ISSN: 0973-7510

E-ISSN: 2581-690X

Research Article | Open Access
Marine-Derived Fungi: A Promising Source of Halo Tolerant Biological Control Agents against Plant Pathogenic Fungi

Tanaporn Chalearmsrimuang1, Siti Izera Ismail2, Norida Mazlan3, Supaporn Suasaard1 and Tida Dethoup1

1Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
2Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Malaysia.
3Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Malaysia.
J Pure Appl Microbiol, 2019, 13 (1):209-223 | Article Number: 5436

https://dx.doi.org/10.22207/JPAM.13.1.22 | © The Author(s). 2019 

Received: 07/01/2019| Accepted: 07/02/2019 | Published: 28/03/2019
Abstract

In this study, twenty marine-derived fungi were evaluated for their antagonistic activities against 10 economically important plant pathogenic fungi and investigated for their halo tolerance on potato dextrose agar (PDA) amended with 1%-25% NaCl. The results of dual culture tests showed that the marine Trichoderma species, T. asperellum and T. harzianum exhibited higher antagonistic effects against all plant pathogens than the other tested fungi, causing percentages of mycelial growth inhibition ranging from 59.31-100%. The results of dilution plate assays revealed that crude extracts of marine-derived fungi in the genera Emericella, Myrothecium, Neocosmospora, Penicillium and Talaromyces displayed great antifungal activity against plant pathogenic fungi at a low concentration of 1 g/L. However, the crude extract of Myrothecium verrucaria showed the best antifungal activity: more than 52% inhibition of five of the tested species of plant pathogenic fungi and complete mycelial growth inhibition of Bipolaris oryzae and Lasiodiplodia theobromae at 1 g/L. All of the tested marine-derived fungi were tolerant to NaCl at concentrations up to 7%. These results revealed marine-derived fungi possess exploitable antagonistic activities against plant pathogenic fungi through antibiosis, competition for nutrients and space and halo tolerance. Moreover, the results from this study showed their potential as novel BCAs for supporting crop production under climatic changes in the future.

Keywords

Antagonistic activities; marine fungi; plant pathogens; halo tolerant fungi.

Introduction

The disadvantages of commercial synthetic fungicides in both organic and conventional farming have led to attempts to find new strategies for controlling plant diseases1,2. Biological control agents are currently held to be a very promising strategy for plant disease management due to their being eco-friendly and non-toxic to consumers and farmers3,4. Finding novel BCAs is required to combat plant disease outbreaks and over come plant pathogen resistance to fungicides. The search for promising BCAs has mostly been conducted by screening terrestrial, endophytic, entomopathogenic microbes while studies of antagonistic microbes from marine environments are still limited. Marine invertebrates present a rich source of bioactive metabolites5,6. Moreover, they are also the major hosts of symbiotic microorganisms such as actinomyces, bacteria and fungi7,8. Marine-derived fungi are often associated with marine organisms and substrata such as sponges, corals, tunicates, higher algae, sea grasses, mangroves, molluscs, woody substrates and drift wood 9,10.

In our ongoing search for bioactive compounds from marine-derived fungi, we isolated a number of fungi from sponges, corals and sea fans, among which was a novel fungal species recently reported11. Several novel metabolites and the antimicrobial activity of marine-derived fungi isolated from marine invertebrates collected from Thai waters against human and plant pathogens have been reported by our group12-15. Fungi isolated from marine environments, particularly from sponges, have shown great potential as important sources of pharmacologically active metabolites and biological activities which have great potential for the development of new drugs as well as new agrochemical substances16-18.They have also been reported to be more important producers of novel natural products and bioactive compounds than other microorganisms 19-23.

These new bioactive compounds are attracting researchers to attempt to isolate fungi from marine environments. These fungi have previously been isolated from soils and plants in different locations and climates. To date, studies of diversity in marine organisms have led to the isolation of hundreds of fungal species belonging to Ascomycetes, Deuteromycetes, Zygomycetes and Mitosporic fungi 24-28. Most of them were previously reported as terrestrial fungi, and they were able to grow on media both with and without the addition seawater 11,16.The fungi and the marine invertebrate/ plant relationship is still unclear; however, sponge derived fungi have classified into three groups: sponge-generalists, sponge-associates and sponge-specialists27,29.

In our previous study, we reported the in vitro antifungal activity of five marine-derived fungi against 10 economically important plant pathogens. Among these,the extract of Talaromyces trachysporus isolated from the marine sponge Clathria reinwardtii had great mycelial growth inhibition capability on Pythium aphanidermatum even at the low concentration of IC50 100 ppm10. Besides this, other researchers have investigated the antibacterial and antifungal properties of marine-derived fungi against plant pathogens30-32. For example, several Trichoderma spp. were isolated from the Mediterranean sponge, Psammocinia sp., and were evaluated for their antagonistic activity against three plant pathogenic fungi, Botrytis cinerea, Rhizoctonia solani and Alternaria alternata. The results showed that all the tested fungi extracts displayed antagonistic activity in dual plate assays. T. atroviride and T. asperelloides effectively reduced the incidence of R. solani damping-off disease of beans and also induced defense responses in cucumber seedlings against Pseudomonas syringae pv. lachrimans33.

