Safe utilization of farmland soil with cadmium pollution: strategies and deliberations.
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 263-271.
The cadmium pollution in farmland soils of China has posed a potential threat to food safety and human health. Based on the analysis of sources, status and harms of cadmium pollution in farmland soils, we summarized related technologies and application status of safe utilization of farmland soils with Cd pollution, including screening of low cadmium accumulation crop,
in situ immobilization technology, agronomic measures and comprehensive prevention and control patterns. Meanwhile, the safe utilization of farmland soils with cadmium pollution faces many problems including significant regional differences, insufficient pollution source control, incomplete process interception, limited prevention and control technologies and lack of risk evaluation system. Furthermore, corresponding suggestions were proposed towards the control of farmland soils with cadmium pollution in China.
Straightforward method for preparing a user-customizable DNA marker by improved overlap extension polymerase chain reaction.
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 272-277.
DNA marker is a set of standards that are used to indicate the approximate molecular-mass size of certain DNA fragment which separated by agarose gel electrophoresis. In the present study, a straightforward method for preparing a user-customizable DNA marker by an improved overlap extension polymerase chain reaction (PCR) was established. The DNA fragment, containing the identical sequence in its
and 5 ' ends, was amplified by a conventional PCR. In the following denaturation and annealing process performed by the overlap extension PCR, two single-stranded DNA in the 3 ' end of which had complementary base sequences could form double-stranded DNA. In the extension process, these single-stranded DNA was utilized as both template and primer. The number of small fragments of DNA decreased and the number of large fragments of DNA increased gradually with the increased number of PCR cycles. The products of different cycles of overlap extension PCR were taken for agarose gel electrophoresis. The optimal PCR cycle or optimal combination of different PCR cycles was chosen according to the results of electrophoresis. Then 100 bp DNA ladder marker was obtained by the overlap extension PCR under the chosen condition. There was no need for purification of PCR products as the amplified fragments could be directly used in the agarose gel electrophoresis. The bands of prepared DNA marker were clear and accurate, and could be used for molecular studies. In sum, this method for DNA marker production is simple, time saving, cost effective, byproduct free and user-customizable in comparison with current ones widely used in most laboratories. 3 '
Effects of agro-photovoltaic integrating system on field illumination and sweet potato growth
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 288-295.
In order to investigate the effects of establishment of photovoltaic (PV) panels on field illumination conditions and sweet potato growth in an agro-photovoltaic integrating system, we used wooden boards to simulate the PV panel construction. The simulated PV assembly had a set of standard parameters, which were an average height of 1.6 m, a slope of 22°, a width of 3 m and a length of 9 m. A two-year field experiment was carried out, and three sweet potato cultivars of “Xinxiang” (Xx) , “Zheshu 13” (ZS 13) and “Zheshu 77” (ZS 77) were planted under the PV panel shading and CK (without PV panels) treatments. The results showed that under the sunny weather condition, the illumination intensity was 80-400 μmol/(m
2?s), which was higher than the light compensation point of the tested cultivars and slightly lower than the light saturation point of Xx and ZS 13. The variation of field light intensity in cloudy days of summer and autumn was similar to that in the sunny days. The light intensity of observation points under the PV panel shading treatment in rainy days was only slightly higher than the light compensation point of the tested cultivars. At the same light intensity, the net photosynthetic rate and stomatal conductance of sweet potato leaves under the shading treatment decreased significantly, and the intercellular CO 2 concentration increased significantly. After the PV panel shading treatment, the maximum leaf area per plant decreased significantly, and the leaf area of Xx, ZS 13 and ZS 77 decreased by 18.94%, 45.54% and 56.26%, respectively. The leaf mass of per area for the three cultivars also decreased significantly. The dry matter accumulation trend of the three cultivars after shading treatment was the same as that of the CK, but the total amount decreased significantly. Compared with the CK, the yield of sweet potato per unit area decreased by 39.25%, 33.70% and 23.60% for Xx, ZS13 and ZS 77, respectively, but the total yield of Xx was still the highest under the shading treatment. In summary, the agro-photovoltaic integrating system formed by the construction of photovoltaic panels in the farmland has some adverse effects on the field light intensity and sweet potato growth, but the economic benefits per unit area are greatly increased. Thus, the crop yield can be increased by increasing density of sweet potato seedlings and reasonable fertilizer, and the agro-photovoltaic integrating system has a good prospect of popularization.
Aseptic germination and bulblet formation of
Ixiolirion tataricum (Pall.) Herb. seeds. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 296-305.
