Schematic diagram of the research 83 - - - 0.61 -

5.6 Morphological characters of Diaporthe endophytica on C. calisaya. for 7 days in PDA. 51 5.7 Morphlogical characters of Diaporthe sp. on C. calisaya for 7 days in PDA. 51 5.8 Maximum-parsimony tree showing the relationship between endophytic Fusarium sp. based on the sequnces of ITS of rDNA and Penicillium citrinum isolate AX4602 is taken as outgroup 53 5.9 Maximum-parsimony tree showing the relationship between endophytic Fusarium sp. based on the sequnces of EF1 –α and Penicillium citrinum isolate AX4602 is taken as outgroup 54 5.10 Morphological character of Fusarium oxysporum M16 on C. calisaya for 7 days in PDA. 55 5.11 Morphological characters of Fusarium incarnatum M34 on C. calisaya for 7 days in PDA. 55 5.12 Morphological characters of Fusarium incarnatum M66 on C. calisaya for 7 days in PDA. 56 5.13 Morphological characters of Fusarium incarnatum M67 on C. calisaya for 7 days in PDA. 56 5.14 Morphological characters of Fusarium solani M93 on C. calisaya for 7 days in PDA. 57 5.15 Morphological characters of Fusarium solani M97 on C. calisaya. for 7 days in PDA 57 6.1 Clusters of endophytic Diaporthe spp. based on the similarity on their alkaloid profile 68 LIST OF APPENDICES

