Journal of Henan Agricultural Sciences

Application Progress of CRISPR/Cas9 Technology in Crop Genetic Breeding

JIAO Yaolei, WANG Chunsheng, QU Shuo, SUN Shanshan, ZHU Tingting, ZHAO He, WANG Piwu

2021, 50(7): 1-7. DOI: 10.15933/j.cnki.1004-3268.2021.07.001

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CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISRP‑associated nuclease 9）is a new type of genome⁃directed editing technology after ZFNs（zinc finger nucleases）and TALENs（transcription activator like effector nucleases）.Compared with the previous two generations of technology，it has the characteristics of simplicity and high efficiency.CRISPR/Cas9 is not only a basic research tool，but also has become one of currently useful molecular breeding tools，and important progress has been made in crop genetic improvement. The structure，classification，action mechanism of the CRISPR/Cas9 system and its application progress in crop quality improvement，yield enhancement，resistance breeding and male sterile material selection were reviewed，its existing problems were discussed，and its application prospects were prospected.

Advances in the Influence of Ecological Factors on the Content of Saponins and Planting Patterns of Paris polyphylla var. chinensis

HU Ke, TIAN Meihua, TANG Anjun

2021, 50(7): 8-14. DOI: 10.15933/j.cnki.1004-3268.2021.07.002

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The species of Paris polyphylla var. chinensis is one of the Chinese traditional medicinal materials.However，the wild resources of this species are sharply decreasing due to excessive unscientific collection and digging，even already resulting in such a phenomenon that the wild P.polyphylla var.chinensis of a few natural distribution areas has disappeared.Artificially planting P. polyphylla var.chinensisis is largely increasing，which has been considered as an effective way to solve the contradiction between supply and demand of Paris resources. The medical part of this medical species is mainly the rhizome and the saponin content in the dry rhizomes must satisfy the official standard in China. Based on the research and investigation in recent years，the difference of saponin content in the rhizome and aboveground part of P. polyphylla var. chinensis，the influences of ecological factors and planting patterns were summarized and analyzed in this paper. Seeds seed coats and leaves of this species also contain abundant saponins，however，lower than that of the rhizome. And the climatic factors and soil factors are the leading ecological factors affecting the saponin content in the rhizome. In practice，planting P.poluphylla var. chinensis under forest and in shade shed is major type at present，and appropriate shading treatments and canopy density can improve both the growth of the whole plant and its rhizome. Inaddition，some main problems occurring in studying saponins and planting P.poluphylla var. chinensis were commented on，and a few study directions worth exploring in the future were put forward.

Effects of Reduced Application of Nitrogen and Phosphorus on Yield and Nutrient Utilization of Spring Wheat under Drip Irrigation of Water⁃Fertilizer Integration

ZHANG Lei, LI Huaisheng, LIU Jinxia, WANG Heya, LIANG Fei, WANG Guodong, LI Jinke, YANG Jie

2021, 50(7): 15-22. DOI: 10.15933/j.cnki.1004-3268.2021.07.003

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No fertilization treatment（T1），supervised fertilization treatment（T2），supervised fertilization without nitrogen fertilizer treatment（T3⁃N），supervised fertilization without phosphorus fertilizer treatment（T4⁃P） and farmers’routine fertilization treatment（T5） were set up to study the effects of reduced application of nitrogen and phosphorus on yield and nutrient utilization of spring wheat under drip irrigation of water⁃fertilizer integration，so as to provide scientific basis for reduced application of fertilizer for spring wheat under drip irrigation of water⁃fertilizer integration. The results showed that reasonablewater and fertilizer operation could increase dry matter accumulation of spring wheat under drip irrigation. The dry weight of spring wheat of T3⁃N and T4⁃P treatments was similar and high，followed by T2 treatment.The nitrogen and phosphorus contents of grain，glume，stem and leaf of T2，T3⁃N and T4⁃P treatments were higher than those of T5 and T1 treatments. The yield of spring wheat of T2 treatment significantly increased by 20.07% and 33.44% compared with T5 and T1 treatments respectively.Compared with T5 treatment，the application rate of nitrogen fertilizer of T2 treatment decreased by 25%，and the application rate of phosphate fertilizer of T2 treatment decreased by 112%，while the yield of spring wheat of T2 treatment did not reduce，even increased. Compared with the farmers’routine fertilization treatment，the fertilization treatments，（T2，T3⁃N，T4⁃P）with water and fertilizer integration could improve the agronomic efficiencies，partial productivities and utilization efficiencies of nitrogen and phosphorus fertilizers. In general，fertilization treatments with water and fertilizer integration can improve spring wheat yield and fertilizer utilization efficiency，reduce the amount of fertilizer application，and achieve the purpose of reduction of fertilizer without reduction of yield.

