Regeneration of Cold Desert Pine of N.W. Himalayas (India) A Preliminary Study

Transcription

1 Regeneration of Cold Desert Pine of N.W. Himalayas (India) A Preliminary Study T. N. Lakhanpal Sunil Kumar Abstract The cold desert pine of India, Pinus gerardiana (Wall.) has been subjected to overexploitation because of the commercial value of its edible seeds and ethnic uses. Regeneration is deficient. Preliminary studies conducted by inoculating the seedlings with mycorrhiza show great promise in establishment and performance of the seedlings. Pinus gerardiana, commonly and commercially known as chilgoza and/or neoza pine, is a forest tree restricted in India to dry inner valleys of the Northwest Himalayas (1,600 to 3,000 m elevation). It occurs in Kinnaur (Satluj Valley) and Pangi in Himachal Pradesh (Ravi and Chenab Valleys) extending westward to Kashmir, Afghanistan, and Northern Baluchistan. Neoza pine grows gregariously, forming forests of a somewhat open type, though it sometimes forms moderately dense pole crops. It is mixed with deodar in varying proportions in the region outside the influence of monsoons. The annual precipitation (about 250 to 270 mm) is received mainly in the form of snow during winter. It endures severe winter cold. The summer temperature within its habitat, however, seldom exceeds 39 C. The neoza pine makes little demand on the fertility of the soil and is capable of growing on very dry hillsides with shallow soils. Pinus gerardiana is well known for its edible seed. The seed (chilgoza) is eaten as dry fruit which is rich in oil, starch, and albumenoids. Seeds are obtained from cones which are still green. The cones are gathered from the trees, heaped up, and burned to open them, after which the seeds are picked out. Much damage is apt to be done to the trees during cone collection. The natural regeneration of this pine is deficient. There are a number of factors responsible for poor natural regeneration. First, since chilgoza is a cash crop, the rights holders remove almost all the cones for seed collection leaving none for germination; second, whenever seeds are left, they are damaged by rodents, birds, and reptiles; third, there is high seed mortality during drought; fourth, the big seed does not embed into loose sandy soil with poor soil moisture; and In: Roundy, Bruce A.; McArthur, E. Durant; Haley, Jennifer S.; Mann, David K., comps Proceedings: wildland shrub and arid land restoration symposium; 1993 October 19-21; Las Vegas, NV. Gen. Tech. Rep. INT-GTR-315. Ogden, UT: U.S. Department of Agriculture, Forest Service, Intermountain Research Station. T. N. Lakhanpal is Professor, Department of Bio-Sciences, Himachal Pradesh University Shimla , H.P. (India). fifth, heavy and unrestricted sheep and goat grazing causes a lot of damage to young seedlings (Chauhan 1986). All these factors reduce the chances of natural regeneration of this pine. Severe biotic interference and lack of regeneration in this pine may result in the extinction of this species (Kumar 1986; Sehgal and Chauhan 1989). For regeneration, it has been suggested that areas bearing chilgoza pine should be closed for a period of 30 years for rights holders. Artificial regeneration has been achieved at a number of places both by sowing and planting of nursery raised plants at Kalpa, Ralli (Kilba Range), Akpa (Morrand Range), Shongtong, and Purbani (Kalpa Range) (Chauhan 1986). However, no attention has ever been paid to the use of mycorrhiza for artificial inoculation of chilgoza pine seedlings. Saplings are usually planted after they attain a height of about 5 to 10 cm and are 3 to 4 years old. This article reports the first attempts of artificial mycorrhizal inoculation of Pinus gerardiana seedlings. Materials and Methods The mycobiont was isolated from the natural mycorrhizal roots following Marx et al. (1982) and pure cultures were raised following Mikola (1973). For raising cultures, Martins (1950), White s modified (Vasil 1959), and Potato Dextrose Peptone-Agar (Rawlings 1933) media were used. For artificial inoculation, two inoculum sources, forest soil (soil from the natural range of chilgoza pine) and pure culture of the mycobiont were used. The former involves the incorporation of about 10 to 20 percent