Journal of Henan Agricultural Sciences

Research Progress on Mechanism and Application of Reductive Soil Disinfection（RSD）in Prevention and Control of Continuous Cropping Obstacles of Facility Soil and Vegetable

WANG Guangyin, GUO Weili, CHEN Bihua, PAN Feifei, HUANG Xinqi, CAI Zucong, ZHOU Jianhua, GU Guilan

2023, 52(1): 1-10. DOI: 10.15933/j.cnki.1004-3268.2023.01.001

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Reductive soil disinfestation（RSD） is an environment‑friendly method，which has the characteristics of inhibiting soil‑borne pathogens and improving soil physicochemical properties.It has been successfully applied to prevent and control the continuous cropping obstacle in facility soil and vegetable production.In order to provide theoretical reference for the mechanism and application study of RSD to prevent and control soil continuous cropping obstacles，this paper summarizes the mechanism research progress of RSD to prevent and control soil continuous cropping obstacle，introduces the specific application of RSD in facility soil and vegetable production in recent years，and prospects the future research and application of RSD.

Effects of Foliar Zinc Stages on Concentration and Distribution of Microelement in Different Summer Maize Varieties

ZHANG Panpan, QIAO Jiangfang, LI Chuan, ZHANG Meiwei, NIU Jun, HE Jiawen

2023, 52(1): 11-23. DOI: 10.15933/j.cnki.1004-3268.2023.01.002

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The effects of foliar zinc（Zn）stages［no foliar Zn（Zn0），foliar Zn of 1∶1 at seedling and jointing stage（Zn1），foliar Zn of 1∶1 at jointing and flare opening stage（Zn2）and foliar Zn at flare opening stage（Zn3），4.5 kg/ha ZnSO4·7H2O］on the content and distribution of cuprum（Cu），manganese（Mn），ferrum（Fe）and Zn of 10 summer maize varieties were studied in field，and the relationship between grain yield and microelement content was explored，so as to provide scientific basis for the rational application of Zn fertilizer in summer maize production. The results showed that compared with Zn0 treatment，Zn2 treatment increased the content of Cu and Zn and accumulation of Cu，Fe and Zn in content and HI of Mn and Fe. Dika 653 had the highest Cu content in grains，Gushenyu 66 had the highest Mn and Zn contents in grains，Longping 638 and Weike 702 had the highest Fe content in grains，which were 9.71，9.56，50.74 and 51.83 mg/kg，respectively.Dika 653，Qiule 618，Weike 702 and Dedan No.5 had the highest HI of Cu，Mn，Fe and Zn，respectively. Compared with Zn0 treatment，Zn1，Zn2 and Zn3 treatments significantly increased the yield，the yields of Zn2 and Zn3 treatments were higher，which increased by 3.2% and 4.7% respectively，and there was no significant difference between the two treatments. The yield had significant or extremely significant negative correlations with Zn content in grains and contents of Cu and Mn in the other tissues，Zn content in grains had significant or extremely significant positive correlations with Cu content in grains and contents of Cu and Mn in the other tissues，and there were significant positive correlations between Mn content and Fe content in grains，between Cu content and Mn content in the other tissues. By comprehensive consideration，foliar Zn of 1∶1 at jointing and flare opening stage could enhance the Cu，Fe and Zn accumulation in grains and yield，achieve the goal of simultaneous increase of grain yield and mineral element nutrient quality，and thus could be promoted in the maize production.

Analysis on Crop Yield and Nitrogen and Phosphorus Balance of Different Crop+Crayfish Models

TANG Wei, CHEN Can, HUANG Huang, ZHOU Tiansong, WANG Ren

2023, 52(1): 24-33. DOI: 10.15933/j.cnki.1004-3268.2023.01.003

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Six treatments were set up，including rice‑crawfish continuous cropping（RC），rice monoculture（R），lotus‑crawfish co‑cropping（LC），seed lotus monoculture（L），water bamboo‑crawfish co‑cropping（ZC）and water bamboo monoculture（Z），the crop yield，nitrogen and phosphorus uptake，and nitrogen and phosphorus balance of different crop+crawfish models were studied，so as to provide a theoretical basis for reasonable regulation of nutrient cycle and balance of ecological planting and breeding system and promoting sustainable development of farmland system.The results showed that there was no significant difference in yield between LC，ZC treatments and the corresponding crop monoculture treatments except that the yield of RC treatment significantly decreased compared with that of R treatment because of no fertilization.As for nitrogen and phosphorus absorption of crops，the phosphorus absorption of rice grains of RC treatment significantly decreased by 17.02% compared with R treatment，and the nitrogen absorption of naked water bamboo and water bamboo straw of ZC treatment significantly increased by 31.47% and 21.16% compared with Z treatment，respectively. Except these there was no Compared with the corresponding crop monoculture treatment，the different crop+crayfish treatments not only increased the input and output of nitrogen and phosphorus，but also increased the surplus of nitrogen and phosphorus in the farmland ecosystem.Therefore，in order to improve the utilization rate of nitrogen and phosphorus in farmland and reduce the environmental pollution risk caused by nitrogen and phosphorus，it is necessary to appropriately reduce the input of fertilizer and feed in farmland to ensure the sustainable development of ecological agriculture.

