Effects of Marine Antagonistic Fungi against Plant Pathogens and Rice Growth Promotion Activity

Ten marine-derived fungi crude extracts, namely Emericella stellatus KUFA0208, Eupenicillium parvum KUFA0237, Neosartorya siamensis KUFA0514, N. Talaromyces flavus KUFA 0119, T. macrosporus KUFA 0135, T. trachyspermus KUFA0304, Trichoderma asperellum KUFA 0559, T. asperellum KUFA 0559 and T. harzianum KUFA 0631 were determined for their fungicidal activity against five rice pathogens in vitro. The results showed that the extracts of E. stellatus KUFA0208 and N. siamensis KUFA0514 exhibited the best antifungal activity, causing complete cessation of the mycelial growth of Alternaria padwickii, Bipalaris oryzae, Fusarium semitectum, Pyricularia oryzae and Rhizoctonia solani at 10 g/L. The N. siamensis KUFA0514 extract was fractioned and antifungal compounds were found in the fractions derived from petroleum-ether and chloroform (7: 3) evidenced by inhibition zones against the mycelial growth of A. padwickii around the disc containing each fraction. Moreover, in rice growth promotion tests, diluted cultural broth of T. asperellum KUFA 0559 and T. harzianum KUFA 0631 were found to strongly promote rice shoot and root elongation; however, higher concentrations of all marine fungal broths resulted in significantly reduced rice seedling growth rather than promotion. Meanwhile, Trichoderma showed great indole-3-acetic acid (IAA) production leading to the optimum IAA values of 45.38 and 52.30 µg/ml at 11 and 13 days after inoculation, respectively. The results of this study indicated that marine fungi are promising agents having antagonistic mechanisms involving antibiosis production and plant growth promotion and may be developed as novel biocontrol agents for rice disease management.

Lasiodiplodia theobromae. 25 Furthermore, many crude extracts isolated from marine fungi isolated from Thai waters showed potent antifungal activity on plant pathogens at 1 g/ L using dilution plate assays. 26 In addition, these marine fungi were found able to tolerate salinity at NaCl concentrations of up to 10% by increasing osmotic pressure. 22,27 Moreover, many studies have also reported the efficacy of marine fungi as potent biological control agents in plant disease management in many countries. For instance, Trichoderma strains previously isolated from Mediterranean Psammocinia sp. sponges showed high mycoparasitic potential in reducing dampingoff disease severity on beans caused by Rhizoctonia solani and also induced defense responses in cucumber seedlings in reducing Pseudomonas syringae pv. lachrimans infection. 27 The culture filtrate of marine T. longibrachiatum SFC100166 was found to effectively reduce the severity of rice blast, tomato late blight and gray mold diseases in tomato and rice. 28 It also produced the bioactive compounds spirosorbicillinol B, bisvertinolone, bisorbicillinol and epoxysorbicillinol, which effectively suppressed the incidence of tomato late blight disease, caused by P. infestans. 29 Moreover, the direct applications of the marine fungi, T. longibrachiatum and T. asperelloides as seed treatments resulted in a greater yield and significant increases in maize growth parameters as well as reduced late wilt disease severity caused by Magnaporthiopsis maydis. 30 Plant diseases are considered a main constraint in crop production which cause yield losses both qualitatively and quantitatively. Many studies have been made to find effective approaches to plant disease management such as searching for biological control agents and novel fungicides and developing integrated pest management. 21,[30][31][32] In addition, eco-friendly approaches have been sought to minimize negative effects on humans, animals and the environment. Research and development work is being conducted on soil, plant and marine microbes as biological control agents to replace chemical pesticides. While many marine fungi have been evaluated for their antagonistic activity against plant pathogens, for lots of them there still has been no assessment of their antagonistic activity against plant disease control and or their capabilities in plant growth promotion. The main purpose of this study was the evaluation of antagonistic activity of marine-derived fungi against five rice pathogenic fungi in vitro and in planta. The investigation of production of the auxin phytohormone, IAA by marine-derived fungi was conducted to assess the effectiveness of marine-derived fungi culture broth to enhance rice germination. In addition, this study was intended to reveal the hidden antagonistic properties of plant pathogenic fungi and also explore the ability of IAA production of marine-derived fungi leading to the development of novel biological control agents which will serve as alternative fungicides for combating plant diseases. The objectives in this study were: 1. to evaluate the in vitro antagonistic activity of ten marine-derived fungi against rice pathogens, 2. to investigate the secondary metabolites from the marine-derived fungus, Neosartorya siamensis KUFA 0514, and 3. to study the effects of marine-derived fungal broths on rice seed germination promoting activity and their IAA production.

