Antifungal Activities of Chaetomium Spp. Against Fusarium Wilt of Tea

Vol. 52, 2016, No. 1: 10–17 Plant Protect. Sci. doi: 10.17221/34/2015-PPS Antifungal Activities of Chaetomium spp. against Fusarium Wilt of Tea Nguyen HUU PHONG1,2, Wattanachai PONGNAK 1 and Kasem SOYTONG 1 1Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand; 2Northern Mountainous Agriculture and Forestry Science Institute, Phu Tho, Vietnam Abstract Huu Phong N., Pongnak W., Soytong K. (2016): Antifungal activities of Chaetomium spp. against Fusarium wilt of tea. Plant Protect. Sci., 52: 10–17. An isolate of Fusarium denominated as NHP-Fusa-2 from tea wilt and root-rot diseased sample collected in Vietnam was identified as Fusarium oxysporum based on molecular analysis of translation elongation factor-1α sequence. Interestingly, it is the first time F. oxysporum is reported as a causal pathogen of wilt and root-rot disease of tea in Vietnam. Chaetomium spp. were investigated to control F. oxysporum NHP-Fusa-2 in in vitro test. Three antagonists (Ch. cupreum CC3003, Ch. globosum CG05, and Ch. lucknowense CL01) inhibited mycelial growth by 31.69–34.03% and reduced conidial production of the pathogen by 67.25–75.92% in the bi-culture antagonistic test after 30 days with non-significant difference in their effect. All the crude extracts of these antagonists significantly inhibited mycelial growth and conidial production of NHP-Fusa-2. MeOH extract of CC3003 was more effective in conidial inhibition of the pathogen than the others, the effective dose (ED50) was 85.30 μg/ml of which, it was hexane (49.32 μg/ml) and EtOAc extract (62.17 μg/ml) in the case of CG05 and CL01, respectively. Keywords: Chaetomium species; Fusarium oxysporum; growth; sporulation; inhibition Chaetomium, a strictly saprophytic fungus that several plant pathogens (Di Pietro et al. 1992; Park et belongs to Ascomycota of the family Chaetomiaceae, al. 2005; Charoenporn et al. 2010; Sibounnavong was reported to be one of the largest genera of et al. 2012). Ascomycetes with more than 300 species worldwide Root-rot is found to be one of very dangerous (von Arx et al. 1986). They are normally found in diseases of tea in Vietnam. The disease is usually soil and organic compost, degrade cellulose and not detected until several neighbouring tea bushes other organic materials relying on producing lytic have been seriously affected and died. With the enzymes (Soytong et al. 2001). Many species of progressing disease the tea leaves become yellow, Chaetomium with the potential as biological control wilted, and die, leading to final death of the whole agents suppress the growth of bacteria and fungi bush (Nguyen 2001; Zeiss & Braber 2001). With through competition, mycoparasitism, antibiosis, a 1% loss of bushes which is owed to root disease, or their various combinations (Zhang & Yang in the 10th years the loss of the potential crop would 2007). Until now, more than 200 compounds with be 10% (Hainsworth 1952). In Kenya, the loss of a wide range of bioactive effects have been isolated tea bushes caused by root disease could reach up to from Chaetomium spp., and many of them exhibited 50% (Onsando et al. 1997). antifungal activity against plant pathogenic fungi This study aimed to isolate the pathogenic fungus (Zhang et al. 2012). Some species of Chaetomium causing wilt of root-rot disease of tea in Vietnam, and have been reported to work as antagonists against to evaluate antifungal potential of antagonistic fungi, Supported by the King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand, Grant No. KREF 125601. 10 Plant Protect. Sci. Vol. 52, 2016, No. 1: 10–17 doi: 10.17221/34/2015-PPS namely Chaetomium cupreum CC3003, Ch. globosum denaturation at 94°C for 1 min 25 s followed by CG05, and Ch. lucknowense CL01, against the isolated 35 cycles of denaturation at 95°C for 35 s, annealing pathogen in in vitro test. at 61°C for 55 s, and elongation at 72°C for 1 min 30 s; final extension was at 72°C for 10 minutes. The PCR product was sent to the 1st BASE DNA Sequencing MATERIAL AND METHODS Division (Singapore Science Park II, Singapore) for sequencing with the same primers. The partial DNA Isolation of the pathogen. Samples of tea wilted sequencing data was used as a blast query against the of root-rot disease were collected from tea plants database, which is publicly accessible at Fusarium- showing the symptoms at Northern Mountainous ID (http://isolate.fusariumdb.org), for identifying Agricultural and Forestry Science Institute (Vietnam) the isolate to species included within the database. in 2013. The pathogen was isolated by transfer- NHP-Fusa-2 was also identified via phylogenetic ring surface sterilised diseased tissues to water agar analysis by adding sequences of related taxa, which (WA) containing streptomycin, then the growing were downloaded from Fusarium-ID database, to the mycelium tip of it was sub-cultured and purified in DNA sequence alignment using MEGA Version 6.06 potato dextrose agar (PDA) to get the pure culture software (Tamura et al. 2013). (NHP-Fusa-2). Pathogenicity test. NHP-Fusa-2 was tested for DNA extraction and identification of the patho- its pathogenicity with tea cuttings using a method gen. DNA of NHP-Fusa-2 was extracted using the of Bhagat and Chakraborty (2010), which was method described by Liu et al. (2000). A small lump modified. The inoculum (chaff-grains) of the patho- of the fungal mycelia was added to an Eppendorf gen was prepared following the method of Leslie tube containing 500 µl of lysis buffer (400 mM of and Summerell (2006). Chaff and grain were mixed Tris-HCl (pH 8.0), 60 mM EDTA (pH 8.0), 150 mM together at the ratio of 5 : 1, respectively, the mix- NaCl, and 1% of sodium dodecyl sulphate), then left ture was added into a 2-litre beaker up to a 0.5-litre at room temperature (RT) for 10 min, then 150 µl of level, and tap water was added up to a 1-litre level. potassium acetate (60 ml of 5 M potassium acetate, The beaker was mixed thoroughly and placed at 5°C 11.5 ml of glacial acetic acid, and 28.5 ml of distilled overnight. After that, the water was drained out, and water, pH 4.8) was added. Afterward, the tube was the chaff-grain mixture was dried out. The drained vortexed briefly and spun at 10 000 rpm for 1 min, mixture was inserted into an Erlenmeyer flask which the supernatant layer was transferred to a new tube was filled up to a height of approximately 5 cm, then and spun again under the same conditions. The new autoclaved for 15 min on each two successive days supernatant layer was transferred into another tube, before inoculating with the pathogen’s conidial sus- and an equal volume of isopropyl alcohol was added. pension (105 conidia/ml) at the rate of 2 ml of fungal The contents of this tube was mixed briefly and suspension per 250 ml of chaff-grain mixture. The spun at 10 000 rpm for 2 minutes. Subsequently, the inoculum material was shaken daily for the 3–4 first supernatant layer was discarded to get DNA pellet, days and incubated at 25°C until the material in which was later washed in 300 µl of 70% ethanol the flask was completely colonised (14 days). Sub- and spun at 10 000 rpm for 1 min, ethanol was then sequently, the culture was removed from the flask poured off. The DNA pellet was air-dried and dis- and air-dried at RT overnight. The dried substrate solved in 50 µl of 1X Tris-EDTA buffer (BioThema, of the inoculum was mixed with sterilised soil at Handen, Sweden). the ratio of 1 : 8, respectively. One-year tea cuttings To identify the isolate, a polymerase chain reaction were separately planted in plastic pots containing (PCR) was performed with the primer pair of ef1 1 kg sterile soil each, and regularly watered for two (forward primer: 5'-ATGGGTAAGGA(A/G)GACAA- weeks before inoculation. 100 g of the fungal soil GAC-3') and ef2 (reverse primer: 5'-GGA(G/A) mixture was added carefully into the rhizosphere GTACCAGT(G/C)ATCATGTT-3') (Geiser et al. area of the tea cutting. A pot planted with a tea cut- 2004). The PCR mixture with a total volume of 50 µl ting without inoculation by the pathogen served as a consisted of 1X PCR buffer, 3.5 mM MgCl2, 0.16 mM control. After inoculation the disease symptoms were each dNTP, 1.75 U Taq polymerase, 0.2 µM of each observed daily. The pathogen was then re-isolated primer, 2 µl of DNA template, and deionised water from feeder roots of the infected inoculated tea cut- (top up to 50 µl). Cycling conditions were: initial tings to confirm the pathogenic isolate. 11 Vol. 52, 2016, No. 1: 10–17 Plant Protect. Sci. doi: 10.17221/34/2015-PPS concentrations of the crude extracts, stock crude Bi-culture antagonistic test. The antagonistic fungi, namely Chaetomium cupreum strain CC3003, extracts of each concentration were weighed, then Ch. globosum strain CG05, and Ch. lucknowense dissolved in 2% dimethyl sulfoxide, and added to strain CL01, were tested to inhibit NHP-Fusa-2 us- molten PDA before autoclaved at 121°C (15 psi) ing bi-culture technique following the method of for 20 minutes. To perform the assay, a mycelia Charoenporn et al. (2010). A mycelial disc (5 mm disc (3 mm in diameter) of the pathogen at the in diameter) of NHP-Fusa-2 was placed singly (as actively growing edge of the culture was placed in controls) or oppositely to the mycelia disc of each the centre of a 5-cm-diameter Petri dish containing of the above-mentioned antagonistic fungi in Petri PDA with the predetermined crude extract at each dishes (9 cm in diameter) containing PDA.

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