Conservation of Pinus Gerardiana Through Mycorrhizal Biotechnology

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Conservation of Pinus gerardiana through Mycorrhizal Biotechnology

(GBPI/IERP-NHMS/15-16/29/23, Dated: 31.03.2016)

Annual Progress Report

(April, 2017 to March, 2018)

Submitted to G.B. Pant National Institute of Himalayan Environment & Sustainable Development, Almora Under Integrated Eco-Development Research Programme (IERP)

HIMALAYAN FOREST RESEARCH INSTITUTE (Indian Council of Forestry Research & Education) Conifer Campus, Panthaghati SHIMLA – 171 013 (H.P.)

Appendix IV

PROFORMA FOR ANNUAL PROGRESS REPORT (IERP PROJECTS)

1. Project Title : Conservation of Pinus gerardiana through mycorrhizal biotechnology 2. Name of Principal : Dr. Ashwani Tapwal, Scientist –E (PI), Investigator and Project Dr. K.S. Kapoor, Scientist-G (Co-PI) Staff Sh. Yogesh Thakur (PA) 3. GBPIHED project sanction : GBPI/IERP-NHMS/15-16/29/23 letter No. Date of sanction: 31.03.2016 4. Total outlay sanctioned : Rs. 16,84, Lakh 4.1 Duration: 3 Years 5. Date of start : May, 2016 6. Grant received during the : Rs. 2,59,186/- year 7. Expenditure incurred : Rs. 3,41,046.64 during the year 8. Bound area of research : Biodiversity Conservation and Ecological Security 9. Sub area of the project : Mycorrhizal Biotechnology

10. Approved objectives of the project:

i. Survey, collection and identification of ectomycorrhizal symbionts of Pinus gerardiana across different altitudinal zones. ii. Pure culture isolation, multiplication and application of dominant ectomycorrhizal fungus with P. gerardiana seedlings. iii. Evaluation of field performance of artificially inoculated Pinus gerardiana seedlings in natural as well in out of provenance zone.

11. Methodology: i. Study area: Pinus gerardiana forests of Kinnaur district, Himachal Pradesh.

Kinnaur is a tribal district of Himachal Pradesh, located in Northeast corner of the state bordering a Tibet to the east (Plate-1: a-c). It consists of very high mountains, deep valleys, gorges, glaciers and rivers. The climate of area is

characterized by extremes in temperature coupled with excessive dryness, dry and highly evaporative wind. During prolonged winters, which usually starts from the end of September, area receive heavy to moderate snowfall which continues up to May and hence, snow remains the only form of precipitation. Bare mountains exhibit typical desert colouration of rocks due to their peculiar weathering.

Plate-1 (Study area)

a b .

ii. Nursery trials: Nursery trails were conducted in collaboration with state forest department in Kinnaur district in December, 2016. Growth parameters of the seedlings like shoot height, shoot volume, root volume, collar diameter, root length and per cent mycorrhizal roots, shoot height, collar diameter, root volume, per cent ectomycorrhizal infection, and root & shoot dry weight are being recorded at the interval three months. iii. Microscopic characterization of host-fungus relationship: Transverse sections of the mycorrhizal roots of artificially inoculated seedlings were cut, temporary

mounts were prepared and observed under microscope for colour/thickness/ texture of the fungal mantle, extraradical hyphae and Hartig net. iv. Rhizosphere soil analysis: Rhizosphere soil of control and inoculated seedlings was collected at the interval of three months and analysed for available carbon, nitrogen, phosphorus, potassium etc. following Walkley and Black (1934), Subbiah and Asija (1956), Bray and Kurtz (1945) and Neutral normal ammonium acetate extractant method for the estimation of organic carbon, nitrogen, phosphorus and potassium respectively. v. Training: A training programme was organised for a period of two days with the specific objective of capacity building of stakeholders on the application of mycorrhizae in forestry. vi. Field Trials: Seedlings will be transplanted to field conditions in winter and their survival, establishment and growth parameter will be recorded.

