Resource, Diversity And Taxonomy Of Endophytic Pestalotiopsis On Camellia Sinensis In Hangzhou

Camellia sinensis (L.)O. Kuntze is a woody evergreen plant also a major cash crop in Hangzhou area. Endophytic fungi are a major part of biodiversity naturally and endophytic Pestalotiopsis were a major group of endophytic fungi.The investigation on the resource, diversity and taxonomy of endophytic Pestalotiopsis on Camellia sinensis was carried out. The result was as follows:1 Ten species of endophytic Pestalotiopsis on Camellia sinensis in Hangzhou were identified based on Steyaert (1949) , Guba (1961) and Nag Raj (1993). They are Pestalotiopsis microspora, Pestalotiopsis clavispora, Pestalotiopsis hetewcornis, Pestalotiopsis virgatula, Pestalotiopsis fici, Pestalotiopsis sinensis, Pestalotiopsis versicolor, Pestalotiopsis photiniae, Pestalotiopsis theae, Pestalotiopsis algeriensis. It was found that the resource of endophytic Pestalotiopsis on Camellia sinensis was abundant. That was isolated from the twig, foliage, flower of Camellia sinensis, and at least three endophytic Pestalotiopsis species were found from each cultuvars of Camellia sinensis.2 Diversity of endophytic Pestalotiopsis2.1 The endophytic Pestalotiopsis on the three cultuvars of Camellia sinensisTen species of endophytic Pestalotiopsis were found from the cultivar. Longjingchangye, but only three species of that were from the cultivars juhuachun and longjing43, respectively.2.2 The colonization of endophytic Pestalotiopsis on Camellia sinensisThe colonization frequencies of endophytic Pestalotiopsis in leaves of each cultivar (Iongjing43, longjingchangye and juhuachun) in leaves were 0.27%, 0.83% and 1.9%, respectively. That in twigs of the three cultivars were 4.2%, 3.8% and 6% respectively.2.3 The Dominant species and Shannon-Wiener index of endophytic Pestalotiopsis on Camellia sinensisThe dominant species of endophytic Pestalotiopsis in Camellia sinensis were Pestalotiopsis theae, P. clavispora and P. hetewcornis. The Shannon-Wiener index of endophytic Pestalotiopsis in the three cultivars of Camellia sinensis were 0.7663,0.8360 and 2.0614, respectively. The species richness index of endophytic Pestalotiopsis in them were 10, 3 and 3, respectively. The coefficient of community of endophytic Pestalotiopsis between Camellia sinensis cv. Longjing 43 and Camellia sinensis cv. juhuachun was a middle similarity, that between Camellia sinensis cv. juhuachun and Camellia sinensis cv. longjingchangye was a low similarity.2.4 The seasonal change of endophytic Pestalotiopsis colonization frequencies on Camellia sinensisThe colonization frequencies on Camellia sinensis reached the highest in the winter (January) (0.92%), and the lowest in the summer (August) (0.2%) .3.1 Molecular phylogenetic relationship in genus of PestalotiopsisA molecular phylogenetic tree was generated by neighbor joining method in ClustalX (1.81) with the ITS sequence which included the ITS1 and ITS2 sequences of 50 Pestalotiopsis strains. Pestalotiopsis strains were firstly divided into two branches A and B: A branch with darkly brown (dresden brown to olivaceous black) colored median cells (concolorous or versicolorous) of conidia (Ridgway, 1912);B branch with brownishly (yellow ocher to tawny-olive) colored median cells concolorous of conidia. A branch was further divided into 2 obvious subbranches (A) and A2), A] subbranch included Pestalotiopsis species with knobbed apical appendages, but A2 did not include. P. clavispora, P .photiniae, P. zonata, P. versicolor, P gracilis and P. foedans of A) subbranch were intersectant and the morphology of these species was superposable. P. theae and P. fid of A2 subbranch were together;B branch was further divided into 2 subbranches (Bi and B2), P .microspora, P. neglecta, and P .algeriensis of B] branch were close to each other;B2 branch was further divided into 2 subbranches (B2i and B22), the subbranche B2i included only a strains P.sinensis;All strains P.heterocornis of B22 subbranche were close to each other. Based on the molecular phylogenetic tree, the following morphological characters of colored median cells were confirmed to be significantly important for the taxonomy: 1) type of colored median cells of conidia;2) the characters of apical appendages including knobbed or unknobbed terminal of conidia. The taxonomy based on the length and width of conidia should be combined with the subbranches of molecular phylogenetic tree and the modality of the strains.3.2 Evaluation of the genus of Pestalotiopsis according to the morphological character and molecular phylogenetic tree.P. davispora, P. photiniae, P. zonata, P. versicolor, P. gradlis and P. foedans of Aj subbranch were intersectant and the morphology of these species was superposable, the conidia were versicolorous, colors of the two upper colored median cells were snuff color or olivaceous black, the colored median cell was tawny-olive on the base of conidia, they produced conidia [21~27X6~8um] with 2~4 apical appendages (15~32) um and most 3 apical appendages, minority had 2 apical appendages, According to the morphological character and molecular phylogenetic tree, Ai was considered as Pestalotiopsis versicolor. B2 branch was further divided into 2 subbranches (B2i and B22). B2i branch had a strain P. sinensis;All P. heterocornis strains of B22 branch were together, the colored median cells of conidia [19.0—25.2x4.9~5.7 um] were versicolorous, yellow ocher, the apical appendages [11.3~19.6 um] had unknobbed terminals and were branched, it was considered as Pestalotiopsis microspora. the colored median cells of conidia [18~25X5~7 um] of P. microspora, P. neglecta, P. algeriensis which belonging to Bi branch were concolorous and most 2 or 3 apical appendages, According to the morphological character and molecular phylogenetic tree, B2i and B22 were considered as only a species, Pestalotiopsis microspora.