Ixiolirion tataricum (Pall.) Herb. is a unique and elite plant germplasm resource in China. The distribution of I. tataricum is limited in Xinjiang Uygur Autonomous Region. Ixiolirion. tataricum has strong resistance, high ornamental and medicinal values, which are of great potential in breeding. In this study, the seeds of I. tataricum collected from Xinjiang Uygur Autonomous Region were used as materials to compare the effects of sterilization method, culture conditions, gibberellin A 3 (GA 3) pretreatment and storage conditions on the aseptic germination and bulblet formation. The results showed that 30 s treatment with 75% ethanol followed by 5 min treatment with 2% NaClO was the best sterilization method for I. tataricum with 100% sterile seedlings. Using this sterilization method, the germination rate was more than 94.0% at 4 ℃, while almost no seed germinated at 25 ℃. Normally, seeds could germinate after storage for six months in the refrigerator at 4 ℃, but completely lost germination capacity at room temperature. We also found that MS+6-benzylaminopurine (6-BA) 1.0 mg/L+1-naphthylacetic acid (NAA) 0.2 mg/L+sucrose 30 g/L was the best germination medium for I. tataricum, which was beneficial to bulblet formation. Germinated in this medium, shoots could eventually expanded into bulbs after being transferred to light culture at 25 ℃ and then transferred to MS medium containing 60 g/L sucrose. This study provides an experimental basis for in vitro conservation of I. tataricum and to enhance the development and utilization of wild resources in China.
Secretory expression of recombinant human acetylcholinesterase gene in
Pichia pastoris and evaluation of its sensitivity to pesticides Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 317-324.
Acetylcholinesterase (AChE) is a key enzyme for the detection of organophosphorus and carbamate pesticide residues by enzyme inhibition method. In this study, human acetylcholinesterase (hAChE) was secreted and expressed by
Pichia pastoris expression system, and its sensitivity to eight pesticides was evaluated, which laid the foundation for large-scale industrial production of highly sensitive enzyme sources. Primers F1 and R1 were designed according to the published gene sequence of hAChE in GenBank. Then the hAChE gene coding region sequence was amplified by high fidelity PCR using pReceiver-M02- hAChE-ORF plasmid as the template, and the recombinant expression vector pPIC9K- hAChE was constructed by inserting into the vector pPIC9K through double enzyme digestion of SnaB I and Avr Ⅱ. The transformant was selected to sequence and verify the accuracy of the sequence. The linearized pPIC9K- hAChE by Sal I was electro-transformed into P. pastoris GS115. The recombinant strains were screened by minimal dextrase medium (MD) plate and G418 resistance gradient plate. Finally, the positive strain colony was screened on the yeast extract peptone dextrose medium (YPD) plate with 4.00 mg/mL G418 resistance and was identified by polymerase chain reaction (PCR). The expression of the recombinant strain was induced by 0.5% methanol for 144 h. The sodium dodecyl sulfonate (SDS)-polyacrylamide gel electrophoresis map of the fermentation supernatant showed an obvious protein band at 65 kDa, and the size was consistent with the expected hAChE protein. The purified hAChE was obtained using a Q-Sepharose FF chromatography. The content of hAChE protein in the fermentation supernatant was 1.330 mg/mL and accounted for 5.58% in total protein by Bradford method. The highest enzyme activity reached 1 080 nmol/(min?mL) after purification. By preliminary calculation of the 50% inhibitory concentration (IC 50) of the eight common pesticides of the fermentation supernatant, it was found that trichlorfon＞dichlorvos＞arbofuran＞malathion＞dimethoate＞Methamidophos＞Carbaryl＞chlorpyrifos, suggesting that hAChE is most sensitive to chlorpyrifos.
Effects of digested pig slurry application on agronomic trait, yield and forage quality of
indica rice Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 325-331.
