Regeneration of Cold Desert Pine of N.W

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.) fifth, heavy and unrestricted sheep and goat grazing causes has been subjected to overexploitation because of the commercial a lot of damage to young seedlings (Chauhan 1986). All value of its edible seeds and ethnic uses. Regeneration is deficient. these factors reduce the chances of natural regeneration Preliminary studies conducted by inoculating the seedlings with of this pine. Severe biotic interference and lack of regener- mycorrhiza show great promise in establishment and performance ation in this pine may result in the extinction of this species of the seedlings. (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 Pinus gerardiana, commonly and commercially known a number of places both by sowing and planting of nursery as ‘chilgoza’ and/or ‘neoza’ pine, is a forest tree restricted raised plants at Kalpa, Ralli (Kilba Range), Akpa (Morrand in India to dry inner valleys of the Northwest Himalayas Range), Shongtong, and Purbani (Kalpa Range) (Chauhan (1,600 to 3,000 m elevation). It occurs in Kinnaur (Satluj 1986). Valley) and Pangi in Himachal Pradesh (Ravi and Chenab However, no attention has ever been paid to the use of Valleys) extending westward to Kashmir, Afghanistan, mycorrhiza for artificial inoculation of chilgoza pine seed- and Northern Baluchistan. lings. Saplings are usually planted after they attain a Neoza pine grows gregariously, forming forests of a some- height of about 5 to 10 cm and are 3 to 4 years old. This what open type, though it sometimes forms moderately article reports the first attempts of artificial mycorrhizal dense pole crops. It is mixed with deodar in varying pro- inoculation of Pinus gerardiana seedlings. portions 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 habi- Materials and Methods tat, however, seldom exceeds 39 °C. The neoza pine makes The mycobiont was isolated from the natural mycorrhizal little demand on the fertility of the soil and is capable of roots following Marx et al. (1982) and pure cultures were growing on very dry hillsides with shallow soils. raised following Mikola (1973). Pinus gerardiana is well known for its edible seed. The For raising cultures, Martins (1950), White’s modified seed (chilgoza) is eaten as dry fruit which is rich in oil, (Vasil 1959), and Potato Dextrose Peptone-Agar (Rawlings starch, and albumenoids. Seeds are obtained from cones 1933) media were used. For artificial inoculation, two which are still green. The cones are gathered from the inoculum sources, forest soil (soil from the natural range trees, heaped up, and burned to open them, after which of chilgoza pine) and pure culture of the mycobiont were the seeds are picked out. Much damage is apt to be done used. The former involves the incorporation of about 10 to the trees during cone collection. to 20 percent of soil inoculum by volume in the experimen- The natural regeneration of this pine is deficient. There tal pots prior to transplanting. In the latter case mycobiont are a number of factors responsible for poor natural regener- was isolated from the ectomycorrhiza itself. ation. First, since ‘chilgoza’ is a cash crop, the rights holders After four weeks, when seedlings reached the cotyledon remove almost all the cones for seed collection leaving none stage, they were picked up from the experimental beds and for germination; second, whenever seeds are left, they are planted in sterilized plastic pots containing thermally ster- damaged by rodents, birds, and reptiles; third, there is high ilized soil. A sufficient amount of inoculum was taken from seed mortality during drought; fourth, the big seed does not the culture tubes and mixed with sterilized soil. A thin embed into loose sandy soil with poor soil moisture; and layer of inoculum was spread on the topsoil. The inoculum was also put at the planting hole as an additional safe- guard to ensure that every seedling receives the inoculum (Mikola 1969). When mixing inoculum with potting mix- ture, 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 In: Roundy, Bruce A.; McArthur, E. Durant; Haley, Jennifer S.; Mann, were kept in temperature- and moisture-controlled cham- David K., comps. 1995. Proceedings: wildland shrub and arid land resto- ration symposium; 1993 October 19-21; Las Vegas, NV. Gen. Tech. Rep. bers in the greenhouse. The seedlings’ characteristics, like INT-GTR-315. Ogden, UT: U.S. Department of Agriculture, Forest Service, green luster on the foliage, height, growth, and root develop- Intermountain Research Station. T. N. Lakhanpal is Professor, Department of Bio-Sciences, Himachal ment, were noted during the course of experiments. Shoot Pradesh University Shimla- 171005, H.P. (India). height was recorded at the end of the experiments. The

