30 Axenic Culture of Symbiotic Fungus Piriformospora

30 Axenic Culture of Symbiotic Fungus Piriformospora indica Giang Huong Pham, Rina Kumari, Anjana Singh, Rajani Malla, Ram Prasad, Minu Sachdev, Michael Kaldorf, François Buscot, Ralf Oelmüller, Rüdiger Hampp, Anil Kumar Saxena, Karl-Heinz Rexer, Gerhard Kost and Ajit Varma

1 Introduction A large number of media compositions are available in the literature for the cultivation of various groups of fungi, but almost no literature is available for axenic cultivation of symbiotic fungi. In this chapter, we have made efforts to provide the documentary evidence for growth and multiplication of Piriformospora indica (see Chap. 15, this Vol. for characteristic features of the fungus). This new fungus named P. indica, due to its characteristic spore morphology, improves the growth and overall biomass production of different plants, herbs and trees, etc., and can easily be cultivated on a number of complex and synthetic media (Varma et al. 1999, 2001; Singh An et al. 2003a, b). Significant quantitative and morphological changes were detected when the fungus was grown on different nutrient compositions with no apparent negative effect on plants. It is relevant to mention here that different media can be used to understand the morphological and functional properties, or to test possible biotechnological applications.

2 Morphology Young mycelia were white and almost hyaline, but inconspicuous zonations were recorded in other cultures. The mycelium was mostly flat and submerged into the substratum. Hyphae were thin-walled and of different diameters ranging from 0.7 to 3.5 mm. The hyphae were highly interwoven, often adhered together and gave the appearance of simple intertwined cords. The hyphae often showed anastomoses and were irregularly septated. They often intertwined and overlapped each other. In older cultures and on the root surface, hyphae were often irregularly inflated, showing a nodose to coralloid

Plant Surface Microbiology A. Varma, L. Abbott, D. Werner, R. Hampp (Eds.) © Springer-Verlag Berlin Heidelberg 2004

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Giang Huong Pham et al.

Fig. 1. An overall view of P. indica, grown on solidified MYP medium for 7 days. Note the distinct hyphal coils (h) and pear-shaped chlamydospores (c) Bar = 20 µm. By courtesy of Oliver Blechert

Fig. 2. P. indica colonized maize root segment covered by numerous chlamydospores on the surface and scattering away from the root. Enlarged view of chlamydospores showing nuclei. Chlamydospores were stained with DAPI and observed in epifluorescence. Different optical planes were assembled in one picture using the IMPROVISION software package (IMPROVISION, Govenny, UK)

30 Axenic Culture of Symbiotic Fungus Piriformospora indica

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shape and granulated dense bodies were observed. Many cells contained more than one nucleus. Chlamydospores were formed from thin-walled vesicles at the tips of the hyphae. The chlamydospores appeared singly or in clusters and were distinctive due to their pear-shaped appearance (Fig. 1). The chlamydospores were (14-) 16–25 (-33) mm in length and (9-) 10–17 (-20) mm in width. Figure 2 shows the maize root colonization. The cytoplasm of the chlamydospores was densely packed with granular material and usually contained 8–25 nuclei (Fig. 2, inset). Very young spores had thin, hyaline walls. At maturity, these spores had walls up to 1.5 mm thick, which appeared two-layered, smooth and pale yellow. Neither clamp connections nor sexual structures could be observed.

3 Taxonomy of the Fungus Different kinds of substrates were tested to induce sexual development, such as young and mature leaves of Cynodon dactylon and pollen grains, oat meal, potato, carrot or tomato dextrose agar and soil-on-agar culture methods. No apparent adverse affect was seen on cultivation in light. It is not necessary to grow the fungus in the dark. Growth under light and dark conditions did not promote sexuality. May be the fungus is heterothallic in nature and one has to work for compatible strains. Since all these efforts did not lead to the desired results, there were only a few features to characterize the fungus morphologically and group it according to the classical species concept. In order to obtain more information about the systematic position of the new fungus, the ultrastructures of the septal pore and the cell wall were examined. The cell walls were very thin and multilayered structures. The septal pores consisted of dolipores with continuous parenthosomes. The dolipores were very prominent, with a multilayered cross wall and a median swelling mainly consisting of electron-transparent material. The electron-transparent layer of the cross walls extended deep into the median swellings, but did not fan out. In median sections of the septal pores, the parenthosomes were always straight and had the same diameter as the corresponding dolipore. Parenthosomes were flat discs without any detectable perforation. The parenthosomes consisted of an electron-dense outer layer, which showed an inconspicuous dark line in the median region. The parenthosomes were in contact with the ER membranes, which were mostly found near the dolipore (Verma et al. 1998). The ultrastructural data proof that P. indica is a menber of the Hymenomycetes (Basidiomycota). Studies on the moleclar phylogeny will help to reveal the closest relatives of this species (Fig. 3). Interestingly, immunological characterization showed a strong cross-reactivity with the members of Zygomycota (Glomerales) instead of species of Basidiomycota (Table 1). This aspect needs further critical appraisal.

