Accepted Article
Received Date : 22-Jul-2014 Accepted Date : 13-Aug-2014 Article type Editor:
: Research Letter
Paolina Garbeva
Fusarium oxysporum induces the production of proteins and volatile organic compounds by Trichoderma harzianum T-E5
Fengge Zhang1, Xingming Yang1,2, Wei Ran∗1,2, Qirong Shen1,2
1
National Engineering Research Center for Organic-based Fertilizers, Nanjing
Agricultural University, Nanjing, 210095, China 2
Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization,
Nanjing Agricultural University, Nanjing, 210095, China
Corresponding author: Wei Ran
∗
Corresponding author E-mail address: [email protected] (W. Ran)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12582 This article is protected by copyright. All rights reserved.
Accepted Article
Postal address: College of Resources and Environmental Sciences, Nanjing Agricultural University, 210095, Nanjing, Jiangsu Province, P. R. of China.
E-mail address: [email protected] Tel: 0086-025-84396212 Fax: 0086-025-84396824
Abstract Trichoderma species have been used widely as biocontrol agents for the suppression of soil-borne pathogens. However, some antagonistic mechanisms of Trichoderma are not well characterized. In this study, a series of laboratory experiments were designed to characterize the importance of mycoparasitism, exoenzymes and volatile organic compounds (VOCs) by T. harzianum T-E5 for the control of F. oxysporum f. sp. cucumerinum (FOC). We further tested if these mechanisms were inducible and upregulated in presence of FOC. The results were as follows: T-E5 heavily parasitized FOC by coiling and twisting the entire mycelium of the pathogen in dual cultures. T-E5 growing medium conditioned with deactivated FOC (T2) showed more proteins and higher cell wall-degrading enzyme activities than T1, suggesting that FOC could induce the upregulation of exoenzymes. The presence of deactivated FOC (T2’) also resulted in the upregulation of VOCs that 5 and 8 different types T-E5-derived VOCs were identified from T1’ and T2’, respectively. Further, the excreted VOCs in T2’ showed significantly higher antifungal activities against FOC than T1’. In conclusion, mycoparasitism of T-E5
This article is protected by copyright. All rights reserved.
Accepted Article
against FOC involved mycelium contact and the production of complex extracellular substances. Together, these data provide clues to help further clarify the interactions between these fungi. Keywords: Mycoparasitism; Proteins; SDS-PAGE; Volatile organic compounds; Head-space solid-phase micro-extraction (HS-SPME)
Introduction Cucumber (Cucumis sativus) is one of the most important economic crops cultivated and consumed worldwide. However, when continuous cropping occurs in the same field over many years, fusarium wilt disease can easily threaten the crop. This disease is caused by the soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum (FOC), and it will result in severe plant damage and economic losses for farmers. The pathogen typically infects the cucumber vascular system and then the plants develop necrotic lesions on their stems and leaves. Fusarium wilt develops, and the cucumber plants die within a few days. Traditionally, chemical controls can effectively protect plants from infectious pathogens (Compant et al., 2005). However, the drawbacks of the chemical fungicides are obvious when pathogen resistance to pesticides, food safety and environmental quality are considered. Therefore, finding an effective and environment-friendly strategy to control fusarium wilt disease appears essential and urgent for the development of the cucumber industry. Biocontrol by applying antagonistic microorganisms instead of chemicals has been studied widely for the management of fusarium wilt disease and other soil-borne diseases (Cao et al., 2012; Zhao et al., 2012). Several soil fungal Trichoderma species have been approved as effective biocontrol agents in the inhibition of many soil-borne This article is protected by copyright. All rights reserved.
Accepted Article
pathogens (Margolles Clark et al., 1996). Recently, Trichoderma strains have been used in the biocontrol of fusarium wilt and damping-off diseases of cucumber (Chen et al., 2011; Huang et al., 2011). The biocontrol effect of Trichoderma species is achieved by a variety of antagonistic mechanisms. Mycoparasitism, characterized by the direct attack of one fungus against another, is considered an important antagonistic mechanism of Trichoderma species (Benitez et al., 2004; Mendoza-Mendoza et al., 2003). The induced production of some extracellular substances (e.g., cell wall-degrading enzymes, antifungal VOCs) is also regarded as momentous mechanisms of Trichoderma against fungal pathogens. In mycoparasitism, Trichoderma grows towards the pathogen’s hypha by paralleling or coiling (Huang et al., 2011) and generates cellular attachments. The mycoparasite then excretes various extracellular cell wall-degrading enzymes, such as chitinases, β-1, 3-glucanases, proteases and xylanases, and penetrates the pathogen hyphae to absorb nutrients (Harman et al., 2004). Lastly, the entire pathogen system collapses with mycelial cell-wall breakdown and leakage of the inner cytoplasm. However, few studies have been designed to link mycoparasitism to the induction of extracellular enzymes and VOCs using growth substrates that originate from a plant pathogen.VOCs are the end products of many metabolic pathways, and they include many classes, such as sesquiterpenes, alcohols and ketones, among others (Korpi et al., 2009). The report of Stoppacher et al. (2010) showed that these volatile metabolites take part in varying biological processes. For example, VOCs can significantly inhibit the mycelial growth and spore germination of F. oxysporum f. sp. cubense (Yuan et al., 2012). The determination of volatile fungal metabolites is typically accomplished using gas chromatographic mass spectrometry (GC-MS). There are many reports (Bruce et al., 2000; Polizzi et al., This article is protected by copyright. All rights reserved.
Accepted Article
2011) that have examined Trichoderma VOCs although little research has been conducted on the induction of Trichoderma VOCs to gain insight into mycoparasitism and the antagonism effects of the VOCs on FOC. These advances will facilitate a better understanding of the antagonistic mechanisms of Trichoderma species. The biocontrol fungus T. harzianum T-E5 was reported to significantly suppress fusarium wilt of cucumber in our previous pot experiments (Zhang et al., 2013). The objective of this study was to investigate the mechanisms used by T. harzianum T-E5 in antagonizing fusarium wilt. The possible mechanisms, mycoparasitism and productions of proteins and VOCs that underlie the T-E5 biocontrol process for fusarium wilt were evaluated in vitro. Materials and methods Microorganisms, deactivated FOC cell walls and media Trichoderma harzianum T-E5 (CCTCC No. AF2012011, China Center for Type Culture Collection) was used throughout this study (Zhang et al., 2013a; Zhang et al., 2013b). The soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum (FOC, Agricultural Culture Collection of China (ACCC) No.30220) was provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Both strains were usually cultured on potato dextrose agar (PDA) medium. The conidia suspensions of FOC were prepared based on Zhang et al. (2013a). The conidia concentration was determined using hemocytometer counts, and the final concentration was 1.48 × 107 CFU mL-1. The preparation of deactivated FOC cell walls was according to El-Katatny et al. (2000) and Yang et al. (2009). The minimal medium (10 g C-source, 6.9 g NaH2PO4, 2.0 g KH2PO4, 1.4 g (NH4)2SO4, 1.0 g peptone, 0.3 g MgSO4.7H2O and 0.3 g urea in 1 L distilled water, This article is protected by copyright. All rights reserved.
Accepted Article
pH 5.0) and solid minimal medium (20 g agar in 1 L minimal medium) were used in the followed experiment. T1 was the minimal medium supplemented with glucose as a C-source, T2 was the minimal medium supplemented with deactivated FOC cell walls as a C-source; T1’ and T2’ were the solid form of T1 and T2. Dual cultures and observations of mycoparasitism T. harzianum T-E5 antagonism of FOC was evaluated in dual culture tests (Huang et al., 2011). A PDA plate inoculated with FOC only was used as a control. All plates were incubated at 28 °C for 7 days. For observations of mycoparasitism, T-E5 and FOC were inoculated in dual cultures. We placed some small cover slips at the boundary of the two strains. After 3 days of dual culture, some of the cover slips were chosen for observation of interactions using an optical microscope (OLYMPUS CX21, Tokyo, Japan). The other cover slips were fixed using 2.5% glutaraldehyde and then observed using a scanning electron microscope (HITACHI S-4800 FESEM, Tokyo, Japan). Imitation of the biocontrol environment T. harzianum T-E5 conidia suspensions were prepared according to FOC. A 2-ml T-E5 conidia suspension (2.27 × 107 CFU mL-1) was inoculated into 250 ml T1 and T2 minimal medium, respectively. After inoculation, T1 and T2 were incubated in a shaking incubator at 28 °C/180 rpm for 96 h. The cultures were collected every day after inoculation (24, 48, 72 and 96 h), and they were filtered using filter paper (Whatman No.1, φ11 cm) and then a 0.45-μm filter membrane. The filtrates were used for enzyme activities assays and protein extraction. Determination of protein content and enzyme activities This article is protected by copyright. All rights reserved.