102 seedlings were harvested, taking care that all root ends Discussion remained intact. Data on root lengths, number of laterals, total short roots (including both uninfected and mycorrhizal) The seedlings of Pinus gerardiana inoculated with my- and total mycorrhizal roots were recorded for individual corrhizal symbiont showed a 67.6% increase in mycorrhizal seedlings. Since mycorrhizal roots exhibit repeated dichot- development. The inoculated seedlings were highly ectomy- omy in this plant, the branch was counted as one mycorrhiza. corrhizal. The number of bifurcate roots which developed Ten-power magnification was used to classify short roots four months after inoculation showed a threefold increase. as mycorrhizal or uninfected. Shoulders (1972) observed that inoculated slash pine seed- The mycobiont was isolated from the rhizoplane of Pinus lings had four times as many bifurcate roots at lifting as gerardiana seedlings and seedlings were inoculated with uninoculated seedlings. Trappe (1967) and Harley (1969) the culture. The development and estimation of mycorrhiza pointed out that bifurcate or dichotomously branched in inoculated seedlings are presented in table 1. short roots are not irrefutable evidence of mycorrhizal in- The seedlings inoculated with the mycobiont attained fection, nor is their absence concrete proof that roots are better shoot height, root length, stem diameter, total root not infected. shoot, fresh weight, and high shoot-root fresh weight ratio. Inoculation markedly increased the intensity of infection The shoot height, root length, and fresh weight shoot-root and also enhanced the survival. The abundance of bifur- ratio was significantly greater (at the 0.01 level) in the in- cated roots on seedlings appeared to be a useful index of oculated seedlings; the stem diameter was also significantly transplanting survival. Nonmycorrhizal seedlings (table 1) higher (at the 0.05 level). Development of mycorrhiza in grew pale and remained stunted in contrast to mycorrhizal inoculated seedlings results in green luster on the foliage; seedlings, which grew vigorously and acquired bright green they are easily distinguished from uninoculated control luster. Similar observations have been reported from various seedlings which remained pale green. parts of the world following inoculation of soil with pure cul- There was a significant difference (at the 0.05 level) in tures of ectomycorrhizal fungi (Fassi et al. 1969; Theodorou the mycorrhizal counts between inoculated and uninoculated and Bowen 1970; Theodorou 1971; Vozzo and Hacskaylo control seedlings. The mycorrhizal counts show that all the 1977; Lamb and Richards 1974). Kormanik et al. (1977) seedlings which were inoculated developed ectomycorrhizal also reported that inoculation of plants with mycorrhizal infection. None of the plants in the uninoculated controls fungi normally caused a striking increase in growth. developed any mycorrhizal short roots. Seedlings inocu- There was a significant increase in the shoot height of lated with mycobiont had 67.60% mycorrhizal short roots mycorrhizal seedlings compared to nonmycorrhizal seed- and 32.40% uninfected short roots. The total number of lings (table 2). The fresh weight as well as dry weight of short roots (144 maximum) was higher in inoculated seed- shoots and roots of mycorrhizal plants was higher than lings than in the uninoculated seedlings (96 maximum). 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 12.5 8.6 4.2 12.0 8.1 20.1 1.48 11.6 8.3 4.6 13.5 7.9 21.4 1.70 Pale 13.0 8.5 4.5 13.0 7.8 20.8 1.54

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 80 0 80 0 96 0 96 0 92 0 92 0

Basidiomycetous 48 (37%) 82 (63%) 130 100 hyphae 46 (32.40%) 96 (67.69%) 142 100 54 (39.89%) 88 (61.11%) 144 100

*P 0.05 = significant; **P 0.01 = highly significant.

