Journal of Henan Agricultural Sciences

Contents

2023, 52(11): 0-0.

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Progress on Application of bHLH Transcription Factors in Cold Tolerance Genetic Engineering of Plants

QI Xueli, LI Ying, LI Chunying, HAN Liupeng, ZHAO Mingzhong, ZHANG Jianzhou

2023, 52(11): 1-9. DOI: 10.15933/j.cnki.1004-3268.2023.11.001

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Plants often encounter cold stress，which influences the growth and geographical distribution，and decreases yield and quality of plants. bHLH（basic helix⁃loop⁃helix）family is the second largest transcription factor family in plant，which plays an important role in regulation of tolerance to cold stress.This paper elaborated the structure and the application of MYC（avian myelocytoma virus）⁃like bHLH transcription factor ICE［inducer of CBF（C⁃repeat binding factor） expression］ and other bHLH transcription factors in plant cold tolerance genetic engineering，so as to provide some references for the utilization of bHLH transcription factors in cold tolerance genetic improvement and breeding.

Research Progress on the Effect of Organic Carbon on the Formation of Soil Aggregates

ZHOU Jiahao, CHU Junjie, SUN Wanchun, ZOU Ping, YU Qiaogang, MA Junwei, YANG Jun

2023, 52(11): 10-20. DOI: 10.15933/j.cnki.1004-3268.2023.11.002

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Soil organic carbon（SOC），as an important substance involved in biogeochemical cycles，plays an important role in the formation and stability of soil aggregates.The definition，characteristics and formation theory of soil aggregates were reviewed，the turnover process of SOC in aggregates was analyzed，and the role and influence of SOC quantity，SOC structure，microbial residue carbon and SOC loss in the formation of soil aggregates were reviewed.Suggestions on the cultivation of healthy soil were put forward from the perspective of the synergistic relationship between soil carbon sink and aggregates，and the future research direction of soil aggregates was prospected in order to provide reference for the sustainable development of soil resources.

Effect of Reduction of Nitrogen Fertilizer Rate on Yield and Nitrogen Utilization of Wheat⁃Maize Rotation System in Yellow⁃Cinnamon Soil Area

LI Lingyun, HE Ailing, YANG Huanhuan, LIU Gaoyuan, GUO Zhongyi, DU Jun

2023, 52(11): 21-32. DOI: 10.15933/j.cnki.1004-3268.2023.11.003

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In order to investigate the optimum reduction of nitrogen fertilizer rate of the wheat⁃maize rotation system，from 2018 to 2020，different fertilization measures were considered in the yellow⁃cinnamon soil area，and a total of 7 treatments were set up，including no nitrogen fertilizer（CK），conventional fertilization（FP，N 225 kg/ha），optimized fertilization（CF，N 180 kg/ha），conventional nitrogen reduction by 10%（90%FP，N 202.5 kg/ha），conventional nitrogen reduction by 20%（80%FP，N 180 kg/ha），conventional nitrogen reduction by 30%（70%FP，N 157.5 kg/ha），combined application of controlled loss urea and common urea with 7∶3（CRU，N 180 kg/ha），and the effect of reduction of nitrogen fertilizer rate on grain yield and nitrogen utilization under wheat⁃maize rotation system was explored. The results showed that in the two crop rotation cycles，the yields of wheat and maize of CF treatment were the highest，followed by CRU treatment，there was no significant difference between CRU treatment and CF treatment. Compared with FP treatment，the yields of wheat under CF treatment and CRU treatment increased by 3.62% and 2.57%，and yields of maize increased by 3.53% and 1.85% in the two crop rotation cycles，respectively；the yields of wheat and maize of 80%FP treatment decreased，but there was no significant difference.The yields of wheat and maize of the treatment with conventional nitrogen reduction by 30% decreased compared with FP treatment in the two crop rotation cycles.In the two crop rotation cycles，the contents of organic matter，total nitrogen and hydrolyzed nitrogen of CF and CRU treatments were higher than the other treatments in soil，there was no difference between the two treatments，and the content of hydrolyzed nitrogen of 70%FP treatment generally significantly decreased compared with FP treatment.The nitrogen recovery rate，agronomic efficiency of nitrogen and partial productivity of nitrogen of wheat and maize of CF and CRU treatments were higher，there was no difference between the two treatments，but were all significantly higher than FP treatment in the two crop rotation cycles. Among them，the nitrogen recovery rates of wheat and maize of CRU treatment increased by 32.1%—50.8% and 41.7%—44.2%，respectively；the agronomic efficiency of nitrogen increased by 31.7%—35.6% and 29.5%—31.9%，respectively；the partial productivity of nitrogen increased by 27.4%—28.9% and 26.9%—27.7%，respectively.Overall，for the wheat⁃maize rotation system in yellow⁃cinnamon soil area，optimized fertilization（CF treatment）and combined application of controlled loss urea and common urea with 7∶3（CRU treatment）under conventional nitrogen reduction by 20% could improve crop yield and nitrogen use efficiency，while conventional nitrogen reduction by 30% risked reduction of yield.Comprehensively considered，combined application of controlled loss urea and common urea with 7∶3 with one⁃time fertilization is the best fertilization measure in the wheat⁃maize rotation system in the yellow⁃cinnamon soil area.