1.1 Schematic diagram of the research 83

2.1 Cultural characteristics of the endophytic fungi from

C. calisaya on PDA

83

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

4.1 Alkaloid profiles of endophytic Diaporthe spp. Colletotrichum spp., and others endophytic fungi 93 4.2 Cinchona alkaloid production of endophytic fungi in C. calisaya 109 434 4.3 HPLC analysis of quinine, quinidine, cinchonine and cinchonidine standard 112 4.4 HPLC analysis of endophytic fungi Cercospora sp. on quinine, quinidine, cinchonine and cinchonidine product 113 4.5 HPLC analysis of endophytic fungi Diaporthe sp. of quinine, quinidine, cinchonine and cinchonidine product 113 4.6 HPLC analysis of endophytic fungi Fusarium spp. of quinine, quinidine, cinchonine, cinchonidine product 115 4.7 GeneBank ITS and EF accession number of Fusarium spp. included in this study 116 4.8 GeneBank ITS, EF, ACT, CAL, and HIS accession number of Cercospora spp. included in this study 119 4.9 GeneBank ITS and EF1 –α accession number of Diaporthe sp., D. cinchonae, D. endophytica of fungal isolates included in this study 122 4.10 List of abbreviation of endophytic genera 128 1 GENERAL INTRODUCTION Quina tree Cinchona spp., Rubiaceae is a medicinal plant, native to the Andes forest of Western South America. Local people used this plant for fever treatment. In 1820, Pelletier and Caventou extracted an alkaloid substance called quinine, from the Cinchona bark Achan et al. 2011. Untill now, quinine is used as a primary drug for the treatment of severe malarial illness caused by Plasmodium falciparum infection WHO 2014 in about 80 of malaria endemic countries. Quinine is also used as a food colorant, beverage flavor and raw material for some other chemical industries Santoso et al. 2004. Therefore, quinine is one of the world’s economically important substances from plant. As the demand for the quina bark has increased, Cinchona was domesticated and cultivated out side South America. Cultivation was started in the British and Dutch colonial area in Eastern Africa, Indonesia, Ceylon, India, and the Philippines Dawson 1991. Weddell in 1848, brought some seeds of Cinchona calisaya Wedd. [syn. C. ledgeriana Howard Bern. Moens ex Trimen], from Bolivia to Paris. These are the first quina tree grown in Europe, and later the seeds were also sent to Algiers and Java Taylor 1975. In 1852, during Dutch colonization in Indonesia, Quina seeds were sent from Bolivia to Java. The seeds were first planted in two places, Cibodas Botanical Garden and Gambung, West Java, Indonesia Tao Taylor 2011. Quina trees grow well in the tropics. At present, the area of Quina plantation in Indonesia is approximately 3.886 hectares which previously planted 12.000 hectares Susilo 2011 and the largest area is in Java. The quinine industry in Indonesia has existed before the Second World War and Indonesia had been recognized as the worlds largest supplier of quinine Susilo 2011. The bark production from the plantation only field 30 –50 of demand of the industry Susilo 2011. This supply shortage is filled with the imported bark flake. Even though, Quina plantation in Indonesia is now under replanting program, an alternative source for quinine production has to be investigated. Therefore, it is necessary to explore natural resources for quinine production other than the Quina tree. One of the promising resources is endophytic fungi. Endophytes are microbes, that for at least one periode of their life cycle, inhabit the internal plant tissues without causing apparent harm to the host Petrini 1991. A higher diversity of endophyte was found in tropical plants Banerjee 2011. Endophytic fungi that have been examined to date are found in all major plant lineages such as trees, shurbs, grasses, and ferns Arnold et al. 2001. Almost all plant species are inhabiting one or more endophytic organisms Tan Zou 2001. These colonize both vegetative leaves, petioles, bark or stems and reproductive parts of their hosts Arnold et al. 2003, 2007, Qiu et al. 2008. Endophytic fungi are able to produce secondary metabolite compounds as their host Petrini et al. 1992. Based on that report, various type of endophytic fungi that are potential have been explored intensively. Endophytic fungi can be used in biotechnology as new pharmaceutical compounds, biocontrol agents, and induce plant metabolite production for plant health Bacon White 2000. Bioprospecting analyses are usually performed either after or along with a bio- inventory program. By using of bio-inventory, a large number of endophytic fungi has been reported from various plant species Nalini et al. 2014 including many ethnomedical plants Strobel Daisy 2003. For example Artemisia capillaris, A. indica and A. lactiflora Asteraceae became the source of endophytic Alternaria, Colletotrichum, Phomopsis dan Xylaria Huang et al. 2009 and Colletotrichum sp. endophyte of A. annua has the capability to produce secondary metabolites that act as anti –microbes Lu 2000. Suwannarach 2013 found Col. gloeosporioides, Col. acutatum, Phomopsis spp., Guignardia mangiferae and xylariaceous taxa from Cinnamomm bejolghota. Phoma-like species endophytes of Cin. mollisimum showed cytotoxic and antifungal activities Santiago et al. 2011. Orlandelli 2012 isolated the predominant Bipolaris from Piper hispidum and Garcia et al. 2012 obtained Cochliobolus, Alternaria, Curvularia, Diaporthe, Phomopsis and Phoma from the medicinal plant Sapindus saponari. Guignardia mangiferae, Fusarium proliferatum, and Col. gloeosporioides from Taxus media produce taxol, a potency anti –cancer drug Xiong et al. 2013. Another fungus, Col. gloeosporioides from Justicia gandarusa was also found to be capable of producing taxol Gangadevi Muthumary 2008. Aspergillus, Cladosporium, Fusarium, Nectria, Penicillium and Verticillium were isolated from Panax ginseng and Fusarium spp. were reported to produce saponin as having an antimicrobial substance Wu et al. 2013. Therefore, the exploration of endophytic fungi from Quina tree would provide the opportunity to get a potency strain for producing quinine. Quina tree including C. calisaya may have endophytic fungi. The diversity of endophytic fungi on Cinchona plant has not been intensively studied. The information available is scattered and limited to only that from bark and young stem Simanjuntak et al. 2002, Mumpuni et al. 2004, Winarno 2006, Maehara 2010. Some of these endophytic fungi have been screened for secondary metabolites such as cinchona alkaloids quinine, quinidine, cinchonine and cinchonidine. The endophytic Diaporthe from Cinchona bark can produce these substances Maehara et al. 2012, 2013 since bark contains the highest quinine level besides other alkaloids such as quinidine, cinchonine, and cinchonidine Taylor 1975. Quinine is not only present in the bark of Cinchona, but also as reported by Simanjuntak et al. 2002 and Shibuya et al. 2003, that the bark of Cinchona contains the highest concentration of quinine, while root, stem, twig, leaf, and flower contain quinines in various concentrations. Whilest the information on fungal endophyte from bark and young stem is available, that from other organ is still lacking. Therefore, study on the community structure of fungal endophyte from all organs of Cinchona is carried out to get a more comprehensive information on its diversity and further to analyze its potency to produce quinine. As the bio-prospect of the endophyte is attractive for industry, the fungal isolate as the agents of metabolites production should be obtained and identificated accurately. Therefore, the fungal endophytes community is estimated using a culture-dependent method. Limitations of this method have been reported such as sterilia mycelia of fungal cultures making the identification is difficult Huang et al. 2008, Guo et al. 2000. This limitation is overcome to some extent by applying molecular approach on the bases of a single analysis, a multigene analyses Zhang et al. 1998, Huang et al. 2009 or a polyphasic approach Samson et al. 2007. However, Ganley et al. 2004 considered that the identification of endophytic fungi using available taxonomic method is often difficult as most of the endophytic fungi isolates tend to form complex species. Secondary metabolite profiling of exo –metabolites can be used as to determine characteristics in classification and identification Frisvad et al. 2008; Bhagobaty Joshi 2011; Frisvad 2015. Fungal chemotaxonomy based on secondary metabolites has been used successfully in ascomycetous fungi such as Alternaria, Aspergillus, Fusarium, Hypoxylon, Penicillium, Stachybotrys, and Xylaria Frisvad et al. 2008. Yet, the presence of secondary metabolite, such as quinine substances in endophytic fungi from C. calisaya has not been known. Furthermore, the taxonomical value of that secondary metabolite has not been known either. Therefore, distribution of the secondary metabolite from endophytic fungi of Cinchona is studied. In this study, the secondary metabolites was investigated the presence of the cinchona alkaloid. The information on the diversity of endophytic fungi from C. calisaya and its potential to produce cinchona alkaloids is still limited. A comprehensive study on the aspect of biodiversity of endophytes of C. calisaya and their potency for secondary metabolites necessary to be studied covering: a. Phylogenetic study of culturable endophytic fungi using ITS rDNA region. b. Community structure of the fungal endophytes and their distribution within host organs. c. Discovery of the novel taxa that will contribute to the data of the fungal biodiversity of the potensial indigenous species. d. Analyses of the potential endophytes to produce the cinchona alkaloid quinine, quinidine, cinchonine and cinchonidine. e. Evaluation of polyphasic approach for species delimitation of endophytic fungi by incorporating data. The research was conducted by following schematic diagram Appendix 1 that was designed for achieving the aims of this research, which are: a. To get an inventory of the culturable endophytic fungi from the whole organs of C. calisaya with a correct scientific name. The culturable of endophytic in C. calisaya can be the first data of culture collection in Quina tree. b. To investigate the diversity and community structure of fungal endophyte in C. calisaya. The community structure can provide an information about the diversity, composition, and dominance of endophytic fungi in C. calisaya. c. To analyse the capability of the endophytic fungi, in particular the dominant one to produce alkaloid substances. The capability of the endophytic fungi can be use to produce cinchona alkaloid and replacing the bark of Quina tree. By understanding the diversity of endophytes of C. calisaya and its potency to produce secondary metabolites. It is expected that knowledge on the endophyte- plant associations especially those on the roles of endophyte in secondary metabolite production of the host, will be improved and the opportunity to manipulate their potential for human benefit will be increased. This research is expected to have impacts on: a. The increase of indigenous fungal culture collection Indonesia with accurate identity deposited in culture collection such as InaCC and IPBCC. b. An overview on biodiversity of indigenous fungal endophytes indigenous Indonesia from C. calisaya. c. Information on alkaloid profiles of endophytic fungi from C. calisaya. d. Potential strains for further investigation in the purpose of sustainable production of quinine and other related substances. e. Contribution to publications at least one national publication, one manuscript for international publication under review, and two manuscripts for international publication submitted. The dissertation comprises 7 chapters. The background of this study and objectives are described in chapter 1. Chapter 2 elucidates phylogenetic study of endophytic fungi of C. calisaya with ITS rDNA region. Chapter 3 is about the community structures of fungal endophytes in C. calisaya. Chapter 4 is about the alkaloid profile of endophytic fungi from C. calisaya. The determination of novel species candidat using a multigene approach in chapter 5. Finally, General discussion and conclusion are in chapter 6 and 7. 2 PHYLOGENETIC STUDY OF ENDOPHYTIC FUNGI OF CINCHONA CALISAYA INTRODUCTION Quina Cinchona spp. is an important medicinal plant that have been used since the 16 th century. Quina tree species that is widely cultivated in Indonesia is Cinchona calisaya. Currently, C. calisaya is used as a section stock for multiplication of commercial Quina clone for quinine production. Studies on endophytic fungi from Cinchona plant have been reported by several researchers in Indonesia. All the research were mainly dealt with those from bark and young stem Simanjuntak et al. 2002, Shibuya et al. 2003, Mumpuni et al. 2004 and Winarno 2006. However, fungal endophyte may occupy leaves, stems, petioles, barks, and roots from many Angiosperm taxa of tropical plants Benerjee 2011. Some researches on endophytic fungi from medicinal plants such as leaves, stems and inflorescences of Artemisia capillaris, A. indica, A lactiflora Huang et al. 2009, and Annona squamosa Lin et al. 2010, Piper hispidum leaves Orlandelli 2012, bark and leaves of Taxus media Xiong et al. 2013, root of Panax ginseng Wu et al. 2013 have been reported. Therefore, all organs of medicinal plants can be source of endophytic fungi. Previous study on fungal endophytes of Cinchona spp. is restricted on those from certain part of the tree, and none of them concerned with fungal endophyte from all part of the tree. Exploration of fungal endophyte C. calisaya from the whole part of the tree was conducted in this study. Most of the isolate have not been identified into species and when identification was made they mainly used morphological approach or combined with BLAST search. Thus, none of those research is on based phylogenetic. A phylogenetic based approach had strengthened our classification and identification. These study reports the diversity and phylogenetic relationship of endophytes from C. calisaya. Diversity of endophytes in their hosts is the key to understand their role in endophyte-plant associations. Phylogenetic study have been applied for studying the diversity endophytic such as reported by Huang et al. 2009, Jeewon et al. 2013, and Gokul Raj et al. 2014. None of these reports are fungal endophytes from Quina tree. Furthermore, classification systems of fungi were previously based on morphology. Characters of sexual and asexual reproduction have been traditional basis of this system. However, in the current system, phylogenetic relationships is being used by molecular systematics. Molecular characters have been essential for phylogenetic analysis when morphological characters are convergent, reduced, or missing among the taxa considered Hibbet et al. 1998. These are very useful especially when the specimen found is sterile. Use of molecular characters allows identification of mycelia sterilia and asexual fungi to be placed among their closest relatives teleomorph. As information on the diversity of of endophytic fungi from the whole part of the tree is still lacking, exploration of fungal endophyte diversity of Quina tree using phylogenetic approach is conducted using culture dependent approach. This study aims 1 to identify and quantify of endophytic fungi associated with C. calisaya 2 to verify of morphotype based on ITS rDNA sequence MATERIALS AND METHODS Specimen collections Specimens were collected from Quina tree grown in Quina germ plasm orchard managed by the Research Center for Tea and Quina, Gambung, West Java, Indonesia 29 September 2012. Sampling location 7°835.78S, 107°3059.55E, 1400 m asl. Samples collected include flowers, leaves, petioles, stems, barks, and roots of C. calisaya. Five pieces of each healthy organs from 5 individual plants were taken and placed in zipped plastic bags. The plastic bags were sealed and labelled with the name of the host, collection site, date, and collectors. All specimens were kept in ice boxes prior to isolation in the laboratory. Isolation The isolation protocol of endophytic referred to the method described by Mostert et al. 2001 with modification. The samples were first washed in running tap water, than surface-sterilized using 70 ethanol for 1 minute, followed by soaking in sodium hypochlorite 3 for 2 minutes, and 70 ethanol for 20 seconds. The samples were rinsed 3 times in sterile distilled water, and dried with sterile paper for at least 6 hours. The sterile distilled water of the final rinse was poured onto the agar medium as a quality control of sterilization process. After drying, samples were cut into segments approximately 1 × 1 cm and placed on the surface of malt extract agar MEA Difco, USA 4 segmentspetri dish. All petri dish were incubated at room temperature 27 o C. Three replications were made for each sample. The growth of endophytic fungi mycelium were observed every day, for about 30 days. The growing colonies were purified using hyphal tip isolation method to get a pure culture.The working cultures were kept in potato dextrose agar PDA Difco, USA agar slant. The culture stock were preserved in glycerol- trehalose, kept in –80 °C in Institut Pertanian Bogor Culture Collection. Microscopic observation Colony characters of each isolate was determined from 14 days old culture grown on PDA.The colony characteristics observed include diameter, color of the surface and reverse, margin, texture and zonation. Microscopic structures, such as conidia, conidiophores were examined by using Olympus BX53 light microscope OLYMPUS, Japan under 1000× magnification using immersion oil Barnett Hunter 1998. DNA extraction Fungal isolates was grown in 5 ml of PDB medium for 3 –5 days in an incubator shaker at 100 rpm in room temperature. Pellets was taken and put in a microtube containing 1 mL mili Q water and then centrifuged at 10.000 rpm for 10 minutes. DNA isolation will be performed using the Kit Phytopure tm GE according to the manufacturer’s protocol. After centrifugation, the supernatant was discarded and the pellet was taken using a sterile pestle and then added with 300 µL reagent 1 SDSwater lysis and homogenized. Then, 3 µL RNAse added and incubated for 30 minutes at 37 °C in waterbath. The 200 µL reagent 2 will be added and shaked for 10 minutes. The mixture was incubated for 10 minutes at room temperature and then incubated in freezer for 20 minutes. Mixture was added with 250 µL chloroform, 250 µL phenol, and then centrifuged at 10.000 rpm for 10 minutes. Isopropanol in concentration of half volume of the supernatant was added into supernatant, and centrifuged at 10.000 rpm for 10 minutes. The supernatant discarded. The pellet was added by 100 µL ethanol 99 and then centrifuged at 10.000 rpm for 10 minutes. The supernatant was discarded and dried for 30 minutes. The pellet was added by 50 µ L nuclease free water. PCR amplification and sequencing Amplification was done using Polymerase Chain Reaction PCR method performed in a 25 µ L reaction volume as follow: 10 µ L nuclease free water, 12.5 µL DreamTaq ® green master mix Thermo scientific, USA, 0.5 µL of forward and reverse primer, 0.5 µ L DMSO, and 1 µ L DNA template. The primer used for all strains is ITS, and the second primer varies among fungal genera found. The primer pairs of ITS5 forward 5’–TCCTCCGCTTATTGATATGC –3’ and ITS4 reverse 5–TCCGTAGGTGAACCTGCGC–3 White et al. 1990 were used to amplify the Internal Transcribed Spacer ITS region including 5.8S rDNA. The PCR condition was 90 seconds at 95 °C for pre-denaturation followed by 35 cycles of 30 seconds at 95 °C denaturation, 30 seconds at 55 °C for annealing, 90 seconds at 72 °C for extension, and 5 minutes at 72 °C for final extension. All PCR reactions were conducted using T100 thermal cycler Bio-Rad, USA. PCR products were electrophorised in a 1 wv agarose gel soaked in 1× TAE buffer at 100 Volt for 30 minutes. 1 kb DNA ladder was used as a marker during the electrophoresis. The gel was soaked in EtBr ethidium bromide for 30 minutes prior to UV light examination using Gel Doc XR system Bio –Rad, USA. Purified PCR products were sent to 1stBASE Malaysia for sequencing. Phylogenetic analysis Nucleotide sequences obtained from the respective primer pairs ITS5 and ITS4 were examined and refined by direct examination using Chromas Pro 1.41 software Technelysium Pty Ltd., Australia. Newly ITS sequences of endophytic from C. calisaya were aligned with sequence from NCBI using MUSCLE Edgar 2004 implemented in MEGA 6 Tamura et al. 2013. Species was used as outgroup in analyses. Regions designated as ambiguously aligned were excluded from the analyses. GeneBank accession number, strain code, and taxon names used in this study. Phylogenetic analysis was conducted using the Maximum Parsimony MP method in PAUP 4.0b10 Swofford 2002 on the bases either single or multigene depending on the genera. The heuri stic search option using the ‘Tree-Bisection- R econstruction’ TBR algorithm with 1000 random sequence additions was performed to find the optimum tree. The stepwise addition option set as random and maximum tree number was set at 500. Tree length TL, consistency index CI, retention index RI, related consistency index RC, and homoplasy index HI were also calculated. The strength of the internal branches of the phylogenetic tree in MP analysis was tested with bootstrap BS analysis Felsenstein 1985 using 1000 replications. BS values of 50 or higher than that are shown. Random sequence addition was used in the bootstrap analysis. All sites were treated as unordered and unweighted, and gaps treated as missing data. The partition homogeneity test Farris et al. 1994 with 1000 replicates. TreeGraph 2 software Stöver Müller 2010 was used to refine the phylogenetic tree. RESULTS In total 687 endophytic fungal isolates consisting 123 isolates from leaves, 67 isolates from petioles, 239 isolates from twigs, 49 isolates from barks, 56 isolates from roots, 17 isolates from flowers, and 136 isolates from fruits were obtained from 700 segments of C. calisaya. These cultures are grouped into 96 morphotypes Appendix 2.1; 2.2 on the basis of their colony morphology and growth rate. Most of these morphotypes are mycelia sterilia. Therefore, molecular techniques on the bases of ITS5 –5.8S–ITS4 sequence is used as primary approach for identifying them to either generic or specific level. ITS5 –5.8S–ITS4 sequences of 96 morphotype were compared to 73 corresponding sequences of reference fungal taxa extracted from the genbank database and was rooted to Saccharomyces cerevisiae strain ATCC 18824T and CBS 1171 Table 2.1. In this non-coded dataset, there were 860 total variable characters that composed of 230 constant characters, 65 variable characters parsimony-uninformative and 565 variable parsimony – informative characters. Heuristic search resulted most parsimonious trees with length = 4541, consistency index CI = 0.300, retention index RI = 0.759, rescaled consistency index RC = 0.228, and homoplasy index HI = 0.700. One of the most parsimonious tree is shown Fig. 2.2. The phylogenetic analysis showed that all morphotypes are included in the Ascomycota. This analysis not only showed that the diversity of the endophytic fungi associated with C. calisaya but also illustrate the phylogenetic placement of these endophytes within Ascomycota tree. Sodariomycetes 78.2 represents the largest group, followed by Dothidiomycetes 14.1 and Eurotiomycetes 7.7 . The fungal endophytes from C. calisaya is distributed in 2 main clades. The first main clade is a monophyletic Pestalotiopsis Sordariomycetes, Xylariales, Amphisphaeria-ceae that supported with 100 bootstrap BS value. Strain M80 is within this clade, thus this identified as Pestalotiopsis sp. Table 2.1 GeneBank ITS accession numbers strains of fungal endophytes and sequence used in this study No Species Strain Genbank accesion number 1 Aspergillus sp. M19 IPBCC 15.1255 LC040923 2 As. versicolor M27 IPBCC 15.1256 LC040926 3 As. sydowii M55 IPBCC 15.1257 LC040927 4 As. sydowii M62 IPBCC 15.1258 LC040925 5 As. versicolor M47 IPBCC 15.1259 LC040924 6 Cercospora sp. M18A IPBCC 14.1189 LC040921 7 Cercospora sp. M18B IPBCC 14.1190 LC040922 8 Cladosporium oxysporum M25 IPBCC 15.1260 LC040920 9 Colletotrichum sp. M1 IPBCC 15.1268 LC040907 10 Colletotrichum sp. M3 IPBCC 15.1267 LC040911 11 Colletotrichum sp. M6 IPBCC15.1266 LC040917 12 Col. aenigma M2 IPBCC 15.1262 LC040909 No Species Strain Genbank accesion number 13 Col. gloeosporioides M4 IPBCC 15.1269 LC040912 14 Col. gloeosporioides M7 IPBCC 15.1270 LC040919 15 Col. acutatum M57 IPBCC 15.1271 LC040908 16 Col. arxii M53 IPBCC 15.1272 LC040916 17 Col. boninense M28 IPBCC 15.1264 LC040914 18 Col. boninense M45 IPBCC 15.1264 LC040914 19 Col. brasiliense M76 IPBCC 15.1265 LC040913 20 Col. crassipes M30 IPBCC 15.1263 LC040918 21 Col. crassipes M82 IPBCC 15.1261 LC040910 22 Diaporthe sp. M9 IPBCC 15.1286 LC041055 23 Diaporthe sp. M12 IPBCC 15.1309 LC041062 24 Diaporthe sp. M13 IPBCC 15.1292 LC041020 25 Diaporthe sp. M14 IPBCC 15.1291 LC041028 26 Diaporthe sp. M15 IPBCC 15.1308 LC041049 27 Diaporthe sp. M22 IPBCC 15.1307 LC041030 28 Diaporthe sp. M23 IPBCC 15.1303 LC041042 29 Diaporthe sp. M33 IPBCC 15.1282 AB899787 30 Diaporthe sp. M38 IPBCC 15.1284 LC041019 31 Diaporthe sp. M39 IPBCC 15.1338 LC041060 32 Diaporthe sp. M41 IPBCC 15.1311 