Introduction
The
soil pH relates closely to microorganism activity (Song et al.
2019), conversion and release of soil nutrient elements (Schwamberger and Sims 1991), and effective
micro-element in the soil (Deng et al. 2017). Tobacco (Nicotiana
tabacum L.) could normally grow in the soil of pH 4.5~8.5 (Cao 1991); yet different pH is bound to affect its
growth. The volume, dry weight and vitality of tobacco root increased with
ascending pH in hydroponic culture in pH 4.5~8.5; nevertheless, declining to
the lowest at pH 8.5 (Xu et al.
2004). In another study, tobacco growth varied as the reversed parabolic curve,
and dry matter accumulation of plant reached to the top at pH 6.5 (Zhu et al. 2012). The
soil pH was adjusted from 5.0 to pH 5.5~6.6 by putting calcium oxide on soil,
which greatly facilitated the root growth of tobacco (Tang and Xiong 2002). In
root extension period, tobacco grows more prosperously under weak acid
conditions comparing with medium and weak alkaline ones, which was unveiled by
adjusting to pH environment using sand cultivation (Ren et al. 1995). Different tobacco varieties have different demands of
pH for optimum root growth (Zhou et
al. 2000). Slightly acid condition (pH 5.5~6.5) was beneficial to leaf dry
matter accumulation in the stage of the root extension period, and
comparatively higher pH (7.0~8.0) was more beneficial to leaf dry matter
accumulation in the middle and late stage of flue-cured tobacco (Han et al. 1992). Another
research demonstrated that soil pH affects photosynthetic features of
tobacco leaves and inner activity of the protective enzymes as well (Wang et al. 2005).
In recent years, research institutions and agricultural experts have
paid attention to the issues relating tobacco growing in strong acidic and
alkaline conditions due to soil acidification and alkalization phenomenon in
some tobacco-growing area. However, the focus of available research was to see
the effects of soil pH on tobacco growth and no inclusive studies were done to
unveil the mechanisms behind this growth inhibition. Therefore, this study was
designed to evaluate the effects of solution pHs on root morphology,
above-ground growth, dry matter accumulation and allocation, photosynthesis and
anti-oxidants metabolism. The findings of this study will help to explore the
mechanisms of tobacco growth reduction at strong acidic and alkaline
conditions, and optimization of soil pH conducive for high-quality tobacco
production.
Materials and Methods
Experimental material
The tobacco variety (Nicotiana tabacum L.) used in the experiment was K326,
which was first planted on the holed foam board with nutrient solution under
it. The nutrient solution adopted improved Hoagland formula:
calcium nitrate tetrahydrate 945 mg/L, potassium nitrate 506 mg/L, ammonium
nitrate 80 mg/L, potassium phosphate monobasic 136 mg/L, magnesium sulfate 493
mg/L, iron salt solution 2.5 mL, trace element solution 5 mL. Tobacco seedlings
at four leaves stage were transplanted to another bigger foam board.
Experimental treatments
Tobacco was grown in hydroponic solution culture at
varying solution pH levels i.e.,
solution pHs 4, 5, 6, 7 and 8. There are 30 plants used for each treatment.
Experiment was laid out following completely randomized design (CRD) with three
replications. The tobacco seedlings after one-week pre-nurture, almost in the
same shape, were transplanted in rectangle opaque plastic box fixed by the
orderly holed foam board, whose roots were put in the nutrient solution and
supplied with oxygen by a pump every day. Nutrient solution was renewed every
week according to the set pH and measured by IQ 150 pH Meter produced by
Spectrum Corp and adjusted to the targeted value by utilizing diluent HCl or
NaOH solution.