Creating of Wheat Taaap3 Mutant with Low Fertilizer Tolerance Using CRISPR/Cas9 Technology

LI Yan, CHEN Yanyan, HUA Xia, FANG Yuhui, WANG Yumin, GONG Chen, QI Xueli, HU Lin

2023, 52(11): 33-41. DOI: 10.15933/j.cnki.1004-3268.2023.11.004

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In order to create new wheat（Triticum aestivum L.）germplasm with low fertilizer tolerance and high yield，the CRISPR/Cas9 technology was utilized to edit the amino acid permeability enzyme gene TaAAP3 in wheat，the types of mutants were determined，the yields of mutants under low fertilizer condition were analyzed，and new wheat materials with low fertilizer tolerance and high yield were screened.The results showed that three mutants of TaAAP3 gene were obtained，including one monogenomic homozygous mutant and two trigenomic homozygous mutants.The main mutations were insertion of single base and insertion or deletion of small fragment of DNA，resulting in frameshift mutations that terminated translation prematurely and caused the protein sequence completely altered.The expression level of TaAAP3 gene in the leaves of Taaap3 mutants seedlings was significantly lower than that of wild type，and the expression level of TaAAP3 in mutants with trigenomic mutations was significantly lower than that with monogenomic mutation.The grain yields of the three Taaap3 mutants were significantly higher than that of wild type under low fertilizer condition，and the lower expression level of TaAAP3 gene，the greater increase of grain yield.Overall，knocking out the TaAAP3 gene in wheat significantly reduces its expression level，and significantly increases grain yield under low fertilizer condition.

Effects of Rice⁃Poultry Symbiosis on Growth Characteristics and Yield Formation of Rice Population

MA Xuehu, MA Yunjun, HUANG Huang, CHEN Can, WANG Ren, ZHANG Yin, WU Shihao, ZHANG Quan, ZHANG Qin

2023, 52(11): 42-48. DOI: 10.15933/j.cnki.1004-3268.2023.11.005

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Three treatments，namely rice⁃chicken symbiosis（RC），rice⁃duck symbiosis（RD），and rice monoculture（CK），were set up to study the effects of rice⁃poultry symbiosis on the growth characteristics and yield formation of rice populations，and identify the optimal rice field poultry farming mode，so as to provide a theoretical basis for the promotion and application of rice field poultry farming.The results showed that the symbiosis of rice and poultry generally reduced rice plant height，aboveground dry weight，leaf area index and photosynthetic potential，and increased seed setting rate，but the impact on yield showed opposite results between two years.Compared with CK，the rice yield under RC and RD treatments increased by 9.26% and 4.75% respectively in 2021 with significant difference in RC treatment，decreased by 10.20% and 15.04% respectively in 2022 with no significant difference in RC treatment. Compared with RD treatment，RC treatment improved the leaf area index，population growth rate，photosynthetic potential and yield of rice.In summary，the rice⁃chicken symbiosis model has high promotion value.

Screening and Analysis of SSR Markers for Evaluating Sesame Germplasm Oil Content Level

XU Fangfang, ZHENG Lei, JU Ming, MA Qin, LI Chun, DUAN Yinghui, ZHANG Xianmei, MIAO Hongmei