LC041054 33 Diaporthe sp. M42 IPBCC 15.1277 LC041035 34 Diaporthe sp. M43 IPBCC15.1276 AB899784 35 Diaporthe sp. M44 IPBCC 15.1293 LC041052 36 Diaporthe sp. M48 IPBCC 15.1310 LC041053 37 Diaporthe sp. M52 IPBCC 15.1300 LC041024 38 Diaporthe sp. M59 IPBCC 15.1284 LC041023 39 Diaporthe sp. M65 IPBCC 15.1290 LC041057 40 Diaporthe sp. M69 IPBCC 15.1287 LC041056 41 Diaporthe sp. M70 IPBCC 15.1304 LC041034 42 Diaporthe sp. M72 IPBCC 15.1340 LC041038 43 Diaporthe sp. M74 IPBCC 15.1306 LC041029 44 Diaporthe sp. M79 IPBCC 15.1289 LC041052 45 Diaporthe sp. M85 IPBCC 15.1284 LC041023 46 Diaporthe sp. M89 IPBCC 15.1288 LC041058 47 Diaporthe sp. M91 IPBCC 15.1283 LC041044 48 Diaporthe sp. M96 IPBCC 15.1305 LC041048 49 D. beckhausii M37 IPBCC 15.1275 LC041024 50 D. beckhausii M73 IPBCC 15.1273 LC041016 51 D. beckhausii M54 IPBCC 15.1274 LC041031 52 D. eucalyptorum M46 IPBCC15.1294 AB899785 53 D. eucalyptorum M81 IPBCC15.1295 LC041021 54 D. eucalyptorum M56 IPBCC 15.1296 LC041022 55 D. endophytica M90 IPBCC 15.1312 AB899789 No Species Strain Genbank accesion number 56 D. endophytica M20 IPBCC 15.1315 LC041025 57 D. helianthi M21 IPBCC 15.1314 LC041026 58 D. ganjae M71 IPBCC 15.1313 LC041037 59 D. hongkongensis M31 IPBCC 15.1278 AB899786 60 D. hongkongensis M36 IPBCC 15.1279 LC041046 61 D. infecunda M63 IPBCC 15.1316 LC041032 62 63 D. infecunda M68 D. litchicola M78 IPBCC 15.1317 IPBCC 15.2997 LC041033 AB899788 64 D. lithicola M88 IPBCC 15.1298 LC041036 65 D. palmicola M11 IPBCC 15.1339 LC041017 66 D. phaseolorum M40 IPBCC 15.1318 LC041040 67 D. phaseolorum M10 IPBCC 15.1319 LC041043 68 D. pseudomangiferae M24 IPBCC 15.1299 LC041041 69 D. psoraleae-pinnatae M94 IPBCC 15.1320 LC041018 70 D. psoraleae-pinnatae M92 IPBCC 15.1321 LC041045 71 D. psoraleae-pinnatae M84 IPBCC 15.1322 LC041047 72 D. psoraleae-pinnatae M32 IPBCC 15.1323 LC041050 73 D. psoraleae-pinnatae M77 IPBCC 15.1324 LC041039 74 Fusarium incarnatum M34 IPBCC 15.1253 LC026132 75 F. incarnatum M66 IPBCC 15.1251 LC026133 76 F. incarnatum M67 IPBCC 15.1252 LC026134 77 F. solani M93 IPBCC 15.1248 LC026135 78 F. solani M97 IPBCC 15.1249 LC026136 79 F. solani M8 IPBCC 15.1247 LC026137 80 F. oxysporum M16 IPBCC 15.1250 LC026138 81 Gliocladiopsis tenuis M49 IPBCC 15.1325 LC040899 82 llyonectria sp. M64 IPBCC 15.1329 LC040895 83 Leptosphaerulina chartarum M83 IPBCC 15.1331 LC040928 84 L. chartarum M87 IPBCC 15.1330 LC040929 85 Neofusicoccum chordaticola M17 IPBCC 15.1332 LC040894 86 Penicillium citrinum M51 IPBCC 15.1333 LC040893 87 Pestalotiopsis sp. M80 IPBCC 15.1334 LC040892 88 Peyronellaea coffeae arabicae M58 IPBCC 15.1336 LC040891 89 Phoma sp. M50 IPBCC 15.1335 LC040890 90 Phomopsis tersa M95 IPBCC 15.1337 LC041027 91 Phyllosticta capitalensis M35 IPBCC 15.1326 LC040896 92 Phy. capitalensis M29 IPBCC 15.1327 LC040897 93 Phy. capitalensis M61 IPBCC 15.1328 LC040898 94 Pyrigemmula aurantiaca M99 IPBCC 15.1341 LC040889 95 Trichoderma atroviride M26 IPBCC 15.1342 LC040888 96 T. hamatum M98 IPBCC 15.1343 LC040887 Table 2.2 GeneBank ITS accession number of reference strains used in this study No Species Strain Genbank accesion number ITS 1 As. persii CBS 112795 FJ491580 2 As. Persii NRRL 35669 EF661399 3 As. sclerotiorum NRRL 415 EF661400 4 As. versicolor CCF 3690 FR733851 5 As. versicolor NRRL 239 EF652449 6 As. sydowii LT3 001 2 GQ229078 7 As. sydowii CBS 593 65 AB267812 8 Cercospora cf brunkii CBS 132657 JX143559 9 Cer. kikuchii CBS 128 27 DQ835070 10 Cladosporium oxysporum ATCC 76499 AF393720 11 Cla. oxysporum CBS 125991 HM148118 12 Col. acutatum IMI 348160 AJ536200 13 Col. aenigma C1253 4 JX010244 14 Col. aenigma CBS 132458 KC566727 15 Col. boninense MAFF305972 JX010292 16 Col. brasilliense CBS 128501 JQ010292 17 Col. crassipes CBS 112988 FN557348 18 Col. gloeosporioides CBS 119204 JX010150 19 Col. arxii CBS 132 511 KF687716 20 Col. arxii CBS 169 59 KF687717 21 D. beckhausii CBS 138 27 KC343041 22 D. endophytica CBS 133811 KC343065 23 D. eucalyptorum CBS 132525 NR 120157 24 D. ganjae CBS 180 91 KC343112 25 D. helianthi CBS 592 81 KC343115 26 D. hongkongensis CBS 115448 KC343119 27 D. infecunda CBS 133812 KC343126 28 D. litchicola BRIP 54900 JX862533 29 D. phaseolorum CBS 113425 KC343174 30 D. phaseolorum CBS 116019 KC343175 31 D. psoraleae pinnatae CPC 21638 KF777159 32 D. pseudomangiferae CBS 101339 KC343181 33 F. equisetti NRRL 26419 GQ505688 34 F. incarnatum MAFF 236521 AB586988 35 F. oxysporum CBS 171 31 U28161 FOU28161 36 F. oxysporum CBS 133023 KF255448 37 F. solani LSL 1 KF826493 38 F. solani NRRL 28579 DQ094383 39 Gliocladiopsis tenuis CBS 114148 JQ666070 40 Gli. tenuis STE U706 AF220981 No Species Strain Genbank accesion number ITS 41 Gli. sagariensis CBS 199 55 JQ666063 42 Glomerella acutate IMI 117617 AF411700 43 Guignardia mangiferae CBS 123404 FJ538333 44 G. mangiferae UFMGC8 5025 JN159689 45 llyonectria anthuriicola CBS 564 95 NR 121494 46 l. vitis CBS 129082 JF735303 47 Leptosphaerulina chartarum TSS 152 KJ398148 48 Neofusicoccum chordaticola MUCC297 EU301020 49 N. chordaticola T NR 119487 50 Penicillium citrinum P 1637 JQ316514 51 P. citrinum NRRL 1841 NR 121224 52 Pestalotiopsis adusta ICMP 6088 NR 111788 53 Pes. microspora CBS 364 54 AF377292 54 Pes. neglecta LK29 DQ000992 55 Peyronellaea coffeae arabicae CBS 123398 FJ426994 56 Pey. coffeae arabicae CBS 123380 FJ426993 57 Phoma glomerata XSD 41 EU273521 58 Pho. herbarum CBS276 37 JF810524 59 Pho. palmicola CP1 KF496903 60 Pho. tersa GRMP 42 JQ818195 61 Phy. capitalensis CBS 119720 FJ538340 62 Pyrigemmula aurantiaca CPC 18064 HM241693 63 Trichoderma atroviride CBS 142 95 F456917 64 T. atroviride NBRC 101776 NR 077207 65 T. hamatum CPK 2317 49 22 69 4200 66 T. hamatum Tri 612 4 KC 747811 67 T. hamatum DAOM 167 057 Z48816 68 Saccharomyces cereviseae ATCC 18824 KC881067 69 Sacch. cereviseae CBS 1171 ABO 18043 Ex-type cultures are in bold Abbreviation see appendix 4.10 The second clade is a polyphyletic group composed of 3 classes of Ascomycota i.e. Sordariomycetes, Dothidiomycetes and Eurotiomycetes. Sordariomycetes is a polyphyletic group composed 8 sub-clades i.e. 2 sub-clades of Fusarium spp., one sub-clade of Gliocladiopsis-Ilyonectria Hypocreales, Nectriaceae with 100 BS and Colletotrichum sub-clade Melanconiales, Melanconiaceae with 100 BS; Trichoderma Hypocreales, Hypocreaceae subclade with 100 BS, Pyrigemmula Sordariales, Chaetosphaeriaceae sub- clade with 91 BS, and Diaporthe Diaporthales. Diaporthaceae sub-clade with 98 BS. Three morphotypes M34, M66, and M67 representing F. incarnatum and one morphotype M16 representing F. oxysporum are within Fusarium clade. In other Fusarium clade there are 3 morphotypes of F. solani M8, M94, and M97. Within Ilyonectria clade there is one morphotype of Ilyonectria anthuriicola M64 and within Gliocladiopsis clade there is one morphotypes of G. tenuis M49. Thirteen morphotypes of Colletotrichum representing 7 species i.e. Colletotrichum sp. M1, M3 and M6 Col. acutatum M57, Col. aenigma M2, Col. arxii M53, Col. boninense M28 and M5, Col. brassiliense M76, Col. crassipes M30 and M82 and Col. gloeosporioides M4 and M47 are within Colletotrichum clade. In the Trichoderma clade, there are one morphotype each of T. atroviridae M26 and T. hamatum M98. Pyrigemulla clade is represented by one morphotype of P. aurantiaca M69. Within Diaporthe clade, there are 27 morphotypes representing Diaporthe sp. M9, M12, M13, M14, M15, M22, M23, M33, M38, M39, M41, M42, M43, M44, M48, M52, M59, M65, M69, M70,M72, M74, M79,M85, M89, M91, M96, D. beckhausii M37, M54 and M73, D. endophytica M90 and M20, D. eucalyptorum M46, M56, and M81, D. ganjae M71, D. helianthi M21, D. hongkongensis M31 and M36, D. infecunda M63 and M68, D. pseudo mangiferae M24, D. palmicola M11 and M72, D. phaseolorum M10 and M40, D. litchicola M78 and M88 and D. psoraleae-pinnatae M32, M77, M84, M92, and M94. Within Eurotiomycetes Eurotiales, Trichocomaceae clade 100 BS there are two sub-clades. Penicillium sub-clade 100 BS consisting P. citrinum M51 and Aspergillus sub-clade 57 BS consisting of Aspergillus sp. M19, As. sydowii M62, As. versicolor M27, M47 and M55. Dothideomycetes is a polyphyletic group composed of 4 sub-clades. In this group, Phoma sp. M50 Pleosporales is a sister clade of Phoma-Peyronelleae clade with 100 BS. The strain is tentatively identification Phoma since the BLAST search result showed that this is the closest genus. Peyronelleae coffeae- arabicae M58 Didymellaceae Pleosporales and Leptosphaerulina chartarum M83 and M87 Leptosphariaceae, Pleosporales form one sub-clade 81 BS with their reference strains. Neofusicoccum chordaticola M17 with their reference strains and Phyllosticta capitalensis M29, M35, and M61 with their reference strains Botryosphaeriaceae, Botryosphaeriales each forming one sub-clade with 100 BS. Two other subclades are Cladosporium sub-clades Davidiellaceae, Capnodiales and Cercospora Mycosphaerellaceae, Capnodiales subclades with 100 BS. Cladosporium oxysporum M25 and Cercospora sp. M18A B forming clades with a strong BS 100 . This study showed that molecular identification based on ITS sequences to some extend can be used to verify morphological identification of unknown endophytes. Of the 96 morphotypes, morphotypes of As. versicolor, Colletotrichum spp., Diaporthe spp., F. oxysporum, F. solani, Phy. capitalensis superfluous within species. This suggested that ITS sequences can be used to identify certain species, and others only to generic level. About 18 genera of endophytic fungi are listed in this study. They include Aspergillus, Cercospora, Cladosporium, Colletotrichum, Diaporthe, Fusarium, Gliocladiopsis, Ilyonectria, Leptosphaerulina, Neofusicoccum, Penicillium, Pestalotiopsis, Phoma, Phomopsis, Phylosticta, Pyrigemmula, Peyronellaea, and Trichoderma. All genera, except Diaporthe, Penicillium and Phomopsis are reported for the first time as endophytic fungi from C. calisaya. Totally, this study reported 42 taxa of endophytic fungi C. calisaya i.e. Aspergillus sp., As. sydowii, As. versicolor, Sordariomycetes Sordariomycetes Figure 2.1 Maximum Parsimony phylogenetic tree showing the relationship between endophytic fungi from all of plant organ C. calisaya and related fungi and plants based on the sequences of 5.8S of rDNA. Tree was rooted with Saccharomyces cerevisae KC881067 and AB018043. Bootstrap values 50 1000 replicates are shown at the branches. Dothidiomycetes Eurotiomycetes 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, Fusarium incarnatum, F. oxysporum, F. solani, Gliocladiopsis tenuis, Ilyonectria sp., Leptosphaerulina chartarum, Neofusicoccum chordaticola, Penicllium citrinum, Pestalotiopsis sp., Phoma sp., Phomopsis palmicola, Pho. tersa, Phyllosticta capitalensis, Pyrgemulla aurantiaca, Peyronellaea coffeae arabicae, Trichoderma hamatum, and T. atroviridae. Some species of endophytic fungi can be found in almost all organs of plants, and some others were only found in one organ. There are 11 species found only one organ such as Col. arxii M53 was isolated from the root, Col. acutatum M57 fruit, Col. aegnima M2 fruit, D. eucalyptorum M81 bark, Diaporthe sp. M91 bark, D. litchicola M88 fruit, Ilyonectria sp. M64 root, L. chartarum M87 leaf, P. citrinum M51 fruit, Pho. tersa M95 twig, Pyr. aurantiaca M99 root. These species are organ –specific. Phylogenetic study indicated that close related species might occur in different organs. The phylogenetic study describes distribution and adaptation of microhabitat specificity. DISCUSSION This study provides a complete representation of fungal diversity within a healthy tree of C. calisaya, since this study reports the diversity of fungal endophytes from all organs of the plant. In contrast, previous study were restricted on those from twig and bark only Simanjuntak et al. 2002, Shibuya et al. 2003, Mumpuni et al. 2004, Maehara et al. 2010. As most of the isolates are sterilia mycelia, molecular approach is chosen for identification. According to Hyde Soytong 2007 problems associated with identification of mycelia sterilia could be solved using DNA-based analysis, even though molecular analysis alone has its own limitations. In this study, ITS region is used considering this region has been decided as the marker for fungi. The use of ITS sequences also has limitations in phylogenetic analysis, as this noncoding ITS sequence is fast evolving with many variable characters. It is usually difficult to achieve a perfect sequence alignment at high taxonomic levels. Meanwhile, it has been shown that some of sequences down loaded from GenBank for comparative analysis may not be accurate in the identification Hyde Soytong 2007. It is suggested that using different gene sequences can resolve this type of difficulties in the phylogenetic analysis of the fungi. Phylogenetic analyses was done using representative strains of each morphotypes. Grouping on the bases of morphotypes usually suggested the number of species. Strains collected in this study were first grouped into 96 morphotypes, however phylogenetic study indicates that one clade may contain several morphotype. Thus indicates that morphotype does not correspond with the number of species. Guo et al. 2003 mentioned that fungi can not be identified based on traditional morphological techniques, they can only be sorted into different groups based on similar cultural characters. In contrast, Lacap et al. 2003 stated that morphotyping has been useful in estimating fungal numbers and the species is the basic unit in biodiversity. Endophytic fungi consists of Ascomycetes and Basidiomycetes. Although Ascomycota and Basidiomycota have endophytic members, most reports stated that endophytic fungi primarily belong to Ascomycetes and its anamorphs Davis et al. 2003, Arnolds 2007, Khan et al. 2010. All fungal endophytes found in this study belongs to Ascomycota and no basidiomycetous strains were found. Nevertheless, the existence of basidiomycetous endophytes from Cinchona were reported by Mumpuni et al. 2004 and Maehara et al. 2011. This study reported 18 genera of endophytic fungi from C. calisaya. of those genera found in this study, only Diaporthe, Phomopsis, and Penicillium which have been reported Maehara et al. 2010 in C. ledgeriana syn. of C. calisaya. Therefore, Aspergillus, Cercospora, Cladosporium, Colletotrichum, Fusarium, Gliocladiopsis, Ilyonectria, Leptosphaerulina, Neofusicoccum, Peyronellaea, Pestalotiopsis, Phoma, Phyllosticta, Pyrigemmula and Trichoderma were new records of endophytic fungal genera in Cinchona. In C. calisaya, genera Diaporthe is the dominant endophytes represented by 130 isolates, followed by genera Colletotrichum 117 isolates. Phomopsis anamorph of Diaporthe was reported as dominant in Cinchona ledgeriana Maehara et al. 2010. Colletotrichum and Phomopsis were frequently identified as dominant endophytes in various plants Cannon Sommons 2002, Devarajan Suryanarayanan 2006, Huang et al. 2008, Costa et al. 2012. The Coelomycetes such as Phoma, Phomopsis, and Phyllosticta are common endophytic fungi Avekamp et al. 2008. Many species of fungi were commonly described as endophytes, but others could be found occasionally colonizing the host tissue and were isolated only once or twice in several samples Siqueira et al. 2011. A single endophytic fungal species that form relationships with two related plant species or demonstrate a preference for one particular host is categorized as having host selectivity Cohen 2006. Benerjee 2011 stated that endophyte fungal diversity had been isolated from leaves, stems, petioles, barks, and roots from many Angiosperm taxa of tropical plants including from medicinal plant. Research of endophytic fungi from medicinal plants have been intensively done previously. Endophytic fungi mostly from orders of Diaporthales, Clavicipitales, and Xylariales were obtained from Annona squamosa Lin et al. 2010. From Artemisia capillaris, A. indica and A. lactiflora, some endophytic fungi such as Alternaria, Colletotrichum, Phomopsis dan Xylaria were obtanined Huang et al. 2009. Suwannarach 2012 found Col. gloeosporioides, Col. acutatum, Phomopsis spp., G. mangiferae and xylariaceous taxa from Cinnamomum bejolghota. Orlandelli 2012 isolated Bipolaris which was the most dominant in the Piper hispidum. Garcia et al. 2012 obtained Cochliobolus, Alternaria, Curvularia, Diaporthe, Phomopsis and Phoma from the medicinal plant Sapindus saponari. Guignardia and Colletotrichum were the dominant genera from Taxus media Xiong et al. 2013. Nectria, Aspergillus, Fusarium, Verticillium, Penicillium, Cladosporium were isolated from Panax ginseng Wu et al. 2013. Alternaria sp., Neurospora sp., Phomopsis sp., and Phoma sp., were the dominant endophytes in Acer ginnala Qi et al. 2012. The phylogenetic study of the endophytes of C. calisaya enriches the diversity information of endophytic fungi from tropical medicinal plants. CONCLUSION All plant organs of C. calisaya hosted endophytic fungi. In total, 687 isolates were obtained from 5 healthy plants. These isolates were divided into 96 morphotypes. Phylogenetic study showed that the morphotypes represented 18 genera and 42 species of Ascomycota. Of these genera, 15 genera are the first reported and mainly belong to Sordariomycetes. Phylogenetic taxa can inhabit different microhabitat. 3 COMMUNITY STRUCTURES OF FUNGAL ENDOPHYTES IN CINCHONA CALISAYA INTRODUCTION Endophytic fungi that widely distributes within the plant tissues are rich in species diversity Qiu et al. 2008. Endophytes are considered as important component of biodiversity and distribution of endophytic fungi in each host plant are different. Diversity of endophyte was expected to be higher in tropical plants Banerjee 2011 including those in medicinal plant. Medicinal plants are known to harbour endophytic fungi that associated with the production pharmaceutical substances Zhang et al. 2006. Therefore, it is important to explore endophytic fungi in the medicinal plant such as Quina tree Cinchona spp in order to enable pharmaceutical substance production. While C. calisaya syn. C. calisaya var. legderiana is one of the best clone of cultivated Quina tree for quinine production in Indonesia. Recently, the plantation area has been reduced drastically Susilo, 2011. Therefore, alternative agent for quinine and other active metabolites production should be investigated. Hence, the study of fungal endophyte community from all organs of C. calisaya is initiated. Prior to this study, some fungal endophytes of Cinchona from Indonesia have been reported. However, most researches on endophytic fungi of Cinchona spp. focused on isolation from certain part of the plant and screening their potential for secondary metabolites production. Eventhough some of these endophytic fungi from bark has been studied for their metabolite products Simanjuntak et al. 2002, Winarno 2006, Maehara et al. 2010, no comprehensive study covering the fungal endophytes community structures in the whole part of C. calisaya has been done. Information on fungal endophytes community in Cinchona is important as bases for understanding the endophyte roles in relation to secondary metabolic production of their host. Despite its limitation, culture based method was selected for studying the community structure of endophytic fungi as this method will also provide genetic resource material for secondary metabolites biotechnology. Therefore, this study was aimed to analyse the community structure of endophytic fungi in bark and other plants organs leaf, petiole, twig, root, flower, and fruit of C. calisaya to elucidate the diversity, species dominance and distribution endophytic fungi in different organs. MATERIALS AND METHODS Isolation and identification of the fungi Specimen collection, fungal endophyte isolation and identification were done following protocol that was described in page 6. Data analysis Community structures were expressed as diversity, colonization rate and frequency of occurrence of the species in each plant organs. One colony is considered as one individual fungus. Shannon-Wiener diversity index H’ was employed to evaluate and compare the diversity of fungal communities between different organ of C. calisaya plant. H’ was calculated according to the following formula: k H’ = - ∑pi × lnpi i=1 where k is the total of fungal species, and pi is the proportion of individuals that species i contributes to the total Tao et al. 2012. Colonization rate was calculated as a percentage of the total number of segments colonized by the fungi divided by total number of segments observed. While frequency of occurence of an endophytic species was calculated to estimate the species dominance and distribution of the fungi. The fungal occurrence in different organ was calculated using the following formula: Number of strains in species-i Frequency of occurrence of species-i FO = ---------------------------------- × 100 Total number of strains found Unweighted pair group method with arithmetic mean UPGMA cluster analysis was performed using Jaccard’s coefficient using Multi-Variate Statictical Package software MVSP version 3.13r. on the bases of diversity index, the presence of the fungi, and frequency of its occurence. Dendrogram of community relatedness was reconstructed on the bases of similarity distance of Jac card’s coefficient. RESULTS Phylogenetic analyses using ITS4 –5.8S–ITS5 region is able to identify 96 representative isolates of the morphological groups from the 687 fungal isolates. The community assemblages of the endophytic fungi belongs to Ascomycota Table 3.1, in which mostly are members of Sordariomycetes. Table 3.1 Endophytic Ascomycota isolated from C. calisaya Class Species name Eurotiomycetes Aspergillus sp., As. sydowii, As. versicolor, P. citrinum Dothidiomycetes Cercospora sp, Cladosporium oxysporum, Phyllosticta capitalensis, Leptosphaerulina chartarum, Neofusicoccum chordaticola, Phoma sp., Peyronellaea coffeae arabicae Sodariomycetes Colletotrichum spp., Col. acutatum, Col. aenigma, Col. arxii, Col. boninense, Col. brasiliense, Col. crassipes, Col. gloeosporioides, Diaporthe spp., D. beckhausii, D. eucalyptorum, D. endophytica, D. infecunda, D. ganjae, D. hongkongensis, D. helianthi, D. litchicola, D. phaseolorum, D. pseudomangiferae, D. psoraleae-pinnatae, Fusarium incarnatum, F. oxysporum, F. solani, Gliocladiospsis tenuis, Ilyonectria sp., Pestalotiopsis sp., Phomopsis palmicola, Pho. tersa, Pyr. aurantiaca, Trichoderma hamatum, T. artroviride Diaporthe were the most frequently isolated species 55.3 , followed by Colletotrichum 15.4 and Fusarium 9.3 . The remaining species contributes to 20 of the total species. Only about 49.3 of the total segments studied occupied by the endophytic fungi and the remaining about 50.7 were free from fungal endophytes Fig 3.1. Of those occupied by the fungi, the segments hosted either one fungus or more. This means that the fungus may not be grown continuously within the organ tissue. Of the organs studied, twig was the most preferred organ being colonized 12.4 , followed by fruit 8.9 , leaf 8.8 and root 7.6 . The other organs were colonized less frequent between 1.4 –5.7 . Although endophytic fungi occurred in all plant organs,their diversity in each plant organ varied. Leaf and fruit H’=3.0 bore the most diverse endophytic fungi, followed by bark H’=2.9, twig and petiole H’=2.8. While flower H’=1.7 and root H’=1.6 contained less diverse endophytic fungi Fig. 3.2. The number of taxa within organs leaf, fruit, bark, twig and petiole with high diversity index was also different from the ones flower and root with low diversity index. Within high diversity index organs, about 25 –36 taxa were found, while only 7–15 taxa occurred in low diversity index organs. Figure 3.1 Colonization rate of endophytic fungi in various organs of C. calisaya The frequency of occurrence FO of the endophytic fungi within all organs shows positive correlation with their colonization rate in that respective organ. For example twig harboured the highest fungal population 34.8 and showed the highest colonization rate. Fruits host less fungal endophyte population 19.8 , and followed by leaf 17.9 , petiole 9.8 , root 8.2 , bark 7.5 , and flower 2.5 Fig. 3.3, and their colonization rate in those organ were 8.9 , 8.8 , 7.6 , 5.7 , 4.5 and 1.4 , respectively. In the twig, 239 isolates belonging to 11 genera of 24 species were obtained, followed by fruit 136 isolates, 13 genera, 26 species; leaf 123 isolates, 12 genera, 27 species; petiole 67 isolates, 11 genera, 19 species; root 56 isolates, 10 genera, 13 species; bark 49 isolates, 10 genera, 22 species and flower 17 isolates, 5 genera, 7 species. Figure 3.2 The Shannon-Wiener diversity index H’ of endophytic fungi in each plant organ Distribution of fungal endophytes are different among organs Figure 3.3. There are 11 species of Diaporthe and an unidentified group of Diaporthe called Diaporthe spp. Among the species found, Diaporthe spp. exhibits the widest distribution in the plant organs, and it was dominantly found in twig 16.2 . In the other organs fruit, leaf, petiole, bark, root and flower the frequency of Diaporthe spp. occurrence were 5.2 , 4.8 , 2.9 , 1.9 , 0.4 , 0.1 , respectively. The plant organs harbored 1 –9 species of Diaporthe, of which twig and fruit contain the most diverse species of Diaporthe spp. Other than Diaporthe spp., the most common species within some organs are unique. For instance, Neofussicoccum chordaticola was common fungi on the twig with FO about 