Antifungal screening of endophytic fungi from Ginkgo biloba for discovery of potent anti-phytopathogenic fungicides

Authors

  • Yu Xiao,

    1. Jiangsu Key Laboratory of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
    2. Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
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  • Hong-Xia Li,

    1. Jiangsu Key Laboratory of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
    2. Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
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  • Cong Li,

    1. Jiangsu Key Laboratory of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
    2. Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
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  • Jian-Xin Wang,

    1. Jiangsu Key Laboratory of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
    2. Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
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  • Jun Li,

    1. Jiangsu Key Laboratory of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
    2. Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
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  • Ming-Hua Wang,

    1. Jiangsu Key Laboratory of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
    2. Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
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  • Yong-Hao Ye

    Corresponding author
    1. Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    • Jiangsu Key Laboratory of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
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Correspondence: Yong-Hao Ye, College of Plant Protection, Jiangsu Key Laboratory of Pesticide Science, Nanjing Agricultural University, Nanjing 210095, China. Tel./fax: +86 25 84395479; e-mail: yeyh@njau.edu.com

Abstract

Many endophytic fungi have been found to synthesize bioactive compounds to defend host plants against pathogenic organisms. Here we performed anti-fungal bioassay of 80 endophytic fungi isolated from Ginkgo biloba. Fifteen endophytes were active against at least one of the selected fungi, Fusarium graminearum, Sclerotinia sclerotiorum and Phytophthora capsici, using the agar diffusion method. The most bioactive strain CDW7 was identified as Chaetomium globosum by microscopic examination and ITS rRNA gene sequence data. Culture broth of CDW7 diluted 3-fold completely inhibited the mycelial growth and conidia germination of F. graminearum in vitro. Therefore, Fusarium head blight, a common disease in wheat and barley associated with Fusarium spp., was used to test the anti-phytopathogenic activity in vivo. The fermentation broth of CDW7 resulted in a protective efficacy of 54.9% and curative efficacy of 48.8%. Followed by a bioassay-guided approach, 1,2-benzenedicarboxaldehyde-3,4,5-trihydroxy-6-methyl (flavipin) was isolated and demonstrated to significantly inhibit the growth of several plant-pathogenic fungi, especially F. graminearum with an EC50 value of 0.73 μg mL−1 comparable to the commonly used fungicide carbendazim, indicating that it could be used as a fungicide or as a lead compound of new fungicides.

Introduction

Fungal diseases of plants cause huge losses in food production. Fusarium head blight (FHB) causes reduced grain yield and quality and kernel contamination with mycotoxins such as deoxynivalenol, nivalenol, T-2 toxin and zearalenone, which are harmful to livestock and threaten human food safety (Wang et al., 2010). In China, Fusarium graminearum is the main cause of FHB; for over 30 years it has been controlled largely by the application of carbendazim (Chen et al., 2007a). Synthetic fungicides, the cheap and common approach for the control of plant diseases, may produce harmful side effects, such as severe environmental pollution and the development of multi-resistant strains. For example, the continued use of carbendazim to control FHB is threatened by the development of carbendazim-resistance in F. graminearum populations (Zhang et al., 2009). There is an urgent need to develop more effective and environment-friendly fungicides, especially those with novel modes of action to control FHB potently and safely.

Endophytic fungi have been recognized as a potential source of bioactive secondary metabolites (Schulz et al., 2002; Debbab et al., 2011). More recently, attention has been paid to screening for antimicrobial endophytic fungi and their secondary metabolites (Phongpaichit et al., 2006; Xing et al., 2011). Ginkgo biloba, one of the oldest species on earth with fossil records dating back more than 200 million years, has long been used in China as a traditional medicine for various ailments (Chen et al., 2007a, b). Previous studies reported that G. biloba had fewer pests and diseases than other gymnosperms (Cao, 2007), which may be caused in part by its associated endophytes, as many endophytes were found to be able to synthesize bioactive compounds to defend host plants against pathogenic fungi and bacteria (Schulz et al., 2002). However, endophytes from G. biloba have scarcely been studied for their abilities to inhibit phytopathogenic microorganisms. Therefore, we report the anti-phytopathogenic assay of the endophytes associated with G. biloba and the characterization of flavipin, a potent anti-phytopathogenic molecule from CDW7, the most active strain against tested phytopathogenic fungi.