Testing program and methods
Three samples of tobacco seedlings were taken in each
treatment, first after 10 days, and then every 7 days at 17, 24, 31, 38 and 45
days after transplanting. Root indexes including length, volume, diameter,
surface area, and tip number of roots were measured by the root-analyzing
system of LA-2400 multi-parameter. The root vitality was measured by TTC (2, 3,
5-triphenyte-trazoliumchloride) (Li 2000). The indexes of above-ground growth
and dry matter including plant height, stem thickness, leaf number, maximum
leaf length and width (maximum leaf area = length × width × 0.6345) were
measured according to Investigation and Measurement Method of Tobacco Agronomic
Characteristics (YC/T14-2010). The physiological and biochemical indexes and
photosynthetic indexes in leaves were taken from fully expanded sec-sequenced
leaf. To be specific, MDA (malondialdehyde) content (Cakmak and Marschner
1992), indicated as μmol/g, FW
(fresh weight), was measured by Thiobarbituric acid method and SOD (superoxide
dismutase) activity was measured by Pyrogallol auto-oxidation method (Wei
2000). The SPAD-502plus portable chlorophyll tester (Japan Konica Minolta Co.,
Ltd.) was used to note chlorophyll SPAD value. The net photosynthetic rate of
tobacco leaves was recorded by LI-6400XT Portable Photosynthesis Meter at
9:00~11:00 a.m. on sunny days (Wei 2000).
Statistical analysis
Collected
data were analyzed by Microsoft Excel 2003 and SPSS 17.0 following one-way ANOVA
for each sampling date to check overall significance of data. Moreover,
treatments means were separated using Duncan’s Multiple Range Test at 5%
probability level (Steel et al.
1997).
Results
Root traits
Different solution pHs had significant effect on roots
traits (root length, root volume, root surface area, root diameter, root tip
number and root vitality) of tobacco grown in hydroponic conditions at all
sampling dates i.e., 10, 17, 24, 31,
38 and 45 days after transplanting (DAT) (Table 1; Fig. 1). Strong acidic
conditions (solution pH 4) resulted in substantial reduction in entire roots traits
(root length, root volume, root surface area, root diameter, root tip number
and root vitality) at all sampling dates i.e.,
10, 17, 24, 31, 38 and 45 DAT with the only exception of non-significant effect
of solution pHs on root vitality at 10 DAT (Table 1; Fig. 1). Nonetheless,
neutral to slightly alkaline conditions, solution pH 8 in particular, improved
tobacco root length, root volume, root surface area, root diameter, root tip
number and root vitality recorded at 10, 17, 24, 31, 38 and 45 DAT (Table 1;
Fig. 1).
Above-ground
growth of tobacco seedlings
Table 1: Comparison of tobacco
root traits under different solution pH
Days |
Medium |
Root length (cm) |
Root volume (cm3) |
Root surface
area (cm2) |
Root diameter (mm) |
Roots tip number |
10 |
pH 4 |
170 ± 36.9b |
1.27 ± 0.34b |
65.9 ± 14.2a |
0.81 ± 0.14a |
636 ± 85.3b |
pH 5 |
195 ± 34.0a |
1.40 ± 0.13a |
63.2 ± 6.74a |
0.93 ± 0.11a |
826 ± 15.0a |
|
pH 6 |
203 ± 29.1a |
1.54 ± 0.44a |
60.7 ± 24.9a |
0.85 ± 0.22a |
898 ± 64.5a |
|
pH 7 |
199 ± 21.1a |
1.56 ± 0.89a |
75.9 ± 21.8a |
0.84 ± 0.13a |
889 ± 78.8a |
|
pH 8 |
188 ± 30.0 a |
1.53 ± 0.66a |
