Changes in Meteorological Factors Generating Field Cold Stress
It can be seen from Fig. 1 that the experimental base located in Jianji village of Laojun Mountain Town showed significant fluctuations in temperature and rainfall in August: the daily highest and lowest temperature varied in the ranges of 21.5 to 41.9 °C and 9.4 to 17.1 °C, respectively, with the temperature difference in the range of 12.1 to 24.6 °C; the daily precipitation varied within 0 to 21.6 mm. The maximum diurnal temperature difference (30.1 °C) was recorded on 12 August; however, no rainfall occurred thereon; the minimum diurnal temperature difference (8.7 °C) was found on 1 August, accompanying with 5.9 mm of precipitation. According to the survey results of local field cold stress, it can be found that substantial cooling accompanying meteorological disasters (including rainfall and a small amount of hail) appeared on 16 and 17 August; in the next six days, tobacco showed a range of symptoms caused by cold stress (Fig. 2). The tobacco leaves in the middle and upper parts suffered the most serious damage. The surface color of the tobacco leaves changed from normal to dark green (within 1 to 2 d), to purple red (within 2 to 3 d), then dark red (within 3 to 4 d), and finally off-white (within 4 to 6 d). Eventually, a large area of scalded leaves was found, showing poor flue-curing availability.
To explore the specific causes for cold stress, the meteorological data collected inside and outside the greenhouse on 16 to 17 August were analyzed (Fig. 3). It can be found from Fig. 3 that the temperature outside the greenhouse (natural conditions) varied within 9.4 to 34.8 °C, with a difference of 25.4 °C; the temperature inside the greenhouse varied between 12.5 to 36.2 °C, with a difference of 23.7 °C. The rainfall occurring in the cooling period was concentrated between 18:00 to 22:00 on 16 August, with a total precipitation of 5.2 mm. The tobacco leaves inside and outside the greenhouse in the experimental base were sampled on 19 August (Fig. 4). The 10th leaf (counted from the bottom to up) was sampled. It can be seen from Fig. 4 that the tobacco leaves inside the greenhouse showed no significant symptoms caused by cold stress, except for having a small number of brown spots on leafstalk; tobacco leaves outside the greenhouse exhibited significant red and puce plaques from the middle part of leaves to the leafstalk and the leaves turned from green to yellow on the whole. It was judged that tobacco leaves outside the greenhouse were subjected to cold stress.
Comparison of Slices of Tobacco Leaves Inside and Outside the Greenhouse
As shown in Fig. 5 and Table 1, the thicknesses of laminae, palisade tissues, and spongy tissues and the ratio of the thickness of palisade tissues to that of spongy tissues (tissue ratio) of fresh tobacco leaves from inside the greenhouse showed a significant difference (P < 0.05) from those outside.
As shown in Table 1, the thickness of the laminae, palisade tissues, spongy tissues, upper epidermis and lower epidermis of fresh tobacco leaves outside the greenhouse was lower than that inside the greenhouse, the values inside the greenhouse were separately 151.76%, 105.19%, 175.90%, 91.97% and 222.35% greater than those outside, which showed a significant difference. On the contrary, in terms of the ratio of the thickness of palisade tissues to that of spongy tissues (tissue ratio) of fresh tobacco leaves, the value outside the greenhouse was 25.27% greater than that inside the greenhouse.
Table 1
Parameters of microstructures of fresh tobacco leaves in treatments inside and outside the greenhouse
Treatment
|
Thickness of lamina (µm)
|
Thickness of upper epidermis (µm)
|
Thickness of lower epidermis (µm)
|
Thickness of palisade tissues (µm)
|
Thickness of spongy tissues (µm)
|
Ratio of thickness of palisade tissues to that of spongy tissues (tissue ratio)
|
inside the greenhouse
|
335.09A
|
30.37A
|
30.72A
|
110.31A
|
162.12A
|
0.68A
|
outside the greenhouse
|
133.10B
|
15.82B
|
9.53B
|
53.76B
|
58.76B
|
0.91A
|
The different capital letters indicate significant between different treatments at the same stage (P < 0.05). Values represent the averages of three biological replicates.