Effects of Cutting off Top 3rd Leaf on Yield and Quality of Malting Barley

LIU Xiaoning, PAN Yongdong, BAO Qijun, ZHANG Huayu, ZHAO Feng

2021, 50(7): 23-29. DOI: 10.15933/j.cnki.1004-3268.2021.07.004

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Abstract：In order to investigate the influence of top 3rd leaf on yield and quality of malting barley，the top 3rd leaf of Ganpi No.6 was cut off at heading stage in 2016—2017，and the effects of cutting off the top 3rd leaf on dry aboveground weight，grain yield，grain quality and brewing quality were analyzed. The results showed that cutting off the top 3rd leaf decreased the dry weight of top fourth leaf，internode below ear and total internode，increased dry weight of flag leaf，top 2rd leaf and their leaf sheaths，spike weight，1 000⁃grain weight and yield，and the yield increased by 10.7% in 2017. Cutting off the top 3rd leaf increased protein content，starch content（2017）and screening rate of top grain of spike，and significantly decreased thin and sterile grain rates of middle and top grains of spike，so improved the grain quality of malting barley. The protein content of top grain of spike increased by 8.3%（2017），and thin and sterile grain rate of top grain of spike decreased by 22.2%—37.5%. Cutting off the top 3rd leaf significantly decreased β⁃glucanase content，saccharifying time（2017） and chroma，significantly increased diastatic power，soluble nitrogen content and Kolbach index，so improved the brewing quality of malting barley. The β⁃glucanase content decreased by 23.6%—32.6%，diastatic power increased by 16.3%—18.1%，and soluble nitrogen content increased by 16.1%—22.0%. In conclusion，cutting off top 3rd leaf can promote the growth of flag leaf and top 2rd leaf，which synthesize more photosynthetic products，so increases grain yield；cutting off top 3rd leaf can increase protein content of top grain of spike，diastatic power and soluble nitrogen content，and decrease thin and sterile grain rate，β⁃glucanase content，saccharifying time and chroma，so improves malting barley quality.

Effects of Sorghum⁃Soybean Intercropping on Soil Moisture Distribution and Water Use Efficiency

LIANG Xiaohong, CAO Xiong, ZHANG Ruidong, LIU Jing, HUANG Minjia, WANG Aiai

2021, 50(7): 30-38. DOI: 10.15933/j.cnki.1004-3268.2021.07.005

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To explore the effects of sorghum⁃soybean intercropping on water use efficiency，field experiments were conducted in 2018 and 2019，seven treatments were designed，which were monoculture of high⁃stalk sorghum Jinza 22（G1），monoculture of low⁃stalk sorghum Jinza 34（G2），monoculture of soybean（D，Jindou 40 in 2018，Jindou 25 in 2019），intercropping of 2 rows of sorghum and 2 rows of soybean（2G1∶2D，2G2∶2D），intercropping of 2 rows of sorghum and 4 rows of soybean（2G1∶4D，2G2∶4D），and the distribution of 0—100 cm soil moisture，water use efficiency of leaf，dry biomass，yield of different treatments and water equivalent ratio（WER）of intercropping system were studied. The results showed that the soil water content（0—100 cm）of soybean monoculture treatment in the growth period was higher than those of sorghum⁃soybean intercropping and sorghum monoculture treatments，and the soil water content in sorghum strip was lower than that in soybean strip. Compared with the corresponding sorghum monoculture treatments，the average soil water content of Jinza 22⁃soybean intercropping treatment increased，and that of Jinza 34⁃soybean intercropping treatment decreased，especially in the 40—80 cm soil layer. Compared with the soybean monoculture treatment，the leaf water use efficiency of soybean of 2G1∶2D，2G2∶2D，2G1∶4D and 2G2∶4D treatments decreased by 19.86%，16.14%，6.67%，5.26% for two years respectively，and the leaf water use efficiency advantage of soybean of 2∶4 treatment performed better than that of 2∶2 treatment. The water use efficiencies of dry biomass and yield of sorghum of 2G1∶2D and 2G1∶4D treatments were not significantly different from G1 treatment. But compared with G2 treatment，the water use efficiencies of dry biomass and yield of sorghum of 2G2∶2D treatment significantly decreased by 24.02% and 24.67% in 2019，and the water use efficiency of yield of sorghum of 2G2∶4D treatment significantly decreased by 37.48% and 35.81% in 2018 and 2019 respectively. Compared with soybean monoculture treatment，the average water use efficiency of dry biomass of soybean of 2G1∶2D treatment decreased by 50.71% for two years，the water use efficiency of yield of soybean of 2G1∶2D treatment significantly decreased by 60.49% in 2019，and the water use efficiency of dry biomass and yield of soybean of 2G2∶2D treatment significantly decreased by 42.92%and 60.85% in 2019 respectively. The water equivalent ratios of all intercropping treatments were more than 1，and 2G1∶4D treatment was the highest，indicating that the sorghum⁃soybean intercropping system increased cropland water use efficiency. There was no significant difference in water equivalent ratio among intercropping treatments in 2018. The water equivalent ratios of 2∶4 treatments were significantly higher than those of 2∶2 treatments，and 2G1∶4D，2G2∶4D treatments increased by 51.18%，48.28% compared with 2G1∶2D，2G2∶2D in 2019 respectively. In conclusion，sorghum⁃soybean intercropping increased cropland water use efficiency，and 2G1∶4D treatment was optimal under the experimental condition.