Genome Size，Ploidy and Phylogeny of Cyperus esculentus L.

WANG Huiwei, ZHU Shixin, ZHANG Xinyou, WANG Yan, YANG Tiegang, ZHANG Xiangge, WANG Shufeng, LI Chunxin

2023, 52(1): 34-41. DOI: 10.15933/j.cnki.1004-3268.2023.01.004

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In order to clarify the genome characteristics of the main varieties and characteristic germplasm of Cyperus esculentus L. in the Huang Huai region and the phylogenetic relationship between them and their related species，the genome size and ploidy of six C. esculentus L. materials were evaluated by flow cytometry and genome survey analysis，and the phylogenetic relationship between them and their related groups was analyzed based on the sequences of the external transcribed spacer（ETS） and internal transcribed spacer（ITS） of ribosomal DNA. The results of flow cytometry analysis showed that the genomes of six C.esculentus L. materials were 0.808 6—0.858 5 Gb，with an average of 0.826 4 Gb；the results of genome survey analysis showed that the genome size，proportion of repetitive sequences，GC content and heterozygous ratio of the three tuber types of C.esculentus L. were 0.697 9 Gb，81.02%，34.7% and 0.28% for Yuyousha 2，0.778 7 Gb，84.45%，36.4% and 0.08% for Yuyousha 3.and 0.790 6 Gb，83.75%，34.9% and 0.19% for YYS‑4，respectively；genome survey analysis and pollen staining results showed that the three tuber types of C.esculentus L. were triploid. Phylogenetic analysis showed that six C. esculentus L. were clustered into C.esculentus L.branch，this branch was closely related to the branch composed of C.rotundus L. and C.glomeratus L.，they formed sister group，and formed C4 plant branch with C.iria L. ，C.glomeratus L. and C.cuspidatus H.B.K.

Evaluation of the Effect of Fertilizer Operation Mode on Yield and Quality of Flue‑cured Tobacco in Western Henan Based on Fuzzy Mathematics

CHEN Peiyu, SHI Qiuhuan, MA Junhong, LI Lihua, LI Haohao, SONG Zhengxiong, DAI Xiaoyan

2023, 52(1): 42-52. DOI: 10.15933/j.cnki.1004-3268.2023.01.005

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This experiment studied the effects of different fertilization modes on the growth and quality of flue‑cured tobacco，to provide a reference for optimizing the formula fertilization and improving the comprehensive evaluation system of flue‑cured tobacco quality in the tobacco area of western Henan.With the local main cultivar Qinyan 96 as the material，the L9（34）orthogonal design was carried out for three fertilizer factors of nitrogen，phosphorus and potassium，the effects of different fertilization modes on the accumulation of dry matter，photosynthetic properties，yield and quality of flue‑cured tobacco were compared，and fuzzy mathematics and weighting method were applied to comprehensively evaluate the appearance quality，chemical composition availability index（CCUI）and economic effect index（ECI）of tobacco leaves. The results showed that with the increase of nitrogen dosage，the net photosynthetic rate of tobacco plants increased first and then decreased；medium nitrogen treatment could increase the content of total sugar and reducing sugar in the middle and upper leaves after curing，and coordinate the ratio of sugar to nicotine；in high nitrogen treatment，the content of two sugars in the middle and upper leaves decreased，and the content of nicotine in the middle leaves increased significantly. Under the same nitrogen application level，increasing the application of potassium fertilizer could effectively improve the photosynthetic index of flue‑cured tobacco；under the high nitrogen level，the sugar content，output value and ECI value of the middle and upper leaves after roasting in the high potassium treatment were significantly increased compared with those in the low potassium treatment.Under the same nitrogen level，the chlorine content of flue‑cured tobacco in high phosphorus treatment was significantly reduced，the potassium chlorine ratio was significantly increased，the appearance quality and the total amount of aroma substances were significantly improved. The results of fuzzy analysis showed that when the nitrogen fertilizer level was the same，increasing the application of phosphorus and potassium fertilizer could improve the appearance quality，CCUI and ECI values of flue‑cured tobacco to varying degrees.The tobacco leaves treated with medium nitrogen had higher scores，among them，T5（N2P2K3）treatment significantly increased the dry matter accumulation，photosynthetic index and CCUI value of flue‑cured tobacco，and the output value was significantly increased by 96.22% compared with T8（N3P2K1）treatment，the ECI value and comprehensive evaluation score were the highest. Under the conditions of this study，the application of nitrogen，phosphorus and potassium fertilizers at 60.0，120.0，354.0 kg/ha，respectively，is conducive to achieving the best yield and quality benefits of flue‑cured tobacco，and could
be used as the recommended fertilization mode in western Henan tobacco region.

