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Efficacy Assessment of Antifungal Metabolites from Chaetomium

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Efficacy Assessment of Antifungal Metabolites from Chaetomium Biological Control 64 (2013) 90–98 Contents lists available at SciVerse ScienceDirect Biological Control journal homepage: www.elsevier.com/locate/ybcon 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 anal- ysis. Both in vitro and in planta bioassay experiments showed that chaetoglobosin A displayed potent bio- control 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 sec- ondary 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 be- tween the two inoculation sites of strain No.05 and fungal plug 2.
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