Efficacy Assessment of Antifungal Metabolites from Chaetomium

Home , Chaetomium globosum, Setosphaeria, Setosphaeria turcica

Biological Control 64 (2013) 90–98

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Biological Control

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Efﬁcacy assessment of antifungal metabolites from Chaetomium globosum No.05, a new biocontrol agent, against Setosphaeria turcica

Guizhen Zhang a,1, Fengting Wang a,1, Jianchun Qin a, Di Wang a, Jingying Zhang b, Yanhua Zhang a, ⇑ Shihong Zhang a, Hongyu Pan a,

a College of Plant Sciences, Jilin University, Changchun, Jilin 130062, China b College of Environmental and Resource Sciences, Jilin University, Changchun, Jilin 130026, China

highlights graphical abstract

" One of the very few reports on biological control of northern corn leaf blight (NCLB). " Strain No.05 strongly reduce disease on detached maize leaves and on seedlings. " Two antifungal substances are obtained from strain No.05 by bioassay-guided isolation. " Chaetoglobosin A displays potent suppression of Setosphaeria turcica both in vitro and in planta.

article info abstract

Article history: Northern corn leaf blight (NCLB), an important and potentially destructive corn foliar disease, is caused Received 19 June 2012 by Setosphaeria turcica. The intent of this study was to evaluate antifungal metabolites from Chaetomium Accepted 16 October 2012 globosum (Cg) strain No.05 to suppress NCLB in maize. This strain significantly suppressed mycelial Available online 29 October 2012 growth of numerous phytopathogenic fungi especially S. turcica on potato dextrose agar medium. The secondary metabolites of the strain inhibited mycelial growth and conidial germination of S. turcica. Keywords: When co-inoculated at three droplets (5 lL/droplet) of conidial suspension (5 104 conidia/mL) on each Setosphaeria turcica 8-cm-long detached leaf, 20% culture filtrates completely suppressed disease incidence of northern corn Biological control leaf blight. The application of the culture filtrates at 2 h post-inoculation (hpi) of S. turcica in greenhouse Chaetomium globosum Culture filtrate studies showed a 81.9% inhibition of NCLB on the seedlings, while culture filtrates applied before path- Secondary metabolites ogen inoculation showed even higher rates of disease reduction. The application of the culture filtrates Chaetoglobosin had no observed effects on the treated maize leaves or seedlings. Two active compounds, isolated from the extracts, were identified as chaetoglobosin A and chaetoglobosin C based on the spectroscopic analysis. Both in vitro and in planta bioassay experiments showed that chaetoglobosin A displayed potent biocontrol efficiency against S. turcica. To the best of our knowledge, this is the first report of the evaluation of the inhibitory effects of C. globosum and chaetoglobosin A against S. turcica both in vitro and on detached maize leaves. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction

⇑ Corresponding author. Fax: +86 431 86758762. Northern corn leaf blight (NCLB), caused by the heterothallic E-mail addresses: [email protected] (G. Zhang), [email protected] (F. Wang), ascomycete, Setosphaeria turcica (Luttrell) Leonard and Suggs [ana- [email protected] (J. Qin), [email protected] (D. Wang), morph: Exserohilum turcicum (Pass.) Leonard and Suggs], is a major [email protected] (J. Zhang), [email protected] (Y. Zhang), foliar disease of maize (Zea mays L.) and is prevalent in most maize [email protected] (S. Zhang), [email protected] (H. Pan). 1 These authors contributed equally to this work and are considered co-ﬁrst production regions worldwide (Perkins and Pedersen, 1987; authors. Raymundo and Hooker, 1981; Zhang et al., 2012a). The disease