4.1 . In the leaf, Diaporthe spp. 4.8 and Colletotrichum spp. 2.3 were the most common taxa. In the flower, Col. brasiliense was the most common with 0.9 FO. Fusarium oxysporum 5.1 was the most common fungal endophytes in the root. However, the most common fungal endophyte species in the petiole, fruit and bark was not unique in which Diaporthe spp. were the most common fungi with frequency of occurrence 2.9 , 5.2 and 1.9 , respectively. As the diversity, and the distribution of the fungal species is varies among organs, their assemblage forming different community structure in each plant organ. Based on UPGMA analyses, with similarity index 0.5 as the cutting score, endophytic fungal communities can be divided into four clusters Figure 3.4. The fungal community in twig was close to those in leaf and petiole with similarity index 0,52. The fungal communities in bark and fruit were also closely similar. While those in root and flower from distinct communities. The close similarity among fungal endophytes community from leaf, petiole, and twig were possibly due to the presence of two predominant species, i.e. Colletotrichum spp. and Diaporthe spp. The flower and root were apparently separate due to differences in fungal endophytes composition, there were exclusively species i.e. only Col. gloeosporioides in flower and T. atroviride. Ilyonectria sp., G. tenuis in the root. Figure 3.3 Frequency of occurrence of endophytic fungal species in each plant organs Figure 3.4 Cluster fungal endophyte community in C. calisaya 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 1.3 4.1 1.5 5.1 1.2 0.6 2.2 0,4 0,4 2 1.3 1.0 2.8 1.3 4.8 2.9 16.2 1.9 0,9 5.2 1.5 1.5 0.9 2.3 1.3 0.7 2.2 0.7 1.3 Fre quenc y of occ ur enc e Plant organs Aspergillus sp. As. sydowii As. versicolor Cercospora sp. Cladosporium oxysporum Colletotrichum spp. Col. arxii Col. acutatum Col. aenigma Col. boninense Col. brasilliense Col. crassipes Col. gloeosporioides Diaporthe spp. D. beckhausii D. endophytica D. eucalyptorum D. infecunda D. hongkongensis D. helianthi D. ganjae D. litcthicola D. phaseolorum D. pseudomangiferae D. psoraleae-pinnatea Fusarium incarnatum F. oxysporum F. solani Gliocladiopsis tenuis Phylosticta capitalensis Ilyonectria sp. Leptosphaerulina chartarum Neofusicoccum chordaticola Penicillium citrinum Phomopsis tersa P. palmicola Pestalotiopsis sp. Phoma sp. Peyronellaea coffeae arabicae Pyrigemmula aurantiaca Trichoderma hamatum T. atroviride DISCUSSION Endophytic fungi and higher plants have evolved together as distinct systems in which each organism benefits the other. In C. calisaya, diverse fungal endophytes were detected occupying its organs, which means that many endophytic fungi evolve with the host. The host organs provides microhabitat suitable for the fungi. When the colonization of endophytic fungi was significantly different in each organ, indicates that each organ has different structure and nutrient. The colonization was higher in twig 12.4 than in leaf, petiole, bark, root, flower, and fruit. On the contrary, the colonization in leaves of Piper hispidum was dominat over twig as leaves exposed to a high abundance of inoculum in the air Orlandelli et al. 2011; Garcia et al. 2012. The colonisation of twig was appoximately one fourth of the total organs occupied by the fungi. The colonization of endophytic fungi in the flower was the lowest 1.4 . The type of tissue in twig and flower are presumably different and therefore, determined the different endophytic community as suggested by Collado et al. 2000, Wang Guo 2007, Guo et al. 2008, Qiu et al. 2008, Khan et al. 2010, and Sun et al. 2011. This is strongly supported by the UPGMA analyses. Endophytic assemblages of twig is placed in different cluster from those from flower. Besides the characteristic of the host tissue, the colonization of endophytes may be influenced by other factors, such as plant age and the climate condition Arnold Herre 2003. In this study, the tree observed 30 –40 years old. Although, this study was not concerned with the community structure of fungal endophytes in tree and leaf of different age, but the study showed that the fungal endophytes existed within very old trees. Hilarino et al. 2011, Suryanarayanan Thenarasan 2004 found that the old leaves support more endophyte than relatively younger leaves. The Gambung area is located in the highland in the tropic. The climate condition during sampling such as temperature 15 –25 o C, relative humidity 80 −92 , rainfall 3743 – 4043 mm per year and soil pH between 44 –5.5 supported the endophytic life. Arnold et al. 2001 said that the most endophyte species are rich in tropical trees and Arnold Lutzoni 2007 stated that the percentage of colonized tissue decreased with increased latitude. The Shannon –Wiener diversity index H has been applied as the measurement of fungal diversity Kumaresan Suryanarayanan et al. 2001, Kumar Hyde 2004, Tao et al. 2012, Wearn et al. 2012. Fungal endophyte diversity in the leaf, fruit, twig, petiole, and bark were nearly twice that of the root and flower. Leaf, fruit, twig, petiole and bark hosted more diverse endophytes than in root and flower because they contain photosynthate which is readily available for the fungi. The organ with thicker tissue such as bark will provide a suitable micro- environment for fungal development. Pinnoi et al. 2006 suspected that the cell wall, that was thicker on the wood, produced higher nutrition particularly cellulose that would support the growth of fungi. The Shannon –Wiener index takes into account the number and the evenness of the species, therefore the index increased either by having additional unique species, or by having higher species evenness Banerjee et al. 2006. This study found that leaf, fruit and twig had the highest index, and hosted L. chartarum, P. citrinum, D. litchicola, and Pho. tersa as unique fungal inhabitat. Flower had the smallest index and lacks of unique fungus. Surprisingly, root has relatively low diversity index, but hosted Pyr. aurantiaca and Ilyonectria sp. as unique endophytic species. Similar situation is reported by Tao et al. 2008 whom stated that diversity of endophytic fungi within leaves of Bletilla ochracea was higher than those within root and the fungal communities within leaf was significantly different from those in root. Qiu et al. 2008 studied that the fungal endophyte diversity in twigs of 6 medicinal plants was much higher than in leaves. The endophyte species diversity increased when the dominant species was excluded from the analysis Suryanarayanan et al. 2003. Distribution of endophytic fungi in all parts of plant organs is significantly different. Twig haboured more endophytic fungi than the other organs. In leaves of Quercus ilex, Collado et al. 2000 reported that there was a higher number of endophytic fungi in twigs than in leaves while the diversity indices of endophytic fungi were higher in leaves than in twigs. This is in contrast to the finding of Wearn et al. 2012 in which the fungal endophyte community in grass root was greater than that in leaf. The ability of endophytes to penetrate from one part to another part of the organ tissues and the ability of endophytic fungi to adapt to its microhabitat may determine the distribution of the fungus in the tree. The adaptive process may favour the coexistence of endophyte species in the same organ. Host organ can affect the diversity of its endophyte as i individual taxa might have special capacity for utilizing or surviving within a specific substrate; ii fungal communities iii hosts or ecological sites, for example, below-or above-ground, have different limited factors such as humidity, chemistry structure, temperature that affect the fungal communities and diversity Wang Guo 2007. The species composition and frequency of endophytes were found to variety with different organs. The majority of endophytic fungi obtained at C. calisaya were obtained distributed in all of the plant organs uniformly. Diaporthe spp. were predominant in C. calisaya, and the second predominant endophytic fungus was Colletotrichum sp., which colonize higher in the twig than in other parts. Both of those species were isolated from almost every part of the plant organs. This result conformed to Maehara et al. 2010 whom firstly reported that Phomopsis spp., and Diaporthe spp. were predominant in Cinchona ledgeriana, while Gomes et al. 2013 considered Diaporthe as a common endophyte in almost all plant. The similar result was reported by Kumaresan Suryanarayanan 2002 whom found that Phomopsis and Colletotrichum were predominant in distantly related host plant. Dongsheng et al. 2011 also found that Colletotrichum spp., and Phomopsis spp., were predominant in Dendranthema indicum. Besides Nodulisporium, and Pestalotiopsis, Colletotrichum, and Phomopsis were predominant in 12 trees in Iwokrama Forest Reserve, Guyana Cannon Simmons 2002. Some endophyte genera such as Phyllosticta, Xylaria Okane et al. 2003; 2008, Phomopsis Gomes et al. 2013 and Colletotrichum occur in a wide variety of distantly-related host plant Kumaresan Suryanarayanan 2002. Environmental and geographic factors, such as temperature, moisture, altitude, host species, and plant tissue could influence the species composition and the distribution of the endophytic fungi Wang Guo 2007. In this study, one species of endophytic fungi could be haboured more than one organ. Wearn et al. 2012 also stated that fungal endophytes exhibit plant and tissue specificity. Difference in endophytic assemblages in different types of plant tissue might be a reflection of individual tissue preferences in the dominant taxa and might indicate their capacity for utilizing or surviving on a specific substrate Rodrigues 1994. Further, these factors may be important such as weathering of the leaf cuticle, tissue texture, and changes in the tissue physiology and chemistry Petrini Carroll 1981. Change in leaf biochemistry influence endophytic of composition in the organ with consequences for endophyte distribution Fernandes et al. 2011. Endophytic fungi have been shown to be tissue-recurrent in several studies Cannon Simmons 2002. Despite, our results indicated that different fungal communities occur within different organs, more samples repeated sampling including more number of individual plants as replication should be considered to minimize the bias of available data. CONCLUSION The present study provides firsth and information on the community structure of fungal endophytes in C. calisaya describing the diversity, dominance, colonization rate and the distribution of the fungal species within the host organs. Based on Shannon-Wiener diversity index, leaf and fruit hosted the most diverse endophytic fungi followed by twig, petiole and bark. Flower and root contained the least diverse endophytes. Twig was the most preferred organ being colonized by fungal endophytes. Diaporthe spp. were the predominant fungal endophyte, that widely distributed in all organs. The second predominant fungi were Colletotrichum spp and not organ-specific. Some other fungi are organ-spesific. Neofussicoccum sp. was only found in twig. Col. gloeosporioides and Phy. capitalensis were unique to leaf, while Col. brasiliense was unique to flower. Fusarium oxysporum was specific to the root. Based on Jaccard’s coefficient similarity with 50 similarity as a cut off criterion, the fungal endophyte community was clustered into 4 groups. The first group contained fungal endophyte community from leaf, twig, and petiole. The second group composed of those in fruit and bark, while root and flower forming two distinct groups. 4 ALKALOID PROFILE OF ENDOPHYTIC FUNGI FROM CINCHONA CALISAYA INTRODUCTION Bark of Quina tree Cinchona spp. is known to contain alkaloids Taylor 1975. This bark is traditionally used by the local people in Andes for fever and malarial treatment. When extract alkaloids is available, the extracted chinchona alkaloid has replaced the used of the bark for malarial treatment. Four cinchona alkaloids i.e quinine 1, quinidine 2, cinchonine 3 and cinchonidine 4 are known to be effective against malaria Achan 2011. However, the problem of the supply and the finding of the synthetic antimalarial drug chloroquin has replaced the used of quinine as the main drug for malaria. The emergence of chloroquin resistance caused quinine regain the promotion for main drug for malaria. The side effect quinine cinchonism has replaced the use of it by artemisin. Nowadays, artemisin resistance occurs and in the future quinine is recommended to be used treatment of severe Plasmodium falciparum infection Achan, 2011. Besides malarial treatment, quinine can be used as raw material in pharmacy, food colorant, and beverage flavor Santoso et al. 2004. Therefore, quinine needed in the treatment of infection of P. palciparum. Quinine industry in Indonesia that has been established before Second World War relied on the bark supply. The bark supplied by the Quina plantation in Indonesia only 30 –50 demand of the industry Susilo 2011. The remaining is fulfilled by imported bark flakes. Although Quina plantation has a replanting program to increase bark production, alternative agents for quinine production have to be investigated. Endophytic fungi have been recognized to produce bioactive compounds originally from their host Strobel et al. 1993, Zhao et al. 2010. For example, taxol is produced by Taxus brevifolia but also be produced by various fungal endophytes from different medicinal plants Gangadevi et al. 2008a, 2008b, Visalakchi Muthumary 2010. Artemisin is produced by A. annua and endophytes may produce artemisin as well. Endophytic fungi of Cinchona spp. have been reported by several researchers Simanjuntak et al. 2002, Mumpuni et al. 2004, Winarno, 2006, Maehara et al. 2010, a single strain of a closely related Diaporthe phaseolorum, an endophytic fungi from C. ledgeriana syn. of C. calisaya, was reported to produce quinine and other cinchona alkaloids Maehara et al. 2012. The inventory of endophytic fungi from all part of the Quina tree C. calisaya has been done. The isolates has been preserved for long term and kept in culture collection and yet whether these strains are capable to produce cinchona alkaloids are not known. It is, therefore, this study aims at 1 analyzing the capability of the fungal endophytes strain to produce alkaloids, and 2 examining certain prospective strains for cinchona alkaloids production. Figure 4.1 Chemical structures of cinchona alkaloid MATERIALS AND METHODS Fungal strains collection The fungal strains used in this study Table 2.1 covering 96 taxa and 6 other taxa of Diaporthe obtained from different sampling time from similar location. The working cultures were subcultures in PDA and incubated for a week at room temperature 27 o C. Cultures of 7 days old are used for further investigation. Alkaloid production and extraction Three pieces of mycelial plug 0.5 × 0.5 cm 2 were inoculated into flask 500 mL containing 200 mL PDB with an initial pH of 6.0. The culture were incubated with in static condition for 21-d at room temperature 27 o C. The mycelial mass was separated from the filtrate using Whatman paper no. 1. The secondary metabolites were extracted separately both from biomass and filtrate by adding 100 mL vv chloroform CHCl 3 Simanjuntak et al. 2002; Winarno 2006 and 0.1 N NaOH vv then homogenized using a separating funnel and allowed to stand a few seconds to form two layers. The upper layer was taken out and the same volume of new chloroform was given to extract the remaining metabolites. Each fraction was then collected and concentrated with a rotary evaporator Buchi, Switzerland at a temperature of 45 °C and 60 rpm rotation, up to concentrated extracts obtained. The extract stored at 4 °C as stock solution for alkaloid analysis. Analysis of alkaloids compounds by high performance liquid chromatography HPLC HPLC analyses were done in two steps. In the first step, the analyses were done for the 96 taxa. The concentration of the quinine, quinidine, cinchonine and cinchonidine standard is 100 mgL -1 . The fractions were analyzed quantitatively by PerkinElmer HPLC with 200 UV-Vis detector, using Ascentis C18 column, with KH 2 PO 4 20 mM pH 2.5: CH 3 CN = 75 : 25 as eluent, at flow rate of 1.20 mL min -1 and detected in 234 nm wave length. It was assumed that all peaks emerge during this analyses are alkaloids that are soluble in the chloroform. The concentration of quinine was calculated as follows: Area of samples Concentration of quinine mgL -1 = ------------------------- × [standard] Area of standar The other cinchona alkaloids were determined qualitatively. In the second step, the analyses were done for the prospective genera. Concentration of the quinine, quinidine, cinchonine and cinchonidine standards were 1 mgL -1 . HPLC analyses used Shimadzu HPLC machine with cosmosil 5C18- MS-II 4.6 × 150 mm column mobile phase, methanol : acetonitril = 80 : 20 as eluent with flow rate 1 mLmin -1 , at 40 o C, detection wavelength 210 nm. The concentration of cinchona alkaloids was calculated based on the above formula. RESULT Alkaloid Profile of Endophytic Fungi Analyses of the alkaloid profile were done only on the representative strains of each morphotype 96 taxa. Of the 96 taxa, all taxa produced chloroform soluble alkaloids Appendix 4.1. Base on their unique retention time Rt, about 80 different alkaloids were detected from all taxa. One strain might produce 2 –38 alkaloids which meant that these akaloid did not exist as a single component in the crude extract of the metabolites. The alkaloids mostly detected between Rt 1.4 –3.0 and the number of alkaloids detected between those Rt are about 2 –10 compound in each strain. In the first step analyses, the peak of the quinine standard appeared at Rt 1.9, while the other standard such as quinidine was detected at Rt 1.5, cinchonine at Rt 0.8, and cinchonidine Rt 0.9. Quinine is detected in about 44 taxa from 96 taxa Fig. 4.1 and their concentration is between 0.6 –186.6 mgL -1 . Quinine is produced in endophytic Aspergillus spp., Cercospora sp. Colletotrichum spp., Diaporthe spp., Fusarium spp., N. chordaticola, L. chartarum, P. citrinum, Pestalotiopsis sp., Pho. tersa, Phy. capitalensis and T. artroviridae Fig 4.1. Other strains examined that belongs to Cladosporium, Gliocladiopsis, Ilyonectria, Peyronella, Pyrigemulla and Phoma did not produce qunine and other cinchona alkaloids. Of those quinine producing strains, Diaporthe contains the highest number strains that produced qunine 24 strains, followed by Fusarium 5 strains and Colletotrichum 3 strains. All members of some genera, for example Cercospora, Leptosphaerulina, Neofusicoccum and Penicillium produced quinine. However, the number of strains found within these genera is small, consisiting only 1 –2 strains. When the genera consisting larger member, not all strains produced quinine. About 5.2 Fusarium 5 strains collections produced quinine. While Diaporthe is the dominant taxa 25 strains, about 25 strains produced quinine. Other cinchona alkaloids were detected in less number of strains Appendix 4.2. Quinidine was detected in extract of 37 strains covering Aspergillus spp., Cercospora sp. Colletotrichum spp., Diaporthe spp., Fusarium spp. N. chordaticola, L. chartarum spp., Gli. tenuis, P. citrinum, Phoma sp., Pho. tersa, and Phy. capitalensis. Cinchonine was produced by D. phaseolorum M10 and D. pseudomangiferae M88, whereas cinchonidine was produced by Col. boninense M28, Diaporthe sp. M12, and D. pseudomangiferae M78. Cinchona Alkaloid Profile of Novel Genera Of the strains producing cinchona alkaloids, only Cercospora, Diaporthe and Fusarium were selected for other cinchona alkaloid analyses for various reason. Cercospora is interested since this genus has ever been reported to cause leaf spot in Cinchona and an endophytic strain may have an antiplasmodial property. Diaporthe is the dominant genus and has ever reported to produce quinine. Fusarium spp. is of interest since Fusarium sp., F. incarnatum and F. oxysporum were found to live as endophytes in mangrove that had antimalaria properties. Cinchona Alkaloids of Cercospora Cercospora sp. M18A and M18B that was isolated from petiole of C. calisaya produced quinine only, while Cercospora sp. M18B that was also isolated from petiole produced both quinine and cinchonidine Table 4.1, Appendix 4.4. The production of quinine is relatively comparable to the amount of the standard used Appendix 4.3. In contrast, cinchonidine production was very low. This indicates that different strain of Cercospora sp. M18A and B produce different alkaloids at the same condition of production process. This is also the first report of endophytic Cercospora sp. producing quinine and cinchonidine from C. calisaya. Table 4.1 Cinchona alkaloids production of endophytic Cercospora sp. Cinchona alkaloid of Diaporthe Most of endophytic Diaporthe species from C. calisaya produced quinine, upon cultivation in a PDB medium. Quinine is present in 25 strains out of 49 strains of endophytic Diaporthe spp. studied. The concentration is between 0.6 mgL -1 and 186.8 mgL -1 Fig 4.2. Quinine is the major cinchona alkaloids in 4 strains tested, i.e. Diaporthe sp. M13, M21, M63 and M70. Of those quinine-producing strains, three strains i.e. M13, M70, and M21 produced relatively high concentration of quinine, i.e. 186.8 mgL -1 , 155.2 mgL -1 , 134.2 mgL -1 respectively. The number of Diaporthe strains produced cinchona alkaloids is less than those produced quinine. Sixteen strains of Diaporthe produced quinidine, 2 strains produced cinchonine and 2 strains produced cinchonidine. Similar pattern of alkaloids production were detected during cinchona alkaloids production by Diaporthe sp., D. cinchonae, and D. endophytica originated from C. calisaya. All of these strains were able to produce quinine Table 4.2; Apendix 4.5, but only a certain strains produced other cinchona alkaloids. Diaporthe sp. InaCC-F235 and D. cinchonae InaCC-F2310 were able to produce cinchonidine, while other strain of D. cinchonae InaCC-F239 and InaCC-F236 produced cinchonine in low consentration. Alkaloids Rt Cercospora sp. M18A M18B Area Concentration mgL -1 Area Concentration mgL -1 Quinine 2.2 5771 1.2 3590 0.7 Quinidine 1.0 - - - - Cinchonine 1.7 - - - - Cinchonidine 1.9 - - 1007 0.002 Figure 4.2 Quinine production of endophytic fungi in C. calisaya 32 Cinchona alkaloids of Fusarium The pattern of cinchona alkaloid production in Fusarium spp. is similar to the other 2 genera. Quinine is produced in higher concentration than the cinchona alkaloids. In this study, 5 Fusarium species were capable of producing quinine. All of the strains did not produce quinidine and cinchonine, but all except F. solani M93 produced cinchonine Table 4.3; Apendix 4.6. Based on the cinchona alkaloid detected, quinine production of Fusarium spp. are not similar to those of Diaporthe spp. Fusarium spp. could produce quinine and cinchonine, but Diaporthe spp. produced quinine, cinchonine, and cinchonidine. DISCUSSION Some of the 96 taxa of fungal endophyte examined produced cinchona alkaloids, particularly quinine. Other cinchona alkaloids were produced by fewer taxa, because they are endophyte from C. calisaya, which produce cinchona alkaloids. The product of bioactive substance by endophytes are related to the independent evolution of the endophytic fungi, which may have incorporated genetic information from plants Pimentel et al. 2011. In their transitory association, the fungal endophyte give benefit to the host plant by adapting to its habitat, promoting plant gowth and protecting the plants from biotic and abiotic stress Schulz Boyle 2005; Rodriguez et al. 2008, besides producing bioactive secondary metabolites Lu et al. 2000; Guo et al. 2006; Barik et al. 2010; Maehara et al. 2012 with unique structure including alkaloid, flavonoids, phenolic acids, quinones, steroids, terpenoids, xanthones Tan Zou 2001. It was predicted that about 300.000 species of plants in terrestrial ecosystem is likely to associate with more than one species of bacterial and fungal endophytes Strobel Daisy 2003. With their capability in producing various bioactive compounds, fungal endophytes has gained more attention in discovery of new secondary metabolites, or as an alternative source to replace plants as bioactive plant secondary metabolites producer due to their ability to synthesize the same natural products produced by the plant Kusari Spiteller 2011. One of the earlier report indicating cinchona alkaloids production by fungal endophytes was published by Simanjuntak et al. 2002. In further report, common endophyte fungus belonging to the genus Diaporthe anamorph: Phomopsis was reported as fungal endophyte capable of producing quinine Maehara et al. 2012. Different genera of endophytic fungi such as Arthrinium, Fomitopsis, Penicillium, Schizophyllym, and Xylaria were also reported as potential quinine producer Shibuya et al. 2003, Agusta et al. 2005, Maehara et al. 2010. Table 4.2 Cinchona alkaloid production of endophytic Diaporthe spp. Table 4.3 Cinchona alkaloid production of endophytic Fusarium spp. Alkaloids Quinine Quinidine Cinchonine Cinchonidine Rt 2.2 2.6 1.9 1.7 Strain Area Concentration mgL -1 Area Concentration mgL -1 Area Concentration mgL -1 Area Concentration mgL -1 Diaporthe sp. InaCC-F235 5162 1.0 - - - - 1003 2.1 x 10 -2 D. cinchonae InaCC-F236 4264