These data showed that marine-derived fungi, and especially marine sponge-associated fungi, are a promising source of antagonist microbes which may be useful in developing as novel BCAs to control plant diseases. However, their antimicrobial properties were mostly demonstrated for pharmaceutical purposes; thus, the evaluation of antagonistic activity against plant pathogens in this study may provide more information concerning the value and potential of marine-derived fungi in crop protection. The purpose of this study was to evaluate the antagonistic activities and  halo tolerance of twenty selected marine-derived fungi collected from Thai waters against ten plant pathogenic fungi in vitro.

MATERIALS AND METHODS

Sponge samples
The marine sponge samples were collected from coral reefs at two locations in Thailand: Samaesan Island, Chonburi Province in Eastern Thailand and Similan Island, Phang Nga Province, in Southern Thailand, by scuba- diving at a depth of 10-15meters during 2011-2016 (Table 1). The samples were placed in plastic bags containing natural seawater and were stored in ice and in a refrigerator for later analysis.

Table (1):
Fungi isolated from marine invertebrates used in this study.

Marine–derived fungus
KUFA
Accession No.
Sponge
Location
Arthrinium xenocordella
1018
KY041870
Unidentified marine sponge No. 1
Samaesan Island, Chonburi
Eurotium chevalieri
0464
KY942148
Rhabdermia sp.
Similan Island, Phang Nga
Emericella foveolata
1003
KY041869
Xestospongia testudinaria
Samaesan Island, Chonburi
Emericella nidulans
0031
MF614160
Mycale armata
Samaesan Island, Chonburi
Emericella rugulosa
1002
KY041871
Acanthella sp.
Samaesan Island, Chonburi
Emericella variecolor
0261
MF614163
Xestospongia testudinaria
Samaesan Island, Chonburi
Hamigera avellanea
0450
KY942147
Acanthella sp.
Samaesan Island, Chonburi
Hamigera terricola
0214
KU500029
Xestospongia testudinaria
Samaesan Island, Chonburi
Geosmithia lavendula
0319
KY942145
Stylissa flabelliformis
Samaesan Island, Chonburi
Myrothecium verrucaria
0192
KY942146
Mycale sp.
Samaesan Island, Chonburi
Neocosmospora vasinfecta var. vasinfecta
1004
KY041868
Mycale sp.
Samaesan Island, Chonburi
Penicillium aculeatum
0201
MF614161
Xestospongia testudinaria
Samaesan Island, Chonburi
Neosartorya fischeri
0107
KY942143
Rhabdermia sp.
Similan Island, Phang Nga
Neosartorya pseudofischeri
0061
KY942144
Hyrtios erecta
Similan Island, Phang Nga
Neosartorya quadricincta
0081
KT201525
Xestospongia testudinaria
Samaesan Island, Chonburi
Neosartorya tsunodae
0052
KT201524
Aka coralliphaga
Similan Island, Phang Nga
Talaromyces tratensis
0091
KT728350
Mycale sp.
Samaesan Island, Chonburi
Talaromyces stipitatus
0207
KU500028
Stylissa flabelliformis
Samaesan Island, Chonburi
Trichodermaasperellum
0677
KY942142
Mycale sp.
Samaesan Island, Chonburi
Trichodermaharzianum
0689
MF614160
Hyrtios erecta
Similan Island, Phang Nga

Isolation of fungi from marine sponges
The sponge sample tissues were washed three times with sterilized sea water and cut into pieces of 0.5 x 0.5 cm under aseptic conditions. Five pieces of each marine sponge were placed on a Petri dish plate containing 15 mL malt extract agar (MEA) medium mixed with70% sea water and 0.003% streptomycin sulphate, and then incubated at room temperature for 7 days. Hyphal tips emerging from sponge pieces were cut and transferred to MEA slants for further identification. The pure cultures were maintained at the Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand under the code KUFA.

Marine-derived fungi identification
The identification of the fungi was based on morphological characteristics as observed from the growth pattern, color and texture on MEA. Colony characteristics were examined under a stereoscopic microscope, and microscopic characteristics were thoroughly investigated under light and scanning electron microscopes afterwards. The fungi were further identified by molecular techniques using ITS primers. DNA was extracted from young mycelia following a modified Murray and Thompson method 34.Universal primer pairs ITS1 and ITS4 were used for ITS gene amplification35. The gene sequences of the marine-derived fungi were submitted to the BLAST program for alignment and compared with those of fungal species in the NCBI database (http://www.ncbi.nlm.nih.gov/). Their ITS gene sequences were deposited in GenBank with accession numbers as shown in Table 1.

In vitro antagonistic activity testing of the marine-derived fungi against plant pathogenic fungi by the dual culture method
Twenty marine-derived fungi were selected for testing of their antagonistic activity against ten species of plant pathogenic fungi (Table 2). The marine-derived fungi and plant pathogenic fungi were cultured on separate Petri dish plates containing PDA and incubated at room temperature for 7 days. A mycelial plug of marine-derived fungus and a mycelial plug of plant pathogenic fungus were cut from the colony margin with a sterile steel borer (0.5 cm diam.) and placed on PDA as a dual culture, 7 cm apart. The Petri dish plates of the dual culture assay were incubated at room temperature for 3 days for Sclerotium rolfsii and Rhizoctonia solani, and for 14 days for the other species. A mycelial plug of each plant pathogenic fungus was placed on a separate PDA plate to serve as a control. The inhibition levels were calculated by using the formula: [(x-y)/ x] x 100, where x = the colony radius of the plant pathogenic fungi in the control, and y = the colony radius of the plant pathogenic fungi in the dual culture test. Each treatment was performed with five replicates and repeated three times.