103 Table 2—Shoot height of 8-month-old mycorrhizal and Table 4—Shoot/root ratio per plant of 8-month-old mycorrhizal and nonmycorrhizal seedlings of Pinus gerardiana Wall. nonmycorrhizal Pinus gerardiana Wall. seedlings.

Mycorrhizal Nonmycorrhizal ‘t’ value Mycorrhizal Nonmycorrhizal ‘t’ value Sample (Mean ± SE) (Mean ± SE) (df = 8) Sample (Mean ± SE) (Mean ± SE) (df = 8) ------cm------Fresh weight shoot/root ratio 1 17.2 ± 0.28 12.1 ± 0.28 2.65* 1 6.28 ± 0.21 4.54 ± 0.21 2.17* 2 16.8 ± 0.32 11.2 ± 0.32 2.65* 2 5.91 ± 0.26 5.21 ± 0.26 2.65* 3 16.4 ± 0.31 10.2 ± 0.31 3.00* 3 6.28 ± 0.24 5.16 ± 0.24 2.35** 4 14.7 ± 0.32 8.2 ± 0.32 2.25* 4 5.82 ± 0.18 3.86 ± 0.18 1.75 NS 5 17.4 ± 0.36 8.4 ± 0.36 3.00** 5 6.78 ± 0.22 4.42 ± 0.22 3.00* 6 16.8 ± 0.28 9.2 ± 0.28 2.65* 6 6.36 ± 0.24 4.36 ± 0.24 1.65* 7 16.2 ± 0.30 8.8 ± 0.30 2.65** 7 6.14 ± 0.23 4.08 ± 0.23 2.35** 8 17.2 ± 0.31 8.6 ± 0.31 1.75** 8 6.36 ± 0.24 4.62 ± 0.24 1.75* 9 17.4 ± 0.28 9.1 ± 0.28 3.00** 9 6.52 ± 0.25 4.15 ± 0.25 1.65 NS 10 16.6 ± 0.34 8.5 ± 0.34 2.25* 10 6.64 ± 0.24 4.36 ± 0.24 2.60* SE = standard error about mean; df = degree of freedom; *P 0.05 = signifi- Dry weight shoot/root ratio cant; **P 0.01 = highly significant. 1 3.98 ± 0.20 2.82 ± 0.20 2.25** 2 4.38 ± 0.17 3.16 ± 0.17 2.65** 3 4.32 ± 0.18 3.26 ± 0.18 2.40* 4 4.28 ± 0.21 2.17 ± 0.21 1.50 NS from table 4 that the shoot/root ratio for both fresh weight 5 4.36 ± 0.24 3.67 ± 0.24 3.25* and dry weight was significantly higher in mycorrhizal 6 4.08 ± 0.22 2.87 ± 0.22 3.00* plants. 7 3.68 ± 0.21 2.67 ± 0.21 2.54* However, there were no obvious differences in soil nutri- 8 4.28 ± 0.24 2.64 ± 0.24 3.75** ents (organic carbon percentage, total nitrogen percentage, 9 4.26 ± 0.21 3.07 ± 0.21 2.65* available phosphorus and available potassium) and pH of 10 4.17 ± 0.18 2.94 ± 0.18 3.00* the soils. The soils were low in nitrogen, available phospho- SE = standard error; df = degree of freedom; *P 0.05 = significant; rus and available potassium. The pH of unsterilized soil **P 0.01 = highly significant; NS = nonsignificant. was more acidic as compared to sterilized soil (table 5). Significant differences were obtained in the percentage of nitrogen, phosphorus, potassium, calcium, and magne- was significant at the 0.01 probability level (table 5). The sium accumulated in needles of the mycorrhizal and non- level of potassium, calcium, and magnesium in the needles mycorrhizal seedlings. Needles of the mycorrhizal seed- of mycorrhizal seedlings was significantly higher compared lings generally showed the higher concentration of these to nonmycorrhizal seedlings. elements (table 6). The percentage of nitrogen accumula- The total nutrient percentage in shoots and roots was tion in the needles varied from 0.95 to 0.98 in mycorrhizal higher in mycorrhizal seedlings compared to nonmycor- seedlings and from 0.72 to 0.76 in the nonmycorrhizal seed- rhizal seedlings (table 7). The difference in accumulation lings. The difference was significant at the 0.05 probability of phosphorus in mycorrhizal and nonmycorrhizal seed- level. lings was threefold; the difference was significant at the The gain in phosphorus by the needles of mycorrhizal 0.01 level. Nitrogen, potassium, calcium, and magnesium seedlings was three times that of nonmycorrhizal seedlings. are significantly higher in the roots and shoots of mycor- Phosphorus levels in the mycorrhizal needles varied from rhizal plants at the 0.05 probability level. 1.27 to 1.28 percent, whereas in nonmycorrhizal needles Inoculation of seedlings with mycorrhizal fungi clearly in- phosphorus varied from 0.39 to 0.43 percent. The difference creases 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 ------1 1.76 1.15 0.66 0.46 1.62 0.96 0.36 0.21 2 1.84 0.98 0.47 0.37 1.71 0.72 0.29 0.19 3 1.90 0.86 0.61 0.42 1.58 0.64 0.40 0.24 4 1.86 0.95 0.86 0.38 1.38 0.82 0.32 0.26 5 1.56 1.20 0.94 0.46 1.48 0.86 0.32 0.23 6 1.64 1.16 0.96 0.51 1.62 0.78 0.36 0.21 7 1.75 0.88 0.90 0.32 1.70 0.98 0.32 0.27 8 1.82 0.96 0.65 0.46 1.56 0.96 0.31 0.26 9 1.58 1.05 0.70 0.42 1.35 0.94 0.28 0.21 10 1.72 1.22 0.98 0.42 1.68 0.72 0.36 0.24

