Molecular phylogenetics and anti-Pythium activity of endophytes from rhizomes of wild ginger congener, Zingiber zerumbet Smith

D. Keerthi1, R. Aswati Nair1, D. Prasath2
1School of Biotechnology, National Institute of Technology Calicut (NITC), Calicut, India
2Division of Crop Improvement and Biotechnology, Indian Institute of Spices Research (IISR), Calicut, India

Tóm tắt

Zingiber zerumbet, a perennial rhizomatous herb exhibits remarkable disease resistance as well as a wide range of pharmacological activities. Towards characterizing the endophytic population of Z. zerumbet rhizomes, experiments were carried out during two different growing seasons viz., early-June of 2013 and late-July of 2014. A total of 34 endophytes were isolated and categorized into 11 morphologically distinct groups. Fungi were observed to predominate bacterial species with colonization frequency values ranging from 12.5 to 50 %. Among the 11 endophyte groups isolated, molecular analyses based on ITS/16S rRNA gene sequences identified seven isolate groups as Fusarium solani, two as F. oxysporum and one as the bacterium Rhizobium spp. Phylogenetic tree clustered the ITS sequences from Z. zerumbet endophytes into distinct clades consistent with morphological and sequence analysis. Dual culture assays were carried out to determine antagonistic activity of the isolated endophytes against Pythium myriotylum, an economically significant soil-borne phytopathogen of cultivated ginger. Experiments revealed significant P. myriotylum growth inhibition by F. solani and F. oxysporum isolates with percentage of inhibition (PoI) ranging from 45.17 ± 0.29 to 62.2 ± 2.58 with F. oxysporum exhibiting higher PoI values against P. myriotylum. Using ZzEF8 metabolite extract, concentration-dependent P. myriotylum hyphal growth inhibition was observed following radial diffusion assays. These observations were confirmed by scanning electron microscopy analysis wherein exposure to ZzEF8 metabolite extract induced hyphal deformities. Results indicate Z. zerumbet endophytes as promising resources for biologically active compounds and as biocontrol agents for soft rot disease management caused by Pythium spp.

Tài liệu tham khảo

Ainsworth GC, Sparrow FK, Sussman AS (1973) The fungi: an advanced treatise. In: Ainsworth GC, Sussman AS (eds) A taxonomic review with keys: ascomycetes and fungi imperfecti, vol IV(A). Academic Press, New York Aly AH, Debbab A, Proksch P (2011) Fungal endophytes: unique plant inhabitants with great promises. Appl Biochem Biotechnol 90:1829–1845 Arnold AE, Herre EA (2003) Canopy cover and leaf age affect colonization by tropical fungal endophytes: ecological pattern and process in Theobroma cacao (Malvaceae). Mycologia 95(3):388–398 Arnold AE, Mejía LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, Herre EA (2003) Fungal endophytes limit pathogen damage in a tropical tree. PNAS 100:15649–15654 Aswati NR, Thomas G (2007) Isolation, characterization, diversity analysis and expression studies of resistance gene candidates (RGCs) from Zingiber spp. Theor Appl Genet 116:123–134 Backman PA, Sikora RA (2008) Endophytes: an emerging tool for biological control. Biol Control 46(1):1–3 Bashan Y, Diab S, Okon Y (1982) Survival of Xanthomonas campestris pv. vesicatoria in pepper seeds and roots in symptomless and dry leaves in non-host plants and in the soil. Plant Soil 68:161–170 Brown DW, Proctor RH (2013) Diversity of polyketide synthases in Fusarium. In: Brown DW, Proctor RH (eds) Fusarium: genomics, molecular and cellular biology. Caister Academic Press, UK, pp 143–164 Brundrett MC (2006) Understanding the roles of multifunctional mycorrhizal and endophytic fungi. In: Schulz BJE, Boyle CJC, Sieber TN (eds) Microbial root endophytes. Springer, Germany, pp 281–293 Buchenauer H (1998) Biological control of soil-borne diseases by rhizobacteria. Z Pflanzenk Pflanzen 105:329–348 CABI (2014) Zingiber zerumbet (shampoo ginger). CAB International, Wallingford. www.cabi.org/cpc Castillo U, Strobel GA, Ford EJ, Hess WM, Porter H, Jensen JB, Albert H et al (2002) Munumbicins, wide spectrum antibiotics produced by Streptomyces munumbi, endophytic on Kennedia nigriscans. Microbiology 148:2675–2685 Chakravarthi BVSK, Das P, Surendranath K, Karande AA, Jayabaskaran C (2008) Production of paclitaxel by Fusarium solani isolated from Taxus celebica. J Biosci 32:1–9 Chen C, Bauske EM, Mussan G, Rodriguez-Kabana R, Kloepper JW (1995) Biological control of Fusarium wilt on cotton by use of endophytic bacteria. Biol Control 5:83–91 Clay K (1988) Fungal endophytes of grasses: a defensive mutualism between plants and fungi. Ecology 69:10–16 Conn VM, Walker AR, Franco CMM (2008) Endophytic actinobacteria induce defense pathway in Arabidopsis thaliana. Mol Plant Microbe Interact 21:208–218 Cook D, Beaulieu WT, Mott IW, Riet-Correa F, Gardner DR, Grum D et al (2013) Production of the alkaloid swainsonine by a fungal endosymbiont of the Ascomycete order Chaetothyriales in the host Ipomoea carnea. J Agric Food Chem 61:3797–3803 Damodaran NP, Dev S (1968) Studies in sesquiterpenes—XXXIX. Structure of humulenols. Tetrahedron 24:4133–4142 Deng BV, Liu KH, Chen WQ, Ding XW, Xie XC (2009) Fusarium solani, Tax-3, a new endophytic taxol-producing fungus from Taxus chinensis. World J Microbiol Biotechnol 25:139–143 Dev S (1960) Sesquiterpenes. XVI. Zerumbone, a monocyclic sesquiterpene ketone. Tetrahedron 8:171–180 Ding X, Liu K, Deng B, Chen W, Li W, Liu F (2013) Isolation and characterization of endophytic fungi from Camptotheca acuminata. World J Microbiol Biotechnol 29:1831–1838 Dudeja SS, Giri R, Saini R, Suneja-Madan P, Kothe E (2012) Interaction of endophytic microbes with legumes. J Basic Microbiol 52:248–260 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17(6):368–376 Fitch WM (1971) Towards defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20:406–416 Gange AC, Eschen R, Wearn JA, Thawer A, Sutton BC (2012) Differential effects of foliar endophytic fungi on insect herbivores attacking a herbaceous plant. Oecologia 168:1023–1031 Ghini R, Monica M, Ambrosoli R, Barberis E, Garibaldi A, Piedade SMS (2000) Fusarium oxysporum strains as biocontrol agents against Fusarium wilt: effects on soil microbial biomass and activity. Pesquisa Agropecuária Brasileira 35(1):93–101 Gibert A, Volaire F, Barre P, Hazard L (2012) A fungal endophyte reinforces population adaptive differentiation in its host grass species. New Phytol 194(2):561–571 Gómez-Lama Cabanás C, Schilirò E, Valverde-Corredor A, Mercado-Blanco J (2014) The biocontrol endophytic bacterium Pseudomonas fluorescens PICF7 induces systemic defense responses in aerial tissues upon colonization of olive roots. Front Microbiol 5:427 Greenhalgh R, Fielder DA, Morrison LA, Charland JP, Blackwell BA, Savard ME, ApSimon JW (1989) Secondary metabolites of Fusarium species: apotrichothecene derivatives. J Agric Food Chem 37(3):699–705 Gutierrez RM, Gonzalez AM, Ramirez AM (2012) Compounds derived from endophytes: a review