62.0 ± 16.4a |
0.96 ± 0.11a |
923 ± 67.9a |
|
17 |
pH 4 |
206 ± 50.9c |
2.29 ± 0.61c |
75.2 ± 19.5c |
1.02 ± 0.05b |
686 ± 84.0c |
pH 5 |
243 ± 50.5b |
2.32 ± 0.73c |
87.8 ± 27.8b |
1.16 ± 0.16a |
879 ± 26.6b |
|
pH 6 |
256 ± 27.2 b |
2.53 ± 0.47b |
93.9 ± 20.8a |
1.22 ± 0.12a |
961 ± 79.3a |
|
pH 7 |
309 ± 70.0 a |
2.78 ± 0.99ab |
98.8 ± 13.8a |
1.24 ± 0.10a |
969 ± 87.6a |
|
pH 8 |
297 ± 64.4 a |
3.36 ± 0.65a |
101 ± 11.7a |
1.23 ± 0.29a |
1035 ± 91.5a |
|
24 |
pH 4 |
226 ± 65.6d |
2.36 ± 0.85d |
85.8 ± 23.0c |
1.05 ± 0.06c |
746 ± 79.3c |
pH 5 |
297 ± 44.4c |
2.85 ± 0.24cd |
98.6 ± 19.5c |
1.26 ± 0.19b |
936 ± 31.8b |
|
pH 6 |
301 ± 15.9b |
3.06 ± 0.12c |
103 ± 16.0b |
1.48 ± 0.33a |
1014 ± 75.6b |
|
pH 7 |
412 ± 87.1a |
3.77 ± 0.29b |
107 ± 17.7b |
1.41 ± 0.17a |
1115 ± 99.7b |
|
pH 8 |
421 ± 78.1a |
4.17 ± 0.66a |
114 ± 27.1a |
1.49 ± 0.40a |
1319 ± 94.4a |
|
31 |
pH 4 |
322 ± 33.6d |
2.66 ± 0.46c |
92.7 ± 17.9c |
1.12 ± 0.12c |
784 ± 81.4c |
pH 5 |
362 ± 69.9c |
3.06 ± 0.36b |
109 ± 7.55c |
1.49 ± 0.24b |
970 ± 86.3b |
|
pH 6 |
416 ± 39.7b |
3.16 ± 0.61b |
114 ± 6.46b |
1.75 ± 0.05a |
1056 ± 42.6b |
|
pH 7 |
457 ± 24.5ab |
3.13 ± 0.03b |
116 ± 21.8b |
1.60 ± 0.48a |
1162 ± 56.8b |
|
pH 8 |
499 ± 62.1a |
4.70 ± 0.94a |
121 ± 15.3a |
1.70 ± 0.75a |
1443 ± 75.3a |
|
38 |
pH 4 |
365 ± 97.5c |
2.95 ± 0.22c |
103 ± 18.8c |
1.21 ± 0.07c |
900 ± 106c |
pH 5 |
460 ± 11.8b |
3.36 ± 0.60b |
123 ± 6.89b |
1.69 ± 0.28b |
1081 ± 89.7b |
|
pH 6 |
484 ± 43.6b |
3.38 ± 0.87b |
137 ± 18.0b |
1.57 ± 0.14b |
1459 ± 107a |
|
pH 7 |
559 ± 24.0ab |
3.67 ± 0.92b |
138 ± 23.1b |
1.72 ± 0.00ab |
153 ± 46.1a |
|
pH 8 |
592 ± 55.8a |
5.02 ± 0.78a |
158 ± 29.1a |
1.85 ± 0.10a |
1602 ± 73.1a |
|
45 |
pH 4 |
385 ± 59.0c |
3.17 ± 0.87d |
111 ± 13.7c |
1.39 ± 0.13c |
931 ± 73.5c |
pH 5 |
520 ± 39.0 b |
3.94 ± 0.23c |
152 ± 11.1b |
1.44 ± 0.18c |
1174 ± 42.1b |
|
pH 6 |
582 ± 83.9ab |
4.60 ± 0.94b |
157 ± 21.5b |
1.68 ± 0.08b |
1540 ± 44.7a |
|
pH 7 |
607 ± 82.5a |
5.36 ± 0.58a |
161 ± 14.4b |
1.65 ± 0.21b |
1611 ± 93.6a |
|
pH 8 |
616 ± 33.2a |
5.34 ± 0.53a |
182 ± 28.9a |
2.00 ± 0.36a |
1644 ± 91.7a |
Means ± standard deviation with different letters, in
the same group, differ significantly from each other at P ≤ 0.5 according to Duncan’s Multiple Range Test
Fig. 1:
Effect of solution pH on tobacco root
vitality
Bar charts represent means ± standard deviation and means
with different letters differ significantly from each other at 5% probability
level
Different solution pHs had significant effect on plant
height, stem thickness and maximum leaf area of tobacco grown in hydroponic
conditions at all sampling dates i.e.,
10, 17, 24, 31, 38 and 45 DAT with the exception of non-significant effect on
plant height at 10 DAT and on stem thickness at 10 and 17 DAT. However,
solution pH had non-significant effect on tobacco leaf number at all sampling
dates (Table 2). Tobacco grown in strong acidic conditions
(solution pH 4) and alkaline conditions (pH 8) observed significant decrease in
plant height, stem thickness and leaf area; however, tobacco grown under
slight acidic to neutral conditions (pH 6~7) recorded more plant height, stem
thickness and leaf area at all sampling dates (Table 2).