Effects of Different Dosages of AFD on Photosynthetic Characteristics and Boll Formation of Cotton

LI Wenshan, ZHANG Junyao, WANG Jiayong, LI Yabing, XU Wenxiu, XU Qinghua, LOU Shanwei, TANG Jianghua

2023, 52(1): 53-60. DOI: 10.15933/j.cnki.1004-3268.2023.01.006

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In this study，the effects of plant growth regulator AFD on photosynthetic characteristics，boll‑forming rule and yield formation of cotton were investigated，and the optimal drip application amount of AFD was selected. Field trials were conducted from April to October 2021. Seven treatments were set as 0 g/ha（CK），1 050 g/ha（A），1 350 g/ha（B），1 650 g/ha（C），1 950 g/ha（D），2 250 g/ha（E），2 550 g/ha（F）.They were applied on July 6 at the beginning of flowering（15%）and at July 14 at the full flowering（85%）.The effects of different dosages of AFD on photosynthetic characteristics，boll‑forming rule and yield of cotton were analyzed. The results showed that the SPAD value increased firstly and then decreased from July 10 to August 10，and basically reached the maximum value on August 1，in the order of D>E>C>B>A>CK>F. Leaf area index（LAI）showed a trend of first increasing and then decreasing with the advance of growth process，and each treatment basically reached the maximum value at the full boll stage，presenting as D>E>C>F>B>A>CK. Both Pn and Ci of cotton in blooming stage were greater than those in full boll stage. Pn showed an increasing trend with the increase of the dosage of AFD in blooming stage，while Ci showed an opposite change pattern. AFD had obvious effect on promoting boll formation rate and inhibiting boll shedding rate. When the treatment level was 1 650 g/ha，the boll number per plant and single boll weight were significantly increased compared with the control，and the grain yield was the highest.In conclusion，1 650 g/ha was the best dose of AFD in this study.

Temporal and Spatial Distribution Characteristics of Soil Moisture and Its Response to Rainfall in Sloping Farmland in Black Soil Area

HOU Shutao, WANG Shuo, WANG Yiang, LIU Huanjun, MENG Linghua, SONG Mengning, HOU Yonghua

2023, 52(1): 61-72. DOI: 10.15933/j.cnki.1004-3268.2023.01.007

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In order to reveal the temporal and spatial distribution characteristics of soil moisture in different soil layers and different point positions of sloping farmland and its response to atmospheric rainfall，the sloping farmland in typical black soil area was taken as the research object，the 0—100 cm soil moisture content and atmospheric precipitation data of five slope positions including upper slope，middle section and lower section of western slope，middle section and lower section of eastern slope were obtained by in‑situ monitoring method during the growing period of maize. And the rainfall response characteristics of different slope positions in the field scale after rainfall were exploded.The results showed that soil moisture distribution in sloping farmland was uneven. In terms of horizontal distribution，the average soil moisture content in the lower section of the western slope was the highest（34.63%），while the average soil moisture content in the upper slope was the lowest（30.00%），and the average soil moisture variation coefficient in the upper section was the largest（20.38%）；In the vertical distribution，soil moisture content increased with the deepening of soil layer，and the coefficient of variation decreased.Influenced by seasonal drought and maize growth，soil water storage in the field showed a downward trend during the monitoring period，and there were differences in the response characteristics of soil water storage between different point positions and between the early and late drought periods.Soil water storage in the late drought period was more strongly responsive to rainfall.The variation trend of soil moisture content in the 10 cm soil layer was consistent with the rainfall，and the response time was the earliest，but with the deepening of soil layer，the response time lagged. The response of soil moisture to rainfall was affected by preferential flow，including lateral flow recharge of soil and rapid infiltration of large pores formed by maize roots. There were differences in the response of soil moisture to rainfall among different point positions. Because the upper slope was at the top of the slope and the slope was large，the response to rainfall was not significant. The median section of the western slope was strongly responsive to rainfall due to the change of soil physical properties caused by rainfall erosion.The lower part of the slope was affected by the water supply from the upper slope，and the water subsided slowly.