Effect of Sowing Date on Dry Matter Accumulation, Transportation and Yield of Different Foxtail Millet Cultivars

LI Junxia , FAN Yongqiang, DAI Shutao, ZHU Cancan, HAN Yanli, QIN Na, WANG Yanhui, SONG Yinghui

2021, 50(7): 39-47. DOI: 10.15933/j.cnki.1004-3268.2021.07.006

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In order to determine the suitable sowing date of summer sowing foxtail millet cultivars in Henan Province，four main cultivars，Yugu 17，Yugu 23，Yugu 28 and Yugu 29，were used to analyze change rules of growth period，dry matter accumulation and transportation of various vegetative organs before anthesis and yield under five sowing dates（June 11，June 19，June 26，July 3 and July 10）. The results showed that with the delay of sowing date，the days from seedling to heading of foxtail millet were shortened，while the days from heading to ripening were prolonged. With the delay of sowing date，dry weight of vegetative organs at anthesis and mature stages firstly decreased and then increased. All foxtail millet cultivars had relatively higher dry matter translocation amount of vegetative organs before anthesis under the first three sowing dates. The transportation rate of each vegetative organ before anthesis showed stem sheath>leaf> stem，while the contribution rate of vegetative organs to grain was just opposite，showing stem sheath<leaf<stem. Under different sowing dates，the transportation rate of total dry matter of vegetative organs before anthesis ranged from 17.26% to 45.83%，which showed a trend of first increase and then decrease with the delay of sowing date，while contribution rate of vegetative organs to grain ranged from 8.83% to 25.17%，which didn’t show a clear pattern. Further analysis showed that spike length and diameter were not significantly affected by sowing date，and their differences were mainly due to the characteristics of cultivars，while grain weight per spike and 1 000⁃grain weight decreased obviously with the delay of sowing date. The grain yield of all foxtail millet cultivars decreased with the delay of sowing date，and different cultivars had different responses to the delay of sowing date，the optimum sowing dates of Yugu 17 and Yugu 23 were before June 19，while the optimum sowing dates of Yugu 28 and Yugu 29 were before June 26 and July 3，respectively. On the whole，the suitable sowing date is before June 19 for summer foxtail millet cultivars in Henan Province，and sowing date can be advanced or delayed appropriately according to the cultivar characteristics.

Effects of Waterlogging Stress and Exogenous Abscisic Acid on Stem Physiology and Yield of Adzuki Bean Seedlings

LI Wan, LI Bo, XIANG Hongtao, HE Ning, WANG Manli, LIU Jia

2021, 50(7): 48-56. DOI: 10.15933/j.cnki.1004-3268.2021.07.007

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In order to provide theoretical basis for adzuki bean waterlogging resistance cultivation，this study investigated the effects of waterlogging stress and pre⁃spraying abscisic acid on stem physiological indexes and yield of adzuki bean by waterlogging for five days at seedling stage，using adzuki bean varieties Longxiaodou 4（LXD4）and Tianjinhong（TJH）under potted conditions，with spraying distilled water and normal soil moisture as control（CK）. The results showed that the contents of H2O2，MDA，proline，soluble sugar，soluble protein and the activities of SOD，POD and CAT in the stem of adzuki bean were increased under waterlogging stress at seedling stage. Compared with CK，the yields of LXD 4 and TJH were significantly decreased by 8.40% and 9.91% after five days of waterlogging，respectively.content，significantly increase activities of POD and CAT in the stem，further increase the SOD activity in the stem of LXD4，and increase the content of proline，soluble protein and soluble sugar of TJH stem.Pre⁃straying ABA significantly increased the yield of LXD4 and TJH after four days of waterlogging by 6.95% and 4.46%，respectively. In summary，waterlogging stress caused the accumulation of H2O2 and membrane lipid peroxidation in the stem of adzuki bean，induced the increase of protective enzyme activity and osmotic adjustment substance content to resist stress，and finally led to the decrease of yield. Exogenous ABA could increase the activity of protective enzymes and the content of osmotic adjustment substances，improve antioxidant capacity and alleviate the harm of waterlogging stress.

Effects of Zn Contamination on Fe Oxidation and Reduction in Calcareous Paddy Soil