Belowground and aboveground dry weight of tobacco
seedlings
Different solution pHs had significant effect on
belowground root dry weight and its ratio, and dry weight of above-ground stem
and leaf and its ratio in hydroponic conditions at all sampling dates i.e., 10, 17, 24, 31, 38 and 45 DATS.
Slight acidic to neutral conditions (pH 6~7) observed the greatest belowground
root dry weight and dry weight of above-ground stem and leaf, which dropped to
the least grown in strong acidic conditions (pH 4) at all sampling dates (Fig.
2–3). There was an obvious increase in the ratio of root to whole plant, and a
substantial reduction in the ratio of stem and leaf to whole plant with
increasing solution pH at all sampling dates (Fig. 2–3).
Membrane-lipid peroxidation and
protective enzyme activities of tobacco leaves
Table 2: Comparison
of above ground parts tobacco growth under different solution pH
Days |
Medium |
Plant height
(cm) |
Stem
thickness (cm) |
Maximum
leaf area (cm2) |
|
10 |
pH 4 |
13.0 ± 1.42 |
2.00 ± 0.08 |
4.50 ± 0.58 |
125 ± 13.7b |
pH 5 |
11.7 ± 1.24 |
2.13 ± 0.19 |
5.00 ± 0.00 |
123 ± 8.60b |
|
pH 6 |
12.4 ± 1.64 |
1.93 ± 0.17 |
4.25 ± 0.50 |
150 ± 28.1a |
|
pH 7 |
13.0 ± 4.72 |
2.18 ± 0.36 |
4.25 ± 0.96 |
146 ± 30.7a |
|
pH 8 |
11.8 ± 1.67 |
1.83 ± 0.21 |
4.25 ± 0.50 |
122 ± 15.6b |
|
17 |
pH 4 |
13.4 ± 2.17b |
2.15b ± 0.21 |
5.75 ± 0.50 |
164 ± 24.7c |
pH 5 |
17.3 ± 2.71a |
2.55a ± 0.30 |
6.50 ± 0.58 |
179 ± 35.7b |
|
pH 6 |
17.3 ± 1.55a |
2.60a ± 0.29 |
7.00 ± 0.00 |
181 ± 39.4b |
|
pH 7 |
17.0 ± 2.92a |
2.68a ± 0.22 |
7.00 ± 0.00 |
207 ± 22.8a |
|
pH 8 |
13.5 ± 1.59b |
2.53a ± 0.21 |
6.75 ± 1.50 |
157 ± 36.7c |
|
24 |
pH 4 |
14.8 ± 0.61c |
2.23 ± 0.13c |
7.50 ± 0.58 |
178 ± 20.9c |
pH 5 |
17.6 ± 1.33b |
2.80 ± 0.24b |
7.50 ± 0.58 |
193 ± 14.0b |
|
pH 6 |
18.6 ± 1.22b |
2.95 ± 0.17ab |
7.75 ± 0.50 |
224 ± 35.9a |
|
pH 7 |
21.9 ± 1.88a |
3.03 ± 0.17a |
7.25 ± 0.50 |
208 ± 34.4ab |
|
pH 8 |
18.1 ± 1.44b |
2.85 ± 0.06b |
7.00 ± 0.00 |
178 ± 40.8c |
|
31 |
pH 4 |
15.6 ± 1.03c |
2.83 ± 0.17b |
8.75 ± 0.96 |
165 ± 20.9b |
pH 5 |
18.6 ± 2.58b |
2.83 ± 0.17b |
9.75 ± 0.50 |
208 ± 17.1a |
|
pH 6 |
20.8 ± 0.89b |
2.98 ± 0.17ab |
9.75 ± 0.50 |
232 ± 27.4a |
|
pH 7 |
24.1 ± 1.19a |
3.15 ± 0.13a |
10.0 ± 0.00 |