Materials and methods

Isolation and identification of strains

Healthy leaves and small branches of G. biloba were collected from plants from April to September 2009 located in Taixing and Nanjing in Jiangsu Province and Chengdu in Sichuan Province, P.R. China. The samples were then stored in valve bags at 4 °C. A total of 80 endophytic fungi were isolated according to the previously reported method of Liu et al., (2004). Morphological identification was referenced by Wei, (1979). Strain CDW7 was isolated from the leaf of the plant and further identified based on the analysis of the DNA sequence of the ITS1-5.8S-ITS2 (Phongpaichit et al., 2006). The size and shape of perithecium, hyphae and ascospores of strain CDW7 grown on PDA were measured by microscopy. An Olympus IX71 microscope with an Olympus DP72 lens using an image-por express version 6.0.0.319 (Media Cybernetics Inc.) program was employed to capture images. The morphological characteristics of the fungus were also observed through a scanning electron microscope (SEM) (S-3400N II, Hitachi, Japan) following the method reported by Qiu et al., (2010).

Preparation of fungal fermentation broth

Endophytic fungal isolates were grown on PDA medium (potato 200 g, dextrose 20 g, agar 20 g, H2O 1000 mL) at 25 °C for the appropriate number of days. Eight pieces (5 mm diameter) of mycelial agar plugs removed from the edge of young cultures of endophytic fungi were each inoculated into 250-mL Erlenmeyer flasks with 100 mL PD broth medium, followed by shaking (150 rpm) continuously for 12 days at 25 ± 1 °C. The broth culture was filtered to separate the culture broth and mycelia. The culture broth was refiltered with a 0.22-μm bacterial filter to obtain the sterile fermentation broth, which was subsequently kept at 4 °C.

Extraction and fractionation of CDW7 culture

To discover the antifungal compounds produced by strain CDW7, the culture broth was extracted three times with petroleum ether (A, 0.5 g), dichloromethane (B, 2.5 g), ethyl acetate (C, 4.0 g), 1-butanol (D, 3.4 g) in turn and dried by a rotary evaporator to yield each organic phase extract. The bioactive dichloromethane phase was chromatographed on silica gel (200 g, 100–200 mesh) using a stepwise gradient of CHCl3 : MeOH (v/v, 1 : 0–0 : 1) to give four fractions (A1, 0.1 g; A2,1.5 g; A3, 0.5 g; A4, 0.3 g) based on TLC. Fraction A2 was then separated on a Sephadex LH-20 column eluted with CHCl3 : MeOH (v/v, 1 : 1), followed by recrystallization to yield yellow crystal (100 mg), which was identified as flavipin (1,2-benzenedicarboxaldehyde-3,4,5-trihydroxy-6-methyl) with its 1H-NMR and 13C-NMR spectrum corresponding to that reported by James et al., (2002).

Methods to evaluate antifungal activity

Agar diffusion method

Fungal culture broth (200 μL) was transferred to an Oxford cup (6 × 7.8 × 10 mm) to screen antifungal activity against three common pathogenic fungi – F. graminearum, Sclerotinia sclerotiorum and Phytophthora capsici according to the method reported by Taechowisan et al., (2005). Briefly, fungi blocks (5 mm in diameter) were made with a cork borer from the edge of the fresh fungi cultured on PDA medium and subsequently placed in the central of new PDA culture plates, cultivating at 25 °C. When the colony diameter reached 3 cm, 200 μL of fungal culture broth was added into Oxford cups placed 1 cm away from the edge of the colony and then cultivated at 25 °C. PD broth and cycloheximide (10 μg per cup) were negative and positive controls, respectively. Inhibition zones were measured to assess antimicrobial activity.

Determination of the inhibitory activity of CDW7 metabolites against the mycelial growth of F. graminearum in vitro

A 5-mm diameter mycelial disk cut from the edge of an actively growing culture (3 days old) of F. graminearum was placed in the center of a 9-cm diameter Petri dish containing PDA plus fermentation broth of CDW7 or its organic extracts. The fermentation broth was diluted 3-, 5-, 10- and 20-fold respectively; the concentration of each organic phase extract obtained from solution extractions was 10 μg mL−1. After 3 days at 25 °C, the colony diameter of each strain was measured with the original mycelial disk diameter (5 mm) subtracted from this measurement. Percentage inhibition was calculated as (1-a/b) × 100, where a is the colony diameter in Petri dishes with metabolites and b is the mean colony diameter in Petri dishes without metabolites. Carbendazim (10 μg mL−1) was used as the positive control.

