การประชุมวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 57 สาขาพืช

Screening of Endophytic Fungi as Antagonistic Agents Against Rice Disease Pathogens

Nge-Htwe Su-Han1, Onuma Piasai1* and Nattawut Boonyuen2

ABSTRACT Fungal diseases which are occurring in the rice fields are one of the most destructive problems and cause significant yield losses in rice production. Disease control by using fungicides are very harmful to environment and human beings. Nowadays, an effective control measure without damaging the environment and human health is needed for long term agriculture. This study aims to examine the antimicrobial activity of endophytic fungi isolated form rice against rice pathogens in vitro. Among 21 rice endophytic fungal species isolated from rice, four fungal species including cupreum, Nodulisporium-like sp., Talaromyces stipitatus and Trichoderma asperellum were used as antimicrobial agents to control three rice pathogenic fungi: , and Helminthosporium oryzae. In dual culture test, Trichoderma asperellum had the highest antimicrobial activity against all three test pathogens within 5 days after incubation (DAI). Nodulisporium-like sp. and Talaromyces stipitatus expressed high inhibitory growth of Rhizoctonia solani. In evaluating ethyl acetate crude extracts of four fungal cultures at four different concentrations, Talaromyces stipitatus exhibited strong mycelial growth inhibition on Magnaporthe grisea at highest concentration (10,000 ppm). It indicated that Nodulisporium-like sp., Talaromyces stipitatus and Trichoderma asperellum could be used as antimicrobial agents to control rice pathogenic fungi: Magnaporthe grisea and Rhizoctonia solani and further studies are needed to identify new potential sources which can be utilized as bio-fungicides in the field conditions.

Keywords: endophytic fungi, rice, antagonist, mycelial growth inhibition *Corresponding author; e-mail address: [email protected] 1 Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900 2 BIOTEC, National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Road, Khlong Luang, Pathum Thani 12120

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INTRODUCTION Diseases of rice are controlled by using various kinds of chemical pesticides which are not only effective but also hazardous to natural environment (Gavrilescu, 2005). The effective way to control pests and diseases without harming the human health and without damaging the environment is needed for sustainable agriculture. Nowadays, biological control by the use of fungal endophytes is increasing interests and is one of the best strategies for disease management to substitute the chemical pesticides. Endophytic fungi are microorganisms that live inside plant tissues without causing any deleterious symptoms (Cao et al., 2016). They spend the whole or parts of their life cycle colonizing inter- and/or intracellular spaces of healthy plant tissues (Rodriguez et al., 2008). In addition, fungal endophytes play a major role in plant growth-promoting activities, such as nitrogen fixation and the production of plant growth regulators (Zheng et al., 2017). These fungi also produce antibiotic and antifungal substances that protect against insects, nematodes and plant diseases (Amin, 2016). The aims of this study were to investigate and evaluate the antagonistic activities of selected rice endophytic fungi against rice disease pathogens in vitro.

MATERIALS AND METHODS 1. Sample collection Eight selected sites of rice field were surveyed on October, 2016 and January, 2017 including 4 sites in the Si Prachan and Sam Chuk districts, Suphanburi. Samples of Pathumthani 1 and Rice Department 47 rice cultivars were randomly collected and transported to the laboratory.

2. Isolation and purification of endophytic fungi Three hundred and twenty segments (1x1cm2) from healthy leaves and 320 segments from healthy roots of two rice cultivars collected from 8 sites were used to isolate endophytic fungi. Each segment was washed with 70% ethanol (w/v) for 2 min, followed by sodium hypochlorite solution for 5 min, 70% ethanol for 30 s and finally two rinses in sterile distilled water. Sterilized leaf and root segments were placed in 9 cm petri dishes containing potato dextrose agar (PDA). Petri dishes were incubated at 28ºC for 1–2 weeks (Naik et al., 2009). Fungi growing from the plant tissues were transferred to fresh PDA medium for further study.

3. Identification of endophytic fungal species Fungal identification were based on the morphology of the fungal culture and the characteristics of spores as well as molecular analysis. Total genomic DNA was extracted directly from mycelium using a CTAB method modified and previously described by Boonyuen et al. (2011). The genomic DNA was amplified by using polymerase chain reaction (PCR) in a 50 μL reaction

70 การประชุมวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 57 สาขาพืช mixture. Two regions of rDNA sequences including the internal transcribed spacer (ITS) and large subunit (LSU) were amplified using primer pair ITS5 and ITS4 (White et al., 1990), and LROR and LR7 (Vilgalys and Hester, 1990), respectively.