2023, 52(11): 49-56. DOI: 10.15933/j.cnki.1004-3268.2023.11.006

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In order to establish the molecular marker identification technology system related to sesame oil content level，we used 24 SSR markers closely related to sesame oil content developed by ourselves to conduct rapid PCR identification and validation analysis on 48 sesame germplasms with different oil content.The results showed that the oil content of 48 sesame germplasms ranged from 29.81% to 57.86%，with average oil content of 46.91%.The analysis of variance of SSR markers showed that 24 SSR markers showed polymorphic alleles in 48 samples，including Hs393（4/2），Hs635（2/2），Hs4082（2/1）and Hs4089（2/2）. These four SSR marker polymorphic loci were significantly（P<0.05） or very significantly（P<0.001）associated with the sesame oil content level. Among the 48 samples tested，a total of 12 samples carried excellent alleles of the four SSR markers mentioned above，with oil content ranging from 52.74% to 57.86%，all of which belonged to high oil content samples.In conclusion，these four SSR markers，Hs393，Hs635，Hs4082，and Hs4089，can be used for identifying and evaluating the oil content level of sesame germplasms and breeding materials，providing technical support for establishing and improving the molecular marker⁃assisted breeding technology system related to sesame quality.

Effects of Combined Application of Water⁃retaining Agent and Antitranspirant on Soil Moisture，Chemical Composition and Carbon and Nitrogen Metabolism of Yunyan 87

HAN Yating, GUO Tianjiao, JIA Guotao, ZHAO Hongchao, HUANG Wuxing

2023, 52(11): 57-65. DOI: 10.15933/j.cnki.1004-3268.2023.11.007

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Western Henan is the main production area of high⁃quality tobacco leaf in our country.In recent years，because of perennial drought，the tobacco leaf ripens too late，the quality declines and so on.Therefore，water⁃retaining agent and antitranspirant were used as materials，the effects of combined application of the two on soil moisture content，the growth period of Yunyan 87，chemical composition of tobacco leaf，key enzyme activity and relative expression of related genes of carbon and nitrogen metabolism were studied.The results showed that，the middle and upper leaves could be harvested in advance by applying water⁃retaining agent and antitranspirant，and the growth period in field was 19 days earlier than that of conventional cultivation.The application of water⁃retaining agent and antitranspirant increased the contents of total sugar，reducing sugar and potassium，decreased the contents of total nitrogen，nicotine and chlorine，and increased the ratio of potassium to chlorine and sugar to base in the upper leaves of flue⁃cured tobacco.Among them，the chemical composition of tobacco leaves treated with water⁃retaining agent and antitranspirant was more harmonious.The application of water⁃retaining agent and antitranspirant increased the soil moisture content.The effect of single application of water⁃retaining agent was better than that of single application of antitranspirant，and the combination of them was the best treatment.Compared with conventional cultivation，the activities of key enzymes of carbon and nitrogen metabolism（NR，GS，SPS，SS）and the expression levels of related genes（NtNR，NtGS，NtSPS，NtSS）were significantly decreased under the treatment of water⁃retaining agent and antitranspirant.In conclusion，the application of water⁃retaining agent and antitranspirant can alleviate the problem of late ripening of tobacco plants and improve the quality of tobacco leaves.The combination of water⁃retaining agent and antitranspirant has the best effect.

Genetic Diversity Analysis of Ganoderma lucidum Germplasm Resources Based on ITS and ISSR

XU Lin, ZHANG Rui, WANG Sheng, LI Jintao, LIU Linling, YAN Meixia

2023, 52(11): 66-74. DOI: 10.15933/j.cnki.1004-3268.2023.11.008

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In order to clarify the genetic diversity of Ganoderma lucidum germplasm resources cultivated in Jilin Province，18 main Ganoderma lucidum strains in Jilin Province were used as test materials，the internal transcribed spacer sequence analysis and antagonism test were applied，and inter⁃simple sequence repeat molecular markers were combined for identification and genetic diversity analysis.The phylogenetic tree based on ITS sequence showed that all the 18 Ganoderma lucidum strains were grouped together with Ganoderma lingzhi，and were subdivided into four groups.Antagonism was found among 79.7% of Ganoderma lucidum strains.The strains were divided into seven groups based on ITS sequence analysis and antagonism test results.The results of ISSR genetic diversity analysis showed that the genetic similarity coefficient of 18 Ganoderma lucidum isolates ranged from 0.382 4 to 0.974 4，with an average of 0.652 9.When the similarity coefficient was about 0.679，the isolates could be divided into seven groups.In summary，the classification results based on ISSR molecular markers were consistent with the classification results based on ITS sequence analysis+antagonism test，and 18 ganoderma lucidum strains showed obvious genetic diversity.