Accepted Article
The protein content of the two filtrates was assayed using a Bicinchoninic acid (BCA) protein assay kit (Dingguo Changsheng Biotechnology, Co. Ltd., Beijing, China). The activities of chitinases, β-1, 3-glucanases, cellulases, and xylanases were determined according to previous researches (Boller and Mauch., 1988; Pan et al., 1991; Liao et al., 2012). Protease activity was determined using a protease activity assay kit (Abvona Biotechnology, Co. Ltd., Taiwan), following the manufacturer’s instructions. Extraction of proteins and SDS-PAGE The solid ammonium sulfate precipitation method was used to extract crude proteins, and the entire extraction process was completed in an ice bath (Liao et al., 2012). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed following the procedures of Laemmli (1970) with the Mini-Protean II system (Bio-Rad, USA). Antagonistic assay of VOCs from T-E5 against F. oxysporum f. sp. cucumerinum For the antagonistic assay of T-E5 VOCs against FOC, a fresh 5-mm-diameter mycelial disc of T. harzianum T-E5 was inoculated into the center of a Petri dish which containing T1’ and T2’ minimal medium respectively. The lid of each plate was replaced by the bottom of a plate containing 1/4 PDA inoculated with a 5-mm-diameter mycelial disc of FOC. Then the two plates were sealed together using adhesive tape and incubated at 28 °C for 7 days in the dark. The plate with FOC only was used as control and three replicates were designed for both T1’ and T2’. The colony diameters of FOC were measured every day after incubation and compared with those of control plates.
This article is protected by copyright. All rights reserved.
Accepted Article
Collection of VOCs using head-space solid-phase micro-extraction (HS-SPME) and GC-MS analysis The VOCs of T-E5 grown in T1’ and T2’ were collected using head-space-solid-phase micro-extraction (HS-SPME) (Strobel et al., 2001). An SPME fiber of 50/30 µm divinylbenzene (DVB)/ polydimethylsiloxane (PDMS) (Supelco, Vienna, Austria) was selected as an appropriate fiber coating for extractions. A fresh 5-mm-diameter T-E5 mycelial disc was inoculated into a vial (200 ml) containing 50 ml T1’ minimal medium. In parallel, a SPME syringe with a fiber (50/30 µm DVB/ PDMS) was inserted into the headspace of the vial. The vial was placed at 28 °C, and the fiber was then exposed to absorb the volatiles during the growth process of T-E5. Seven days after inoculation, the syringe containing the volatiles was inserted into a gas chromatography-mass spectrometer (GC-MS). The volatiles bound to the SPME fibers were desorbed at 220 °C for 1 min using a HP-5MS column (Agilent, Waldbronn, Germany). Helium was used as a carrier gas at a constant flow rate of 1 ml/min. The following oven program was used for GC-MS detection: 40 °C for 2 min, then a ramp to 200 °C at a rate of 10 °C/min followed by an additional ramp to 260 °C at a rate of 25 °C/min and then hold for 5 min. The mass spectrometry detector (MSD) parameters were: electron ionization mode, 70 eV; source temperature, 220 °C; quadrupole, 150 °C; full scan with 50 to 500 m/z. The chromatographic peaks of the VOCs were compared with entries in the Mainlib and Replib databases. The VOCs that showed mass spectra with match factors ≥90% were considered identified substances. The VOCs of T-E5 grown in T2’ medium was collected and analyzed according to the above procedures.
This article is protected by copyright. All rights reserved.
Accepted Article
Statistical analysis Data were analyzed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). All data were calculated and analyzed statistically using analyses of variance (ANOVA) and Duncan’s multiple range tests (p≤0.05). The MSD Chemstation G1701EA E.01.00.237 software was employed for GC-MS analyses. The figures were drawn using Sigmaplot 11.0. Results Dual cultures and mycelial interaction The growth of T-E5 was faster than FOC, as shown in Fig. 1A, the petri dish inoculated with FOC only was filled with FOC mycelia at 7 days after inoculation. However, in the dual cultures, the T-E5 mycelium inhibited the growth of FOC, and at 7 days after inoculation, FOC was covered by the T-E5 mycelium completely. The mycelia of T-E5 and FOC in dual cultures that had made contact were observed three days after inoculation. As shown in Fig. 1B, T-E5 mycelium was obviously dense and twisted around the FOC mycelium by close coiling under optical photomicrographs. Scanning electron micrograph was taken for detailed investigation of the interaction. The T-E5 mycelia also observed tightly encircled the FOC mycelium by coiling and parallel growth (Fig. 1C). Analysis of extracellular enzyme activities T-E5 was capable of secreting five enzymes (chitinase, β-1, 3-glucanase, protease, xylanase and cellulase) on both C-source media (Table 1). On the whole, the enzymes in T2 exhibited more amount than T1, especially chitinase and β-1, 3-glucanase. Moreover, cellulase and xylanase with minute quantities were difficult to detect in This article is protected by copyright. All rights reserved.
Accepted Article
this study. The enzyme activities analysis revealed significant differences between chitinase, β-1, 3-glucanase and proteases, and insistently higher activities of chitinase, β-1, 3-glucanase, and proteases were detected from T2 than those from T1 (0-96h). However, there was no significant difference between the two treatments in cellulase and xylanase activities at any sampling time. SDS-PAGE analysis The extracellular proteins obtained from T1 and T2 treatments were harvested and separated using SDS-PAGE. As shown in Fig. 2, the band number and density from T2 treatment was more and higher than those from T1 treatment. The molecular weights of the secreted proteins were concentrated in the range of 25 to 130 kDa, and there was little protein distributed beyond 130 kDa. After staining by R-250, six and twelve major bands were detected in T1 and T2 treatments, respectively, and many minor bands were also found in the gel. Inhibitory activity of T-E5 VOCs The mycelial radial growth of FOC was analyzed to investigate the inhibitory activity of T-E5 VOCs. When compared with control, the VOCs produced by T-E5 showed significant antifungal activity against FOC for the colony diameters of FOC were significantly lower than in control plates 5 days after inoculation (Fig. 3A). The FOC colony in the control plate (8.23 cm) almost covered the entire petri dish at 7 days after inoculation, whereas the colony diameter of FOC in T1’ and T2’ plates were 7.10 and 6.48 cm, respectively. Moreover, the colony diameter in T2’ was significantly decreased 6 days after inoculation, as compared with T1’. Correspondingly, the inhibition rate of T-E5 VOCs against FOC in T2’ was significantly higher than T1’after 6th day (Fig. 3B). This article is protected by copyright. All rights reserved.
Accepted Article
Identification of VOCs The VOC profiles of T1’ and T2’ treatments were different (Fig. 4A, B): some compounds were T2’ specific while some were both produced in the two treatments. A total of 5 and 8 primary fungal metabolites were identified in T1’ and T2’, respectively. The VOCs in Fig. 4 are marked a-e and A-H with respect to retention time. They were identified as (a) pentadecane, (b) α-cubebene, (c) hexahydrofarnesol, (d) pristane and (e) verticillol, and (A) 2,4-di-tert-butylphenol, (B) β-bisabolene, (C) α-curcumene, (D) lignocerane, (E) nerolidol, (F) verticillol, (G) biformen (6CI) and (H) 2,6,10-trimethylundeca-5,9-dienal, respectively (Table 2). Discussion Trichoderma spp. has been widely used as a potent biocontrol agent for a variety of phytopathogenic diseases (Yedidia et al., 2003; Mahalakshmi et al., 2013). The response of Trichoderma to soil-borne fungi (e.g., Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsli) mainly includes these known mechanisms: competition, mycoparasitism, production of antibiotic compounds and induced resistance. Many reports demonstrated that Trichoderma had strong competition for nutrition or space (Sivan and Chet, 1989) or secreted some metabolites (El-Hasan et al., 2008) to suppress the growth of Fusarium. In the present study, T. harzianum T-E5 outgrew FOC, and it inhibited the growth of FOC effectively, which has also been observed in other studies (Chen et al., 2011; Tseng et al., 2008). Trichoderma is frequently used as mycoparasite (Benhamou and Chet, 1996) and biocontrol agent against several plant pathogens. Some reports (Benhamou and Chet, 1993; Lopez-Mondejar et al., 2010) have shown that the mycoparasitism process used by T. harzianum against pathogens, during This article is protected by copyright. All rights reserved.