12. Work Plan

S. No. Activities Year-1 Year-2 Year-3

Discussion with officials of state forest 1. department for implementation of project  activities.

Survey, collection of mycorrhizal fungi 2.  across different altitudinal zones.

Identification and in vitro culturing of 3.   mycorrhizal fungi

Microscopic characterization of host-fungus    4. relationship.

5. Nursery experiment and maintenance.   

6. Observation on growth parameters and   mycorrhizal association of seedlings in nursery.

7. Transplantation of seedlings to field   conditions in different area including out of

provenance zone.

8. Observation on establishment and   performance of seedlings in field

9. Training and demonstration of mycorrhizal  biotechnology to the officials of State Forest Department

10. Data compilation and project completion  report

13. Salient research achievements as per work plan/objectives covered during the year

i. Study area and species: Pinus gerardiana commonly known as ‘Chilgoza’ and /or ‘Neoza’ pine, is named after its discoverer, captain Gerard, is a tree restricted in dry inner valleys of North-West Himalayas, where it grows in between altitudes of 1,600m to 3,000m above msl. It grows gregariously, forming forests of somewhat open type, though moderately dense pole crops are also met with. It occurs in Kinnaur (Satluj Velley) and in Pangi (Ravi and Chenab valleys) of Himachal Pradesh extending westwards to Kashmir, Afghanistan and Northern Baluchistan. Soil of the study area was sandy loam having lots of pebbles and rocks. The soil was acidic with pH varying from 5.60-6.95. ii. Nursery trials: Nursery trails were laid in the natural distribution zone of Pinus gerardiana at Akpa, district Kinnaur, Himachal Pradesh, in collaboration with State Forest Department. The observations on establishment of mycorrhizal association and growth characters of seedlings were recorded at the interval of three months. The results as described as follows: a. Effect of artificial inoculation of mycorrhizae on the growth of seedlings

Growth of the inoculated seedlings was better as compared to control (Table-1; Fig.- 1; Plate-2 a-e)). The per cent increase in the growth of three month old to one old year inoculated seedlings over the control varied from 7.64-55.37% in different parameters, the details are summarised as follows: Shoot height (15.23-25.72%), Shoot fresh weight (7.64-55.04%), Shoot dry weight (7.86-55.37%), Shoot volume (27.27-51.35%), Collar diameter (18.09-39.08%), Mycorrhizal roots (0.00-51.25%),

Root length (16.70-28.54%), Root fresh weight (20.93-55.31%), Root dry weight (0.22-47.43%), Root volume (27.16-46.94%).

Table-1 Growth parameters of inoculated and control seedlings in nursery

Growth parameters Treatment Observations on growth of seedling 3 month 6 month 9 month 12 month Shoot height (cm) Control 3.12 8.46 9.52 9.85 Inoculated 4.18 10.97 11.23 13.26 Root length (cm) Control 20.98 39.45 59.60 61.41 Inoculated 29.36 47.36 77.40 82.14 Collar diameter (mm) Control 2.40 3.24 3.57 3.78 Inoculated 2.93 4.57 5.86 5.94 Mycorrhizal roots (%) Control 0.00 24.13 35.12 38.12 Inoculated 5.00 49.50 57.50 64.48 Shoot fresh weight (mgs) Control 763.27 1090.50 1289.29 1560.3 Inoculated 826.41 1549.60 1884.75 3470.16 Shoot dry weight (mgs) Control 372.07 410.90 631.15 491.7 Inoculated 403.83 529.70 814.06 1101.8 Root fresh weight (mgs) Control 454.98 2245.87 2362.42 5878.1 Inoculated 575.42 3351.90 5285.80 7764.8 Root dry weight (mgs) Control 90.13 1225.97 1954.34 1957.68 Inoculated 90.33 2332.00 3254.98 2932.6 Shoot Volume (ml) Control 1.60 1.80 3.80 4.61 Inoculated 2.20 3.70 6.40 7.95 Root Volume (ml) Control 2.28 3.80 7.30 8.14 Inoculated 3.13 5.30 10.50 15.34

b. Effect of mycorrhizal inoculations on the CNPK content of rhizosphere soil

Rhizosphere soil of artificially inoculated and control seedlings were analysed for available phosphorus, nitrogen, potassium and carbon. The results revealed considerable increase in availability of the tested nutrients in the soil in artificially inoculated soil as compared to control (Table-2; Fig.-2). The phosphorus content showed an increase from 39.30-59.60%, whereas nitrogen (13.98-26.76%),

