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

ISSN: 0973-7510

E-ISSN: 2581-690X

Abstract
Keywords
Introduction
Materials and Methods
Results
Discussion
Declarations
References

Research Article | Open Access

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

Tanaporn Chalearmsrimuang¹, Supaporn Suasa-ard¹, Arom Jantasorn² and Tida Dethoup^1,3

¹Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.

²Bodhivijjalaya College, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok 10110, Thailand.

³Graduate School of Integrated Sciences for Life, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Hiroshima, Japan.

J Pure Appl Microbiol. 2022;16(1):402-418 | Article Number: 7445

https://doi.org/10.22207/JPAM.16.1.35 | © The Author(s). 2022

Received: 20/11/2021 | Accepted: 04/01/2022 | Published online: 21/02/2022

Issue online: March 2022

Abstract

Ten marine-derived fungi crude extracts, namely Emericella stellatus KUFA0208, Eupenicillium parvum KUFA0237, Neosartorya siamensis KUFA0514, N. spinosa KUFA 0528, 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.

Keywords

Antagonist fungi, marine fungi, rice disease control, rice seedling growth

Introduction

Marine invertebrates are multicellular animals such as sponges, corals and sea fans. Some marine invertebrates can produce a diversity of bioactive compounds that are used in the pharmaceutical and agrochemical industries.^1-4Especially, marine sponges are considered as an important source of natural products for cytotoxic, antibiotic and anti-cancer compounds.^5-7 In addition, marine sponges are the host of various symbiotic microorganisms such as archaic, bacteria, cyanobacteria, fungi and microalgae which are sources of various natural products.^8-10

Marine-derived fungi can be found in the marine ecosystem growing together with marine invertebrates. Marine fungi can be classified into many groups on the basis of many various criteria; in one such classification, marine fungi are divided into two main groups: obligate marine fungi and facultative marine fungi.^11-12 The potential uses of the fungi from marine resources as producers of novel antimicrobial metabolites has been reported by numerous studies.^13-18 Marine-derived fungi isolated from Thailand have been proven to be a great source of novel metabolites and various chemical groups of natural products have been recorded.^2,3,19-20 Some of these are sources of novel biological control agents against plant pathogenic fungi.^21-23 In our previous research, we discovered that many potent marine fungi isolated from marine invertebrates possess potential antagonistic activities such as antibiosis production and competition for space and nutrients against important pathogens of economic crops. For instance, Talaromyces tratensis KUFA 0091, which was isolated from the marine sponge, Mycale sp., possessed high potential biological activity in controlling rice pathogens, namely, Alternaria padwickii, Curvularia lunata, Fusarium moniliforme, and Bipolaris oryzae.^21,24 The ethyl acetate crude extract of T. tratensis displayed mycelial inhibition of B. oryzae at concentrations increasing from 100 ppm to 10,000 ppm, with values ranging from 52.36% – 100% inhibition in vitro.²¹ Under field conditions, the application of the spore suspension and its crude extract effected reduction of disease incidence of up to 60%.^21,24 Moreover, this marine fungus proved to be a potent agent in controlling post-harvest disease stem end rot on mango fruit caused by 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 damping-off 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.^21,30-32 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.

Materials and Methods

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 method³³ 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.

Fractionation of the crude extract of Neosartorya siamensis KUFA 0514
The crude extract of N. siamensis KUFA 0514 showed the best antifungal activity in inhibiting the rice pathogen growth in vitro, thus, it was studied further to identify its bioactive compounds using column chromatography. Forty grams of the extract 40 g was applied to a silica gel column (200 g), and eluted with petroleum ether (petrol)–chloroform (CHCl₃), and CHCl₃-acetone, 300-500 mL/ fractions. The fractions were analyzed by analytical TLC and combined, according to their composition, as follows: 1-68 CHCl₃-petrol (1:1), 69-195 CHCl₃-petrol (7:3), 196-318 CHCl₃-petrol (9:1), 319-420 CHCl₃-acetone (9:1) and 421-485 CHCl₃-acetone (4:1).

In vitro antifungal activity test of fractions of Neosartorya siamensis KUFA 0514 against 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 Joshi³⁵ 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 twenty-four 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 marine-derived 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 H₂SO₄, 250 ml of distilled water, 7.5 ml of 0.5 M FeCl₃.6H₂O) 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).