Accepted Article
which the recognition process was important and was the precondition necessary to damage the pathogen. The specific lectin on the surface of the host cell played an important role in the recognition process of T. harzianum for pathogens (Neethling and Nevalainen., 1996). The mycoparasitism process of coiling, which depends on recognition, was detected clearly in both optical and SEM observations in our research. This phenomenon may indirectly demonstrate that T. harzianum T-E5 was capable of recognizing the FOC lectin effectively and could then continue the process of parasitism. The cell wall-degrading enzymes involved in mycoparasitism comprise primarily of various hydrolases. During our experiments, we evaluated the activities of major extracellular enzymes produced by T-E5 in two different media. Based on the results that activities of chitinase, β-1, 3-glucanase and protease produced in T2 were significantly higher than those produced in T1, we conclude that the expression of extracellular enzymes depends on the type of C-source, and the deactivated FOC cell walls was capable of inducing T-E5 to secrete more proteins. El-katatny et al. (2000) also revealed that deactivated cell wall was more effective in inducing the production of extracellular enzymes. All of these results are consistent with the protein profiles of the crude protein samples obtained from the cultures maintained in glucose or deactivated FOC cell walls determined using SDS-PAGE. Research from Tseng et al. (2008) also showed that deactivated pathogenic mycelia induced T. harzianum to produce both more proteins and greater levels of these proteins. The study of Daniela et al. (2009) demonstrates that VOCs are considered as a potential direct long distance mechanism for antagonistic action. However, the antagonism induced by VOCs themselves produced by Trichoderma species during the process of biological control has been poorly studied. Here, the VOCs showed antifungal activities and were proven to be useful for biological control. In our study, This article is protected by copyright. All rights reserved.
Accepted Article
the excreted volatile metabolites showed significantly higher antifungal activities against FOC when T-E5 was cultured in a biocontrol environment (T2’) than in T1’ (Fig. 3). Moreover, the antagonism of T-E5 VOCs occurred as a significant inhibition of FOC mycelial growth but not by killing FOC, which is consistent with the results of Wang et al. (2013). The whole biocontrol effects of VOCs were closely related with the antifungal activity of each component. Thus, the VOCs that showed antifungal activities against FOC were further detected and identified by GC-MS. Different Trichoderma species or different culturing conditions can induce the production of different VOCs. Different media (T1’ and T2’) influenced the types of volatile metabolites produced greatly, which is consistent with the report of Polizzi et al. (2011). The types and compositions of VOCs were similar in part to those reported by Stoppacher et al. (2010) and Gal-hemed et al. (2011), which mainly included alkanes, alkenes and alcohols. For example, some alkenes showed significant antifungal activities against pathogens, which is primarily because the structures of these compounds are multifaceted and they typically exhibit antimicrobial and antiviral activities (Fraga, 2008). All of these results prove a potential biocontrol mechanism of T-E5 VOCs against FOC. In conclusion, T. harzianum T-E5 is a potent agent against F. oxysporum f. sp. cucumerinum, a property linked to the direct parasitism, production of exoenzymes and VOCs, which helps to better understand the biocontrol process of T. harzianum T-E5. These antifungal traits are furthermore induced and upregulated in the presence of FOC. We will exploit the molecular antagonism mechanisms involved in suppressing fusarium wilt disease in future research.
This article is protected by copyright. All rights reserved.
Accepted Article
Acknowledgements This work was supported financially by the Innovative Research Team Development Plan of the Ministry of Education of China (IRT1256), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the 111 project (B12009), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Chinese Ministry of Science and Technology (2011BAD11B03) and the Chinese Ministry of Agriculture (201103004).
References Benhamou N, Chet I (1993) Hyphal interactions between Trichoderma harzianum and Rhizoctonia solani - ultrastructure and gold cytochemistry of the mycoparasitic process. Phytopathology 83: 1062-1071. Benhamou N, Chet I (1996) Parasitism of sclerotia of Sclerotium rolfsii by Trichoderma harzianum: Ultrastructural and cytochemical aspects of the interaction. Phytopathology 86: 405-416. Benitez T, Rincon, AM, Limon MC, Codon AC (2004) Biocontrol mechanisms of Trichoderma strains. Int Microbiol 7: 249-260. Boller T, Mauch F (1988) Colorimetric assay for chitinase. Meth Enzymol 161: 430-435. Bruce A, Wheatley RE, Humphris SN, Hackett CA, Florence MEJ (2000) Production of volatile organic compounds by Trichoderma in media containing different amino acids and their effect on selected wood decay fungi. Holzforschung 54: 481-486. Cao Y, Xu Z, Ling N, Yuan Y, Yang X, Chen L, Shen B, Shen Q (2012) Isolation and identification of lipopeptides produced by B. subtilis SQR 9 for suppressing Fusarium wilt of cucumber. Sci Hortic 135: 32-39. Chen L, Yang X, Raza W, Li J, Liu Y, Qiu M, Zhang F, Shen Q (2001) Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers. Appl Microbiol Biotechnol 89: 1653-1663. Compant S, Duffy B, Nowak J, Clement C, Barka EA (2005) Use of plant growth-promoting bacteria for biocontrol of plant diseases: Principles,
This article is protected by copyright. All rights reserved.
Accepted Article
mechanisms of action, and future prospects. Appl Environ Microbiol 71: 4951-4959. Daniela M, Simone B, Maria LG, Angelo G (2009) Volatile organic compounds: a potential direct long-distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35. Environ Microbiol 11: 844-854. El-Katatny MH, Somitsch W, Robra KH, El-Katatny MS, Gubitz GM (2000) Production of chitinase and beta-1,3-glucanase by Trichoderma harzianum for control of the phytopathogenic fungus Sclerotium rolfsii. Food Technol Biotech 38: 173-180. El-Hasan A, Walker F, Buchenauer H (2008) Trichoderma harzianum and its metabolite 6-Pentyl-alpha-pyrone suppress fusaric acid produced by Fusarium moniliforme. J Phytopathol 156: 79-87. Fraga BM (2008) Natural sesquiterpenoids. Natural Product Reports 25: 1180-1209. Gal-Hemed I, Atanasova L, Komon-Zelazowska M, Druzhinina IS, Viterbo A, Yarden O (2011) Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculturE. Appl Microbiol Biotechnol 77: 5100-5109. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species Opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2: 43-56. Huang X, Chen L, Ran W, Shen Q, Yang X (2011) Trichoderma harzianum strain SQR-T37 and its bio-organic fertilizer could control Rhizoctonia solani damping-off disease in cucumber seedlings mainly by the mycoparasitism. Appl Microbiol Biotechnol 91: 741-755. Korpi A, Jarnberg J, Pasanen AL (2009) Microbial volatile organic compounds. Crit Rev Toxicol 39: 139-193. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680. Lee YG, Chung KC, Wi SG, Lee JC, Bae HJ (2009) Purification and properties of a chitinase from Penicillium sp LYG 0704. Protein Expres Purif 65: 244-250. Liao HP, Xu CM, Tan SY, Wei Z, Ling N, Yu GH, Raza W, Zhang RF, Shen QR, Xu YC (2012) Production and characterization of acidophilic xylanolytic enzymes from Penicillium oxalicum GZ-2. Bioresour Technol 123: 117-124. Lopez-Mondejar R, Anton A, Raidl S, Ros M, Antonio Pascual J (2010) Quantification of the biocontrol agent Trichoderma harzianum with real-time TaqMan PCR and its potential extrapolation to the hyphal biomass. Bioresour Technol 101: 2888-2891. This article is protected by copyright. All rights reserved.
