Diversity Of Plant Endophytic Pestalotiopsis In Hainan And Molecular Phylogenetics Of Pestalotiopsis

More than two hundred Pestalotiopsis species have been recorded, most of that were repoted as plant pathogens. However, Pestalotiopsis is a major group of endophytic fungi. More than fourty six species of Pestalotiopsis have been recorded as endophytes since Espinosa-Garcia et al. firstly reported Pestalotiopsis funerea as an endophyte on Sequoia sempervirens inl990.As a part work of the research projects of "The investigation of resource and diversity of endophytic Pestalotiopsis and its taxonomic study" and "The molecular systematics of Pestalotiopsis and allied genera" supported by National Natural Science Foundation of China, the investigation on diversity of plant endophytic Pestalotiopsis in Hainan was carried out, and the taxonomy and molecular phylogenetics of the genus of Pestalotiopsis were studied.1 Endophytic Pestalotiopsis species in HainanTotaly 43 Pestalotiopsis species were isolated and identified from 95 host plant species belonging to 42 families, of which Pestalotiopsis hainanensis as a new endophyte and six species formerly disposed in the genus Pestalotia were illustrated and recombined. Concurrently, 16 Pestalotiopsis species are new records as entophytes,.1.1 The new species and new combinations of Pestalotiopsis1) Pestalotiopsis hainanensis A.R. Liu, T. Xu et L.D. Guo. sp. nov. Fungal Diversity, 2006. (in press)Colonies on PDA white, cottony, margin nearly round;acervuli developed in mycelia and gave rise to black spore mass, punctate, discrete, and developed on the carnation leaves on PDA, scattered, irregular 50.7-221 μm (x|- = 102.9 μm) in diam. Conidia fusiform, erect or slight curving, 5-celled, 19-22 × 5-6 μrn, slight constricted at septa;intermediate coloured cells subcylindrical, thick-walled, smooth, usually approximate concolorous, together 13-15 μrn long;exterior hyaline cells small, trigonal, bearing 1 setula, rarely 2 or 3 setulae, short, 1-10 μm long;basal appendage often absent.Habitat: In living stem of Podocarpus macrophyllus(Thunb.) D. Don.Pestalotiopsis hainanensis formed a terminal clade in the strict consensus trees which generated from ITS(ITS1 & ITS2 including 5.8S gene) and beta-tubulin 2 gene(tub2) sequences of several Pestalotiopsis species.2) Pestalotiopsis bicolor (Ellis & Everhart) A.R. Liu, T. Xu et L.D. Guo. comb. nov. Mycosystema, 2006 (in press).Habitat: In living leaves of Hyophorbe lagenicaulis Mart.3) Pestalotiopsis cinchonae (Zimm.) A.R. Liu, T. Xu et L.D. Guo. comb. nov. Mycosystema, 2006 (in press).Habitat: In living leaves of Coeos nucifera L., Cyrtostachys renda Bl., Lucuma nervosa A. DC. and Kandelia candel (L.) Druce and in living twigs of Podocarpus macrophyllus (Thunb.) D. Don.4) Pestalotiopsis gaurae (Guba) A.R. Liu, T. Xu et L.D. Guo.comb. nov. Mycosystema, 2006 (in press).Habitat: In living twigs of Camella oleifera Abel.5) Pestalotiopsis lambertiae (Petrak) A.R. Liu, T. Xu et L.D. Guo. comb. nov. Mycosystema, 2006 (in press).Habitat: In living leaves of Dracontomelon duperreanum Pierre and Neodypsis decaryi Jum.6) Pestalotiopsis pampeana (Speg.) A.R. Liu, T. Xu et L.D. Guo. comb. nov. Mycosystema, 2006 (in press).Habitat: In living leaves of Pachira macrocarpa Walp.7) Pestalotiopsis pandani (Verona) A.R. Liu, T. Xu et L.D. Guo. comb. nov. Mycosystema, 2006 (in press).Habitat: In living leaves of Cocos nucifera L. 1.2 New records of entophytic Pestalotiopsis in ChinaPestalotiopsis: P. acaciae (Thiim.) K. Yokoy. & S. Kaneko apud Kaneko,P. albo-maculans (P. Henn.) Y.X. Chen,P. algeriensis (Sacc. & Berl.) W.P. Wu,P. aloes (Trinchieri) J.X. Zhang & T. Xu,P. calabae (Westend.) Steyaert,P. carveri (Guba) P.L. Zhu. Ge et Xu,P. coffeae (Zimm.) Y.X. ChenPfici Steyaert,P.foedans (Sacc. & Ellis) Steyaert,Pfuchsiae (Thiim.) Y.X. Chen,P. milletiae (Laughton) Y.X. Chen & G. Wei,P. pauciseta (Sacc.) Y.X. Chen, P. sorbi (Pat.) X.A. Sun & Q.X. Ge, P. vismiae (Petr.) J.X. Zhang & T. Xu, P. westerdijkii Steyaert,P. zahlbruckneriana (Hem.) P.L. Zhu, Q.X. Ge & T. Xu0 2 Diversity of plant endophytic Pestalotiopsis in Hainan2.1 The colonization of plant endophytic PestahtiopsisColonization frequencies of endophytic Pestalotiopsis varied with the host plants. That varied from 0.8% to 27.2% on seven species of Palmae and from 2.5% to 21.7% in nine mangrove plants. The defference of Pestalotiopsis colonization frequencies between some palmaceous plant were significant but there was no significant difference in mangrove plants except in Ceriops tagal (Perr.) C.B.Rob, and in Bruguiera gymnorrhiza (L.) Poir.Colonization frequencies of endophytic Pestalotiopsis were different in different tissue of the same plant. Colonization frequencies of endophytic Pestalotiopsis in twigs were relatively higher than that in leaves.Colonization frequencies of endophytic Pestalotiopsis in plant was influenced by