of soil inoculum by volume in the experimental pots prior to transplanting. In the latter case mycobiont was isolated from the ectomycorrhiza itself. After four weeks, when seedlings reached the cotyledon stage, they were picked up from the experimental beds and planted in sterilized plastic pots containing thermally sterilized soil. A sufficient amount of inoculum was taken from the culture tubes and mixed with sterilized soil. A thin layer of inoculum was spread on the topsoil. The inoculum was also put at the planting hole as an additional safeguard to ensure that every seedling receives the inoculum (Mikola 1969). When mixing inoculum with potting mixture, care was taken to secure even distribution of the inoculum. After inoculation, roots were sampled periodically to estimate the number of mycorrhiza. The inoculated pots were kept in temperature- and moisture-controlled chambers in the greenhouse. The seedlings characteristics, like green luster on the foliage, height, growth, and root development, were noted during the course of experiments. Shoot height was recorded at the end of the experiments. The 102

2 seedlings were harvested, taking care that all root ends remained intact. Data on root lengths, number of laterals, total short roots (including both uninfected and mycorrhizal) and total mycorrhizal roots were recorded for individual seedlings. Since mycorrhizal roots exhibit repeated dichotomy in this plant, the branch was counted as one mycorrhiza. Ten-power magnification was used to classify short roots as mycorrhizal or uninfected. The mycobiont was isolated from the rhizoplane of Pinus gerardiana seedlings and seedlings were inoculated with the culture. The development and estimation of mycorrhiza in inoculated seedlings are presented in table 1. The seedlings inoculated with the mycobiont attained better shoot height, root length, stem diameter, total root shoot, fresh weight, and high shoot-root fresh weight ratio. The shoot height, root length, and fresh weight shoot-root ratio was significantly greater (at the 0.01 level) in the inoculated seedlings; the stem diameter was also significantly higher (at the 0.05 level). Development of mycorrhiza in inoculated seedlings results in green luster on the foliage; they are easily distinguished from uninoculated control seedlings which remained pale green. There was a significant difference (at the 0.05 level) in the mycorrhizal counts between inoculated and uninoculated control seedlings. The mycorrhizal counts show that all the seedlings which were inoculated developed ectomycorrhizal infection. None of the plants in the uninoculated controls developed any mycorrhizal short roots. Seedlings inoculated with mycobiont had 67.60% mycorrhizal short roots and 32.40% uninfected short roots. The total number of short roots (144 maximum) was higher in inoculated seedlings than in the uninoculated seedlings (96 maximum). Discussion The seedlings of Pinus gerardiana inoculated with mycorrhizal symbiont showed a 67.6% increase in mycorrhizal development. The inoculated seedlings were highly ectomycorrhizal. The number of bifurcate roots which developed four months after inoculation showed a threefold increase. Shoulders (1972) observed that inoculated slash pine seedlings had four times as many bifurcate roots at lifting as uninoculated seedlings. Trappe (1967) and Harley (1969) pointed out that bifurcate or dichotomously branched short roots are not irrefutable evidence of mycorrhizal infection, nor is their absence concrete proof that roots are not infected. Inoculation markedly increased the intensity of infection and also enhanced the survival. The abundance of bifurcated roots on seedlings appeared to be a useful index of transplanting survival. Nonmycorrhizal seedlings (table 1) grew pale and remained stunted in contrast to mycorrhizal seedlings, which grew vigorously and acquired bright green luster. Similar observations have been reported from various parts of the world following inoculation of soil with pure cultures of ectomycorrhizal fungi (Fassi et al. 1969; Theodorou and Bowen 1970; Theodorou 1971; Vozzo and Hacskaylo 1977; Lamb and Richards 1974). Kormanik et al. (1977) also reported that inoculation of plants with mycorrhizal fungi