Effect of Biochar Combined with Natural Minerals on Reducing Substances in Paddy Soil

FU Xihao, ZHU Jianqiang, FAN Xianpeng, ZHANG Zhiyi, HE Shaohua, NI Chengfan, WU Maoqian

2023, 52(11): 75-84. DOI: 10.15933/j.cnki.1004-3268.2023.11.009

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In order to investigate the change of soil redox status after straw returning and the recluction effect of biochar combined with natural minerals on soil reducing substances in paddy field，the corn straw biochar（B Ⅰ），tobacco straw biochar（B Ⅱ）and natural minerals（S）were combined into conditioning agents in different proportions.The pot planting was used to study the effects on soil total reducing substances，active reducing substances，Fe²⁺，Mn²⁺ and yield of paddy.The results showed that，straw returning increased soil reducing matter content at seedling stage to tillering stage.The average contents of total reducing matter，active reducing matter and ferrous ion in RS（straw⁃returning）treatment increased by 1.18，1.83 cmol/kg and 194.22 mg/kg，respectively，compared with CK（without straw⁃returning）treatment. Corn straw biochar was better than tobacco straw biochar in reducing soil reducing substances.In S₅BⅠ₅（S∶BⅠ=5∶5），S₃BⅠ₇（S∶BⅠ=3∶7）and S0BⅠ₁₀（S∶BⅠ=0∶10），the average contents of total reducing substances and active reducing substances were decreased by 0.13—0.20 cmol/kg and 0.23—0.60 cmol/kg compared with RS，respectively.The average ferrous content of S₃BⅠ₇ was 110.41 mg/kg lower than that of RS treatment.Compared with RS treatment，except for S₃BⅡ₇（S∶BⅡ=3∶7），corn straw biochar and tobacco straw biochar combined with natural minerals reduced soil solution ferrous content in the range of 29.78—54.60 μg/kg and 8.58—33.57 μg/kg near rice roots.Compared with RS treatment，the average pH value of soil solution with different compound adjusters was increased by 0.06—0.29.The effective spikes and solid grain weight of S₃BⅠ₇ treatment were 4.0 points/hole and 12.4 g/hole higher than RS treatment，respectively.In conclusion，straw returning to the field can increase soil reducing substances in the early stage of rice growth，and the combination of biomass charcoal and natural minerals can effectively reduce the production rate of soil reducing substances and Fe²⁺，improve the pH of soil solution，and ultimately increase the effective spikes and yield of rice.The effect of corn straw biochar is better than that of tobacco straw biochar，and the mixture of corn straw biochar and natural mineral at 7∶3 has better effect.

Effects of Different Organic Fertilizers on Soil Nitrogen Mineralization and Enzyme Activity in the Tobacco Growing Areas of Central Henan and Western Yunnan

LIU Wei, ZHAO Yuanyuan, CHEN Xiaolong, WEI Jianyu, LI Juan, XIE Tianqi, SHI Hongzhi

2023, 52(11): 85-93. DOI: 10.15933/j.cnki.1004-3268.2023.11.010

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In order to explore the effects of different types of organic fertilizers on nitrogen mineralization dynamics and enzyme activity in typical tobacco planting soils in central Henan and western Yunnan，the soils with different organic fertilizers（plant organic fertilizer，sesame cake fertilizer，biochar organic fertilizer）in the two areas under the conditions of equal nitrogen（100 mg/kg）input and 30℃ were studied in laboratory.The changes in soil nitrogen mineralization parameters and soil enzyme activity during different periods were analyzed. Different organic fertilizers increased soil mineral nitrogen content and mineralization rate.In the early stage of cultivation（7 d），sesame cake fertilizer treatment had the highest soil mineral nitrogen content in central Henan and western Yunnan，with 66.40 and 108.53 mg/kg，respectively. In the later stage of biochar organic fertilizer treatment（7—28 d），the mineralization amount was relatively larger and the mineralization rate was faster.Biochar organic fertilizer effectively improved soil urease and N⁃acetyl content⁃β⁃D⁃glucosidase activity，while sesame cake fertilizer had a significant enhancing effect on protease and sucrase activity. Correlation analysis results showed that N⁃acetyl⁃β⁃D⁃glucosidase activity was significantly positively correlated with the contents of nitrate nitrogen，ammonium nitrogen and mineral nitrogen，and most of the enzyme activities were significantly correlated with each other. According to the physical and chemical characteristics of soil in different tobacco growing areas and the nitrogen mineralization characteristics of soil after applying organic fertilizer，applying sesame cake fertilizer to high organic matter soil in western Yunnan can effectively control the mineralization rate，and increase biological fixation of the soil；Adding biochar organic fertilizer to the low organic matter tobacco planting soil in central Henan can effectively improve nitrogen mineralization ability and the mineral nitrogen content of the soil.