the natural environmental condition. Colonization frequencies of the endophytes in Palmae and Podocarpaceae in April 2004 was significantly higher than that in April 2005. It was rainless in most area of Hainan during the period from September 2004 to September 2005. The drought and high temperature would be the main reason for the significant variation.2.2 Species composition of endophytic Pestalotiopsis on host plantsSpecies composition of endophytic Pestalotiopsis varied in different familes of plants. Twenty species of endophytic Pestalotiopsis were found in Palmae, eleven species in Rhizophoraceae, nine species in Podocarpaceae and eight species in Planchonellae. The species composition of the endophytes varied even -among different plants of the same family. For example, eight species of endophytic Pestalotiopsis were in Roystonea regia (H.B.K) Cook., seven species in Zalacca wallichiana Salacca., five species in Chrysalidocarpus lutesens H. Wendl. and Neodypsis decaryi Jum., respectively, but only one to three species in other plants of Palmae.The dominant species of endophytic Pestalotiopsis were different among the families of plants investigated. They were Pestalotiopsis clavispora, Pestalotiopsiszonata and Pestalotiopsis virgatula in Palmae;Pestalotiopsis adusta in Rhizophoraceae;Pestalotiopsis virgatula and Pestalotiopsis zonata in Podocarpaceae;Pestalotiopsis paeoniae in Planchonellae.2.3 Shannon-Wiener index of endophytic PestalotiopsisShannon-Wiener index of endophytic Pestalotiopsis in Palmae, Rhizophoraceae, Podocarpaceae and Planchonellae changed from 1.4775 to 2.5013. Evenness index changse from 0.3624 to 1.0431. Richness index of endophytic Pestalotiopsis had no correlation with Shannon-Wiener index and Evenness index. For example, the Richness index of Palmae was highest among the four plant families. However, It's Shannon-Wiener index and Evenness index was lower than Rhizophoraceae.2.4 The species similarity comparability of endophytic Pestalotiopsis among the four plant families.The coefficient of community of endophytic Pestalotiopsis among the four plant families was less than 0.5 showing low similarity. 3 Molecular Phylogenetic relationship between Pestalotiopsis and allied genera1) The phylogenetic trees generated from the ITS region (ITS1 & ITS2 including 5.8S), 28S gene and beta-tubuling gene {tub!) sequences revealed the relationship between Pestalotiopsis and allied genera. Pestalotiopsis and Truncatella was the dependent genera. Monochaetia and Seiridium was very similar which always assembled as the same clade. It is proposed that those two genera should be merged into one genus: Monochaetia. The relationship between Pestalotiopsis and Monochaetia (including Seiridium) was more closerthan the relation between Pestalotiopsis and Truncatella.2) Recharacterization of the genus Pestalotiopsis and allied genera based on the molecular phytogeny and morphologyPestalotiopsis: Conidia 5-celled, 3 intermediate coloured cells, apical cell and basal cell hyaline, apical appendage 2 to several, pedicel present or absent.Truncatella: Conidia 4-celled, 2 intermediate coloured cells, apical cell and basal cell hyaline, apical appendage 2 to several, present or absent.Monochaetia: Conidia 5-celled or 6-celled, 3 or 4 intermediate coloured cells, apical cell and basal cell hyaline, 1 apical appendage, pedicel present or absent.3) The phylogenetic trees showed the relationship between teleomorph and anamorph of Pestalotiopsis.4. Molecular phylogenetic relationship in the genus of Pestalotiopsis1) The phylogenetic trees generated from the 80 ITS region (ITS1 & ITS2 including 5.8S), 63 beta-tubuling gene (tub2) sequences and 45 28S gene sequences partitioned the species of Pestalotiopsis into 10 groups corresponding to 10 clades of the trees (Aj-Ag and Bi, B2).2) Evoluating the taxonomic significance of Pestalotiopsis conidia morphological characteristics based on the molecular phylogeny: The type of the intermediate coloured cells of conidia: light (brown to olivaceous) afld dark (umber or fuliginous to brown) was the principal characteristic in taxonomy of Pestalotiopsis. Apical appendage knobbed at the extremities was the secondary key characteristic. Moreover, the position of the apical appendage arising from apical hyaline cell, apical appendage single then branched, the number of apical appendage and the length of conidia were also important characteristics in the taxonomy of Pestalotiopsis. However, pedicel characteristics (presence or absence, branched or not and the length) were useful in taxonomy of Pestalotiopsis.3) Comparing the three genes (ITS1 & ITS2 including 5.8S, beta-tubuling gene and 28S gene ) sequences used in analysis of the molecular phylogenetic relationship of the genus of Pestalotiopsis, it is suggested ITS and beta-tubulin gene (tub2) were more informative and meaningful than 28S in the present study.