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Direct comparison of culture-dependent and culture-independent molecular approaches reveal the diversity of fungal endophytic communities in stems of grapevine (Vitis vinifera)

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Abstract

Grapevines (Vitis vinifera) are colonized by ubiquitous microorganisms known as endophytes, which may have advantageous or neutral effects without causing disease symptoms. Certain endophytes are uncultivable, so culture-independent approaches such as next generation sequencing (NGS) can help for a better understanding of their ecology and distribution. To date, there are no studies which directly link NGS results with taxa derived from a culturing approach, integrating morphological and multi-gene phylogenetic analysis of endophytes. In this study, a culture-dependent and high-resolution culture-independent approach (next generation sequencing) were used to identify endophytes in grapevine stems. In the culture-dependent approach, a total of 94 isolates were recovered from 84 of 144 healthy grapevine stem fragments (colonization rate = 58.3%). The study is unique as we used subsets of combined multi-gene regions to identify the endophytes to species level. Based on each multi-gene phylogenetic analysis, 28 species belong to 19 genera (Acremonium, Alternaria, Arthrinium, Ascorhizoctonia, Aspergillus, Aureobasidium, Bipolaris, Botryosphaeria, Botrytis, Chaetomium, Cladosporium, Curvularia, Hypoxylon, Lasiodiplodia, Mycosphaerella, Nigrospora, Penicillium, Phoma, Scopulariopsis) were identified. A higher number of culturable fungi were obtained from 13 year-old vines, followed by eight and three year-old vines. In the culture-independent approach, a fungal richness of 59 operational taxonomic units (OTU) was detected, being highest in 13 year-old grapevines, followed by eight and three years. Even though the cultivation approach detected lower fungal richness, the results related to stem are consistent for fungal community composition and richness. Comparison of the fungal taxa identified by the two approaches resulted in an overlap of 53% of the fungal genera. Due to interspecific variability of the sequences from NGS, in many cases the OTUs (even with the highly abundant ones) were only assignable to order, family or genus level. Incorporating multi-gene phylogenies we successfully identified many of the NGS derived OTUs with poor taxonomic information in reference databases to the genus or species levels. Hence, this study signifies the importance of applying both culture-dependent and culture-independent approaches to study the fungal endophytic community composition in Vitis vinifera. This principle could also be applied to other host species and ecosystem level studies.

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Acknowledgements

This work was financially supported by Beijing Talent Program for Dr. Jiye Yan, CARS-29, Beijing science and technology project D17110001617002. We thank Dr. Heng Gui for his support to submit Raw Illumina reads to the Sequence Read Archive (SRA) of National Center for Biotechnology Information (NCBI).

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Authors and Affiliations

  1. Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, No. 9 of Shuguanghuayuan Zhonglu, Haidian District, Beijing, 100097, China

    Asha J. Dissanayake, Wei Zhang, Chunyuan Fu, Mei Liu, Qikai Xing, Xinghong Li & Jiye Yan

  2. Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Beijing Academy of Agriculture and Forestry Sciences, No. 9 of Shuguanghuayuan Zhonglu, Haidian District, Beijing, 100097, China

    Asha J. Dissanayake, Wei Zhang, Chunyuan Fu, Mei Liu, Qikai Xing, Xinghong Li & Jiye Yan

  3. Center of Excellence in Fungal Research, Mae Fah Luang University, 333 Muang, Chiang Rai, 57100, Thailand

    Asha J. Dissanayake & Kevin D. Hyde

  4. Department of Soil Ecology, UFZ-Helmholtz Centre for Environmental Research, Halle (Saale), Germany

    Witoon Purahong & Tesfaye Wubet

  5. Beijing Academy of Forestry and Pomology Sciences, No. 12 of Xiangshanruiwangfen, Beijing, 100093, China

    Haiying Xu & Guojun Zhang

  6. German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany

    Tesfaye Wubet

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  1. Asha J. Dissanayake
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  2. Witoon Purahong
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  5. Wei Zhang
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  9. Mei Liu
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Correspondence to Xinghong Li or Jiye Yan.

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Dissanayake, A.J., Purahong, W., Wubet, T. et al. Direct comparison of culture-dependent and culture-independent molecular approaches reveal the diversity of fungal endophytic communities in stems of grapevine (Vitis vinifera). Fungal Diversity 90, 85–107 (2018). https://doi.org/10.1007/s13225-018-0399-3

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