Accepted Article
Mahalakshmi P, Raja I. Y (2013) Biocontrol potential of Trichoderma species against wilt disease of carnation (Dianthus caryophyllus L.) caused by Fusarium oxysporum f. sp. dianthi. J Biopest 6:32-36. Margolles-Clark E, Hayes CK, Harman GE, Pentillä M (1996) Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei. Appl Environ Microb 62: 2145-2151. Mendoza-Mendoza A, Pozo MJ, Grzegorski D, Martinez P, Garcia JM, Olmedo-Monfil V, Cortes C, Kenerley C, Herrera-Estrella A (2003) Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase. Proceedings of the National Academy of Sciences of the United States of America 100: 15965-15970. Neethling D, Nevalainen H (1996) Mycoparasitic species of Trichoderma produce lectins. Can J Microbiol 42: 141-146. Pan SQ, Ye XS, Kuc J (1991) Association of β-1, 3-glucanase activity and isoform pattern with systemic resistance to blue mold in tobacco induced by stem injection with Peronospora tabacina or leaf inoculation with Tobacco mosaic virus. Physiol Mol Plant Pathol 39: 25-39. Polizzi V, Adams A, Picco AM, Adriaens E, Lenoir J, Van Peteghem C, De Saeger S, De Kimpe N (2011) Influence of environmental conditions on production of volatiles by Trichoderma atroviride in relation with the sick building syndrome. Build Environ 46: 945-954. Sivan A, Chet I (1989) The possible role of competition between Trichoderma harzianum
and
Fusarium
oxysporum
on
rhizosphere
colonization.
Phytopathology 79: 198-203. Stoppacher N, Kluger B, Zeilinger S, Krska R, Schuhmacher R (2010) Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride by HS-SPME-GC-MS. J Microbiol Meth 81: 187-193. Strobel GA, Dirkse E, Sears J, Markworth C (2001) Volatile antimicrobials from Muscodor albus, a novel endophytic fungus. Microbiology-SGM 147: 2943-2950. Tseng SC, Liu SY, Yang HH, Lo CT, Peng KC (2008) Proteomic study of biocontrol mechanisms of Trichoderma harzianum ETS 323 in response to Rhizoctonia solani. J Agric Food Chem 56: 6914-6922. Wang BB, Yuan J, Zhang J, Shen ZZ, Zhang MX, Li R, Ruan YZ, Shen QR (2013) Effects of novel bioorganic fertilizer produced by Bacillus amyloliquefaciens W19 on antagonism of Fusarium wilt of banana. Biol Fert Soils 4: 435-446.
This article is protected by copyright. All rights reserved.
Accepted Article
Yang HH, Yang SL, Peng KC, Chaur-Tsuen LO, Liu SY (2009) Induced proteome of Trichoderma harzianum by Botrytis cinerea. Mycol Res 113: 924-932. Yedidia I, Michal S, Zohar K, Nicole B, Yoram K, Ilan C (2003) Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins. Appl Environ Microbiol 69: 7343-7353. Yuan J, Raza W, Shen Q, Huang Q (2012) Antifungal Activity of Bacillus amyloliquefaciens NJN-6 volatile compounds against Fusarium oxysporum f. sp cubense. Appl Environ Microbiol 78: 5942-5944. Zhang FG, Zhu Z, Yang XM, Ran W, Shen QR (2013a) Trichoderma harzianum T-E5 significantly affects cucumber root exudates and fungal community in the cucumber rhizosphere. Appl Soil Ecol 72: 41-48. Zhang FG, Yuan J, Yang XM, Cui YQ, Chen LH, Ran W, Shen QR (2013b) Putative Trichoderma harzianum mutant promotes cucumber growth by enhanced production of indole acetic acid and plant colonization. Plant Soil 368: 433-444. Zhao Q, Dong C, Yang X, Mei X, Ran W, Shen QR, Xu YC (2011) Biocontrol of Fusarium wilt disease for Cucumis melo melon using bio-organic fertilizer. Appl Soil Ecol 47.
Fig. 1 (A) Dual cultures (7 days after inoculation) of T. harzianum T-E5 and F. oxysporum f. sp. cucumerinum; (B) Observation of mycelial interactions between T. harzianum T-E5 and F. oxysporum f. sp. cucumerinum at 72 h using optical microscopy (OLYMPUS CX21, Tokyo, Japan, ×400); (C) Observation of the mycoparasitism of T. harzianum T-E5 against F. oxysporum f. sp. cucumerinum at 72 h using a scanning electron microscope (HITACHI S-4800 FESEM, Tokyo, Japan). Fig. 2 Extracellular protein extracts of T. harzianum T-E5 resolved using 10% SDS-PAGE. M denotes the lane of molecular weight markers. Fig. 3 (A) The colony diameters of F. oxysporum f. sp. cucumerinum in control, T1’ and T2’ treatment plates at different days after inoculation; (B) The inhibition rate of
This article is protected by copyright. All rights reserved.
Accepted Article
T-E5 VOCs against F. oxysporum f. sp. cucumerinum at different days after inoculation. Fig. 4 The GC profiles of volatile components produced by T. harzianum T-E5 in T1’ and T2’ treatments. Peaks of compounds produced in the T1’ treatment with retention times of 12.86, 12.92, 15.38, 15.60, and 16.48 are denoted a-e, respectively. Peaks of compounds produced in the T2’ treatment with retention times of 11.18, 12.02, 13.19, 13.37, 13.88, 16.49, 17.05 and 17.89 are denoted A-H, respectively. NL: Nominal level.
Fig. 1
This article is protected by copyright. All rights reserved.
Accepted Article
Fig. 2
This article is protected by copyright. All rights reserved.
Accepted Article
Fig. 3
This article is protected by copyright. All rights reserved.
Accepted Article
Fig. 4
This article is protected by copyright. All rights reserved.
Accepted Article
Table 1 Extracellular enzyme activities (Units/mg) of T. harzianum T-E5 in two different media at 24, 48, 72, 96 h after inoculation Time
Treatments
Chitinases -1
(U mg )
β-1,3-glucanase s
Proteases -1
Xylanases -1
Cellulases
(U mg )
(U mg )
(U mg-1)
(U mg-1) 24h 48h 72h 96h
T1
3.75±0.49b
2.42±0.36b
1.2±0.12b
0.43±0.07a
1.39±0.12a
T2
11.75±1.02a
18.07±1.17a
5.7±0.61a
0.51±0.05a
1.54±0.09a
T1
3.11±0.37b
3.54±0.41b
2.3±0.22b
0.52±0.02a
1.48±0.11a
T2
24.31±1.21a
20.01±1.18a
12.4±1.19a
0.54±0.05a
1.62±0.13a
T1
1.81±0.21b
4.07±0.58b
3.41±0.46b
0.45±0.09a
1.59±0.12a
T2
30.31±1.54a
23.12±1.29a
6.24±0.89a
0.61±0.07a
1.73±0.15a
T1
1.63±0.09b
3.94±0.46b
3.21±0.33b
0.48±0.05a
1.48±0.11a
T2
36.75±2.31a
24.17±1.54a
5.93±0.74a
0.56±0.03a
1.61±0.14a
Notes: The data were analyzed using Duncan’s ANOVA tests. All values are of the mean of three replicates. Values with different letters within the same column are significantly different, P≤0.05. Numbers following “±” are the standard errors (SEs).
This article is protected by copyright. All rights reserved.
Accepted Article
Table 2 VOCs of T. harzianum T-E5 cultured in T1’ and T2’ medium extracted using SPME were identified by GC-MS Treatments
T1’
T2’
Retention time (min)
Volatile Metabolites markers
names
12.86
a
Pentadecane
12.92
b
α-cubebene
15.38
c
Hexahydrofarnesol
15.60
d
Pristane
16.48
e
Verticillol
11.18
A
2,4-Di-tert-butylphenol
12.02
B
β-Bisabolene
13.19
C
α-curcumene
13.37
D
lignocerane
13.88
E
Nerolidol
16.49
F
Verticillol
17.05
G
17.89
H
Biformen (6CI) 2,6,10-trimethylundeca-5,9-dienal
This article is protected by copyright. All rights reserved.