FAN Haolong, WANG Xugang, SUN Lirong, ZHAO Xu, GUO Dayong, SHI Zhaoyong

2021, 50(7): 57-65. DOI: 10.15933/j.cnki.1004-3268.2021.07.008

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In order to clarify the effect of heavy metal Zn on iron redox process in soil，calcareous paddy soil sampled from the middle reaches of the Yellow River was contaminated with the addition of ZnSO4•7H2O solution，anaerobic slurry incubation was conducted in the experiment to study the dynamic change of Fe（Ⅱ）in the process of incubation，and the redox status of iron was analyzed. The modified BCR protocol was employed to measure Zn morphology in soils before and after incubation under illuminated and dark conditions to discuss phase transformation of reducible Zn. Water soluble organic and inorganic carbon in soils，CO2 and CH4 in the headspace were evaluated using total organic carbon analyzer and gas chromatography respectively to clarify carbon mineralization in soil contaminated with Zn. The results showed that reduction capacity of Fe（Ⅲ）in soils decreased by Zn contamination，and the decreasedamplitude enlarged with elevation of Zn pollution level. Ferrous oxidation appeared in soil incubated under illumination，and was hindered by Zn contamination. Fe（Ⅱ）oxidation amount declined from2.96 mg/g to 0.91—1.71 mg/g，and the decrease enlarged with elevation of contamination level. Velocity of Fe（Ⅱ）oxidation and percentage of Fe（Ⅱ）oxidized both decreased significantly with increase of Zn pollution. Diethylenetriaminepentaacetic acid extractable Zn in contaminated soil decreased after 30 d of incubation，and the extractability was related to incubation conditions. 98.09% and 85.21% of externally added Zn in soils incubated under dark and illuminated conditions could not be extracted by diethylenetriaminepentaacetic acid respectively. In conclusion，both iron reduction and ferrous oxidation in calcareous paddy soils were impeded by zinc contamination，and the inhibition was promoted with elevated Zn pollution level. Externally added Zn in soils incubated darkly would transform from weak acid
extractable Zn to reducible Zn.

Effects of Bio⁃organic Fertilizer Made from Chinese Traditional Herb Residues on the Content of Heavy Metals,Microbial Biomass Carbon and Nitrogen and Enzyme Activity in Hg and Cd Compound Contaminated Soil

CHEN Fen, YU Gao, ZHANG Hongli, WU Hanqian, CHEN Rong, HOU Jianwei

2021, 50(7): 66-75. DOI: 10.15933/j.cnki.1004-3268.2021.07.009

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In order to understand the improvement effect of bio⁃organic fertilizer made from Chinese traditional herb residues（BOFCTHR）on heavy metal contaminated soil，an indoor incubation study was conducted to investigate the effects of BOFCTHR［0（CK），0.75%，1.50% and 3.00%］on soil nutrients contents，available heavy metals contents（HCl⁃Hg，DTPA⁃Cd），microbial biomass carbon，nitrogen and enzyme activity in Hg and Cd compound contaminated soil. The results showed that soil pH，soil organic matter，total N，available P，available K，and microbial biomass carbon contents，catalase activity，urease activity and invertase activity were linearly and positively correlated with the application ratio of BOFCTHR. The contents of HCl⁃Hg and DTPA⁃Cd were linearly and negatively correlated with the application ratio of BOFCTHR. Soil microbial biomass nitrogen tended to increase gradually and then decrease with the increasing application ratio of BOFCTHR. The correlation analysis showed that the contents of HCl⁃Hg and DTPA⁃Cd were negatively related to soil nutrients contents，microbial biomass carbon and enzyme activity（except for microbial biomass nitrogen）. The path analysis results showed that soil catalase activity had the greatest impact on the HCl⁃Hg content，and soil urease activity had the greatest impact on the DTPA⁃Cd content. It is concluded that BOFCTHR can decrease soil heavy metals contents，enhance soil fertility，and it can be used in acidic southern soil.

Effects of Long⁃term Application of Biogas Slurry on Soil Nutrient Content and Enzyme Activity

WU Jicheng, PAN Xiaoying, YANG Yonghui, GAO Cuimin, WANG Yue,

2021, 50(7): 76-86. DOI: 10.15933/j.cnki.1004-3268.2021.07.010

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In order to find out the effect of different application years of biogas slurry on soil nutrient content and enzyme activity，11 treatments with different amount and years of biogas slurry applied were designed according to the source of biogas slurry（breeding enterprises），and the nutrients contents and enzymes activities of soil applied with biogas slurry and the control soil without biogas slurry were studied. The results showed that the contents of organic matter（OM），total nitrogen（TN），total phosphorus（TP）and total potassium（TK）of biogas slurry were relatively low，OM contents were 0.03—0.94 g/L，the average was 0.26 g/L；TN contents were 0.30—2.60 g/L，the average was 1.27 g/L；TP contents were 0.04—0.38 g/L，the average was 0.19 g/L；TK contents were 0.40—1.98 g/L，the average was 0.94 g/L；pH values were 7.40—8.59，the average was 8.11. Long⁃term application of biogas slurry changed pH value to be conducive to soil nutrient release，and increased the nutrients contents in the soil tillage layer.Compared with the control，the OM contents increased by 0.96%—367.11%，TN contents increased by 2.15%—600.00%，TP contents increased by 29.94%—663.27%，and the treatment with 900—1 050 t/ha biogas slurry produced by Henan Kanglong Industrial Group Co.，Ltd. applied for 21 years had the biggest increase range；TK contents increased by 1.14%—28.10%，and the treatment with 900—1 050 t/ha biogas slurry produced by Henan Youlin Industrial Co.，Ltd. applied for 5 years had the biggest increase range；available phosphorus（AP）contents increased by 26.76%—593.99%，hydrolyzed nitrogen（HN）contents increased by 7.49%—420.24%，and the treatment with 900—1 050 t/ha biogas slurry produced by Henan Kanglong Industrial Group Co.，Ltd. applied for 21 years had the biggest increase range；available potassium（AK）contents increased by 15.48%—103.30%，and the treatment with 900—1 200 t/ha biogas slurry produced by Henan Yifa Animal Husbandry Co.，Ltd. applied for 27 years had the biggest increase range. Long⁃term application of biogas slurry significantly increased the activities of soil hydrolase and oxidoreductase. Compared with the control，the protease activities increased by 9.20%—44.69%，sucrase activities increased by 6.41%—41.91%，urease activities increased by 14.47%—42.58%，acid phosphatase activities increased by 13.03%—44.44%，cellulase activities increased by 2.77%—44.65%，nitrate reductase activities increased by 22.00%—54.38%，nitrite reductase activities increased by 1.41%—42.17%dehydrogenase activities increased by 4.94%—47.44%，catalase activities increased by 1.42%—47.67% respectively. There were significantly or extremely significantly positive correlations between soil nutrients contents and enzymes activities. The contents of soil OM，TN，TP，TK，HN，AP and AK were extremely significantly positively correlated with the activities of dehydrogenase and catalase；the contents of TN，HN，AP were extremely significantly positively correlated with the cellulase activity；the contents of OM，AK were significantly positively correlated with the cellulase activity.