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Table 1. Cross-reactivities of polyclonal antisera raised against P. indica (total hyphal homogenate) as determined by ELISA. Optical density (OD405 nm) values are given as the mean of three replicates after correction of control (OD405 nm)±SD. Statistical analysis was done by ANOVA. (n.d., not detectable) Antigens

OD405 nm 1:1600

Source

Piriformospora indica

0.49±0.005

Ajit Varma, JNU, New Delhi

Nonmycorrhizal fungi Agaricus bisporus Beauvaria sp. Candida albicans Cladosporium sp. Cunninghamella echinulata Fusarium solani Rhizoctonia bataticola Rhizoctonia solani Rhizopus sp. Saccharomyces cerevisiae Schizophyllum commune Sclerotinia sclerotiorum Sclerotium solani Ustilago maydis

0.08±0.002 0.003±n.d. ± 0.004 0.11± 0.004±n.d. 0.03±0.001 0.03±0.002 0.04±0.002 0.013±0.001 0.06±0.001 0.17±0.020 0.005±0.004 0.16±0.006 0.05±0.001 0.08±0.007

AK Sarbhoy, IARI, New Delhi AK Sarbhoy, IARI, New Delhi R Prasad, JNU, New Delhi AK Sarbhoy, IARI, New Delhi G Kost, Marburg, Germany AK Sarbhoy, IARI, New Delhi G Kost, Marburg, Germany A K Sarbhoy, IARI, New Delhi AK Sarbhoy, IARI, New Delhi R Prasad, JNU, New Delhi G Kost, Marburg, Germany AK Sarbhoy, IARI, New Delhi G Kost, Marburg, Germany AK Sarbhoy, IARI, New Delhi

Ectomycorrhizal fungi Amanita muscaria Lactarius torminosus Lentinus edodes Paxillus involutus Pisolithus tinctorius Rhizopogon roseolus Rhizopogon vulgaris Suillus variegatus

0.18±0.007 0.03±0.004 0.02±0.001 0.02±0.001 0.15±0.007 0.12±0.019 0.01±0.001 0.003±0.004

T Satyanarayana, South Campus, Delhi University Erika Kothe, Jena, Germany T Satyanarayana, South Campus, Delhi University T Satyanarayana, South Campus, Delhi University Erika Kothe, Jena, Germany T Satyanarayana, South Campus, Delhi University T Satyanarayana, South Campus, Delhi University Erika Kothe, Jena, Germany

Endomycorrhizal fungi Gigaspora margarita Gi. gigantia Glomus caledonium G. coronatium G. geosporura G. intraradices G. lamellosum G. mosseae G. mosseae 376 G. proliferum Scutellospora gilmorei

0.41±0.005 0.46±0.002 0.20±0.039 0.07±0.011 0.16±0.019 0.003±0.003 0.02±0.004 0.15±0.010 0.10±0.027 0.24±0.023 0.40±0.002

Alok Adholeya, TERI, New Delhi KVBR Tilak, IARI, New Delhi François Buscot, Jena, Germany François Buscot, Jena, Germany François Buscot, Jena, Germany François Buscot, Jena, Germany François Buscot, Jena, Germany François Buscot, Jena, Germany François Buscot, Jena, Germany François Buscot, Jena, Germany Ajay Shanker, JNU, New Delhi

AMF-like Sebacina vermifera var sensu 0.39±0.049 Sebacina sp. 0.23±0.013

Karl-Hein Rexer, Marburg, Germany Karl-Hein Rexer, Marburg, Germany

Statistical analysis of the data shows the P values, which are significant (P90 %). The first step of germination was the formation of germ tubes at the protruded zone of the spore, followed by hyphal emergence. Most of the nuclei followed the hyphae and seldom were one or two nuclei left behind in the spore. Soon branching appeared with a short and long branch (Fig. 4).