normally caused a striking increase in growth. There was a significant increase in the shoot height of mycorrhizal seedlings compared to nonmycorrhizal seedlings (table 2). The fresh weight as well as dry weight of shoots and roots of mycorrhizal plants was higher than compared to nonmycorrhizal plants (table 3). It is clear Table 1 Effect of mycorrhizal inoculation on seedlings of Pinus gerardiana Wall. after 6 months of inoculation (mean of five readings). Soil infestation Shoot Root Stem Fresh weight Shoot/root Foliage treatment height length diameter Shoot Root Total ratio luster cm mm gm Control Pale Basidiomycetous 21.2** 14.0** 4.8* 18.0* 11.0* 29.0* 1.63* mycelium 20.3* 13.6* 4.7* 19.5** 10.8** 30.3** 1.80** Green 20.6* 13.2* 5.0* 19.8** 10.5* 30.3** 1.88** Mycorrhizal Counts Number of Number of Total number mycorrhizae uninfected short roots mycorrhizal short roots of short roots development percent Control Basidiomycetous 48 (37%) 82 (63%) hyphae 46 (32.40%) 96 (67.69%) (39.89%) 88 (61.11%) *P 0.05 = significant; **P 0.01 = highly significant. 103

3 Table 2 Shoot height of 8-month-old mycorrhizal and nonmycorrhizal seedlings of Pinus gerardiana Wall. Mycorrhizal Nonmycorrhizal t value Sample (Mean ± SE) (Mean ± SE) (df = 8) cm ± ± * ± ± * ± ± * ± ± * ± ± ** ± ± * ± ± ** ± ± ** ± ± ** ± ± * SE = standard error about mean; df = degree of freedom; *P 0.05 = significant; **P 0.01 = highly significant. from table 4 that the shoot/root ratio for both fresh weight and dry weight was significantly higher in mycorrhizal plants. However, there were no obvious differences in soil nutrients (organic carbon percentage, total nitrogen percentage, available phosphorus and available potassium) and ph of the soils. The soils were low in nitrogen, available phosphorus and available potassium. The ph of unsterilized soil was more acidic as compared to sterilized soil (table 5). Significant differences were obtained in the percentage of nitrogen, phosphorus, potassium, calcium, and magnesium accumulated in needles of the mycorrhizal and nonmycorrhizal seedlings. Needles of the mycorrhizal seedlings generally showed the higher concentration of these elements (table 6). The percentage of nitrogen accumulation in the needles varied from 0.95 to 0.98 in mycorrhizal seedlings and from 0.72 to 0.76 in the nonmycorrhizal seedlings. The difference was significant at the 0.05 probability level. The gain in phosphorus by the needles of mycorrhizal seedlings was three times that of nonmycorrhizal seedlings. Phosphorus levels in the mycorrhizal needles varied from 1.27 to 1.28 percent, whereas in nonmycorrhizal needles phosphorus varied from 0.39 to 0.43 percent. The difference Table 4 Shoot/root ratio per plant of 8-month-old mycorrhizal and nonmycorrhizal Pinus gerardiana Wall. seedlings. Mycorrhizal Nonmycorrhizal t value Sample (Mean ± SE) (Mean ± SE) (df = 8) Fresh weight shoot/root ratio ± ± * ± ± * ± ± ** ± ± NS ± ± * ± ± * ± ± ** ± ± * ± ± NS ± ± * Dry weight shoot/root ratio ± ± ** ± ± ** ± ± * ± ± NS ± ± * ± ± * ± ± * ± ± ** ± ± * ± ± * SE = standard error; df = degree of freedom; *P 0.05 = significant; **P 0.01 = highly significant; NS = nonsignificant. was significant at the 0.01 probability level (table 5). The level of potassium, calcium, and magnesium in the needles of mycorrhizal seedlings was significantly higher compared to nonmycorrhizal seedlings. The total nutrient percentage in shoots and roots was higher in mycorrhizal seedlings compared to nonmycorrhizal seedlings (table 7). The difference in accumulation of phosphorus in mycorrhizal and nonmycorrhizal seedlings was threefold; the difference was significant at the 0.01 level. Nitrogen, potassium, calcium, and magnesium are significantly higher in the roots and shoots of mycorrhizal plants at the 0.05 probability level. Inoculation of seedlings with mycorrhizal fungi clearly increases overall growth and development. In these isolations Table 3 Fresh weight and dry weight of 8-month-old mycorrhizal and nonmycorrhizal seedlings of Pinus gerardiana Wall. Mycorrhizal Nonmycorrhizal Fresh weight Dry weight Fresh weight Dry weight Sample Shoot Root Shoot Root Shoot Root Shoot Root gm

