Antifungal ability of some mangrove endophytic actinomycetes in Viet Nam

JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2016-0067 Natural Sci. 2016, Vol. 61, No. 9, pp. 145-151 This paper is available online at 145 ANTIFUNGAL ABILITY OF SOME MANGROVE ENDOPHYTIC ACTINOMYCETES IN VIETNAM Duong Minh Lam, Phung Thi Ngoc Quyen and Tong Thi Mo Faculty of Biology, Hanoi National University of Education Abstract. Mangroves play many special ecological and practical roles in ecosystems. Microorganisms in mangroves, especially endophytic actinomycetes have increasingly called attention of microbiologists. The number and distribution of actinomycetes are very different in 10 species. In this study, 114 endophytic actinomycetes were isolated from 10 mangrove species in Giao Thuy, Nam Dinh: of which 44 strains were from their fruits, 40 from leaves, 21 from roots; 5 from trunks and 4 from flowers. Antifungal ability of endophytic actinomycetes of mangrove plants are relatively high (i.e. 58/114 shows activities on either Aspergillus Niger or Candida albicans) compared to the results of research on soil actinomycetes. These results demonstrate that the endophytic actinomycetes in mangrove plants are prospective for applied researches. Keywords: Endophytic actinomycetes, mangroves, antifungal. 1. Introduction The actinomycetes are famous for its ecological and practical roles. In recent years, there have increasingly been researches on endophytic actinomycetes and it has been proved that actinomycetes positively effected on plant growth protecting it against pathogenic microorganisms [1, 2] by producing phytohormones, indole-3-acetic acid (IAA), siderophores, or antibiotics [3, 4]. Antifungal ability of actinomycetes is one of the most important research areas of microbiology. Fungal diseases in plants, animals and humans normally require long treatment time and a combination of drugs. According to the World Health Organization, more than 50% of medicines are inappropriately prescribed and dispensed; and more than 50% of patients in developing countries are ineffectively treated as prescribed drugs [5]. The above situation makes drug-resistant microorganisms common, mycosis harder to be treated, besides, new antifungal medicines and strategies are in need. Many studies have been conducted on endophytic actinomycetes and some antifungal substances have been announced with biological activities such as 6-prenylindole [4], naphthomycins A, K [6], hydroxamate [7]. Received January 21, 2016. Accepted October 12, 2016. Contact Duong Minh Lam, e-mail address: duong.minhlam@gmail.com Duong Minh Lam, Phung Thi Ngoc Quyen and Tong Thi Mo 146 In the past few years, several studies on mangrove actinomycetes were conducted on soil, water and plant debris samples in Vietnam. Mai Thi Hang et al. (2007) [8] investigated the anticancer ability of Streptomyces padanus D138 isolated from mangroves in Giao Thuy, Nam Dinh. The anticancer compound showed strong activity on lung cancer cell lines, with a low concentration of IC50 0,091 μg/mL. The result suggested that the S. padanus D138 strain was highly potential for the treatment of lung cancer. The actinomycetes isolated from mangrove soils in Nam Dinh province have been biologically characterized. Of the 128 isolates, 34 strains showed antibiotic activity against Fusarium oxysporum and 2 strains against Pseudomonas aeruginosa [9]. In another study, Streptomyces sp. QN63 isolated from mangroves in Yen Hung, Quang Ninh strongly inhibited growth of a penicillin-resistant Staphylococcus aureus TCV. Anticancer ability of some endophytic actinomycetes in mangrove trees was recently published [9, 10]. Vietnam is a hotspot of biodiversity, however, there has been few researches on endophytic actinomycetes, especially in mangrove plants. It is a so-called open and interesting area for microbiologists to study and response the need of both basic and applied researches. In this study, we presented our research results on antifungal ability of endophytic actinomycetes that were isolated from 10 mangrove plant species in Giao Thuy district, Nam Dinh province. 