5 Cultivation Fungi are heterotrophic for carbon compounds and these serve two essential functions in fungal metabolism. The first function is to supply the carbon needed for the synthesis of compounds which comprise living cells. Proteins,

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nucleic acids, reserve and food materials, etc., would be included here. Second, the oxidation of carbon compounds produces appreciable amounts of energy. Fungi can utilize a wide range of carbon sources such as monosaccharides, disaccharides, oligosaccharides, polysaccharides, organic acids and lipids. Carbon dioxide can be fixed by some fungi, but cannot be used as an exclusive source of carbon for metabolism. P. indica can be successfully cultivated on a wide range of synthetic solidified and broth media, e.g., MMN1/10, modified aspergillus, M4N, MMNC, MS, WPM, MMN, Malt-Yeast Extract, MYP, PDA and aspergillus (Fig. 5). Among the tested media, aspergillus (Kae-

Fig. 4. Chlamydospores of P. indica. a Germinating chlamydospore showing initial branching after 12 h, b mature chlamydospores were germinated on a glass slide coated with thin nutrient agar, photographed after 24 h, c scattered spores and thin, irregular, undulating hyphae


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fer 1977) was the best. However, other media were helpful in carrying out several physiological and molecular experiments (see Chap. 15, this Vol.). Figure 6 shows typical growth on solidified aspergillus medium after 28 days. Rhythmic growth was often recorded. The mycelium stopped its growth for some time and produced a large number of chlamydospores of different dimensions. After 24–48 h, the mycelium started its growth again, producing normal amount of chlamydospores. This resulted in the formation of rythmic rings. The physiological reason for this phenomenon is not yet known, although this tendency has been recorded for several other members of Basidiomycetes. The fungus grows profusely upon shaking broth aspergillus medium. The temperature range of the fungal growth is 25–35 °C; the optimum temperature and pH being 30 °C and 5.8 (4.8–6.8), respectively. Figure 7 gives a view of the cultivation on broth media. Colonies were large and small depicting sea urchin-like radial growth. The maximum surface growth was recorded after 10 days. The colony diameter is indicated in Fig. 8. The fungal biomass is indicated in Table 2. The optimum growth was recorded after 5

Fig. 5. P. indica was grown on the following solidified media. a MS, b WPM, c MMN, d M4N, e PDA, f aspergillus

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Fig. 6. An overall view of P. indica grown on solidified aspergillus medium. Inset shows enlarged view of a small portion. On an agar concentration of 2 % w/v concentric rings often appeared (arrows) indicating the rhythmic growth of the fungus. The black arrows point on the regions with slow growth and high amount of chlamydospores, the white arrows point on thin mycelial mats resulting from fast growth of the hyphae

days with a gradual decrease in fresh and dry biomass after prolong incubation. Linear growth of the fungus on different solidified agar media is represented in Table 3. On modified Melin-Norkrans (MMN) medium sparsely running hyaline hyphae on the agar surface were seen, while on Potato Dextrose Agar deep furrows with strong adhesion to the agar surface were apparent. This sharp mode of growth was not observed when fortified with malt extract and normal aspergillus medium. In contrast to aspergillus medium, shaking conditions on MMN broth medium invariably inhibited the growth. The explanation for this observation is not known. Fungal growth acidifies the medium within 10 days to pH 4.4. Buffered medium prevented the reduction of pH (Table 4). 2-(N-morpholine) ethane sulfonic acid (MES) in the range of 25–100 mM was used.

6 Carbon and Energy Sources Individual sugars were uniformly added to the minimal broth at a rate of 1.0 % (w/v) in all treatments.They were included in the medium separately after sterilization. In all the sugar-supplied media, growth was better than the control (Table 5). There were not many changes in the growth except for rafinose and fructose.There were no changes in the color of the mycelium.Good growth was recorded in media containing maltose followed by xylose, sucrose, rhamnose, arabinose, glucose, lactose and mannose, respectively. The final pH was not altered significantly, but was lower than that of the control (Table 5). In a further study, fungal growth was best when glucose (1 % w/v) was used as a carbon source as compared to sucrose, and followed by fructose. A


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Table 2. The data represent an average of P. indica biomass of 5 replicates grown in 100 ml aspergillus broth medium in 250 ml capacity Erlenmeyer flasks. Incubation was done on a rotary shaker (GFL 3.19, Germany) at 144 rpm at 30 °C Days

Biomass (g)

5 7 10

Fresh

Dry

3.67±0.84 2.99±0.38 2.39±0.01

0.06±0.02 0.06±0.01 0.07±0.01

Fig. 7. Growth of P. indica on aspergillus broth medium under constant shaking condition at 25 °C for 7 days. Colonies of different developmental stages were shown. Mature colonies have the appearance of sea urchins

7

5 5 LM 3.6 ± 0.15

10 7

A 10.2

LM 5.4 ± 0.36

10 A 22.9

LM 7.5 ± 0.16

A 43.9

Fig. 8. A comparative linear growth of P. indica on aspergillus solidified medium. Measurements were made after 5, 7 and 10 days, respectively. Incubation was conducted in dark at 25 °C. Parameter selected was the diameter of 5 replicates of the linear measurement (LM). Readings are given in cm standard deviation and area (A) on agar medium. Statistical analysis of the data showed P