In order to promote ecological breeding and reasonable utilization of digested pig slurry from large-scale biogas project, we designed 14 treatments (N1-N14) to explore the effects of biogas slurry application on agronomic trait, yield and forage quality of
indica rice. The results showed that: 1) The overall growth, yield and quality were better than no nitrogen fertilizer treatment within the range of biogas slurry applied amount (N 40%-480%); and compared with the conventional chemical fertilizer treatment (N2), except that the plant height of N3 treatment and tiller number of N4 treatment were both significantly lower, the plant height, tiller number and effective panicle number in other biogas slurry applied treatments changed no significance ( P＞0.05), presented the trend of increasing first and then decreasing. 2) Compared with the N2 treatment, with the increase of biogas slurry applied amount, the rice yield of N5 and N8 treatments increased by 9.5% ( P＞0.05) and 8.9% ( P＞0.05), respectively. As for the aboveground biomass, N8 and N9 treatments increased by 21.4% ( P＜0.05) and 5.4% ( P＞0.05), respectively. The thousand seed mass of N6-N9 treatments indicated an increasing trend with N7 treatment significantly increasing by 11.3% ( P＜0.05). However, when the biogas slurry applied amount was above N10 (816 t/hm 2), the yield of indica rice decreased, even lower than that of N2 treatment. 3) The protein contents of unpolished rice in N5-N14 treatments were higher than that of N1, with the treatments of N7-N14 markedly higher ( P＜0.05). The amylose contents of unpolished rice in N3-N14 decreased to a certain extent compared with the N2. All in all, application of proper biogas slurry is more conducive to the growth of indica rice than conventional chemical fertilizers in this test. Besides, biogas slurry can effectively improve the yield and forage quality of indica rice when the application amount of biogas slurry is 612 t/hm 2 (N8).
Effects of rice-crab culture on nitrogen leaching in rice fields in the north of China.
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 332-342.
In order to explore the leaching loss of nitrogen (N) in the rice-crab culture system, a field experiment was carried out in Panjin, Liaoning Province. A split-plot design with two factors was arranged in this experiment, with crab or without crab as main factors, with or without N fertilizer as sub-factors. The treatments included: 1) rice monoculture without N fertilizer (R0M); 2) rice-crab culture without N fertilizer (R0C); 3) rice monoculture with N fertilization (with N of 160 kg/hm
2, R1M); 4) rice-crab culture with N fertilizer (with N of 160 kg N/hm 2, R1C). The results showed that NH 4 +-N was the primary form of N in flooding water, accounting for 50.8% of the total N (TN), while NO 3 －-N contributed the most to N in leaching water, accounting for 58.5% of TN. Fertilization could significantly increase the concentrations of microbial biomass nitrogen (MBN) in soil, N in flooding and leaching water ( P＜0.05). The mean concentration of soil MBN in rice-crab field was 17.7% higher than that in rice monoculture. Rearing crab significantly increased the concentration of MBN in soil and reduced leaching losses of NO 3 －-N ( P＜0.05), but had little effect on N concentrations in flooding water and leaching losses of NH 4 +-N and dissolved organic nitrogen (DON). The concentration of NO 3 －-N in the leaching water showed positive correlation with NO 3 －-N in flooding water ( P＜0.01), while the concentration of DON in the leaching water was negatively correlated with the concentration of soil MBN ( P＜0.01). The cumulative TN leaching of R1M and R1C treatments was 7.6% and 6.3%, respectively, which indicated that the N leaching was not the predominant fertilizer N losses. Rearing crab in rice fields could reduce the cumulative TN leaching by 15.0% at fertilized plots ( P＜0.05), and 7.2% at no fertilized plots ( P＞0.05). All in all, the integration of rice and crab cultivation effectively reduces the leaching loss of N fertilizer in rice field.
Effects of super absorbent polymer with different application rates on soil characteristics and flue-cured tobacco growth in Qinba mountain area
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 343-354.
In order to improve soil characteristics, reduce the effect of seasonal drought on the growth of flue-cured tobacco, and promote the application of super absorbent polymer in flue-cured tobacco production, we analyzed the effects of different super absorbent polymer application rates on soil moisture, soil bulk density, flue-cured tobacco growth, conventional chemical composition and economic characteristics. The potassium polyacrylate super absorbent polymer of 45, 60 and 75 kg/hm
2 was separately applied to the tobacco field with yellow brown soil in Qinba mountain area, and no super absorbent polymer was applied as a control. The results showed that the application rate of 75 kg/hm 2 had a relatively large effect on the soil moisture from seedling survival stage to rosette stage of flue-cured tobacco, while the treatment with application rate of 60 kg/hm 2 had a relatively greater effect on soil moisture from flourishing growth stage to after maturity stage. However, different application rates of potassium polyacrylate super absorbent polymer had no significant effect on soil moisture from seedling survival stage to after maturity stage. The soil bulk density of 0-20 and ＞20-40 cm soil layers was significantly reduced by the treatment with application rate of 60 kg/hm 2, but the treatments with application rate of 45 and 75 kg/hm 2 had no significant effect on soil bulk density. All application rates of potassium polyacrylate super absorbent polymer promoted the growth of flue-cured tobacco, increased flue-cured tobacco yield, conventional chemical composition and output value. The proportion of superior tobacco significantly increased, while the proportion of inferior tobacco significantly decreased, and the net output value of the flue-cured tobacco significantly increased by the treatment with application rate of 60 kg/hm 2. In sum, the suitable application rate of potassium polyacrylate super absorbent ploymer is about 60 kg/hm 2 in the tobacco field with yellow brown soil in Qinba mountain area.