1049-9644/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.10.005 G. Zhang et al. / Biological Control 64 (2013) 90–98 91 can cause extensive defoliation during the grain-filling period, cell-free supernatant from the pellet. The cell-free supernatant was resulting in up to 50% or more grain yield loss (Perkins and Peder- passed through a 100-lm pore size filter to further exclude myce- sen, 1987; Raymundo and Hooker, 1981; Zhang et al., 2012a,b). lia and to obtain a filtrate which was used to test antifungal activ- Control of the disease depends mainly on the application of resis- ity and the efficiency of disease suppression in the following tant cultivars together with chemical fungicides. However, breed- bioassays (see below). ing of resistant varieties is time-consuming and does not keep up For disease suppression tests, conidia of S. turcica were har- with the development of new physiological races of the pathogen. vested in 20 mL sterilized 0.01% Tween-20 solution (v/v) by gently Meanwhile, the detrimental effects caused by the use of hazardous scraping fungal mycelia and spores from 7-d-old culture on oat- chemicals for disease control in crops have received increasing meal-tomato agar plates. The conidial suspensions were filtered attention worldwide, due to the facts that more pathogens have through three layers of cheesecloth to remove mycelial fragments become resistant to the used chemical fungicides and the resulted and agar, and adjusted the conidial concentrations of approxi- environmental pollution and ecological imbalances (Seebold et al., mately 5 104 conidia/mL for the following bioassays (see below). 2004; Soytong et al., 2005; Chouvenc et al., 2011; Zhang et al., For the fungal growth inhibition tests, ten phytopathogenic fungi 2012a). Thus, there is a great demand for new methods to supple- from our collections (Table 1) were grown on PDA at 26 ± 0.5 °C ment the existing disease management strategies to achieve better for 3-7 d, then the mycelial plugs were taken from the PDA plates NCLB control. and used in the assay as described below. Applications of biological control agents (BCAs) and their secondary metabolites are important strategies in agricultural control 2.2. Plant materials against plant diseases. With the increased interest in biological control, many BCAs such as Bacillus amyloliquefaciens (Fukumoto) Five common maize (Z. mays L.) cultivars, CI6502 (Pioneer), Priest, Bacillus vallismortis Roberts, Pseudomonas fluorescens M753 (Monsanto), XY335 (Pioneer), XY696 (Pioneer), and ZD958 (Flügge) Migula, Streptomyces globisporus (Krasilnikov) Waksman, (Henan academy of agricultural sciences, China) moderate or Chaetomium globosum Kunze, and Trichoderma harzianum Rifai highly susceptible to NCLB, were used in this study. All in vivo have been demonstrated to play important roles on biological studies were done with the moderate susceptible maize cultivar plant disease control in agriculture due to their potential biological Pioneer XY335, except in Section 2.7. All five maize cultivars seeds activity (Chen et al., 2009; Zhao et al., 2010; Krishnamurthy and were individually sterilized in 0.5% sodium hypochlorite solution Gnanamanickam, 1998; Prabavathy et al., 2006; Bressan, 2003; for 1 min, and rinsed with sterile distilled water. The sterilized Salman and Abuamsha, 2012; Li et al., 2011a,b; Vitale et al., seeds were placed on moistened absorbent paper in enclosed plas- 2011; Soytong et al., 2005). However, effective control of S. turcica tic dishes in an incubator at 26 ± 0.5 °C for 3 d. The germinating with BCAs has remained elusive. seeds were transplanted into 15 cm 16 cm (height diameter) Safer and more environmentally friendly methods, including plastic pots containing approximately 6 kg autoclaved local soil the use of natural substances would be favorably considered for [sand:loam = 1:5 (v/v)] and each pot contained five seeds. The pot- disease management by the public, politicians, and the scientific ted seeds were grown in a greenhouse at 25–30 °C with a 14 h- communities, particularly in those habitats where the use of chem- light/10 h-dark cycle. Leaves were sprayed with tap water twice icals is restricted or banned. To date, more than 200 compounds a day till runoff, and irrigated as needed. The seedlings were fertil- have been identified from Chaetomium spp. and some of the com- ized with 1 g ammonium sulfate per pot at 10 d post-transplant. pounds have been reported to possess significant biological activi- All seedlings were used for bioassay at the 4-leaf stage. ties, such as cytotoxic, enzyme inhibition, and antibiotic (Gunatilaka, 2006; Scherlach et al., 2010; Li et al., 2011b; Qin 2.3. Antagonism of strain No.05 to phytopathogenic fungi et al., 2009a,b; Yang et al., 2011a,b). However, the biological effects of secondary metabolites derived from Chaetomium spp. still re- The dual culture method (Li et al., 2011) was used to evaluate mained untested on NCLB control. the potential antagonisms of strain No.05 against several phyto- In this study, the control effect of NCLB by secondary metabo- pathogenic fungi. Each one of the phytopathogenic fungi and C. lites from C. globosum strain No.05 was investigated. The objectives globosum were co-cultured separately on a 9-cm Petri dish with of this study were: (1) to assess the antifungal activity of the cul- PDA medium at 26 ± 0.5 °C for 5–7 days. The distance between ture filtrates of strain No.05 on the mycelial growth of a variety the two inoculation sites (strain No.05 and other tested fungal of fungi, (2) to investigate the inhibitory efficiency of the secondary plug) on each plate was about 45 mm. In the inoculated control metabolites from strain No.05 against NCLB, (3) to identify the bio- plates, a 12-mm-diameter mycelial plug of a phytopathogenic logical active compounds from C. globosum, and (4) to assess the fungus was inoculated at the side of a PDA plate with sterile antifungal efficacy of the identified compounds against S. turcica 12-mm-diameter PDA plug placed at the edge. When mycelial both in vitro and on detached maize leaves. growth of a fungal isolate had reached the edge of an inoculated control plate, the non-colonized zone of the treatment plates between the two inoculation sites of strain No.05 and fungal plug 2. Materials and methods was measured. Each treatment contains triplicate Petri dishes and the experiment was repeated three times. 2.1. Microorganisms and culture conditions 2.4. Suppression of mycelial growth of S. turcica by culture filtrates of The strain of the fungus (strain No.05) was isolated from fresh strain No.05 barks of Ginkgo biloba L., a medicinal plant growing in Linyi City, Shandong Province, China. After growing on potato dextrose agar The effects of culture filtrate of strain No.05 on mycelial growth (PDA) at 25 °C for 4 days (d), the fresh mycelium of the fungal of S. turcica were determined in liquid culture in 500 ml flasks. The strain was inoculated to a liquid medium (pH 7.0) containing culture filtrates were incorporated into potato dextrose broth 30.0 g oatmeal, 20 g maltose, and 1000 mL water. Fermentation (PDB) to achieve a final concentrations of 0% (negative control), was carried out in 500 mL flasks with 200 mL medium by incubat- 0.1%, 0.2%, 0.5%, 1%, 5%, 10%, and 20% (v/v). Single mycelial plugs ing the flasks at 28 °C for 6 d on a rotary shaker at 200 rpm. Then (9-mm diameter) of S. turcica from 3-d-old PDA cultures were inoc- the culture broth was centrifuged (10,625g, 15 min) to separate the ulated into each flask. Each treatment contains three flasks. After 92 G. Zhang et al. / Biological Control 64 (2013) 90–98

Table 1 Antagonism of the Chaetomium globosum strain No.05 on selected phytopathogenic fungi in dual culture tests on PDA plates.