Spatial‑Temporal Variability of Nutrients and Assessment of Fertility in Tobacco‑Planting Soil in Hilly Areas of Southwest Henan

LI Qian, JIA Xiaoguo, LI Liang, ZHANG Xiang, CHENG Yuyuan, LI Peng

2023, 52(1): 73-85. DOI: 10.15933/j.cnki.1004-3268.2023.01.008

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To clarify the spatial‑temporal variation characteristics of tobacco‑planting soil nutrients in hilly areas of southwest Henan under long‑term field management，the data of tobacco‑planting soil in Neixiang County in 2002 and 2020 were selected as the study object.The spatial‑temporal variation characteristics and spatial distribution patterns of soil nutrients were analyzed by using geostatistics and geographic information system(GIS)techniques.A fuzzy mathematical model was used to comprehensively assess the soil fertility.The results showed that the average pH value of tobacco‑planting soil in Neixiang County decreased by 1.15 from 7.74 to 6.59 over the past 18 years，the average contents of available potassium and water soluble chloride decreased by 7.31% and 34.29%，and the average contents of organic matter，alkali‑hydrolyzable nitrogen and available phosphorous increased by 21.00%，109.77% and 20.77%，respectively.The soil fertility index increased from 0.54 to 0.61，mostly upgrading from grade Ⅲ to grade Ⅱ .Soil pH value，alkali‑hydrolyzable nitrogen and water soluble chlorine content showed significant spatial variation characteristics. Among them，the proportion of tobacco‑planting area with higher pH value and water soluble chlorine content decreased by 79.14 and 16.84 percentage points respectively，and the proportion of tobacco‑planting area with medium and above alkali‑hydrolyzable nitrogen content increased by 97.46 percentage points. The county‑wide soil fertility was mainly good in 2020，and the areas with the same grade were concentrated，with 65.51% and 83.16% of tobacco‑planting areas having higher alkali‑hydrolyzable nitrogen and water soluble chloride content，respectively，indicating that the long‑term scientific management effectively improved the fertility of tobacco‑planting soil in the study area，but there was still a need to further improve the fertility of tobacco‑planting soil by taking site‑specific measures to control nitrogen and reduce chlorine.

Effects of Rainfall Intensity on Nitrogen Leaching from Typical Tobacco Growing Soils in Western Yunnan and Central Henan under Soil Column Simulation

WANG Ge, ZHAO Yuanyuan, CHEN Xiaolong, WEI Jianyu, WANG Zheng, XIE Tianqi, SHI Hongzhi

2023, 52(1): 86-94. DOI: 10.15933/j.cnki.1004-3268.2023.01.009

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The difference of nitrogen leaching loss in different typical tobacco planting soils was compared by indoor soil column simulated rainfall.This experiment selected typical tobacco planting soils in central Henan（Xiangcheng County） and western Yunnan（Eryuan County），set a total rainfall of 600 mm，simulate three different rainfall intensities P1（0.52 mm/h），P2（1.04 mm/h），P3（1.56 mm/h），and study the amount of leaching solution and the leaching of nitrate nitrogen and ammonium nitrogen.The results showed that high rainfall intensity could reduce soil water capacity，and the water capacity of tobacco planting soil in western Yunnan was higher than that in central Henan.The concentration of nitrogen leaching occurred at the initial stage of leaching.The leaching concentration and amount of nitrate nitrogen decreased rapidly with the increase of rainfall，while the ammonium nitrogen decreased slowly.The total amount and rate of nitrate nitrogen and ammonium nitrogen leaching in tobacco planting soil increased with the increase of rainfall intensity.The nitrate nitrogen was the main form of leaching，accounting for 90% on average in the two places.The concentration of nitrate nitrogen leaching in soil was not significantly affected by rainfall intensity，but was significantly affected by the content of residual nitrogen in soil. The amount and rate of nitrogen leaching from tobacco planting soil in western Yunnan were higher than those in central Henan，and the risk of nitrogen leaching pollution was higher.

