www.thaiagj.org Thai Journal of Agricultural Science 2011, 44(2): 123-131 Coniochaeta ligniaria an Endophytic Fungus from Baeckea frutescens and Its Antagonistic Effects Against Plant Pathogenic Fungi 1 1 2 3 1 J. Kokaew , L. Manoch ’*, J. Worapong , C. Chamswarng , N. Singburaudom , N. Visarathanonth1, O. Piasai1 and G. Strobel4 1Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand 2Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Rd., Payathai, Bangkok, 10400, Thailand 3Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand 4Departments of Plant Science, Montana State University, Bozeman, MT 59717, USA *Corresponding author. Email: [email protected] Abstract Coniochaeta ligniaria (KUFC 5891), an endophyte was isolated from leaves of Baeckea frutescens (Myrtaceae) obtained from the Phu Luang wildlife sanctuary, Loei province, Thailand. The fungus was characterized by the characteristics of its colony and the morphological features of its sexual and asexual stages. The fungus in dual culture with various plant pathogens showed strong inhibitory effects, especially to Pythium aphanidermatum, Phytophthora palmivora, Sclerotium rolfsii, and Rhizoctonia oryzae. A crude ethyl acetate extract of a rice based media, at 100 ppm yield completely suppressed P. palmivora with higher concentrations needed to inhibit P. aphanidermatum and other fungal pathogens. This organism and its crude ethyl acetate extract has potential for control of various plant diseases including some of the most important diseases caused by P. palmivora. Keywords: Coniochaeta, endophytic fungi, antagonistic test, plant pathogenic fungi Introduction al., 2004; Kim et al., 2007) Thus, endophytic fungi are expected to be potential sources of new bioactive Endophytic fungi are microbes that colonize living agents and to be useful as agents of biocontrol against internal tissues of plants without causing any plant disease. immediate or negative effects (Bacon and White, Most endophytic fungi were reported from wild 2000). These microorganisms are to be found in plants in the forest (Strobel, 2006). In our virtually every plant on earth (Strobel and Daisy, investigation, we collected many forest tree in Phu 2003). Endophytic fungi have been recognized as a Luang Wildlife Santuary, Loei province the repository of novel secondary metabolites in interesting endophytic fungus Coniochaeta ligniaria phamaceutical and agricultural systems (Hoffman et was isolated from plant where Baeckea frutescens, a al., 2008; Pongcharoen et al., 2008; Strobel, 2006; promising plant. Baeckea frutescens L. (Family Riga et al., 2008; Worapong, 2009). Endophytic fungi Myrtaceae) a small tree which is found in Southeast within plants have been known to produce plant Asia to Australia, including southern China, growth regulatory, antimicrobial, antiviral or Peninsular Malaysia, Sumatra, Borneo and Thailand. insecticidal substances to enhance the growth and B. frutescens grows wild in Thailand, it is found on competitiveness of the host in nature (Wiyakrutta et the beach forest, melaleuca forest and the mountain 124 J. Kokaew et al. Thai Journal of Agricultural Science tops on sandstone from Loei, Surat Thani, Pattani, ellipsoidea (DSM13856) have been found to exhibit Nakhon Si Thammarat, Chanthaburi, Ubon useful biochemical properties the antibiotic Ratchathani and Narathiwat (Smitinand, 2001). It is coniosetin, which has a pronounced antibacterial and a medicinal plant the leaves are also used to treat antifungal action, inhibiting even drug-resistant strains impetigo. In Thailand use the leaves as a tea to of Staphylococcus aureus (Segeth et al., 2003) reduced body aches (Parnell and Chantharanothai, The aims of this study were 1) to study the 2002). Chinese people use the leaves as remedy for morphology of Coniochaeta ligniaria, an endophytic sunstroke and fever. In Malaysia and Indonesia, they fungi isolated from Baeckea frutescens, and 2) to test are used as an ingredient of the traditional medicine for antagonistic activity of Coniochaeta ligniaria given to mothers during confinement (Herbal against some plant pathogenic fungi in vitro. Medicine Research Centre, 2002). Jantan et al. (1998) reported the constituents of the essential oil Materials and Methods including pinenes, terpinene, cineole from leaf of B. frutescens. The methanol extracts of B. frutescens Fungal Isolation exhibited potent antibacterial activity against the The twigs and leaves of Baeckea frutescens cariogenic bacterium Streptococcus mutans (Hwang et (Myrtaceae) were collected from Phu Luang al., 2004). Lu et al. (2008) reported a new flavanol wildlife sanctuary, Loei province, Thailand altitude glycoside, name 6,8-dimethylkaempferol-3-O-alpha- 1,200 m, 17๐ 8' 24" N 101o 39' 54" E 101 (Figure L-rhamnoside from Chinese herb B. frutescens. 