104 Table 5—Nutrient content of sterilized and unsterilized soils in which the inoculum was from the excised mycorrhizal roots. There nonmycorrhizal and mycorrhizal seedlings of Pinus ger- is need to collect associated fungi and try their pure cultures ardiana Wall. were raised. Each figure is the mean of five for mycorrhizal synthesis; it has been reported that differ- readings. ent fungi and their strains differ in their capacity to form Total Available mycorrhiza. Nevertheless, it is conclusively proven that in Organic nitrogen soil nutrients inoculated seedlings the transplanting period will be reduced

Treatment Soil pH carbon content P2O5 K2O almost by a year or so, which if calculated in terms of time, money, and energy is a lot of saving. - - - - -Percent ------lb/acre - - - Unsterilized soil 6.4 0.54 0.32 48 128 Sterilized soil 6.2 0.57 0.34 49 132 References

Chauhan, B. S. 1986. Regeneration in Chilgoza pine. Proc. of conf. on Silviculture. 7 p. Table 6—Elemental compostition of needles of 6-month-old mycor- Fassi, B.; Fontana, A.; Trappe, J. M. 1969. Ectomycorrhizae rhizal and nonmycorrhizal seedlings of Pinus gerardiana formed by Endogone lactiflua with species of Pinus and Wall. Each figure represents the mean of five readings. Pseudotsuga. Mycologia. 61: 412-414. Harely, J. L. 1969. The biology of mycorrhiza. Leonard Hill, Nutrient Mycorrhizal Nonmycorrhizal ‘t’ value London. elements (Mean ± SE) (Mean ± SE) (df = 8) Kormanik, P. P.; Bryan, W. C.; Schultz, R. C. 1977. In: ------Percent ------Vines, H. M., ed. The role of mycorrhiza in plant growth Nitrogen 0.98 ± 0.04 0.74 ± 0.04 0.80* and development. South. Sect. Am. Soc. Plant Physiol. 0.96 ± 0.06 0.72 ± 0.06 0.85* Atlanta, GA. 0.95 ± 0.03 0.76 ± 0.03 1.00* Kumar, P. 1986. Studies on phenotypic variations in natu- Phosphorus 1.28 ± 0.02 0.41 ± 0.02 3.25** ral stands of Pinus gerardiana Wall. In: Kinnaur, H.P. 1.27 ± 0.05 0.43 ± 0.05 2.80* 77, V, XVI P. M.Sc. Dissertation submitted to Dept. of 1.28 ± 0.03 0.39 ± 