Piriformospora indica
Piriformospora indica Sav. Verma, Aj. Varma, Rexer, G. Kost & P. Franken 1998, in Verma et al., Mycologia, 90(5), 896-903 (1998)
Etymology. indica (lat.): pertaining to India.
Diagnosis: In Latin, Fungus crescat non modo in radicibus plantarum vivarum, sed etiam in artificiale, nutrico iure vivat; juvenibus cultivatis myceliis albis et hyalinis, mature zonatis candido vel cremeo colore; hyphis tenui tunicatis, efibulatis, septatis, nonnumquam anastomosis, 0. 7-3.5 1-1m in diametro; in matura cultura hyphis intricates et coralloidibus; in septis hypharum parenthesomatibus continuis et discoidibus super doliporis; chlamydosporis oriundis ab apicibus hypharum, piriformibus (14) 16-25(33) X (9) 12-17(20) µm, levibus, crasse tunicatis ( -1.5 µm); pariete chlamydosporae bistrato, pallide ochraceo colorato; cytoplasma chlamydosporae granulosa et guttulata, 8-25 nucleis.
The fungus grew on a wide range of synthetic and complex media, e.g., on minimal medium (MM1) normally used for in vitro germination of AM fungi with 10% saccharose or glucose as a carbon source, on two different media for Aspergillus sp. (CM and MM2), and on Moser B medium. Young mycelia were white and almost hyaline (Figure 1A), but inconspicuous zonations were recorded in older cultures (Figure 1B). The mycelium was mostly flat and submerged into the substratum. Hyphae were thin-walled and of different diameter ranging 0. 7-3.5 µm (Figure 2A). They often intertwined and overlapped each other (Figure 2B). In older cultures and on the root surface, hyphae were often irregularly inflated, showing a nodose to coralloid shape, and granulated dense bodies were observed (Figure 2C). Hyphae sometimes showed anastomosis and were irregularly septated (Figure 2D). For this reason, many cells contained more than one nucleus. Chlamydospores were formed from thin-walled vesicles at the tips of the hyphae (Figure 3). The chlamydospores appeared singly or in clusters and were distinctive due to their pear-shaped structure. The chlamydospores were (14)16-25(33) µm in length and (9)10-17(20) µm in width. Very young spores had thin, hyaline walls. At maturity, these spores had walls up to 1.5 µm thick, which appeared two layered, smooth and pale yellow. The cytoplasm of the chlamydospores was densely packed with granular materials and usually contained 8-25 nuclei (Figure 4). Neither clamp connections nor sexual structures were observed. Significant quantitative and morphological changes were detected when the fungus was challenged to grow on different media. Shaking during incubation retarded growth in MM1 liquid cultures (7-12 g fresh wt/L after 2 wk at 30 oC), whereas no such negative effect was ever observed during cultivation in any other substrate. In fact, the fungal biomass was considerably enhanced on shaking cultures with CM (sometimes up to 50 g fresh weight/L after 2 wk at 30 C). On Moser B medium the colonies appeared compact and wrinkled with furrows. The mycelium produced defined zonations and a high amount of white aerial hyphae. Hyphae were highly interwoven, often adhered together and gave the appearance of simple cords. New branches emerged irregularly, and at regular intervals the hyphal wall showed some external deposits that stained deeply. Root colonization - Pot cultures of maize were inoculated with the mycelium of the fungus in order to examine its ability to interact with plants. The hyphae colonized the surface of the roots (Figure 5A), and after two weeks the fungus was also observed inside the cortex (Figure 5B). A more detailed analysis of the colonization and the effect on plant growth is currently being done and will be published elsewhere. Ultrastructure -In order to obtain more information about the systematic position of the new fungus, the ultrastructure of the septal pore and the cell wall were examined. The cell walls were very thin and showed multilayered structures (Figure 6, arrow 1). The septal pores consisted of dolipores with continuous parenthesomes (Figure 6, arrow 2). The dolipores were very prominent, with a multilayered cross wall and a median swelling mainly consisting of electron transparent material. The electron-transparent layers of the cross walls extended deep into the median swellings but did not fan out. In median sections of the septal pores, the parenthesomes were always straight and had the same diameteras the corresponding dolipore. Parenthesomes were flat discs without any detectable perforation. The parenthesomes consisted of an electrondense outer layer and a less dense inner layer, which showed an inconspicuous dark line in the median region. The parenthesomes were in contact with the ER membranes, which were mostly found near the dolipore. Molecular studies -In order to get a more precise idea about the closest relatives of P. indica, part of the 18S rRNA was amplified, sequenced and compared with corresponding data on a number of different Basidiomycota from GenBank. Sclerotinia sclerotia (Ascomycota) and Glomus mosseae (Zygomycota) were used as outgroups. Based on the results, a dendrogram of the molecular phylogeny was constructed (Figure 7). This dendrogram indicated the lowest evolutionary distance of the ISS rRNA sequence of the new fungus to members of the Rhizoctonia group (Ceratobasidiales), namely Rhizoctonia solani Kuhn and Thanatephorus praticola (Kotila) Talbot. The significance of a common branch shared by these fungi and P. indica in this reconstructed phylogeny is indicated by the bootstrap value of 61%. When the same analysis was carried out without the Rhizoctonia group, the new fungus did not match up with any other species (data not shown), but occupied its own branch.
Index Fungorum Number: IF446949
Figure 1. Colony growth of Piriformospora indica. An agar disk was transferred onto CM medium and incubated at 30oC. A. 15 da and B. 30 da after inoculation.
Figure 2. Morphology of P. indica hyphae. A. On MM agar; B. on Moser B agar; C. on the surface of a maize root; D. on MM2 agar. Hyphae were photographed under the light microscope without staining (A, B), after trypan blue staining (C) or under the epifluorescense microscope after calcofluor staining (D).
Figure 3. Chlamydospores of Piriformospora indica.
Figure 4. Nuclei in a chlamydospore. Chlamydospores were stained with DAPI and observed by epifluorescense microscopy. Different optical plains were assembled in one picture using the Improvision software package (IMPROVISION, Coventry, UK).
Figure 5. Colonization of maize roots with P. indica stained with chlorazole black E. A. Fungal structures outside roots harvested 2 wk after inoculation. B. Fungal structures within roots harvested 4 wk after inoculation.
Figure 6. Dolipore and parenthesomes of P. indica. Section of hyphae were observed by TEM. Cell wall (arrow 1) and parenthesomes (arrow 2).
Figure 7. Dendrogram of evolutionary distances based on part of the 18S rDNA sequences of different fungi using the neighbor joining method. The comparison was carried out based on the Jukes-Cantor equation. Only those values of a data resampling bootstrap analysis are shown which were necessary for evaluating the taxonomic status of the new fungus. The bar indicates 2% in nucleotide differences.
Reference:
Verma, S., Varma, A., Rexer, K. H., Hassel, A., Kost, G., Sarbhoy, A., ... & Franken, P. (1998). Piriformospora indica, gen. et sp. nov., a new root-colonizing fungus. Mycologia, 90(5), 896-903.
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