5 potassium (20.00-27.59%) and carbon contents also showed an increasing trend from 4.39-38.24%. Table-2 CNPK content of rhizosphere soil of inoculated and control seedlings in nursery

Soil analysis Concentration of CNPK in rhizosphere Treatment soil (PPM) 3 month 6 month Carbon Control 32700 8400 Inoculated 34200 13600 Nitrogen Control 83.159 109.5 Inoculated 96.67 149.5 Phosphorus Control 5.4255 20 Inoculated 8.938 49.5 Potassium Control 34.913 72 Inoculated 48.217 90 iii. Host fungus relationship in the artificially inoculated seedlings Morpho-anatomical characteristics of roots revealed well developed mycorrhizal association in the inoculated seedlings. In inoculated plants mantle was well developed, root hairs were absent and Hartig net formation was recorded in cortex region. Morphological study revealed that the ectomycorrhizal roots are of bifurcate or dichotomous type and occasionally multiforked (corolloid) or monopodial. Every bifurcation of short root is considered as an ectomycorrhiza. Under low power, the mycorrhizal roots were having swollen root tips, covered with hyphal sheath and numerous extraradical hyphae. (Plate-2 f-g). iv. Training of the Stakeholders: Two days capacity building training of the field functionaries of State Forest Department and college students was conducted at the institute. Twenty participants attended the training. During the training, audio visual lectures covering different aspects of mycology and pathology including the importance of mycorrhizae in forests were covered. Laboratory and field exposure to the participants was also conducted to enrich their knowledge on the host- pathogen dynamics and importance of mycorrhizae in the natural forests (Plate-3 a-f).

Fig.-1 Percent increase in the growth parameters of one year old inoculated seedlings over control 60.00

50.00

40.00

30.00 3 month 20.00 6 month

9 month Percent increase in growth Percentincrease growth in 10.00 12 month

0.00

Fig.-2 NPK content (ppm) of control and inoculated rhizosphere soil of 3 & 6 month old sedlings 160

140

120

100 Control (N) Inoculated (N) 80 Control (P) 60 Inoculated (P) Content in ppm Contentppm in Control (K) 40 Inoculated (K) 20

0 1 2

Plate-2 Growth of seedlings

c c c . t t . . t

a b c . . .

c t .

f. c t .

H d e. g. M . a. Three month old seedlings; b. Six month old seedlings; c. Nine month old seedlings; d-e. Twelve month old seedlings; f. Mycorrhizal roots; g. T.S. of Mycorrhizal root (where c- control, t – artificially inoculated seedling; M- mantle, H- Hartig net

Plate-3 Training of stakeholders

a. b.

c. d.

f e.