Table (2):
Antagonistic effects of marine-derived fungi on ten plant pathogenic fungi in dual cultures on PDA.

Marine–derived fungus % Mycelial growth inhibition
AB* BO CG CC FO LT PP PO RS SR
Arthrinium xenocordella 65.19d 42.59ij 45.93c 57.41e 54.81c 29.63g 39.26e 62.59d 0l 11.11f-h
Emericella foveolata 49.17i 44.17i 37.41gh 50.74f 43.70ef 0l 36.67f 48.06g 0l 3.70hi
Emericella nidulans 55.57g 68.89c 44.44cd 51.85f 40.21h 64.8a1 39.44e 56.67e 60.37b 55.57a
Emericella rugulosa 44.44jk 39.26l 37.78gh 39.30i 36.34ij 0l 29.26h 47.41g 0l 19.26d-f
Emericella variecolor 58.89f 51.48g 36.66h 43.70h 37.40i 0l 44.44c 42.97i 19.4h4 0i
Eurotium chevalieri 45.00jk 32.56m 22.78j 31.11k 30.78l 30.5g6 21.74j 39.22j 31.89f 22.2d2
Hamigera avellanea 64.44d 54.82f 48.89b 58.52de 42.96fg 34.74f 41.85d 66.30c 14.82i 11.11f-h
Hamigera terricola 49.74hi 46.67h 45.18c 44.81h 44.44ef 23.31i 27.22i 45.13h 0l 12.17e-g
Geosmithia lavendula 48.89i 39.22l 37.78gh 35.21j 35.22j 41.11c 41.48d 38.85j 32.47f 0i
Myrothecium verrucaria 48.80i 46.67h 48.15b 45.25h 32.96k 60.00b 44.82c 48.89g 43.33c 4.8g-i2
Neocosmospora vasinfecta var. vasinfecta 55.56g 43.70j 42.22e 50.37f 45.18e 26.30h 29.63h 54.44f 27.76g 22.22d
Penicillium aculeatum 48.11i 43.28i 33.14i 35.09j 37.00i 15.62k 34.74g 29.87l 10.31k 0i
Neosartorya fischeri 43.33k 62.59d 40.37f 60.00d 44.41ef 22.08i 41.85d 45.64h 28.51g 0i
Neosartorya pseudofischeri 67.03c 60.91e 45.58c 47.63g 55.18c 42.37c 48.69b 47.81g 37.03e 47.19 b
Neosartorya quadricincta 68.52c 40.74kl 42.92de 58.52de 42.59fg 19.26j 43.70c 56.30e 40.37d 35.93c
Neosartorya tsunodae 45.57j 41.32jk 36.07h 39.58i 41.38gh 20.11j 40.36de 35.67k 12.36j 10.87f-h
Talaromyces tratensis 51.11h 41.11jk 38.89fg 40.00i 42.59fg 37.03e 36.67f 42.96i 0l 15.92d-f
Talaromyces stipitatus 62.51e 47.39h 48.05b 65.24c 49.20d 39.07d 48.26b 61.22d 32.0f7 20.14de
Trichodermaasperellum 83.33a 95.68a 100a 80.00b 72.96b 60.65b 100a 81.15b 71.0a0 61.48a
Trichodermaharzianum 80.25b 92.37b 100a 92.11a 81.12a 59.31b 100a 90.35a 70.25a 68.32a

*AB = Alternaria brassicicola, BO = Bipolaris oryzae, CC = Colletotrichum capsici, CG = C. gloeosporiodes, FO= Fusarium oxysporum, LT = Lasiodiplodia theobromae, PP = Phytophthora palmivora, PO = Pyricularia oryzae, RO = Rhizoctonia oryzae, SR = Sclerotium rolfsii

Values in a column followed by the same letter are not significantly different at p< 0.05, when analyzed using Duncan’s multiple range testof One-Way ANOVA.

Preparation of the marine-derived fungal extracts
The 20 selected marine-derived fungi were evaluated for their antifungal activity against plant pathogenic fungi (Table 3). These fungi were cultured on separate PDA plates and incubated at room temperature for 7 days. Five mycelial plugs of each fungus were cut from a 7-day-old colony margin and inoculated in 500 ml Erlenmeyer flasks containing potato dextrose broth 200 mL, and then incubated on a rotary shaker at 120 rpm for 7 days for preparing spore suspensions. Twenty-five 1,000 ml Erlenmeyer flasks, each containing 300 g cooked rice, were autoclaved at 121oC for 15 min. and then inoculated with approximately 20 mL of mycelial suspension of each fungus. The inoculated flasks were then incubated at room temperature for 30 days, after which 500 mL of ethyl acetate was added to each flask and macerated for 7 days. The ethyl acetate solutions were filtered through filter paper (Whatman No.1) to give the organic solutions and then evaporated under reduced pressure 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 ten plant pathogenic fungi. One gram of each of the crude ethyl acetate extracts of marine-derived fungi was dissolved in 1 mL dimethyl sulfoxide and serially diluted with sterile distilled water to prepare stock solutions of 100 and 10 g/L concentrations. One mL of each stock solution was added to 9 mL of warm PDA, mixed, and poured into the Petri dishes to obtain final concentrations of 10 and 1 g/L. A mycelial plug of each of the ten plant pathogenic fungi was cut 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: [(x-y)/ x] x 100, where x = the colony radius of the plant pathogenic fungi in the control, and y = the colony radius of the plant pathogenic fungi in the presence of the tested crude extract. Each treatment was performed with five replications and repeated three times.