Phytopathogenic fungus Host of origin Origin Inhibition zone (mm)a Setosphaeria turcica Zea mays L. Baicheng, Jilin, China 28.21 ± 1.35a Coniothyrium diplodiella (Speg.) Sacc. Vitis vinifera Xian, Shanxi, China 27.90 ± 2.08a Colletotrichum glocosporioides (Penz.) Penz. and Sacc. Citrus reticulate Banco Guangyuan, Sichuan, China 27.18 ± 1.47a Valsa mali Miyabe et Yamada. Malus pumila mill Xian, Shanxi, China 26.89 ± 1.47a Ceratocystis ﬁmbriata Ellis et Halsted Ipomoea batatas Baoji, Shanxi, China 26.00 ± 1.22a Sclerotinia sclerotiorum (Lib.) de Bary Brassica campestris L. Baicheng, Jilin, China 25.64 ± 0.95a Rhizopus stolonifer Kuhn. Amygdalus persica Linn Changchun, Jilin, China 22.21 ± 1.17b Pellicularia sasakii (Shirai) Ito Oryza sativa Changchun, Jilin, China 22.07 ± 1.33b Cylindrocarpon destructans (Zins) Panax ginseng C.A. Mey Fusong, Jilin, China 20.70 ± 1.10b Fusarium oxysporum f. sp. vasinfectum (Atk) Snyder and hanson Gossypium spp. Changchun, Jilin, China 19.40 ± 1.59c