RESULTS

In vitro antifungal activity test of marine-derived fungi crude extracts against plant pathogenic fungi
The results of evaluation of the antifungal activity of ten marine-derived fungi crude ethyl acetate extracts against the five rice pathogens revealed that the crude extracts displayed increasing effect against plant pathogens in a dose-dependent manner (Table 1). At the highest dose tested, 10 g/L, all fungal extracts except Eupenicillium parvum KUFA0237 and Talaromyces macrosporus KUFA0135 extracts displayed complete mycelial growth inhibition of at least one of the plant pathogens tested while the extract of E. parvum KUFA0237 exhibited the lowest activity. Neosartorya siamensis KUFA0514 crude extract exhibited the greatest antifungal activity, causing 100% mycelial growth inhibition against all tested plant pathogens at 10 g/L except Fusarium semicteactum and also exerted effective antifungal activity in mycelium growth inhibition of Alternaria padwickii, Fusarium semicteactum and Pyricularia oryzae at 1 g/L (Table 1, Fig. 1).

Fig. 1. Effects of marine fungal crude extracts against the mycelial growth of Alternaria padwickii at 10 1 and 0.1 g/ L by using the dilution plate method
A1-A3 = Trichoderma asperellum KUFA 0559
B2-B3 = Trichoderma asperellum KUFA 0559
C1-C3 = Trichoderma harzianum KUFA 0631
D1-D3 = Talaromyces trachyspermus KUFA0304
E1-E3 = Emericella stellatus KUFA0208
F1-F3 = Talaromyces flavus KUFA 0119
G1-G3 = N. spinosa KUFA 0528
H1-H3 = Neosartorya siamensis KUFA0514

Alternaria padwickii was the most sensitive to the marine fungal extracts; the extracts of Emericella stellatus KUFA0208, Neosartorya siamensis KUFA0514, N. spinosa KUFA0528, Talaromyces flavus KUFA0119, T. trachyspermus KUFA0304 and Trichoderma harzianum KUFA 0631 exhibited 100% mycelial inhibition of this fungus at 10 g/L. Meanwhile, the extracts of E. stellatus KUFA0208, N. siamensis KUFA0514, T. trachyspermus KUFA0304, Trichoderma asperellum KUFA 0559 and T. harzianum KUFA 0631 exerted the complete mycelial growth inhibition of B. oryzae at the highest dose tested while E. stellatus KUFA0208 and N. siamensis KUFA0514 showed significant antifungal activity against the pathogen of 55 and 52%, respectively, at 1g/ L.

Table (1):
Effects of marine fungi crude extracts against rice pathogens.

Marine-derived fungus	KUFA	Accession No.	% Mycelia growth inhibition at different concentrations (g/ L)
			Alternaria padwickii		Bipolaris oryzae		Fusarium semitectum		Pyricularia oryzae		Rhizoctonia oryzae
			10	1	10	1	10	1	10	1	10	1
Emericella stellatus	0208	OK663098	100^a	25.47^g	100^a	55.28^a	100^a	25.39^g	100^a	38.21^c	100^a	12.50^d
Eupenicillium parvum	0237	OK663099	95.20^d	27.36^f	90.57^e	23.69^h	67.32^e	0ⁱ	90.27^e	0ⁱ	80.25^f	0^f
Neosartorya siamensis	0514	OK663100	100^a	58.36^a	100^a	52.47^b	95.15^c	48.36^a	100^a	48.69^a	100^a	34.90^b
Neosartorya spinosa	0528	OK663101	100^a	46.72^b	96.50^c	26.95^g	100^a	42.31^c	100^a	45.32^b	95.04^c	25.93^c
Talaromyces flavus	0119	OK663102	100^a	32.57^d	90.24^e	35.60^d	100^a	45.25^b	90.24^e	31.64^e	100^a	38.95^a
Talaromyces macrosporus	0135	OK663103	96.35^c	30.41^e	95.27^d	31.68^f	95.27^c	30.08^f	94.45^d	14.23^h	90.35^d	0^f
T. trachyspermus	0304	OK663104	100^a	39.58^c	100^a	35.95^d	98.07^b	34.15^d	95.48^c	35.69^d	100^a	35.60^b
Trichoderma asperellum	0559	OK663105	95.63^cd	20.15^h	100^a	34.16^e	90.58^d	33.24^e	95.37^c	20.17^f	88.25^e	0^f
Trichoderma hamatum	0629	OK663106	98.25^b	25.38^g	100^a	35.74^d	95.11^c	20.15^h	98.07^b	19.39^g	95.87^b	10.25^e
Trichoderma harzianum	0631	OK663107	100^a	30.65^e	98.35^b	37.33^c	95.38^c	25.49^g	95.25^c	0ⁱ	100^a	25.31^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.