Salt tolerance assay
The selected twenty marine-derived fungi were evaluated for their halo tolerance on PDA amended with NaCl (Sigma-Aldrich®) concentrations at 1%, 3%, 5%, 7%, 9%, 12%, 15%, 17%, 20% and 25%. A mycelial plug of each marine-derived fungus was placed on the center of a PDA plate containing each NaCl concentration and incubated for 30 days at room temperature. The mycelial growth of each marine-derived fungus was observed and recorded at 21 days as compared with the control (0%).  Each treatment was performed with five replications and repeated three times.

Statistical analysis
Due to the non-significant differences between the repeated experiments of each treatment at p< 0.05, data obtained from the repeated experiments were pooled and submitted to analysis of variance (ANOVA), and means were compared by Duncan’s multiple range test(p <0.05), using the statistical program SPSS version 19 (IBM Corporation, Somers, NY).

RESULTS

Antagonistic activity of marine-derived fungi
Twenty marine-derived fungi were selected and identified to species based on morphological and ITS gene analysis and their gene sequences were submitted to Genbank (Table 1). Results of their antagonistic activity against the ten plant pathogenic fungi in the dual cultures on PDA plates are shown in Table 2. Seven of these pathogenic fungi belonged to Ascomycetes (Alternaria brassicicola, Bipolaris oryzae, Colletotrichum capsici, C. gloeosporioides, Fusarium oxysporum, Lasiodiplodia the obromae and Pyricularia oryzae), one to Oomycetes  (Phytophthora palmivora) and two to Agonomycetes (Rhizoctonia solani and Sclerotium rolfsii).

Trichoderma asperellum (KUFA 0677) and T. harzianum (KUFA 0689) displayed the highest effect against all plant pathogenic fungi,causing more than 60.65% mycelial growth inhibition, and they caused 100% inhibition of C. gloeosporioides and P. palmivoraby overgrowing colonies of these pathogens.

Values in a column followed by the same letter are not significantly different at p< 0.05, when analyzed using Duncan’s multiple range test of One-Way ANOVA.

The results on the antagonistic effects of the rest of the selected marine-derived fungi against plant pathogenic fungi belonging to Ascomycetes revealed that ten of the tested fungi displayed potent (> 50% inhibition) antagonistic effect against at least one pathogen belonging to this class. Five fungi, namely A. xenocordella (KUFA1018), E. nidulans(KUFA0031), H. avellanea (KUFA0450), N. vasinfecta var. vasinfecta (KUFA1004) and T. stipitatus (KUFA0207) exhibited effective mycelial growth inhibition against A. brassicicola, C. capsici and P. oryzae with values ranging from 50.37 to 66.30%. Meanwhile, E. nidulans(KUFA0031), N. fischeri (KUFA0107) and N. pseudofischeri (KUFA0061)also displayed potent antagonistic effect against B. oryzae,causing 62-68% mycelial growth inhibition.

Moreover, A. xenocordella (KUFA1018) and N. pseudofischeri (KUFA0061) showed a moderate inhibitory effect, causing 54-55% mycelial growth inhibition of F. oxysporum. Additionally, E. nidulans (KUFA0031) and M. verrucaria (KUFA0192) showed effective action against the mycelial growth of L. theobromae, with inhibition values of 60-64%.

Besides Trichoderma species, the other marine-derived fungi showed a weak effect, causing mycelial growth inhibition of P. palmivora with a value lower than 50%, and only E. nidulans (KUFA0031) exhibited a potent antagonistic effect against R. solani and S. rolfsii, causing mycelial growth inhibitions of 60.37 and 55.57%, respectively. Interestingly, six out of the twenty marine-derived fungi showed antagonistic activity against the tested plant pathogenic fungi by forming zones of inhibition although they caused mycelial growth inhibition lower than 50%. T. tratensis (KUFA0091) displayed the strongest activity with formation of the widest zone of inhibition, 1.2 to 2.2 cm in width, against A. brassicicola, B. oryzae, L. theobromae and P. palmivora. In addition, A. xenocordella,E. rugulosa (KUFA1002), E. foveolata (KUFA1003), N. vasinfecta var. vasinfecta (KUFA1004) and N. pseudofischeri (KUFA0061) showed antagonistic activity by forming zones of inhibition 0.5-1.2 cm in width against some plant pathogenic fungi belonging to Ascomycetes (Fig. 1).