223 ± 39.7a |
|
pH 8 |
21.4 ± 2.89b |
2.85 ± 0.30b |
9.25 ± 0.50 |
210 ± 12.7a |
|
38 |
pH 4 |
15.8 ± 3.51c |
2.90 ± 0.18b |
9.25 ± 1.50 |
185 ± 42.6d |
pH 5 |
20.3 ± 1.45b |
2.95 ± 0.24b |
10.8 ± 0.50 |
210 ± 47.3c |
|
pH 6 |
23.0 ± 2.09b |
3.03 ± 0.15b |
10.8 ± 0.96 |
254 ± 8.16a |
|
pH 7 |
25.6 ± 3.78a |
3.25 ± 0.13a |
10.0 ± 0.82 |
226 ± 15.0b |
|
pH 8 |
21.7 ± 3.07b |
2.95 ± 0.26b |
10.3 ± 0.50 |
224 ± 28.4b |
|
45 |
pH 4 |
21.2 ± 5.10c |
2.90 ± 0.26b |
10.0 ± 0.82 |
195 ± 21.6d |
pH 5 |
25.0 ± 3.21b |
3.00 ± 0.33b |
11.0 ± 0.00 |
221 ± 28.2c |
|
pH 6 |
27.7 ± 1.68ab |
3.20 ± 0.08a |
11.3 ± 0.50 |
270 ± 32.8a |
|
pH 7 |
29.9 ± 2.58a |
3.33 ± 0.35a |
10.5 ± 0.58 |
248 ± 20.7b |
|
pH 8 |
22.0 ± 1.54c |
2.98 ± 0.10b |
10.5 ± 1.00 |
233 ± 46.6c |
Means ± standard deviation with different letters, in
the same group, differ significantly from each other at P ≤ 0.5 according to Duncan’s Multiple Range Test
Fig. 2: Effect
of solution pH on root dry weight and its ratio of tobacco
Bar charts represent means ± standard deviation and
means with different letters differ significantly from each other at 5% probability
level
Different solution pHs had significant effect on MDA and
SOD of tobacco seedlings grown
in hydroponic conditions at all sampling dates i.e., 10, 17, 24, 31, 38 and 45 days after transplanting (DAT).
Acidic to alkaline (pH 5~8) conditions
observed an increase in MDA of
tobacco seedlings, and a decrease in SOD at all sampling dates with rising pH
(Fig. 4). Moreover, solution pH 4 and
pH 8 had more significant effects on MDA and SOD contents than other
solutions (Fig. 4). It was shown
that a strong acid condition did more harm to cell membrane than the alkaline
in the preliminary stage of tobacco transplant, and vice versa in the late
stage.
Chlorophyll content and photosynthetic rate of tobacco
leaves
Different solution pHs had significant effects on SPAD
values (proportional to chlorophyll content) and photosynthetic rate of tobacco
leaves in hydroponic conditions at all sampling dates i.e., 10, 17, 24, 31, 38 and 45 DATS. SPAD values and
photosynthetic rate of tobacco leaves were maximum at pH 6 compared with strong
acidic (pH 4) and alkaline conditions (pH 8) at all sampling dates (Fig. 5).
SPAD values and photosynthetic rate were lowest at pH 8 recorded at 10, 17 and
24 DAT and at pH 4 recorded at 31, 38, and 45 DAT (Fig. 5).
Discussion
Fig. 3: Effect
of solution pH on stem dry weight and stem and leaf dry weight ratio of tobacco
Bar charts represent means ± standard deviation and means
with different letters differ significantly from each other at 5% probability
level
Fig. 4:
Effect of solution pH on MDA content and
superoxide dismutase (SOD) activity of tobacco leaf
Bar charts represent means ± standard deviation and means
with different letters differ significantly from each other at 5% probability
level
Fig. 5:
Effect of solution pH on chlorophyll SPAD
value and photosynthetic rate of tobacco leaf
Bar charts represent means ± standard deviation and means
with different letters differ significantly from each other at 5% probability
level
Results of this hydroponic study unveiled that slightly
acidic to neutral conditions (pH 6~7)
improved root growth and germination, dry weight accumulation, and root
activity of tobacco seedlings. However strong acidic conditions curbed root
growth, and strong acid and alkaline conditions impaired aboveground growth of
tobacco (Tables 1, 2). In another hydronic study as nutrient solution changed
from pH 4.5 to 8.5, tobacco growth observed a reversed parabolic curve, and
maximum dry matter accumulation was recorded at pH 6.5 (Zhu et al. 2012).
Decrease in tobacco growth
under strong acidic conditions might be linked with decreased root activity and
growth. This reduced root vitality and growth might be resulted in poor
nutrient and water uptake leading to impaired above-ground growth and
metabolism (Tong 2005). Besides, the destroyed internal mechanism of tobacco in
strong acidic conditions, and the comparative higher speed of nutrient
consumption than merging, gives rise to the low accumulation of above-ground
dry weight (Li et al. 2009). The
negative effects on tobacco growth and germination under alkaline conditions
might be due to reduced absorption of early nitrogen and the contribution of
nutrient elements for tobacco growth (He and Yuan 2014). Moreover, both pH
extremes (pH 4 and 8) elevated oxidative stress (as was evident from higher
values of MDA contents) and damaged cell membrane (Fig. 4). Elevated membrane
damage coupled with decreased chlorophyll contents resulted in lower
photosynthesis and ultimately less accumulation of biomass.
It is also evident from the
research results that strong acidic (pH 4) and alkaline (pH 8) environment
increased the MDA contents (oxidative damage) on one hand while also decreased
the activity of protective enzymes like superoxide dismutase (SOD) on the other
hand. Therefore, this two-way damage accelerated the membranes damage and
ultimately reduced the carbon fixation potential of tobacco seedlings. Despite
the insignificant influence of environmental pH on inside plant cell pH (Raven
and Smith 1980), solution pH directly or indirectly affected the properties and
state of cell membrane, intracellular matter transport, metabolism, and uptake
of mineral elements by plant roots. If resistance or defense limits of plants
are surpassed, the merging of membrane protective enzyme activity and
protective content would be curbed. As a result, the process of membrane lipid
peroxide was intensified leading to more cell membrane damaged (Wolfe 1991).
Hence these differences of root vitality, membrane-lipid peroxidation, protective
enzyme activity, chlorophyll content and photosynthetic rate of tobacco leaves
under different pH environment were the key reasons to influence tobacco
growth, and the accumulation and allocation of dry matter.
Conclusion
Solution pH has significant impacts on growth and
germination of flue-cured tobacco and physiological and biochemical
characteristics of its leaf. Strong acid and alkaline conditions impaired
tobacco growth due to over generation of reactive oxygen species, decreased
activity of protective enzymes along with cell membrane damage leading to
substantial decline in photosynthesis. Therefore, soils having pH in the range
of 6–7 seemed more suitable for flue-cured tobacco growth.
Acknowledgements
This paper was jointly supported by Agronomy College of Hunan
Agricultural University and Ningxiang Branch of Changsha Tobacco Company.
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