Analysis of Differences and Mechanism of Resistance to Canker among Different Kiwifruit Rootstock‑Scion Combinations

HE Zhanxue, ZHU Taifu, LI Xin, SU Xiaolan, LIU Huimin, WANG Lianchun

2023, 52(1): 95-107. DOI: 10.15933/j.cnki.1004-3268.2023.01.010

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The experiment was carried out with different rootstocks[Zhaotong kiwifruit(A₁)，Xianmai kiwifruit No.2(A₂)，Cuhua kiwifruit(A₃)and Yingmao kiwifruit(A₄)]，different scions[Hongyang kiwifruit(B₁)，Hort16‑A(B₂₎，Guichang kiwifruit(B₃)and Hort16‑A seed propagation plant(B₄)],different concentrations of Pseudomonas syringae pv.actinidiae(Psa)[5×10⁵（C1），5×10⁶（C2），5×10⁷(C3)and 5×10⁸（C₄）cfu/mL）and different inoculation methods［injection inoculation（D₁），trauma inoculation(D₂)，needle inoculation(D₃)and no injury inoculation(D4)].The L₁₆（44）orthogonal test was designed to investigate the effect of different factors on the disease index of kiwifruit canker，and analyze the changes of physiological and biochemical indexes of the plants after inoculation with Psa pathogen and their correlation with the disease index of canker，so as to provide a theoretical basis for future control of kiwifruit canker，use of wild kiwifruit resources and analysis of disease resistance.The results showed that A₁B₄C₄D₄，A₂B₂C₁D₄，A₃B₁C₃D₄ and A4B₃C₂D₄ combinations showed high resistance to canker，A₃B₄C₂D₁ and A₄B₄C₁D₃ combinations showed resistance to canker，A₁B₁C₁D₁，A₁B₂C₂D₂，A₁B₃C₃D₃，A₂B₁C₂D₃，A₂B₃C₄D₁ and A₃B₂C₄D₃ combinations showed tolerance to canker，and A₂B₄C₃D₂，A₃B₃C₁D₂，A₄B₁C₄D₂ and A₄B₂C₃D₁ combinations showed susceptibility to canker；the inoculation method was the main factor affecting the disease index of kiwifruit canker among the four factors，and the optimal combination was A₁B₃C₁D₄(Zhaotong kiwifruit as rootstock,Guichang kiwifruit as scion，Psa concentration of 5×10⁵ cfu/mL，and injury‑free inoculation）. At 10—40 days after inoculation with Psa，the activities of peroxidase(POD)，superoxide dismutase(SOD)，polyphenol oxidase(PPO)and content of soluble sugar in leaves increased first and then decreased，and the contents of proline and malondialdehyde showed continuous upward trend.The disease index was significantly negatively correlated with POD，SOD，PPO activities，soluble sugar content and proline content，and significantly positively correlated with malondialdehyde content.In summary，wound was the main factor causing the occurrence of kiwifruit canker disease，bacterial concentration，scion and rootstock all had different degrees of influence on plant disease resistance，and the resistance of rootstock could effectively enhance the disease resistance of scion.The POD，SOD，PPO activities and contents of soluble sugar，proline and malondialdehyde had linear relationship with the disease index，which could be used as physiological indicators to evaluate the kiwifruit resistance to canker.

Studies on Oviposition Behavior and Oviposition Medium Screening of Natural Enemy Insect Picromerus lewisi

SUN Ganlin, GUO Puyuan, YANG Yun, LI Jianyong, CHEN Bin, CHEN Cong

2023, 52(1): 108-115. DOI: 10.15933/j.cnki.1004-3268.2023.01.011

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To clarify the oviposition behavior of the natural enemy insect Picromerus lewisi and screen the suitable medium for female oviposition，this study was carried out to observe the oviposition behavior of P.lewisi using net room method in laboratory. At the same time，the egg mass concentration，oviposition quantity，oviposition duration and egg hatching rate on four mediums（folded and unfolded） were analyzed，including kraft paper，sulfuric acid paper，A4 printing paper and gauze. The results showed that oviposition behavior of P.lewisi adults was mainly divided into three steps：finding a suitable oviposition site，ovipositing and resting.P.lewisi adults could lay eggs multiple times and eggs were distributed in the form of egg mass.Female adults had higher preference for oviposition on folded gauze.The highest concentration of oviposition on folded gauze was 38.86%，the maximum oviposition quantity and total oviposition quantity were 49.33 and 140.87，respectively，and the duration of oviposition was 38.27 d.Oviposition mediums（folded and unfolded）had no significant effect on the hatching rate of eggs.All of the hatching rate of eggs on four mediums including kraft paper，sulfuric acid paper，A4 printing paper and gauze exceeded 90.00%.In summary，the oviposition quantity of P.lewisi female adults is large and the hatching rate of eggs is high.The folded gauze is an effective oviposition medium for indoor large-scale breeding of P.lewisi.