Determination of the inhibitory activity of CDW7 metabolites against germination of F. graminearum in vitro

Conidia of F. graminearum (1 × 105 CFU mL−1) were prepared with reference to Yu et al., (2011). A 50-μL volume of the conidial suspension was spread on a 9-cm diameter Petri dish containing water agar with fermentation broth of strain CDW7 diluted 3-, 5- and 10-fold. After 8 h at 25 °C in darkness and when the germination rate of the controls was over 85%, the germination rates of the treatments were recorded and the percentage inhibition was calculated as (1-c/d) × 100, where c is the germination rate in Petri dishes with metabolites and c the mean germination rate in Petri dishes without metabolites. Carbendazim (10 μg mL−1) was used as the positive control.

Efficacy of fermentation broth of strain CDW7 against Fusarium head blight (FHB) in vivo

The strain F. graminearum and F. graminearum-susceptible wheat cultivar Yangmai 158 were used to measure the efficacy of the fermentation broth of strain CDW7 in vivo. The wheat was planted on 30 October 2011 in small circular plots (0.2 m in diameter) with 10 plants per plot, and plots at 0.5-m intervals at a field site (7.9 × 4.4 m) at Pailou Experimental Center of Nanjing Agricultural University. Two dilutions (1- and 5-fold) of the fermentation broth were sprayed the day before and the day after artificial inoculation with F. graminearum. Carbendazim (50%WP; Jiangsu Rotam Lanfeng Biochemical Co., Ltd) was co-assayed as the positive control. Each treatment was represented by three replicate plots. The conditions in the field were suitable for FHB development (25–30 °C and 85% relative humidity), and no natural FHB developed in this field. FHB severity was rated 20 days after inoculation using the following scale: 0, no visible symptoms; 1, 1–3%; 2, 4–10%; 3, 11–25%; 4, 26–50%; 5, 51–75%; 6, > 75% of the spikelets showing FHB symptoms (Yu et al., 2011). The disease index was [n(1) × 1 + n(2) × 2 + n(3) × 3 + n(4) × 4 + n(5) × 5 + n(6) × 6]/(number of spikelets × 6) × 100, where n indicates the number of infected plants rated as 1, 2, 3, 4, 5 or 6. Efficacy of the disease control was calculated with the following formula:

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Antifungal activity of flavipin against phytopathogenic fungi in vitro

Flavipin (final concentration 10 μg mL−1) was initially tested for antifungal activity against a wide range of phytopathogenic fungi using the mycelia growth inhibition method described above and displayed significant inhibitory capacity (inhibitory rate over 85%) against five fungi including F. graminearum, S. sclerotiorum, P. capsici, Rhizoctonia solani and Alternaria solani. The median effective concentration (EC50) values were determined by measuring mycelial growth in agar containing flavipin at a series of concentrations. Carbendazim, chlorothalonil and procymidone were used as the positive controls.

Statistical analysis

All the experiments that estimated antifungal activity were conducted in triplicate and statistical analysis of the data was performed by analysis of variance (one-way anova), using spss 16.0 software. A probability value of P ≤ 0.05 was considered to denote a statistical significance difference. Data are presented as mean ± standard deviation (SD).

Results

Identification and screening of endophytic fungi for antifungal activity

A total of 80 endophytic fungi were isolated from G. biloba. Fifty-three of these isolates were identified: 15 species from the genus Alternaria, 12 species from the genus Phomopsis, six species from the genera Colletotrichum and Fusarium, three species from the genus Chaetomium, two species from the genera Sphaeropsis, Guignardia and Botrytis, and one each from the genera Penicillium, Cladosporium, Mucor, Aspergillus and Gloeosporium. Twenty-seven isolates, considered to be sterile mycelia, did not sporulate in the culture medium.

The fermentation broth of 15 fungal isolates inhibited at least one of the microorganisms studied (Table 1). Among the tested extracts, the cultural broth of strain CDW7 inhibited the growth of all three plant-pathogenic fungi considerably. CDW7 was further identified as Chaetomium globosum (GenBank accession number JN588554) based on rRNA gene sequencing of ITS region and microscopic examination. Figure 1 illustrates the obverse colonies of CDW7 growing on a PDA plate for 10 days (a) and 25 days (b). The color varied with the state of sporulation from light brown to blue. Figure 1c–e represents the results of light and scanning electron microscopic studies of strain CDW7. The hyphae were always branching with small thorns on the top growing parts of the mycelia. The perithecium wall was composed of staggered dark brown filaments and perithecia were oval or nearly spherical, 200–400 × 150–280 μm in size. Ascospores were brown, sub-spherical or lemon-shaped (7.5–10.2 × 6.5–7.5 μm). All these observed characteristics were consistent with those of C. globosum (Wei, 1979).