Inhibitory Activity of Difenoconazole to Alternaria alternata from Tobacco and the Changes of Phyllosphere Microbes after Its Application

ZHANG Yi, WANG Hancheng, ZHANG Xinghong, ZHANG Lin, GUO Moyan, XIANG Ligan, CAI Liuti, WANG Feng

2023, 52(11): 94-103. DOI: 10.15933/j.cnki.1004-3268.2023.11.011

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To understand the microcosmic effect of difenoconazole in the control of tobacco brown spot，the toxicity of difenoconazole to Alternaria alternata was determined by the mycelial growth rate test，and the phyllosphere microorganisms community structure and metabolic functions of both diseased and healthy tobacco leaves during different application periods were determined by Illumina high⁃throughput sequencing and Biolog metabolic phenotype techniques，respectively.Results showed that difenoconazole had a potent inhibitory activity against A.alternata，with an average EC₅₀ value for inhibiting mycelial growth of 0.17 mg/L.At the early occurrence stage of the disease，phyllosphere fungi in both diseased and healthy leaves were Ascomycota and Basidiomycota dominance at phylum level，the dominant fungal genera were Altemaria，Cladosporium and Symmetrospora.At the phylum level，the dominant bacteria were Proteobacteria and Firmicutes，and the dominant bacteria genera were Pantoea and Pseudomonas.After the application of difenoconazole WDG 900 g/ha，the relative abundance of the phyllosphere fungus Altemaria in diseased tobacco leaves decreased at 5 and 15 d，increased at 10 d after application，the relative abundance of Cladosporium continued to decrease after application，and the relative abundance of the phyllosphere Didymella in both diseased and healthy tobacco leaves decreased at 5 and 10 d and increased at 15 d after application，the relative abundance of the remaining flora also showed irregular increases and decreases.The relative abundance of Pantoea，Massilia，and Sphingomonas in the phyllosphere of diseased tobacco leaves decreased at 5 and 10 d and increased at 15 d after application，while the relative abundance of Pseudomonas kept increasing after application.The phyllosphere microorganisms metabolic activity of diseased tobacco leaves was weaker than that of healthy tobacco leaves，and the phyllosphere microorganisms in the diseased tobacco leaves were weaker in the metabolism of L⁃threonine，D，L⁃α⁃glycerol，α⁃ketobutyric acid，α⁃cyclodextrin carbon sources，while stronger metabolic activity of healthy tobacco leaves was found for all tested carbon sources except for α⁃ketobutyric acid and 2⁃hydroxy benzoic acid carbon sources.The treatment of difenoconazole decreased the microorganisms metabolic activity of both diseased and healthy tobacco leaves，and the activity gradually recovered with the application time.In summary，the application of difenoconazole can inhibit the growth of A.alternata，change the community structure of phyllosphere microorganisms in diseased leaves，and rapidly reduce the metabolic activity of phyllosphere microorganisms.

Changes of Metabolic Functional Diversity of Potato Rhizosphere Soil Microorganisms under Feeding Stress of Potato Tuber Moth，Phthorimaea operculella Zeller

TANG Fenfen, YANG Weike, XIE Kun, ZHANG Zuyun, LI Na

2023, 52(11): 104-112. DOI: 10.15933/j.cnki.1004-3268.2023.11.012

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In order to study the effects of insect feeding stress on the functional diversity of plant rhizosphere soil microbial community，the effects of potato tuber moth，Phthorimaea operculella Zeller feeding stress on the rhizosphere soil microbial community diversity and soil carbon source utilization of two major potato varieties（Hezuo 88 and Lishu No. 6）in Yunnan Province were tested by Biolog ECO technology，with no pest feeding stress as control.The results showed that potato tuber moth feeding stress increased the average color change rate（AWCD）（96 h），which measures the metabolic activity of root microorganisms，and the AWCD values of using 31 carbon sources by rhizosphere soil microorganisms in two feeding stressed potato varieties all increased，and reached the maximum on the 7th day of feeding stress.At the same time，the diversity indices of Shannon and Simpson of the two feeding stress treatments were higher than those of the control groups，and reached the maximum on the 7th day after feeding stress.The analysis of carbon source utilization showed that under feeding stress，the utilization of various carbon sources was improved by the rhizosphere microorganisms in both two stressed groups although their utilization rates were different.In addition，principal component analysis showed that，the feeding stress changed the community structure of root microorganisms of the two varieties.The above results showed that feeding stress of potato tuber moth increased the functional diversity of potato rhizosphere soil microbial community.