of phytochemistry and pharmacology. Curr Med Chem 19(18):2992–3030 Gutiérrez-Zamora ML, Martínez-Romero E (2001) Natural endophytic association between Rhizobium etli and maize (Zea mays L.). J Biotechnol 91:117–126 Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43(10):895–914 Hasegawa SMA, Shimizu M, Nishimura T, Kunoh H (2006) Endophytic actinomycetes and their interactions with host plants. Actinomycetologica 20:72–81 Hata K, Futai K (1995) Endophytic fungi associated healthy pine needles infested by the pine needle gall midge, Thecodiplosis japonensis. Can J Bot 73:384–390 Hodgson S, Cates C, Hodgson J, Morley NJ, Sutton BC, Gange AC (2014) Vertical transmission of fungal endophytes is widespread in forbs. Ecol Evol. 4(8):1199–1208 Huang WY, Cai YZ, Survesvaran S, Hyde KD, Corke H et al (2009) Molecular phylogenetic identification of endophytic fungi isolated from three Artemisia spp. Fungal Divers 36:69–88 Jaber LR, Vidal S (2010) Fungal endophyte negative effects on herbivory are enhanced on intact plants and maintained in a subsequent generation. Ecol Entomol 35:25–36 Jasim B, Joseph AA, John CJ, Mathew J, Radhakrishnan EK (2014) Isolation and characterization of plant growth promoting endophytic bacteria from the rhizome of Zingiber officinale. 3. Biotech 4:197–204 Kaur R, Kaur J, Singh RS (2010) Nonpathogenic Fusarium as a biological control agent. Plant Pathol J 9(3):79–91 Kavitha PG, Thomas G (2007) Evaluation of Zingiberaceae for resistance to ginger soft rot caused by Pythium aphanidermatum (Edson) Fitzp. Plant Genet Resour Newslett 152:1–4 Kelly KL (1964) Inter-Society Color Council—National Bureau of Standards color name charts illustrated with centroid colors. US Government Printing Office, Washington Kour A, Shawl AS, Rehman S, Sultan P, Qazi PH, Suden P, Khajuria RK, Verma V (2008) Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva. World J Microbiol Biotechnol 24:1115–1121 Kunoh H (2002) Endophytic actinomycetes: attractive biocontrol agents. J Gen Plant Pathol 68:249–252 Kusari S, Pandey SP, Spiteller M (2013) Untapped mutualistic paradigms linking host plant and endophytic fungal production of similar bioactive secondary metabolites. Phytochemistry 91:81–87 Lahlali R, Bajii M, Jijakli MH (2007) Isolation and evaluation of bacteria and fungi as biological control agents against Rhizoctonia solani. Commun Agric Appl Biol Sci 72:973–982 Liu JY, Huang LL, Ye YH, Zou WX, Guo ZJ, Tan RX (2005) Antifungal and new metabolites of Myrothecium sp. Z16, a fungus associated with white croaker Argyrosomus argentatus. J Appl Micobiol 100:195–202 Ma L-J, van der Does HC, Borkovich KA, Coleman JJ, Daboussi M-J et al (2010) Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature 464:367–373 Ma L-J, Geiser DM, Proctor RH, Rooney AP, O’Donnell K, Trail F, Gardiner DM, Manners JM, Kazan K (2013) Fusarium pathogenomics. Annu Rev Microbiol 67:399–416 Mandeel Q, Baker R (1991) Mechanisms involved in biological control of Fusarium wilt of cucumber with strains of nonpathogenic Fusarium oxysporum. Phytopathology 81:462–469 McInroy JA, Kloepper JW (1995) Population dynamics of endophytic bacteria in field-grown sweet corn and cotton. Can J Microbiol 41(10):895–901 Meguro AOY, Hasegawa S, Shimizu M, Nishimura T, Kunoh H (2006) An endophytic actinomycete, Streptomyces sp. MBR-52, that accelerates emergence and elongation of plant adventitious roots. Actinomycetologica 20:1–9 Misaghi IJ, Donndelinger CR (1990) Endophytic bacteria in symptom-free cotton