Introduction

The ECM fungi form symbiotic interactions with roots of forest trees and play a vital role to bring about various benefits to the plant species, including enhancement in the roots absorption surface area ¹, increased uptake of various nutrients ², resistance to pathogenic microorganisms ³ and drought stress ⁴. ECM symbiosis also help to increase the growth and nutrient contents of plants growing in nutrient deficient soils ⁵ and in return, the photosynthetic host tree species supply carbohydrates to their non-photosynthetic ECM fungal partners.

The roots of a single tree in natural forest ecosystems are almost invariably associated with many different ECM fungal species ⁶. Therefore host specificity of ECM fungi is an important part of forest ecosystems, which can be utilized as a tool for managing different forest resources ⁷. Natural hosts of ECM mushroom Tricholoma matsutake include Pinus sylvestris and P. abies ^8,9, P. densiflora and Quercus mongolica in northeastern China and Korean peninsula ^10,11, P. armandii, P. densata, P. wallichiana, P. yunnanensis, Casta-nopsis orthacantha, Q. aquifolioides, Q. pannosa, Q. guyavifolia, Q. semecarpifolia, Lithocarpus spp., and Pasania spp. in southwestern China and Bhutan ^8,11 whereas Amanita muscaria, Laccaria laccata, Pisolithus sp. and Suillus brevipes have been reported to produce ectomycorrhizae with P. patula ¹².

Many systems of in vitro ECM synthesis have been developed by various researchers to examine the potential of different ECM fungi to form symbiosis with different forest tree species ^13-18. ECM synthesis investigations are helpful to decide fungus and host plant compatibility for morphological, anatomical and physiological investigations ¹⁹.

Pinus gerardiana is an important edible nut yielding conifer and is well known in dry fruit trade.  The trade of its seeds significantly contributes to the annual income of most of the tribal families living in the area of its distribution.  In district Kinnaur of Himachal Pradesh alone approximate export value of its annual produce around 18 crores rupees.  It clearly reflects that the tree is not only ecologically important to the area but also has direct relevance to the economic status of the people residing over there ²⁰. Hence keeping into consideration the immense economic as well as ecological importance of P. gerardiana, the present investigation is aims to know about the ECM symbiosis between two gilled mushrooms and P. gerardiana also provide morphological and anatomical account of the synthesized ECM roots. These ECM fungi could be a very useful tool for the artificial inoculation in the nurseries to develop inoculated ECM seedling which further could also show better establishment in its natural habitats and helps in the reforestation and regeneration programs of P. gerardiana in Himachal Pradesh, India.

MATERIALS AND METHODS

Collection and isolation of pure culture of ECM fungi

The sporophore of A. ceciliae and L. sanguifluus were collected from district Kinnaur of Himachal Pradesh. The cultures were isolated from pileus region of the fresh sporophore of A. ceciliae and L. sanguifluus. Pileus was pulled gently apart and exposed interior tissue was removed with the help of sterilized scalpel and transferred into Petri plates containing PDA and MMN Medium. Then they were incubated at 25^0C and observed regularly for the appearance of culture. The well-grown colonies were subcultured on a PDA medium to obtain pure cultures.

Surface sterilization and seeds germination

Seeds of P. gerardiana were washed for 4 hours under the running tap H₂O and then treated with 10% H₂O₂ for 10 minutes. The H₂O₂ was drained and seeds were rinsed in sterilized distilled H₂O for 2 minutes. Then they were treated with 10% NaClO and Tween-20 and kept for 30 minutes. After that washed 5-6 times using sterilized distilled H₂O. Then seeds were placed in sterilized H₂O for 24 hours. After that seeds were put on sterilized wet filter paper in petri-plates and placed in low temperature for 24 hours before put in the seed germinator.