Fig. 1. Antagonistic effects of marine-derived fungi (left) on plant pathogenic fungi (right) in dual cultures on PDA plates.Talaromyces tratensis KUFA0091 vs A. brassicicola (A1), B. oryzae (A2), L. theobromae(A3),P. palmivora(A4)Emericella rugulosa KUFA1002 vs A. brassicicola (B1), C. gloeosporiodes (B2), F. oxysporum (B3), P. oryzae (B4) C. Neocosmospora vasinfecta var. vasinfecta KUFA1004 vs A. brassicicola (C1), C. capsici (C2), C. gloeosporiodes (C3), F. oxysporum(C4) D. Arthrinium xenocordella KUFA1018 vs A. brassicicola (D1), C. capsici (D2), C. gloeosporiodes (D3),F. oxysporum(D4) E. Trichoderma harzianum KUFA0677 vs A. brassicicola (E1), P. palmivora (E2), P. oryzae (E3),R. oryzae (E4)

Antifungal activity of marine-derived fungi
The result of testing the antifungal activity of marine-derived fungi crude ethyl acetate extracts against the ten plant pathogenic fungi revealed that the crude extracts displayed increased effect against plant pathogens when the concentration increased (Table 3). At the highest dose tested, 10 g/L, all fungal extracts except E. chevalieri (KUFA0464), G. lavendula (KUFA0319), and N. pseudofischeri (KUFA0061) extracts exhibited 100% mycelial growth inhibition of at least two of the plant pathogens tested. M. verrucaria (KUFA0192) crude extract displayed the greatest antifungal activity, causing 100% inhibition against all tested plant pathogens at 10 g/L and also complete inhibition of B. oryzae and L. theobromae mycelial growth at 1 g/L.

Table (3):
Antifungal effects of marine-derived fungalextracts on ten plant pathogenic fungi by using the dilution method.

Marine–derived fungal extract % Mycelial growth inhibition at different concentrations (g/L)

 
AB* BO CC CG FO
10 1 10 1 10 1 10 1 10 1
Arthrinium xenocordella 100a 37.78n 100a 38.52o 100a 15k 40j 0u 54.72i 0r
Emericella foveolata 100a 28.61p 100a 49.44j 100a 17.22k 55h 31.67lm 100a 16.94p
Emericella nidulans 44.44lm 21.11u 36.66p 0x 51.67e 18.61k 32.22klm 11.11p 17.40p 0r
Emericella rugulosa 75.50de 63.06i 100a 55.28i 100a 0m 100a 34.72k 100a 25o
Emericella variecolor 100a 78.14d 100a 0x 100a 72.77d 100a 78.88d 100a 83.70c
Eurotium chevalieri 35.17o 0s 29.18s 11.76w 24.22j 15.76k 30.25lm 12.31op 37.14m 10.32q
Hamigera avellanea 66.66h 43.33lm 63.04j 23.70t 100a 23.70j 100a 30m 66.66e 35.25m
Hamigera terricola 60.12j 0s 60.45h 35.47qr 100a 30.04i 72.59e 39.25j 74.10d 45.32k
Geosmithia lavendula 54.10k 0s 71.42e 0x 74.12d 14.36k 68.21f 0u 57.84fh 31.22n
Myrothecium verrucaria 100a 44.41lm 100a 100a 100a 72.72d 100a 73.70e 100a 87.77b
Neocosmospora vasinfecta var. vasinfecta 78.89d 67.78h 42.78l 44.72k 100a 46.94f 68.33f 45.22i 49.72j 38.06m
Penicillium aculeatum 82.14c 24.11q 35.36qr 0x 87.61b 48.30ef 84.64c 32.56klm 58.97f 0r
Neosartorya fischeri 100a 42.32m 100a 0x 100a 18.50k 100a 20.20n 100a 35.25m
Neosartorya pseudofischeri 75.50e 45.43l 80c 40.75m 82.94c 35.47h 95.50b 20.59n 55.41hi 10.50q
Neosartorya quadricincta 35.51on 0s 21.18u 0x 5.73i 0m 0u 0u 15.32p 0r
Neosartorya tsunodae 100a 26.67p 65.83f 34.17r 100a 16.39k 100a 14.44o 36.39m 0r
Talaromyces tratensis 100a 37.78n 100a 55.92i 100a 18.61k 100a 4.44q 100a 7.50u
Talaromyces stipitatus 28.33p 5.56r 87.78b 40.02mn 45f 4.17lm 38.33j 0u 30.83n 0r
Trichodermaasperellum 88.89b 0s 77.22d 44.44k 80.28c 0m 32.96kl 0u 41.11l 6.94u
Trichodermaharzianum 70.48f 20.17u 65.42f 14.37v 100a 15k 45.87i 0u 31.89n 0r