2. Content 2.1. Materials and methods Materials: plant samples including: flowers, fruits, leaves, trunks, roots of Sonneratia caseolaris, Sonneratia apetala, Ipomoea pes-caprae, Lumnitzera racemosa, Rhizophora apiculata, Acanthus ilicifolius, Bruguiera gymnorrhiza, Sesuvium portulacastrum, Clerodendrum inerme and Avicennia marina); Aspergillus niger and Candida albicans were isolated and kept at Micro- biology and Bio-technology Department, HNUE. Samples collection: A selection of healthy plants were collected and washed with sea water in the mangrove and taken to the laboratory within 24 hours, then kept at 4 o C for isolation. Surface sterilization: 10 cm pieces of samples were continuously washed under tape-water for 10 min. then sunk in 70% ethanol and 2% sodium hypoclorite for 5 minutes each. The samples were then washed 5 times with sterilised water. Isolation: 5 grams of samples were grounded in 2 mL of H2O2, 45 mL of 0. 9%NaCl; 100 µL of ground samples was spred on different culture media including S Agar, Starch-Casein Agar, Gause II, IM2, with an addition of 30 mg of nalidixic acid, 20 mg of ketoconazole and 50 mg of cycloheximide. All plates were kept at 28 o C for actinomycetes checking from 7 - 30 days [11]. Antifungal test: actinomycetes strains were grown in Gause II liquid culture for 7 days to take liquid for testing fungal inhibition ability. Fungal spores had been spread on an agar plate of PDA medium then a well of 1 cm diameter on agar was prepared. 100 µL of 7-days liquid culture was added into the well, kept at 4 o C for 12 hours and then incubated at 28 o C for 2 - 3 days to check inhibition zone [9]. Morphological studies: actinomycete morphologies were described under light and electron microscopes using characteristics of conidiophores and conidia. Antifungal ability of some mangrove endophytic actinomycetes in Vietnam 147 2.2. Results and discussions 2.2.1. Isolation There were 114 strains of endophytic actinomycetes isolated from 10 studied mangrove plant species. The number of isolates is mostly from roots (21), leaves (40) and fruits (44). However, the distribution of the isolates in parts, or in plant species was greatly different (Table 1). Among 44 strains isolated from fruits, 32 in Sonneratia apetala and the rest 12 strains from Avicennia marina. Similarly, the results showed something special when leaves from 5 species gave isolates while the other five did not (Table 1). Table 1. Isolation results of endophytic actinomycetes from 10 mangrove tree species Host plants Roots (strain/total) Trunks (strain/total) Leaves (strain/total) Flowers (strain/total) Fruits (strain/total) Sonneratia caseolaris 14/14 Sonneratia apetala 2/36 2/36 32/36 Ipomoea pes-caprae 1/5 3/5 1/5 Lumnitzera racemosa 2/2 Rhizophora apiculata 20/20 Bruguiera gymnorrhiza 2/2 Acanthus ilicifolius 1/1 Sesuvium portulacastrum 2/2 Clerodendrum inerme 2/4 2/4 Avicennia marina 16/28 12/28 Total 21/114 5/114 40/114 4/114 44/114 In many other studies, leaves are normally the most diverse parts for endophytic actinomycetes. These results from 10 mangrove plant species showed somehow a different picture within 21 from roots, 40 from leaves, 44 from fruits, 5 from trunks and 4 from flowers. Sonneratia caseolaris gave 14 isolates from leaves. All other samples of this plant species did not give any strain to the studies. More interesting results were shown in Sonneratia apetala when 32/36 strain of these species were isolated from fruits. The question for this plant species is how such a big difference in endophytic actinomycetes in leaves and fruit exists. What was the change from flowers to fruits when none of the isolates was from flowers? Similar questions should also pose for Avicennia marina. The six plant species are not rich in endophytic actinomycetes which include: Ipomoea pes-caprae (5 isolates), Lumnitzera racemosa (2 isolates), Bruguiera gymnorrhiza (2 isolates), Acanthus ilicifolius (1 isolates), Sesuvium portulacastrum (2 isolates) and Clerodendrum inerme (4 isolates). The research results are new, and meaningful in pointing Duong Minh Lam, Phung Thi Ngoc Quyen and Tong Thi Mo 148 out the richness of endophytic actinomycetes in studied plant species, hence suggesting researchers to focus and select plant species for future studies. Endophytic actinomycete distribution is an interesting subject. Comparing with some other studies, 114 strains from 10 mangrove plant species is a relative low number of strains isolated. Huang et al. (2012) [12] isolated 280 endophytic actinomycete strains from 12 medicinal plant species in Hainan island, China including 154 strains from roots, 73 strains from trunks, and 53 from leaves. Zhang et al. (2012) [13] isolated 65 endophytic actinomycete strains from 4 plant species of Achyranthes bidentata, Paeonia lactiflora, Radix platycodi and Artemisiae argyi. 