Isolation of fungi from marine sponges
The sample tissues of the sponge Mycale sp. were collected at Samaesan Island (12°34′36.64″ N 100°56′59.69″ E), Chonburi province, Thailand, in April 2016 at a depth of 5-10 m. by scuba diving. It was identified by taxonomy analysis by Dr. Sumaitt Putchakarn, Institute of Marine Science, Burapha University, Thailand. The sponge was surface disinfection with 0.01% sodium hypochlorite solution for 1 min, then washed twice by sterilized seawater, and then let it dried on sterile filter paper. The sponge was cut into small pieces (5 × 5 mm) and placed on potato dextrose agar (PDA) medium plate mixed with streptomycin sulfate (0.1%) to prevent bacterial contamination, and incubated at 28 °C for 7 days. The hyphal tips emerging from sponge pieces were individually transferred onto PDA slants and maintained as pure cultures under code KUFA for further identification.

Marine-derived fungi identification
The identification of the fungi was based on morphological characteristics and molecular analysis as described previously. 22 The fungal morphological characteristics observed were the colony characteristic and growth, color and texture on malt extract agar and microscopic characteristics found through examination with stereoscopic and light microscopes. The isolated fungi were further identified by molecular techniques. DNA was extracted from young mycelia according to a modified Murray and Thompson method 33 using universal primer pairs ITS1 and ITS4 for ITS gene amplification. 34 Their accession numbers are shown in Table 1.

Preparation of the marine-derived fungal extracts
The 10 marine-derived fungi were determined for their antifungal activity against plant pathogenic fungi (Table 1). Each fungus was cultured and extracted their metabolites according to Dethoup et al. 21 Briefly, they were cultured on PDA plates and incubated for 7 days. For preparing spore suspensions, five mycelial plugs of each fungus were removed of a 7-day-old colony and inoculated in potato dextrose broth (PDB), and then incubated on a rotary shaker at 120 rpm for 7 days. The mycelial suspension of each fungus were inoculated in 1 L Erlenmeyer flasks, containing 300 g cooked rice and incubated at room temperature for 1 month, after which 500 mL of ethyl acetate was filled to each flask and macerated for 7 days. The ethyl acetate solutions were filtered through cheesecloth and then evaporated using a rotary evaporator at 45°C and 220 mbar, to obtain a crude ethyl acetate extract of each marine-derived fungus.
In vitro antifungal activity test of marine-derived fungi crude extracts against plant pathogenic fungi The dilution plate method was used for the evaluation of the in vitro antifungal activity against five rice plant pathogenic fungi, namely, Alternaria padwickii KUFA0011, Bipalaris oryzae KUFA0012, Fusarium semitectum KUFA0013, Pyricularia oryzae KUFA0014 and Rhizoctonia solani KUFA0015 which were kept as culture collection at Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand. To prepare stock solutions, a gram of each of the crude extracts was dissolved in 1 mL dimethyl sulfoxide and serially diluted with sterile distilled water. One mL of each stock solution was added to 9 mL of warm PDA, mixed, and poured into the Petri dishes to give final concentrations of 10, 1 and 0.1 g/L. A mycelial plug of each of the five plant pathogenic fungi was obtained from a 7-day-old colony margin with a sterile steel borer and transferred to a PDA plate containing one of the concentrations of each crude extract. All the Petri dishes were incubated at room temperature for 14 days. A PDA plate void of the fungal crude extract was used as a control. The inhibition levels were calculated using the formula as previously studied. 26 All treatments were done with five replications and repeated twice.

Alternaria padwickii
The combined fractions obtained from N. siamensis KUFA 0514 extract were diluted with methanol and distilled water to final concentrations of 10, 5 and 1 g/ L. The pathogen A. padwickii was cultured on PDA for 10 days and then distilled water was poured on the PDA culture plates and the spores were scraped with a steel needle to obtain a spore suspension. After that, the spore suspension was mixed with warm PDA and poured into a sterile Petri dish. Sterile filter paper 0.5 cm in size was dipped in the combined fraction at each concentration and placed on the prepared PDA plates mixed with the Alternaria spore suspension with three discs per plate containing 10, 1 and 0.1 g/L. The inhibition zone around the disc was measured using a vernier caliper at 10 days after inoculation. Each treatment was performed with three replicates. Distilled water and mancozeb at 10, 1 and 0.1 g/L were used as the negative and positive controls, respectively.