plants. Phytopathology 80:808–811 Mohana Kumara P, Zuehlke S, Priti V, Ramesha BT, Shweta S et al (2012) Fusarium proliferatum, an endophytic fungus from Dysoxylum binectariferum Hook.f, produces rohitukine, a chromane alkaloid possessing anti-cancer activity. Antonie Van Leeuwenhoek 101:323–329 Murakami A, Takahashita D, Kinoshita T, Koshimizu K, Kim HW, Yoshihiro A, Nakamura Y, Jiwajinda S, Tereo J, Ohigashi H (2002) Zerumbone, a Southeast Asian ginger sesquiterpene, markedly suppress free radical generation, proinflammatory protein production, and cancer cell proliferation accompanied by apoptosis: the α, β-unsaturated carbonyl group is a prerequisite. Carcinogenesis 23(5):795–802 Nel B, Steinberg C, Labuschagne N, Viljoen A (2006) The potential of non-pathogenic Fusarium oxysporum and other biological control organisms for suppressing Fusarium wilt of banana. Plant Pathol 55:217–223 Nesic K, Ivanovic S, Nesic V (2014) Fusarial toxins: secondary metabolites of Fusarium fungi. Rev Environ Contam Toxicol 228:101–120 Owen NL, Hundley N (2004) Endophytes—the chemical synthesizers inside plants. Sci Prog 87(2):79–99 Panaccione DG, Beaulieu WT, Cook D (2014) Bioactive alkaloids in vertically transmitted fungal endophytes. Funct Ecol 28(2):299–314 Paparu P, Dubois T, Coyne D, Viljoen A (2009) Dual inoculation of Fusarium oxysporum endophytes in banana: effect on plant colonization, growth and control of the root burrowing nematode and the banana weevil. Biocontrol Sci Technol 19:639–655 Parisot D, Devys M, Barbier M (1990) Naphthoquinone pigments related to fusarubin from the fungus Fusarium solani (Mart.) Sacc. Microbios 64(258):31–47 Qin S, Xing K, Jiang JH, Xu LH, Li WJ (2011) Biodiversity, bioactive natural products and biotechnological potential of plant-associated endophytic actinobacteria. Appl Microbiol Biotechnol 89:457–473 Quadt-Hallmann A, Benhamou N, Kloepper JW (1997) Bacterial endophytes in cotton: mechanisms of entering the plant. Can J Microbiol 43:577–582 Rahman MA, Begum MF, Alam MF (2009) Screening of Trichoderma isolates as a biological control agent against Ceratocystis paradoxa causing pineapple disease of sugarcane. Mycobiology 37:277–285 Rodriguez R, Redman R (2008) More than 400 million years of evolution and some plants still can’t make it on their own: plant stress tolerance via fungal symbiosis. J Exp Bot 59:1109–1114 Rodriguez RJ, White JF, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330 Rogers SO, Bendich AJ (1994) Extraction of total cellular DNA from plants, algae and fungi. Plant Mol Biol Manual D1:1–8 Ruslay S, Abas F, Shaari K, Zainal Z, Sirat HM, Israf DA, Lajis NH (2007) Characterization of the components present in the active fractions of health gingers (Curcuma xanthorrhiza and Zingiber zerumbet) by HPLC–DAD–ESIMS. Food Chem 104(3):1183–1191 Saikkonen K, Saari S, Helander M (2010) Defensive mutualism between plants and endophytic fungi? Fungal Divers 41(1):101–113 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425 Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686 Sette LD, Passarini MRZ, Delarmelina C, Salati F, Duarte MCT (2006) Molecular characterization and antimicrobial activity of endophytic fungi from coffee plants. World J Microbiol Biotechnol 22:1185–1195 Shimizu M, Nakagawa Y, Sato Y, Furumai T, Igarashi Y, Onaka H, Yoshida R, Kunoh H (2000) Studies of endophytic Actinomycetes (I) Streptomyces sp. isolated from rhododendrons and its antifungal activity. J Gen Plant Pathol 66:360–366 Shiono