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PLOS ONE  2011 

Characterization of Five Fungal Endophytes Producing Cajaninstilbene Acid Isolated from Pigeon Pea [Cajanus cajan (L.) Millsp.]

DOI: 10.1371/journal.pone.0027589

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Five fungal endophytes (K4, K5, K6, K9, K14) producing Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-c?arboxylicacid) were isolated from the roots of pigeon pea [Cajanus cajan (L.) Millsp.]. CSA is responsible for the prominent pharmacological activities in pigeon pea. The amount of CSA in culture solution varied among the five fungal endophytes. K4 produced the highest levels of CSA (1037.13 μg/L) among the endophytes tested after incubation for five days. Both morphological characteristics and molecular methods were used for species identification of fungal endophytes. The five endophytic isolates were characterized by analyzing the internal transcribed spacer (ITS) rRNA and β-tubulin genes. The K4, K5, K9 and K14 strains isolated from pigeon pea roots were found to be closely related to the species Fusarium oxysporum. K6 was identified as Neonectria macrodidym. The present study is the first report on the isolation and identification of fungal endophytes producing CSA in pigeon pea. The study also provides a scientific base for large scale production of CSA.


[1]  Fujita K, Kai Y, Takayanagi M (2004) Genotypic variability of pigeonpea in distribution of photosynthetic carbon at low phosphorus level. Plant Sci 166: 641–649.
[2]  Tang Y, Wang B, Zhou XJ (1999) Effect of external application of herbal cajani preparation on the fibronection content during healing process of open wound. J Guangzhou U Tradit Chin Med 16: 302–304.
[3]  Kong Y, Fu YJ, Zu YG, Liu W, Wang W, et al. (2009) Ethanol modified supercritical fluid extraction and antioxidant activity of cajaninstilbene acid and pinostrobin from pigeonpea [Cajanus cajan (L.) Millsp.] leaves. Food Chem 117: 152–159.
[4]  Liu W, Zu YG, Fu YJ, Kong Y, Ma W, et al. (2010) Variation in contents of phenolic compounds during growth and post-harvest storage of pigeon pea seedlings. Food Chem 121: 732–739.
[5]  Grover JK, Yadav S, Vats VJ (2002) Medicinal plants of India with anti-diabetic potential. J Ethnopharmacol 81: 81–100.
[6]  Morton JF (1976) The pigeon pea (Cajanus cajan Millsp.), a high protein, tropicalbush legume. HortScience 11(1): 11–19.
[7]  Sun SM, Song YM, Liu J (1995) Studies on the pharmacology of Cajanin preparation. Chinese Traditional and Herbal Drugs 26(3): 147–148.
[8]  Fu YJ, Zu YG, Liu W, Efferth T, Zhang NJ, et al. (2006) Optimization of luteolin separation from pigeonpea [Cajanus cajan (L.) Millsp.] leaves by macroporous resins. J Chromatogr A 1137(2): 145–152.
[9]  Fu YJ, Zu YG, Liu W, Zhang L, Tong MH, et al. (2008) Determination of vitexin and isovitexin in pigeonpea using ultrasonic extraction followed by LC–MS. J Sep Sci 31(2): 268–275.
[10]  Wu N, Fu K, Fu YJ, Zu YG, Chang FR, et al. (2009) Antioxidant activities of extracts and main components of pigeonpea [Cajanus cajan (L.) Millsp.] leaves. Molecules 14: 1032–1043.
[11]  Duker-Eshun G, Jaroszewski JW, Asomaning WA, Oppong-Boachie F, Christensen SB (2004) Antiplasmodial constituents of Cajanus cajan. Phytother Res 18: 128–130.
[12]  Zu YG, Fu YJ, Liu W, Hou CL, Kong Y (2006) Simultaneous determination of four flavonoids in pigeonpea [Cajanus cajan (L.) Millsp.] leaves using RP-LC-DAD. Chromatographia 63: 499–505.
[13]  Zheng YY, Yang J, Chen DH, Sun L (2007) Effects of the stilbene extracts from Cajanus cajan L. on ovariectomy-induced bone loss in rats. Acta Pharm Sinica 42: 562–565.
[14]  Cooksey CJ, Dahiya JS, Garratt PJ, Strange RN (1982) Two novel stilbene-2-carboxylic acid phytoalexins from Cajanus cajan. Phytochemistry 21(12): 2935–2938.
[15]  Chong JL, Poutaraud A, Hugueney P (2009) Metabolism and roles of stilbenes in plants. Plant Sci 177: 143–155.
[16]  Petrini O (1991) Fungal endophytes of tree leaves. In: Andrews JH, Hirano SS, editors. Microbial Ecology of Leaves. Springer, New York. pp. 179–197.
[17]  Herre EA, Knowlton N, Mueller UG, Rehne SA (1999) The evolution of mutualisms: exploring the paths between conflict and cooperation. Trends Ecol Evol 14(2): 49–53.
[18]  Redman RS, Sheehan KB, Scout RG, Rodríguez RJ, Henson JM (2002) Thermotolerance generated by plant/fungal symbiosis. Science 298: 1581.
[19]  Arnold AE, Mejia LC, Kyllo D, Rojas EI, Maynard Z, et al. (2003) Fungal endophytes limit pathogen damage in a tropical tree. PNAS 100: 15649–15654.
[20]  Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, et al. (2005) The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. PNAS 102: 13386–13391.
[21]  Li WK, Hu ZB (2005) Endophytes and natural medicines. Chin J Nat Med 4: 193–199.
[22]  Poland WSW, Reinhard K, Thomas P, Eva M, Heidrum A (2007) Anti-Candida metabolites from endophytic fungi. Phytochemistry 68: 886–892.