Table 1. Antifungal activities of fermentation broth from endophytes associated with Ginkgo biloba
Strain Fusarium graminearum Sclerotinia sclerotiorum Phytophthora capsici
  1. Only positive results are reported.

  2. The assay was conducted by agar diffusion method. −: no inhibition; inhibitory zone +: < 1 mm; ++: 1–5 mm; +++: > 5 mm.

CDP15+++
CDW7++++++++
CDW9++
NJP17+
NJSC13++
NJSC8++
NJSP73+
NJSW2++++
NJWP9++
NJWP38+++
NJWP72++
NJWP10+++
TXCP41++++
TXP58++
TXP84+
Cycloheximide+++++++
Figure 1.

Morphological characteristics of strain CDW7. The obverse colonies of CDW7 growing on a PDA plate for (a) 10 days and (b) 25 days. Light micrographs of (c) perithecium and (d) hyphae and ascospores. (e) Scanning electron micrographs of hyphae and ascospores.

Fermentation broth of C. globosum, CDW7, against F. graminearum

Chaetomium globosum CDW7 was selected for further antifungal study against F. graminearum. As shown in Table 2, Cultural broth of CDW7 diluted 3-fold completely inhibited the mycelial growth of F. graminearum, whereas 5-, 10- and 20-fold dilutions strongly inhibited growth. The germination of conidia was also fully inhibited by the 3-fold-diluted cultural broth of CDW7. These strong activities of fermentation broth of CDW7 against F. graminearum in vitro indicated CDW7 could be used to control Fusarium head blight in vivo. The inoculated plants in negative control plots were infected at a disease rate of 77.5% and a disease index of 41.0. When treated with the cultural broth of strain CDW7, both the disease rate and index significantly declined (Table 3). Overall, control of FHB in vivo was considerable with metabolites of CDW7, giving a protective efficacy of 54.9% and a curative efficacy of 48.8%, as compared with 81.7% and 80.5% in the positive control.

Table 2. Fermentation broth from strain CDW7 against Fusarium graminearum
TreatmentsGermination rate (%)Germination inhibition rate (%)Mycelial growth (mm)Mycelial growth inhibition rate (%)
  1. Carbendazim was used as the positive control and PD broth as the negative control.

  2. Statistical analysis of the data was performed by analysis of variance (one-way anova). A probability value of P ≤ 0.05 was considered to denote a statistically significance difference. Different letters in superscript indicate significant differences within stains and references.

Fermentation broth (dilution times)
30g1005.0 ± 0.0a100
548.3 ± 1.4d45.411.0 ± 1.0b92.1
1079.1 ± 2.7b10.715.3 ± 0.6c86.8
2018.3 ± 1.2d82.9
Positive control (μg mL−1)
1040.0 ± 2.5e54.85.0 ± 0.0a100
Negative control88.5 ± 1.2a81.5 ± 1.5e
Table 3. Efficacy against Fusarium head blight of fermentation broth from strain CDW7
TreatmentsDisease rate (%)Disease indexEfficacy (%)
  1. Carbendazim (50% WP) was used as the positive control and PD broth as the negative control.

  2. Statistical analysis of the data was performed by analysis of variance (one-way anova). A probability value of P ≤ 0.05 was considered to denote a statistically significance difference. Different letters in superscript indicate significant differences within stains and references.

Protection (dilution times)
147.5 ± 1.5cd18.5 ± 1.4e54.9
550.0 ± 2.4c30.0 ± 1.0c26.8
Positive control (mg per plot)
2020.0 ± 1.0f7.5 ± 1.3f81.7
Therapy (dilution times)
145.0 ± 2.5d21.0 ± 1.8d48.8
565.0 ± 2.0b33.0 ± 1.0b19.5
Positive control (mg per plot)
2025.0 ± 1.8e8.0 ± 1.3f80.5
Negative control77.5 ± 2.5a41.0 ± 2.0a

Antifungal activity of organic phase extracts and flavipin against phytopathogenic fungi in vitro

As demonstrated in Fig. 2, the petroleum ether and dichloromethane extracts (10 μg mL−1) exhibited considerable activity against F. graminearum with an inhibitory rate of 59.20% and 71.64%, respectively, demonstrating the existence of antifungal compounds. Flavipin was characterized as the most active metabolite from the dichloromethane extract and significantly inhibited the growth of F. graminearum, S. sclerotiorum, P. capsici, R. solani and A. solani, with EC50 values ranging from 0.73 to 12.35 μg mL−1 (Table 4).