4. Endophytic fungi screening 4.1 Antifungal activity by dual culture technique Three species of rice pathogenic fungi including Bipolaris oryzae (KUFCP101), Magnaporthe grisea (KUFCP102) and Rhizoctonia solani (KUFCP103) were isolated previously from rice disease samples using tissue transplanting method. Dual culture technique was adopted on PDA plates for antagonistic test against three pathogens. Five day old mycelial disks (5 mm diameter) of test pathogens were placed on one corner of petri plates containing PDA medium. Each treatment was done with three replications. Plates were incubated at 28ºC for 5 days and percentages of inhibitions were calculated according to the formula: D1–D2 / D1 x 100, D1 indicated colony diameter of plant pathogenic fungi (mm) in the control and D2 colony diameter of plant pathogenic fungi (mm) in the dual culture test (Rabha et al., 2014).

4.2 Determination of mycelial inhibition by poisoned food technique After screening endophytic fungi by dual culture technique, selected fungal species were used to prepare the crude extract. The endophytic species were cultured at 28˚C on autoclaved rice in 1,000 mL flasks (200 g of rice in 200 ml of water). After 30 days incubation, 800 mL of ethyl acetate was added to the culture and all flasks were kept for 14 days. The organic phase was evaporated to dryness under reduced pressure with a rotary evaporator. A dark brown viscous mass of crude ethyl acetate extract was collected and kept at 28ºC until used (Kokaew et al., 2011). Dilution plate method was applied for the evaluation of the anti-mycelial growth of plant pathogenic fungi. Each 1 g of crude extract was dissolved in 10 ml of ethanol to prepare a stock solution of 100,000 ppm, which was serially diluted to prepare four different concentrations (10000, 1000, 100 and 10 ppm). Then, 1 ml from each solution was added into 9 ml of warm PDA, mixed and poured into the middle of Petri dishes. After getting cold, the plates were inoculated with young mycelia of the plant pathogenic fungi, and incubated at 25 ºC for 7–14 days. The PDA petri dishes with ethanol and with sterilized water were used as positive and negative controls. Each treatment was performed with three replications. The inhibition rates were calculated as the same procedure of dual culture technique. RESULTS AND DISCUSSION 1. Endophytic fungi from rice

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A total of 242 sporulating rice endophytic fungi including 21 species belonging to 17 genera and 10 families were isolated from rice in this study. Twenty-four isolates of four antagonistic fungal species including Chaetomium cupreum, Nodulisporium-like sp., Talaromyces stipitatus and Trichoderma asperellum, which have high potential to control rice disease pathogens according to the many papers and reviews (Cao et al., 2016; Samuels and Hebbar, 2015) were selected for antifungal activity against three rice pathogenic fungi (Figure 1, Table 1).

10 µm 10 µm 10 µm 10 µm A B C D Figure 1 Morphological characteristics of four endophytic fungal species: perithecia with sterile hairs of Chaetomium cupreum (A), conidia and conidiophores of Nodulisporium-like sp. (B), cleistothecia with asci and ascospores of Talaromyces stipitatus (C), conidia and conidiophores of Trichoderma asperellum(D). 2. Activities of endophytic fungi as antagonistic agents The results indicated that Trichoderma asperellum (ERR11-12) showed completely inhibited the mycelial growth of 3 test pathogens (Table 1) within 5 days after incubation. The mycelium growth of T. asperellum is very quick and within 5 days, it completely covered the whole plates (Table 1). The study of Samuels and Hebbar (2015) revealed that Trichoderma spp. as biological control agent due to its unique characteristics, such as competition, , antibiotics and inducing resistance of the plant. Three isolates of Talaromyces stipitatus (ERR10-7, ERR11-5 and ERR12-4) showed 98–100% of inhibitory growth against R. solani, whereas they cannot express high inhibitory growth on other two pathogens, M. grisea and B. oryzae (Table 1). Talaromyces as endophytes are prolific producers of diverse bioactive secondary metabolites, such as cytotoxic norsesquiterpene peroxides and coumarins (Zhai et al., 2016). Runlin et al. (2017) reported that endophytic , T. stipitatus SK-4 which were isolated from the leaves of a mangrove plant Acanthus ilicifolius and its crude extract exhibited significant antibacterial activity against Bacillus subtilis. Although two isolates of Chaetomium cupreum (ERL13-9 and ERL9-9) cannot show high inhibitory growth on all test pathogens, they produced antibiotic compounds to protect the pathogens growth. They showed 39– 63% antagonism on the test pathogens by forming clear inhibition zones. Three isolates of Nodulisporium-like sp. (ERL13-6, ERL16-1, ERL16-13) revealed strong antagonistic activities on M. grisea and R. solani whereas they cannot show high inhibition on B. oryzae. Two isolates (ERL13-6 72 การประชุมวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 57 สาขาพืช and ERL16-13) showed 99% inhibition on R. solani whereas all three isolates revealed 62–77% on M. grisea. (Table 1). Cao et al. (2016) reported that Nodulisporium sp. can produce a metabolite, sporothriolide, as the antifungal activity against R. solani and Sclerotia sclerotiorum and it inhibited spore germination of Magnaporthe oryzae in vitro and in vivo. Table 1 Percent inhibition of 24 endophytic fungi against three rice pathogens in dual culture test on PDA at 14 days after incubation.