 
Marine – derived fungal extract % Mycelial growth inhibition at different concentrations ( g/L )
LT PP PO RS SR
10 1 10 1 10 1 10 1 10 1
Arthrinium xenocordella 100a 0j 100a 19.63l 100a 0j 100a 0m 100a 9.17n
Emericella foveolata 67.22d 0j 100a 39.17h 100a 18.89i 50.00ef 6.67i 51.48ef 16.39m
Emericella nidulans 100a 55.78e 100a 57.44e 100a 77.22bc 57.40d 0m 100a 49.44f
Emericella rugulosa 100a 36.39h 100a 64.44d 100a 34.07h 100a 12.22k 100a 29.44j
Emericella variecolor 100a 0j 100a 0m 100a 0j 100a 0m 100a 0o
Eurotium chevalieri 0j 0j 0m 0m 0j 0j 10.12k 0m 21.35l 0o
Hamigera avellanea 100a 5.55j 100a 0m 100a 33.33h 100a 66.66c 100a 25.92k
Hamigera terricola 100a 0j 55.55ef 30.12j 51.14f 12.17ij 85.40b 22.81j 74.35c 32.17i
Geosmithia lavendula 24.92i 0j 64.65d 22.57k 62.03de 0j 47.25fh 0m 58.22d 0o
Myrothecium verrucaria 100a 100a 100a 35.92i 100a 52.17ef 100a 24.51j 100a 0o
Neocosmospora vasinfecta var. vasinfecta 68.89d 25.28i 100a 0m 68.61cd 52.50ef 51.67e 10.00k 31.39ij 0o
Penicillium aculeatum 97.21a 32.58h 100a 56.82e 86.21b 11.25ij 47.39fh 28.00i 59.21d 0o
Neosartorya fischeri 0j 0j 100a 0m 100a 33.33h 100a 45.42h 100a 53.75e
Neosartorya pseudofischeri 87.25b 45.81f 75.25c 20.37kl 74.22c 35.47h 65.40c 45h 72.57c 20.35l
Neosartorya quadricincta 0j 0j 86.95b 36i 0j 0j 100a 0m 95.56b 0o
Neosartorya tsunodae 76.94c 0j 100a 20.28kl 100a 0j 100a 4.72i 100a 42.22h
Talaromyces tratensis 76.39c 0j 100a 53.33f 100a 32.50h 21.94j 0m 100a 0o
Talaromyces stipitatus 100a 0j 100a 0m 100a 15.56i 100a 0m 100a 0o
Trichodermaasperellum 64.44d 0j 100a 0m 100a 0j 100a 0m 100a 0o
Trichodermaharzianum 50.11ef 0j 100a 0m 100a 0j 100a 0m 100a 0o

*AB = Alternaria brassicicola, BO = Bipolaris oryzae, CC = Colletotrichum capsici, CG = C. gloeosporiodes, FO= Fusarium oxysporum, LT = Lasiodiplodia theobromae, PP = Phytophthora palmivora, PO = Pyricularia oryzae, RO = Rhizoctonia oryzae, SR = Sclerotium rolfsii

Values in two columns of each pathogen followed by the same letter are not significantly different at p< 0.05, when analyzed using Duncan’s multiple range testof One-Way ANOVA.

At 1 g/L, the crude extracts of seven marine-derived fungi: E. nidulans (KUFA0031), E. rugulosa (KUFA1002), E. variecolor (KUFA0261), N. vasinfecta var. vasinfecta (KUFA1004), N. fischeri (KUFA0107), P. aculeatum (KUFA0201)and T. tratensis (KUFA0091) displayed significant antifungal activity against plant pathogenic fungi,causing more than 50% inhibition of at least one plant pathogenic fungus. Among them, E. variecolor (KUFA0261) showed great inhibition (72-83%) of the mycelial growth of A. brassicicola, C. capcisi, C. gloeosporioides and F. oxysporum whereas E. rugulosa (KUFA1002)extract exhibited an antifungal effect on A. brassicicola, B. oryzaeand P. palmivoraof 55-64% and E. nidulans extract caused 55-72% inhibition of L. theobromae, P. palmivora and P. oryzae. Furthermore, T. tratensis (KUFA0091) extract displayed promising antifungal effect against the mycelial growth of B. oryzae and P. palmivora causing 53-55% inhibition at 1 g/L. P. aculeatum (KUFA0201)and N. fischeri(KUFA0107)extracts exhibited 56 and 54% inhibition of mycelial growth of P. palmivora and S. rolfsii, respectively.

Values in two columns of each pathogen followed by the same letter are not significantly different at p< 0.05, when analyzed using Duncan’s multiple range test of One-Way ANOVA.

Halo tolerance of marine-derived fungi
The result of testing the salt tolerance of marine-derived fungi on PDA amended with NaCl at different concentrations is shown in Table 4. All marine-derived fungi exhibited NaCl tolerance, being able to grow on PDA amended with NaCl up to 7%, but none of them were able to grow on PDA amended with NaCl at 20% and 25%. Five of them showed high tolerance to NaCl, being able to grow slowly on PDA amended with NaCl at 15%,and another six species were able togrow at 10% NaCl concentration. The effects of NaCl on fungal growth observed included inhibition of fungal growth compared with the controls when NaCl’s concentrations were increased except inE. chevalieri (KUFA0464). Moreover, the teleomorphic species of Penicillium and Aspergillusex hibited only the anamorphic state, producing conidiophores without cleistothecial formation.

Table (4):
NaCl tolerance of marine-derived fungi.

Marine – derived fungus Mycelial growth of marine – derived fungi on PDA amended with NaCl