Identification，Characterization，and Expression Analysis of VvCAX Gene Family in Grapevine

CUI Xiaoyue, SHANG Hongquan, LÜ Zhongwei, LOU Yusui, ZHANG Ke

2023, 52(11): 113-123. DOI: 10.15933/j.cnki.1004-3268.2023.11.013

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CAX（Ca²⁺/H⁺exchanger antiporter）is an important cation transporter，which is essential for plant growth and development and resistance to abiotic stress.In our study，a total of five VvCAX genes were identified from Vitis vinifera by bioinformatics analysis，named VvCAX1—VvCAX5 in turn according to the position of each member on the chromosomes，and their physical and chemical properties，secondary structure，tertiary structure，transmembrane domain，gene structure，conserved motif，and phylogenetic tree were analyzed.The results showed that members of the VvCAX gene family were all hydrophobins，which were mainly localized to plasma membrane and vacuole，and contained 10—11 transmembrane domains and four typical functional domains.The secondary structure was dominated by α⁃helix and random coil，and the tertiary structure was very similar and monomer.Phylogenetic analysis showed that VvCAX was divided into two subfamilies，and the structure of VvCAX in the same subfamily was similar，while the conserved motifs of VvCAX in different subfamilies were not alike.Seven pairs of VvCAX genes underwent fragment replication events during the evolution of grapes.The interaction proteins of VvCAX were mainly related to calcium transport，calcium channel，and ion transport.The analysis of upstream regulatory factors and promoter cis⁃acting elements showed that the VvCAX genes might be involved in hormone and stress response by the regulation of transcription factors such as MYB and ERF，which was verified by expression pattern analysis.The expression levels of all VvCAX genes were affected by ABA.The members of the ⅠA subfamily responded to wound，downy mildew，drought，and low temperature，while the members of the ⅠB subfamily were mainly related to high temperature，so VvCAX genes of the ⅠA subfamily were more valuable for further research.

Influence Factors of Anther Culture and Ploidy Identification of Regenerated Plants in Pepper

CHENG Zhifang, SHI Yanyan, LI Li, HAN Ya’nan, DONG Xiaoyu, CHANG Xiaoke, YAO Qiuju

2023, 52(11): 124-132. DOI: 10.15933/j.cnki.1004-3268.2023.11.014

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In order to improve the anther culture efficiency of pepper，14 genotypes of pepper were used as test materials to investigate the effects of different concentrations of carbon source combination，activated carbon，silver nitrate and hormone on anther culture of pepper.The ploidy of regenerated plants was determined by DNA flow cytometry.The results showed that all the 14 genotypes were able to develop embryos，and 13 genotypes were able to develop seedlings. Among them，the embryo rate and seedling rate of Huazhixiu No.1 were the highest，which were 63.33% and 23.88%，respectively.The best carbon source was 10 g/L sucrose+20 g/L maltose.When 2 g/L activated carbon and 6 mg/L AgNO₃ were added to MS medium，the anther culture efficiency was the highest，and the highest embryo rate and seedling rate were 29.63% and 15.20%，espectively.The best plant growth regulators were 1.000 mg/L 2，4⁃D+ 1.50 mg/L KT and 0.001 mg/L 2，4⁃D + 0.05 mg/L KT.The highest embryo rate and seedling formation rate of the genotypes treated by 1.000 mg/L 2，4⁃D+1.50 mg/L KT were 63.33% and 23.88%，respectively.The highest embryo rate and seedling rate of the genotypes treated by 0.001 mg/L 2，4⁃D+0.05 mg/L KT were 20.00% and 10.54%，respectively.The ploidy of 383 regenerated plants was determined.279 plants were haploid，87 plants were diploid，2 plants were triploid，2 plants were tetraploid，1 plant was mixed（haploid and diploid）and 12 plants were aneuploid.Haploid and diploid accounted for 72.85% and 22.72% respectively in the regenerated plant population.In conclusion，the natural doubling rate of regenerated plants in anther culture of pepper is low，and the ploidy of regenerated plants induced by the same anther is usually the same.