Percent growth inhibition (%) Endophytic fungi Code B. oryzae M. grisea R. solani Chaetomium cupreum ERL9-9 39.25±0.18c2/ 57.05±0.33a2/ 49.90±0.34b2/ ERL13-93/ 39.07±0.74c2/ 63.71±1.08a2/ 50.48±0.52b2/ Nodulisporium-like sp. ERL16-1 42.07±1.53ab1/ 77.25±0.51a1/ 19.34±0.16h ERL13-63/ 47.35±3.45a1/ 69.60±2.30b1/ 99.40±0.23a1/ ERL4-6 42.07±4.49ab1/ 66.66±1.56bc1/ 0.00±0.00 ERL16-13 37.19±1.22ab1/ 62.35±1.70cd1/ 99.03±0.17a1/ ERL4-3 39.83±2.64ab1/ 60.59±0.00cd1/ 90.29±0.47c1/ ERL13-2 32.49±3.33bc1/ 55.68±0.78de1/ 47.66±0.44d ERL4-4 38.20±3.32ab1/ 55.29±0.00de1/ 26.95±0.43f ERL13-4 33.90±1.24bc1/ 55.29±0.68de1/ 95.18±0.30b1/ ERL4-1 32.71±1.01bc1/ 54.11±3.27ef1/ 24.92±0.08g ERL11-2 29.46±1.01c1/ 53.33±0.39fg1/ 89.87±0.11c1/ ERL16-2 30.88±0.81bc1/ 51.37±1.09fg1/ 30.58±0.43e ERL2-1 29.35±0.85c1/ 50.19±0.39fg1/ 29.46±0.01e ERL11-1 30.48±0.61bc1/ 47.05±0.00g1/ 19.35±0.21h Talaromyces stipitatus ERR10-7 26.85±0.89a1/ 49.41±0.19bc1/ 100.00±0.00a1/ ERR11-5 30.07±1.58a2/ 51.95±1.53ab1/ 98.06±0.35a1/ ERR12-43/ 29.47±0.34a1/ 50.39±0.19bc1/ 100.00±0.00a1/ ERR10-5 28.44±1.05a1/ 52.15±1.41ab1/ 94.26±0.36b1/ ERR8-8 28.44±0.01a2/ 37.44±0.19e2/ 100.00±0.00a1/ ERR15-1 27.63±1.68a1/ 46.66±1.17b2/ 94.11±0.23b1/ ERR10-8 27.02±0.53a2/ 49.01±1.41bc1/ 94.15±0.30b1/ ERL6-5 23.20±5.07a 60.97±1.17a1/ 90.97±1.17c1/ Trichoderma asperellum ERR 11-123/ 100.00±0.00a1/ 100.00±0.00a1/ 100.00±0.00a1/ The same letters in each column mean not statistically different (Tukey test, ∞=0.05). Numbered followed by the ± is standard deviation (SD). 1/ Overgrwoth mechanism over plant pathogenic fungi. 2/ Inhibition of plant pathogenic fungi by forming clear zone. 73 สาขาพืช การประชุมวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 57

3/ Means selected endophytic fungi for poison food technique ERL = Endophytic fungi from rice leaf, ERR = Endophytic fungi from rice root

After screening endophytic fungi by dual culture test, one isolate of each fungal species which expressed high antagonistic activity on three rice disease pathogens including C. cupreum (ERL13-9), Nodulisporium-like sp. (ER13-6), T. stipitatus (ERR12-4) and T. asperellum (ERR11-12) were selected and applied for evaluate the activity using poison food technique (Table 2).