In vitro ECM synthesis

ECM synthesis was carried out by using in vitro synthesis experiments ²¹. For this 70ml of fungal nutrient medium, 90ml of vermiculite and 10ml peat was used in each 200ml test tube. The mixture of test tubes was autoclaved and then for treatments a disc of 5mm fungal culture was placed in test tube under aseptic conditions. For both treatments and uninoculated controls 10 replicates were used and treatments were kept for mycelium colonization. After that aseptically germinated P. gerardiana seedling was put into each treatments and control placed in the growth chamber and observed regularly for ECM synthesis. After 5 months seedlings pulled out from test tubes and checked for mycorrhization by following the methods of Fortin et al. ²².

Confirmation of ECM synthesis

The confirmation of ECM synthesis was done following Moore et al. ²³. For this a minute inoculum of vermiculite, peat mixture was removed from test tubes and inoculated on Petri plates containing nutrient medium. The synthesized ECM roots were detached from root system of the seedlings, surface sterilized and small pieces of ECM roots were inoculated on nutrient medium. Then Petri plates were observed for the mycelial growth and fungal colonies formed from both were compared with original ECM fungal culture colonies to confirm the ECM synthesis.

Morpho-anatomical characterization of ectomycorrhiza

Morphological as well as anatomical detail of ECM roots was made by following Zak ²⁴. The various growth characteristics e.g. form of ectomycorrhiza, colour of the fungal mantle, thickness of mantle etc. were recorded. The fixation ECM roots was done in F.A.A. solution and then preserved by using 70% alcohol. Anatomical details were worked out in both fresh as well as from preserved material. The sectioning of ECM roots was done following Johansen ²⁵.

Effect of in vitro ECM synthesis on seedlings growth

The five P. gerardiana seedlings were lifted randomly from each treatment and control. The observations on various growth parameters e.g. shoot length, root length, total number of short roots, shoot fresh weight, root fresh weight, shoot dry weight and root dry weight were recorded.

Statistical analysis of the data

The data was statistically analyzed, with the help of ANOVA test and Tukey’s multiple compression test was used to determine HSD (honestly significant difference) values for significance among various mean values.

Results

Isolation of pure culture and sub culturing of ECM fungi

The pure cultures of A. ceciliae was isolated on PDA medium. The colonies form dense mycelial mat and are white in colour. The pure culture of L. sanguifluus has been isolated on MMN medium. The colony of mycelium form concentric zones during its growth. The margin of the colony was irregular. Sub culturing of ECM mushrooms was carried out on PDA medium.

In vitro ECM synthesis

The pure cultures of ECM mushrooms (A. ceciliae and L. sanguifluus) were anlysed for their potential of ECM synthesis with the roots of P. gerardiana seedlings (Figure 1A and Figure 2A). The experiments  revealed the  successful in vitro mycorrhization between the host and two ECM mushrooms tested. The treated seedling showed the formation of many branched short lateral roots which ultimately leads to the development of ectomycorrhizae (Figure 1B and Figure 2B). The A. ceciliae and L. sanguifluus formed dark brown (Figures 1C,D) and yellowish brown types (Figures 2C,D) of ECM roots respectively. The description of synthesized ECM roots is given in Table 1.

Table 1: Morpho-anatomical description of Pinus gerardiana ECM roots synthesized with Amanita ceciliae and Lactarius sanguifluus

Effect of in vitro ECM Synthesis on the seedlings growth

The seedlings of P. gerardiana were evaluated for various growth characteristics after the completion of in vitro experiments with both ECM fungi tested.  The results on different growth characteristics are presented in Table 2. The results revealed that in both treatments (A. ceciliae and L. sanguifluus)  there was a significant difference(P<0.01 and P<0.05) on the overall growth characteristics of the seedlings as compared to the control (Table 2). Therefore in vitro ECM synthesis have significant effect on the seedlings growth and development.