Y, Tsuchinari M, Shimanuki K, Miyajima T, Murayama T, Koseki T, Laatsch H, Takanami K, Suzuki K (2007a) Fusaristatins A and B, two new cyclic lipopeptides from an endophytic Fusarium sp. J Antibiot (Tokyo) 60(5):309–316 Shiono Y, Tsuchinari M, Shimanuki K, Miyajima T, Murayama T, Koseki T, Laatsch H, Takanami K, Suzuki K (2007b) Fusaristatins A and B, two new cyclic lipopeptides from an endophytic Fusarium sp. J Antibiot 60:309 Singh CB, Nongaleima Kh, Singh BS, Ningombam S, Lokendrajit N, Singh LW (2012) Biological and chemical properties of Zingiber zerumbet Smith: a review. Phytochem Rev 11:113–125 Strobel GA (2003) Endophytes as sources of bioactive products. Microbes Infect 5:535–544 Sturz AV, Matheson BG (1996) Populations of endophytic bacteria which influence host-resistance to Erwinia induced bacterial soft rot in potato tubers. Plant Soil 184:265–271 Sturz AV, Christie BR, Matheson BG, Nowak J (1997) Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol Fertil Soils 25:13–19 Sulaiman MR, Padzil A, Shaari K, Khalid S, Shaikmossadeq W, Shahmohamad A, Ahmad S, Akira A, Israf D, Lajis N (2010) Antinociceptive activity of Melicope ptelefolia ethanolic extract in experimental animals. J Biomed Biotechnol Article ID: 937642 Surette MA, Sturz A, Lada RR, Nowak J (2003) Bacterial endophytes in processing carrots (Daucus carrota L. var. sativus): their localization, population density, biodiversity and their effects on plant growth. Plant Soil 253:381–390 Taechowisan T, Lu C, Shen V, Lumyong S (2005) Secondary metabolites from endophytic Streptomyces aureofaciens CMUAC 130 and their antifungal activity. Microbiology 151:1691–1695 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep 18:448–459 Tayung K, Barik BP, Jha DK, Deka DC (2011) Identification and characterization of antimicrobial metabolite from an endophytic fungus, Fusarium solani isolated from bark of Himalayan yew. Mycosphere 2(3):203–213 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680 Thongchai T, Peberdy JF, Saisamorn L (2003) Isolation of endophytic actinomycetes from selected plants and their antifungal activity. World J Microbiol Biotechnol 19:381–385 Tushar Basak S, Sarma GC, Rangan L (2010) Ethnomedical uses of Zingiberaceous plants of northeast India. J Ethanopharmacol 132(1):286–296 U’ren JM, Lutzoni F, Miadlikowska J, Laetsch AD, Arnold AE (2012) Host and geographic structure of endophytic and endolichenic fungi at a continental scale. Am J Bot 99(5):898–914 van Buren AM, Andre C, Ishimaru CA (1993) Biological control of the bacterial ring rot pathogen by endophytic bacteria isolated from potato. Phytopathology 83:1406 Vimala S, Norhanom AW, Yadav M (1999) Anti-tumour promoter activity in Malaysian ginger rhizobia used in traditional medicine. Br J Cancer 80:110–116 White JF, Bacon CW (2012) The secret world of endophytes in perspective. Fungal Ecol 5:287–288 Xu L, Zhou L, Zhao J, Li J, Li X, Wang J (2008) Fungal endophytes from Dioscorea zingiberensis rhizomes and their antibacterial activity. Lett Appl Microbiol 46:68–72 Yob N, Jofrry SM, Affandi M, Teh L, Salleh M, Zakaria Z (2011) Zingiber zerumbet (L.) Smith: a review of its ethnomedicinal, chemical, and pharmacological uses. Evid Based Complement Alternat Med 2011:1–12 Zhang HW, Song YC, Tan RX (2006) Biology and chemistry of endophytes. Nat Prod Rep 23:753–771 Zimmerman NB, Vitousek PM (2012) Fungal endophyte communities reflect environmental structuring across a Hawaiian landscape. Proc Natl Acad Sci USA 109(32):13022–13027