Comparison of Water Loss Rates of Tobacco Leaves Inside and Outside the Greenhouse in the Flue-curing Process
As shown in Fig. 6 and Fig. 7, under different curing stages, there were significant differences (P < 0.05) in water loss rate and moisture content of tobacco leaves; under the same curing stage, only at 48 °C showed a significant difference (P < 0.05) in moisture content of tobacco leaves inside and outside the greenhouse.
With the increase of the flue-curing time, the moisture contents and water loss rates of tobacco leaves inside and outside the greenhouse slowly changed at first, then rapidly varied and finally changed slowly again. The moisture content of tobacco leaves inside the greenhouse was generally higher than that outside, while the water loss rate of the former was lower than that of the latter. In terms of the moisture content, the changes in the moisture content of the tobacco leaves in treatments inside and outside the greenhouse from 25 °C (fresh tobacco leaves) to 54 °C were 83.28 to 24.95% and 78.42 to 12.72%, respectively. The moisture content was greatly changed in the flue-curing process at 42 to 54 °C: the moisture contents of tobacco leaves inside and outside the greenhouse at 42 °C were separately 177% and 362% higher than those at 54 °C; the moisture contents of tobacco leaves in the curing process at 42 °C, 48 °C, and 54 °C inside the greenhouse were separately 17.80%, 123.19%, and 96.15% higher than those outside.
As for the water loss rate, its change amplitudes for tobacco leaves inside and outside the greenhouse at 38 to 54 °C were 16.14 to 92.66% and 35.3 to 97.3%, respectively; the water loss rates of tobacco leaves inside and outside the greenhouse at 48 °C were separately 382% and 161% higher than those at 38 °C; the water loss rates of tobacco leaves outside the greenhouse in the curing process at 38 °C, 42 °C, and 48 °C were separately 118.71%, 27.86%, and 18.26% higher than those inside the greenhouse.
Comparison of SPAD Values and Plamochromic Pigments in Tobacco Leaves Inside and Outside the Greenhouse During Flue-curing
As shown in Table 2, the SPAD values and plamochromic pigments in tobacco leaves inside and outside the greenhouse in different flue-curing stages both presented a significant difference (P < 0.05).
In the flue-curing process, the values of the SPAD, chlorophyll a, and chlorophyll b in tobacco leaves inside the greenhouse were all greater than those outside the greenhouse. The three indices of tobacco leaves inside and outside the greenhouse tended to decrease rapidly at first and then slowly decreased. They were degraded at the fastest rate at 38 °C and then the rate of degradation decreased rapidly, then tended to stabilize. The SPAD values of tobacco leaves inside the greenhouse were 29.79 to 172.57% greater than those outside the greenhouse, and were the lowest and the highest in fresh tobacco leaves and in leaves flue-cured at 38 °C, respectively; the values of the chlorophyll a in tobacco leaves inside the greenhouse were 148.94 to 1153.91% greater than those outside the greenhouse, in which the lowest and the highest values were found in fresh tobacco leaves and in leaves flue-cured at 42 °C, respectively; the values of the chlorophyll b content in tobacco leaves inside the greenhouse were 73.91 to 628.26% larger than those outside the greenhouse, with the lowest and the highest values at 52 °C and 42 °C, respectively.
The values of the lutein and β-carotene content in tobacco leaves inside the greenhouse were all greater than those outside. Under flue-curing, the lutein and β-carotene contents in tobacco leaves inside and outside the greenhouse rose at first and then decreased. The two indices inside the greenhouse reached their maxima separately at 42 °C and 48 °C while the outside of were lutein and β-carotene content maximized at 54 °C. The chlorophyll contents of tobacco leaves inside and outside the greenhouse separately varied between 150.30 to 297.14 µg g− 1 and 93.28 to 139.42 µg g− 1, in which the value inside the greenhouse was 48.21 to 164.57% greater than that outside. Moreover, the index was separately minimized and maximized at 54 °C and 42 °C. The contents of β-carotene in tobacco leaves inside and outside the greenhouse separately varied between 1940.38 and 4489.59 µg g− 1 and 1207.53 to 1888.94 µg g− 1. The content of β-carotene in tobacco leaves inside the greenhouse was 60.69 to 149.34% greater than that outside. The values were separately maximized and minimized in fresh tobacco leaves and in flue-cured leaves at 48 °C.