Arsenic Content and Spatial Distribution in Paddy Soil Profile of Typical Parent Material in Hunan Province

SHI Qiang, WANG Cuihong, BU Siyi, ZENG Ying, LI Xiaojing, OUYANG Ningxiang

2021, 50(7): 87-100. DOI: 10.15933/j.cnki.1004-3268.2021.07.011

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In order to guide the prevention and control of arsenic pollution in farmland and improve the basic database of heavy metals in farmland，the changes and spatial distribution characteristics of arsenic content in paddy soil profile of typical parent material in Hunan Province were studied. Based on the soil classification，59 typical sections and 332 soil samples were excavated from the paddy soils of Hunan typical parent materials，namely，alluvial sandy soil，grey clayey soil，yellow clayey soil，reddish yellow clayey soil，purple clayey soil and granitic sandy soil. The As content，pH，particle classification composition，organic matter and free iron oxide were detected and analyzed，with a focus on the section characteristics and spatial distribution of As in paddy soil and their relationship with physical and chemical properties of soil.The results showed that the content of total arsenic in the whole soil profile was 2.03—31.16 mg/kg，with an average of 12.30 mg/kg，mostly in the range of 2.00—18.00 mg/kg. The contents of total arsenic in alluvial sandy soil，grey clayey soil，yellow clayey soil，reddish yellow clayey soil，purple clayey soil and granitic sandy soil were 16.78，15.01，13.86，12.24，8.44，7.40 mg/kg. The arerage contents of total arsenic in the cultivated layer and the bottom layer were 12.09 mg/kg and12.34 mg/kg，respectively.On the whole，the contents of total arsenic in soils distributed in Eastern Hunan and in a few soil samples in Southern Hunan and Western Hunan were higher.The average contents of available arsenic in paddy soil were 0.19 mg/kg in the whole layer，0.26 mg/kg in the surface layer and 0.17 mg/kg in the bottom layer，respectively.The content of available arsenic was the highest in grey clayey soil，lowest in granitic sandy soil，and similar in other paddy soils.The contents of organic matter and free iron oxide in paddy soil were the main soil factors to determine the total arsenic content.In summary，most of the paddy soils tested in Hunan Province based on soil classification were at safe arsenic levels.

Pathogen Identification of Tobbaco Fusarium Root Rot and Its Sensitivity to Five Fungicides

LIU Lijia, LI Fangfang, HE Lei, PENG Yufu, DING Yongle, SUN Jutao

2021, 50(7): 101-109. DOI: 10.15933/j.cnki.1004-3268.2021.07.012

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In order to screen out effective pesticides for the control of tobacco Fusarium root rot，and reduce the impact of the disease on the yield and quality of tobacco leaves，the methods of microscopic observation，evolutionary tree analysis，and inoculation identification were used to conduct biological identification，molecular identification and pathogenicity identification of the pathogen NC⁃11 isolated from the roots of tobacco Fusarium root rot plants in Xiangcheng County，Henan Province. Five fungicides were selected for virulence test，and the best fungicide among them was tested for field control effect. The results showed that the pathogen NC⁃11 isolated from the roots of tobacco plants with Fusarium root rot disease was Fusarium oxysporum. There were significant differences in the inhibitory effects of the five tested fungicides on the mycelial growth of Fusarium oxysporium. The inhibitory effect of metalaxyl⁃M·fludioxonil 62.5 g/L FSC was the best，its EC50 was 0.186 2 mg/L，and the inhibition rate on mycelial growth was up to 97.27%；Famoxadone·mancozeb 68% WG had the worst inhibitory effect，its EC50 was 203.152 2 mg/L，when the mass concentration was 1 000.00 mg/L，the inhibition rate on mycelial growth was only 66.53%. Field verification results showed that 46 days after the first application in the field，the relative control effect of metalaxyl⁃M·fludioxonil 62.5 g/L FSC reached 72.69%.Therefore，metalaxyl⁃M·fludioxonil 62.5 g/L FSC can be used as the first choice for the prevention and control of tobacco Fusarium root rot.