The extracts of E. stellatus KUFA0208, N. spinosa KUFA0528 and T. flavus KUFA0119 caused 100% mycelial growth inhibition of Fusarium semicteactum whereas the extracts of both marine Neosartorya spp. exhibited complete mycelial growth inhibition of P. oryzae at 10 g/L. Furthermore, N. siamensis KUFA0514 extract displayed a promising antifungal effect against the mycelial growth of F. semicteactum and P. oryzae causing 48% inhibition at 1 g/L. In addition, E. stellatus KUFA0208, N. siamensis KUFA0514, T. flavus KUFA0119, T. trachyspermus KUFA0304 and T. harzianum KUFA 0631 exhibited 100% mycelial inhibition of R. solani at 10 g/L but all fungal extracts showed low antifungal activity against this pathogen at low concentrations (Table 1).

Antifungal activity of N. siamensis KUFA 0514 fractions against Alternaria padwickii
The total of 525 fractions were collected from the crude extract of Neosartorya siamensis KUFA 0514 through column chromatography as follows: Fr. 1-68 CHCl₃-petrol (1:1), 69-195 CHCl₃-petrol (7:3), 196-318 CHCl₃-petrol (9:1), 319-420 CHCl₃-acetone (9:1), 421-484 CHCl₃-acetone (4:1) and 486-525 CHCl₃-acetone (1:9). They were further analyzed by analytical TLC and combined according to their composition to A1-A30 and then their antifungal activities were evaluated using a paper disc method as shown in Fig. 2. The results showed that the fractions of A8-A14 and A16 derived from combined fractions 69-74, 75-84, 85-88, 89-93, 94-98, 99-118, 119-162, and 179-195, respectively, exhibited inhibition zones against the mycelial growth of A. padwickii at 5 and 10 g/ L (Fig. 2). Clear inhibition zones were observed in treatments A8-A14 and A16 of 1.87, 1.70, 2.00, 1.60, 1.47, 1.33, 0.40 and 1.23 cm at 5 g/ L and 2.4, 2.2, 2.2, 2.00, 1.9, 1.7, 1.27 and 1.27 cm at 10 g /L, respectively. However, the fractions showed no activity at the low concentration of 1 g/ L whereas the mancozeb caused clear inhibition zones of 2.1, 1.8 and 1.2 cm at 10, 5 and 1 g/ L, respectively.

Fig. 2. Effects of fractions obtained from Neosartorya siamensis KUFA 0017 crude extracts of 1, 5, and 10 g/ L against A. padwickii compared with controls.
A1-A6 = fractions 1-25, 26-35, 35-45, 46-50, 51-56 and 57-61
A7-A12 = fractions 62-68, 69-74, 75-84, 85-88, 89-93 and 94-98
A13-A18 = fractions 99-118, 119-162, 163-178, 179-195, 196-203 and 204-318
A19-A24 = fractions 212-222, 223-239, 240-318, 319-360, 361-382 and 383-405
A25-A30 = fractions 406-409, 410-420, 421-446, 447-460, 461-480 and 481-484
A31 = fraction 485-525 B1 = negative control (water) B2 = positive control (mancozeb)

Effects of marine-derived fungi culture broth on rice seed germination
The culture broths of Emericella stellatus KUFA0208, Neosartorya siamensis KUFA0514, 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 0.47 cm whereas T. harzianum KUFA 0631 broth caused a shoot height of 0.45 cm at the same concentration. Meanwhile, T. asperellum KUFA 0559 broth at the low concentrations of 0.01 and 0.001 g/ L exhibited great stimulation of rice root elongation to lengths of 1.21 and 0.97 cm, respectively (Tables 2-3, Fig. 3).

Table (2):
Effects of the culture broths of five species of marine-derived fungi at different concentrations on promotion of rice shoot elongation after 48 hours.

Treatment		Shoot length (cm) at concentrations (g/ L)
Treatment	0	0.001	0.01	0.1	1	10	100
Emericella stellatus KUFA 0208	–	0.15^kl	0.14^l	0.15^kl	0.14^l	0.12^m	0.10ⁿ
Neosartorya siamensis KUFA 0514	–	0.24ⁱ	0.25^hi	0.26^gh	0.42^c	0.37^d	0.34^e
Neosartorya spinosa KUFA 0528	–	0.15^kl	0.10ⁿ	0.12^m	0.08^o	0.16^k	0.12^m
Trichoderma asperellum KUFA 0559	–	0.11^mn	0.21^j	0.42^c	0.47^a	0.28^f	0.27^fg
Trichoderma harzianum KUFA 0631	–	0.24ⁱ	0.46^ab	0.38^d	0.45^b	0.42^c	0.27^fg
Negative control	0.16^k	–	–	–	–	–	^–