Received Date : 22-Jul-2014 Accepted Date : 13-Aug-2014 Article type Editor:
: Research Letter
Paolina Garbeva
Fusarium oxysporum induces the production of proteins and volatile organic compounds by Trichoderma harzianum T-E5
Fengge Zhang1, Xingming Yang1,2, Wei Ran∗1,2, Qirong Shen1,2
1
National Engineering Research Center for Organic-based Fertilizers, Nanjing
Agricultural University, Nanjing, 210095, China 2
Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization,
Nanjing Agricultural University, Nanjing, 210095, China
Corresponding author: Wei Ran
∗
Corresponding author E-mail address: [email protected] (W. Ran)
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12582 This article is protected by copyright. All rights reserved.
Accepted Article
Postal address: College of Resources and Environmental Sciences, Nanjing Agricultural University, 210095, Nanjing, Jiangsu Province, P. R. of China.
E-mail address: [email protected] Tel: 0086-025-84396212 Fax: 0086-025-84396824
Abstract Trichoderma species have been used widely as biocontrol agents for the suppression of soil-borne pathogens. However, some antagonistic mechanisms of Trichoderma are not well characterized. In this study, a series of laboratory experiments were designed to characterize the importance of mycoparasitism, exoenzymes and volatile organic compounds (VOCs) by T. harzianum T-E5 for the control of F. oxysporum f. sp. cucumerinum (FOC). We further tested if these mechanisms were inducible and upregulated in presence of FOC. The results were as follows: T-E5 heavily parasitized FOC by coiling and twisting the entire mycelium of the pathogen in dual cultures. T-E5 growing medium conditioned with deactivated FOC (T2) showed more proteins and higher cell wall-degrading enzyme activities than T1, suggesting that FOC could induce the upregulation of exoenzymes. The presence of deactivated FOC (T2’) also resulted in the upregulation of VOCs that 5 and 8 different types T-E5-derived VOCs were identified from T1’ and T2’, respectively. Further, the excreted VOCs in T2’ showed significantly higher antifungal activities against FOC than T1’. In conclusion, mycoparasitism of T-E5
This article is protected by copyright. All rights reserved.
Accepted Article
against FOC involved mycelium contact and the production of complex extracellular substances. Together, these data provide clues to help further clarify the interactions between these fungi. Keywords: Mycoparasitism; Proteins; SDS-PAGE; Volatile organic compounds; Head-space solid-phase micro-extraction (HS-SPME)
Introduction Cucumber (Cucumis sativus) is one of the most important economic crops cultivated and consumed worldwide. However, when continuous cropping occurs in the same field over many years, fusarium wilt disease can easily threaten the crop. This disease is caused by the soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum (FOC), and it will result in severe plant damage and economic losses for farmers. The pathogen typically infects the cucumber vascular system and then the plants develop necrotic lesions on their stems and leaves. Fusarium wilt develops, and the cucumber plants die within a few days. Traditionally, chemical controls can effectively protect plants from infectious pathogens (Compant et al., 2005). However, the drawbacks of the chemical fungicides are obvious when pathogen resistance to pesticides, food safety and environmental quality are considered. Therefore, finding an effective and environment-friendly strategy to control fusarium wilt disease appears essential and urgent for the development of the cucumber industry. Biocontrol by applying antagonistic microorganisms instead of chemicals has been studied widely for the management of fusarium wilt disease and other soil-borne diseases (Cao et al., 2012; Zhao et al., 2012). Several soil fungal Trichoderma species have been approved as effective biocontrol agents in the inhibition of many soil-borne This article is protected by copyright. All rights reserved.
Accepted Article
pathogens (Margolles Clark et al., 1996). Recently, Trichoderma strains have been used in the biocontrol of fusarium wilt and damping-off diseases of cucumber (Chen et al., 2011; Huang et al., 2011). The biocontrol effect of Trichoderma species is achieved by a variety of antagonistic mechanisms. Mycoparasitism, characterized by the direct attack of one fungus against another, is considered an important antagonistic mechanism of Trichoderma species (Benitez et al., 2004; Mendoza-Mendoza et al., 2003). The induced production of some extracellular substances (e.g., cell wall-degrading enzymes, antifungal VOCs) is also regarded as momentous mechanisms of Trichoderma against fungal pathogens. In mycoparasitism, Trichoderma grows towards the pathogen’s hypha by paralleling or coiling (Huang et al., 2011) and generates cellular attachments. The mycoparasite then excretes various extracellular cell wall-degrading enzymes, such as chitinases, β-1, 3-glucanases, proteases and xylanases, and penetrates the pathogen hyphae to absorb nutrients (Harman et al., 2004). Lastly, the entire pathogen system collapses with mycelial cell-wall breakdown and leakage of the inner cytoplasm. However, few studies have been designed to link mycoparasitism to the induction of extracellular enzymes and VOCs using growth substrates that originate from a plant pathogen.VOCs are the end products of many metabolic pathways, and they include many classes, such as sesquiterpenes, alcohols and ketones, among others (Korpi et al., 2009). The report of Stoppacher et al. (2010) showed that these volatile metabolites take part in varying biological processes. For example, VOCs can significantly inhibit the mycelial growth and spore germination of F. oxysporum f. sp. cubense (Yuan et al., 2012). The determination of volatile fungal metabolites is typically accomplished using gas chromatographic mass spectrometry (GC-MS). There are many reports (Bruce et al., 2000; Polizzi et al., This article is protected by copyright. All rights reserved.
Accepted Article
2011) that have examined Trichoderma VOCs although little research has been conducted on the induction of Trichoderma VOCs to gain insight into mycoparasitism and the antagonism effects of the VOCs on FOC. These advances will facilitate a better understanding of the antagonistic mechanisms of Trichoderma species. The biocontrol fungus T. harzianum T-E5 was reported to significantly suppress fusarium wilt of cucumber in our previous pot experiments (Zhang et al., 2013). The objective of this study was to investigate the mechanisms used by T. harzianum T-E5 in antagonizing fusarium wilt. The possible mechanisms, mycoparasitism and productions of proteins and VOCs that underlie the T-E5 biocontrol process for fusarium wilt were evaluated in vitro. Materials and methods Microorganisms, deactivated FOC cell walls and media Trichoderma harzianum T-E5 (CCTCC No. AF2012011, China Center for Type Culture Collection) was used throughout this study (Zhang et al., 2013a; Zhang et al., 2013b). The soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum (FOC, Agricultural Culture Collection of China (ACCC) No.30220) was provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Both strains were usually cultured on potato dextrose agar (PDA) medium. The conidia suspensions of FOC were prepared based on Zhang et al. (2013a). The conidia concentration was determined using hemocytometer counts, and the final concentration was 1.48 × 107 CFU mL-1. The preparation of deactivated FOC cell walls was according to El-Katatny et al. (2000) and Yang et al. (2009). The minimal medium (10 g C-source, 6.9 g NaH2PO4, 2.0 g KH2PO4, 1.4 g (NH4)2SO4, 1.0 g peptone, 0.3 g MgSO4.7H2O and 0.3 g urea in 1 L distilled water, This article is protected by copyright. All rights reserved.
Accepted Article
pH 5.0) and solid minimal medium (20 g agar in 1 L minimal medium) were used in the followed experiment. T1 was the minimal medium supplemented with glucose as a C-source, T2 was the minimal medium supplemented with deactivated FOC cell walls as a C-source; T1’ and T2’ were the solid form of T1 and T2. Dual cultures and observations of mycoparasitism T. harzianum T-E5 antagonism of FOC was evaluated in dual culture tests (Huang et al., 2011). A PDA plate inoculated with FOC only was used as a control. All plates were incubated at 28 °C for 7 days. For observations of mycoparasitism, T-E5 and FOC were inoculated in dual cultures. We placed some small cover slips at the boundary of the two strains. After 3 days of dual culture, some of the cover slips were chosen for observation of interactions using an optical microscope (OLYMPUS CX21, Tokyo, Japan). The other cover slips were fixed using 2.5% glutaraldehyde and then observed using a scanning electron microscope (HITACHI S-4800 FESEM, Tokyo, Japan). Imitation of the biocontrol environment T. harzianum T-E5 conidia suspensions were prepared according to FOC. A 2-ml T-E5 conidia suspension (2.27 × 107 CFU mL-1) was inoculated into 250 ml T1 and T2 minimal medium, respectively. After inoculation, T1 and T2 were incubated in a shaking incubator at 28 °C/180 rpm for 96 h. The cultures were collected every day after inoculation (24, 48, 72 and 96 h), and they were filtered using filter paper (Whatman No.1, φ11 cm) and then a 0.45-μm filter membrane. The filtrates were used for enzyme activities assays and protein extraction. Determination of protein content and enzyme activities This article is protected by copyright. All rights reserved.