Effects of Imidacloprid on Resistance Selection and Activity of Detoxifying Enzyme in Bradysia difformis Frey

ZHANG Rui, HE Chao, LI He, SHEN Dengrong, YUAN Shengyong, LI Xun

2021, 50(7): 110-116. DOI: 10.15933/j.cnki.1004-3268.2021.07.013

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Bradysia difformis is one of the main pests of edible fungi. In order to investigate the resistance risk and biochemical mechanism of B.difformis to imidacloprid，resistance selection and realized heritability（h²） of B.difformis to imidacloprid were analyzed in the laboratory，and the detoxifying enzyme activities of B.difformis treated with sublethal doses of imidacloprid（LC₁₀，LC₂₀ and LC₄₀）were determined. The results showed that the resistance ratio of B. difformis was 4.09 times after 21 generations of resistant selection，and resistance level of B.difformis was sensitivity drop to imidacloprid.The realized heritability（h²） of B.difformis to imidacloprid was 0.091 3，it would take 15.9—20.1generations to achieve 10⁃fold resistance under the selection pressure of 80% to 90%. With the increase of the sublethal dose of imidacloprid，the activities of mixed function oxidase O⁃demethylation and carboxylesterase in susceptible strain and resistant strain of B.difformis increased gradually，and both reached the peak value at 48 h after LC40 treatment，at this time，the activities of mixed function oxidase O⁃demethylation in susceptible strain and resistant strain increased by 63.81% and 119.42%，and the activities of carboxylesterase in susceptible strain and resistant strain increased by 18.40% and 48.13%，respectively，compared with blank control. These results indicated that the resistance risk of B.difformis to imidacloprid was low，and the enhanced activities of mixed function oxidase and carboxylesterase were closely related to the metabolic resistance of B. difformis to imidacloprid.

Study on the Rapid Identification Method of Alternaria alternata in Imported Wheat

LI Yiran, XUE Jiaxin, LIANG Liang, LAN Xi, QIN Jinyi, GUO Jiaqi, SUN Hao

2021, 50(7): 117-124. DOI: 10.15933/j.cnki.1004-3268.2021.07.014

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Fungal diseases are usually found in wheat during port quarantine. In order to detect pathogenic microorganisms carried by imported grain more quickly and simply，the genetically stable phospholipid fatty acid（PLFA）in the microbial cell membrane was used as an important identification marker. Based on the observation of microbial LB culture and DNA sequencing analysis,it was determined that the strain isolated and purified from the diseased part of imported wheat was Alternari alternata,which was one of the main pathogens of crops. After PLFA analysis，the significant difference in response value was compared，and the biological sampling range for detecting this strain was 0.070—0.090 g. After correlation analysis and redundancy analysis，it was determined that the characteristic fatty acids of Alternaria alternata were 15∶0 iso，15∶0 anteiso，16∶1 w7c and 18∶2 w6，9c. By analyzing the correlation between the biological sampling amount and the PLFA response value，the models of Y1 =0.047 + 6.183 × 10-5 × C15：0 anteiso + 4.330 × 10-6 × C18：2w6，9c + 1.386 × 10-7 × C16：1w7c and Y2 = 0.036 +2.864 × 10-5 × C15：0 iso were established. Y1 is the biomass calculated by regression of 15：0 anteiso，18：2 w6，9c and 16：1 w7c；Y2 is the biomass calculated by 15：0 iso regression.This model can predict the amount of pathogenic microorganisms carried by wheat，and provides theoretical data and referencesfor establishing quality quarantine and testing standards for imported grains.

Identification and Sequence Analysis of the MdLAC Gene Family Members in Apple

YANG Yang, LIU Yuanyuan

2021, 50(7): 125-135. DOI: 10.15933/j.cnki.1004-3268.2021.07.015

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In order to understand the function and phylogenetic relationship of the apple laccase（MdLAC）gene family，the members of the MdLAC gene family were identified by the bioinformatics method，at the same time，the physical and chemicalproperties，phylogenetic relationships，gene structure，conserved structure，amino acid sequence and chromosome location were analyzed. The results showed that 77 MdLAC gene family members were identified in apple. The length of the amino acid sequence of encoded proteins ranged from 323 to 611，the molecular weight ranged from 36.29 to 67.87 ku，and the isoelectric point（pI） ranged from 4.65 to 9.92. The results of subcellular localization prediction showed that most of the MdLAC proteins were located outside the cell，the phylogenetic tree analysis showed that the MdLAC gene family had 10 branches，and the conservative structure analysis showed that the MdLAC gene family was conservative in structure. Chromosome mapping revealed that 77 MdLAC genes were unevenly distributed on 18 chromosomes.