In vitro seed germination promoting activity test of marine-derived fungi in rice Rice seed germination promotion testing
A modification of the methodology of Bhagobaty and Joshi 35 was used for evaluating the effects of five marine-derived fungi, namely, Emericella stellatus KUFA0208, Neosartorya siamensis KUFA0514, Neosartorya spinosa KUFA 0528, Trichoderma asperellum KUFA 0559 and Trichoderma harzianum KUFA 0631, on rice seed germination. Each marine-derived fungus was cultured in PDB containing 0.5 % w/v L-tryptophan, and incubated using a rotary shaker at 150 rpm for seven days. Afterward, the fungal culture broth of each fungus was filtered through a 0.2-micron syringe filter and designated as a stock solution of 100 g/ L ppm concentration. The stock solution was serially diluted by PDB to five concentrations of 0.001, 0.01, 0.1, 1 and 10 g/L. Rice seed variety KMDL 105 was surface-disinfected by soaking in 3% sodium hypochlorite solution for 3 min and then washed three times in sterile distilled water. The treated rice was then soaked in a crude fungal culture broth in each concentration for twentyfour hours. Rice seeds were placed on wet filter paper in a sterile Petri dish and incubated at room temperature for four days. Rice seed soaked in PDB was used as the control. The shoot and root elongation lengths were assessed after 24, 48, 72 and 96 hours. Each treatment was performed with three replicates.

Determination of IAA production by marinederived fungi
The determination of IAA production by marine-derived fungi was conducted following the method of Bhagobaty and Joshi. 35 Five marine-derived fungi, namely, Emericella stellatus KUFA0208, Neosartorya siamensis KUFA0514, Neosartorya spinosa KUFA 0528, Trichoderma asperellum KUFA 0559 and Trichoderma harzianum KUFA 0631 were cultured in 200 ml of PDB with 0.5 % w/v L-tryptophan, and then incubated with a rotary shaker at 150 rpm at room temperature for twenty-four hours. One mL of culture broth of each marine-derived fungus was transferred to a 1.5 ml microfuge tube and centrifuged at 13,000 rpm for five minutes. The supernatants were mixed with 4 ml of Salkowshi's reagent (150 mL of concentrated H 2 SO 4 , 250 ml of distilled water, 7.5 ml of 0.5 M FeCl 3 .6H 2 O) and stored at room temperature for 20 min. before analysis of absorbance at 535 nm. The concentration of IAA was then analyzed by comparison with a standard curve. An assessment was conducted every day for two weeks. Each treatment was performed with three replicates.

Statistical analysis
All experiments were carried out twice. Data were statistically analyzed by one-way analysis of variance (ANOVA) and the means were calculated with Duncan's multiple range test (p < 0.05), using the statistical program SPSS v.19 (IBM Corporation, Somers, NY).

Effects of marine-derived fungi culture broth on rice seed germination
The culture broths of Emericella stellatus KUFA0208, Neosartorya siamensis KUFA0514,

c
Means followed by the same letter in each row do not significantly differ at p < 0.05, when analyzed using Duncan's test of one-way ANOVA.
Neosartorya spinosa KUFA 0528, Trichoderma asperellum KUFA 0559 and T. harzianum KUFA 0631 were selected and their rice seed germination promoting activities were tested at 0.001, 0.01, 0.1, 1, 10 and 100 g/L. The results showed that the culture broths of all tested marine-derived fungi stimulated rice germination after 48 hr. of incubation (Tables 2-7). However, the rice growth parameter values of both rice root elongation and rice shoot height dramatically increased at the low concentrations of 0.001 to 1 g/L, but at the concentrations of 10 and 100 g/ L, all fungal broths significantly reduced rice seedling growth rather than promoting it. At 48 hr. after inoculation, Trichoderma strains showed potent activity on rice growth, the T. asperellum KUFA 0559 broth at 1 g/ L resulted in the best rice shoot height of 0. After 72 hr. of incubation, the culture broths of both Trichoderma also displayed significant enhancement of rice seedling growth, the treatment with T. asperellum KUFA 0559 broth at 0.1 g/L displayed the best promotion of rice growth activity causing the greatest rice growth of 0.76 and 1.96 cm of rice shoot height and root elongation, respectively (Tables 4-5, Fig.  3). Corresponding with the results at 48 and 72 hr., after 96 hr. of incubation, the treatment with T. asperellum KUFA 0559 broth at 1 g/L resulted in the best rice growth promotion causing the greatest rice shoot height of 1.19 cm and inducing the best root elongation of 2.49 cm at 0.1 g/L (Tables 6-7, Fig. 3).