Effects of Silicon and Selenium on Photosynthesis and Antioxidant Enzyme System of Cucumber Seedlings under Cadmium Stress

YU Minmin, FAN Wenhua, LIU Fenwu, TIAN Ludan, WANG Gailing, MENG Qinghui

2023, 52(1): 116-124. DOI: 10.15933/j.cnki.1004-3268.2023.01.012

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To explore the effects of silicon（Si）and selenium（Se）on photosynthesis and antioxidant enzyme system of cucumber seedlings under cadmium（Cd） stress in calcareous soil，and provide a theoretical basis for the application of Si and Se in alleviating Cd toxicity，pot experiment was conducted to study the effects of single or simultaneous application of Si（300 mg/kg）and Se（2，4 mg/kg）on cucumber seedling growth，chlorophyll content，photosynthetic parameters and antioxidant enzyme activity under Cd（5 mg/kg）stress. The results showed that Cd stress had obvious toxic effects on plant growth，leaf photosynthesis and antioxidant enzyme activities in cucumber seedlings. Compared with blank control，the chlorophyll content，net photosynthetic rate（Pn），transpiration rate（Tr） and stomatal conductance（Gs）of cucumber leaves under Cd treatment were decreased significantly，the intercellular carbon dioxide concentration（Ci）increased significantly，the activities of peroxidase（POD），superoxide dismutase（SOD） and catalase（CAT） decreased significantly，while the content of malongizldehyde（MDA）increased significantly. Cd toxicity was alleviated under the application of 300 mg/kg Si or 2，4 mg/kg Se，which could promote the growth of cucumber，increase the chlorophyll content and Pn，Tr，Gs and POD，SOD，CAT activities of cucumber leaves，and reduce the Ci and MDA content. The effect of simultaneous application of Si and Se was more obvious than that of single application of Si or Se，and the application of 300 mg/kg Si and 2 mg/kg Se had the best effect. Compared with single application of Cd，Pn，Tr and Gs of cucumber leaves significantly increased by 18.53%，42.67% and 87.50%，respectively，Ci significantly decreased by 35.61%，POD，SOD and CAT activities significantly increased by 87.65%，32.40% and 23.58%，respectively，and MDA content significantly decreased by 42. 68%.The results show that applying Si and Se in calcareous soil can alleviate the toxicity of Cd to cucumber，enhance the photosynthesis of cucumber leaves and improve the activity of antioxidant enzyme system，and the simultaneous application of Si and Se has a certain synergistic effect.

Selection of LncRNAs Reference Genes in Development of Flower Bud in Broccoli

PEI Xuli, FENG Pengyu, TANG Zheng, LI Yamei, JIAO Peng, JING Zange

2023, 52(1): 125-133. DOI: 10.15933/j.cnki.1004-3268.2023.01.013

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In order to preliminarily analyze the expression of LncRNAs during the flower bud development of broccoli，it is very important to screen the stably expressed internal reference genes. In this study，16 candidate internal reference genes were screened by high‑throughput sequencing.The expression levels were detected by qRT‑PCR. And the expression stability of 16 LncRNAs in different parts and different developmental stages of broccoli flower bud was analyzed by geNorm，NormFinder and BestKeeper softwares.The results showed that in different parts of bud，geNorm demonstrated that XLOC_000400，XLOC_008536and XLOC_034059 were suitable reference genes；NormFinder recommended XLOC_021473，XLOC_000400 and XLOC_034059 as reference genes；BestKeeper showed that the expression of XLOC_025578 was the most stable.In different bud development stages，geNorm demonstrated that XLOC_000108，XLOC_039609 and XLOC_021473 were suitable reference genes；NormFinderrecommended XLOC_021473，XLOC_039609 and XLOC_000108 as reference genes； BestKeeper showed that the expression of XLOC_034059 was the most stable. After a comprehensive evaluation，XLOC_000400/XLOC_008536 and XLOC_039609/XLOC_021473 were the most stable reference genes for different parts and different developmental stages，respectively.

Effect of miR‑M11 Gene Editing on Replication of Marek’s Disease Virus in Vitro

WANG Weidong, TENG Man, ZHENG Luping, LIU Jinling, ZHANG Wenkai, LI Linyan, ZHANG Zhihui, FAN Jianming, LUO Jun

2023, 52(1): 134-143. DOI: 10.15933/j.cnki.1004-3268.2023.01.014

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In order to explore the effect of virus‑encoded miRNA precursor miR‑M11 on the in vitro replication ability of MDV，the domestic isolate HN302 of MDV was used as the parental virus，and the CRISPR/Cas9 system was used to targetedly edit and knock out miR‑M11 located in the Mid gene cluster，to construct a miR‑M11 gene editing deletion strain HN302ΔM11（clone number C58‑8‑4）.After PCR identification，DNA sequencing，indirect immunofluorescence assay（IFA），real‑time quantitative PCR（qPCR），and passage stability analysis，it was confirmed that the miR‑M11 encoded by HN302 was precisely edited and completely deleted from the viral genome，and no reverse mutation occurred.The results of the virus proliferation curve assay showed that the proliferation rate of the miR‑M11‑deleted virus strain HN302ΔM11 was significantly higher than that of the parental virus strain HN302（P<0. 05）.In conclusion，miR‑M11 is a non‑essential gene for MDV replication，and its deletion can enhance the invitro replication ability of MDV.