Data represent the means ± standard error (SE) from three independent experiments with triplicate plates examined for each treatment; different superscript letters in the same column indicate statistical significance (P<0.05) according to the Fisher’s Protected Least Significant Difference Test. a The inhibition zones were measured as the distance between the leading edges of two opposing colonies. incubation at 28 °C for 4 d, the mycelial mats were passed through growth chamber. The disease incidence and lesion areas were cal- dried and pre-weighed filter paper, dried at 65 °C for 2 d, and culated to determine the pathogenicity. Length and width of le- weighed again and the net mycelial mats weight was calculated sions were used to estimate the lesion areas using the formula: ðdcdtÞ as inhibition mycelial percent x ¼ ðdtdiÞ 100%, where dc is the (p A B)/4, where A and B refer to the maximum and the mini- mean mycelial weight of negative control sets, dt is the mean mum axis of a lesion, respectively. The disease incidence percent- mycelial weight of treatment sets, and di is the initial mycelial age were calculated by this formula: Disease incidence = (number weight of fungal PDA discs. Three independent experiments were of infection sites of treatment sets/number of inoculum sites of performed. treatment sets) 100%. Three independent experiments were performed with triplicate plates in each treatment. 2.5. Effect of culture filtrates of strain No.05 on germination of conidia of S. turcica 2.7. Biological control efficiency of Cg culture filtrate on detached different maize cultivars leaves Segments of onion (Allium cepa L.) surface were placed onto water agar (2%) plates and inoculated with 5 lL of conidial suspen- To test the effectiveness of Cg, culture filtrates were applied to sions containing 5 104 conidia/mL. The conidia were pre-mixed the susceptible or highly susceptible cultivars of different maize with the culture filtrates ranging in concentration from 0.1% to cultivars, except for Pioneer XY335. These investigations were car- 20% (v/v). Drops of Tween-20 were added at the final concentration ried out on four other maize cultivars, CI6502 (Pioneer), M753 of 0.01% (v/v). The onion segments that had been treated with (Monsanto), XY696 (Pioneer), and ZD958 (Henan academy of agricul- equal amount of conidial suspensions in 0.01% Tween-20 (v/v) tural sciences, China). Segments of each of the maize cultivar leaves solution were used as control. The conidial suspensions (5 104 - (8-cm long) were placed on separate wet paper towel placed on the conidia/mL) with or without 0.01% Tween-20 (v/v) were also ap- bottom of 15-cm Petri dish plates and three droplets (5 lL/droplet) plied in order to investigate the effects of 0.01% Tween-20 (v/v) of conidial suspension (5 104 conidia/mL) which had been pre- germination of conidia of S. turcica. Inhibition conidial germination mixed with the 1% (v/v) culture filtrate were inoculated. Conidial ðdcdtÞ 4 percent =x ¼ ðdtdiÞ 100%. dc is the mean conidial germination of suspensions (5 10 conidia/mL) containing 0.01% Tween-20 (v/ negative control sets; dt is the mean conidial germination of treat- v) but without the culture filtrate were used as positive controls. ment sets; di is the initial conidial germination. All the plates con- PDB medium without the culture filtrate and conidia but contain- taining the onion surface segments were incubated at 26 ± 0.5 °Cin ing 0.01% Tween-20 (v/v) was also dropped on the five different darkness with 100% relative humidity in a growth chamber for maize cultivars leaves and used as negative control. All the plates 20 h. For each replicate, at least 200 spores were counted, and each were incubated at 26 ± 0.5 °C for 72 h in darkness with 100% rela- treatment contained triplicate plates with three onion surface seg- tive humidity in a growth chamber. The disease incidence and le- ments in each plate. The experiment was repeated three times. sion areas were calculated to determine the pathogenicity. Length and width of lesions were used to estimate the lesion areas 2.6. Effect of the culture filtrate of strain No.05 on S. turcica using the formula: (p A B)/4, where A and B refer to the maxi- pathogenicity on detached maize leaves mum and the minimum axis of a lesion, respectively. The disease incidence percentage were calculated by the following formula: The fourth leaf of maize plants was used in the study. Segments Disease incidence = (number of infection sites of treatment sets/ of the detached Pioneer XY335 leaves (8-cm long) were placed on number of inoculum sites of treatment sets) 100%. Three inde- wet paper towel placed on the bottom of 15-cm Petri dish plates. pendent experiments were performed with triplicate plates in each The leaves were inoculated with three droplets (5 lL/droplet) of treatment. conidial suspension (5 104 conidia/mL) which had been pre- mixed with the culture filtrate at concentrations ranging from 2.8. Effect of the culture filtrate of strain No.05 on the infection process 0.1% to 20% (v/v). Conidial suspensions (5 104 conidia/mL) that on detached maize leaves contained 0.01% Tween-20 (v/v) but without the culture filtrates were used as positive controls. PDB medium without the culture In the preventive test, 8-cm long sections of the fourth maize filtrates and conidia but containing 0.01% Tween-20 (v/v) was also leaves were placed on wet paper towel placed on the bottom of dropped on maize leaves and used as negative control. The conidial 15-cm Petri dish plates and treated with the strain No.05 culture suspensions (5 104 conidia/mL) with or without 0.01% Tween-20 filtrate for 12 h before inoculation (hbi). In the curative tests, the (v/v) were also tested in order to investigate the effects of 0.01% culture filtrate amended with 0.01% Tween-20 (v/v) were sprayed Tween-20 (v/v) on S. turcica. All the plates were incubated at on leaves at 3, 6, 12, 24, or 48 h post inoculation (hpi). At least four 26 ± 0.5 °C for 72 h in darkness with 100% relative humidity in a leaf segments were placed in each plate of the triplicate plates and G. Zhang et al. / Biological Control 64 (2013) 90–98 93 each segment was inoculated with three droplets (5 lL/droplet) of mL) in PDB as described above in section 2.5. Each treatment con- conidial suspension (5 104 conidia/mL). In both the preventive tained triplicate plates. The minimal inhibitory concentration and curative tests, maize leaves inoculated with conidial suspen- (MIC) was recorded by reading the lowest concentration that sions containing 0.01% Tween-20 (v/v) but without the culture fil- inhibited visible growth of S. turcica. Effective concentrations for trate were used as positive controls. The leaves sprayed with the half inhibition of fungal mycelial growth (IC50) were calculated PDB containing only 0.01% Tween-20 (v/v) but without the culture according to the correlations between mycelial inhibition percent- filtrate and conidial suspension were used as negative controls. All age and concentrations of chaetoglobosin A (Leroux et al., 1999). the plates were incubated at 26 ± 0.5 °C with 100% relative humid- Three independent experiments were performed. ity and the disease incidence and severity were evaluated at 72 hpi. The disease incidence and lesion areas were calculated to deter- 2.12. Biocontrol efficiency of chaetoglobosin A against S. turcica on mine the pathogenicity. Length and width of lesions were used detached maize leaves to estimate the lesion areas using the formula: (p A B)/4, where A and B refer to the maximum and the minimum axis of a The biocontrol efficiency of chaetoglobosin A against S. turcica lesion, respectively. The disease incidence percentage were calcu- on detached maize leaves was determined as described above in lated by the following formula: Disease incidence = (number of Section 2.7, and 10 different concentrations of chaetoglobosin A infection sites of treatment sets/number of inoculum sites of treat- (0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 lg/mL) were used. PDB with ment sets) 100%. Three independent experiments were per- dimethylsulphoxide (DMSO) and 0.01% Tween-20 (v/v) but with- formed with triplicate plates in each treatment. out S. turcica conidia was used as mock control. The experiment was repeated three times. 2.9. Control of NCLB in greenhouse by the culture filtrates of strain No.05 2.13. General

Maize seedlings and the fungal inoculum were prepared as de- The active compounds have been isolated, purified, and charac- scribed above. In the preventive tests, both the culture filtrates and terized by means of FT-IR, Nuclear Magnetic Resonance (NMR) the fungicide control (tricyclazole, 750 lg/mL, 75% wettable pow- spectroscopy, and mass spectrometry. der, Sanonda Co., Ltd., China) amended with Tween-20 was The optical rotation of the compound was measured on a Per- sprayed on the 4-leaf stage maize seedlings (8 mL/pot). The maize kin-Elmer 141 polarimeter. The melting point was determined on seedlings sprayed with PDB containing Tween-20 instead of the an XRC-1 micro-melting point apparatus and was uncorrected. IN- culture filtrate were used as negative controls. After air-drying FRA-RED spectra were obtained with a Nexus 870 FT-IR with KBr for 24 h, the treated maize seedlings were sprayed with 8 mL of 1 4 pellets. H NMR spectra included Varian Inova 400 (399.95 MHz), conidial suspension (5 10 conidia/mL) of S. turcica per pot. The 13 500 (499.8 MHz), and 600 (600 MHz). C NMR spectra included maize seedlings were incubated in a growth chamber at Varian Inova 500 (125.7 MHz) and 600 (150.9 MHz). Chemical 26 ± 0.5 °C and covered with plastic bags for 3 d to maintain high shifts were measured using tetramethylsilane as an internal stan- humidity. In the curative tests, the culture filtrates and fungicide dard, and d was expressed in ppm. Mass spectra included EI-MS controls were sprayed on seedlings at 2 hpi, and the treated maize at 70 eV with Varian MAT 731, Varian 311A, AMD-402, and high seedlings were incubated under the same conditions as described resolution using perflurokerosine as a standard. Desorption chem- above. Each treatment contained three pots with five seedlings in ical-ionization mass spectrometry (Finnigan corporation; Type: each pot, and the experiment was repeated three times. MAT 95 A; Electron energy:200 eV; Reactant gas:NH3), Column chromatography was carried out on silica gel (200–300 mesh, 2.10. Bioassay-guided isolation of antifungal compounds Merck, German) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden). All solvents used in this study were analytical After incubation at 28 °C for 6 d, the culture broth (50 L) of grade. Fractions were monitored by TLC and spots were visualized strain No.05 was filtered to harvest culture filtrate. The entire cul- by heating silica gel plates sprayed with 10% H2SO4 in ethanol. ture filtrate was extracted three times with ethyl acetate and dried in vacuum to get the total crude extract (27.5 g). The crude extract showing antifungal activity observed by the agar-diffusion method 2.14. Data analysis (Yang et al., 2011a,b) was chromatographed on a silica gel by using the Thin-Layer Chromatography (TLC) assay and eluted with a gra- The incidence and severity of the disease were analyzed by the analyses of variance (ANOVA) using SPSS 13.0 software for Win- dient system of CH2Cl2/MeOH (50:1, 20:1, and 10:1). Five fractions were produced and designated as fractions A to E. Bioassay-guided dows (SPSS, China). Percentage data (i.e., conidial germination fractionation technique (Yang et al., 2011a,b) was used to isolate inhibition, mycelial growth inhibition, disease incidence and con- and identify the antifungal components against S. turcica from cul- trol efficacy) were assessed prior to ANOVA. Mean comparisons ture filtrate of strain No.05. The bioactive fraction of C was deter- were performed by Fisher’s Protected Least Significant Difference mined by antimicrobial screening. Fraction C was (LSD) test (P < 0.05). chromatographed over Sephadex LH-20 (CH2Cl2/MeOH = 6:4, V/V) and then Sephadex LH-20 (MeOH) successively. Then bioactive 3. Results subtractions C-3-2 were repeatedly chromatographed over silica column (CH2Cl2/MeOH = 6:4, V/V) and purified by reversed-phase 3.1. Antagonism of strain No.05 against selected phytopathogenic (ODS) column (MeOH/H2O = 5:5, V/V) to obtain the compounds 1 fungi and 2. Strain No.05 displayed significant inhibitory effects on the 2.11. Evaluation of the antifungal activity of chaetoglobosin A against mycelial growth of various plant pathogens in the in vitro assays. S. turcica in vitro The inhibition zones (Table 1) observed among the different phytopathogenic fungi ranged from 19.40 to 28.21 mm. That data shows The suppression of S. turcica by chaetoglobosin A was carried that strain No.05 exhibited the stronger inhibitory effect against out at nine concentrations (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 lg/ phytopathogenic fungi such as S. turcica, Coniothyrium diplodiella 94 G. Zhang et al. / Biological Control 64 (2013) 90–98

(Speg.) Sacc., Colletotrichum glocosporioides (Penz.) Penz. and Sacc., two treatments of 0% and 0.1% culture filtrate on the spore germi- Valsa mali Miyabe et Yamada., Ceratocystis fimbriata Ellis et Halsted, nation (Fig. 1B). However, with the increased concentration of the Sclerotinia sclerotiorum (Lib.) de Bary, compared with the inhibition culture filtrate ranging from 0.2% to 20%, the conidial germination of four other phytopathogenic fungi (Table 1). inhibition percentage was decreased from 95.0% to 2.6%, respectively. The conidial germination was almost completely inhibited when the spores were treated with the culture filtrate at a concen- 3.2. Suppression of mycelial growth of S. turcica by the culture filtrate tration of 20% (v/v) (Fig. 1B). of strain No.05

3.4. Effects of the culture filtrate of strain No.05 on NCLB development The inhibition efficiency on S. turcica mycelial growth in liquid culture was positively correlated with the concentration of culture The disease incidence was 100% and the average lesion area was filtrate of strain No.05. The average dry weight of S. turcica was 47.6 mm2 in the positive control. There was no significant differ- 5.11 mg/mL in the negative control. With the increased concentra- ence in the disease incidence among the treatments but the aver- tion of the culture filtrate ranging from 0.1% to 2%, the dry weight age lesion area decreased significantly from 31.2 to 1.7 mm2 with of S. turcica decreased significantly from 4.62 to 0.14 mg/mL and the increased culture filtrate concentration ranging from 0.1% to the growth inhibition percentage of S. turcica increased dramati- 10.0% (Fig. 2). Disease symptom was not observed on inoculated cally from 9.59% to 97.26% (Fig. 1A). When the concentration of maize leaves pre-treated with culture filtrate at the concentration the culture filtrate were increased to 5%, 10%, and 20%, much less of 20% (v/v) (Fig. 2). The negative effects were not observed when mycelia were observed in the culture medium and the growth inhi- the leaves were treated with the undiluted culture filtrates of bition of S. turcica was further increased to 98.04%, 99.41%, and strain No.05 (data not shown). 99.80%, respectively (Fig. 1A). The pH value of the medium showed no significant change from 5.50 to 5.86 (data not shown), suggest- 3.5. Evaluation of Cg culture filtrate against S. turcica on detached ing that the observed significant inhibitory effects of the culture fil- maize cultivar leaves trate on S. turcica were not caused by the pH changes.