0.8 -

- - - - - InaCC-F238 2772 0.5 - - - - - - InaCC-F239 5431 1.0 - - 5217 1.34x10 -4 - - InaCC-F2310 4666

0.9 -

- - - 1154 2.4x10 -4 D. endophytica InaCC-F237 3243 0.6 - - - - - - Alkaloids Quinine Quinidine Cinchonine Cinchonidine Rt 2.2 2.6 1.9 1.7 Strain Area Concentration mgL -1 Area Concentration mgL -1 Area Concentration mgL -1 Area Concentration mgL -1 F. incarnatum M34 4754

0.9 -

- 5125 1x10 -4 - - M66 4139

0.8 -

- 1701 3.6x10 -5 - - M67 4159

0.8 -

- - - - - F. oxysporum M16 4639 0.9 - - 1172 2.4x10 -4 - - F. solani M93 4549

0.9 -

- - - - - M97 3419 0.7 - - 1181 2.5x10 -5 - - 34 Cinchona Alkaloids of Fungal Endophyte Cinchona alkaloids of Cercospora Cercospora are commonly considered as plant pathogens. C. kikuchii is the only species known both as plant pathogen and endophyte Tales 2011. The information of endophytic Cercospora has not been widely reported. Cer. kikuchii was reported to be an endophyte of medicinal plant Falopia japonica. This species produced cercosporenes and guanacastane diterpenes Feng et al. 2014, which have antifungal activity. Moreno et al. 2011 found endophytic Mycosphaerella teleomorph of Cercospora in Psychotria horizontalis from Panama that produced cercosporin and its acetylated derivate. These compounds have antiplasmodial against Plasmodium falciparum IC50 1.03 and 2.99 μM. During study of endophytic fungal diversity associated with quinine plant the most important medicinal plant e.g. antimalaria, antibacterial, etc Maehara et al. 2010, Skogman et al. 2012, Wolf et al. 2002, two isolates of Cercospora-like were found. The existence of some endophytic fungi in the stem and bark of quinine plant have been reported by several researchers in Indonesia, however, none of those researchers reported the endophytic Cercospora. Therefore, the findings of endophytic Cercospora from C. calisaya was interesting and to analyze its potential for alkaloid production, particularly quinine, quinidine, cinchonine, and cinchonidine. Two strains of Cercospora sp. was isolated from the petiole and these strain were capable to produce quinine and one strain produced cinchonidine. Cinchona alkaloids of Diaporthe The quinine-producing Diaporthe found in this study were isolated either from leaf, twig, root, flower and bark of C. calisaya, with twig as the most common habitat. This finding indicates that Diaporthe from organs other than young stem Maehara et al. 2011 are also able to produce quinine. Diaporthe sp. M13 from the twig showed the highest quinine production. This indicates that quinine can be produced by non-bark fungus. The highest quinine contents reside in the Cinchona bark Song et al. 2009, it is unexpected that highest quinine –producing fungal endophyte was isolated from non –bark of the Cinchona plant tissue. According to Zhang et al. 2012 various quinoline and isoquinoline alkaloids were produced by endophytic fungi. However, alkaloid-producing Diaporthe is rarely reported. A few examples have been identified, such as D. phaseolorum, an endophytic fungi from C. ledgeriana that excreted quinine Maehara et al. 2012; Diaporthe sp. from Rhizophora stylosa produced isochromophilones Zang et al. 2012; Phomopsis sp. from Allamanda cathartica had lactone alkaloid Nithya et al. 2011; Phomopsis sp. an endophyte fungus from Senna spectabilis produced potential anti-inflammatory, antifungal substances and acetylcholinesterase Chapla et al. 2014. The current study revealed that fungal endophytes isolated from non-bark Cinchona plant tissues were capable in producing quinine in synthetic liquid medium. Based on the HPLC analysis, all isolated from Cinchona fruit produced quinine about 1.0 mgL -1 . This amount is higher than quinine produced by Diaporthe sp. CLF-J AB505415, Diaporthe sp. CLF-M AB505418 and Arthrinium sp. AB505426 Maehara et al. 2012, 2013. According to Maehara et al. 2013, endophytic fungi from Cinchona were reported capable of producing principal cinchona alkaloids such as quinine, quinidine, and cinchonine range from50 µgmL -1 . This study showed that quinine production of Diaporthe spp. range 0.6 – 186.8 mgL -1 , comparing to those reported by Maehara et al. 2012, i.e. 60 –100 µgL -1 . In addition to quinine, other alkaloids such as quinidine, cinchonine and cinchonidine are expected to present in the metabolite extract. When these are present, the concentration are lower than quinine. Winarno 2006 found that endophytic fungi from C. ledgeriana namely LMC-19 produced quinine 0.117 mgL -1 in the media B for 6 days and LMC-29 0.610 mgL -1 in the PDB for 7 days. Antimalarial activity may be exhibited by other chemicals. Isaka et al. 2007, 2010 reported that pullularin A, B and C from culture of endophytic Pullularia sp. and sesquiterpenoids from Xylaria sp. exhibited antimalarial activities. They inhibited Plasmodium falciparum K1 with IC 50 3.6 μgmL -1 . Furthermore, Romero et al. 2008 isolated Butyrolactone V compound from endophytic Xylaria sp., with potential activity against P. falciparum. Haritakun et al. 2010 also isolated endophytic Aspergillus terreus that showed anti-malarial activity with IC 50 7.9 μgmL -1 . Besides quinine, other alkaloid such as quinidine, cinchonine, and cinchonidine are produced by certain strain of Diaporthe upon cultivation in a PDB medium. The same result was reported by Maehara et al. 2012 stated that an endophytic filamentous fungus species of the genus Diaporthe isolated from C. ledgeriana Rubiaceae produced quinine 60 –110 μgL -1 , quinidine 3 –5 μgL -1 , cinchonidine 10 –15 μgL -1 , and cinchonine 15 –20 μgL -1 in a synthetic liquid medium . The concentration of these cinchona alkaloids was close to the concentration of those from endophytic Diaporthe being studied. This suggests that in the same host and fungal species have the ability to produce the same metabolite. Cinchona alkaloid of Fusarium In this study, three Fusarium species are reported as the new fungal endophytes capable of producing quinine, namely, F. incarnatum strain M66 and strain M34, F. oxysporum strain M16, and F. solani strain M97. These Fusarium species were isolated from different type of plant tissue. Fusarium incarnatum strain M66 was originally isolated from fruit, while F. incarnatum strain M34 was isolated from petiole, F. oxysporum strain M16 from bark, and F. solani strain M97 from twig. Although member of Fusarium is more recognized as fungal pathogen on many economically important plants and on human Guarro Gene 1995, O’Donnell et al. 2009, they are also frequently isolated as endophytes from various plants and capable in producing secondary metabolites with medicinal and antimicrobial activities Kour et al. 2008, Li et al. 2008, Deng et al. 2009, Tayung et al. 2011 as showed in this study. CONCLUSION Cinchona alkaloids such as quinine, quinidine, cinchonine and cinchonidine can be produced by many endophytic fungus originated from C. calisaya. About 40 taxa from 96 taxa are capable to produce quinine, 37 taxa are capable to produce quinidine, 2 taxa cinchonine and 3 taxa cinchonidine. Aspergillus spp., Cercospora sp. Colletotrichum spp., Fusarium spp., N. chordaticola, L. chartarum, P. citrinum, Pestalotiopsis sp. Pho. tersa, Phy. capitalensis and T. artroviridae are the first reported as endophyte producing cinchona alkaloids. Cinchona alkaloid were also produced by 21 strains out of 49 Diaporthe spp. Cinchona alkloid of novel genera of Cercospora sp. Diaporthe sp., D. cinchonae, D. endophytica, F. incarnatum, F. oxysporum, and F. solani are capable of producing quinine. The type and concentration of the cinchona alkaloids is strain dependent. 5 DETERMINATION OF NOVEL SPECIES CANDIDATES USING MULTIGENE-APPROACH INTRODUCTION Many research of endophyte have been done recently to evaluate and elucidate the potential of the endophytic fungi applied in biotechnological process focusing on the production of bioactive compunds. Despite this potency, the majority of these fungi remain taxonomically uncharacterized Huang et al. 2009. Efforts on discovery of new secondary metabolites from fungal endophytes must be carried out in line with the fungal endophytes taxonomical study to reveal their identity. This situation also applies to endophytic Cercospora, Diaporthe and Fusarium from C. calisaya. Determination of the fungal name found in this study is needed and done in concurrent with their cinchona alkaloids analyses. Identification of fungus based on morphotypes and phylogenetic study based on ITS are often unsatisfactory since these approach sometimes resulted in many unidentified morphotypes. In case of Diaporthe, Udayanga et al. 2012 and Gomes et al. 2013 stated that the delimitation of species within the genus Diaporthe only proved satisfactory once multigene DNA sequence data were generated. This multigene approach is also used to identify Cercospora Hunter et al. 2006 and Fusarium O’Donnell et al. 2000. Many region can be used in multigene analyses such as ACT, CAL, EF 1- α, and HIS Groenewald et al. 2012. As naming of endophytic Cercospora, Diaporthe and Fusarium was previously done using ITS only, confirmation of this genera that contain proposed novel species was done in this study using selected gene regions. MATERIAL AND METHODS Fungal strains The fungal strains used are those belongs to Cercospora, Diaporthe, and Fusarium. Determination of the fungal identities Determination was done by phylogenetic study of the combined sequence of ITS rDNA see Chapter 2, part of the elongation factor 1- α gene Carbone Kohn 1999 and calmodulin Carbone Kohn 1999, actin Carbone Kohn 1999, and histon Crous et al. 2004; Glass Donaldson 1995. Genomic DNA was extracted following procedure described Chapter 2 Page 7. Amplification of ITS region see page 7, EF1- α, CAL, ACT and HIS gene were done using respective primers Table 5.1 and in certain PCR conditions Table 5.2. Amplification of EF gene was performed in 25 L reaction volumes, each reaction containing nuclease free water 8.75 µ L, Gotaq green master mix Promega, USA 12.5 µL, forward and reverse primer 0.625 µ L for each primer, DMSO 0.5 µ L and DNA templete 2 µL. All PCR reactions were conducted using T100 thermal cycler Bio-Rad, USA. Electrophoresis of the amplicons, sequencing and editing the sequence followed the protocol that have described previously see page 6 –7. Tabel 5.1 Primer sequences were used in this study Regions Primers Sequences Sources ITS ITS4 ITS5 5’-TCCGTAGGTGAACCTGCGC- 3’ 5’-TCCTCCGCTTATTGATATGC-3 White et al. 1990 EF1- α EF1-728F EF1-986R 5’-CATCGAGAAGTTC GAGAAGG-3’ 5’-TACTTGAAGGAACCCTTACC-3’ O’Donnel et al. 2010 CAL CAL-228F CAL-737R 5-CAGTTCAAGGAGGCCTTCTCC-3 5 CATTCTTTCTGGCCATCATGG-3 Carbone Kohn 1999 ACT ACT-512F ACT-783R 5-ATGTGCAAGGCCGGTTTCGC-3 5-TACGAGTCCTTCTGGCCCAT-3 Carbone Kohn 1999 HIS CYLH3F CYLH3R 5’-AGGTCCACGGTGGCAAG-3’ 5’-AGCTGGATGTCCTTGGACTG-3’ Crous et al. 2004 Tabel 5.2 PCR condition Regions Predenaturation Annealing Elongation ITS 1 cycle 5’’ at 95 °C, 35 cycles30’ at 95 °C 30’ at 56 °C 1’’ at 72 °C, 10’’ at 72 °C EF1- α, ACT,HIS, CAL 1 cycle 5’’ at 94°C, 40 cycles 30 ’at 94 °C 30’ at 52 °C 30’ at 72 °C, 7’’ at 72 °C RESULT Identity of Cercospora sp. ITS, EF, ACT, CAL and HIS loci with appoximately 580, 320, 230, 320 and 400 bases, respectively are often used for identification of Cercospora through phylogenetic analyses Groenewald et al. 2012. The data set is combined prior to analyses. The partition of homoegenity test of the data set from five loci showed that no signifivant conflict among the phylogenies of a single data set p = 0.55. The combined data matrix contained 84 taxa including one outgroup taxa and 1764 total characters including alignment gaps. Of these characters, 1257 characters were constant, 353 characters were variable parsimony-informatif, and 154 characters were parsimony-uninformative. Parsimony analysis yielded most parsimonius trees and the best parsimonious tree was generated in 1461 steps TL = 1608 steps, CI = 0.225, RI = 0.639, RC = 0.144 Fig. 5.1. Cercospora sp. M18A and M18B is distinct from other Cercospora species due to forming independent clade 100 BS separated from other clades of Cercospora spp. Fig 5.1. This clade of Cercospora sp. from petiole of C. calisaya is a sister clade of C. cf malloti. Therefore, these sequences represented one species. Cercospora M18A and M18B were mycelia sterilia because no sporulation found in the culture Fig 5.2. The colonies on PDA was slow growing, with aerial mycelium, margins smooth, reaching 25 mm diam at 10-d and 39 mm diam at 14- d. The upper surface is white to pale red after two weeks and in reverse in dark. Stromata present. Conidiophores solitary, arising from the Figure 5.1 Phylogenetic tree based on combination of ITS, EF1 –α, ACT, CAL and HIS genes region representing placement of two strains of Cercospora from petiole of C. calisaya within Cercospora spp. of Groenewald et al. 2012. Bootstrap support ≥ 50 from Maximum Parsimony analysis are shown on the nodes Figure 5.2 Morphological characters of Cercospora sp. on C. calisaya. a,b 10 days culture on PDA. c-d. Stroma, conidiophore Bar a,b = 1 cm c,d = 20 μm upper cells of stromata, straight, subcylindrical to flexuous, unbranches, 40 –100 x 5 –7 µm, septate, thin-walled, smooth; conidia was lacking Fig 5.2. The mycelium is septate, pale brown, branches, with smooth surface, 2 –3 µm wide. Stromata not observed. Identity of Diaporthe spp. Only 39 strain out of 49 strains of endophytic Diaporthe spp. from various organs of C. calisaya were re-identified Table 5.3 since the EF amplification was successful for those strains. Re-identification was done using combination of ITS and EF sequences. Phylogenetic analyses shows that seventeen strains can be identified into species level. These consist of D. Cynaroidis 1, D. endophytica 1, D. ganjae 1, D. gardenia 5, D. litchicola 2, D. phaseolorum 2, D. pseudomangiferae 1, and D. rhoina 4. The remaining 22 strains are still unidentified and they represent 15 clusters. In this combined analyses of ITS and EF 1- α sequences, the data set consisted of 147 taxa with 1280 total characters and Diaporthella corylina CBS 121124 is used as an outgroup. Of these characters, 494 characters were constant, 222 were parsimony uninformative and 564 were parsimony informative. Following a heuristic search using PAUP, 958 most parsimonious trees were retained length=6517 steps, CI=0.236, RI=0.651, RC= 0.154, HI=0.764 of which one is shown in Fig. 5.3. A partition homogeneity test showed that ITS and EF1- α could be combined P=0.647 into single analysis. In the most parsimonious tree, 10 clades are formed. Clade I consists of 23 strains of Diaporthe spp. and within this clade there are 2 sub-clades, and some single lineage groups. Sub-clade I consists of 19 strains of Diaporthe spp. that do not cluster with any reference strains Fig. 5.3. Two strains M78 and M21 of D. litchicola and D. litchicola strain BRIP 54900 form sub-clade II with 100 BS. Diaporthe sp. M24 is in one clade with D. pseudomangiferae CBS 101339 with 85 BS. The other Diaporthe such as strain M36, M95, M65, M70, and M96 form single lineage group that are not clustered with any reference strains, and thus strains could not be identified. Table 5.3 Thirty nine strains of endophytic Diaporthe spp. in C. calisaya that were re-identified in this study Species Source Strain Accesion number ITS EF Diaporthe sp. M9 Twig IPBCC 15.1286 LC041055 LC050454 Diaporthe sp. M14 Twig IPBCC 15.1291 LC041028 LC050469 Diaporthe sp. M31 Twig IPBCC 15.1278 AB899786 AB900129 Diaporthe sp. M33 Twig IPBCC 15.1282 AB899787 AB900130 Diaporthe sp. M36 Leaf IPBCC 15.1279 LC041046 LC050463 Diaporthe sp. M38 Twig IPBCC 15.1284 LC041019 LC050471 Diaporthe sp. M41 Twig IPBCC 15.1311 LC041054 LC050455 Diaporthe sp. M42 Twig IPBCC 15.1277 LC041035 LC050459 Diaporthe sp. M43 Petiole IPBCC 15.1276 AB899784 AB900127 Diaporthe sp. M45 Leaf IPBCC 15.1284 LC041023 LC050466 Diaporthe sp. M46 Petiole IPBCC 15.1294 AB899785 AB900131 Diaporthe sp. M48 Twig IPBCC 15.1310 LC041053 LC050483 Diaporthe sp. M56 Fruit IPBCC 15.1296 LC041022 LC050484 Diaporthe sp. M59 Twig IPBCC 15.1284 LC041023 LC050486 Diaporthe sp. M65 Root IPBCC 15.1290 LC041057 LC050456 Diaporthe sp. M69 Twig IPBCC 15.1287 LC041056 LC050467 Diaporthe sp. M70 Twig IPBCC 15.1304 LC041034 LC050473 Diaporthe sp. M72 Twig IPBCC 15.1280 LC041038 LC050485 Diaporthe sp. M74 Leaf IPBCC 15.1280 LC041029 LC050461 Diaporthe sp. M81 Bark IPBCC 15.1295 LC041021 LC050457 Diaporthe sp. M95 Twig IPBCC 15.1301 LC041027 LC050468 Diaporthe sp. M96 Twig IPBCC 15.1305 LC041048 LC050476 D. cynaroidis M54 Fruit IPBCC 15.1274 LC041031 LC050472 D. endophytica M90 Leaf IPBCC 15.1312 AB899789 AB900123 D. ganjae M71 Twig IPBCC 15.1340 LC041037 LC050478 D. gardeniae M11 Petiole IPBCC 15.1339 LC041017 LC050460 D. gardeniae M22 Leaf IPBCC 15.1307 LC041030 LC050470 D. gardeniae M12 Twig IPBCC 15.1310 LC041062 LC050462 D. gardeniae M35 Petiole IPBCC 15.1275 LC041051 LC050474 D. gardeniae M15 Twig IPBCC 15.1308 LC041049 LC050477 D. litchicola M78 Fruit IPBCC 15.1297 AB899788 AB900128 D. litchicola M21 Fruit IPBCC 15.1314 LC041026 LC050475 D. phaseolorum M10 Fruit IPBCC 15.1319 LC041043 LC050458 D. phaseolorum M40 Twig IPBCC 15.1318 LC041040 LC050482 D. pseudomangiferae M24 Twig IPBCC 15.1299 LC041041 LC050480 D. rhoina M84 Twig IPBCC 15.1322 LC041047 LC050464 D. rhoina M94 Petiole IPBCC 15.1320 LC041018 LC050465 D. rhoina M89 Twig IPBCC 15.1288 LC041058 LC050479 D. rhoina M32 Twig IPBCC 15.1323 LC041050 LC050481 Clade I Clade II Clade III Clade IV Figure 5.3 Maximum-parsimony tree showing a relationship between endophytic fungi of Diaporthe spp. and references based on the sequences of combine between ITS5-5.8S-ITS4 of nuclear rDNA and EF1- α gene. The tree was rooted with Diaporthella corylina CBS 121124. Bootstrap value 50 1000 replicates are shown at the branches. Clade VI Clade V Clade VII Clade VIII Clade X Clade IX Clade II consists of D. rhoina M84, M94, M89, M36 and D. rhoina CBS 14627 that 100 BS. Clade III consists of D. gardeniae M11, M22, M48, M35, M15 and D. gardeniae CBS 288 46 with BS 96 . None of the specimens of Diaporthe is in clade IV. In clade V, strain M90 is identified as D. endophytica since it is in one clade with D. endophytica CBS 13381 and LGMF 919 with 70 BS. Diaporthe strain M10 and M40 are considered as D. phaseolorum, eventhough their clustering with D. phaseolorum CBS 116019 supported with low BS value 53 . Strain M45 is a sister clade to D. melonis CBS 43587 and 50778, and thus is unidentified. Strain M71 is D. ganjae since they form one clade with D. ganjae CBS 18091 BS 100 . In clade VIII, strain M54 form one clade with D. cynaroidis CBS 122676 that is supported by low BS value. None of the specimens fall within clade VI and VII, IX and X. All the organ of plants C. calisaya were inhabited by various fungal endophyte Diaporthe spp. Some endophytic fungi that occupy one or more of tree organ. The twig organ most heavily populated followed by fruits, leaves, petioles, roots and bark with 28, 8, 7, 6, 2, and 1 a number of isolates respectively. D. gardeniae inhabited in the twig, leaf, and petiole organ; D. rhoina found twig and petiole; D. phaseolorum found in the twig and fruit; D. cynaroidis, D. pseudomangiferae, D. ganjae were spesific found in the twig; D. endophytica is specific found in the leaf; Diaporthe spp. were found in several organ such as twig, leaf, petiole, root, bark, and fruit. In other phylogenetic study Fig. 5.4 for the 4 out of 6 strains indicated that the name of D. cinchonae can be given with reasons explained below. Diaporthe cinchonae The partition homogeneity test of the two datasets-ITS and part of EF1- α regions –showed that no significant conflict exist between the phylogenies of the individiual dataset P=0.01. Alignment of the combined regions contained 141 taxa and 1213 total characters, of which 542 characters are constant, 148 characters are variable and parsimony-uninformative, 523 characters are parsimony- informative. The best parsimonious tree was generated in 5508 steps CI=0.245, RI=0.654, RC =0.160, HI=0.755 Fig. 5.4. Six strains of endophytic Diaporthe from C. calisaya were separated into three clades. Sequence of Diaporthe sp. strain InaCC-F235, which was isolated from fruit, nested in the clade contains D. hongkongensis strain CBS 115448 from fruit of Dichroa febrifuga, Diaporthe sp. 7 RG 2013 strain CBS 458.78 from Anacardium occidentale, D. arecae strain CBS 161.64 from fruit of Areca catechu, D. pseudophoenicicola strain CBS 462.69 isolated from dead tops of green leaves of Phoenix dactylifera and CBS 176.77 from dieback symptom of Mangifera indica, Diaporthe sp. 8 RG 2013 strain LGMF925 from Aspidosperma tomentosum, D. pseudomangiferae strain CBS 101339 from Mangifera indica, D. eugeniae strain CBS 444.82 from leaf of Eugenia aromatica, D. arengae strain CBS 114979 from Arenga engleri, D. musigena strain CBS 129519 from leaves of Musa sp. Australia, D. perseae strain CBS 151.73 from young fruit of Persea gratissima, Diaporthe sp. 6 RG 2013 from fruit of Maesa perlarius strain CBS 115595 and CBS 115584, and D. arecae strain CBS 535.75 from fruits of Citrus sp. with 63 BS. In this clade, Diaporthe sp. strain InaCC-F235 is paraphyletic to D. hongkongensis strain CBS 115448. Figure 5.4 Phylogenetic tree based on combination of ITS and partial EF1 –α genes region representing placement of six sequences of Phomopsis spp. from different organs of C. calisaya within Diaporthe spp. of Gomes et al. 2013. Bootstrap support ≥ 50 from Maximum Parsimony analysis are shown on the nodes Four Diaporthe strains from petiole and branch of C. calisaya InaCC-F236, InaCC-F238, InaCC-F239, and InaCC-F2310 formed independent clade separated from other DiaporthePhomopsis sequences with 96 BS. Therefore, members of this clade are proposed as a new species, namely, D. cinchonae sp. nov Fig. 5.4. Strain InaCC-F237 nested in the same clade containing D. endophytica strain LGMF919 and strain CBS 133811 with 81 BS. This clade clearly showed that Diaporthe sp. strain InaCC-F237 belonging to D. endophytica. Another strain, InaCC-F235 becomes sister clade of D. cinchonae, but this could get any specific epithet does not form a clade with strong BS. The species name given to those strains is supported by morphological observation Fig. 5.5, 5.6, 5.7. Morphological comparison among those species Table 5.4 indicates the morphological difference in several characters. Figure 5.5 Morphological characters of Diaporthe cinchonae sp. nov. on C. calisaya. a. 7 days culture on PDA. b. Conidiomata sporulating on PDA with yellowish-light brown exudates. c. Beta and alpha conidia. d. Conidiogenous cells. Bar a = 1 cm c, d, e, f = 10 μm. Etymology –named after the generic name of the host, Cinchona. Conidiomata 316.7 –376.9 × 196.23–370.9 µm, pycnidial, globose to subglobose, covering with mycelia, immersed, outer surface not smooth, blackish, ostiolate, formed after 7 –8 weeks incubation at room temperature, scatterred throughout the colony surface, sometimes the pycnidia arranged in clusters. Conidiophores 0 –1 septate, hyaline to subhyaline, subcylindrical, smooth, rarely branched. Conidiogenous cells 17.5 –24.6 × 1.9–2.2 µm, phialidic, aseptate, tapering toward the apex, slightly curved in the apex, hyaline, smooth, unicellular, unbranched. Alpha conidia 5.4 –5.7 × 1.3–1.6 µm, hyaline, oblong to ellipsoidal, blunt at the apex, with subtruncate base, guttulates. Beta conidia 23.6 –27.1 × 1.6–2.1 µm, spindle shaped, aseptate, hyaline, smooth, curved at one end, apex rounded, with truncate base. Gamma conidia not observed. Culture characteristics –Colonies on PDA white, fast growing, reaching 47– 52 mm diam. after 7 days, forming wavy concentric radial texture, after 2 weeks become pale to grey, and covering the peti dishes 9 cm diam.. In reverse, pale to smoke-grey, sometimes light brown to dark. Figure 5.6 Morphological characters of Diaporthe endophytica on C. calisaya. a. 7 days culture on PDA. b. Conidiomata sporulating on PDA. c –d. Beta conidia. e. Conidiogenous cells. Bar a = 1 cm c,d,e = 20 μm Figure 5.7 Morphological characters of Diaporthe sp.on C. calisaya. a. 7 days culture on PDA. b. Conidiomata sporulating on PDA. c –e. Conidiogenous cells d. Beta conidia. Bar a = 1 cm c,d,e = 20 μm Table 5.4 Comparison of morphological characters of Diaporthe spp. from C. calisaya used in this study Character D. cinchonae D. endophytica Diaporthe sp. Pycnidia 316.7 –376.9 × 196.2 –370.9 µm 363.7 –392.6× 414.6 –454.3 µm 218.3 –234.4 × 160.3– 237.6µm Alpha conidia 5.4 –5.7 × 1.3– 1.6 µm - - Beta conidia 23.6 –27.1 × 1.6 –2.1 µm 17.4 –20.7× 1.9– 2.4 µm 22.1 –24.9 × 1.3–1.6 µm Character D. cinchonae