Effects of Clostridium butyricum on Growth，Intestinal Morphology and Barrier Function of Broilers

LI Wenjia, XU Bin, WEI Fengxian, DENG Wen, MA Huihui, WANG Linyi, BAI Jie, SUN Quanyou, XI Yanyan, LI Shaoyu

2023, 52(1): 144-153. DOI: 10.15933/j.cnki.1004-3268.2023.01.015

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The experiment was conducted to investigate the effects of Clostridium butyricum in diet on growth performance，intestinal morphology and intestinal barrier function of broilers. A total of 324 healthy one‑day‑old Arbor Acres male broilers with the same specifications were randomly divided into 3 treatments with 6 replicates per treatment and 18 broilers per replicate. Control group（CON）was fed with a basal diet，antibiotic group（ANT）and Clostridium butyricum group（CB）were fed with the basal diet containing 10 mg/kg virginiamycin and 1×10⁹ cfu/kg Clostridium butyricum，respectively.The experiment period was 42 days. At the age of 21 and 42 days，the growth performance，intestinal tissue morphology，intestinal mucosal pyroptosis and intestinal barrier related gene mRNA expression abundance of each group were statistically analyzed. The results showed that compared with the CON group，the average daily gain（ADG）and final body weight（FBW）in ANT group and CB group were significantly increased by 6.31% and 8.43%，and 6.26% and 8.31%，respectively.At 42 days，the levels of Caspase‑1，IL‑18 and IL‑1β in CB group were significantly decreased by 24.91%，42.96% and 19.91% compared with the CON group，respectively. The jejunum villus height and villus height/crypt depth（V/C）ratio were significantly increased by 13. 40% and 54.20%，respectively；and the ileum crypt depth was significantly decreased by 32.62%，and the V/C ratio of ileum was significantly increased by 60.76%.At 42 days，the mRNA relative expression levels of IL‑1β，TNF‑α，Caspase‑1 and IL‑18 in jejunum mucosa in CB and ANT groups were significantly lower than those in CON group and the expression levels of ZO‑1 and Claudin‑1 in CB group were significantly up‑regulated，respectively. In conclusion，dietary supplementation of 1×10⁹ cfu/kg C.butyricum can improve the growth performance and feed utilization of broilers，improve the intestinal morphology，enhance the intestinal immune and barrier functions，and alleviate the intestinal cell pyroptosis of broilers to a certain extent.

Effects of Artificial Diet and Mulberry Leaves on Activities of Three Metabolic Enzymes in Bombyx mori

WANG Yongsheng, ZHU Shuifen, CHEN Song, CHEN Yunming, JIANG Ya, FAN Yonghui, YANG Qihui, LI Qiongyan, YANG Hai

2023, 52(1): 154-160. DOI: 10.15933/j.cnki.1004-3268.2023.01.016

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In order to clarify the effects of artificial diet and mulberry leaves on the activities of metabolic enzymes in silkworm，the spectrophotometry was used to detect the changes of alkaline phosphatase （ALP），carboxylesterases（CarE）and acetylcholinesterase（AchE）activities in hemolymph and midgut of Bombyx mori after reared with artificial diet and mulberry leaves from the fifth newly exuviated larvae with different time. Results showed that the ALP activity in hemolymph had no significant change after reared with artificial diet，while its activity showed a trend of increasing gradually after reared with mulberry leaves；the ALP activity in mulberry leaf group was significantly higher than the artificial diet group at 60 h and 72 h.The CarE activity in hemolymph increased gradually with the extension of rearing time both in the mulberry leaf group and artificial diet group，the enzyme activity of artificial diet group was slightly higher than that of mulberry leaf group，but the difference was not significant.In hemolymph，the AchE activity of mulberry leaf group was slightly lower than that of artificial diet group at 12 h and24 h；At 36 h，the AchE activity began to increase in the mulberry leaf group and was higher than that of artificial diet group at 48 h，60 h and 72 h，the AchE activity was 1.52，1.64 and 1.71 times that of artificial diet group，respectively.In midgut of silkworm reared with mulberry leaves，the ALP activity was significantly higher than that in artificial diet group at 36 h and continued to increase with extension of time，the enzyme activity was extremely significantly higher than that in artificial diet group at 72 h，which was 2.13 times that in artificial diet group. In midgut of silkworm reared with artificial diet group，the CarE activity was significantly higher than that of the mulberry leaf group at 24 h and reached the maximum value at 60 h，which was 2.61 times that in mulberry leaf group.At 72 h，the CarE activity showed a decreasing trend，but it was still significantly higher than that of mulberry leaf group.The AchE activity in midgut of silkworm reared with mulberry leaves was significantly higher than that of the artificial diet group at 12 h and continued to increase from 24 h to 48 h.In addition，the AchE activity was extremely significantly higher than that of artificial diet group at 60 h and 72 h，which were 2.68 and 2.84 times that of artificial diet group，respectively.These results indicated that the activities of ALP and AchE in hemolymph and midgut of silkworm reared with artificial diet were significantly lower than those of mulberry leaf group，while the activity of CarE in the midgut was significantly higher than that of the mulberry leaf group.It suggests that the metabolic physiology of silkworm reared with artificial diet is
influenced to some extent，which results in a reduced resistance of silkworm against unfavorable environment.