Accepted Article
The protein content of the two filtrates was assayed using a Bicinchoninic acid (BCA) protein assay kit (Dingguo Changsheng Biotechnology, Co. Ltd., Beijing, China). The activities of chitinases, β-1, 3-glucanases, cellulases, and xylanases were determined according to previous researches (Boller and Mauch., 1988; Pan et al., 1991; Liao et al., 2012). Protease activity was determined using a protease activity assay kit (Abvona Biotechnology, Co. Ltd., Taiwan), following the manufacturer’s instructions. Extraction of proteins and SDS-PAGE The solid ammonium sulfate precipitation method was used to extract crude proteins, and the entire extraction process was completed in an ice bath (Liao et al., 2012). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed following the procedures of Laemmli (1970) with the Mini-Protean II system (Bio-Rad, USA). Antagonistic assay of VOCs from T-E5 against F. oxysporum f. sp. cucumerinum For the antagonistic assay of T-E5 VOCs against FOC, a fresh 5-mm-diameter mycelial disc of T. harzianum T-E5 was inoculated into the center of a Petri dish which containing T1’ and T2’ minimal medium respectively. The lid of each plate was replaced by the bottom of a plate containing 1/4 PDA inoculated with a 5-mm-diameter mycelial disc of FOC. Then the two plates were sealed together using adhesive tape and incubated at 28 °C for 7 days in the dark. The plate with FOC only was used as control and three replicates were designed for both T1’ and T2’. The colony diameters of FOC were measured every day after incubation and compared with those of control plates.
This article is protected by copyright. All rights reserved.
Accepted Article
Collection of VOCs using head-space solid-phase micro-extraction (HS-SPME) and GC-MS analysis The VOCs of T-E5 grown in T1’ and T2’ were collected using head-space-solid-phase micro-extraction (HS-SPME) (Strobel et al., 2001). An SPME fiber of 50/30 µm divinylbenzene (DVB)/ polydimethylsiloxane (PDMS) (Supelco, Vienna, Austria) was selected as an appropriate fiber coating for extractions. A fresh 5-mm-diameter T-E5 mycelial disc was inoculated into a vial (200 ml) containing 50 ml T1’ minimal medium. In parallel, a SPME syringe with a fiber (50/30 µm DVB/ PDMS) was inserted into the headspace of the vial. The vial was placed at 28 °C, and the fiber was then exposed to absorb the volatiles during the growth process of T-E5. Seven days after inoculation, the syringe containing the volatiles was inserted into a gas chromatography-mass spectrometer (GC-MS). The volatiles bound to the SPME fibers were desorbed at 220 °C for 1 min using a HP-5MS column (Agilent, Waldbronn, Germany). Helium was used as a carrier gas at a constant flow rate of 1 ml/min. The following oven program was used for GC-MS detection: 40 °C for 2 min, then a ramp to 200 °C at a rate of 10 °C/min followed by an additional ramp to 260 °C at a rate of 25 °C/min and then hold for 5 min. The mass spectrometry detector (MSD) parameters were: electron ionization mode, 70 eV; source temperature, 220 °C; quadrupole, 150 °C; full scan with 50 to 500 m/z. The chromatographic peaks of the VOCs were compared with entries in the Mainlib and Replib databases. The VOCs that showed mass spectra with match factors ≥90% were considered identified substances. The VOCs of T-E5 grown in T2’ medium was collected and analyzed according to the above procedures.
This article is protected by copyright. All rights reserved.
Accepted Article
Statistical analysis Data were analyzed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). All data were calculated and analyzed statistically using analyses of variance (ANOVA) and Duncan’s multiple range tests (p≤0.05). The MSD Chemstation G1701EA E.01.00.237 software was employed for GC-MS analyses. The figures were drawn using Sigmaplot 11.0. Results Dual cultures and mycelial interaction The growth of T-E5 was faster than FOC, as shown in Fig. 1A, the petri dish inoculated with FOC only was filled with FOC mycelia at 7 days after inoculation. However, in the dual cultures, the T-E5 mycelium inhibited the growth of FOC, and at 7 days after inoculation, FOC was covered by the T-E5 mycelium completely. The mycelia of T-E5 and FOC in dual cultures that had made contact were observed three days after inoculation. As shown in Fig. 1B, T-E5 mycelium was obviously dense and twisted around the FOC mycelium by close coiling under optical photomicrographs. Scanning electron micrograph was taken for detailed investigation of the interaction. The T-E5 mycelia also observed tightly encircled the FOC mycelium by coiling and parallel growth (Fig. 1C). Analysis of extracellular enzyme activities T-E5 was capable of secreting five enzymes (chitinase, β-1, 3-glucanase, protease, xylanase and cellulase) on both C-source media (Table 1). On the whole, the enzymes in T2 exhibited more amount than T1, especially chitinase and β-1, 3-glucanase. Moreover, cellulase and xylanase with minute quantities were difficult to detect in This article is protected by copyright. All rights reserved.
Accepted Article
this study. The enzyme activities analysis revealed significant differences between chitinase, β-1, 3-glucanase and proteases, and insistently higher activities of chitinase, β-1, 3-glucanase, and proteases were detected from T2 than those from T1 (0-96h). However, there was no significant difference between the two treatments in cellulase and xylanase activities at any sampling time. SDS-PAGE analysis The extracellular proteins obtained from T1 and T2 treatments were harvested and separated using SDS-PAGE. As shown in Fig. 2, the band number and density from T2 treatment was more and higher than those from T1 treatment. The molecular weights of the secreted proteins were concentrated in the range of 25 to 130 kDa, and there was little protein distributed beyond 130 kDa. After staining by R-250, six and twelve major bands were detected in T1 and T2 treatments, respectively, and many minor bands were also found in the gel. Inhibitory activity of T-E5 VOCs The mycelial radial growth of FOC was analyzed to investigate the inhibitory activity of T-E5 VOCs. When compared with control, the VOCs produced by T-E5 showed significant antifungal activity against FOC for the colony diameters of FOC were significantly lower than in control plates 5 days after inoculation (Fig. 3A). The FOC colony in the control plate (8.23 cm) almost covered the entire petri dish at 7 days after inoculation, whereas the colony diameter of FOC in T1’ and T2’ plates were 7.10 and 6.48 cm, respectively. Moreover, the colony diameter in T2’ was significantly decreased 6 days after inoculation, as compared with T1’. Correspondingly, the inhibition rate of T-E5 VOCs against FOC in T2’ was significantly higher than T1’after 6th day (Fig. 3B). This article is protected by copyright. All rights reserved.
Accepted Article
Identification of VOCs The VOC profiles of T1’ and T2’ treatments were different (Fig. 4A, B): some compounds were T2’ specific while some were both produced in the two treatments. A total of 5 and 8 primary fungal metabolites were identified in T1’ and T2’, respectively. The VOCs in Fig. 4 are marked a-e and A-H with respect to retention time. They were identified as (a) pentadecane, (b) α-cubebene, (c) hexahydrofarnesol, (d) pristane and (e) verticillol, and (A) 2,4-di-tert-butylphenol, (B) β-bisabolene, (C) α-curcumene, (D) lignocerane, (E) nerolidol, (F) verticillol, (G) biformen (6CI) and (H) 2,6,10-trimethylundeca-5,9-dienal, respectively (Table 2). Discussion Trichoderma spp. has been widely used as a potent biocontrol agent for a variety of phytopathogenic diseases (Yedidia et al., 2003; Mahalakshmi et al., 2013). The response of Trichoderma to soil-borne fungi (e.g., Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsli) mainly includes these known mechanisms: competition, mycoparasitism, production of antibiotic compounds and induced resistance. Many reports demonstrated that Trichoderma had strong competition for nutrition or space (Sivan and Chet, 1989) or secreted some metabolites (El-Hasan et al., 2008) to suppress the growth of Fusarium. In the present study, T. harzianum T-E5 outgrew FOC, and it inhibited the growth of FOC effectively, which has also been observed in other studies (Chen et al., 2011; Tseng et al., 2008). Trichoderma is frequently used as mycoparasite (Benhamou and Chet, 1996) and biocontrol agent against several plant pathogens. Some reports (Benhamou and Chet, 1993; Lopez-Mondejar et al., 2010) have shown that the mycoparasitism process used by T. harzianum against pathogens, during This article is protected by copyright. All rights reserved.