D. endophytica Diaporthe sp.

Gamma conidia Absent Absent Absent Culture on PDA white, wavy and zonate concentric radial texture, after 2 weeks releasing the exudates fluid a pale brown colour in the medium. In reverse pale brown white, concentric radial texture, with non – wavy edge, after 2 weeks become pale to grey. In reverse, pale to smoke – grey, sometimes light brown to dark whiteconcentric with variegated or irregular wavy at the edge, after 3 weeks become white to grayish. In reverse smoke – gray to brown Microhabitat branch, petiole Leaf Fruit Identity of Fusarium spp. Based on the molecular analysis, the partition homogeneity test of the two datasets-ITS and part of EF1- α regions showed significant conflict exist between the phylogenies of the individiual dataset P 0.01. Therefore, tree constructed using separate data sets. In the parsimony analysis of ITS sequence dataset, the alignment contained 49 sequences and 489 total characters, of which 257 characters are constant, 60 characters are variable and parsimony-uninformative, 172 characters are parsimony-informative. All characters have equal weight. The best parsimonious tree was generated in 555 steps CI=0.647, RI=0.862, RC0.558, HI = 0.353. The endophytic Fusarium sequences from C. calisaya were divided into three distinct lineages Fig. 5.8. Fusarium sp. M34, M66 and M67 nested in the clade containing F. equisetti, F. incarnatum F. equisetti MAFF 236434, MAFF 236723, NRRL 26419T, and F. incarnatum MAFF 236521 with 99 BS.Strain M16 forms monophyletic clade with members of F. oxysporum F. oxysporum CBS 127.73 and CBS 133023T BS = 83 , and M93 and M97 nested in the clade containing F. solani sequences F. solani strain CBS 132898, strain NRRL 28579T, and F. solani f. mori strain MAFF 238538 with 100 BS. The alignment of partial EF1- α composed of 49 sequences and 345 total characters, of which 72 characters are constant, 61 characters are variable and parsimony-uninformative, 212 characters are parsimony-informative. All characters have equal weight. The best parsimonious tree was generated in 892 steps CI=0.565, RI=0.792, RC=0.448, HI=0.435. The placement of 6 endophytic Fusarium sequences in the phylogenetic tree generated from partial EF1- α dataset Fig 5.8 is similar to that of the ITS tree. Fusarium sp. M34, M66 and M67 nested in the clade containing F. equisetti, F. incarnatum with 92 BS. Strain M16 forms monophyletic clade with F. oxysporum clade BS=100 . Strain M93 and M97 are nested in the F. solani clade with 100 BS. Based on the phylogenetic trees generated from ITS and partial EF1 –α gene regions, the sequence of Fusarium M16 is determined as F. oxysporum. Two other strains i.e. M93 and M97 are determined as F. solani. However, these datasets were failed to resolve the species name of Fusarium spp. M34, M66 and M67. Figure 5.8 Maximum-parsimony tree showing the relationship between endophytic Fusarium sp. based on the sequnces of ITS of rDNA. Bootstrap value 50 1000 replicates are shown at the branches. Penicillium citrinum isolate AX4602 is taken as outgroup Figure 5.9 Maximum-parsimony tree showing the relationship between endophytic Fusarium sp. based on the sequnces of EF1- α. Bootsrap value 50 1000 replicates are shown at the branches. Penicillium citrinum isolate AX4602 is taken as outgroup Figure 5.10 Morphological character of Fusarium oxysporum M16 on C. calisaya. a and b. 7 days culture on PDA, c. Chlamydospore. d. Macroconidia and microconidia, d. Phialid. Bar a,b = 1 cm c,d = 20 μm Figure 5.11 Morphological characters of Fusarium incarnatum M34 on C. calisaya. a,b. 7 days culture on PDA. b. Chlamydospore, d. Microconidia. Bar a,b = 1 cm c,d = 10 μm In order to resolve the identity of Fusarium sp. M34, strain M66 and M67, separate phylogenetic analysis based on partial EF1- α sequence involving these three sequences with 42 sequences belonging to F. equisetti –incarnatum was conducted. F. asiaticum strain NRRL 26156 GenBank accession number: AF212452 was used as outgroup. The alignment of this dataset composed of 46 Figure 5.12 Morphological characters of Fusarium incarnatum M66 on C. calisaya. a,b. 7 days culture on PDA, c. Chlamydospore, d. Microconidia, e. Phialides. Bar a,b = 1 cm c,d = 20 μm, e = 10 μm Figure 5.13 Morphological characters of Fusarium incarnatum M67 on C. calisaya. a. 7 days culture on PDA, b. Chlamydospore. c. Conidiogenous cell, d. Conidiophore, e. Phialid, f. Microconidia. Bar a = 1 cm b,c,d,e,f = 20 μm sequences and 255 total characters included in the analysis, of which 144 charactersare constant, 43 characters are variable and parsimony-uninformative, 68 characters are parsimony-informative. All characters have equal weight. The best parsimonious tree was generated in 204 steps CI=0.667, RI=0.851, RC=0.567, HI =0.333. The phylogenetic tree showed that Fusarium spp. M34, M66 and M67 formed monophyletic clade with F. incarnatum NRRL 34004 GQ505628 BS = 66.6 . This clade nested within the large monophyletic clade containing sequences belong to F. incarnatum sensu stricto BS=81.2 . Based on this analysis, Fusarium spp. M34, M66 and M67 are determined as F. incarnatum. The phylogenetic study was supported by morphological observation on colony and microscopic characteristics Fig 5.10 –5.15. The morphological difference between species were found Table 5.5. Figure 5.14 Morphological characters of Fusarium solani M93 on C. calisaya. a,b. 7 days culture on PDA, b. Macroconidia and microconidia, c. Phialid. Bar a = 1 cm c,d = 2 0 μm. Figure 5.15 Morphological characters of Fusarium solani M97 on C. calisaya. a. 7 days culture on PDA. b. Chlamydospore, c. Phialid, d. Conidiophore, e. Macroconidia and microconidia. Bar a = 1 cm b,c,d,e = 10 μm Table 5.5 Morphological comparison of the structures of three endophytic fungi Fusarium species associated with C. calisaya Morphological F. incarnatum M66 F. incarnatum M34 F. incarnatum M67 F. oxysporum M16 F. solani M93 F. solani M97 characteristics Microconidia 8.7−11.04 × 13.69−45.23 2.93−3.88 × 6.12−8.63 2.47−4.65×6.04−21.56 8.75−12.89 × 25.41− 34.63 2.90 − 5.21 x 13.18−17.21 2.90−8.72 X 8.89−20.90 oblong, aseptate oval, aseptat oblong, aseptat ovale, aseptate ovale, aseptat ovale, aseptat Macroconidia − 3.61−4.15× 11.9−20.9 − 8.49−11.75× 31.92−70.43 19.91 –38.63×3.35–5.91 3.35−11.36×19.91−62.64 − oblong-elliptical, aseptat − obovate, 3 –6 septat obovate, septat 4-6 obovate, aseptat Conidiophore − − long and single − long and single long and single Phialides µm Monophialide − Monophialide − monophialide monophialide 1.4 ×4.3 − 1.4× 6.4 − 3.3 ×5.86 2.7×9.31 Chlamydospore present, globular, present, obovoid present, globular, present, globular, present, ovale, present chain+ or single, intercalar and terimal Intercalar Intercalar intercalar, terminal intercalar ovale, intercalar 5.3−7.9 ×7.1−9.7 6.3×8.5 3.1−4.3× 3.3−4.9 6.1−10.6 ×5.4−11.4 4.5−6.5 ×7.3−8.1 4.6−8.8×6.5−9.2 Colour white to pink white to pink White purpledark purple white to cream white Size cm of colony 4.8 4.2 5.2 4.3 5.8 4.8 Mycelium cottony, aerial cottony, aerial cottony, aerial cottony, immersed cottony, aerial cottony, immersed 57 DISCUSSION Cercospora The first published paper on Cer. cinchonae of the quina plant was written by Ellis Everth 1887, then Crous Braun 2003 has changed its name to Pseudocercospora cinchonae. Boedijn 1962 had described two new species Cer. cinchonicoea and Cer. cinchonicola based on specimen from Quina plant in Indonesia. Braun 2001 moved Cer. cinchonicola to Pseudocercospora cinchonicola. All this species areas plant pathogens. In this study, Cercospora sp. M18A and M18B that were isolated from healthy C. calisaya petiole in Java, Indonesia are endophytes. Based on phylogenetic analysis with the ITS data matrix contained 10 genera of Cercosporoid s. str, Cercospora sp. M18A and 18B form monophyletic within the genus of Cercospora. In fact, Cercospora sp. M18A and M18B to which the name Cercospora sp. applies, apparently are not Cercospora cinchonae Ellis Everth 1887 and Cer. cinchonicola, Boedijn 1962, since the last two names have characteristics of Pseudocercospora. Therefore Cercospora M18A, M18B were considered as a new species, Cer. cinchonae. These species was the first reported endophytic Cercospora in C. calisaya from Indonesia. The combination of the morphological and phylogenetic elucidation of new proposed taxa in Cercopsora is quite important to misidentification. The phylogenetic tree generated from maximum parsimony analysis by combining five genes loci rDNA region showed that the monophyletic of Cercospora with 55 bootstrap value. The Cercospora sp M18A and Cercospora sp. M18B formed a monophyletic clade with 100 bootsrap value. These strains were included in one species. This clade appeared as a sister group C. cf. malloti, C. kikuchii, C. cf. richardiicola, C. cf. sigesbeckiae clade with 65 bootstrap value which indicates a close relatioship between one and others. The phylogenetic tree sequences showed that two sequences of Cercospora sp. 18A and 18B from petiole formed independent clade which separated from other Cercospora sequences included in the analysis 65 BS. The phylogenetic tree is obtained by using the combined sequence data of five genomic loci. The Cercospora sp. M18A and M18B are distinct genetically from other Cercospora sequences. Therefore, members of this clade are proposed as a new species based on phylogenetic analysis. Taylor et al. 2000 developed the Genealogical Concordance Phylogenetic Species Recognition GCPSR concept to definethe limits of sexual species, using the phylogenetic concordance of multiple unlinked genes. This concept has proved greatly useful in fungi, because it is more finely discriminating than other species concepts, as several species can not be recognised due to the lack of distinguishing morphological characters or mycelia sterilia Cai et al. 2011. Cercospora sp. M18A and M18B genes had been analysed with ITS, EF, CAL, ACT and HIS because it was not identity with morphological characters. The adoption of genealogical concordance for species recognition in Cercospora sp. to distinguish species otherwise possible to identify due to mycelia sterilia. Cercospora cinchonicola caused disease in the Quina plant was first reported in Indonesia and identification based on morphological characters by Boedijn 1962. Morphological characteristics of Cer. cinchonicola were stromata small, flattened, blackish brown 15−40 µm in diameter, fascicles fairly dense. Conidiophore brown, with a short side branch, sparingly septate, geniculate, tip bluntly rounded 20−60 x 3−3.5 µm. Conidia pale olivaceous, narrowly obclave, mildy curved, indistincly multiseptate, base truncate, tip rounded, 64−110 × 3−4 µm. Cercospora sp. M18 having Stromata small, flattened, brown. Conidiophores clustered, arising from the upper cells of stromata, straight, subcylindrical to flexuous, unbranches, 40−100 × 5−7 µm, septate, thin-walled, smooth, brown; conidia was not observated. Unfortunately, in this study conidia was not found in Cercospora sp. as morphological characteristics. Therefore, Cercospora can not be identified as the same species with Cer. cinchonicola, because they were found within the same plant host. Many Cercospora species that are morphologically similar proved to be genetically distinct, and several isolates that were formerly identified based on their host, were show to represent different taxa To-Anun et al. 2011. Boedijn 1962 pointed out that the large number of the Cercospora species was due to the presence of a wide variety of vascular plants in Indonesia. He reported 90 species of Cercospora from 109 host plant species, including those from Cinchona plant. In Thailand, various hosts such as crops, weeds and ornamentals plants could associate with cercosporoid fungi. There were one hundred and six species have been recorded from several locations Meeboon et al. 2007; Nakashima et al. 2007. Cercospora species are frequently classified according to host. Highly host-specificity species, e.g. Cercospora beticola from sugar beet or Beta vulgaris Den Breeyen et al. 2006 , Cercospora christellae from weed Christella parasitica from northern Thailand To-anun et al. 2010. Cercopsora habenariicola from leaf of Habenaria susannae L. R. Br. Orchidaceae Meeboon et al. 2007 and Cercospora brassicicola from leaves of Brassica oleracea L. Brassicaceae in Thailand Meeboon et al. 2005 were noted. Groenewald et al. 2005 considered that Cercopsora was not host spesific because some of Cercospora can infect a wide host range e.g. C. apii complex can infect Apium graveolens celery and Beta vulgaris sugar beet, Cercospora cf. citrulina can infect Musa sp., Citrullus lanatus, and Momordica charanthia. Groenewald et al. 2012 stated that some species were found to be limited to a specific host genus and other genus have a wide host range. The stimulate sporulation media had given banana leaves and plant organs quinine on the surface of the agar media could not form sporulation. More detailed studies are required to describe the morphological characters of Cercospora sp. M18 as endophytic fungi. In this study Cercopora sp. could not be found conidia in the artifisial media. Several problematic endophytic fungi could not distinct morphological characters or sterility, e.g., Diaporthe Gomes et al. 2013, Colletotrichum Damm et al. 2012. Vathakos and Walters 1979 observed that Cercospora kikuchii in the Senescent Soybean Plant Agar SSPA could be exposed abudant sporulation on alternating dark and light periods and 8 d to Gro-lux lamp, but it did not occur in continuous darkness. More conidia can be produced if transfer of spores rather than mycelium resulted in cultures. Goode Brown 1979 postulated that the ability of some Cercospora spp. to sporulate for a few generations in artificial culture indicated that those isolates have a genetic component for sporulation. The Cercospora are considered as plant pathogens, C. kikuchii is the only species known both as plant pathogen and endophyte Tales 2011. Little information of endophytic Cercospora were found. During study of endophytic fungal diversity associated with C. calisaya, the most important medicinal plant e.g. antimalaria, antibacterial, etc Maehara et al. 2001, Skogman et al. 2012, Wolf et al. 2002, two isolates of Cercospora-like were found. The existence of some endophytic fungi in the stem and bark of quinine plant have been reported by several researchers in Indonesia, however, none of those researchers reported the endophytic Cercospora. Therefore, the findings of endophytic Cercospora from C. calisaya was interesting and to analyze its potential for alkaloid production, particularly quinine, quinidine, conchonine, and cinchonidine. Diaporthe The identification of genera Diaporthe using ITS sequence is problematics. The application of molecular phylogenetic analysis has resolved problems in determination of DiaporthePhomopsis species complex Rensburg et al. 2006; Udayanga et al. 2012; Gomes et al. 2013. Majority of these reports involving utilization of sequences from 18S, ITS and 28S rDNA region Zhang et al. 1998; Niekerk et al. 2005, and in combination with DNA sequence generated from part of the elongation factor 1- α gene EF1- α Rensburg et al. 2006; Thompson et al. 2011, β–tubulin and calmodulin Gomes et al. 2013. Gomes et al. 2013 reported that several taxa of Diaporthe are host-specific, while some species have wide host ranges. These include members of Diaporthe living in plant tissue as endophyte. Therefore, identification of dominant endophytic Diaporthe spp. is of importance. Identification of the Diaporthe into species require additional genes other than ITS. Identification based on ITS rDNA is commonly used to identify to the species level, especially if the endophytic fungus does not sporulate Zhang et al. 1998. Identification by ITS sequences can be used as a morphological identification verification, against unidentified endophytic fungus based on morphological characters Huang et al. 2009. Therefore, further identification of Diaporthe spp. is done by using a combination of rDNA ITS regions and EF1-  gene. Phylogenetic analysis showed that only 17 of the 39 strains of endophyte Diaporthe found can be named species, while the remaining 22 strains can not be named species. Multigene analysis led to the name of the species based on the ITS approach can not be used. Gomes et al. 2013 use more than one gene for identification of molecular Diaporthe using a combination of ITS sequences, EF1- , ACT Actin, CAL Calmodulin and HIS Histon. The results of molecular analyzes of strains Diaporthe spp. by using combination of ITS and EF1- α left 22 strains unidentified. Identification is based on a combination approach ITS and EF can change the status of naming species based on ITS approach. The name of D. litichola M78, D. pseudomangiferae M24, D. phaseolorum M10 and M40, D. ganjae M71 unchanged, whereas D. helianthi M21 becomes D. litchicola M21, D. psoraleae-pinnatae M32, M84, and M94 under ITS phylogenetic tree are moved into D. rhoina M32, M84, and M94. Further, Phomopsis palmicola M11 moved into D. gardeniae M11, D. beckhausii became D. gardeniae M35 M35, D. beckhausii M54 became D. cynaroidis M54, while Diaporthe sp. M12, M15, M22 are identified as D. gardeniae M12, M15 and M22, Diaporthe sp. M90 become D. endophytica M90. Diaporthe sp. M89 is identified as D. rhoina M89. Identification using multigene approach is usually more accurate than identification by a single gene. The use of molecular identification through polyphasic approach by using multigene and secondary metabolites analysis is supposed to enhance the accuracy of Diaporthe spp. identification. Meanwhile, profile clustering is not in accordance with phylogenetic groupings. This indicates that alkaloids profile can not be considered as criterion included in polyphasic approach as the alkaloid production is each strain- dependent. No new species can be proposed for any Diaporthe spp. in the phylogenetic study. The current study on the six Diaporthe strains included for cinchona alkaloids analyses revealed that at least, three distinct lineages of Diaporthe living inside the tissue of C. calisaya, i.e., D. cinchonae inhabiting branch and petiole, D. endophytica inhabiting leaf, and Diaporthe sp. InaCC-F235 inhabiting fruit. Diaporthe endophytica was also reported as common endophyte inhabiting S. terebinthifolia leaf, M. ilicifolia petiole, and G. max seed in Brazil Gomes et al. 2013. The existence of more than one species of endophytic Diaporthe in the single host plant species was not uncommon as several authors previously had reported. For example, about 15 Phomopsis spp. were determined from grapevines Niekerk et al. 2005, at least four Diaporthe taxa occurred in fennel Foeniculum vulgare Santos Phillips 2009, as well as in soybean Glycine max Santos et al. 2011 and in sunflower Helianthus annuus Thompson et al. 2011, respectively. These information demonstrate that implementation of host-based species concept in determination DiaporthePhomopsis must be avoided Hyde et al. 2010. The existence of more than one species of DiaporthePhomopsis within one host or the possibility of single species of DiaporthePhomopsis has more than one host will definitely affects the management of plant diseases caused by this fungus in the future. Although the phylogenetic analysis clearly showed differences among D. cinchonae, D. endophytica, and Diaporthe sp. strain InaCC-F235, however, the morphological characteristics of these isolates are similar. Slightly differences were found on pycnidial size and β-conidia. The size of β-conidia of D. cinchonae 23.6– 27.1 × 1.6 –2.1 µm was found slightly larger than D. endophytica 17.4–20.7 × 1.9– 2.4 µm and Diaporthe sp. strain InaCC-F235 22.1 –24.9 × 1.3–1.6 µm. However, the colonies characteristics of these 3 species were apparently distinct. Diaporthe cinchonae formed wavy and zonate concentric radial textures, which was distinct from isolates of D. endophytica and Diaporthe sp. strain InaCC-F235 having concentric radial texture with non-wavy edge and concentric with variegated or irregular wavy at the edge, respectively. These data showed that morphological characters such as conidiomatal structure and conidial size are unreliable in determining DiaporthePhomopsis complex into species level. In addition, many species of DiaporthePhomopsis only produce α- or β-conidia, and only about 20 of Phomopsis species have a known teleomorph Rehner Uecker 1994. The molecular phylogenetic analysis is greatly needed to advance more specific genetic evidence to support taxonomic differences, because of limited morphological characters in differentiating species. Gomes et al. 2013 also previously reported that many morphologically similar DiaporthePhomopsis species were proved to be genetically distinct. The combination of morphology and cultural characters with molecular analyses will raise precise identification and description for DiaporthePhomopsis species from a range of host plants. The current study also indicates that tissue types seems to be important microhabitats for specific DiaporthePhomopsis inhabiting the same host. However, more sequences are necessary to be included in the analysis to justify this hypothesis. Due to the considerable overlap of morphological features among the available species within DiaporthePhomopsis, Santos and Phillips 2009 suggested that the phylogenetic species concept developed by Taylor et al. 2000 is probably best fits to determine members of this fungal group into species level. Several problematic endophytic fungi lacking of distinct morphological characters or sterility, e.g., Diaporthe Gomes et al. 2013, Colletotrichum Damm et al. 2012, and Phyllosticta Glienke et al. 2011 have been resolved. However, controversies regarding how many genes sufficient for determination of fungi into species level using this approach are still continue. In DiaporthePhomopsis complex, phylogenetic analyses of sequence from ITS region as primary region, and in combination with HIS or TUB regions were recommended Udayanga et al. 2012; Gomes et al. 2013. Combination of ITS and TEF1 sequences were also common in identification of DiaporthePhomopsis complex Ash et al. 2010; Santos et al. 2011; Thompson et al. 2011. In the case of Diaporthe sp. strain InaCC-F235, identification using combined sequences of ITS and TEF1‒ was proven insufficient. Although the phylogenetic tree sequences showed that Diaporthe sp. strain InaCCF235 did not link to any other sequences included in the analysis, however, it is not enough evidence to propose this strain as a novel species. Furthermore, slight morphological differences of this strain to closely related species have also been found insufficient to support phylogenetic data for species determination. Additional sequence data from morphologically similar endophytic Diaporthe isolates from C. calisaya or additional sequence data from other gene regions are necessary to resolve the identity of this endophytic fungus. The phylogenetic tree generated from combination of ITS and part of EF1- α sequence showed that four sequences of D. cinchonae form an independent clade within the large clade containing Diaporthe sp. strain InaCC-F235, D. hongkongensis, D. arecae, D. pseudophoenicicola, D. pseudomangiferae, D. eugeniae, D. arengae, D. musigena, D. perseae, D. arecae, and several species of Diaporthe on different hosts. Within this clade, D. hongkongensis was the closest sequence to D. cinchonae. Based on morphological characteristics, D. cinchonae differs from D. hongkongensis by having larger pycnidia vs. up to 200 μm diam. of D. hongkongensis, longer beta conidia vs. 18 –22 × 1.5–2 μm of D. hongkongensis, and lacking of gamma conidia at least during this study. The phylogenetic tree generated from combination of ITS and part of EF 1- α sequence analyses clearly showed that this strain belonging to D. endophytica 81 BS. The type species of D. endophytica was first published from the leaf of Schinus terebinthifolia Raddi Anacardiaceae Gomes et al. 2013 . This species is also recorded as endophyte in petiole of Maytenus ilicifolia Mart. ex Reissek Celastraceae and in seed of Glycine max L. Merr. Fabaceae. This is the first report of D. endophytica in C. calisaya from Indonesia. Fusarium Phylogenetic analysis of quinine-producing Fusarium species showed that there are more than one species of the genus Fusarium exist as endophyte within plant tissues of C. calisaya. The finding of more than one species from a single fungal genus occupy the same species of host plant, in fact, was not uncommon as several authors had previously reported Niekerk et al. 2005; Santos Phillips 2009; Santos et al. 2011; Thompson et al. 2011. The current study also found that a single species of endophyte can occupies different type of plant organs as showed by several morphotypes of F. incarnatum isolated from petiole strain M34, fruit strain M66 and bark strain M67. Fusarium is hyphomycetes soil-borne fungi that causes the most economically important plant diseases. Fusarium sp. is cosmopolitan, occurs on a wide range of host plants as plant pathogenic, such as tomatoes Kim et al 2007; Ignjatov et al. 2012, banana Dita et al. 2010, maize Rodriguez et al. 2007. Fusarium also causes diseases in corn, figs, pine, rice and sorgum Nelson et al. 1993. Some species are known to cause diseases in animals and humans, which is important to study because those species remove mycotoxins Monds et al. 2005. The identification of the occurrences of endophytic Fusarium sp. in C. calisaya has not gained much attention, compared to pathogenic Fusarium sp., although there have been reports on Fusarium sp. as the symptom of infections in several types of plant. Therefore, this study was conducted to isolate endophytic Fusarium sp. from all parts of the plant organ of C. calisaya. The association between the endophytic fungus and the plant host may also switch from mutualistic to pathogenic depending on several factors Moricca Ragazzi 2008. The endophyte fungi can infect healthy tissues as biotrophs, but fungus may infect the host after a latent period or become a saprophyte when then host plant dies. Therefore, the association between the fungal endophyte and the host plant is like transitory Bacon Yates 2006. The identification of the occurences of Fusarium spp. in different forms is often confusing due to their similar morphological features. Morphological characters to identify the species is problematic because mycelia pigmentation, shape and size of conidia are unstable and dependent on the composition of media and environmental condition Guo et al. 2001. The differentiation of genes in DNA sequences has been used to support morphological identification of Fusarium sp. Phylogenetic analysis of DNA sequences has been used to distinguish genetic relationships among closely related Fusarium sp. Molecular techniques for fungal identification within species have been used via the ITS region and other gene sequences. O’Donnell et al. 1998, Watanabe et al. 2011 stated that the rDNA clu ster region β-tubulin gene β-tub, the elongation factor 1- α gene EF1-α, and aminoadipate reductase gene lys2, have been used as genetic markers for the phylogenetic analysis of Fusarium sp. The most of Fusarium are classified as saprotrophic, phytopathogenic, and endophyte Barik et al. 2010. Several Fusarium spp. isolated as endophyte from medicinal plants and other plants have been reported to produce metabolites activity, such as grass family Gramineae Sieber 2002, Camptotheca acuminate Wang et al. 2006, Pinus massoniana Lamb Wang et al. 2008, Dracaena cambodiana and Aquilaria sinensis Gong Guo 200, Annona squamosa, Taxus chinensis Deng et al. 2009, and Axonopus compressus Zakaria Ning 2013. Endophytic F. oxysporum and F. solani have been found on the roots of several vegetables Kim et al. 2007, while F. solani have been found on the roots of Rhizophoraceae mangrove trees Xing Guo 2011. There has been no report of the research on endophytic fungus Fusarium sp. and its potential to produce quinine, which was isolated from the entire plant organs of C. calisaya. CONCLUSION The identity reconfirmation Cercospora based on amplyfy region ITS, EF1- α ACT, CAL and HIS analysis, Cercospora was identifified as Cercospora sp. The genera of Diaporthe and Fusarium based on ITS and EF1- α analysis were identified Diaporthe sp. D. cinchonae, D. endophytica, F. incarnatum, F. oxysporum, and F. solani. The Cercospora sp., Diaporthe sp. and D. cinchonae are candidate for new species, but appropriate morphological features are needed to support this proposal. Alkaloid profile clustering of Diaporthe spp. is not in accordance with phylogenetic groupings. 6 GENERAL DISCUSSION BIODIVERSITY OF ENDOPHYTIC FUNGI FROM

C. CALISAYA

Biodiversity as part of the community structure on certain habitat can be investigated by conventional approach using culture dependent method and metagenomics approach based on molecular study. Culture dependent method for analyzing endophytic fungi allows mycologists to enumerate the number of strains within an ecological niche. The expression of true population number of the endophyte is debatable Efriwati et al. 2014, but culture based method create an opportunity to further explore on the potential of the culture for bioprospecting approach. In other way, we can say that the exploration on the endophyte using culture dependent method is a bases for research on the bioprospect of the fungus. This strategy is taken for this study for various reason. Besides planting Cinchona tree is problematics, the demand of bark for quinine production is in shortage. Quinine is still needed worldwide as WHO recommended this as a primary drug to cure severe malaria disease caused by Plasmodium falciparum in the future. About 687 strains collected represent 96 morphotypes. Some of the strains are sterile. According to Huang et al. 2008, these sterilia mycelia are widely distributed among 27 medicinal plants out of 29 host plants used for screening endophytic fungi. About 27 were sterilia mycelia and these fungi could not be identified due to lacking of morphological characters. Therefore phylogenetic study based on ITS rDNA is taken for first approach for fungal identification. Molecular techniques have been successfully employed in phylogenetic analysis for the identification of morphospecies by applying rDNA sequences Guo et al. 2003; Wang et al. 2005. Ribosomal DNA sequences analysis of endophyte was used to verify the morphotype of different groups and to resolve problems of the identification associated with endophytic fungi Lacap et al. 2003. These two approaches are adopted in this study. Verification of the morphotypes using molecular approach is noted as very important in this study. For example D. phaseolorum M40 and D. phaseolorum M10 represent different morphotypes, but molecular identification showed that they are one species. A similar situation was detected in Col. gloeosporioides, D. infecunda, D. pasalorae, F. incarnatum and others. This indicates that species may have morphological plasticity particularly in their colony characteristics. In view of diversity, the result of this study indicates that the diversity of fungal endophytes in Cinchona was greater than reported before Simanjuntak et al.2002, Shibuya et al. 2003, Mumpuni et al. 2004 and Winarno 2006, Maehara et al. 2012. About 18 genera of Ascomycetes are found. Among those, except Diaporthe, Phomopsis and Penicillium are the first reported as endophytes in C. calisaya. However, these genera has been reported as endophytes of other host plant. Aspergillus terreus, As. alternate, and As. niger were found within Withania somnifera Khan et al. 2010. F. solani, F. oxysporum, Phoma eupyrena, and Petalotiopsis sp. were found within the Crataeva magna, A. indica. T. harzianum was found within Hollarrhena antidycenterica Tajesvi at al. 2006. Penicillium sp. was found within Vitex negundo Banerjee et al. 2006. This study found that Diaporthe is dominant genera, followed by Colletotrichum. Other studies supported this finding. Endophytic Colletotrichum, and Phomopsis anamorph of Diaporthe have a wide host range Cannon Simmons 2002; Murali et al. 2006; Hyde et al. 2009. Fungal endophytes are frequently isolated from organ of medicinal plant that used to cure disease for health Selim et al. 2012. In this study, endophytic fungi were found in all organs of C. calisaya. Fungal dominance and distribution related to organ- specificity have been discussed. Organ specificity was shown by certain fungus, in contrast to others that widely distributed. This study may reveal possible physiological differentiation corresponding to ecological adaptation within species. The greater colonization of certain parts of plant may be related to more complex anatomical structure and susceptibility to infection Cannon Simmons 2002. The endophytic fungi, such as Colletotrichum Kumar et al. 2015, Cercospora Crous Braun 2003, Diaporthe Uecker 1988, Fusarium Ignajatov et al. 2012, and Phyllosticta Glienke et al. 2011 are known to live as plant pathogens, occurring on a wide range of host plants. This study found the same genera as endophytes. This phenomenon illustrated the lifestyle of fungi. The endophytic fungi having a host- switching strategy that permits dispersal and persistence when a primary host is unavailable or when the fungi alternates between host taxa. One endophyte exists as endophyte and other as saprobic or pathogenic Davis et al. 2003. Interaction in plant endophytes can be regarded as an unbalanced status of a symbiotic when the host is stress and in physiological or ecological conditions favors for virulence. Endophytes within certain plants could be pathogen against other plants, depending on the balance between pathogenicity and endophytism of the microorganism in different host Schulz Boyle 2005. LINKING BIODIVERSITY TO BIOPROSPECT OF ENDOPHYTIC FUNGI FROM