Analysis of Combining Ability and Heredity for Plant Yield Traits in Pepper

HAN Ya’nan, CHANG Xiaoke, CHENG Zhifang, ZHANG Tao, YAO Qiuju

2021, 50(7): 136-144. DOI: 10.15933/j.cnki.1004-3268.2021.07.016

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In order to evaluate the combining ability and heredity for plant architecture and yield components，and improve the efficiency of selecting elite combinations of pepper（Capsicum annuum L.）,five parental lines（C1—C5）with different characters were used to develop 20 hybrid crosses by Griffing complete diallel crossing design，and nine agronomic traits of 25 genetypes were determined to analyze heterosis，combining ability and heritability．The results showed that the heterosis indexes of yield per plant，fruit number per plant，plant width and fruit length were higher，with the averages of up to 145.98%，131.35% 122.51% and 121.20%，respectively. The coefficients of variation of yield per plant and fruit number per plant were 31.80% and 17.78%，respectively. Analysis of combination ability showed that C2 and C4 with high general combination ability（GCA）were the optimal lines in single fruit weight and single plant yield，and the crosses C4×C5，C1×C2，C2×C3 and C4×C5，C3×C2 were determined as the excellent combinations in weight and single plant yield respeitively based on special combination ability（SCA）and reciprocal（REC）effects. The high values of broad⁃sense heritability and narrow⁃sense heritability for yield per plant and fruit width indicated that these traits were mainly controlled by additive effects，and the environmental variances of stem diameter and flesh thickness were higher than their genetic variances. The coefficients between major traits and yield per plant suggested that the yield per plant was positively correlated with single fruit weight，fruit length and fruit number per plant. In conclusion，the parental lines C2,C4 and the crosses C3×C2，C4×C5 are suitable for cultivating highly⁃yielding varieties. Pepper breeding should be focused on single fruit weight，fruit length and fruit number per plant.

Photosynthetic Regulation Mechanism of Red and Blue LED Light Quality on Growth of Pepper Seedlings

LIU Yubing, CHEN Haiyan, WANG Junwei, HUANG Ke, LI Jie, LIU Mingyue

2021, 50(7): 145-153. DOI: 10.15933/j.cnki.1004-3268.2021.07.017

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The low temperature and weak light in winter and spring in the middle and lower reaches of the Yangtze River severely restricted the growth and development of pepper seedlings. In this paper，the effect of light quality on the growth and development of pepper seedlings was analyzed，and the regulation mechanism from photosynthetic physiology was discussed to provide new ideas and methods for pepper seeding under low temperature and weak light condition.Using pepper Xingshu 215 as the test material,five LED light quality treatments，R/B=1∶1（RB），R/B=3∶1（3RB），R/B=6∶1（6RB），R/B=9∶1（9RB）,and red light（R），were set and white light was used as control. Light intensity was 150 μmol（/m2·s），and light period was 12 h/12 h.The parameters of growth，photosynthetic and chlorophyll fluorescence characteristics of pepper seedlings were measured.Results showed that the plant height and hypocotyl length of pepper seedlings increased with increasing of R/B ratio. The dry and fresh weights of the aboveground and underground parts of 3RB and 6RB treatments were higher than those of R treatment.Under the same light intensity or CO2 concentration，the Pn value of pepper leaves of 3RB treatment was always higher than that of the control and other treatments. The values of carboxylation efficiency（CE）,maximum carboxylation rate（Vmax）,maximum electron transfer rate（Jmax）,triose phosphate utilization rate（TPU）of 3RB treatment were all the highest，but no significant difference was observed between 3RB and 6RB treatments. As the R/B ratio increased，the values of Fo and Fm increased，but no significant difference in the Fv/Fm value existed between the treatments. The values of NPQ under R&B combination light treatments were significantly higher than that of R treatment. Under R treatment，the values of ΔK，ΔJ，ΔI，ΔL were all above zero，while the values of ΔK，ΔJ，ΔI，ΔL were all below zero under R&B combination light treatments 3RB and RB.In conclusion，3RB and 6RB treatments can significantly improve the ability of pepper seedling leaves to absorb and capture light quantum，protect the integrity of the photosynthetic system membrane，smooth the photosynthetic electron transfer，increase the values of Amax，light saturation point，CE，Vmax，Jmax，TPU of pepper leaves，improve the protective ability of photosynthetic structure，reduce light inhibition，and improve the dry matter accumulation of the aboveground and underground parts. R/B=3∶1 and 6∶1 can provide a better light environment for the growth and development of pepper seedlings，which will provide a reference for the cultivation of pepper seedlings under low temperature and weak light condition.

Expression and Identification of PCV2b Virus⁃Like Particles with Neutralizing Epitopes in Tandem in Insect Cells

REN Chunxiao, FENG Hua, ZHANG Teng, JIANG Min, LIU Yunchao, JIN Qianyue, ZHANG Gaiping