Accepted Article
which the recognition process was important and was the precondition necessary to damage the pathogen. The specific lectin on the surface of the host cell played an important role in the recognition process of T. harzianum for pathogens (Neethling and Nevalainen., 1996). The mycoparasitism process of coiling, which depends on recognition, was detected clearly in both optical and SEM observations in our research. This phenomenon may indirectly demonstrate that T. harzianum T-E5 was capable of recognizing the FOC lectin effectively and could then continue the process of parasitism. The cell wall-degrading enzymes involved in mycoparasitism comprise primarily of various hydrolases. During our experiments, we evaluated the activities of major extracellular enzymes produced by T-E5 in two different media. Based on the results that activities of chitinase, β-1, 3-glucanase and protease produced in T2 were significantly higher than those produced in T1, we conclude that the expression of extracellular enzymes depends on the type of C-source, and the deactivated FOC cell walls was capable of inducing T-E5 to secrete more proteins. El-katatny et al. (2000) also revealed that deactivated cell wall was more effective in inducing the production of extracellular enzymes. All of these results are consistent with the protein profiles of the crude protein samples obtained from the cultures maintained in glucose or deactivated FOC cell walls determined using SDS-PAGE. Research from Tseng et al. (2008) also showed that deactivated pathogenic mycelia induced T. harzianum to produce both more proteins and greater levels of these proteins. The study of Daniela et al. (2009) demonstrates that VOCs are considered as a potential direct long distance mechanism for antagonistic action. However, the antagonism induced by VOCs themselves produced by Trichoderma species during the process of biological control has been poorly studied. Here, the VOCs showed antifungal activities and were proven to be useful for biological control. In our study, This article is protected by copyright. All rights reserved.
Accepted Article
the excreted volatile metabolites showed significantly higher antifungal activities against FOC when T-E5 was cultured in a biocontrol environment (T2’) than in T1’ (Fig. 3). Moreover, the antagonism of T-E5 VOCs occurred as a significant inhibition of FOC mycelial growth but not by killing FOC, which is consistent with the results of Wang et al. (2013). The whole biocontrol effects of VOCs were closely related with the antifungal activity of each component. Thus, the VOCs that showed antifungal activities against FOC were further detected and identified by GC-MS. Different Trichoderma species or different culturing conditions can induce the production of different VOCs. Different media (T1’ and T2’) influenced the types of volatile metabolites produced greatly, which is consistent with the report of Polizzi et al. (2011). The types and compositions of VOCs were similar in part to those reported by Stoppacher et al. (2010) and Gal-hemed et al. (2011), which mainly included alkanes, alkenes and alcohols. For example, some alkenes showed significant antifungal activities against pathogens, which is primarily because the structures of these compounds are multifaceted and they typically exhibit antimicrobial and antiviral activities (Fraga, 2008). All of these results prove a potential biocontrol mechanism of T-E5 VOCs against FOC. In conclusion, T. harzianum T-E5 is a potent agent against F. oxysporum f. sp. cucumerinum, a property linked to the direct parasitism, production of exoenzymes and VOCs, which helps to better understand the biocontrol process of T. harzianum T-E5. These antifungal traits are furthermore induced and upregulated in the presence of FOC. We will exploit the molecular antagonism mechanisms involved in suppressing fusarium wilt disease in future research.
This article is protected by copyright. All rights reserved.
Accepted Article
Acknowledgements This work was supported financially by the Innovative Research Team Development Plan of the Ministry of Education of China (IRT1256), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the 111 project (B12009), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Chinese Ministry of Science and Technology (2011BAD11B03) and the Chinese Ministry of Agriculture (201103004).
References Benhamou N, Chet I (1993) Hyphal interactions between Trichoderma harzianum and Rhizoctonia solani - ultrastructure and gold cytochemistry of the mycoparasitic process. Phytopathology 83: 1062-1071. Benhamou N, Chet I (1996) Parasitism of sclerotia of Sclerotium rolfsii by Trichoderma harzianum: Ultrastructural and cytochemical aspects of the interaction. Phytopathology 86: 405-416. Benitez T, Rincon, AM, Limon MC, Codon AC (2004) Biocontrol mechanisms of Trichoderma strains. Int Microbiol 7: 249-260. Boller T, Mauch F (1988) Colorimetric assay for chitinase. Meth Enzymol 161: 430-435. Bruce A, Wheatley RE, Humphris SN, Hackett CA, Florence MEJ (2000) Production of volatile organic compounds by Trichoderma in media containing different amino acids and their effect on selected wood decay fungi. Holzforschung 54: 481-486. Cao Y, Xu Z, Ling N, Yuan Y, Yang X, Chen L, Shen B, Shen Q (2012) Isolation and identification of lipopeptides produced by B. subtilis SQR 9 for suppressing Fusarium wilt of cucumber. Sci Hortic 135: 32-39. Chen L, Yang X, Raza W, Li J, Liu Y, Qiu M, Zhang F, Shen Q (2001) Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers. Appl Microbiol Biotechnol 89: 1653-1663. Compant S, Duffy B, Nowak J, Clement C, Barka EA (2005) Use of plant growth-promoting bacteria for biocontrol of plant diseases: Principles,
This article is protected by copyright. All rights reserved.
Accepted Article
mechanisms of action, and future prospects. Appl Environ Microbiol 71: 4951-4959. Daniela M, Simone B, Maria LG, Angelo G (2009) Volatile organic compounds: a potential direct long-distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35. Environ Microbiol 11: 844-854. El-Katatny MH, Somitsch W, Robra KH, El-Katatny MS, Gubitz GM (2000) Production of chitinase and beta-1,3-glucanase by Trichoderma harzianum for control of the phytopathogenic fungus Sclerotium rolfsii. Food Technol Biotech 38: 173-180. El-Hasan A, Walker F, Buchenauer H (2008) Trichoderma harzianum and its metabolite 6-Pentyl-alpha-pyrone suppress fusaric acid produced by Fusarium moniliforme. J Phytopathol 156: 79-87. Fraga BM (2008) Natural sesquiterpenoids. Natural Product Reports 25: 1180-1209. Gal-Hemed I, Atanasova L, Komon-Zelazowska M, Druzhinina IS, Viterbo A, Yarden O (2011) Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculturE. Appl Microbiol Biotechnol 77: 5100-5109. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species Opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2: 43-56. Huang X, Chen L, Ran W, Shen Q, Yang X (2011) Trichoderma harzianum strain SQR-T37 and its bio-organic fertilizer could control Rhizoctonia solani damping-off disease in cucumber seedlings mainly by the mycoparasitism. Appl Microbiol Biotechnol 91: 741-755. Korpi A, Jarnberg J, Pasanen AL (2009) Microbial volatile organic compounds. Crit Rev Toxicol 39: 139-193. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680. Lee YG, Chung KC, Wi SG, Lee JC, Bae HJ (2009) Purification and properties of a chitinase from Penicillium sp LYG 0704. Protein Expres Purif 65: 244-250. Liao HP, Xu CM, Tan SY, Wei Z, Ling N, Yu GH, Raza W, Zhang RF, Shen QR, Xu YC (2012) Production and characterization of acidophilic xylanolytic enzymes from Penicillium oxalicum GZ-2. Bioresour Technol 123: 117-124. Lopez-Mondejar R, Anton A, Raidl S, Ros M, Antonio Pascual J (2010) Quantification of the biocontrol agent Trichoderma harzianum with real-time TaqMan PCR and its potential extrapolation to the hyphal biomass. Bioresour Technol 101: 2888-2891. This article is protected by copyright. All rights reserved.