Comparative Analysis of the Microbial Community Structure in Litopenaeus vannamei Intestine and Surrounding Environment from Different Culture Patterns

HOU Qiang, ZHOU Xin, ZHANG Jianxiong, MA Ruixin, WU Jiang’ai, CUI Ya’nan, CHEN Yu, ZHENG Baiqin, ZHANG Lifang

2023, 52(11): 133-145. DOI: 10.15933/j.cnki.1004-3268.2023.11.015

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The present study aimed to explore the microbial community structure in Litopenaeus vannamei intestine and surrounding environment of different culture patterns in Tangshan area of Heibei Province，and to contrast the variations between them.16S rRNA high⁃throughput sequencing technology combined with bioinformatics methods was used to analyze the microbial diversity，community structure composition and variations in L.vannamei intestine，surrounding water and sediment from different culture patterns including freshwater intensive culture（FI）and freshwater mixed culture（FM）.The results showed that sequences of FI and FM patterns were clustered into 6 077 and 7 961 operational taxonomic units（OTUs）respectively.Sediment samples had significantly higher microbial diversity than water and shrimp intestine samples from both patterns.Compared with FI pattern，diversity in sediment samples of FM pattern was higher.Proteobacteria and Bacteroidetes were the most prevalent phyla across all sample types，while all types of samples varied in terms of the dominating genus and its relative abundance.Compared to shrimp intestine and water samples，the microbial species number and richness in the sediment samples were the highest.Cluster analysis showed that the composition of the microbial communities in shrimp intestines resembled sediment samples more than water samples.The research showed that the number of microbial species in L.vannamei intestine and surrounding environment from two kinds of freshwater culture patterns in Tangshan remained constant while the relative abundance and diversity of the microorganisms rose with time.In comparison to the FI pattern，the FM pattern contained a greater number of microbial species in each type of samples，and the relative abundance of species，especially Vibrio，changed more significantly at the final stage of culture.

Safety Evaluation of Extracts of Fagopyrum dibotrys Qian Zhong and the Effect on Intestinal Flora of Rats

ZHANG Tao, ZHOU Sixuan, TANG Yuanjiang, TAO Xiaoyan, LU Yuxi, DU Chunlin, WANG Lu, QI Weijin, GUO Yunxun, PU Tongcan

2023, 52(11): 146-156. DOI: 10.15933/j.cnki.1004-3268.2023.11.016

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In order to investigate the safety of extracts of Fagopyrum dibotrys Qian Zhong and the effectt on intestinal flora of rats，acute toxicity and subchronic toxicity tests were conducted to evaluate the safety of the extracts，and high⁃throughput sequencing was used to compare the effect on intestinal flora of rats.In acute toxicity test，the minimum lethal dose of aqueous extract and alcohol extract of Fagopyrum dibotrys Qian Zhong to mice was greater than 5.00 g/kg.In subchronic toxicity test，there was no adverse effect on the body weight of rats in each dose group of extracts，and the high⁃dose of alcohol extract could cause death of some rats，while the other groups had no adverse effect on the appearance and behavior of rats.Compared with the control group，in the low dose alcohol extract group，the white blood cell and lymphocyte were significantly increased（P<0.05）；in the medium dose alcohol extract group，total protein（P<0.01），urea nitrogen and spleen coefficient of female rats（P<0. 05） were significantly decreased；in the high⁃dose ethanol extract group，there was no significant change in triglyceride（P>0.05），but it had significant effect on other blood biochemical indexes，and spleen coefficient of female rats was significantly decreased（P<0.01）. In the high⁃dose aqueous extract group，total protein was significantly decreased（P<0.01），and triglyceride was significantly increased（P<0.05）. Histopathological test showed that high dose of aqueous extract and alcohol extract had certain effects on the structure of
liver and kidney in rats. High⁃throughput sequencing results showed that compared with the control group，the abundance of intestinal flora in the aqueous extract group decreased，and in the alcohol extract group increased；the diversity and species evenness of intestinal flora in the aqueous extract group and the alcohol extract group all decreased.The dominant bacteria were Bacteroidota and Firmicutes in aqueous extract，alcohol extract and control groups.The optimal genera respectively were Ligilactobacillus（9.06%），Bacteroides（37.67%）and Alloprevotella（5.91%）.LEfSe analysis showed that the relative abundance of beneficial bacteria such as Bacteroides in aqueous extract group and Lactobacillus in alcohol extract group increased.The test suggests that the extracts of Fagopyrum dibotrys Qian Zhong have no toxicity according to acute toxicity classification standard of exogenous chemicals. Combined with subchronic toxicity test and pathological test，high⁃dose aqueous extract（40 g/kg）and alcohol extract（34 g/kg）have certain effects on the structure of liver and kidney in rats.The extracts of Fagopyrum dibotrys Qian Zhong can change the structure of rat intestinal flora and regulate the type and quantity of flora.

