CALISAYA Colony of the endophytic fungi from C. calisaya on PDA 87

M78, F. solani M8, F. oxysporum M16, Leptosphaerulina chartarum M83, L. chartarum M87, Penicillium citrinum M51, Phomopsis tersa M95, Phyllosticta capitalensis M35 capable produce quinine and quinidine. D. phaseolorum M10 and D. pseudomangiferae M88 produce quinine and cinchonine. Col. boninense M28 produce quinine and cinchonidine, D. pseudomangiferae M78 produce quinine, quinidine and cinchonidine. The finding on the capability of D. phaseolorum from Cinchona is supported by the report of Maehara et al. 2012 whom found endophytic D. phaseolorum in C. ledgeriana that produced quinine. Cinchona alkaloids profiling was done in this study Appendix 4.1. According to Kubicek et al. 2003; Fisvard et al. 2008 secondary metabolites profiling were required as a component of polyphasic to identify endophytic fungi. Based on Jaccard similarity indices resulted from Unweighted Pair Group Method with Arithmetic average UPGMA analyses on cinchona alkaloid profile of 39 strains of Diaporthe, 23 clusters Fig 6.1 are formed using similarity index of 0.44 as the cutting score. This grouping is hardly depicted the phylogenetic relationship Fig. 5.3. Of those cluster, only Diaporthe sp. M70 –96 group is in accordance with their phylogenetic clustering. Therefore, this result indicates that alkaloid production is not species dependent but strain dependent. Further, alkaloid profile characteristic has been proved to be ambiguous for Diaporthe. Bhagobaty Joshi 2011 studied metabolite profiling of endohytic fungi of ethno-pharmacologically important plant of Meghalaya. Two isolates, RS07OS and RS07OC, had the same metabolite profiles, but had different molecular identity. The same endophytic species may have different metabolic profiles because of differences in their biological activities. Diaporthe sp. was capable to produce cinchona alkaloids from C. calisaya, while Diaporthe sp. from Rhizophora stylosa was capable to produce isochromophilones Zang et al. 2012; Phomopsis sp. from Allamanda cathartica was capable to produce lactone alkaloids Nithya et al. 2011; Phomopsis sp., an endophyte fungus from Senna spectabilis was potential anti-inflammatory, antifungal, and acetylcholinesterase Chapla et al. 2014. This indicated the importance to study the host-endophytes relationship and the effects of endophytic metabolic production within the host plants, which depend on the environment. Therefore, the host plants and their environment are important to study the endophytes metabolites production. All organs of Quina tree were inhabited by endophytic fungi and might produce quinine. Quinine based industries commonly use stems and barks to obtain the quinine extract. However, this study reveals that Diaporthe from other organs than stems and bark were able to produce quinine. Endophytic fungi have been explored in C. calisaya as a source of new taxa diversity and cinchona alkaloid characters and the culture collected had been preserved in long term manner and kept in the InaCC, LIPIMC, and IPBCC. Therefore, long term research on these strain would be possible for sustainable quinine production. 7 GENERAL CONCLUSION Endophytic fungi in C. calisaya phylogenetically related to Ascomycetes. A total of 687 endophytic fungal strains were collected from 700 segments of organs fruits, leaves, roots, petioles, barks, and flowers of C. calisaya. They consist of 96 morphotypes. Phylogenetic analysis using the ITS rDNA region identified them into 18 genus of 42 taxa of Ascomycota in which Sodariomycetes is the largest group, followed by Dothidiomycetes and Eurotiomycetes. These are Aspergillus sp., As. sydowii, As. versicolor, Cercospora sp., Cladosporium oxysporum, Colletotrichum spp., Col. acutatum, Col. aenigma, Col. arxii, Col. boninense, Col. brasiliense, Col. crassipes, Col. gloeosporioides, Diaporthe spp., D. beckhausii, D. endophytica, D. eucalyptorum, D. ganjae, D. helianthi, D. hongkongensis, D. infecunda, D. litchicola, D. phaseolorum , D. pseudomangiferae, D. psoraleae-pinnatae, F. incarnatum, F. oxysporum, F. solani, G. tenuis, I. anthuricola, L. chartarum, N. chordaticola, Penicillium citrinum, Pestalotiopsis sp., Phoma sp., Pho. palmicola, Pho. tersa, Phy. capitalensis, Pyr. aurantiaca, Pey. Coffeae arabicae, T. hamatum, and T. atroviridae. C. calisaya host different species of one endophytic fungal genus. Phylogenetically related taxa may inhabit different microhabitat organ. However, organ-specific phenomenon is also existed. The community structure of fungal endophyte in each organ varied. The community structure of above ground organ is different from below ground organ. Based on diversity index, leaves and fruits hosted the most diverse endophytic fungi, followed by barks, twigs, petioles, flowers, and roots. Twigs were the most colonized organ, followed by fruits, leaves, petioles, roots, barks, and fruits. Diaporthe is the dominant taxa. Alkaloids profiling based on HPLC analyses of soluble alkaloids in choloform of 96 morphotypes indicates that all morphotypes produced alkaloids with 2-38 compound in each morphotypes. Among those compounds are quinine, quinidine, cinchonine and cinchonidine. About 44 strains of 96 experimented strains were able to produce quinine. Diaporthe 24 strains is the most widely qunine producing taxa, followed by Fusarium 5 strains and Colletotrichum 3 strains. Among prospective strains for quinine production, Diaporthe sp. M13, M70, and D. litchicola M 21 are the most promising strains with thousand fold capacity for quinine production comparing to the one that has ever been published. Determination of some prospective strains using combination of either ITS rDNA and ACT, TUB, HIS or ITS rDNA and EF1- α approach resulted Cercospora sp. M18, D. cinchonae InaCC F-236, InaCC F-238, InaCC-F239, and strain InaCC F-2310 as candidates of new species. In general, alkaloid profile clustering is not in accordance with phylogenetic groupings. This indicates that alkaloids profile cannot be considered as criterion included in polyphasic approach as the alkaloid production is each strain-dependent.