IAA production by the marine-derived fungi
The Emericella stellatus KUFA0208, Neosartorya siamensis KUFA0514, Neosartorya spinosa KUFA 0528, Trichoderma asperellum KUFA 0559 and Trichoderma harzianum KUFA 0631 were evaluated for their IAA production. The IAA production was observed every day during a fourteen-day incubation. The IAA production of all the marine fungi dramatically increased from day 1 to 9, and the maximum IAA production was observed after 10-12 days of incubation, and then following this, production dramatically decreased. However, the results indicate that T. asperellum KUFA 0559 and T. harzianum KUFA 0631 displayed great IAA production leading to IAA concentrations of 45.38 and 52.30 µg/ml after 11 and 13 days of incubation, respectively (Figs. [4][5]. Neosartorya siamensis KUFA0514 exhibited moderate IAA production with produced the highest IAA value of 29.78 µg/mL after 10 days of incubation (Fig.  6) whereas E. stellatus KUFA0208 and N. spinosa KUFA 0528 showed low IAA production, with maximum production of IAA only 21.39 and 20.99 µg/ mL, after 10 and 11 days of incubation, respectively ( Fig. 7-8).

disCussiON
The antagonist fungi bring various antagonistic mechanisms to bear against plant pathogens including mycoparasitism, antibiosis production, plant growth promotion and competition for space and nutrients. In this study, we evaluated the antibiosis production of marine  fungi on five rice pathogens and determined their rice growth promotion and IAA production in vitro and in vivo. In the antibiosis production tests, the extracts of N. siamensis KUFA0514 and E. stellatus KUFA0208 exerted great antifungal activity, causing 100% mycelial growth inhibition against almost all of the tested plant pathogens at 10 g/L whereas E. parvum KUFA0237 extract showed the lowest antifungal activity against the tested pathogens even at the highest dose.
At the low concentration of 1 g/L, the all tested crude fungal extracts showed low activity in inhibition of mycelial growth on all rice pathogens. However, the N. siamensis KUFA0514 displayed significant antagonistic activity against the mycelial growth of A. padwickii and B. oryzae by 58 and 52%, respectively. However, to the best of our knowledge, there are no reports of antagonistic activity of this fungus against plant diseases. This fungus was reported as a new species from soils in Thailand 36 and then its secondary metabolites were identified. The seven new indole alkaloids and 4-dihydroxy-3-methylacetophenone, tryptoquivaline, tryptoquivalines L, H, F as well as fiscalins A and C were isolated from the culture of the fungus Neosartorya siamensis KUFC 6349. 37 The chevalone C, nortryptoquivaline, fiscalin A, epi-fiscalin A and, epi-neofiscalin A were able to cause significant cell death by inducing significant DNA damage and displayed an interesting antiproliferative activity and cell death induction, which may lead to their use as chemotherapeutic agents. 38 In this study, we found that the fractions obtained from elution with petroleum ether mixed   with chloroform exhibited antifungal effects against the growth of A. padwickii evidenced by the clear inhibition zones around the discs containing the fractions, which means the antibiosis compounds produced by this fungus were of low polarity and diluted well in petroleum ether or chloroform. Emericella species are one of main bioactive compound producers in inhibition of pathogen growth. In this study, the crude extract of E. stellatus KUFA0208 also displayed potent antifungal activity by complete mycelial growth inhibition of A. padwickii, B. oryzae, P. oryzae and R. solani at 10 g/ L and 55% inhibition of B. oryzae at 1 g/ L. The results in this study corresponding with previous studies which reported that the crude extracts of three Emericella species showed strong antagonistic activity against the mycelial growth of tested plant pathogenic fungi at 10 g/ L, causing 72-83% inhibition of A. brassicicola, C. capcisi, C. gloeosporioides and F. oxysporum. 