Identification of Lasioderma serricorne in Tobacco Leaf Raw Materials Based on Improved YOLOv3 Algorithm

ZHANG Weizheng, CHEN Saiyueyang, WANG Yanling, TIE Jinxin, DING Jia, LI Meng, LI Canlin, SU Xiaoke, GAN Yong

2023, 52(11): 157-166. DOI: 10.15933/j.cnki.1004-3268.2023.11.017

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In order to solve the problem of low efficiency and inaccuracy in manual recognition of tobacco beetles（Lasioderma serricorne）in tobacco leaf raw materials，and achieve accurate and intelligent recognition of tobacco beetles，a tobacco beetle recognition model was proposed based on the improved YOLOv3 algorithm.Firstly，Random Mix data augmentation technology was used to expand the tobacco beetle dataset；By introducing the K⁃means++ algorithm to recluster anchor boxes，YOLOv3’s recognition ability for tobacco beetles was improved；SIoU Loss was used to improve the Loss function of YOLOv3 bounding box to improve the accuracy of model positioning and accelerate model convergence；Finally，a feature refinement module was added to filter conflict information and optimize the accuracy of the model in identifying small targets in the tobacco beetle dataset.The results showed that，the improved YOLOv3 network model achieved average detection accuracy，recall，F1 score，and mAP of 93.26%，88.25%，0.90，and 94.59% for tobacco beetles in the test set，respectively，increased by 12.21 percentage points，11.79 percentage points，0.12，and 12.40 percentage points compared to the existing YOLOv3 algorithm，providing support for the effective recognition of tobacco beetles.

Study on Automatic Classification Method of Wheat Lodging Based on Deep Learning

ZANG Hecang, WANG Congsheng, ZHAO Qiaoli, ZHAO Qing, ZHANG Jie, LI Guoqiang, ZHENG Guoqing

2023, 52(11): 167-173. DOI: 10.15933/j.cnki.1004-3268.2023.11.018

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Lodging is a key factor restricting wheat yield. Aiming at the problems of time⁃consuming and laborious statistics of wheat lodging area，single lodging classification method and low model prediction accuracy，this study used UAV remote sensing platform to obtain wheat lodging visible light image，self⁃built wheat lodging image data set，and used segmentation models U⁃Net，PSPNet，DeepLabv3+，ACSNet to automatically extract wheat lodging area.The results showed that ACSNet had a better segmentation effect on wheat lodging detection by comparing the four methods of wheat lodging area extraction，and its Pre，Rec，DSC and IoU were 87.5%，91.7%，87.0% and 88.6%，respectively，FLOPs were lower，with strong accuracy and robustness.After testing，the prediction results of wheat lodging by ACSNet were close to the real results，and the average relative error of recognition was 4.5%.Thus，ACSNet can effectively extract wheat lodging information，providing support for UAV remote sensing assessment of wheat disaster area and loss.

Discrimination of Redried Leaves of Four Tobacco Varieties Based on SNP Markers

YANG Jinchu, TONG Zhijun, LI Meng, ZHAO Xu, XU Yongming, FENG Yingjie, YANG Zongcan, QU Peng, LI Yue, SUN Jiuzhe, ZHANG Ke

2023, 52(11): 174-180. DOI: 10.15933/j.cnki.1004-3268.2023.11.019

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To efficiently discriminate varieties of redried tobacco leaves，single nucleotide polymorphism（SNP）molecular marker was explored and used to construct DNA fingerprinting.The whole genome sequencing technology was used to discover the SNP sites of four varieties of redried tobacco leaves including Yunyan 87，Honghuadajinyuan，K326 and NC102，and the allele specific primers for PCR were designed and used for allele typing. The results showed that 21.5 Gb data were obtained from the whole genome sequencing of four tobacco leaf samples，and a total of 27 137 SNP sites were detected.Based on 53 SNP sites，570 allele specific primers of different mismatch types were designed，and multiple polymorphic sites were successfully screened to achieve allelic typing of four varieties. SNP loci 35，45 and 51 were found to be polymorphic among different varieties.For loci 35，45 and 51，SNP genotypes were T/T，C/C and T/T for Honghuadajinyuan，C/T，C/T and C/C for K326，and C/T，C/T and C/T for NC 102，respectively.For Yunyan 87，there was no amplification in loci 35，and the SNP genotypes in loci 45 and 51 were C/T and C/T.The molecular ID of each tobacco variety was constructed based on the genotype of three SNP loci，and the four varieties of redried tobacco leaves could be accurately discriminated.

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