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Conservation of Pinus Gerardiana Through Mycorrhizal Biotechnology

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Conservation of Pinus Gerardiana Through Mycorrhizal Biotechnology 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 1 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 . c 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 2 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 3 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%), 4 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
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