No lesions were observed on the five different maize cultivars 3.3. Effects of the culture filtrate of strain No.05 on conidial CI6502 (Pioneer), M753 (Monsanto), XY335 (Pioneer), XY696 (Pio- germination of S. turcica neer), and ZD958 (Henan academy of agricultural sciences, China) leaves on negative controls. However, disease incidence of the po- The effects of the culture filtrate of strain No.05 on the conidial sitive controls and treatments of all these different cultivars leaves germination of S. turcica were examined on detached maize leaves. was 100% after incubation at 26 ± 0.5 °C for 72 h. When inoculated Results showed that 0.01% Tween-20 (v/v) was sufficient to pro- with 1% culture filtrate on detached maize cultivars CI6502 (Pio- mote the adherence of the conidial suspension to the leaf surface neer), M753 (Monsanto), XY335 (Pioneer), XY696 (Pioneer), and and showed no negative effect on the conidial germination of S. ZD958 (Henan academy of agricultural sciences, China) leaves, the turcica. After incubation at 26 ± 0.5 °C for 18 h, no significant differ- disease incidence was 100%, and the average lesion area was ence on S. turcica conidial germination was detected between the 27.0, 14.3, 20.7, 18.0, and 19.1 mm2, respectively. These areas were significantly lower than the corresponding positive controls, i.e., 69.3, 45.3, 48.3, 42.4, and 47.6 mm2, respectively (Fig. 3).

3.6. Effect of the culture ﬁltrate of strain No.05 on pathogenicity process of S. turcica

The disease incidence and the average lesion area on the control leaves was 100% and 47.5 mm2 after incubation at 26 ± 0.5 °C for 72 h, respectively, whereas no lesions were observed on these leaves sprayed with 100% culture filtrate at 12 hbi. On these leaves treated with 100% culture filtrate at 3, 6, and 12 hpi, the disease incidence was 55.6%, 88.9%, and 97.8% (Fig. 4A), and the average lesion area was 2.9, 10.6, and 15.9 mm2, respectively (Fig. 4B). The disease incidence in both treatments was 100% and the average lesion area increased from 28.6 to 38.7 mm2 on the leaves that were treated with the culture filtrate at 24 and 48 hpi, respectively (Fig. 4).

3.7. Effect of the culture ﬁltrate of strain No.05 on maize seedling NCLB in greenhouse

The culture filtrate of strain No.05 was effective for controlling NCLB in the controlled environments. Compared with the negative control, the NCLB control efficiencies were 88.1% and 83.2% when the maize seedlings were treated with 100% culture filtrate and Fig. 1. Suppression of Setosphaeria turcica by the culture filtrates of Chaetomium the fungicide control (tricyclazole, 750 lg/mL) at 24 hbi, respec- globosum strain No.05. (A) The effects of the culture filtrate on mycelial growth of S. tively. Similar results were obtained when the culture filtrate or turcica were determined in PDB liquid culture and incubated at 28 ± 0.5 °C for 4 d. tricyclazole were applied curatively at 2 hpi. Compared with the (B) The effects of the culture filtrates on conidial germination of S. turcica were control, the NCLB control efficiencies of the two treatments were determined on onion segment surface after 20 h of inoculation at 26 ± 0.5 °C. Data represent the means from three independent experiments with triplicate plates in 81.9% and 80.9% when the maize seedlings were treated with the each treatment. culture filtrate and the fungicide tricyclazole at 2 hpi, respectively. G. Zhang et al. / Biological Control 64 (2013) 90–98 95

Fig. 3. Biological control efficacy of Cg culture filtrate on detached maize cultivars leaves after 72 h of inoculation at 26 ± 0.5 °C. The maize cultivars in these treatments were CI6502 (Pioneer), M753 (Monsanto), XY335 (Pioneer), XY696 (Pioneer), and ZD958 (Henan academy of agricultural sciences, China). Lesion areas (mm2) are the means with standard errors from three independent experiments with triplicate pots in each treatment in each experiment. indicates significance (P < 0.01) according to LSD test.

Fig. 2. Control of NCLB on detached maize leaves by different concentrations (%, v/ v) of the culture ﬁltrate from Chaetomium globosum strain No.05 after 72 h of inoculation at 26 ± 0.5 °C. The culture ﬁltrate concentrations in these treatments were 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 lg/ml and mock control (water without S. turcica conidia), respectively. (A and B) Disease incidence (%) (A) and lesion area (mm2) (B) were the means from three independent experiments with triplicate pots in each treatment. (C) A representative sample of detached maize leaves from the bioassay. Representative image was taken from a single experiment. Three independent experiments gave similar results.

3.8. Identiﬁcation of bioactive compounds in the culture ﬁltrate of strain No.05

Two active compounds were isolated from crude extract of this fungus culture by using the activity-directed fractionation ap- proach. The structures of the fractionations were identified as chaetoglobosin A (compound 1, 1.322 g) and chaetoglobosin C (compound 2, 0.017 g) based on the analysis of the Mass Spectrom- etry (MS) and Nuclear Magnetic Resonance (NMR) data, the comparison with those previously reported data (Sekita et al., 1973, 1976; Silverton et al., 1976), and the standard spectra of the known compounds. The inhibition result showed that these two compounds displayed high efficiency at the volume of 20 lg/disc (0.4 lL 50 mg/mL) compared with the Hygromycin B control at the volume of 200 lg/disc (4 lL 50 mg/mL) against S. turcica (Fig. 5). Fig. 4. The development of Setosphaeria turcica after treatment with 100% culture filtrate of Chaetomium globosum strain No.05 on detached maize leaves at the 2 3.9. Evaluation of the biological activity of chaetoglobosin A in vitro indicated time points. Disease incidence (%) (A) and lesion area (mm ) (B) are the means from three independent experiments with triplicate pots in each treatment in each experiment. (C) A representative sample of detached maize leaves from the The average dry weight of S. turcica mycelia was 5.11 mg/mL in bioassay. Representative image was taken from a single experiment. Three the control. With the concentration of chaetoglobosin A increased independent experiments gave similar results. 96 G. Zhang et al. / Biological Control 64 (2013) 90–98

Fig. 5. The inhibitory effects of chaetoglobosin A and chaetoglobosin C against Setosphaeria turcica. The used concentrations of chaetoglobosin A (CA) and chaetoglobosin C (CC) in the assay were the same as 20 lg/disc. Data in each histogram are expressed as means ± SE from three independent experiments (A). Representative image was taken from a single experiment (B). Three independent experiments generated similar results. HB: positive control (Hygromycin B, 200 lg/disc); control indicates DMSO control (0.4 lL/disc, negative control). All the paper discs in all treatments were dried at clean bench for 24 h. and indicate signiﬁcance at P < 0.05 and P < 0.01, respectively.

from 0.1 lg/mL to 2 lg/mL in the treatments, the average dry 4. Discussion weight of S. turcica mycelia decreased significantly (P < 0.05) from 4.69 to 0.07 mg/mL, while the percentage of inhibition of S. turcica As a long-term protective management integrated with other growth increased significantly from 8.22% to 98.63%. Mycelial cultural control measures, C. globosum has been successfully ap- showed slow growth as the concentrations of chaetoglobosin A in- plied to infected soils to control fruit epidemics in several species, creased from 2 lg/mL to 10 lg/ml and no growth was observed at including durian (Durio zibethinus L.) and black pepper (Piper ni- the concentrations of 20 lg/mL or higher. The MIC was less than gram L.) harmed by Phytophthora palmivora, root rots in tangerine 0.1 lg/ml, while the IC50 for chaetoglobosin A on S. turcica mycelial (Citrus reticulata Blanco) caused by P. parasitica and strawberry growth was 0.3730 lg/ml, calculated based on the curve regression (Fragaria spp.) by P. cactorum, wilt in tomato (Lycopersicon esculen- concentration(x) y = 1.7031 log10 + 5.7295, determined by probit tum L.) caused by Fusarium oxysporum f. sp. Lycopersici, and basal analysis of the inhibition mycelial percents (y)(i2 = 0.9532) rot in corn caused by Sclerotium rolfsii (Pietro et al., 1992; Soytong (Leroux et al., 1999). The pH values of the medium varied from et al., 2005; Christian and John, 1983; Zhang et al., 2010). Although 5.25 to 6.33, indicating that the observed significant inhibitory the antagonistic activity of C. globosum against S. turcica has been effects of chaetoglobosin A on S. turcica were not caused by the reported on PDA plates (Wang et al., 2012), to our best knowledge, pH changes. our study is the first report of the application of C. globosum to control S. turcica on corn leaves. In the present study, the Cg strain 3.10. Effects of the biological activity of chaetoglobosin A on detached No.05 growing on PDA strongly inhibited the mycelial growth of maize leaves numerous phytopathogenic fungi, and its antifungal activity was sufficient to inhibit the growth of the tested phytopathogenic fungi The disease incidence in the positive control (0 lg/mL) was including S. turcica, C. diplodiella, C. glocosporioides, V. mali, C. fim- 100%. When the pathogen was exposed to chaetoglobosin A at briata, S. sclerotiorum (Table 1). the concentrations ranging from 0.2 to 10 lg/mL, no significant dif- Chaetomium is a genus containing many species within the fun- ference in disease incidence was observed, while the average lesion gal family Chaetomiaceae (Ascomycota) with over 100 terrestrial- area decreased significantly from 26.6 to 2.3 mm2 (the average le- and marine-derived species (Udagawa et al., 1997) Some species sion area of the control was 47.1 mm2). Disease symptom was not of Chaetomium (e.g., C. globosum and C. cupreum) can produce resis- observed on the inoculated maize leaves treated with chaetoglob- tant substances that can prevent wilt in seedlings of grains and osin A at the concentration of 100 lg/mL. There was no observed Pythium aphanidermatum (Eds. Fitzp) in sugarcane, reduce tomato negative effects on plant tissues in the negative control. wilt (caused by Fusarium spp.) and scab in apple (caused by G. Zhang et al. / Biological Control 64 (2013) 90–98 97

