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. 2010 Mar;76(5):1642-52.
doi: 10.1128/AEM.01911-09. Epub 2009 Dec 28.

Identity, diversity, and molecular phylogeny of the endophytic mycobiota in the roots of rare wild rice (Oryza granulate) from a nature reserve in Yunnan, China

Zhi-Lin Yuan  1 , Chu-Long Zhang, Fu-Cheng Lin, Christian P Kubicek
Affiliations

Identity, diversity, and molecular phylogeny of the endophytic mycobiota in the roots of rare wild rice (Oryza granulate) from a nature reserve in Yunnan, China

Zhi-Lin Yuan et al. Appl Environ Microbiol. 2010 Mar.

Abstract

Rice (Oryza sativa L.) is, on a global scale, one of the most important food crops. Although endophytic fungi and bacteria associated with rice have been investigated, little is known about the endophytic fungi of wild rice (Oryza granulate) in China. Here we studied the root endophytic mycobiota residing in roots of O. granulate by the use of an integrated approach consisting of microscopy, cultivation, ecological indices, and direct PCR. Microscopy confirmed the ubiquitousness of dark septate endophytes (DSEs) and sclerotium-like structures in root tissues. Isolations from 204 root segments from 15 wild rice plants yielded 58 isolates, for which 31 internal transcribed spacer (ITS)-based genotypes were recorded. The best BLAST match indicated that 34.5% of all taxa encountered may represent hitherto undescribed species. Most of the fungi were isolated with a very low frequency. Calculation of ecological indices and estimation of taxon accumulation curves indicated a high diversity of fungal species. A culture-independent approach was also performed to analyze the endophytic fungal community. Three individual clone libraries were constructed. Using a threshold of 90% similarity, 35 potentially different sequences (phylotypes) were found among 186 positive clones. Phylogenetic analysis showed that frequently detected clones were classified as Basidiomycota, and 60.2% of total analyzed clones were affiliated with unknown taxa. Exophiala, Cladophialophora, Harpophora, Periconia macrospinosa, and the Ceratobasidium/Rhizoctonia complex may act as potential DSE groups. A comparison of the fungal communities characterized by the two approaches demonstrated distinctive fungal groups, and only a few taxa overlapped. Our findings indicate a complex and rich endophytic fungal consortium in wild rice roots, thus offering a potential bioresource for establishing a novel model of plant-fungal mutualistic interactions.

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Figures

FIG. 1.
FIG. 1.
A longitudinal section of O. granulate roots. Mature melanized (A) and blue-stained (B) microsclerotia occupy intracellularly root cortex cells. Bar = 20 μm.
FIG. 2.
FIG. 2.
The extensive colonization of dark septate endophytes and other root endophytic fungi in wild rice roots (shown in longitudinal section, bar = 20 μm). (A) Heavy colonization of blue-stained hyphae in epidermal cells with some chlamydospore-like structures; (B) cooccurrence of melanized hyphae and developing sclerotium-like structures in or on the root cortex layer; (C) colonization of melanized hyphae in epidermis and cortex, and hyphae growing along the epidermis or cortex parallel to the longitudinal axis of the roots; (D) other trypan blue-stained endophytic fungi colonize intracellularly within root cortex; (E) cooccurrence of melanized hyphae and other blue-stained endophytic fungi; (F) initiation, development, and formation of microsclerotia.
FIG. 3.
FIG. 3.
The frequency of 31 different ITS-based genotypes determined from total cultured fungi. * denotes undescribed fungal species. Genus and/or species names of identified fungi are indicated above the corresponding column.
FIG. 4.
FIG. 4.
Taxon accumulation curves illustrating observed genotypic or phylotypic richness and estimated total richness (based on bootstrap estimates) of endophyte communities in wild rice roots. (A) culturing method; (B) direct clone library sequencing method. The 95% confidence intervals for each curve are also shown.
FIG. 5.
FIG. 5.
Neighbor-joining phylogenetic tree showing the placement of all the phylotypes based on the sequences of 5.8S of rDNA. The Kimura two-parameter model is used for pairwise distance measurement. The tree is rooted with Rhizopus microsporus (a zygomycete, EU798703). Only bootstrap values of >50% (1,000 replicates) are shown at the branches.
FIG. 6.
FIG. 6.
Position of two isolates of culturable DSEs (Harpophora sp. and Periconia macrospinosa) on the phylogenetic trees, as inferred based on ITS1-5.8S-ITS2 sequence. Maximum-parsimony bootstrap values of >50% are indicated above branch nodes. Number of bootstrap replicates = 1,000. Each tree is rooted with corresponding outgroup. Macro- and microscopic features of each DSE are also indicated. * denotes the described DSE species.
FIG. 7.
FIG. 7.
Phylogeny of Rhizoctonia-related species using maximum parsimony analysis based on ITS1-5.8S-ITS2 sequence (consistency index [CI] = 0.7828, rescaled consistency index [RC] = 0.6697, retention index [RI] = 0.8556, homoplasy index [HI] = 0.2172). Tree length = 907. Bootstrap values of >50% are shown above branch nodes. Agaricus bisporus (AF465404) is designated the outgroup.
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