microbiol. Bio-technol. 18:817– 820.

36. Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Ken-erley CM, Monte E, Mukherjee PK, Zeilinger S, Grigoriev IV, KubicekCP. 2011. Trichoderma: the genomics of opportunistic success. Nat. Rev.Microbiol. 9:749 –759.

37. Subramanian V. 2008. Functional genomics of xenobiotic detoxifyingfungal cytochrome P450 system. Ph.D. thesis, University of Cincinnati,Cincinnati, OH.

38. John RP, Tyagi RD, Prevost D, Brar SK, Pouleur S, Surampalli RY.2010. Mycoparasitic Trichoderma viride as a biocontrol agent against Fus-arium oxysporum f. sp. adzuki and Pythium arrhenomanes and as a growthpromoter of soybean. Crop Prot. 29:1452–1459.

39. Mouria B, Ouazzani-Touhami A, Douira A. 2007. Effect of Trichodermastrains on the growth of tomato plants in greenhouses and their aptitudeto colonize roots and substrate. Phytoprotection 88:103–110.

40. He S, Lin W, Chen R. 2002. Induction of sesquiterpene cyclase geneexpression and antioxidant enzymes regulated by exongenous salicylicacid in leaves of Capsicum annuum. Chin. J. Appl. Ecol. 13:569 –572.

41. Malarz J, Stojakowska A, Kisiel W. 2007. Effect of methyl jasmonate andsalicylic acid on sesquiterpene lactone accumulation in hairy roots ofCichorium intybus. Acta Physiol. Plant. 29:127–132.

42. Yin SH, Mei L, Newman J, Back K, Chappell J. 1997. Regulation ofsesquiterpene cyclase gene expression— characterization of an elicitor-and pathogen-inducible promoter. Plant Physiol. 115:437– 451.

43. Carreras-Villaseñor N, Sánchez-Arreguín JA, Herrera-Estrella AH.2012. Trichoderma: sensing the environment for survival and dispersal.Microbiology 158:3–16.

44. Hogan DA. 2006. Talking to themselves: autoregulation and quorumsensing in fungi. Eukaryot. Cell 5:613– 619.

45. Adams TH, Wieser JK, Yu JH. 1998. Asexual sporulation in Aspergillusnidulans. Microbiol. Mol. Biol. Rev. 62:35–54. (Erratum, 62:545.)

Conidiation Induction in Trichoderma by Sesquiterpene

March 2013 Volume 79 Number 6 aem.asm.org 1913

on August 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from

46. Motohashi K, Hashimoto J, Inaba S, Khan ST, Komaki H, Nagai A,Takagi M, Shin-ya K. 2009. New sesquiterpenes, JBIR-27 and -28, iso-lated from a tunicate-derived fungus, Penicillium sp. SS080624SCf1. J.Antibiot. 62:247–250.

47. Tanaka S, Wada K, Katayama M, Marumo S. 1984. Isolation of sporo-gen-AO1, a sporogenic substance from Aspegillus oryzae. Agric. Biol.Chem. 48:3189 –3191.

48. Leeder AC, Palma-Guerrero J, Glass NL. 2011. The social network:deciphering fungal language. Nat. Rev. Microbiol. 9:440 – 451.

49. Calvo AM, Wilson RA, Bok JW, Keller NP. 2002. Relationship betweensecondary metabolism and fungal development. Microbiol. Mol. Biol.Rev. 66:447– 459.

50. Nemcovic M, Jakubíková L, Víden I, Farkaš V. 2008. Induction ofconidiation by endogenous volatile compounds in Trichoderma spp.FEMS Microbiol. Lett. 284:231–236.

51. Rodríguez-Urra AB, Jiménez C, Nieto MI, Rodríguez J, Hayashi H, UgaldeU. 2012. Signaling the induction of sporulation involves the interaction of twosecondary metabolites in Aspergillus nidulans. ACS Chem. Biol. 7:599–606.

Wang et al.

1914 aem.asm.org Applied and Environmental Microbiology

on August 27, 2019 by guest

http://aem.asm

.org/D

ownloaded from