C. CALISAYA

Enormous potential of fungal endophyte diversity as source of cinchona alkaloids discovery is demonstrated in this study comparing to all prior studies on the endophyte of Cinchona. Endophytic fungi have greatly contributed to the diversity of secondary metabolites, including the production of the chemical components of the characteristics of the host plants. According to Guo et al. 2008 endophytic fungi were capable of synthesizing bioactive compounds that could be used as potential source of pharmacy. Tan Zou 2001 stated that this might occur because of genetic recombination of endophytic fungi within their host plants in the evolutionary process. Germaine et al. 2004 and Zhang et al. 2006 reported that endophytic fungi lived in and adapted to plant tissues by transferring some genetic information DNA of the host plants. All cinchona alkaloids is detected in the tested strains. As. versicolor M55, As. sydowii M62, Colletotrichum sp. M1, Col. boninense M45, Diaporthe sp. M39, Diaporthe sp. M65, Diaporthe sp. M6, D. eucalyptorum M56, D. Pseudomangeferae Figure 6.1 Clusters of endophytic Diaporthe spp. based on the similarity on their alkaloid profile 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. RECOMMENDATION Further research should be done for the prospective strains and the candidates of new species to ascertain its possibility to be used as biotechnological agents for quinine production and for the publication of new species proposal. 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EF1 –α, ACT, HIS, TUB SAMPLING MORHOTYPING SELECTION OF REPRESENTATIVE STRAIN FOR EACH MORPHOTYPE Culture collection Morphotype 1. Community Structures 2. Organ specificity 3. Novel species 4. ALKALOID PROFILING 4. Alkaloid profile of representative strains QUININE DETECTION STRAIN SELECTION BIOPROSPECTING 5. Potential strain for quinine and its related compound production Leaf, petiole, twig, bark, root, flower and fruit samples Appendix 2.1 Cultural characteristics of the endophytic fungi from C. calisaya on PDA Isolate code Size cm of colony Shape Colour Elevation Texture Mycelium Edge Density Zonallity Effect on medium after 7 days Above Reverse 4-7-1-C2-M1 4.3 Circular White White Flat Felty Immersed Entire Medium − − 1-7-1-A1-M2 3.3 Circular Grey Grey Flat Cottony Aerial Entire Medium − − 3-7-2-D2-M3 4.6 Circular White Cream Raised Cottony Immersed Entire Dense − − 5-7-4-D1-M4 4.7 Circular Greyish Grey Flat Velvety Immersed Entired Dense − − 1-7-5-D2-M5 4 Circular Orange Orange Flat Velvety Immersed Undulate Medium − Yellow 2-1-3-A4-M6 4.5 Circular Pale Grey Cream Flat Velvety Immersed Entire Medium − − 3-3-5-D3-M7 3.5 Circular Cream Cream Flat Velvety Immersed Entire Medium − − 4-2-1-B1-M8 3.3 Circular White White Flat Cottony Aerial Entire Dense − − 3-3-5-C1-M9 4.2 Circular White Pale yellow Flat Velvety Immersed Undulate Medium − Yellow 1-7-1-B2-M10 4.8 Circular Cream Orange Flat Cottony Aerial Entire Dense − − 4-2-1-A1-M11 4.8 Circular Cream Pale orange Flat Cottony Aerial Entire Dense − Yellow 3-3-2-D4-M12 4.8 Circular with White Pale orange Raised Cottony Aerial Entire Dense − − Concentric radials 5-3-1-C3-M13 3.4 Circular Pale grey Dark grey Flat Velvety Immersed Undulate Medium − Brown 5-3-1-D3-M14 4 Circular with White Pale yellow Flat Velvety Immersed Entire Dense − Pale yellow Concentric radials 2-3-4-A3-M15 4.8 Circular with Cream Cream Flat Cottony Aerial Entire Dense − − Concentric radials 1-4-2-A2-M16 2.6 Circular Pale purple Dark purple Raised Powdery Aerial Entire Dense − − 3-1-1-A2-M17 4.8 Circular Cream Cream Flat Powdery Immersed Entire Dense − − 5-2-2-C1-M18 2.4 Circular Cream Black Raised Cottony Immersed Entire Spare − − 1-7-4-B2-M19 4.3 Circular Maroon Maroon Flat Powdery Immersed Entire Dense − Marron 5-1-5-B1-M20 4 Circular Cream Orange Flat Felty Immersed Entire Dense − Orange 1-7-5-B1-M21 4.8 Circular White White Flat Cottony Aerial Entire Dense − Light brown 5-1-2-A1-M22 2.8 Irregular with concentric radials Cream cream Raised Cottony Immersed Undulate Medium − - 1-3-1-A1-M23 3.5 Circular with Cream Cream Flat Felty Aerial Undulate Dense − − Concentric radials 1-3-3-C3-M24 3.7 Circular Greyish white Blackish grey Flat Velvety Aerial Undulate Medium − − 4-7-2-B2-M25 4 Circular Dark grey Black Raised Velvety Aerial Entire Dense − − 2-5-5-C1-M26 4.8 Circular Dark green Cream Flat Powdery Immersed Entire Dense − − 8 4 Continue… Isolate code Size cm of colony Shape Colour Elevation Texture Mycelium Edge Density Zonallity Effect on medium after 7 days Above Reverse 2-3-3-B4-M27 4.2 Irregular White Blackish orange Flat Velvety Immersed Undulate Dense − Light-yellow 2-7-2-C3-M28 3.4 Circular Raised Cottony Aerial Entire Dense − − 5-2-5-C1-M29 3.7 Circular Black Black Raised Powdery Aerial Entire Dense − − 1-7-2-A4-M30 2.5 Circular White cream to black Raised Velvety Immersed Entire Spare − − 1-7-2-C3 –M31 3 Circular with concentric radials White Orange Umbonate Cottony Aerial Undulate Medium − Light yellow 1-3-2-C1-M32 3.2 Circular Ornamented Grey Black Umbonate Velvety Aerial Undulate Medium + − 4-3-1-C2-M33 4.2 Circular Cream Orange Flat Velvety Aerial Undulate Dense - − 4-2-2-C1-M34 4 Circular with concentric radials Pink to white Pink to white Flat Cottony Aerial Entire Dense − − 2-2-2-B1-M35 4.7 Circular Black Black Raised Powdery Immersed Entire Dense − − 3-1-1-A7-M36 4.7 Circular Cream Orange Raised Cottony Aerial Entire Dense − − 5-4-2-A3-M37 4.8 Circular White Light yellow Raised Cottony Aerial Entire Dense − − 2-3-1-B1-M38 4.8 Circular Cream Orange Raised Velvety Aerial Entire Dense − − 1-3-4-B3-M39 3.7 Irregular White Dark brown Umbonate Velvety Aerial Undulate Medium − Yellow 1-3-5-A1-M40 3.8 Circular White Orange Raised Velvety Aerial Undulate Medium − Pale yellow 5-3-3-A2-M41 2.8 Circular White Cream to orange Raised Cottony Aerial Entire Spare − 3-3-1-B4-M42 2.7 Circular Cream Cream Flat Velvety Immersed Entire Spare − − 3-1-1-A6-M43 3.2 Irregular Cream Dark brown Flat Cottony Aerial Entire Spare − Yellow 2-1-3-D1-M44 4.6 Circular Light grey Orange Flat Velvety Immersed Entire Dense − Brown 2-1-1-A3-M45 3 Circular with spot yellow Light grey Cream to orange Flat Velvety Aerial Entire Medium − − 1-3-1-A3-M46 2.6 Irregular with concentric radials Cream Blackish orange Flat Velvety Aerial Entire Spare − − 5-1-1-B2-M47 3 Circular Greenish Cream Flat Cottony Aerial Entire Medium − Brown 3-3-2-D2-M48 3 Circular White Cream to orange Flat Cottony Aerial Entire Medium − − 1-5-4-B2-M49 3.5 Circular Light brown Dark brown Umbonate Fluffy Aerial Entire Dense − Yellow 4-2-1-A2-M50 3.6 Irregular Maroon Maroon Flat Velvety Immersed Undulate Medium − Maroon 1-7-4-B4-M51 3.8 Irregular Cream Light yellow Flat Velvety Immersed Undulate Medium − Yellow 1-1-5-A5-M52 2.3 Circular Cream Black Raised Velvety Immersed Entire Spare − − 3-5-2-B1-M53 3.7 Circular Grey Black Flat Velvety Aerial Entire Dense − − 3-7-4-D1-M54 3 Circular Cream Brown to black Flat Powdery Immersed Undulate Medium − Brown 8 5 Continue. .. Isolate code Size cm of colony Shape Colour Elevation Texture Mycelium Edge Density Zonallity Effect on medium after 7 days Above Reverse 4-7-4-C1-M55 3.5 Circular with concentric radials White Orange to black Flat Velvety Immersed Undulate Medium − − 1-7-4-C3-M56 2.7 Irregular Light brown Orange Flat Powdery Immersed Undulate Medium − Brown 3-7-4-C3-M57 3 Circular Light grey Cream Raised Powdery Aerial Entire Dense − − 2-3-4-B2-M58 1.8 Circular Grey Greyish brown Flat Velvety Immersed Entire Spare − − 3-3-1-A1-M59 2.8 Circular Grey Blackish orange Flat Cottony Aerial Dentate Spare − − 3-4-4-B1-M61 2.5 Circular White Orange to brown Flat Velvety Immersed Entire Spare − Light Yellow 1-2-4-B2-M62 3.4 Circular Cream Black Umbonate Felty Immersed Entire Medium − Yellow 1-4-1-A1-M63 1.8 Irregular White White Raised Velvety Immersed Undulate Spare − − 2-5-4-D2-M64 2.2 Circular White Black Flat Felty Immersed Entire Spare − − 1-4-2-D2-M65 2.2 Circular White Light grey Flat Felty Immersed Entire Spare − − 1-7-3-B1-M67 3.2 Irregular Orangish white Orangish white Flat Cottony Aerial Undulate Dense − − 4-4-1-A1-M68 2.3 Circular White White Raised Velvety Immersed Entire Spare − − 1-7-2-C3-M69 3 Circular White Orange to brown Raised Velvety Aerial Entire Medium − − 2-3-3-D4-M70 3.8 Circular Pale grey Orange flat Velvety Immersed Undulate Medium − − 5-3-3-D2-M71 4.6 Irregular White Light orange with black spot Flat Felty Immersed Curied Dense − − 1-3-5-A3-M72 3.5 Circular White Light brown Flat Cottony Aerial Entire Medium − − 5-3-5-C1-M73 3.8 Circular White to grey Orange to black Flat Cottony Aerial Undulate Medium − − 3-3-2-D2-M74 2.5 Circular Cream Brown Umbonate Velvety Immersed Undulate Spare − Orange 1-4-1-A3-M76 4.3 Circular White to dark purple Orange Flat Velvety Immersed Entire Medium − − 1-1-1-A5-M77 2.2 Circular White Light orange Umbonate Cottony Aerial Undulate Spare − − 4-3-3-A4-M78 4.8 Circular Grey Black Flat Velvety Immersed Undulate Medium − − 2-2-2-B1-M79 4.2 Circular White Orange to brown Flat Velvety Immersed Entire Medium − − 1-7-5-B2-M80 4.3 Circular Blackisa grey Orange to black Flat Felty Immersed Entire Medium − − 3-4-4-C1-M81 3.3 Circula with radial concentric White Orange Flat Velvety Immersed Entire Medium − Light yellow 3-3-3-C2-M82 3.7 Circular White Orange Flat Velvety Immersed Lobate Medium − yellow 2-1-5-B2-M83 3.7 Circular Light grey Grey Flat Velvety Immersed Entire Medium − − 8 6 Continue... Isolate code Size cm of colony Shape Colour Elevation Texture Mycelium Edge Density Zonallity Effect on medium after 7 days Above Reverse 4-1-2-B1-M84 2.2 Iregular Brown Black Umbonate Powdery Immersed Undulate Spare − − 2-3-4-B5-M86 2.4 Circular with fimbriate Cream Black Flat Velvety Immersed Undulate Spare − − 5-6-3-A1-M87 4.3 Circular Cream Cream Flat Velvety Immersed Entire Medium − Orange 4-1-2-B2-M88 2.2 Iregular Brown Brown to black Flat Felty Immersed Undulate Spare − − 1-7-5-D1-M89 2.5 Circular Cream Light brown Flat Felty Immersed Entire Spare − − 4-3-5-D2-M90 3.5 Iregular with lobate border White to orange Orange to black Flat Cottony Immersed Lobate Medium − Orange 1-3-4-A4-M91 4 Circular Cream White to black Flat Velvety Immersed Lobate Medium − Orange 5-2-4-D1-M92 3.9 Circular with curcentric radial Greyish white Dark brown Raised Velvety Immersed Entire Medium − Orange 5-1-1-C2-M93 3.1 Circular Cream Brown Raised Cottony Aerial Entire Medium − Orange 4-3-5-A3-M94 3.7 Circular imbricate colony Brownish Brown to black Flat Cottony Aerial Curied Mediuim − Orange 3-2-4-A2-M95 4.6 Circular Reddish white Light brown Flat Velvety Immersed Entire Dense − Orange 5-3-3-C3-M96 4.2 Circular concentric radial Cream Light yellow Flat Velvety Immersed Undulate Dense − Yellow 1-3-4-B2-M97 4.5 Circular Cream Cream Flat Cottony Aerial Entire Medium _ − 3-3-1-A2-M98 4.7 Iregular Green Cream Flat Powdery Immersed Undulate Medium − − 3-5-4-A1-M99 2.7 Iregular Brown Brown Flat Velvety Immersed Entire Spare − − 8 7 Appendix 2.2 Colony of the endophytic fungi from C. calisaya on Potato Dextrosa Agar Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony 1 25 49 74 2 26 50 76 3 27 51 77 4 28 52 78 8 8 Continue… Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony 5 29 53 79 6 30 54 80 7 31 55 81 8 32 56 82 89 Continue … Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony 9 33 57 83 10 34 58 84 11 `35 59 85 12 36 61 87 90 Continue … Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony 13 37 62 88 14 38 63 89 15 39 64 90 16 40 65 91 91 Continue… Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony 17 41 66 92 18 42 67 93 19 43 68 94 20 44 69 95 Continue d… Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony Morphotype Upper side of the colony 21 45 70 96 22 46 71 97 23 47 72 98 24 48 73 99 Bar = 1 cm 93 Apendix 4. 1a Alkaloid profiles of endophytic fungi Diaporthe spp., Colletotrichum spp. and others endophytic fungi Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M9 M10 M11 M12 M13 M14 M15 M20 M21 M22 M23 M24 M31 M32 M33 0.1 - - - - - - - - - - - 0.4 0.4 - - - 15.31 0.86 1.98 0.2 1.09 - - - - - - - - - - - - - - - - - - 0.3 - - - - - - - - - - - - - - - - - - - 0.4 - - - - - - 0.01 - - - - - - - - - - - - 0.5 - - - - - - - - - - - - - - - - - - - 0.6 - - - - - - - - - - - - - - - - - - - 0.7 - - - - - - - - - - - - - - - - - 1.86

0.8 -

- 97.21 - -

0.74 -

- - - - - - - - - - - - 0.9 - - - 100 - - - 3.37 - - - - - - - - - - - 1.2 - - - - - - - - - - - - - - - - - - 1.3 - - - - - - - - - - - - - - - - - - - 1.4 94.17 - - - - - - 34.73 - - - - - - - - - 2.72 0.15 1.5 - - - - - - - - 11.73 - - - - - - - - - - 1.6 - - - - - - 9.46 - - - 22.25 22.25 - - - - 0.68 1.05 1.7 - - - - 32.29 4.86 - - - 40.08 - - - - - - - 15.32 - 1.8 - - - - - - 0.63 - 12.08 - - - - 18.95 - - - - -

1.9 -

- - - 24.44

1.32 -

46.65 -

- - 14.14 14.14

81.05 0.61

0.61 -

- 22.95 2 - - - - - - - - 11.27 - 30.76 - - - - - - - - 2.1 - - - - - - - - - - - - - - 2.53 2.53 84.69 35.51 - 2.2 - - - - 35.23 4.32 - - - - 28.29 - - - - - - - 8.43 2.3 - - - - - - - - - - - - - - - - - - - 2.4 - - - - - - - - 10.49 - - - - - 3.65 3.65 - - 7.21 2.5 2.57 - - - - - - - - - 40.95 - - - - - - - - 2.6 - - - - - - - - 5.05 - - 15.7 15.7 - - - - - - 2.7 - - - - - - - - 41.28 - - - - - - - - - - 2.8 - - - - - - - - - - - - - - 2.63 2.63 - - 58.22 2.9 - - - - - - - - - - - - - - - - - - - 3 - - - - - 9.26 - - - - - 38.06 38.06 - - - - - - 3.1 - - - - - - - - - - - - - - - - - - - 3.2 - - - - - - - - - - - - - - - - - - - 3.3 - - - - - 10.77 8.06 - - - - - - - - - - - - 3.5 - - - - - - 3.79 - - - - - - - - - - - - 3.6 - - - - - - - - - - - - - - - - - - - 3.7 - - - - - - - - - - - - - - - - - - - 3.8 - - - - - - - - - - - - - - - - - - -

3.9 -

- - - - 14.78 - 1.51 - - - - - - - - - - - Continue… Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M9 M10 M11 M12 M13 M14 M15 M20 M21 M22 M23 M24 M31 M32 M33 4 - - - - - - 3.31 - - - - - - - - - - - - 4.4 - - - - - - - - - - - - - - - - - - - 4.6 - - - - - - - - - - - - - - - - - - - 4.8 - - - - - - - - - - - - - - - - - - - 4.9 - - - - - - - - - - - - - - - - - - - 5 - - - - - 7.73 - - - - - - - - 89.46 89.46 - - - 5.1 - - - - - - - - - - - - - - - - - - - 5.2 - - - - - - - - - - - - - - - - - - - 5.3 - - - - 8.04 - - - - - - - - - - - - - - 5.4 - - - - - - - - - - - - - - - - - - - 5.5 - - - - - 5.12 - - - - - - - - - - - - - 5.6 - - - - - - - - - - - - - - - - - - - 5.7 - - - - - - 84.2 - - - - - - - - - - - - 5.8 - - - - - 5.12 - - - - - - - - - - - - - 5.9 - - - - - - - - - - - - - - - - - - - 6 - - - - - - - - - - - - - - - - - - - 6.1 - - - - - - - - - - - - - - - - - - - 6.2 - - - - - - - - - - - - - - - - - - - 6.3 - - - - - - - - - - - - - - - - - - - 6.4 - - - - - - - - 8.09 - - - - - - - - - - 6.5 - - - - - - - 3.8 - - - - - - - - - - - 6.6 - - - - - - - - - - - - - - - - - - - 6.7 - - - - - - - - - - - - - - - - - - - 6.9 - - - - - - - - - - - - - - - - - - - 7.2 - - - - - - - - - - - - - - - - - - - 7.3 - - - - - - - - - 27.37 - - - - - - - - - 7.5 - - 2.61 - - - - - - - - - - - - - - - - 7.6 - - - - - - - - - - - - - - - - - - - 7.7 - - - - - - - - - - - 9.45 9.45 - - - - - - 7.8 - - - - - - - - - - - - - - - - - - - 7.9 - - - - - - - - - - - - - - - - - - - 8 - - - - - - - - - - - - - - - - - - - 8.1 - - - - - - - - - - - - - - - - - - - 8.2 - - - - - - - - - - - - - - - - - - - 8.3 - - - - - - - - - - - - - - - - - - - 8.4 - - - - - - - - - - - - - - - - - - - 95 Continue… Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M9 M10 M11 M12 M13 M14 M15 M20 M21 M22 M23 M24 M31 M32 M33 8.5 - - - - - - - - - - - - - - - - - - - 8.6 - - - - - - - - - - - - - - - - - - - 8.8 - - - - - - - - - - - - - - - - - - - 8.9 - - - - - - - - - - - - - - - - - 43.05 - 9 - - - - - - - - - - - - - - - - - - - 9.7 - - - - - - - - - - - - - - - - - - - 9.8 - - - - - - - - - - - - - - - - - - - Continue… Area Rt Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M36 M37 M38 M39 M40 M41 M42 M43 M44 M46 M48 M52 M54 M56 M59 0.1 - - - - 0.04 0.36 0.37 5.65 6.55 0.97 - 4.98 1.16 - 0.19 - - - 1.4 0.2 1.09 - - - - - - - - - - - - - - - - 0.58 - 0.3 - - - - - - - - - 1.38 - - - - - - - - - 0.4 - - - - - - - - - - - - - - - - - - - 0.5 - - - - - - - - - - - - - - - - - - - 0.6 - - - - - - - - - - - - - - - - - 0.67 - 0.7 - - - - - - - - - - - - - - - - 3.08 - - 0.8 - -

97.21 -

- - - - - - - - - - - - - - - 0.9 - - - 100 - - - - - - - - - - - - - - - 1.2 - - - - - - - - - - - - - - - - - - - 1.3 - - - - - - - - - - - - - - 27.1 - - - - 1.4

94.17 -

- - - 14.91 - 0.11 - - - - - - - - - - - 1.5 - - - - 28.44 - - 0.59 0.21 - - - 16.48 - - - 96.94 0.24 0.81 1.6 - - - - - - 9.36 - - - - - - - - - - - - 1.7 - - - - - 27.85 11.16 - 7.52 - - - - 0.03 8.58 - - 1.5 - 1.8 - - - - - 9.52 - - - 24.82 21 74.24 - - 19.99 - - - 29.06 1.9 - - - - - 20.81 - 93.66 - 72.83 - - - - - - - 2.4 - 2 - - - - - - - - 4.26 - - 20.78 14.19 - - - - 6.43 - 2.1 - - - - - - - - - - - - 9.48 0.8 - - - - 27.1 2.2 - - - - - - - - 22.48 - - - - - 11.26 - - 5.86 - 2.3 - - - - - - - - - - - - - - - - - - - 2.4 - - - - - - - - - - - - 4.26 - - - - - - Continue… Area Rt Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M36 M37 M38 M39 M40 M41 M42 M43 M44 M46 M48 M52 M54 M56 M59 2.5 2.57 - - - - 5.32 - - - - 7.97 4.62 - - - - - 41.64 2.6 - - - - - - - - - - - - - - - - - - - 2.7 - - - - - 10.55 - - 10.86 - - - 16.65 74.76 - - - - - 2.8 - - - - - - - - - - - - - - - - - - - 2.9 - - - - - - - - - - - - - - - - - - - 3 - - - - - - - - - - 3.75 - - - - - - - - 3.1 - - - - - 10.47 - - - - - - - - - - - 81.77 - 3.2 - - - - - - - - - - - - - - 10.61 - - - - 3.3 - - - - - - - - - - - - - - - - - - - 3.5 - - - - - - - - - - - - - - - - - - - 3.6 - - - - - - - - - - - - - - - - - - - 3.7 - - - - - - - - - - - - - - - - - - - 3.8 - - - - - - - - 0.25 - - - - - - - - - -