Venturia spp.), and also inhibit the growth of Rhizoctonia solani was recently characterized from T. harzianum Rifai, showing that Kuhn, Botrytis spp. (Soytong et al., 2005). The culture filtrate of trichodermin had significantly protective effect on early blight on Cg strain No.05 inhibited the S. turcica mycelial growth and conid- tomato and damping-off on cucumber (Chen et al., 2007). To date, ial germination in vitro, suggesting the presence of antifungal sub- a great number of antimicrobial compounds, e.g., alkaloids, pep- stances in the culture filtrate. In this study, when the detached tides, steroids, terpenoids, phenols, quinones, and flavonoids, have maize leaves were inoculated with S. turcica conidia (at the con- been found (Scherlach et al., 2010), and it is believed that searching centration of 5 104 conidia/mL) that suspended in the PDB med- for natural products could be a promising way to solve the problem ium containing 10% culture filtrate of strain No.05, the disease that living beings are becoming resistant to some commonly used incidence of NCLB was completely suppressed. As shown in drugs and meet the emergent demand of discovering highly effec- Fig. 3, the disease symptoms on maize leaves that were sprayed tive BCAs with low toxicity and no environmental impact (WHO, with 1% culture filtrate on different maize cultivars, i.e., CI6502 2002; Yu et al., 2010). The control of plant diseases with antimicro- (Pioneer), M753 (Monsanto), XY335 (Pioneer), XY696 (Pioneer), bial substances as described in this study would be one of the solu- and ZD958 (Henan academy of agricultural sciences, China), demon- tions for biological plant pest management. strated that the biocontrol efficacy of Cg culture filtrate against The inhibitory efficiency of the antifungal substances produced NCLB could be applied to susceptible or highly susceptible maize from C. globosum can be enhanced through optimization of fermen- cultivars. Similar results were observed on the maize seedlings tation conditions, introduction of mutagenesis, or genetic selection treated with the culture filtrate, further indicating that strain of desired traits. Further studies are needed to confirm the control No.05 contains active antifungal substances. efficiency of strain No.05 in the field. It is expected that more anti- Different biocontrol strains of Chaetomium spp. have shown dif- fungal substances could be extracted from this strain and further ferent antifungal activities depending on whether they were tested characterizations of chaetoglobosin A are required to study its in the laboratory or in the field and on the used methods (Pietro instability, security, durability, and efficiency, in comparison with et al., 1992; Heye and Andrews, 1983; Vitale et al., 2011). As shown other fungicides on NCLB control in the field. in Fig. 4, the disease symptoms on maize leaves sprayed with 100% culture filtrate at different time intervals (1, 3, 6, 12, 24, and 48 hpi) demonstrated that the culture filtrate of strain No.05 could Acknowledgments provide strong protective and curative treatments against NCLB. Meanwhile, in the pot experiments, the treatments with the intact The authors gratefully thank Dr. Qingming Qin (College of Plant seedlings in greenhouse further demonstrated that C. globosum Sciences at Jilin University) and Dr. Fengjie Sun (Biology School of strain No.05 could be applied to NCLB control and is suitable for Science and Technology Georgia Gwinnett College, US) for their crop protection. valuable suggestions on data analyses and critical comments on It has been reported that C. globosum produces various types of the revision of this manuscript. The authors would also like to biochemical compounds, including chaetoglobosins (Jiao et al., thank two peer- reviewers who provided constructive comments 2004; Ding et al., 2006; Zhang et al., 2010), cytoglobosins A–G on an earlier draft of this article and Bss. Hongcheng, Huawen (Cui et al., 2010), steroids, azaphilones, and chaetoviridins A and Mo, Jiankun Wang, Ye Tian, Zhenhua Bi, and Drs. Guihua Li and C(Qin et al., 2009a), pyrones and chaetoglocins A and B (Ge et al., Yan Wang (Jilin University, Changchun, China) for their valuable 2011), orsellides and globosumones A–C (Bashyal et al., 2005). input and assistance in greenhouse and laboratory work. This work Our study showed that the antifungal substances in the culture fil- was supported by the grants from the project of National Key Tech- trate of strain No.05 resulting in effective suppression of the maize nology R&D Program in the 12th Five year Plan of China disease caused by S. turcica are are chaetoglobosin A and chaeto- (2012BAD19B04), Ministry of Agriculture Public Benefit Industry globosin C (Fig. 5). Chaetoglobosin A, known as cytochalasan alka- of the People’s Republic of China (20113016), National Natural Sci- loids, exhibits strong cytotoxicity on various of human cancer cell ence Foundation of China (31101394), and the International Coop- lines and P388 murine leukemia cell line (Jiao et al., 2004; Sekita eration Projects in Changchun City and Jilin Province (10GH26, et al., 1973; Scherlach et al., 2010). Chaetoglobosin A and C not only 20100723). showed phytotoxicity against alfalfa seedlings, antibacterial activity against Helicobacter pylori and Staphylococcus aureus (Jiao et al., 2004; Ichihara et al., 1996), but also exhibited highly toxic ef- References fects to mouse and rat cells and chick embryos (Ohtsubo et al., 1978; Vesely´ et al., 1995), and acute toxic effects against Hela cells Bashyal, B.P., Wijeratne, E.M., Faeth, S.H., Gunatilaka, A.A., 2005. 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