Microbial Associates of Hippophae rhamnoides (Seabuckthorn)
Seabuckthorn (Hippophae rhamnoides), a multipurpose shrub and native of higher Himalayas is also known as cold desert gold due to its high potential as a bio resource for wetland reclamation, soil erosion, food medicinal and cosmetic industries. Studies conducted on the microbial associates of this plant revealed the presence of 26 fungal species in its rhizosphere. Three fungal entophytes (i.e., Aspergillus niger, Mortierella minutissima and a sterile mycelium) and four species of VAM spores (i.e., Glomus albidum, Glomus fasciculatum, Glomus macrocarpum and Gigaspora margariata) has also been isolated from different plant parts (Root, stem, leaves and bark) and soil samples, respectively.
Seabuckthorn (Hippophae rhamnoides, Family Elaeagnaceae) is a multipurpose deciduous, dioecious, thorny and nitrogen fixing shrub-tree growing widely on high altitude regions of Himachal Pradesh, Jammu and Kashmir, Sikkim and Uttranchal. It is tolerant of extremes of temperature (43 to +45°C), resistant to drought conditions and well adapted to the salinity and alkalinity. Because of its benefits to the mountain communities, it is called wonder plant or cold desert gold (Kumar, 2003; Jodha et al., 1992). Seabuckthorn is supposed to be a store house of nutrients and many items like jams, soft drinks, sauces, pickles, jelly and biscuits are made from it. Its fruits are good source of vitamins. A part from this it is used as fire wood, as fencing plant on river side for soil conservation purpose (Naithani, 2004).
Research on medicinal properties and other aspects of Seabuckthorn has received much attention in recent past ((Gurevick, 1956; Gurmeet, 1997; Dorjey, 1998; Eccleston et al., 2002; Kumar, 2003; Singh, 2006; Yang and Kallio, 2006), but there is little information available on the rhizosphere, VAM and endophytic fungi of this plant. This communication records observations on such microbial association of Seabuckthorn so as to suggest strategies (by inoculating with suitable mycorrhizal fungi) for growing it on degraded and abandoned land for sustainable development of the traditional mountain societies.
MATERIALS AND METHODS
Site of sample collection: Various samples were collected from District Lahaul-Spiti, of Himachal Pradesh (December, 2004-July, 2005), lying between North latitude 33°44' and 33° 42' and east latitude 76°56' and 78°41'. This District is referred to as cold desert and composed of rugged and different mountain terrains, which is a part of great Himalayan region.
Morphoanatomy of root: Root samples were collected from three different plants from different localities and were stored in F:A:A: (Formaldehyde: Acetic acid: Alcohol) in the ratio 5:5:9. For morphological study, root color, root diameter, presence or absence of nodule, size of nodule and any change in morphology due to an ectomycorrhiza were taken in to account. To reveal anatomical detail, thin sections of these roots were double stained and observed (Marx and Davey, 1969).
Isolation and identification of rhizosphere fungi: Rhizosphere soil samples were collected from three plants in sterile polythene bags. The isolation of rhizosphere fungi from these was done by dilution plate method of Wakesman (1927) and Warcup (1950). This method includes shaking of 1 g soil in 100 mL of distilled water followed by further dilutions under asceptic conditions. Various dilutions were cultured on Potato Dextrose Agar medium and Czapeks Dox medium (Raper and Thom, 1949). Subculturing was done and the fungi were identified following Barnett and Hunter (1972), Gilman (1967) and Nagmani et al. (2005).
VAM spores isolations: Isolation of VAM spores was done by Wet sieving
and Decanting Technique (Gerdemann and Nicolson, 1963). A pinch of soil was
shaked in distilled water and was filtered through sieves 300, 200 and 100 μm
followed by filtration through Whattman No. 1 filter paper. This filter paper
was observed directly under binocular microscope. Each spore was picked with
micro needle and identified following (Mortan, 1988; Schenck and Perez, 1988;
Trappe, 1982; Walker, 1983, 1986).
Isolation of endophytes: To isolate endophytes, various plant parts (i.e., roots, leaves, bark, stem) collected from different plants were screened out following Hot water treatment and Three step methods. Small pieces of tissue were treated with 25, 50 and 75% methanol for 5, 3 and 2 min, respectively. Then these were inoculated on half strength PDA medium. Pure cultures of each endophyte were subcultured at regular intervals.