Table 2 In vitro inhibitory effect of four endophytic fungal crude extracts on the mycelial growth of three rice pathogenic fungi. Concentrations (ppm) Endophytic fungi Rice pathogens 10,000 1,000 100 10 Chaetomium cupreum ERL 13-9 M. grisea 54.36c 47.11c 36.56b 35.43c B. oryzae 39.00e 26.34d 8.50d 2.60c R. solani 0.00h 0.00g 0.00g 0.00d Nodulisporium-like sp. ERL 13-6 M. grisea 67.93b 49.05a 44.93a 37.78ab B. oryzae 20.00f 14.60e 2.4f 2.36d R. solani 0.00h 0.00g 0.00g 0.00d Talaromyces stipitatus ERR 12-4 M. grisea 73.12a 45.29c 38.39b 37.54ab B. oryzae 51.36d 26.57d 5.14e 2.43c R. solani 20.24f 0.00g 0.00d 0.00g Trichoderma asperellum ERR 11-12 M. grisea 68.95b 45.29c 38.52b 39.02a B. oryzae 14.06g 8.33f 8.16d 2.63c R. solani 0.00h 0.00g 0.00g 0.00d Means followed by the same letter do not significantly different at p<0.05, when analyzed using Tukey test of one-way analysis of variance.

All species showed strong antimicrobial activity at 10,000 ppm on M. grisea revealing 54% to 73% inhibition. They cannot express high antagonistic activity on B. oryzae even on 10,000 ppm. The crude extracts of all four endophytic fungal species cannot show high antimicrobial activities against R. solani at all concentrations. The crude extract at 10,000 ppm, C. cupreum revealed 54.36% antifungal activity against M. grisea, while in B. oryzae, it showed 39% inhibitory rate. The extract of Nodulisporium-like sp. showed 67.93% inhibition in M. grisea and 20.00% in B. oryzae. The extract of T. stipitatus exhibited 73.12% inhibition of the mycelium growth in M. grisea and 51.36% in B. oryzae. Interestingly, this species even showed 20.24% antimicrobial activity on R. solani. The crude extract of T. asperellum exhibited 68.95% antifungal activity in M. grisea, whereas it showed 14.06% 74 การประชุมวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 57 สาขาพืช inhibition in B. oryzae. Among four crude extracts at 10,000 ppm, T. stipitatus crude extract can reveal strong inhibitory growth on all three rice disease pathogens, expressing 73.12%, 51.36% and 20.24% on M. grisea, B. oryzae and R. solani, respectively (Table 2 and Figure 2).

Magnaporthe grisea Bipolaris oryzae Rhizoctonia solani 80 67.93 73.12 68.95 70 60 54.36 51.36 50 39.00 40 30 20.00 20.24 20 14.06

Mycelial growth inhibitiongrowth Mycelial 10 0.00 0.00 0.00

% 0 Chaetomium cupreum Nodulisporium-like sp. Talaromyces stipitatus Trichoderma asperellum Concentrations (10,000 ppm) Figure 2 In vitro inhibitory effect of four fungal crude extract at 10,000 ppm concentrations on three rice pathogenic fungi. CONCLUSION In conclusion, antimicrobial activity of endophytic fungi in dual culture test, Trichoderma asperellum can express high inhibition on all three test pathogens and Nodulisporium-like sp. and Talaromyces stipitatus have high antagonistic activities against two rice disease pathogens, Magnaporthe grisea and Rhizoctonia solani. In poison food technique, Nodulisporium-like sp., Talaromyces stipitatus and Trichoderma asperellum can reveal high inhibitions on Magnaporthe grisea at 10,000 ppm concentrations. The inhibition rates differed significantly depending on the types of endophytes. The present study is the preliminary study for rice disease controls and further study on chemical composition and secondary metabolites of these effective endophytic fungi isolated from rice are needed.

ACKNOWLEDGEMENTS The research reported in this study was supported by the SEARCA (Southeast Asia Regional Centre for Graduate Study and Research in Agriculture) scholarship organization.

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