The anatomical details of synthesized ECM roots with both ECM mushrooms tested showed the formation fungal mantle and Hartig net (Figures 1F and Figures 2F). In case of A. ceciliae, the thickness of fungal mantle was 25-30 µm whereas in case of  L. sanguifluus it was 15-20 µm (Table 1).  The seedlings kept as control were non-mycorrhizal with prominent root hairs and without ECM anatomical features in the transverse section of their roots. The culture colonies of A. ceciliae was reisolated on PDA and Hagem’s nutrient medium from both synthesized ECM roots and peat moss, vermiculite mixture (Figure 1E). Similarly the culture colonies of L. sanguifluus was reisolated on PDA and MMN nutrient medium from both synthesized ECM roots and peat moss, vermiculite mixture (Figure 2E)

The culture colonies of A. ceciliae and L. sanguifluus reisolated were found to have similar characteristics while compared with the culture colonies isolated from the fresh sporophores of both ECM mushrooms. Thus both (A. ceciliae and L. sanguifluus) confirming the synthesis of ECM roots with P. gerardiana seedlings.

Figure 1: In vitro ECM mycorrhization between Pinus gerardiana and Amanita ceciliaes: A. Test tubes showing  P. gerardiana seedlings (Larger one with inoculation treatment with A. ceciliae pure culture, smaller one is control).   Click here to view Figure

Figure 2: In vitro ECM mycorrhization between Pinus gerardiana and Lactarius sanguifluus: A. Test tubes showing  P. gerardiana seedlings (Larger one with inoculation treatment with L. sanguifluus pure culture, smaller one is control).   Click here to view Figure

Table 2: Effect of artificial inoculation on the growth of Pinus gerardiana seedlings.

** p<0.01; * p≤0.05; ^NS; Non-Significant differences as revealed through one way ANOVA and Tukey’s HSD multiple comparison test.

Discussion

During the present study two ECM mushrooms A. ceciliae and L. sanguifluus were isolated into pure culture on PDA and MMN medium respectively. The findings of the present study are supported by the results of several previous researches that reported mycelial growth of ECM fungi on various nutrient media. Phlebopus portentosus isolated on (Murashige and Skoog Agar) MS medium ²⁶, Scleroderma sinnamariense prefer (Fungus-Host) FH medium for mycelial growth ²⁷, Rhizopogon roseolus and Tricholoma matsutake cultures show best mycelia growth on PDA medium ²⁸, Pisolithus microcarpus was isolated on (Pridham-Gottlieb Modifled by Kuek) PGK medium ²⁹, Similarly Endo et al. ³⁰  isolated specimens of B. Edulis on MA medium and subculturing was done successfully on (Malt Extract Agar) MA and (Malt Yeast Agar) MYA nutrient media.  However, Agueda et al. ³¹  isolated Boletus aereus, B. edulis, B. pinophilus and B. reticulatus on BAF nutrient medium. The pure cultures are greatly affected by the changes made in cultivation medium and most of the ECM Boletale mushrooms favour media containing malt such as MA and MMN ²⁹.

The genus Pinus is a host for numerous ECM fungi and these fungi made symbiotic association with the roots of this conifer ³². During the present investigations in vitro ectomycorrhizae of P. gerardiana were synthesized with two ECM mushrooms (A. ceciliae and L. sanguifluus). Similarly mycorrhization between Picea abies and Cenococcum geophilum was achieved in asceptic conditions by inoculating mycelum on MMN medium ³³. Vaario et al. ³⁴ achived ECM in vitro synthesis between Abies firma and Pisolithus tinctorius. Pinus densiflora is found to be formed ECM association in laboratory conditions with broad range of ECM fungi ^35,36.

In Tibet, Tricholoma matsutake synthesized mycorrhizae in coniferous and fagaceous hosts tree in natural habitat and synthesized Hartig net in the ECM roots of P. densiflora ³⁵. In Canada T. matsutake synthesized mycorrhizae in pine and oak hosts trees ³⁷ and also formed typical Hartig net in P. densiflora ECM roots.

In similar study Laiye et al. ³⁸ utilized vermiculite, modified MMN medium and peat moss for the in vitro synthesis of ECM roots in the two Larch spp. seedlings with various ECM fungi. More recently, ECM in vitro synthesis of Cedrus deodara with Rhizopogon himalayensis ¹⁷, Pinus gerardiana with Boletus edulis  and Suillus scibricus ¹⁸ was achived successfully.