14. Summary of progress: Till date fifty fruiting body of macro-fungi were collected. Identification of the collected specimens was carried out by following standard taxonomical manuals and websites. The identified macro-fungi belong to 13 genera representing 22 species. The dominant genus was Scleroderma (5 species), followed by Lycoperdon and Geastrum (3 species each) and rest of genera were represented by one species each. Pure cultures of mycorrhizal fungi are being maintained on potato dextrose agar. Nursery trails were established at Akpa, district Kinnaur in collaboration with State Forest Department. The mycorrhizal inoculum of Scleroderma polyrhizum was applied during seed sowing in the month of December, 2016. Nursery trials were later maintained and growth characteristic were recorded at interval of three months. Growth parameters of the seedlings like shoot height, shoot volume, root volume, collar diameter, root length and per cent mycorrhizal roots were higher in inoculated seedlings as compared to control. Growth of the inoculated seedlings was recorded better in comparison to control. The growth parameters recorded at the interval of three months and it was observed that percent increase in the growth of one old year inoculated seedlings over the control varied from 25-55% in different parameters, the details are summarised as follows: Shoot height (25.72%), Shoot fresh weight (55.04%), Shoot dry weight (55.37%), Shoot volume (42.01%), Collar diameter (36.36%), Mycorrhiza formation (40.88%), Root length (25.24%), Root fresh weight (24.30%), Root dry weight (33.24%), Root volume (46.94%). Morpho- anatomical characteristics of roots revealed well developed mycorrhizal association in the inoculated seedlings. In inoculated plants mantle was well developed, root hairs were absent and Hartig net formation was recorded in cortex region. The rhizosphere soil analysis revealed that the phosphorus content was increased from 39.30-59.60%, nitrogen (13.98-26.76%), potassium (20.00-27.59%) and carbon content increased from 4.39-38.24%. Two days capacity building trainings on application of mycorrhizae in forestry and insect- pest and disease management were conducted to the stakeholders. Exposure visits of participants to nursery of HFRI and forest were also conducted during training programme to enrich their knowledge on the host-pathogen dynamics and importance of mycorrhizae in the natural forests.

15. New observations

50 macro-fungi were found associated with Pinus gerardiana and accordingly, their cultures are being maintained. Mass inoculum of Scleroderma polyrhizum was raised and nursery bags were artificially inoculated at the time of seed sowing. The results revealed better growth in the artificially inoculated seedlings as compared to control. Since, no systematic work has been done so far on the artificial inoculation of Pinus gerardiana seedlings with S. polyrhizum; hence the present findings may provide specific guidelines for future studies. 16. Innovations

Because of wide occurrence of Scleroderma polyrhizum –a mycorrhizal fungi- in P. gerardiana forests, mass inoculum of this fungus was produced. The studies revealed the better growth of artificially inoculated seedlings, which help in reducing retention period of the seedlings in nurseries and better chances of their survival in field conditions. The outcome will help the HPSFD for production and subsequent plantation of tall and healthy seedling in the field.

17. Research work which remains to be done under the project referring to Objectives to be covered

Objective Research work which remains to be i. Survey, collection and identification  Proposed work has been done. of ectomycorrhizal symbionts of Pinus gerardiana across different altitudinal zones. ii. Pure culture isolation, multiplication  Proposed work has been done. and application of dominant ectomycorrhizal fungus with P. gerardiana seedlings. iii. Evaluation of field performance of  Field transplantation of seedlings. artificially inoculated Pinus  Observation on the seedlings gerardiana seedlings in natural as establishment and growth in field well in out of provenance zone. conditions.  Training of stakeholders.

Publication: Tapwal, A., Kapoor, K.S., Thakur, Y., and Kumar, A. (2017). Ectomycorrhizal diversity in Pinus gerardiana forests and further assessment of its suitability. In: HP Science Congress organised by HIMCOSTE, Shimla from 20-21 November, 2017 (poster presentation).