Morphological studies on the roots of Hippophae rhamnoides Linn: Hippophae rhamnoides Linn. is a non-leguminous nitrogen fixing plant. It has stout, deep feeding and dichotomously branched roots. The roots are brown in colour. Average diameter of roots ranges from 4 mm-1.5 cm. Roots are nodulated. Nodules actually represent modified lateral root. Each nodule is highly branched and varies from 4.0-9.0 mm in size. Nodule harbours an actinorhizal microorganism called Frankia, which help in nitrogen fixation.
Anatomical features of roots of seabuckthorn: In transverse section, the roots of Hippophae rhamnoides Linn. showed outer most torned bark layer which is deep brown in colour. Next to it somewhat squarish yellow coloured cells of cork are seen. In these layers a network of branching filaments were seen. Next to cork layers, are present cortical cells, which are loosely arranged with large intercellular spaces in between.
The intercellular spaces of cortex reveal the presence of branching filaments. Some of filaments penetrate the cortical cells and form vesicles within the cells. These vesicles are formed by an Actinomycete; Frankia and these were present in large number in the cortical cells. The presence of Frankia in the root nodules of Seabuckthorn confirms the Actinorhizal type of association between both the partners. Inter stellar infection is absent (Fig. 1a and b).
Qualitative assessment of mycoflora from rhizosphere of Hippophae rhamnoides Linn. during Winter season (December, 2004): Fungi were isolated from rhizosphere of Hippophae rhamnoides Linn. using dilution plate technique. During winter season 22 species of fungi were isolated. These fungal isolates fall into 13 genera. One isolate is represented by sterile mycelia.
The different fungal genera isolated from soil samples collected in winter season are Absidia, Alternaria, Aspergillus, Cladosporium, Cunninghamella, Epicoccum, Fusarium, Macrophomina, Mucor, Penicillium, Rhizoctonia, Rhizopus and Trichoderma.
The genus Penicillium is represented by four species (i.e., P. funiculosum, P. nigricans, P. chrysogenum and Penicillium sp.). The genera Aspergillus and Fusarium are represented by three species each (i.e., A. flavus, A. niger, A. versicolor, F. moniliforme, F. oxysporum and Fusarium sp.). The genus Cladosporium is represented by two species (i.e., C. herbarum and C. sphaerospermum). Other nine genera (i.e., Absidia, Alternaria, Cunninghamella, Epicoccum, Macrophomina, Mucor, Rhizoctonia, Rhizopus and Trichoderma) are represented by one species each (i.e., Absidia ramosa, Alternaria alternata, Cunninghamella echinulata, Epicoccum nigrum, Macrophomina phaseoli, Mucor hiemalis, Rhizoctonia solani, Rhizopus negricans and Trichoderma viride).
The fungi isolated from rhizosphere soil samples of Hippophae rhamnoides Linn. belong to the fungal subdivisions Zygomycotina, Ascomycotina, Deuteromycotina and Basidiomycotina. Out of these four genera belong to Zygomycotina (Absidia, Cunninghamella, Mucor and Rhizopus), two belong to Ascomycotina (Aspergillus and Penicillium), seven to Deuteromycotina (Alternaria, Epicoccum, Fusarium, Rhizoctonia, Cladosporium, Macrophomina and Trichoderma). Basidiomycotina is represented by a single sterile mycelium. The maximum representatives are of Deuteromycotina (7 genera), followed by Zygomycotina (4 genera) and Ascomycotina (2 genera) which in return are followed by Basidiomycotina (Table 1). This study shows that during winter season, maximum number of species is that of Penicillium followed by Aspergillus and Fusarium while subdivision Deuteromycotina is represented most.
Qualitative assessment of mycoflora from rhizosphere soil samples of Hippophae
rhamnoides Linn. during Rainy season (July, 2005): The samples of soil
collected during July, 2005 (rainy season), from Lahaul Spiti district of Himachal
Pradesh revealed the presence of 26 species of fungi.
||TS Root of Hippophae rhamnoides Linn. Showing Branching Filaments and Vesicles of Frankia (a and b) Glom macrocarpum (c), Glomus fasciculatum
||Distribution of fungal genera isolated from the rhizosphere
of Hippophae rhamnoides Linn. during winter season into different
subdivisions of fungi
||Comparison of occurrence of different fungal species during
winter and rainy season
|+: Indicating presence of the fungus in a particular season,
: Indicating absence of the fungus in a particular season
These fall into 16 genera. The fungal genera isolated from the rhizosphere
soil samples of Seabuckthorn collected during rainy season are Absidia,
Alternaria, Aspergillus, Cephalosporium, Cladosporium,
Cunninghamella, Drechslera, Epicoccum, Fusarium,
Macrophomina, Mucor, Penicillium, Rhizoctonia, Rhizopus,
Stachybotrys and Trichoderma.