Accepted Article
Mahalakshmi P, Raja I. Y (2013) Biocontrol potential of Trichoderma species against wilt disease of carnation (Dianthus caryophyllus L.) caused by Fusarium oxysporum f. sp. dianthi. J Biopest 6:32-36. Margolles-Clark E, Hayes CK, Harman GE, Pentillä M (1996) Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei. Appl Environ Microb 62: 2145-2151. Mendoza-Mendoza A, Pozo MJ, Grzegorski D, Martinez P, Garcia JM, Olmedo-Monfil V, Cortes C, Kenerley C, Herrera-Estrella A (2003) Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase. Proceedings of the National Academy of Sciences of the United States of America 100: 15965-15970. Neethling D, Nevalainen H (1996) Mycoparasitic species of Trichoderma produce lectins. Can J Microbiol 42: 141-146. Pan SQ, Ye XS, Kuc J (1991) Association of β-1, 3-glucanase activity and isoform pattern with systemic resistance to blue mold in tobacco induced by stem injection with Peronospora tabacina or leaf inoculation with Tobacco mosaic virus. Physiol Mol Plant Pathol 39: 25-39. Polizzi V, Adams A, Picco AM, Adriaens E, Lenoir J, Van Peteghem C, De Saeger S, De Kimpe N (2011) Influence of environmental conditions on production of volatiles by Trichoderma atroviride in relation with the sick building syndrome. Build Environ 46: 945-954. Sivan A, Chet I (1989) The possible role of competition between Trichoderma harzianum
and
Fusarium
oxysporum
on
rhizosphere
colonization.
Phytopathology 79: 198-203. Stoppacher N, Kluger B, Zeilinger S, Krska R, Schuhmacher R (2010) Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride by HS-SPME-GC-MS. J Microbiol Meth 81: 187-193. Strobel GA, Dirkse E, Sears J, Markworth C (2001) Volatile antimicrobials from Muscodor albus, a novel endophytic fungus. Microbiology-SGM 147: 2943-2950. Tseng SC, Liu SY, Yang HH, Lo CT, Peng KC (2008) Proteomic study of biocontrol mechanisms of Trichoderma harzianum ETS 323 in response to Rhizoctonia solani. J Agric Food Chem 56: 6914-6922. Wang BB, Yuan J, Zhang J, Shen ZZ, Zhang MX, Li R, Ruan YZ, Shen QR (2013) Effects of novel bioorganic fertilizer produced by Bacillus amyloliquefaciens W19 on antagonism of Fusarium wilt of banana. Biol Fert Soils 4: 435-446.
This article is protected by copyright. All rights reserved.
Accepted Article
Yang HH, Yang SL, Peng KC, Chaur-Tsuen LO, Liu SY (2009) Induced proteome of Trichoderma harzianum by Botrytis cinerea. Mycol Res 113: 924-932. Yedidia I, Michal S, Zohar K, Nicole B, Yoram K, Ilan C (2003) Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins. Appl Environ Microbiol 69: 7343-7353. Yuan J, Raza W, Shen Q, Huang Q (2012) Antifungal Activity of Bacillus amyloliquefaciens NJN-6 volatile compounds against Fusarium oxysporum f. sp cubense. Appl Environ Microbiol 78: 5942-5944. Zhang FG, Zhu Z, Yang XM, Ran W, Shen QR (2013a) Trichoderma harzianum T-E5 significantly affects cucumber root exudates and fungal community in the cucumber rhizosphere. Appl Soil Ecol 72: 41-48. Zhang FG, Yuan J, Yang XM, Cui YQ, Chen LH, Ran W, Shen QR (2013b) Putative Trichoderma harzianum mutant promotes cucumber growth by enhanced production of indole acetic acid and plant colonization. Plant Soil 368: 433-444. Zhao Q, Dong C, Yang X, Mei X, Ran W, Shen QR, Xu YC (2011) Biocontrol of Fusarium wilt disease for Cucumis melo melon using bio-organic fertilizer. Appl Soil Ecol 47.
Fig. 1 (A) Dual cultures (7 days after inoculation) of T. harzianum T-E5 and F. oxysporum f. sp. cucumerinum; (B) Observation of mycelial interactions between T. harzianum T-E5 and F. oxysporum f. sp. cucumerinum at 72 h using optical microscopy (OLYMPUS CX21, Tokyo, Japan, ×400); (C) Observation of the mycoparasitism of T. harzianum T-E5 against F. oxysporum f. sp. cucumerinum at 72 h using a scanning electron microscope (HITACHI S-4800 FESEM, Tokyo, Japan). Fig. 2 Extracellular protein extracts of T. harzianum T-E5 resolved using 10% SDS-PAGE. M denotes the lane of molecular weight markers. Fig. 3 (A) The colony diameters of F. oxysporum f. sp. cucumerinum in control, T1’ and T2’ treatment plates at different days after inoculation; (B) The inhibition rate of
This article is protected by copyright. All rights reserved.
Accepted Article
T-E5 VOCs against F. oxysporum f. sp. cucumerinum at different days after inoculation. Fig. 4 The GC profiles of volatile components produced by T. harzianum T-E5 in T1’ and T2’ treatments. Peaks of compounds produced in the T1’ treatment with retention times of 12.86, 12.92, 15.38, 15.60, and 16.48 are denoted a-e, respectively. Peaks of compounds produced in the T2’ treatment with retention times of 11.18, 12.02, 13.19, 13.37, 13.88, 16.49, 17.05 and 17.89 are denoted A-H, respectively. NL: Nominal level.
Fig. 1
This article is protected by copyright. All rights reserved.
Accepted Article
Fig. 2
This article is protected by copyright. All rights reserved.
Accepted Article
Fig. 3
This article is protected by copyright. All rights reserved.
Accepted Article
Fig. 4
This article is protected by copyright. All rights reserved.
Accepted Article
Table 1 Extracellular enzyme activities (Units/mg) of T. harzianum T-E5 in two different media at 24, 48, 72, 96 h after inoculation Time
Treatments
Chitinases -1
(U mg )
β-1,3-glucanase s
Proteases -1
Xylanases -1
Cellulases
(U mg )
(U mg )
(U mg-1)
(U mg-1) 24h 48h 72h 96h
T1
3.75±0.49b
2.42±0.36b
1.2±0.12b
0.43±0.07a
1.39±0.12a
T2
11.75±1.02a
18.07±1.17a
5.7±0.61a
0.51±0.05a
1.54±0.09a
T1
3.11±0.37b
3.54±0.41b
2.3±0.22b
0.52±0.02a
1.48±0.11a
T2
24.31±1.21a
20.01±1.18a
12.4±1.19a
0.54±0.05a
1.62±0.13a
T1
1.81±0.21b
4.07±0.58b
3.41±0.46b
0.45±0.09a
1.59±0.12a
T2
30.31±1.54a
23.12±1.29a
6.24±0.89a
0.61±0.07a
1.73±0.15a
T1
1.63±0.09b
3.94±0.46b
3.21±0.33b
0.48±0.05a
1.48±0.11a
T2
36.75±2.31a
24.17±1.54a
5.93±0.74a
0.56±0.03a
1.61±0.14a
Notes: The data were analyzed using Duncan’s ANOVA tests. All values are of the mean of three replicates. Values with different letters within the same column are significantly different, P≤0.05. Numbers following “±” are the standard errors (SEs).
This article is protected by copyright. All rights reserved.
Accepted Article
Table 2 VOCs of T. harzianum T-E5 cultured in T1’ and T2’ medium extracted using SPME were identified by GC-MS Treatments
T1’
T2’
Retention time (min)
Volatile Metabolites markers
names
12.86
a
Pentadecane
12.92
b
α-cubebene
15.38
c
Hexahydrofarnesol
15.60
d
Pristane
16.48
e
Verticillol
11.18
A
2,4-Di-tert-butylphenol
12.02
B
β-Bisabolene
13.19
C
α-curcumene
13.37
D
lignocerane
13.88
E
Nerolidol
16.49
F
Verticillol
17.05
G
17.89
H
Biformen (6CI) 2,6,10-trimethylundeca-5,9-dienal
This article is protected by copyright. All rights reserved.