Allocation analysis of natural and cultivated grassland resources of eco-family pasture based on the theory of grassland agro-ecosystem discordance
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 355-364.
In order to achieve sustainable development in pastoral areas, based on the theory of grassland agro-ecosystem discordance, we selected the meteorological factors in the study area of Otog Front Banner in recent 30 years and the water, soil, grass, and livestock in the area in recent years as the objects of study to discuss how to make a grassland resource allocation plan. The result showed that the natural environment in the study area was harsh and the annual precipitation was very limited; the groundwater resources were abundant but unevenly distributed, and the water consumption for agriculture and animal husbandry exceeded the expected limit. The proportion of natural grassland was obvious, but the overgrazing was serious, and the ecosystem was fragile, and the contradiction of herbage resources was prominent. The most important problem was time paradox, followed by space paradox, and finally by species paradox in the development of family ranches. Meanwhile, in the time paradox, it was necessary to reasonable matching cultivation of grassland resources according to the shortage period and surplus period of natural grassland; in the space paradox, on the one hand, developing high-yielding and high-quality cultivated grasslands to replace natural grassland functions in the towns of Aolezhaoqi and Chengchuan, and on the other hand, it was necessary to strictly control over the stock capacity of natural grassland, develop short-term fattening and improve turnover efficiency in the towns of Aangsu and Shanghaimiao.
Determination and ecological risk assessment of phthalic acid esters in marine sediments
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 365-375.
We proposed a new method for the determination of 16 phthalic acid esters (PAEs) in marine sediments using gas chromatography-mass spectrometry (GC-MS) combined with ultrasonic extraction and dispersive solid phase extraction cleanup. Moreover, the contents of PAEs in marine sediments of Sanmen Bay, Taizhou and waters of Nanji Island, Wenzhou were detected and their ecological risk was assessed. Under the optimized condition, the linearity of this method ranged from 1 to 1 000 μg/L, with regression coefficients ranging between 0.999 1 and 0.999 9. The limits of detection were in the rang of 0.10-0.25 μg/kg. The recoveries of spiked 16 PAEs at different concentration levels were in the range of 75%-116%, with relative standard deviations of 3.1%-7.2%. The results showed that the total concentration of phthalate esters ranged from 297.43 to 550.25 μg/kg. Diisobutyl phthalate (DIBP), di-
n-butyl phthalate (DBP) and di (2-ethylhexyl) phthalate (DEHP) were the predominant compounds in all detected seafood, accounted for 88.7%-96.7% of the total PAEs. Marine sediment’s ecological risk was assessed with the methods of sediment quality guidline and risk quotient. The sediment quality guidline results showed the contents of PAEs in the sediments were all lower than the environmental risk limits, which indicated low ecological risk. The risk quotient values of PAEs were in the following order: DBP＞DIBP＞DEHP, and DBP was high risk to algae and medium risk to crustaceans and fish, and DIBP was medium risk to crustaceans, and DEHP was low risk to aquatic organisms. Overall, the risk of PAEs on algae was the largest, followed by crustaceans, minimal risk for fish, and the health risk of Sanmen Bay, Taizhou was slightly higher than that of waters of Nanji Island, Wenzhou.
Identification for internal reference genes in different periods of granulosa cells of Tianfu meat geese.
Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 376-384.
In order to screen out the most stable reference genes in different periods of granulosa cells in goose, we selected 10 candidate reference genes (
GAPDH, ACTB, TUB, UBC, HMBS, SDH, 18S, 28S, TBP, HPRT1) to determine the relative expression levels by the real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The expression stabilities of 10 candidate reference genes in nine different stages of granulosa cells were systematically analyzed by delta- C T, qbase +, NormFinder and BestKeeper, respectively. The results of RT-qPCR melting curve and PCR amplification showed that the 10 candidate reference genes were specifically amplified. By constructing a standard curve, between C q value and the logarithm of relative copy number exhibited a good linear relationship in the serial dilution concentration gradient. Based on the evaluation results of four different algorithms, SDH, HMBS and 18S were found to be three of the most stable reference genes, but UBC, GAPDH and TUB were three of the least stable reference genes in the different periods of granulosa cells. Therefore, the most stable internal reference genes were SDH and HMBS in granulosa cells at different developmental stages, and it could get more accurate normalization of RT-qPCR data by geometric averaging of the most stable reference genes.