 The successes was achieved to germinate spores ³⁹, obtaining pure culture mycelia, in vitro synthesis of ectomycorrhizae ⁴⁰ and artificial shiro formation in potted culture system containing granite based soil substrate ⁴¹.

ECM fungi had a wide range of host specificities as reported by  Kumla et al. ⁴² and synthesized  in vitro ectomycorrhizae in two tree species viz. Eucalyptus camaldulensis and Pinus kesiya with single ECM fungus Pisolithus orientalis. Pure culture of P. tinctorius had potential for ECM association with Pinus taiwanensis in aseptic systems, but the association has not been reported in natural conditions ⁴³. Gomes et al. ⁴⁴ reported that cultures of P. arhizus isolated from fruiting bodies form symbiotic association with Quercus suber and also had the potential to synthesize mycorrhiza with Arbutus unedo under laboratory and nursery conditions. The ectomycorrhizae were appears mainly on short lateral apices ⁴⁵.

The results of the morpho-anatomical study were similar to the previous reports of Samson and Fortin ⁴⁶ to synthesized grayish brown to pinkish grey, whitish and yellow coloured ECM roots in Larix laricina with two ECM fungal genus Fuscoboletinus and Suillus. In vitro synthesis of ECM roots between S. sibiricus and P. wallichiana was achieved successfully in vessels and formed ECM roots were bifurcate to coralloid and creamish yellow in colour ⁴⁷. Garcia-Rodriguez et al. ⁴⁸  carried out synthesis to ECM roots in Eucalyptus urophylla and Pinus greggii with Pisolithus tinctorius, the synthesized ectomycorrhizae were simple, 1.0-2.6 mm long, yellow brown to bright yellow in colour and fungal mantle was 15.0 µm in thickness whereas ECM roots formed in Pinus greggii were bright yellow to yellowish brown in colour, dichotomously divided and rarely monopodial,.

The ECM roots produced during in vitro experiments in the present study showed similar anatonical features as that observed in many previous studies carried out by Kumla et al. ⁴² and observed that Pisolithus orientalis has ability to synthesized fungal mantles under in vitro conditions with Pinus kesiya and Eucalyptus camaldulensis.The both fungal mantles were plectenchymatous and similar to the mantle synthesized by Pisolithus tinctorius with Abies firma and Quercus ilex, Q. coccifera ⁴⁹, Pisolithus microcarpus with Eucalyptus grandis ⁵⁰ and Pisolithus aurantioscabrosus with Shorea sp. ⁵¹, Cedrus deodara with Rhizopogon himalayensis ¹⁷, Suillus scibiricus, Boletus edulis with Pinus gerardinan ¹⁸. Similar fungal mantle is synthesized by Boletus edulis with Cistus spp. ³¹ and Picea abies ⁵².

The culture of A. ceciliae and L. sanguifluus were reisolated from both synthesis substrate mixture as well as from synthesised ECM roots. They have similar characteristics as that of original cultures isolated from the fruiting bodies, thus validate the successful in vitro synthesis of ectomycorrhizae with these two ECM mushrooms. Similarly, Scleroderma aurantium was reisolated from both peat vermiculite mixture and synthesized ECM roots on MMN nutrient medium ⁵³. The fungal cultures were also reisolated from both synthesis experiment substrate mixture and from synthesized ECM roots to verify symbiotic interactions ^47,16,17.

Conclusion

The present study has synthesized ECM roots of P. gerardiana under laboratory conditions by using in vitro experiments with the pure cultures of two ECM mushrooms (A. ceciliae and L. sanguifluus). The synthesized ectomycorrhizae with A. ceciliae and L. sanguifluus were dark brown and yellowish  brown in colour respectively. The T.S. of ECM roots showed a typical ECM anatomy.

Acknowledgments

The authors are thankful to Department of Bio-Sciences, Himachal Pradesh University, Summer Hill, Shimla H.P. for providing the related support to compile this research work. 

Conflıct of Interest

The authors declare that there is no conflict of interest

Fundıng Sources

There are no funding Sources

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