0.03 3.00** Forestry, Dr. Y. S. Parmar University of Horticulture Potassium 0.63 ± 0.04 0.43 ± 0.04 2.58 and Forestry, Solan, H. P. 0.67 ± 0.04 0.44 ± 0.04 2.25** Lamb, R. J.; Richards, B. N. 1974. Survival potential of 0.72 0.05 0.49 0.05 2.65** ± ± sexual and asexual spores of ectomycorrhizal fungi. Calcium 0.36 ± 0.06 0.32 ± 0.06 2.60* Trans. Br. Mycol. Soc. 54: 371-378. 0.39 ± 0.03 0.36 ± 0.03 2.48** Martin, J. P. 1950. Use of acid rose bengal and streptomy- 0.37 ± 0.04 0.38 ± 0.04 1.90* cin in a plate method for estimating soil fungi. Soil Sci. Magnesium 0.30 ± 0.02 0.21 ± 0.02 3.25** 69: 215-232. 0.28 0.02 0.22 0.02 3.00* ± ± Marx, D. H.; Ruehle, J. L.; Kenney, D. S.; Cordell, C. E.; 0.28 ± 0.04 0.26 ± 0.04 3.20* Riffle, J. W.; Molina, R. J.; Pawnk, W. H.; Mavratil, S.; SE = standard error about mean; df = degree of freedom; *P 0.05 = signifi- Tinus, R. W.; Goodwin, O. C. 1982. Commercial vegetative cant; **P 0.01 = highly significant.

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 = 8 ------Percent ------Nitrogen 0.98 0.76 1.74 ± 0.02 0.68 0.50 1.18 ± 0.02 1.65* 0.96 0.82 1.78 ± 0.03 0.72 0.53 1.25 ± 0.02 2.65* 0.98 0.81 1.71 ± 0.02 0.64 0.54 1.18 ± 0.02 1.75* Phosphorus 1.17 0.98 2.15 ± 0.06 0.39 0.26 0.65 ± 0.06 2.00** 1.18 0.93 2.11 ± 0.05 0.32 0.27 0.59 ± 0.05 3.65** 1.11 0.89 2.00 ± 0.07 0.36 0.30 0.66 ± 0.07 3.25* Potassium 0.54 0.46 1.00 ± 0.01 0.24 0.18 0.42 ± 0.01 2.30* 0.52 0.50 1.02 ± 0.02 0.26 0.21 0.47 ± 0.02 3.25** 0.57 0.54 1.11 ± 0.01 0.25 0.20 0.25 ± 0.01 2.65* Calcium 0.32 0.30 0.62 ± 0.03 0.24 0.22 0.46 ± 0.04 2.65* 0.34 0.32 0.66 ± 0.04 0.21 0.18 0.39 ± 0.04 2.65* 0.31 0.33 0.64 ± 0.01 0.23 0.21 0.44 ± 0.01 3.00** Magnesium 0.28 0.26 0.54 ± 0.02 0.18 0.16 0.34 ± 0.02 1.65 NS 0.24 0.23 0.47 ± 0.02 0.20 0.17 0.37 ± 0.02 2.65* 0.23 0.21 0.44 ± 0.03 0.18 0.13 0.31 ± 0.03 2.65* SE = standard error; df = degree of freedom; *P 0.05 = significant; **P 0.01 = highly significant; NS = nonsignificant.

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