2.2.2. Antifungal ability Antifungal ability of the studied strains was tested on Aspergillus niger and Candida albicans. The results were shown in the Table 2. Table 2. Screening for antifungal ability of isolated strain Test fungi Positive strains Strains (D - d) ≥ 10 mm Strains (D - d) ≥ 15 mm Aspergillus niger 56/114 34/114 19/114 Cadida albicans 58/114 34/114 9/114 It is surprising that there were almost 50% of the strains showed antifungal activities on A. niger (56/114 strains) and C. albicans (58/114 strains) including 19 strains with strong activities (inhibition zone is more than 15 mm diameters) against A. niger, and 8 ones against C. albicans. These results showed that endophytic actinomycetes were high valuable sources for antifungal drug screening. Table 3 showed more details of fungal activities of some selected strains. The results presented in Table 3 showed that the number of strains (the ratio of strongly positive strains) producing antifungal activities was smaller compared to that of actinomycetes in soils [9, 14]. However, 25/114 strains were remarkable when compared with some recent research results worldwide. Huang et al. (2012) [12] reported that 54/280 strains showed activities against Candida albicans ATCC10231. Hong et al. (2009) [15] isolated actinomycetes from soil and mangrove trees and identified to 13 genera, mostly Micromonospora and Streptomyces, of which 5% of the strains against Candida albicans. Gangwar et al. (2015) [3] isolated 64 strains of endophytic actinomycetes from Emblica officinalis Gaertn, 15 strains showed activity against at least 1 of the tested fungi. Many studies demonstrated that the endophytic actinomycetes in medicinal plants possessed antifungal characteristics. Streptomyces aureofaciens CMUAc130 isolated from Thai ginger showed a strong activity against common plant fungal pathogen Colletotrichum musae and Fusarium oxysporum [16]. Within the scope of the results presented here, the strains that should be focused for further studies are ND2-1C11, ND2-1C3, ND2-1C6, ND2-2E26, and ND2-2E29 showed both activities on A. niger and C. albicans. Antifungal ability of some mangrove endophytic actinomycetes in Vietnam 149 Table 3. Strains showed strong activities on A. niger and C. albicans No. Strains C. albicans D - d (mm) A. niger D - d (mm) No. Strains C. albicans D - d (mm) A. niger D - d (mm) 1 ND1 - 2E1 10 15 14 ND2 - 2E13 15 14 2 ND1 - 2E2 12 15 15 ND2 - 2E15 10 18 3 ND2 - 11C8 11 5 16 ND2 - 2E20 11 20 4 ND2 -12A18 0 5 17 ND2 - 2E24 12 16 5 ND2 - 1C10 15 15 18 ND2 - 2E26 12 22 6 ND2 - 1C11 13 20 19 ND2 - 2E27 13 15 7 ND2 - 1C3 12 20 20 ND2 - 2E29 13 20 8 ND2 - 1C4 15 15 21 ND2 - 2E41 18 12 9 ND2 - 1C5 15 15 22 ND2 - 2E42 15 16 10 ND2 - 1C6 15 20 23 ND2 - 4C1 12 15 11 ND2 - 1C7 15 15 24 ND2 - 7C6.1 13 12 12 ND2 - 2C1 10 15 25 ND2 - 7C6.2 12 12 13 ND2 - 2E1 11 15 Based on morphological and biochemical characteristics (Figure 1), the strain ND2-1C6 was identified into the genus Streptomyces. Studies on antifungal properties, biotechnology process of the strains and its applications are necessary. Figure 1. Microscopic and electron pictures of conidiophores and spore chain of ND2-1C6 Duong Minh Lam, Phung Thi Ngoc Quyen and Tong Thi Mo 150 3. Conclusion From 10 species of mangrove plants, 114 endophytic actinomycete strains were isolated. The distribution of actinomycetes among plant species, parts of a tree was different. Almost 50% of the isolates showed activity against Candida albicans and Aspergillus niger. However, 25 strains showed strong activity against test fungi with inhibition zone diameter of more than 15 mm. Comparison to other studies, these results showed that endophytic actinomycetes in mangrove trees in Giao Thuy, Nam Dinh were strong and worth studying in antifungal activities. Acknowledgements. This study was funded by the Ministry of Education and Training for the mangrove Gene Conservation Project. REFERENCES [1] Crawford DL, Lynch JM, Whipps JM, Ousley MA. 1993. Isolation and characterization of actinomycete antagonists of a fungal root pathogen. 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