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

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 yearold 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.