1). Plant samples were placed in plastic bags in ice The fungus, Coniochaeta (Sacc) Cook was box and brought to the laboratory. After isolation, originally introduced by Saccardo in 1882 as a the samples were kept in the herbarium at Kasetsart subgenus of Rosellina (Xylariaceae). Most species of University. The surface sterilize method was the genus differ from the xylariaceous genus employed to isolate endophytic fungi. (Li et al., Roseilinia by their non-stromatic ascomata and lack 2005; Radu and Chen, 2002). A random sample of an amyloid apical apparatus in the ascus (Garcia et from each plant consisting of an asymptomatic leaf al., 2006). The species of Coniochaeta and their was taken. Leaf portions were thoroughtly washed anamorphs occur on dung, wood or bark of trees, soil, in running tap water, after which they were surface leaves and leaf litter and rarely in non- woody host sterilized by submerging in 70% ethanol for 2 min. plants like Gramineae (Lopez-Archilla et al. 2004, After drying, each leaf was divided into four Asgari et al. 2007), In addition some species, such as segments and placed on water agar (WA) Lecythophora haffmannii (teleomorph Coniochaeta supplemented with 50 mg L-1 streptomycin to ligniaria) and L. mutabilis are also known as human suppress bacterial growth. All the plates were pathogens involved and septic shock (de Hoog et al., incubated at room temperature for 3-4 weeks. 2000; Drees et al., 2007; Taniguchi et al., 2009). They Emerging fungi were transferred to fresh potato have also been isolated from food, such as butter dextrose agar (PDA) plates, incubated for 1 week, (Samson et al., 2004). On the other hand, Coniochaeta and periodically checked for purity. Figure 1 Healthy plant sample Baeckea frutescens collected from Phu Luang wildlife Sanctuary, Loei province. Vol. 44, No. 2, 2011 Coniochaeta ligniaria from Baeckea frutescens effects against plant pathogenic fungi 125 Induction of Spores and Ascospores of the fungus at the distances 0.5, 1.0 and 1.5 cm. Endophytic fungi isolate KUFC5891 can not All petri dishes were incubated at room produce conidia and ascospores. To induce temperature (28oC) and the colony diameters were sporulation, the fungus KUFC 5891 was cultivated recorded 24, 48, 72 hr after incubation. The on PDA. Double-autoclaved grapevine wood inhibition levels were calculated by using the pieces were placed onto the surface of synthetic formula: G1-G2/G1x 100, when G1 indicated colony nutrient-poor agar medium (SNA; Nirenberg 1976) radius of plant pathogenic fungi in control and G2 and then the hyphal tip from culture was indicated colony radius of plant pathogenic fungi in transferred to the SNA incubated at 28°C for 1 the dual culture test (Intana et al., 2003). Each week (anamorphs) and 3-4 weeks (teleomorphs) treatment was performed with two replicates. (Damm et al., 2010). Crude Extract Test Morphological Studies One gram of dark brown crude extract of Macroscopic features were studied including KUFC5891 was dissolved in 10 ml of ethyl acetate colony growth pattern, color, texture, and fungal (1,000,000 ppm). Then the stock solution was growth rate was measured on PDA. Colony serially diluted to four concentrations (10-1, 10-2, characteristics and pigment production were noted 10-3, 10-4). Each concentration of crude extract was after 7 days of growth (Crous et al., 2009) added to 9 mL of warm PDA, mixed, and poured incubation at 28°C. Colony colors were rated into petri dishes. The young mycelia of the various according to Rayner (1970). Conidia, ascomata, asci plant pathogenic fungi (Table 1) were transferred to and ascospores were determined from the inducing the PDA plates containing various concentrations grapvine on SNA. Microscopic preparations were of the crude extract solution. All petri dishes were made in water, with 30 measurements per structure, incubated at 28°C. The colony diameters were and morphological characteristics were examined recorded 24, 48, 72 h after incubation. The under stereo and light microscopes (Olympus BH-2 inhibition levels were calculated by comparison with Normaski Interference Contrast). Camera with a control. lucida drawings were employed of conidiophores, conidia, ascomata, asci and ascospores. Results and Discussion Preparation of Crude Extract Identification of the Endophyte The endophytic isolate KUFC 5891 was cultured Culture characteristic: colonies of KUFC 5891 on autoclaved rice in 1,000 ml flasks (200 g of rice in were slow growing on PDA, reaching 1.5-2.0 cm in 200 ml of water) and incubated at room temperature diameter after 7 days at 28oC no sporulation on (28ºC). After incubation for 30 days, 800 ml of ethyl PDA (Figure 3A) Conidiophores and conidia were acetate was added to the culture for 3 days. The found on SNA surface grapevine twig after 7 days organic phase was evaporated to dryness under incubation. B. Anamorph state were similar to the reduced pressure with a rotary evaporater. A dark genus Lecythophora described by Webber (2002) brown viscous mass of crude ethyl acetate extract but our isolation size 3.5-4.5 x 1.5-2.0 µm (Figures was collected and kept at room temperature (28ºC) 2A and 2B). until used (Dethoup et al., 2007). Lecythophora anamorph: The fungus produced conidia on SNA in 7 days.