Table 4. EC50 of flavipin against plant-pathogenic fungi
Pathogenic fungiFlavipin (μg mL−1)Positive controls (μg mL−1)
CarbendazimChlorothalonilProcymidone
Sclerotinia sclerotiorum 3.68 ± 0.230.15 ± 0.03
Fusarium graminearum 0.73 ± 0.090.50 ± 0.08
Rhizoctonia solani 2.62 ± 0.151.42 ± 0.14
Phytophthora capsici 2.76 ± 0.124.48 ± 0.40
Alternaria solani 12.35 ± 0.902.19 ± 0.10
Figure 2.

Different organic phase extracts (10 μg mL−1) against Fusarium graminearum: (a) petroleum ether phase; (b) dichloromethane phase; (c) ethyl acetate phase; (d) 1-butanol phase; (e) negative control.

Discussion

Gingko biloba owes its pharmacological reputation to the fact that it produces bioactive metabolites. Gingko biloba extracts, mainly containing ginkgo flavonoid and ginkgolide, have marked protective effects on cardio-cerebral vascular and central nerve systems (Wang et al., 2006a). In this study, we explored its potential application in agriculture by investigating the anti-phytopathogenic activity of its associated endophytes. Interestingly, 15 endophytic fungi isolated from G. biloba exhibited potent antifungal activities, indicating the prospect that endophytic fungi from G. biloba could be developed as a new reliable source for antifungal compounds.

Chaetomium globosum CDW7 was further studied because of its potent anti-fungal activities. Previous studies reported that C. globosum was a remarkable producer of bioactive natural products, such as cytotoxic globosumones (Bashyal et al., 2005), cytotoxic benzaldehyde derivative chaetopyranin (Wang et al., 2006ab), mycotoxic chaetoglobosins (Ding et al., 2006), and polyhydroxylated steroids (Qin et al., 2009). Flavipin was previously reported to be a metabolite of Aspergillus flavipes, Aspergillus terreus (Raistrick & Rudman, 1956), Epicoccum spp. (Bamford et al., 1961) and C. globosum (James et al., 2002) and was discovered to be antagonistic against nematodes (James et al., 2002) and Monilinia laxa (Madrigal et al., 1991). In this study, flavipin was characterized as the bioactive component responsible for the anti-phytopathogenic activities of the cultural broth of strain CDW7 C. globosum. Flavipin displayed potent broad-spectrum antifungal activity, particularly against F. graminearum comparable to carbendazim, suggesting that flavipin could be used as a broad-spectrum antifungal agent or at least as a hit or lead compound for novel fungicides.

CDW7 exhibited the latent activity against the mycelia growth and conidia germination of F. graminearum, suggesting its application in the control of FHB, a plant disease mainly caused by the infection of Fusarium spp. (Chen et al., 2007a). Inhibition of the conidia germination and mycelia growth of Fusarium spp. plays a vital role in the prevention and control of FHB. The fermentation broth of strain CDW7, diluted 3-fold, completely inhibited the germination of conidia and mycelia growth of Fusarium spp. tested in vitro, and exhibited a favorable protective effect in the in vivo test. The in vivo potency of the fermentation broth of strain CDW7 was less than that of carbendazim. However, it should be noted that the in vivo results in this study were from the raw broth without any optimization or fractionation. It could be expected that the in vivo potency of CDW7 would be enhanced by optimization of the culture conditions and/or fractionation of the culture broth targeting the increase in productivity of the bioactive components in the final product. A follow-up study is in progress and the results will be reported in a separate paper.

Acknowledgements

This work was co-supported by the National Basic Research Program of China (2010CB126104), the National Natural Science Foundation of China (30901854), Fundamental Research Funds for the Central Universities (KYZ201107), Special Fund for Agro-scientific Research in the Public Interest (201303023) and the Open Research Fund Program of Jiangsu Key Laboratory of Pesticide Science (NYXKT201202).

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