Grading of Apple Based on K‑means Clustering and Improved MLP

WANG Yingchao, ZHANG Jingjing, JIA Donglin, ZHOU Tengfei

2023, 52(1): 161-171. DOI: 10.15933/j.cnki.1004-3268.2023.01.017

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In order to accurately realize apple classification with multi‑feature fusion，a classification method of Fuji apples based on K‑means clustering and improved MLP was proposed. The method mainly included image preprocessing，brightness equalization，background segmentation，feature weighting and improved MLP classification network. Firstly，the image quality of apple was improved by means of mean filtering algorithm and brightness equalization operation；then background segmentation was performed by means of K‑means clustering algorithm；on the basis of fruit body and background segmentation，the fruit diameter，fruit shape，color，defect and texture features of apple were extracted in turn；then Pearson correlation analysis and artificial preference weight were used to comprehensively weight the feature data set to simulate the artificial grading scene；finally，the feature data was sent to the improved MLP neural network to complete the apple grading.Through the grading test of 400 graded apples，the accuracy rate reached 94.25%，which verified the feasibility and accuracy of the grading method.This method corresponds to the systematic apple grading standard，and has the advantages of strong timeliness and complete detection indicators.

Effects of Different Post Ripening Treatments on the Baking Quality of Upper Leaves of Flue‑cured Tobacco

XUAN Dongdong, JIA Hongfang, GUO Shiping, XIE Liangwen, WU Deyang, QIN Yanqing, YANG Xingyou, FENG Changchun

2023, 52(1): 172-179. DOI: 10.15933/j.cnki.1004-3268.2023.01.018

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In order to study the effect of post ripening treatment on the baking quality of upper leaves of flue‑cured tobacco and improve the availability of upper leaves，in this study，the upper leaves of normal treatment（CK），12 h post ripening treatment（H1），24 h post ripening treatment（H2）and 36 h post ripening treatment（H3）were used as test materials.The effects of post ripening treatment on the baking quality of upper leaves were analyzed from the aspects of cell biology and molecule by using transmission electron microscope and qRT‑PCR. The main results were as follows：（1）Compared with the normal treated tobacco leaves，the browning speed and degree of post ripening treated tobacco leaves in H2 decreased during the curing process，the proportion of medium and high grade tobacco increased，the proportion of mild browning tobacco leaves decreased by 34.39%，the proportion of moderate and above browning decreased by 43.02%，and the proportion of high‑grade tobacco increased by 17.29%.（2）After post ripening，the tissue structure of upper leaves was loose and the number of starch granules decreased；The contents of reducing sugar and total sugar in flue‑cured tobacco treated with H2 increased by 53.33% and 16.33% respectively，and the starch content in flue‑cured tobacco treated with H3 decreased by 5.61%.The results showed that the macromolecular substances in the upper leaves of post ripening treatment were degraded greatly，and the internal chemical components were more coordinated.（3）The activity of ester‑oxygenase（LOX） in post ripening tobacco leaves increased significantly，and the content of fatty acids decreased significantly.The results showed that the maturity of post ripening tobacco leaves was improved，and the content of lipid metabolism substrate was reduced during the curing process，and the degree of browning was reduced.（4）The expression analysis of key genes of glucose metabolism and lipid metabolism showed that the expressions of lipid transporter gene 1（LTP1），MYB family gene 48（MYB48），wax synthesis gene 10（CER10），sucrose synthase gene（SS），sucrose invertasegene（INV） and pyrophosphorylase gene（AGP） in post ripening tobacco weresignificantly down regulated.The results showed that the sugar metabolism and lipid synthesis of postripening tobacco leaves were inhibited during curing，which had a positive effect on the inclusions and color changes of upper leaves during curing.

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