23 The crude extracts of E. rugulosa and E. nidulans isolated from soils displayed strong antifungal activity against C. gloeosporiodes and F. oxysporum f.sp. lycopersici with ED 50 values 5.98 and 1000 µg/ mL, respectively. [39][40][41] The crude extracts of three species of marine Talaromyces were evaluated for their antifungal activity in this study, and it was found that the extracts of T. flavus KUFA 0119 and T. trachyspermus KUFA0304 displayed high antagonistic activity in the mycelial growth inhibition of rice pathogens whereas the extract of T. macrosporus KUFA 0135 showed low antagonistic activity against the pathogens' growth. Species of Talaromyces have been reported from various habitats including soil, plant and marine environments and many studies have reported their antagonistic activity against plant disease. For instance, the crude ethyl acetate extract of T. trachyspermus, which was isolated from the marine sponge Clathria reinwardtii obtained from a coral reef in Thailand, exhibited effective mycelial growth inhibition of Alternaria brassicicola,   Collectrotrichum capsici, Helminthosporium maydis, Pythium aphanidermatum, Rhizoctonia solani and Sclerotium rolfsii with IC 50 values ranging from 100-186 ppm and also displayed complete mycelial growth inhibition of all tested plant pathogenic fungi at a concentration of 10,000 ppm. 26 Talaromyces tratensis KUFA 0091 exhibited effective inhibition of mycelial growth of Bipolaris oryzae, causing 40.89% inhibition using dual culture assay. In addition, the ethyl acetate crude extract of T. tratensis showed potent mycelial inhibition of B. oryzae at low concentration. Its crude extract and spore suspension had great effect against rice dirty panicle disease, reducing incidence by up to 60% under field conditions. 21 However, the extracts of two marine Trichoderma showed low to moderate antifungal activity against rice pathogens in this study. The T. harzianum KUFA 0631 extract exerted 100% inhibition on mycelial growth of A. padwickii and R. solani at 10 g/ L whereas both the T. asperellum KUFA 0559 and T. hamatum KUFA 0629 extracts exhibited complete inhibition on mycelial growth of B. oryzae, but they had low activity at low concentrations. These results correspond with a previous report of the antifungal effect of T. atroviride, which had the lowest activity against the olive pathogens, Phytopthora megasperma, P. inundata and Verticillium dahlia, when compared with others. 42 Moreover, the marine fungi were tested for their rice growth promotion on rice var. KMDL105 and their production of IAA, which is a well-known plant growth hormone affecting plant cell elongation and cell division. 43 Rice seedlings are susceptible to soil pathogens and easily infected by both pre and post-emergence pathogens in soil. The results in this study found that the treatment with T. asperellum KUFA 0559 and T. harzianum KUFA 0631 broths resulted in good rice growth promotion, enhancing rice shoot height and root length when compared with the untreated control. The results in this study correspond with previous studies which found that Trichoderma species exerted the plant growth stimulation in many crops when applied as seed or foliar treatments. For instance, seed treated with the formulated T. erinaceum improved the germination rate of rice and enhanced vigour by improving chlorophyll content and resulted in high rice yield. 44 Trichoderma harzianum-bioprimed rice was reported to enhance rice growth in droughtstressed soils by triggering various respond mechanisms via many pathways. 45 Moreover,  many Trichoderma species were reported as a potent producers of IAA and enzymes which enhanced plant tolerance to biotic and abiotic stress. Trichoderma longibrachiatum T6 (TL-6) significantly improved the growth of wheat and enhanced wheat tolerance to NaCl stress by reducing the transcriptional level of ethylene synthesis genes expression, and increasing IAA production gene expression. 46 Trichoderma harzianum strain T-A66 had a strong promotion effect on bitter gourd seedlings growth and induced disease resistance against bitter gourd wilt caused by F. oxysporum, by inducing IAA, quick H 2 O 2 bursts and callose deposition, as well as increasing antioxidant enzyme activities and phenolic compound content. 47 Treatment with T. virens stimulated mungbean seedling growth, increasing total biomass, root weight, and root length as well as improving chlorophyll content and IAA-synthase in leaves and roots. 48 Results from this study indicated that the marine-derived fungi tested had potent antagonistic activities against rice pathogens via many antagonistic mechanisms including antibiosis production resulting in potent mycelial growth inhibition of rice pathogens and plant growth promotion. The crude extract of marine fungi, N. siamensis KUFA0514 had promising antifungal activity against rice pathogen growth by producing low-polarity antifungal compounds which dissolved well in petroleum-ether or chloroform. Trichoderma asperellum KUFA