at different concentrations
0% 1 % 3 % 5 % 7 % 10 % 15 %
Arthrinium xenocordella 9 9 9 9 9 7.2 ± 0.16 –1/
Emericella foveolata 9 9 9 9 8.4 ± 1.97 7.84 ± 0.21 3.4± 0.22
Emericella nidulans 9 9 9 9 9 6.2± 0.34 3.52± 0.24
Emericella rugulosa 9 9 9 9 9 5.5± 0.11 –
Emericella variecolor 9 9 9 7.21 ± 0.59 4.25± 0.74 3.43± 0.89 2.3± 0.18
Eurotium chevalieri 2.34 ± 0.24 2.64 ± 0.20 3.28 ± 0.32 3.38 ± 0.18 3.46 ± 0.21 3.14 ± 0.15 3.27 ± 0.20
Hamigera avellanea 9 9 9 9 9 4.56 ± 0.06 –
Hamigera terricola 9 9 9 9 5.5 ± 0.25 2.9 ± 0.23 –
Geosmithia lavendula 9 9 9 9 9 7.54 ± 0.04 1.57± 0.03
Myrothecium verrucaria 9 9 9 6.54 ± 1.12 4.5 ± 0.19 – –
Neocosmospora vasinfecta var. vasinfecta 9 9 9 9 7.5 ± 0.58 4.62 ± 0.29 –
Penicillium aculeatum 9 9 9 5.37 ± 0.85 1.32 ± 0.28 – –
Neosartorya fischeri 9 9 9 9 9 – –
Neosartorya pseudofischeri 9 9 9 9 9 – –
Neosartorya quadricincta 9 9 9 9 9 – –
Neosartorya tsunodae 7.13 ± 0.57 6.58 ± 0.41 5.67 ± 0.27 4.36 ± 0.26 2.07 ± 0.15 – –
Talaromyces tratensis 9 9 5.68 ± 0.38 3.59 ± 0.29 2.74 ± 0.21 1.38 ± 0.09 –
Talaromyces stipitatus 9 9 9 7.08 ± 0.97 3.64 ± 0.35 – –
Trichodermaasperellum 9 9 9 9 6.5 ± 1.54 – –
Trichodermaharzianum 9 9 9 9 5.5± 0.87 – –

1/ No growth was observed.

Discussion

The antagonistic activity of the selected twenty marine-derived fungi against plant pathogenic fungi and their halo tolerance were evaluated. The preliminary results of the dual culture assay showed that among the twenty marine-derived fungi tested, Trichoderma species, T. asperellum and T. harzianum exhibited higher antagonistic effect against all the plant pathogens than the other marine-derived fungi since they caused percentages of mycelial growth inhibition in the range 59.31-100%.  Both Trichoderma species showed antagonistic effects on plant pathogenic fungi via overgrowing colonies of plant pathogenic fungi. Trichoderma species are a common genus in various hosts and are the well-known BCAs which act by means of various mechanisms against plant pathogenic fungi including mycoparasitism and producing cell-wall degrading enzymes and antifungal substances36-37. According with our results, for example, Trichoderma strains which were isolated from the Mediterranean sponge, Psammocinia sp. collected in Israel showed coiling mycoparasitism on mycelium of Fusarium equiseti when tested on PDA dual cultures 18 and Trichoderma atroviride and T. asperelloides extracts effectively reduced the incidence of R. solani damping-off disease of beans and also induced defense responses in cucumber seedlings against Pseudomonas syringae pv. lachrimans33. It is without a doubt that Trichoderma strains are great antagonists and diverse in habitats even in marine environments. Besides, the salt tolerant strains of Trichoderma have been investigated for their activity against plant pathogens to develop BCAs applied in crop protection for application in arid and saline soil areas 33, 38,39.

In contrast, Trichoderma crude extracts showed high antifungal effect on plant pathogens only at the highest concentration, 10 g/L, and they displayed low to medium activity against all the tested plant pathogens at 1 g/L. These results accord with a previously reported of the antifungal effect of an entomopathogenic strain of Trichoderma atroviride was lowest against the olive pathogens, Verticillium dahlia, Phytopthora megasperma and Phytopthora inundata40.

However, six out of the twenty marine-derived fungi displayed antagonistic effects by forming zones of inhibition against the tested plant pathogenic fungi although the average percentage of their mycelial growth inhibition was lower than 50%. For example, Talaromyces tratensis (KUFA 0091) displayed the strongest activity, forming the widest zone of inhibition, 1.2 to 2.2 cm in width, against A. brassicicola, B. oryzae, L. theobromae and P. Palmivora (Fig. 1). Moreover, E.rugulosa (KUFA1002), E. foveolata (KUFA1003), N. vasinfecta var. vasinfecta (KUFA1004) and Neosartorya pseudofischeri showed antagonistic activity by forming zones of inhibition in the range of 0.5-1.2 cm in width against some phytopathogenic fungi belonging to Ascomycetes (Fig. 1). These findings showed that these marine-derived fungi produced and released antifungal substances which inhibited the growth of the plant pathogenic fungi.

The results of the dilution plate assay confirmed their production of antifungal substances.  Crude extracts of eight marine-derived fungi in the genera Emericella, Myrothecium, Neocosmospora, Penicillium and Talaromyces displayed great antifungal activity against the plant pathogenic fungi at a low concentration of 1 g/L. The crude extract of M. verrucaria showed the best antifungal activity, causing more than 52-100% inhibition of five of the tested plant pathogenic fungus species at 1 g/L. This result is in accordance with a previous study which reported that crude ethyl acetate extract of Myrothecium sp. associated with the marine sponge, Axinella sp., was a potential producer of antifungal compounds against Sclerotinia sclerotiorum, a causal agent of stem rot in various crops 41. Meanwhile, the crude extracts of three Emericella species including E. nidulans, E. rugulosa and E. variecolor showed high inhibition of the mycelial growth of eight of the tested plant pathogenic fungi at 1 g/L. Among them, E. variecolor extract displayed the greatest inhibition, causing 72-83% inhibition of A. brassicicola, C. capcisi, C. gloeosporioides and F. oxysporum, whereas E. rugulosa extract exhibited antifungal effects on A. brassicicola, B. oryzae and P. palmivora of 55-64%, and E. nidulans extract caused 55-72% inhibition of L. theobromae, P. palmivora and P. oryzae.  Emericella species are common soil fungi and have been reported as antibiosis producers against plant pathogens.  For example, crude extracts of soil strains of E. rugulosa and E. nidulans showed great antifungal effects against F. oxysporum f.sp. lycopersici and C. gloeosporiodes with ED50 values 5.98 and 1000 µg/mL, respectively 42-43. A few studies reported the antifungal effects of E. variecolor extracts on plant pathogens. For instance, crude extracts of soil strains of E. nidulans, E. rugulosa and E. variecolor were evaluated the antifungal activity and they inhibited by 45-63% the mycelial growth of A. brassicicola, Curvularia lunata, C. capsici, C. gloeosporioides, F. oxysporum, Helminthosporium sp., Pestalotiopsis sp. and P. palmivora in vitro. When compared with our results, the extract of a marine strain of E. variecolor displayed higher antifungal activity against plant pathogens than that of the extract obtained from a soil strain, for it exhibited 72-83% inhibition of A. brassicicola, C. capcisi, C. gloeosporioides and F. oxysporum44.

The results in this study also showed that T. tratensis crude extract displayed a promising antifungal effect against the mycelial growth of B. oryzae and P. palmivora, causing 53-55% inhibition at 1 g/L. This is similar to our previous study in which we reported that the crude ethyl acetate extract of Talaromyces trachyspermus (KUFA 0021) exhibited the most effective mycelial growth inhibition of A. brassicicola, C. capsici, H. maydis, Pythium aphanidermatum, R. solani and S. rolfsii with IC50 values of 100-186 ppm and displayed total inhibition of mycelial growth on all plant pathogenic fungi at the highest concentration tested, 10 g/L10.

The results of this study also reveal that at 1 g/L, P. aculeatum and N. fischeri extracts exhibited 56 and 54% inhibition of mycelial growth of P. palmivora and S. rolfsii, respectively. Similar to our findings, Shen et al.32reported the antimicrobial activity of marine-derived Penicillium oxalicum strain O312F crude extract, which displayed strong antifungal activity against A. brassicicola and F. graminearum.  In addition, the antifungal activity of Penicillium citrinum isolated from a marine sponge, Callyspongia diffusa, collected in the Gulf of Mannar, on the southeast coast of India45. Penicillium citrinum crude extract also displayed strong antifungal activity against nine plant pathogenic fungi, including Alternaria alternata, Botrytis cinerea, Cercospora theae, Fusarium udum, F. oxysporum, Macrophomina phaseolina, Poria hypolateritia, Phomopsis thae and R. solani. The result of this study also reveals that N. vasinfecta var. vasinfecta extract exhibited 52-67% inhibition of mycelial growth of A. brassicicola and P. oryzae at 1 g/L; however, it is not suitable for development as a BCA since it was reported as a causal agent of soybean stem rot 46,47.

The result of testing the halo tolerance of the marine-derived fungi on PDA amended with NaCl at different concentrations showed that all marine-derived fungi exhibited NaCl tolerance, being able to grow on PDA amended with NaCl up to 7%. The tested genera Emericella, Hamigera and Geosmithia showed higher NaCl tolerance than the other tested fungal genera. There are a few studies of salt tolerance and mechanisms in marine fungi for example; marine isolates of Trichoderma atroviride and T. asperelloides were reported tolerate NaCl at 3%33. The thick cell wall and large numbers of vacuoles in marine fungal cells may help these fungi adapt to marine environments48and the increase of the multi-functional cell-wall proteins hydrophobins may played a key role in salt tolerance in eukaryotes49. Although the tested marine-derived fungi could grow on media amended with NaCl, the effects of NaCl on fungal growth and their sporulation were observed in all except E. chevalieri (KUFA0464), which is not surprising because the genus Eurotium is a well-known halophilic and/or xerophilic fungi which is often found in salty food and hypersaline areas 50,51. These observations corresponded to a previous report which found that NaCl caused abnormal conidiophore production in Aspergillus species 52.

Climatic changes such as higher temperatures and drought will result in increased soil salinity, which is predicted to affect plant pathogen growth, development and survival rates as well as modify their pathogenicity leading to changes in disease severity on crops 53-54. Hence, new BCAs with halo tolerant properties should be urgently sought. In this effort, the results from this preliminary study showed that marine-derived fungi are the promising sources of BCAs for application in crop production in normal and salty soil areas and in arid-zone agriculture as well as in supporting crop production under climatic changes in the future.

Results from this study indicate that some of the marine-derived fungi tested in this study possess antagonistic mechanisms including competition for space and nutrients as well as antibiosis production resulting in inhibition the mycelial growth of plant pathogenic fungi. They also possess halo tolerance which made it possible for them to grow on media amended with 7% NaCl. These data suggested that they are potential BCAs which may be promising alternatives to the use of synthetic fungicides to control plant diseases in normal and salty soil areas and in arid-zone agriculture. However, further studies are needed to identify antifungal substances responsible in inhibiting mycelial growth of plant pathogenic fungi as well as to evaluate their biocontrol potential against plant disease under greenhouse and field conditions.

Acknowledgments

The authors wish to thank the Graduate School, Kasetsart University and the Kasetsart University Research Development Institute (KURDI) for financial support of this project.

Conflict of Interest

The authors declare that there is no conflicts of interest.

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