4 Table 5 Nutrient content of sterilized and unsterilized soils in which nonmycorrhizal and mycorrhizal seedlings of Pinus gerardiana Wall. were raised. Each figure is the mean of five readings. Total Available Organic nitrogen soil nutrients Treatment Soil ph carbon content P 2 O 5 K 2 O Percent lb/acre Unsterilized soil Sterilized soil Table 6 Elemental compostition of needles of 6-month-old mycorrhizal and nonmycorrhizal seedlings of Pinus gerardiana Wall. Each figure represents the mean of five readings. Nutrient Mycorrhizal Nonmycorrhizal t value elements (Mean ± SE) (Mean ± SE) (df = 8) Percent Nitrogen 0.98 ± ± * 0.96 ± ± * 0.95 ± ± * Phosphorus 1.28 ± ± ** 1.27 ± ± * 1.28 ± ± ** Potassium 0.63 ± ± ± ± ** 0.72 ± ± ** Calcium 0.36 ± ± * 0.39 ± ± ** 0.37 ± ± * Magnesium 0.30 ± ± ** 0.28 ± ± * 0.28 ± ± * SE = standard error about mean; df = degree of freedom; *P 0.05 = significant; **P 0.01 = highly significant. the inoculum was from the excised mycorrhizal roots. There is need to collect associated fungi and try their pure cultures for mycorrhizal synthesis; it has been reported that different fungi and their strains differ in their capacity to form mycorrhiza. Nevertheless, it is conclusively proven that in inoculated seedlings the transplanting period will be reduced almost by a year or so, which if calculated in terms of time, money, and energy is a lot of saving. References Chauhan, B. S Regeneration in Chilgoza pine. Proc. of conf. on Silviculture. 7 p. Fassi, B.; Fontana, A.; Trappe, J. M Ectomycorrhizae formed by Endogone lactiflua with species of Pinus and Pseudotsuga. Mycologia. 61: Harely, J. L The biology of mycorrhiza. Leonard Hill, London. Kormanik, P. P.; Bryan, W. C.; Schultz, R. C In: Vines, H. M., ed. The role of mycorrhiza in plant growth and development. South. Sect. Am. Soc. Plant Physiol. Atlanta, GA. Kumar, P Studies on phenotypic variations in natural stands of Pinus gerardiana Wall. In: Kinnaur, H.P. 77, V, XVI P. M.Sc. Dissertation submitted to Dept. of Forestry, Dr. Y. S. Parmar University of Horticulture and Forestry, Solan, H. P. Lamb, R. J.; Richards, B. N Survival potential of sexual and asexual spores of ectomycorrhizal fungi. Trans. Br. Mycol. Soc. 54: Martin, J. P Use of acid rose bengal and streptomycin in a plate method for estimating soil fungi. Soil Sci. 69: Marx, D. H.; Ruehle, J. L.; Kenney, D. S.; Cordell, C. E.; Riffle, J. W.; Molina, R. J.; Pawnk, W. H.; Mavratil, S.; Tinus, R. W.; Goodwin, O. C Commercial vegetative Table 7 Nutrient content of shoots of 6-month-old mycorrhizal and nonmycorrhizal seedlings of Pinus gerardiana Wall. Each figure is the mean of five readings. Mycorrhizal Nonmychorrizal t value Nutrient Shoot Root Total ± SE Shoot Root Total ± SE df = Percent Nitrogen ± ± * ± ± * ± ± * Phosphorus ± ± ** ± ± ** ± ± * Potassium ± ± * ± ± ** ± ± * Calcium ± ± * ± ± * ± ± ** Magnesium ± ± NS ± ± * ± ± * SE = standard error; df = degree of freedom; *P 0.05 = significant; **P 0.01 = highly significant; NS = nonsignificant. 105

5 inoculum of Pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on container grown tree seedlings. For. Sci. 28: Mikola, P Application of mycorrhizal symbiosis in forestry practice. In: Marks, G. C.; Kozlowski, T. T., eds. Ectomycorrhizae; their ecology and physiology. Acad. Press, NY: Rawlings, G. B Phytopathological and botanical research methods. John Wiley and Sons, London. Sehgal, R. N.; Chauhan, V Pinus gerardiana the threatened pine of India; life support species, biological diversity and genetic resources news, Commonwealth Science Council (In press). Shoulders, E Mycorrhizal inoculation, influences, survival growth and chemical composition of slash pine seedlings. South. Forest Exp. Stn., New Orleans, LA USDA For. Serv. Res. Pap. SO p. Theodorou, C.; Bowen, G. D Mycorrhizal responses of radiata pine in experiments with different fungi. Aust. Forest. 34: 183. Theodorou, C.; Bowen, C. L Effects of non host plants on growth of mycorrhizal fungi of radiata pine. Aust. For. 35: Trappe, J. M Pure culture synthesis of Douglas-fir mycorrhizae with species of Hebeloma, Suillus, Rhizopogon and Astraeus. For. Sci. 13: Vasil, I. K Cultivation of excised anthers in vitro. J. Exp. Bot. 10: Vozzo, J. A.; Hacskaylo, E Inoculation of Pinus caribaea with ectomycorrhizal fungi in Puerto Rico. For. Sci. 17: