Regeneration of Cold Desert Pine of N.W.
Himalayas (India)—A Preliminary Study
T. N. Lakhanpal
Sunil Kumar
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.
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
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
In: Roundy, Bruce A.; McArthur, E. Durant; Haley, Jennifer S.; Mann,
David K., comps. 1995. 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- 171005, H.P. (India).
102
Discussion
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).
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
treatment
Control
Basidiomycetous
mycelium
Shoot
height
Root
length
Stem
diameter
Shoot
Fresh weight
Root
Total
Shoot/root
ratio
Foliage
luster
20.1
21.4
20.8
1.48
1.70
1.54
Pale
29.0*
30.3**
30.3**
1.63*
1.80**
1.88**
Green
- - - - - - cm - - - - - -
mm
- - - - - - - - - gm - - - - - - - - -
12.5
11.6
13.0
8.6
8.3
8.5
4.2
4.6
4.5
12.0
13.5
13.0
14.0**
13.6*
13.2*
4.8*
4.7*
5.0*
18.0*
19.5**
19.8**
21.2**
20.3*
20.6*
Number of
uninfected short roots
8.1
7.9
7.8
11.0*
10.8**
10.5*
Mycorrhizal Counts
Number of
Total number
mycorrhizal short roots
of short roots
mycorrhizae
development
percent
Control
80
96
92
Basidiomycetous
hyphae
48 (37%)
46 (32.40%)
54 (39.89%)
0
0
0
82 (63%)
96 (67.69%)
88 (61.11%)
*P 0.05 = significant; **P 0.01 = highly significant.
103
80
96
92
0
0
0
130
142
144
100
100
100
Table 2—Shoot height of 8-month-old mycorrhizal and
nonmycorrhizal seedlings of Pinus gerardiana Wall.
Sample
Mycorrhizal
(Mean ± SE)
1
2
3
4
5
6
7
8
9
10
17.2 ± 0.28
16.8 ± 0.32
16.4 ± 0.31
14.7 ± 0.32
17.4 ± 0.36
16.8 ± 0.28
16.2 ± 0.30
17.2 ± 0.31
17.4 ± 0.28
16.6 ± 0.34
Nonmycorrhizal
(Mean ± SE)
Table 4—Shoot/root ratio per plant of 8-month-old mycorrhizal and
nonmycorrhizal Pinus gerardiana Wall. seedlings.
‘t’ value
(df = 8)
Sample
Mycorrhizal
(Mean ± SE)
1
2
3
4
5
6
7
8
9
10
6.28 ± 0.21
5.91 ± 0.26
6.28 ± 0.24
5.82 ± 0.18
6.78 ± 0.22
6.36 ± 0.24
6.14 ± 0.23
6.36 ± 0.24
6.52 ± 0.25
6.64 ± 0.24
1
2
3
4
5
6
7
8
9
10
3.98 ± 0.20
4.38 ± 0.17
4.32 ± 0.18
4.28 ± 0.21
4.36 ± 0.24
4.08 ± 0.22
3.68 ± 0.21
4.28 ± 0.24
4.26 ± 0.21
4.17 ± 0.18
- - - - - - - - - - - cm- - - - - - - - - - -
Nonmycorrhizal
(Mean ± SE)
‘t’ value
(df = 8)
Fresh weight shoot/root ratio
12.1 ± 0.28
11.2 ± 0.32
10.2 ± 0.31
8.2 ± 0.32
8.4 ± 0.36
9.2 ± 0.28
8.8 ± 0.30
8.6 ± 0.31
9.1 ± 0.28
8.5 ± 0.34
2.65*
2.65*
3.00*
2.25*
3.00**
2.65*
2.65**
1.75**
3.00**
2.25*
4.54 ± 0.21
5.21 ± 0.26
5.16 ± 0.24
3.86 ± 0.18
4.42 ± 0.22
4.36 ± 0.24
4.08 ± 0.23
4.62 ± 0.24
4.15 ± 0.25
4.36 ± 0.24
2.17*
2.65*
2.35**
1.75 NS
3.00*
1.65*
2.35**
1.75*
1.65 NS
2.60*
Dry weight shoot/root ratio
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
2.82 ± 0.20
3.16 ± 0.17
3.26 ± 0.18
2.17 ± 0.21
3.67 ± 0.24
2.87 ± 0.22
2.67 ± 0.21
2.64 ± 0.24
3.07 ± 0.21
2.94 ± 0.18
2.25**
2.65**
2.40*
1.50 NS
3.25*
3.00*
2.54*
3.75**
2.65*
3.00*
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
Sample
Fresh weight
Shoot
Root
Dry weight
Shoot
Root
Nonmycorrhizal
Fresh weight
Dry weight
Shoot
Root
Shoot
Root
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - gm - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1
2
3
4
5
6
7
8
9
10
1.76
1.84
1.90
1.86
1.56
1.64
1.75
1.82
1.58
1.72
1.15
0.98
0.86
0.95
1.20
1.16
0.88
0.96
1.05
1.22
0.66
0.47
0.61
0.86
0.94
0.96
0.90
0.65
0.70
0.98
0.46
0.37
0.42
0.38
0.46
0.51
0.32
0.46
0.42
0.42
104
1.62
1.71
1.58
1.38
1.48
1.62
1.70
1.56
1.35
1.68
0.96
0.72
0.64
0.82
0.86
0.78
0.98
0.96
0.94
0.72
0.36
0.29
0.40
0.32
0.32
0.36
0.32
0.31
0.28
0.36
0.21
0.19
0.24
0.26
0.23
0.21
0.27
0.26
0.21
0.24
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.