Table 2
SPAD values and contents of plamochromic pigments in tobacco leaves inside and outside the greenhouse in various stages of flue-curing
Treatment
|
Sampling temperature
(°C)
|
Continuous curing time (h)
|
SPAD
|
Chlorophyll a
(µg g− 1)
|
Chlorophyll b (µg g− 1)
|
Lutein (µg g− 1)
|
β-carotene (µg g− 1)
|
Inside the greenhouse
|
Fresh tobacco leaves
|
——
|
36.2Aa
|
286.06Aa
|
202.99Aa
|
150.30Ab
|
1940.38Ab
|
38
|
23.5
|
14.9Ab
|
105.95Ab
|
42.72Ab
|
248.59Aa
|
3657.98Aa
|
42
|
16.5
|
9.8Abc
|
64.20Ab
|
26.80Ab
|
297.14Aa
|
4127.06Aa
|
48
|
15
|
8.0Abc
|
45.77Ab
|
17.01Ab
|
245.15Aab
|
4489.59Aa
|
54
|
22.5
|
6.2Ac
|
13.33Ab
|
4.40Ab
|
206.64Aab
|
3524.01Aa
|
Initially flue-cured tobacco leaves
|
——
|
——
|
43.75Ab
|
17.42Ab
|
249.00Aa
|
3794.68Aa
|
Outside the greenhouse
|
Fresh tobacco leaves
|
——
|
30.6Ba
|
114.91Ba
|
87.18Ba
|
93.28Aa
|
1207.53Aa
|
38
|
23.5
|
13.4Ab
|
13.37Aa
|
7.94Ab
|
118.40Ba
|
1573.64Ba
|
42
|
16.5
|
9.5Abc
|
5.12Aa
|
3.68Ab
|
112.31Ba
|
1669.27Ba
|
48
|
15
|
7.8Abc
|
4.83Aa
|
3.50Ab
|
115.36Ba
|
1800.61Ba
|
54
|
22.5
|
3.0Ac
|
3.88Aa
|
2.53Ab
|
139.42Aa
|
1888.94Ba
|
Initially flue-cured tobacco leaves
|
——
|
——
|
6.74Aa
|
4.83Ab
|
122.23Ba
|
1783.31Ba
|
The different capital letters indicate significant between different treatments at the same stage (P < 0.05). The different lowercase letters indicate significant between the same treatments at different curing stages. Values represent the averages of three biological replicates.
Comparison of Chemical Compositions and Polyphenols in Tobacco Leaves Inside and Outside the Greenhouse During Curing
It can be seen from Table 3 that conventional chemical composition indexes and polyphenols all presented significant differences inside and outside the greenhouse.
With increased flue-curing time, the starch contents of tobacco leaves inside and outside the greenhouse gradually decreased, exhibiting the fastest reduction in fresh tobacco leaves at 38 °C; the total sugars, reducing sugars, and sugar–nicotine ratio increased at first, then decreased, reaching a peak at 38 °C. Various indices used to classify tobacco leaves were greater outside the greenhouse than inside. The starch contents of tobacco leaves inside and outside the greenhouse separately varied within 1.04 to 33% and 5.4 to 39.94%, in which the index value of tobacco leaves outside the greenhouse was 21.03 to 567.72% greater than that inside. The minimum and the maximum values separately appeared in fresh tobacco leaves and initially flue-cured tobacco leaves. The analyses of the total sugars, reducing sugars, and sugar-nicotine ratio were similar to that for starches.
The chlorogenic acid and rutin content in tobacco leaves inside and outside the greenhouse gradually increased, as described above.