2021, 50(7): 154-160. DOI: 10.15933/j.cnki.1004-3268.2021.07.018

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In order to develop a subunit vaccine that is more efficient to prevent porcine circovirus 2b subtype（PCV2b）infection，the important neutralizing epitope²²⁶LKDPPLNP²³³ on the Cap protein was connected to the C⁃terminal of the cap gene（capE2） in a repeated tandem manner，and the corresponding recombinant proteins were expressed in insect baculovirus expression system.The recombinant proteins CapE2 were identified by means of SDS⁃PAGE，Western blot，dynamic light scattering and transmission electron microscopy observations.Then mice were immunized with purified CapE2 protein，and Cap without the neutralizing epitope，PCV2 subunit vaccine，and PBS were used as controls.Tthe serums post immunization were collected for analyzing the immunogenicity of these target recombinant proteins and the effect of linking neutralizing epitopes on the immunogenicity of Cap protein.The results showed that Cap and CapE2 proteins were successfully expressed in sf21 cells. These purified target proteins could effectively react specifically with the PCV2 monoclonal antibody，and could self⁃assemble to form uniform virus⁃like particles in vitro with a diameter of about 17 nm.Serums antibody test results showed that after the second immunization，the antibody level of CapE2 group increased rapidly，and was significantly higher than other immunization groups after the third immunization. The neutralizing antibody test results showed that the serum antibody titres of the Cap and CapE2 groups were significantly higher than the positive vaccine group，and the neutralization titer of the serum in the CapE2 immunization group could reach up to 1∶2⁹. The recombinant Cap proteins that were connected to two repeating neutralizing epitopes were successfully expressed in insect baculovirus expression system and could self⁃assemble to form virus⁃like particles，which had good immunogenicity and provided a new vaccine candidate for the prevention of PCV2 infection.

Association Analysis of GDF9 Gene Polymorphism and Lambing Size in Five Sheep Populations

SHAO Shuncheng, KANG Xiaolong, YAN Beibei, ZHANG Tianwen, LIANG Peng, ZOU Shifan, MENG Ke, RONG Xuan, QIANG Hao, FENG Dengzhen, LI Xinhai

2021, 50(7): 161-167. DOI: 10.15933/j.cnki.1004-3268.2021.07.019

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In order to explore the genetic effect of GDF9 gene coding region polymorphism and reproductive performance in sheep，five sheep populations of Dorper sheep，hybrid of Tan and Han sheep，hybrid F1 generation，hybrid F2 generation and cross H1 generation were used to detect four missense mutations in GDF9 gene by Sequenom Mass ARRAY® SNP technique，and their association with litter size was analyzed. The results showed that the genotypes of rs425223128，rs597009987 and rs415699275 loci were all homozygous，and there were CC，TC and TT genotypes at rs160076408 locus in five populations. The association analysis between different genotypes of rs160076408 locus and litter size of five populations showed that there was a certain correlation between rs160076408 locus and litter size in hybrid F1 generation population，in which the litter size of TC genotype was significantly higher than that of CC genotype（P<0.05）. It could be seen that the rs160076408 locus was suitable for the breeding of multi⁃lamb trait in the hybrid F1 generation of sheep. Bioinformatics analysis results showed that the theoretical isoelectric point of sheep GDF9 protein was 9.08，the instability coefficient was 54.43，the fat coefficient was 77.31，and the total average hydrophilicity was -0.404. The secondary structure of GDF9 protein changed before and after rs160076408 locus mutation. Protein interaction network analysis showed that GDF9 protein interacted with proteins related to animal reproduction.

Isolation，Identification and Biological Characteristics of Mycoplasma synoviae Strain from Henan Province

XU Yindi, JIAO Wenqiang, WANG Zhifang, LI Haili, ZHANG Qingxian, ZHU Wenhao, WANG Keling

2021, 50(7): 168-173. DOI: 10.15933/j.cnki.1004-3268.2021.07.020

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In order to understand the synovitis infection in large⁃scale chicken farms in Henan Province，a total of 159 samples of lung pericardial effusion and articular fluid were collected from dead chickens with symptoms of tenosynovitis，synovitis，osteoarthritis，balloon inflammation，pericarditis，etc.A total of 25 Mycoplasma synoviae strains were identified by pathogen isolation and culture，16S rRNA gene sequencing and blasting. The separation rate was 15.7%，indicating a high infection rate of chicken synovitis in Henan Province. A strain which was isolated from a certain scale egg farm was named HNMsy1. Virulence test and immunogenicity test were performed on HNMsy1. Results showed that HNMsy1 had strong virulence to chickens. The challenge protection rate of the vaccine prepared by this strain against Mycoplasma synoviae was 100%.

Design and Implementation of Customizable Crop Breeding Data Acquisition System Based on Android

LI Guoqiang, ZHAO Qiaoli, ZANG Hecang, ZHAO Qing, ZHANG Jie, SONG Hui, WANG Meng

2021, 50(7): 174-180. DOI: 10.15933/j.cnki.1004-3268.2021.07.021

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In order to achieve paperless collection of crop breeding data，reduce the secondary data entry process，and improve the efficiency of data collection，a customizable crop breeding data acquisition system based on Android was proposed and developed by using Android Studio development tool and JAVA programming language.The APP architecture adopted a single machine version，and the traits of different crops were stored in the attribute database of the system.The acquisition of traits referred to the investigation standard of agronomic traits of related crops.The APP functions included testmanagement,template customization，data acquisition，data query and data export.Before entering the system，users selected crop types firstly，and created test projects by importing Excel template files.In the template customization module，the APP provided corresponding traits according to the selected categories，andusers selected the traits that needed to be collected at present to be saved as customized templates.Data collection was executed based on the combination of test projects and custom templates.Data storage,query，and export were indexed by the names of test items and custom templates. The APP was preliminarily applied in the investigation of millet core germplasm resources in Anyang Academy of Agricultural Sciences，which improved the data collection efficiency of millet test by 65.18%.The system has the advantages of simple operation,convenient carrying，low popularization cost and shows a broad application prospect.

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