Ectomycorrhizal diversity in Pinus gerardiana forests and further assessment of its suitability Ashwani Tapwal*, K.S. Kapoor, Yogesh Thakur and Akhil Kumar Himalayan Forest Research Institute, Shimla-171013 *Corresponding author: [email protected] ABSTRACT RESULTS Pinus gerardiana Wall. ex D. Don, commonly known as chilgoza or neoza pine, is an ecologically Collection of macro-fungi: and economically important species of Kinnaur district, Himachal Pradesh. It also occurs in small  Twenty six wild fleshy fungi were collected, out of which 14 were identified. patches in Pangi and Bharmour regions of Chamba district. Seeds of P. gerardiana besides rich in  Maximum number of species were recorded in the forest of Skibba followed by Ribba. nutrients also provide a source of income and accordingly, are harvested by the native peoples as one Morphology of P. gerardiana mycorrhizal roots: of their sources of livelihood. However, unscientific and over harvesting including other biotic as  P. gerardiana has a heterozoic root system. well as abiotic factors have become the primary causes of poor regeneration of the said species. It is  Morphologically, mycorrhizal roots were found having typical ectomycorrhizal characteristics like an established fact that in nature, mycorrhizal fungi play an important role in establishment and dichotomous branching, pale yellow colour, swollen root tips and numerous extraradical hyphae on subsequent survival of the seedlings on the forest floor. In view of this, a series of surveys were surface. conducted in the P. gerardiana forests of Kinnaur district (Akpa, Jangi, Lippa, Ribba, Skibba, Kalpa,  Average diameter of mycorrhizal root near the base of dichotomy varied from 0.24 mm to 0.36 mm, Reckong Peo, Thangi) and samples of associated macro-fungi, rhizosphere soil along with whereas at the tips, it varied from 0.11 mm to 0.21mm. mycorrhizal roots were collected. Out of these collections, fourteen macro-fungi namely Lycoperdon pyriforme, Scleroderma hypogaeum, Scleroderma areolatum, Scleroderma polyrhizum, Geastrum Anatomy of Pinus gerardiana mycorrhizal roots: fimbriatum, Geastrum floriforme, Geastrum saccatum and species of Suillus, Boletus, Russula,  In transverse sections, the mycorrhizal root exhibit a thin fungal mantle in the initial stages of Cortinarius, Phallus, Gymnopilus, Morchella etc. were identified. These roots, when further seen development but its thickness increases with age. The fungus was seen to have ensheathed the root tip and analysed morphologically, were found having typical ectomycorrhizal characteristics like completely. dichotomous branching, pale yellow colour, swollen root tips and numerous extra-radical hyphae on  The transverse section of mycorrhizal roots exhibited well developed mantle and Hartig net. The surface. Anatomically, the transverse section of roots exhibited well developed mantle and Hartig net mantle being 30-60 µ thick. formation comprising of 3-5 layers of cortical cells. These observations accordingly, confirm the  Hartig net formation was observed to 3-5 layers of cortical cells. These observations confirm the mycorrhizal associations in the roots of P. gerardiana. The frequency of occurrence of S. polyrhizum mycorrhizal association in the roots of P. gerardiana. which was highest in comparison to other macro-fungi was further selected for the successive  There was intense intercortical infection extending upto the endodermis and prominent intracortical nursery trials aiming at production of quality planting material for better establishment and penetration by the hyphae. The stellar region was free of infection. subsequent survival in the field conditions.  Morpho-anatomical observations confirms the ectomycorrhizal association in the roots of P. Key words: Pinus gerardiana, chilgoza, ectomycorrhiza, macro-fungi, hyphae, Kinnaur. gerardiana. CONCLUSION  It can be concluded that three genera of wild mushroom were associated with P. gerardiana are Mycorrhiza, literally means ‘fungus root’(Frank, 1885) and it represents a mutually beneficial INTRODUCTION Lycoperdon, Scleroderma and Geastrum. symbiotic relationship between the feeder roots of plants and fungi. Mycorrhizae as presently  S. polyrhizum was recorded from all sites and selected accordingly for nursery trials. recognised are of seven categories: Ectomycorrhizae, Endomycorrhizae, Ectendomycorrhizae,  P. gerardiana seedlings tailored through ectomycorrhizal inoculation are expected to grow faster and Arbutoid, Monotropoid, Ericoid and Orchidaceous types (Harley and Smith, 1983). Mycorrhizae survive better after transplantation. increase the survival, growth and development of associated plants by performing essential physiological processes i.e. increased absorption surface, selective ion absorption and accumulation (Jorgenson and Shoulders, 1967; Smith and Read, 2008), and help seedlings to resist infection by certain feeder root pathogens (Marx, 1971). In addition, the mycelia of ECM fungi allow host plants to access carbon and other nutrients (Finlay and Read, 1986; He et al., 2004). Ectomycorrhizae occur on about 10% of the world flora, especially trees belonging to the Pinaceae, Fagaceae, Betulaceae, Salicaceae, Juglandaceae, Myrtaceae and Ericaceae. About 6000 plant species are known to have ectomycorrhizal association with 20000 fungal taxa (van der Heijden et al., 2015). The roots of P. gerardiana are ectomycorrhizal with dichotomous or coralloid type (Lakhanpal and Chaudhary, 1988). The aim of the present study is to investigate the ectomycorrhizal fungi associated with P. gerardiana and identify the mycorrhizal fungus for artificial inoculation.