Table 3
The contents of conventional chemical substances and polyphenols in tobacco leaves treated inside and outside the greenhouse in different curing stages
Treatment
|
Sampling temperature (°C)
|
Continuous flue-curing time (h)
|
Conventional chemical compositions (%)
|
Polyphenols (mg g− 1)
|
|
Total sugar
|
Reducing sugar
|
Total nitrogen
|
Nicotine
|
Potassium oxide
|
Water soluble chloride
|
Starch
|
Protein
|
Sugar–nicotine ratio
|
Nitrogen–nicotine ratio
|
Neochlorogenic acid
|
Chlorogenic acid
|
Caffeic acid
|
Scopoletin
|
Rutin
|
Kaempferol
|
Inside the greenhouse
|
Fresh tobacco leaves
|
——
|
9.36Ac
|
5.20Ab
|
1.81Ac
|
2.33Aa
|
1.93Ab
|
0.12Ab
|
33.00Aa
|
8.46Aa
|
4.35Aa
|
0.82Aab
|
1.56Ac
|
7.25Bc
|
0.12Aab
|
0.12Aab
|
9.69Ab
|
0.11Ab
|
38
|
23.5
|
30.07Aa
|
16.00Ba
|
2.26Aab
|
3.44Aa
|
2.75Aab
|
0.24Aa
|
4.20Bb
|
7.10Ab
|
9.23Ba
|
0.67Aab
|
3.31Aab
|
16.04Ba
|
0.19Aa
|
0.09Ab
|
12.38Aab
|
0.32Aa
|
42
|
16.5
|
18.79Bbc
|
11.53Bab
|
2.63Aa
|
3.18Aa
|
3.57Aa
|
0.14Aab
|
1.40Ab
|
7.84Aab
|
6.04Ba
|
0.83Aab
|
2.71Ab
|
7.44Ac
|
0.05Ab
|
0.20Aa
|
8.42Ab
|
0.31Aa
|
48
|
15
|
21.65Bab
|
11.98Bab
|
2.39Aab
|
2.80Aa
|
2.98Aa
|
0.18Aab
|
2.44Ab
|
7.70Aab
|
7.84Ba
|
0.85Aab
|
3.60Aab
|
12.35Aabc
|
0.23Aa
|
0.10Ab
|
11.52Aab
|
0.15Ab
|
54
|
22.5
|
20.48Bab
|
10.18Bab
|
2.19Abc
|
3.35Aa
|
3.44Aa
|
0.14Aab
|
1.03Ab
|
7.26Aab
|
6.58Ba
|
0.66Ab
|
4.01Aa
|
14.62Aab
|
0.21Aa
|
0.16Aab
|
14.06Aa
|
0.16Ab
|
Initially flue-cured tobacco leaves
|
——
|
21.32Aab
|
12.64Bab
|
2.32Aab
|
2.75Aa
|
3.12Aa
|
0.19Aab
|
1.27Ab
|
7.45Aab
|
7.63Aa
|
0.87Aa
|
3.43Aab
|
10.42Abc
|
0.23Aa
|
0.10Ab
|
9.59Bb
|
0.15Ab
|
Outside the greenhouse
|
Fresh tobacco leaves
|
——
|
7.60Ab
|
3.79Ab
|
1.53Ab
|
2.12Aa
|
1.43Aa
|
0.06Aa
|
39.94Aa
|
6.87Ba
|
3.61Ab
|
0.73Aa
|
1.30Ab
|
14.15Abc
|
0.21Aab
|
0.13Aab
|
11.1Aa
|
0.10Ab
|
38
|
23.5
|
36.95Aa
|
25.06Aa
|
1.44Bb
|
1.89Ba
|
2.12Aa
|
0.14Aa
|
13.25Ab
|
5.41Bb
|
20.74Aa
|
0.78Aa
|
2.18Bab
|
23.20Aa
|
0.25Aa
|
0.06Ab
|
14.18Aa
|
0.19Ba
|
42
|
16.5
|
36.07Aa
|
27.56Aa
|
1.71Bab
|
2.24Aa
|
1.70Ba
|
0.06Aa
|
8.72Abc
|
6.08Bab
|
16.74Aa
|
0.78Aa
|
1.29Bb
|
9.75Ac
|
0.10Ab
|
0.18Aa
|
10.62Aa
|
0.20Ba
|
48
|
15
|
33.2Aa
|
23.02Aa
|
1.99Ba
|
2.86Aa
|
1.76Ba
|
0.07Ba
|
7.41Abc
|
6.56Aab
|
14.07Aa
|
0.79Aa
|
3.07Aa
|
16.73Ab
|
0.25Aa
|
0.12Aab
|
13.28Aa
|
0.08Ab
|
54
|
22.5
|
36.31Aa
|
23.23Aa
|
1.64Bab
|
2.12Ba
|
1.73Ba
|
0.09Aa
|
5.40Ac
|
5.67Bab
|
17.55Aa
|
0.78Aa
|
2.82Ba
|
18.60Aab
|
0.19Aab
|
0.06Bb
|
12.92Aa
|
0.10Ab
|
Initially flue-cured tobacco leaves
|
——
|
30.88Aa
|
24.42Aa
|
2.00Aa
|
2.58Aa
|
2.07Ba
|
0.06Ba
|
8.48Abc
|
6.82Aa
|
12.11Aab
|
0.79Aa
|
2.62Aa
|
15.69Ab
|
0.29Aa
|
0.11Aab
|
14.37Aa
|
0.08Ab
|
The different capital letters indicate significant between different treatments at the same stage (P < 0.05). The different lowercase letters indicate significant between the same treatments at different curing stages. Values represent the averages of three biological replicates.