In the case of rainy season Penicillium is also represented by four species (i.e., Penicillium funiculosum, P. negricans, P. chrysogenum and Penicillium sp.). The genera Aspergillus and Fusarium are represented by three species each (i.e., A. flavus, A. niger, A. versicolor, F. moniliforme, F. oxysporum and Fusarium sp.). The genera Rhizoctonia and Cladosporium are represented by two species each (i.e., R. solani, Rhizoctonia sp., C. herbarum and C. sphaerospermum).
Other eleven genera i.e., Absidia, Alternaria, Cephalosporium, Cunninghamella, Drechslera, Epicoccum, Macrophomina, Mucor, Rhizopus, Stachybotrys and Trichoderma are represented by single species each (i.e., A. ramosa, A. alternata, C. acrimonium, C. echinulata, D. tetramera, E. nigrum, M. phaseoli, M. hiemalis, R. negricans, S. atra and Trichoderma viride).
These fungi belong to subdivision Zygomycotina, Ascomycotina, Deuteromycotina
and Basidiomycotina. Out of these, four genera belong to Zygomycotina (Absidia,
Cunninghamella, Mucor and Rhizopus), three to Ascomycotina
(Aspergillus, Drechslera and Penicillium), nine to Deuteromycotina
(Alternaria, Cephalosporium, Cladosporium, Epicoccum,
Fusarium, Macrophomina, Stachybotrys and Trichoderma).
||Distribution of fungal genera isolated from the rhizosphere
of Hippophae rhamnoides Linn. during Rainy season into different
subdivisions of fungi
Basidiomycotina is represented by three white sterile mycelia. During rainy
season, the number of fungal species isolated from rhizosphere is more than
that isolated during winter. The new genera reported during rainy season are
Cephalosporium, Stachybotrys and Drechslera. One more species
of Rhizoctonia is reported (Table 2).
The maximum representative fungal genera belongs to Deuteromycotina (nine genera), followed by Zygomycotina (four genera) and Ascomycotina (three genera). Basidiomycotina is also represented by three sterile mycelia (Table 3).
Isolation and identification of VAM fungal spores from rhizosphere of Hippophae rhamnoides Linn.: The soil samples from rhizosphere of Hippophae rhamnoides Linn. were analyzed for the presence of various VAM fungi. The fungal spores were isolated by wet Sieving and Decanting Technique (Gerdemann and Nicolson, 1963) and were identified at Department of Botany, University of Delhi, New Delhi by Prof. K.G. Mukerji.
Spores of four species of VAM fungi were isolated from samples taken during December 2004 (winter season) and July, 2005 (Rainy season). These species belong to two genera namely, Glomus and Gigaspora.
Out of four species isolated, three belonged to Glomus and one to Gigaspora. Thus the genus Glomus was more predominant among these genera. The four species isolated are: Glomus albidum, Glomus fasciculatum, Glomus macrocarpum and Gigaspora margarita.
Relative frequency of occurrence of VAM fungal species: In total, 17
spores of VAM fungi were isolated. Out of these, 13 spores (76.47%) were of
Glomus fasciculatum, 2 spores (11.76%) were of Glomus albidum
and only 1 spore (5.88%) was each of Glomus macrocarpum and Gigaspora
|| Relative percentage frequency of occurrence of VAM fungi
This shows that most abundant genus of VAM fungi in the rhizosphere of Hippophae rhamnoides Linn. is Glomus among two genera isolated i.e., Glomus and Gigaspora during this study. Among the three species of Glomus (i.e., G. albidum, G. fasciculatum and G. macrocarpum), G. fasciculatum is most abundant followed by G. albidum and G. macrocarpum (Table 4).
Observations on the endophytic fungal isolates of Hippophae rhamnoides Linn: Tissues taken from the various parts of the plant of Hippophae rhamnoides Linn. (i.e., leaf, bark, stem and root) were analyzed for the presence of fungal endophytes. In total three fungal endophytes were isolated which fall into two genera (i.e., Aspergillus and Mortierella) and one is represented by a non sporulating mycelium. Each of these (Aspergillus and Mortierella) is further represented by a single species i.e., A. niger and M. minutissima. The isolated endophytes belong to Zygomycotina (M. minutissima), Ascomycotina (A. niger) and Basidiomycotina (non sporulating mycelium) sub-divisions of fungi.
Morphology of roots of Seabuckthorn revealed simple and nodulated roots with no sign of ectomycorhiza, where the root tip becomes swollen and shows dichotomous branching. Harley and Smith (1983) have also concluded that while ectomycorrhizal fungi change the root morphology, VAM fungi do not alter roots shape and size. The nodules on the surface of roots of Seabuckthorn are due to Frankia-actinorhizal association, which is a non-leguminous N2 fixing plant (Singh et al., 2003). The presence of vesicles and inter and intracellular branching filaments in the root cortical cells suggest actinorhizal type of association between Seabuckthorn and Frankia. Similar findings have been reported by earlier workers (Katiyar et al., 1989, Vijaylakshmi and Rao, 1988; Gardner et al., 1984; Gauthier et al., 1983) in most of the angiospermic plants. Regarding rhizosphere fungi of Seabuckthorn, it may be mentioned that it is the first report of its kind.
Slight fluctuation in the number of fungal species was observed during winter and rainy seasons. These rhizosphere organisms experience fluctuations due to change in pH, environmental extremes and root exudates (Molina and Amaranthus, 1990). Many interactions occur between plant root and rhizosphere fungi and the dominance of few of the micro-organisms in the rhizosphere and disappearance of other was also reported by Linderman (1988). The rhizosphere fungi are not merely passive prisoners of the abiotic environment rather they play an important role in the creation and maintenance of favourable growth conditions (Perry et al., 1987). Rhizosphere fungi have been isolated with the aim of their exploitation for the overall establishment and welfare of the plant. Because the organisms present in rhizosphere influence plant growth positively by enhancing nutrient uptake, storage, pathogen suppression, development of soil structure and protection against environmental extremes (Linderman, 1986).
VAM fungi help to increase the soil root interphase area and hence increase the nutrient uptake. These fungi are also known to increase the Phosphate and moisture uptake by the plant. The plants which are infected with mycorrhizal fungi exhibit significant increase in the yield as compared to non mycorrhizal plants (Gerdemann, 1968). The four types of endogenous spores obtained from the rhizosphere of Seabuckthorn indicate adaptability of these fungi to varied soil and environmental conditions. Sagar et al. (1993) and Kaur et al. (1997) while conducting studies on Celtis australis and Grewia optiva, respectively reported the presence of Glomus, Sclerocystis, Acaulospora and Gigaspora from the rhizosphere of these plants.
Endophytes are be being studied extensively since some of them may a source of novel biomolecules of pharmaceutical importance (Dreyfuss and Chapela, 1994). Present investigations have also revealed the presence of three endophytes (i.e., Aspergillus niger, Mortierella minutissima and one sterile mycelium) which were found closely associated with the tissue (roots, leaves, stem and bark) of Hippophae rhamnoides. Clelland et al. (1983) have reported the presence of Glomus fasciculatum as an endophytic fungus of Hippophae rhamnoides. The same fungus has also been isolated from the rhizosphere of Seabuckthorn during present study.
These microbial associates (rhizosphere fungi, VAM spores and endophytes) of Seabuckthorn can be exploited for its mass multiplication with the ultimate aim of transplanting the resultant tailored seedlings in degraded and treeless land of cold desert areas. Stone et al. (2000) have also regarded VAM endophytes as sources of novel metabolites for therapeutics, as potential biocontrol agents and helping the establishment of seedlings at new sites by providing disease resistance and enhancing growth.
Present study stress the need to undertake indepth scientific researches on the role of microbial associates and their exploitation so as to suggest strategies for growing Seabuckthorn on degraded and abandoned land which will not only help them to improve their ecological environment but also help in the sustainable development of the traditional mountain societies.
Thanks are due to the Chairman, Department of Biosciences, Himachal Pradesh University, Summer Hill, Shimla-5 India for providing necessary lab facilities to carry out present work. Authors are grateful to Prof. K.G. Mukerji, Department of Botany, University of Delhi, New Delhi, India for identification of fungi. First author is also thankful to CSIR, New Delhi, India, for providing financial assistance in the form of J.R.F.
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