3.9 -

- - - - - - - - - - - - 23.17 - - - - - 4 - - - - - - - - - - - - - - - - - - - 4.2 - - - - - - - - - - 45.27 - - - - - - - - 4.3 - - - - - - - - - - - - - - - - - - - 4.4 - - - - - - - - - - - - - - - - - - - 4.6 - - - - - - - - - - - - - - - - - - - 4.8 - - - - 71.52 - - - - - - - - - - - - - - 4.9 - - - - - - - - - - - - - - - - - - - 5 - - - - - - - - - - - - - - - - - - - 5.1 - - - - - - - - - - - - - - - - - - - 5.2 - - - - - - - - - - - - 25.41 - - - - - - 5.3 - - - - - - - - - - - - - - - - - - - 5.4 - - - - - - - - - - - - - - - - - - - 5.5 - - - - - - - - - - - - - - - - - - - 5.6 - - - - - - - - - - - - - - - - - - - 5.7 - - - - - - - - - - - - - - - - - - - 5.8 - - - - - - - - - - - - - - - - - - - 5.9 - - - - - - - - - - - - - - - - - - - 6 - - - - - - - - - - - - - - 22.24 - - - - 6.1 - - - - - - - - - - - - - - - - - - - 6.2 - - - - - - - - - - - - - - - - - - - 6.3 - - - - - - - - - - - - - - - - - - - 6.4 - - - - - - - - - - - - - - - - - - - 97 Continue… Area Rt Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M36 M37 M38 M39 M40 M41 M42 M43 M44 M46 M48 M52 M54 M56 M59 6.5 - - - - - - - - - - - - - - - - - - - 6.6 - - - - - - - - - - - - - - - - - - - 6.7 - - - - - - - - - - - - - - - - - - - 6.9 - - - - - - - - - - - - 7.76 - - - - - - 7.2 - - - - - - - - - - - - - 0.95 0.01 - - - - 7.3 - - - - - - - - - - - - - - - - - - - 7.5 - - 2.61 - - - - - - - - - - - - - - - - 7.6 - - - - - - - - - - - - - - - - - - - 7.7 - - - - - - - - - - - - - - 0.02 - - - - 7.8 - - - - - - - - - - - - - - - - - - - 7.9 - - - - - - 79.1 - - - - - - - - - - - - 8 - - - - - - - - - - - - - 0.29 - - - - - 8.1 - - - - - - - - - - 22.01 - - - - - - - - 8.2 - - - - - - - - - - - - - - - - - - - 8.3 - - - - - - - - - - - - - - - - - - - 8.4 - - - - - - - - - - - - - - - - - - - 8.5 - - - - - - - - - - - - - - - - - - - 8.6 - - - - - - - - - - - - - - - - - 0.54 - 8.8 - - - - - - - - - - - - - - - - - - - 8.9 - - - - - - - - - - - - - - - - - - - 9 - - - - - - - - 47.86 - - - - - - - - - - 9.7 - - - - - - - - - - - - - - - - - - - 9.8 - - - - - - - - - - - - - - - - - - - Continue… Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M65 M68 M69 M70 M71 M72 M73 M74 M77 M78 M79 M81 M83 M84 M88 0.1 - - - - - - - - - - 0.65 - 7.77 - 4.03 2.82 - - - 0.2 1.09 - - - - - - 0.71 - 0.91 - 0.05 - - - - - - - 0.3 - - - - - - - - - - - - - - - - - - - 0.4 - - - - - - - - - - - - - - - - - - - 0.5 - - - - 14.93 - - - 4.58 - - - - - - - - - - 0.6 - - - - - - - - - - - - - - - - - - - 0.7 - - - - - - - - - - - - - - - - - 0.58 - 98 Continue… Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M65 M68 M69 M70 M71 M72 M73 M74 M77 M78 M79 M81 M83 M84 M88 0.8 - -

97.21 -

- - - - - - - - - - - - - - - 0.9 - - - 100 - - - - - - - - - 0.1 - - - - - 1.2 - - - - - - - - - - - - - 0.8 - - - - - 1.3 - - - - - 12.52 - 0.11 - - - 9.22 0.4 - 0.29 - - 0.02 1.4 94.17 - - - - - - 48.22 0.01 - 20.11 17.26 - - 68.86 - - - 1.5 - - - - 4.52 - 8.1 - - 4.39 30.68 2.23 0.11 - - - - - 1.6 - - - - - - - - - - - - 5.97 - - - - 0.38 0.02 1.7 - - - - - 26.31 - 11.21 2.13 - - 7.71 - 37.89 - - - 0.43 0.96 1.8 - - - - 30.46 15.37 - 9.5 - - - - 26.14 - 10.09 - - - -

1.9 -

- - - 21.8 - 15.14 26.87 - 14.93 5.97 - - 15.08 - 3.34 - 3.51 0.41 2 - - - - - - - - 1.2 - 24.58 - 42.46 - - 1.33 - - - 2.1 - - - - - - 14.38 - - - - - - - - - - 95.1 - 2.2 - - - - - 24.29 - - - - - - - - 85.59 - - - - 2.3 - - - - - - - - - 6.96 - - - - - - - - 0.77 2.4 - - - - - - - - - - 0.37 0.27 - - - - - - - 2.5 2.57 - - - 9.98 - 9.48 - - - - - - - - - - - - 2.6 - - - - - - 50.34 2.94 - - - - - - - - - - - 2.7 - - - - - - - - - - - - - - - - - - - 2.8 - - - - - 3.6 - - - - - - - 9.42 - - - - - 2.9 - - - - - - - - - - - - - - - - - - - 3 - - - - - - - - - 16.97 3.66 - - - - - - - - 3.1 - - - - - - - 0.43 - - - - - 8.51 - - - - 41.38 3.2 - - - - 5.03 - - - - - - 1.7 - - - - - - - 3.3 - - - - - - - - - - - 0.44 - - - - - - - 3.5 - - - - - - - - - - - - - - - - - - 3.6 - - - - - - - - 33.91 - - 0.06 - - - - - - - 3.7 - - - - - - - - - - - 0.06 - - - - - - - 3.8 - - - - - 4.93 - - - - - - - - - - - -

3.9 -

- - - - - - - - - - - - - - - - - 4 - - - - - - - - - - - 0.48 - 6.52 - - - - - 4.2 - - - - - - - - - - - 0.28 - - - - - - 0.15 4.3 - - - - - - - - - - - 0.12 - - - - - - - 4.4 - - - - - - - - - 53.53 - 0.3 - - - - - - - 4.6 - - - - - - - - - - - - - - - - - - 0.46 4.8 - - - - 6.19 - - - - - - 2.5 - - - - - - - 4.9 - - - - - - - - - - - 0.91 - - - - - - - 99 Continue… Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M65 M68 M69 M70 M71 M72 M73 M74 M77 M78 M79 M81 M83 M84 M88 5 - - - - - - - - - - - 1.49 - - - 23.65 - - 0.12 5.1 - - - - - - - - - - - 1.04 - - - - - - - 5.2 - - - - - - - - - - - 1.75 - - - - - - - 5.3 - - - - - - - - - - - 0.79 - 6.23 - - - - - 5.4 - - - - - - - - - - - 1.64 - - - - - - - 5.5 - - - - - - - - - - - 2.07 - - - - - - - 5.6 - - - - - - - - - - - 2.29 - - - - - - - 5.7 - - - - - - - - - - 5.98 2.3 - - - - - - - 5.8 - - - - - - - - - - - 1.22 - - - - - - - 5.9 - - - - - - - - - - - 3.62 - - - - - - - 6 - - - - - - - - - - - 1.79 - - - - - - - 6.1 - - - - 7.09 - - - - - - - - - - - - - - 6.2 - - - - - 4.72 - - - - - 5.74 - - - - - - - 6.3 - - - - - - - - - - - 1.56 - - - - - - - 6.4 - - - - - - - - - 2.32 - - - - - - - - - 6.5 - - - - - - - - - - - - - - - - - - - 6.6 - - - - - - - - - - - - - - - - - - - 6.7 - - - - - - - - - - - 42.56 - - - - - - - 6.9 - - - - - - - - - - - - - - - - - - - 7.2 - - - - - - - - - - - - - - - - - - 8.45 7.3 - - - - - - - - - - - - - - - - - - - 7.5 - - 2.61 - - - - - 58.18 - - - - - - - - - - 7.6 - - - - - - 0.89 - - - - - - - - - - - - 7.7 - - - - - - - - - - - 2.37 - - - - - - - 7.8 - - - - - - - - - - - - - - - - - - - 7.9 - - - - - - - - - - - - - - - - - - - 8 - - - - - - - - - - - 0.47 - - - - - - - 8.1 - - - - - - - - - - - 0.07 - - - - - - - 8.2 - - - - - - - - - - - - - - - - - - - 8.3 - - - - - - - - - - - - - - - - - - - 8.4 - - - - - - - - - - - 0.03 - - - - - - - 8.5 - - - - - - - - - - - 0.09 - - - - - - - 8.6 - - - - - - - - - - - - - 2.52 - - - - - 8.8 - - - - - - - - - - - 0.02. 0.08 - - - - - - 48.24 8.9 - - - - - - - - - - - - - - - - - - - Continue… Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M65 M68 M69 M70 M71 M72 M73 M74 M77 M78 M79 M81 M83 M84 M88 9 - - - - - - - - - - - 0.09 - - - - - - - 9.7 - - - - - - - - - - - 0.04 - - - - - - - 9.8 - - - - - - 1.67 - - - - 0.07 - - - - - - - Continue… Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M89 M90 M91 M92 M94 M95 M96 0.1 - - - - 4.67 1.98 2.01 1.15 6.25 - 2.45 0.2 1.09 - - - - - - - - 0.77 - 0.3 - - - - - - - - - - - 0.4 - - - - - - - - - - - 0.5 - - - - - - - 5.48 - - - 0.6 - - - - - - - - - - - 0.7 - - - - - - - - - - - 0.8 - -

97.21 -

- - - - - - - 0.9 - - - 100 - - - - - - - 1.2 - - - - - - - - - - - 1.3 - - - - - - - - - - 0.67 1.4

94.17 -

- - - 0.15 1.62 - - - 26.07 1.5 - - - - 0.64 - 5.49 19.78 - 34.98 - 1.6 - - - - 2.43 1.05 - - 0.44 - - 1.7 - - - - - - - - 1.38 12.42 70.81 1.8 - - - - - - - 55.42 0.22 - - 1.9 - - - - - 22.95 - - - 40.04 - 2 - - - - 20.69 - 80.92 - - - - 2.1 - - - - - - - - - - - 2.2 - - - - 39.77 8.43 - - 52.47 - - 2.3 - - - - - - - - - - - 2.4 - - - - - 7.21 9.96 - - - - 2.5 2.57 - - - - - - - - - - 2.6 - - - - - - - - - - - 2.7 - - - - - - - - 7.72 - - 2.8 - - - - 12.35 58.22 - 7.24 - - - 101 Continue… Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M89 M90 M91 M92 M94 M95 M96 2.9 - - - - - - - - - - - 3 - - - - - - - - 26.35 - - 3.1 - - - - 19.43 - - - - - - 3.2 - - - - - - - - - - - 3.3 - - - - - - - - - - - 3.5 - - - - - - - - - - - 3.6 - - - - - - - - - - - 3.7 - - - - - - - - 1.91 - - 3.8 - - - - - - - - - - - 3.9 - - - - - - - - - - - 4 - - - - - - - - - - - 4.2 - - - - - - - - - - - 4.3 - - - - - - - - - - - 4.4 - - - - - - - - - - - 4.6 - - - - - - - - - - - 4.8 - - - - - - - - - - - 4.9 - - - - - - - - 3.01 - - 5 - - - - - - - - - - - 5.1 - - - - - - - - - - - 5.2 - - - - - - - - - - - 5.3 - - - - - - - - - - - 5.4 - - - - - - - - - - - 5.5 - - - - - - - - - - - 5.6 - - - - - - - - - - - 5.7 - - - - - - - - - - - 5.8 - - - - - - - - - - - 5.9 - - - - - - - - - - - 6 - - - - - - - - - 1.3 - 6.1 - - - - - - - - - - - 6.2 - - - - - - - - - 0.33 - 6.3 - - - - - - - - - 0.28 - 6.4 - - - - - - - - - 0.89 - 6.5 - - - - - - - - - - - 6.6 - - - - - - - - 0.26 0.6 - 6.7 - - - - - - - - - - - 6.9 - - - - - - - - - 1.55 - Continue… Rt Area of detected alkaloids Standard Diaporthe strains Quinine Quinidine Cinchonine Cinchonidine M89 M90 M91 M92 M94 M95 M96 7.2 - - - - - - - - - 0.4 - 7.3 - - - - - - - - - - - 7.5 - - 2.61 - - - - - - 0.61 - 7.6 - - - - - - - - - 0.6 - 7.7 - - - - - - - - - 0.55 - 7.8 - - - - - - - - - 0.69 - 7.9 - - - - - - - - - 0.42 - 8 - - - - - - - - - 0.29 - 8.1 - - - - - - - - - 0.19 - 8.2 - - - - - - - - - 0.17 - 8.3 - - - - - - - - - 0.39 - 8.4 - - - - - - - - - 0.35 - 8.5 - - - - - - - - - 0.24 - 8.6 - - - - - - - - - 0.16 - 8.8 - - - - - - - - - 0.19 - 8.9 - - - - - - - - - 0.33 - 9 - - - - - - - - - 0.15 - 9.7 - - - - - - - - - - - 9.8 - - - - - - - - - - - b. Alkaloid profiles of endophytic Colletotrichum spp. Rt Area detected alkaloids Standard Colletotrichum strains Quinine Quinidine Cinchonine Cinchonidine M1 M2 M3 M4 M5 M6 M7 M30 M53 M57 M76 M82 M45 0.1 2.17 - - - 6.81 - - - 0.31 - - - 2.18 - - - - 0.2 - - - - - 0.27 10.23 - - - - - - - 1.51 14.55 - 0.3 1.09 - - - - - - - - - - - - 1.81 - - - 0.7 - - - - 0.03 - - - - - 29.67 - - - - - - 0.8 - -

97.21 -

- - - - - - - - - - - - - 0.9 - - - 100 - - - - - - - - - - - - 0.09 1.0 - - - - 0.03 - - - - - - - 5.24 - - - - 1.2 - - - - 0.04 - - - - - - - - - - - - 1.3 - - - - - - - 1.08 - - - 6.18 - - - - 103 Continue… Rt Area detected alkaloids Standard Colletotrichum strains Quinine Quinidine Cinchonine Cinchonidine M1 M2 M3 M4 M5 M6 M7 M30 M53 M57 M76 M82 M45 1.4 - - - - - - - - - - - - - - - - 1.5 - - - - 1.70 1.17 - - 2.62 - 0.20 - 17.64 - 31.55 0.15 - 1.6 - - - - - - - 7.81 - - - 100.00 - - - - - 1.7 - - - - 46.19 - 19.32 - 0.87 49.15 - - 50.65 41.26 4.49 46.96 - 1.8 - - - - - 45.31 - 2.40 - - 69.30 - - - 23.27 - -

1.9 94.17

- - - 37.80 - 2.33 - 1.64 - - - - - - - - 2.0 - - - - - - - 2.93 3.42 - - - - - - - 0.09 2.1 - - - - - - 33.46 - - - - - - 26.19 - - - 2.2 - - - - - - 15.43 0.92 - 37.13 - - - - 11.79 26.37 - 2.3 - - - - 2.84 29.68 - 3.50 - - - - - - - - - 2.4 - - - - 2.89 - - - - - - - - - - - - 2.5 - - - - - - - - - - - - - - 6.04 - - 2.6 - - - - 0.98 - - - - 7.74 0.83 - - - - - 2.67 2.7 - - - - - - - - - - - - - - 8.70 - - 2.8 - - - - - - - - - - - - - 7.84 - - - 2.9 - - - - - - - - - - - - - - 12.65 - 17.02 3.0 2.57 - - - - - 1.27 - - - - - - - - - -

3.9 -

- - - - - - - - - - - - - - - - 7.5 - - 2.61 - - - - - - - - - - - - - - C. Alkaloid profiles of other endophytic Rt Area detected alkaloids Standard Others strains Quinine Quinidine Cinchonine Cinchonidine M19 M27 M62 M18A M18B M25 M34 M66 M62 M67 M93 M97 0.1 2.17 - - - 3.42 - 2.03 - - 2 39.19 2.00 - - - - 0.2 - - - - - - - - 1.26 - 1.26 - - - - 0.3 1.09 - - - - - - - - - - - - - - - 0.4 - - - - 0.69 - - - - - - - - - - - 0.5 - - - - - - - - - - - - - - - 4.08 0.6 - - - - - - - - - - - - 14.93 - - - Continue… Rt Area detected alkaloids Standard Others strains Quinine Quinidine Cinchonine Cinchonidine M19 M27 M62 M18A M18B M25 M34 M66 M62 M67 M93 M97

0.8 -

- 97.21 - - - - - - - - - - - - -

0.9 -

- - 100 - - - - - - - - - - - - 1.3 - - - - - - - 38.25 38.25 - - - - - - 0.06 1.4 - - - - - - - - - - - - - - - - 1.5 - - - - 31.80 2.7 5.1 5.10 - - - - - - - 1.6 - - - - - 5.68 - 4.8 4.8 - - - 4.52 - - 1.20 1.7 - - - - - 5.07 - 7.02 7.02 - - - - - 37.36 35.77 1.8 - - - - 15.57 - 7.79 7.7 - - - - - - -

1.9 94.17

- - - -

38.87 -

6.66 6.6

96.54 60.81

96.54 30.46

- - 37.01 2 - - - - - - - 30.41 3.41 - - - 21.8 - - - 2.1 - - - - 26.43 - - - - - - - - - - 13.86 2.2 - - - - - - - - - - - - - - 24.77 - 2.3 - - - - - - - - - - - - - - - - 2.4 - - - - - - - - - - - - - - - - 2.5 - - - - - - - - - - - - - - - 2.6 - - - - - - - - - - - 9.98 - - - 2.7 - - - - - - - - - - - - - - 2.8 2.57 - - - - - - - - - - - - - - 2.9 - - - - - 25.47 - - - - - - - - - 8.03 3 - - - - - - - - - - - - - - - 3.1 - - - - - - - - - - - - 5.03 - - - 3.2 - - - - 17.57 - - - - - - - - - - - 3.3 - - - - - - 95.27 - - - - - - - - - 3.5 - - - - - - - - - 0.13 - - - - - - 105 105 Continue… Rt Area detected alkaloids Standard Others strains Quinine Quinidine Cinchonine Cinchonidine M19 M27 M62 M18A M18B M25 M34 M66 M62 M67 M93 M97 3.6 - - - - 0.25 - - - - - - - - - - - 3.7 - - - - - - - - - - - - 6.19 - - - 3.8 - - - - - - - - - - - - - - - - 3.9 - - - - - - - - - - - - - - - - 4 - - - - - - - - - - - - - - - - 4.2 - - - - - - - - - 0.01 - 0.01 7.09 - - - 4.3 - - - - - - - - - - - - - - - - 4.4 - - - - - - - - - 0.01 - 0.01 - - - - 4.6 - - - - - - - - - 0.01 - 0.01 - - - - 4.8 - - - - 4.2 - - - - - - - - - 4.9 - - - - - - - - - 0.01 - 0.01 - - - - 5 - - - - - - - - - 0.01 - 0.01 - - - - 5.1 - - - - - - - - - 0.03 - 0.02 - - - - 5.2 - - - - - - - - - - - - - - 37.87 - 5.3 - - - - - - - - - - - - - - - - 5.4 - - - - - - - - - - - - - - - - 5.5 - - - - - - - - - - - - - - - - 5.6 - - - - - - - - - - - - - - - - 5.7 - - - - - - - - - - - - - - - - Continue… Rt Area detected alkaloids Standard Others strains Quinine Quinidine Cinchonine Cinchonidine M8 M16 M49 M83 M87 M17 M51 M50 M95 M35 M29 M26 M98 M35 0.1 2.17 - - - 0.55 - - - 6.9 59.91 1.3 - - - - - - 0.04 0.2 - - - - - - - - - - - - - - - - - 0.3 1.09 - - - - - - - - - - - - - - - - - 0.4 - - - - - - - - - - - - 10.68 - - - - - 0.5 - - - - - - - - - - - - - - - - - - 0.6 - - - - - 2.12 - - - - - - - - - - - -

0.8 -

- 97.21 - - - - - - - - - - - - - - -

0.9 -

- - 100 - - - - - - - - - - - - - 1.3 - - - - - - - - - - - - 0.07 - - - - - 1.4 - - - - - - - - - - 7.3 - - - - - - - 1.5 - - - - 18.19 22.34 32.69 1.91 2.2 - 49.7 43.16 0.63 5.71 - - - 28.44 1.6 - - - - - - 67.31 - - - - - - - - - 0.03 - 1.7 - - - - 3.48 13.51 - 4.65 - - - - - 10.81 - - - - 1.8 - - - - - 6.78 - - - - - 26.36 20.12 - - - - -

1.9 94.17

- - - 13.13

18.54 -

48.35 62.29

31.07 6.4

- -

11.98 -

48.3 -

- 2 - - - - - - - - - - - - - - - - - - 2.1 - - - - 5.14 - - 21.28 - - 36.06 - - - - - - - 2.2 - - - - - - - - - - - - 28.14 16.87 - - - - 2.3 - - - - 11.75 - - - 11.84 - - 28.65 - - 100.00 - - - 2.4 - - - - - - - 14.97 - - - - - - - - - - 2.5 - - - - - 1.76 - - - - - - 6.09 - - - - - 2.6 - - - - 14.81 - - - - - - - - - 11.3 8 - - 2.7 - - - 9.47 - - 1.09 - 9.4 - - - - - - - - 107 Continue… Rt Area detected alkaloids Standard Others strains Quinine Quinidine Cinchonine Cinchonidine M8 M16 M49 M83 M87 M17 M51 M50 M95 M35 M29 M26 M98 M35 2.8 2.57 - - - - - - - - - - - - - - - 69.45 - 2.9 - - - - - - - - 16.75 - - - - - - - - - 3.1 2.17 - - - - - - - - - - - - 7.3 - - - - 3.3 - - - - - - - - - - 0.02 - - - - - - 3.9 1.09 - - - - - - - - - - - - - - - - - 4.5 - - - - - - - - - - - - - - - - 30.49 - 4.6 - - - - - - - - - - - - - - - - - - 4.8 - - - - - - - - - - - - - - - - - - 4.9 - -

97.21 -

- - - - - - - - - - - - - - 5.2 - - - 100 - - - - - - - - - 47.32 - - - - 5.7 - - - - - 2.70 - - - - - - - - - - - - 6.1 - - - - - - - - - - - - - - - - - - 6.2 - - - - - - - - - - - - - - - - - - 6.3 - - - - - - - - - - - - - - - - - 71. 52 6.5 - - - - - - - - - - - - - - - - - - 6.6 - - - - - - - - - - - - - - - 38.7 6 - - 6.7

94.17 -