Comparison of Activities of PPO and Antioxidant Enzymes in Tobacco Leaves Inside and Outside the Greenhouse During Flue-curing
As can be seen from Fig. 8, under the activities of SOD, POD and CAT, the activity of the three enzymes in the greenhouse was higher than that outside the greenhouse, and showed a trend of increase at first and then decreased. The activity of the three enzymes was the highest at the stage of curing at 38 °C to 42 °C, and then decreased sharply. Meanwhile, under the same curing time, the SOD, POD and CAT enzymes treated in the greenhouse could maintain high activity more effectively than those treated outside the greenhouse. The content of MDA in the treatment inside and outside the greenhouse increased with the progress of curing, and the treatment outside the greenhouse increased sharply after 42 °C and exceeded that of the treatment inside the greenhouse at 48 °C. The PPO activity of both inside and outside the greenhouse treatment showed a trend of a small decrease, then a sharp increase and then a sharp decrease, the indoor treatment increased abruptly at 38 to 42 °C, then decreased sharply, while the out-of-greenhouse treatment increased abruptly at 42 to 48 °C, and then decreased sharply.
Comparison of Economic Traits and Sensory Evaluation of Initially Flue-cured Tobacco Leaves Inside and Outside the Greenhouse
It can be seen from Fig. 9 that there is an obvious difference in the appearance of the first-cured tobacco leaves inside and outside the greenhouse. There is bright color, good opening, no obvious miscellaneous color and hanging ash inside the greenhouse. However, outside the greenhouse, tobacco leaves have gray and dark color, small opening, obvious miscellaneous color and hanging ash on the surface.
As can be seen from Table 4, there are significant differences in the output, output value and average price of tobacco leaves inside and outside the greenhouse. Each index shows that inside the greenhouse is higher than that outside the greenhouse, and the yield inside the greenhouse is 13.45%, 47.32%, 37.32% and 29.85% higher than that outside the greenhouse, respectively. As can be seen from Table 5, the total score of sensory quality of tobacco leaves in the greenhouse was significantly higher than that of tobacco leaves outside the greenhouse by 20.44%.
Table 4
Economic properties inside and outside the greenhouse of flue-cured tobacco
Treatment
|
Yield (kg ha− 1)
|
Output values (dollar ha− 1)
|
Proportions of middle- high-class tobacco (%)
|
Average price (dollar kg− 1)
|
inside the greenhouse
|
2326.8A
|
8179.09A
|
72.71A
|
3.52A
|
outside the greenhouse
|
2050.95B
|
5551.96B
|
52.95A
|
2.71B
|
The different capital letters indicate significant between different treatments at the same stage (P < 0.05). Values represent the averages of three biological replicates.
Table 5
Sensory evaluation of smoking quality inside and outside the greenhouse of flue-cured tobacco
Treatment
|
Aroma note
(10)
|
Aroma quality
(15)
|
Aroma volume
(15)
|
Concentration
(10)
|
Mixed gas
(10)
|
Irritancy
(15)
|
Strength
(5)
|
Cleanness
(10)
|
Moisture
(5)
|
Taste
(5)
|
Total score
(100)
|
inside the greenhouse
|
8.5
|
12.5
|
13.5
|
7
|
7.5
|
12
|
5
|
8.5
|
4
|
4
|
82.5A
|
outside the greenhouse
|
7.5
|
8.0
|
9.0
|
7.0
|
8.0
|
9.5
|
4.0
|
9.0
|
3.0
|
3.5
|
68.5B
|
The different capital letters indicate significant between different treatments at the same stage (P < 0.05). Values represent the averages of three biological replicates.