Treatment
Soil pH
Unsterilized soil
Sterilized soil
6.4
6.2
Organic
carbon
Total
nitrogen
content
- - - - -Percent - - - 0.54
0.57
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.
Available
soil nutrients
P2O5
K2O
- - -lb/acre - - -
0.32
0.34
48
49
128
132
References
Chauhan, B. S. 1986. Regeneration in Chilgoza pine. Proc.
of conf. on Silviculture. 7 p.
Fassi, B.; Fontana, A.; Trappe, J. M. 1969. Ectomycorrhizae
formed by Endogone lactiflua with species of Pinus and
Pseudotsuga. Mycologia. 61: 412-414.
Harely, J. L. 1969. The biology of mycorrhiza. Leonard Hill,
London.
Kormanik, P. P.; Bryan, W. C.; Schultz, R. C. 1977. In:
Vines, H. M., ed. The role of mycorrhiza in plant growth
and development. South. Sect. Am. Soc. Plant Physiol.
Atlanta, GA.
Kumar, P. 1986. 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. 1974. Survival potential of
sexual and asexual spores of ectomycorrhizal fungi.
Trans. Br. Mycol. Soc. 54: 371-378.
Martin, J. P. 1950. Use of acid rose bengal and streptomycin in a plate method for estimating soil fungi. Soil Sci.
69: 215-232.
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. 1982. Commercial vegetative
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
elements
Mycorrhizal
(Mean ± SE)
Nonmycorrhizal
(Mean ± SE)
‘t’ value
(df = 8)
- - - - - - - - -Percent - - - - - - - - - Nitrogen
0.98 ± 0.04
0.96 ± 0.06
0.95 ± 0.03
0.74 ± 0.04
0.72 ± 0.06
0.76 ± 0.03
0.80*
0.85*
1.00*
Phosphorus
1.28 ± 0.02
1.27 ± 0.05
1.28 ± 0.03
0.41 ± 0.02
0.43 ± 0.05
0.39 ± 0.03
3.25**
2.80*
3.00**
Potassium
0.63 ± 0.04
0.67 ± 0.04
0.72 ± 0.05
0.43 ± 0.04
0.44 ± 0.04
0.49 ± 0.05
2.58
2.25**
2.65**
Calcium
0.36 ± 0.06
0.39 ± 0.03
0.37 ± 0.04
0.32 ± 0.06
0.36 ± 0.03
0.38 ± 0.04
2.60*
2.48**
1.90*
Magnesium
0.30 ± 0.02
0.28 ± 0.02
0.28 ± 0.04
0.21 ± 0.02
0.22 ± 0.02
0.26 ± 0.04
3.25**
3.00*
3.20*
SE = standard error about mean; df = degree of freedom; *P 0.05 = significant; **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.
Nutrient
Shoot
Mycorrhizal
Root
Total ± SE
Nonmychorrizal
Shoot
Root Total ± SE
‘t’ value
df = 8
- - - - - - - - - - - - - - - - - - - - - - - Percent - - - - - - - - - - - - - - - - - - - - - Nitrogen
0.98
0.96
0.98
0.76
0.82
0.81
1.74 ± 0.02
1.78 ± 0.03
1.71 ± 0.02
0.68
0.72
0.64
0.50
0.53
0.54
1.18 ± 0.02
1.25 ± 0.02
1.18 ± 0.02
1.65*
2.65*
1.75*
Phosphorus
1.17
1.18
1.11
0.98
0.93
0.89
2.15 ± 0.06
2.11 ± 0.05
2.00 ± 0.07
0.39
0.32
0.36
0.26
0.27
0.30
0.65 ± 0.06
0.59 ± 0.05
0.66 ± 0.07
2.00**
3.65**
3.25*
Potassium
0.54
0.52
0.57
0.46
0.50
0.54
1.00 ± 0.01
1.02 ± 0.02
1.11 ± 0.01
0.24
0.26
0.25
0.18
0.21
0.20
0.42 ± 0.01
0.47 ± 0.02
0.25 ± 0.01
2.30*
3.25**
2.65*
Calcium
0.32
0.34
0.31
0.30
0.32
0.33
0.62 ± 0.03
0.66 ± 0.04
0.64 ± 0.01
0.24
0.21
0.23
0.22
0.18
0.21
0.46 ± 0.04
0.39 ± 0.04
0.44 ± 0.01
2.65*
2.65*
3.00**
Magnesium
0.28
0.24
0.23
0.26
0.23
0.21
0.54 ± 0.02
0.47 ± 0.02
0.44 ± 0.03
0.18
0.20
0.18
0.16
0.17
0.13
0.34 ± 0.02
0.37 ± 0.02
0.31 ± 0.03
1.65 NS
2.65*
2.65*
SE = standard error; df = degree of freedom; *P 0.05 = significant; **P 0.01 = highly significant; NS = nonsignificant.
105
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106