[23]  Stierle A, Strobel G, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science 5105: 214–216.
[24]  Strobel GA (2002) Rainforest endophytes and bioactive products. Crit Rev Biotechnol 22: 315–333.
[25]  Wang Y, Guo LD, Hyde KD (2005) Taxonomic placement of sterile morphotypes of endophytic fungi from Pinus tabulaeformis (Pinaceae) in northeast China based on rDNA sequences. Fungal Divers 20: 235–260.
[26]  Xu LL, Han T, Wu JZ, Zhang QY, Zhang H, et al. (2009) Comparative research of chemical constituents, antifungal and antitumor properties of ether extracts of Panax ginseng and its endophytic fungus. Phytomedicine 16: 609–616.
[27]  Nel B, Steinberg C, Labuschagne N, Viljoen A (2006) Isolation and characterization of nonpathogenic Fusarium oxysporum isolates from the rhizosphere of healthy banana plants. Plant Pathol 55: 207–216.
[28]  Sébastien A, Christelle C, Claude A, Chantal O (2008) Comparative analysis of PR gene expression in tomato inoculated with virulent Fusarium oxysporum f. sp. lycopersici and the biocontrol strain F. oxysporum Fo47. Physiol Mol Plant P 73(1–3): 9–15.
[29]  Booth C (1971) The Genus Fusarium. Commonwealth Agricultural Bureau: Kew, Surrey, England.
[30]  Nelson PE, Toussoun TA, Mararas WFO (1983) Fusarium species: An Illustrated Manual for Identification. University Park, PA: The Pennsylvania State University Press.
[31]  Windels CE (1992) Fusarium. In: Singleton LL, Mihail JD, Rush CM, editors. Methods for Research on Soilborne Phytopathogenic Fungi. American Phytopathological Society Press. St Paul. pp. 115–128.
[32]  Ouellette GB, Bard G (1966) A perennial canker of balsam fir on Anticosti Island. Plant Dis Rep 50: 722–724.
[33]  Schultz ME, Parmeter JR (1990) A canker disease of Abies concolor caused by Nectria fuckeliana. Plant Dis 74: 178–180.
[34]  Brayford D, Honda BM, Mantiri FR, Samuels GJ (2004) Neonectria and Cylindrocarpon: the Nectria mammoidea group and species lacking microconidia. Mycologia 96: 572–597.
[35]  Vasiliauskas R, Stenlid J, Johansson M (1996) Fungi in bark peeling wounds of Picea abies in central Sweden. Eur J Forest Pathol 26: 285–296.
[36]  Rossman AY, Samuels GJ, Rogerson CT, Lowen R (1999) Genera of Bionectria-ceae, Hypocreaceae and Nectriaceae (Hypocreales, Ascomycetes). Stud Mycol 42: 1–260.
[37]  Seifert KA, McMullen CR, Yee D, Reeleder RD, Dobinson KF (2003) Molecular differentiation and detection of ginseng-adapted isolates of the root rot fungus Cylindrocarpon destructans. Phytopathology 93: 1533–1542.
[38]  Bigelis R, He HY, Yang HY, Chang LP, Greenstein M (2006) Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation. J Ind Microbiol Biot 33: 815–826.
[39]  Booth C (1959) Studies of pyrenomycetes. IV. Nectria (part 1). Mycological Papers 73: 1–115.
[40]  Samuels GJ, Brayford D (1993) Phragmosporous Nectria species with Cylindrocarpon anamorphs. Sydowia 45: 55–80.
[41]  Vasiliauskas R, Lygis V, Thor M, Stenlid J (2004) Impact of biological (Rotstop) and chemical (urea) treatments on fungal community structure in freshly cut Picea abies stumps. Biol Control 31: 405–413.
[42]  Xin G, Glawe D, Doty SL (2009) Characterization of three endophytic, indole-3-acetic acid producing yeasts occurring in Populus trees. Mycol Res 113: 973–980.
[43]  Anderson IC, Cairney JWG (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environ Microbiol 6: 769–779.
[44]  O'Donnell K, Kistler HC, Tacke BK, Casper HH (2000) Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab. PNAS 97: 7905–7910.
[45]  Germaine K, Keogh E, Garcia-Cabellos G, Borremans B, Lelie D, et al. (2004) Colonisation of poplar trees by gfp expressing bacterial endophytes. FEMS Microbiol Ecol 48: 109.
[46]  Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep 18: 448–459.
[47]  Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A (2002) Systematics of Basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS Yeast Res 2: 495–517.
[48]  Strobel G, Daisy B (2003) Bioprospecting for Microbial Endophytes and Their Natural Products. Microbiol Mol Biol R 67(4): 491–502.
[49]  Hua X, Fu YJ, Zu YG, Wu N, Kong Y, et al. (2010) Plasma pharmacokinetics and tissue distribution study of cajaninstilbene acid in rats by liquid chromatography with tandem mass spectrometry. J Pharmaceut Biomed 52: 273–279.
[50]  White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, PCR Protocols: a guide to methods and applications. Academic Press, SanDiego. pp. 315–322.
[51]  O'Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7: 103–106.
[52]  Sánchez Márquez S, Bills GF, Zabalgogeazcoa I (2007) The endophytic mycobiota of the grass Dactylis glomerata. Fungal Divers 27: 171–195.
[53]  Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596–1599.
[54]  Saitou N, Nei M (1987) The neighbor-joining method–a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425.
[55]  Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.


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