MATERIALS & METHODS

 Sporocarps wild fleshy fungi associated with P. gerardiana were collected.  Tissue culture method was employed for the isolation / raising of pure cultures on mycorrhizal fungi.  Sporocarps were preserved in FAA (40% Formalin, acetic acid, and 50% alcohol).  Collected mycorrhizal fungi were identified the basis of macroscopic and microscopic characters by following standard keys and monographs. b c d e  Free hand cross sections of both fresh and fixed ectomycorrhizal roots of P. gerardiana were examined under dissecting and compound microscopes for morpho-anatomical studies.  Physicochemical analysis of soil was done by following standard methodology of Soil testing in India (STI, 2011).

REFERENCES

Finlay R.D. and Read, D.J. (1986). The structure and function of the vegetative mycelium of f g h i ectomycorrhizal plants. New Phytol., 103: 157–165. EM Frank, A.B. (1885). Uber die auf wurzelsymbiose beruhende Emährung gewisr.cr. Baume durch unterirdische. Berlin Deutsches Botanische Gessalschaft, 3: 128-145. Harley, J.L. and Smith, S.E. (1983). Mycorrhizal Symbiosis. Academic Press, Toronto. - He X., Critchley, C., Ng, H., Bledsoe, C. (2004). Reciprocal N (15NH4+ or 15NO3 ) transfer between non N2-fixing Eucalyptus maculata and N2-fixing Casuarina cunninghamiana linked by the ectomycorrhizal fungus Pisolithus sp. New Phytol.,163:629–640.

Jorgenson, J.R. and Shoulders, E. (1967). Mycorrhizal root development vital to survival of slash pine FM nursery stock. US Dept. Agric. For. Serv. Tree Plant Notes, 18: 7-11. Lakhanpal, T.N. and Chaudhary, S. (1988). Studies on mycorrhiza of Pinus gerardiana. In: M Mycorrhiza for Green Asia (Eds. Mahadevan, A., Ramman, M. and Natarajan, K.). Alamn Printing Works, Madras (Chennai). pp. 283-285. HN Marx, D.H. (1971). Ectomycorrhizae as biological deterrents to pathogenic root infections. In: j k l Mycorrhizae (Ed. Hacskaylo, E.). US Govt. Printing Office Washington. pp. 81-96. Smith S.E. and Read D.J. (2008). Mycorrhizal symbiosis. 3rd ed. London: Academic Press, London, a. Sites surveyed, Fungal fruit bodies of: b. Scleroderma polyrhizum, c. Geastrum saccatum, d. Russula novelis, e. Suillus fuscotomentosus, Spores of: f. Scleroderma polyrhizum, g. Geastrum saccatum, h. Russula novelis i. Suillus fuscotomentosus, j. U.K. Mycorrhizal root of Pinus gerardiana, k. Ectomycorrhizal root with emanating extraradical hyphae. l. Transverse section of P. Soil Testing in India (STI) (2011). Methods Manual Department of Agriculture & Cooperation. gerardiana root showing mantle and Hartig net. Ministry of Agriculture, Government of India, New Delhi, 70p. (Where: EM= Extramatrical hyphae; FM= Fungal Mantle; HN= Hartig Net; M= Mycorrhizal root tips). Van der Heijden M.G.A., Martin F.M., Selosse M.A. and Sanders I.R. (2015). Mycorrhizal ecology Acknowledgements: The authors are thankful to G.B. Pant National Institute of Himalayan Environment & Sustainable and evolution: the past, the present, and the future. New Phytol 205: 1406–1423. Development, Almora for financial support to the project ‘Conservation of Pinus gerardiana through mycorrhizal biotechnology (GBPI/IERP-NMHS/15-16/29/23).

Signature of PI Dated: