Journal of Henan Agricultural Sciences

Progress on Application of Nano‑gene Vector in Plant Genetic Transformation

AN Yiwei, LIANG Huihui, ZHONG Chongjia, SUN Dihu, SHENG Jiacheng, ZHANG Zhen, LI Hao, GUO Guanghui

2022, 51(12): 1-9. DOI: 10.15933/j.cnki.1004-3268.2022.12.001

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Nano‑gene vector constructed with nanomaterial has special superiority in plant genetic transformation，and has been successfully applied in genetic transformation of a variety of plants.The properties and types of nano‑gene vectors were clarified，the progress on application of inorganic nano‑gene vector，natural polymeric nano‑gene vector and synthetic polymeric nano‑gene vector in plan genetic transformation was reviewed，and the application prospects of nano‑gene vector in plant genetic transformation were forecasted.

Research Progress of Phytoremediation of Heavy Metal Polluted Farmland Soil

LI Xu, CHAO Ying, YAN Xianghui, LUO Feng, YU Jinpeng, LOU Yanhong, ZHUGE Yuping, WANG Hui, PAN Hong, YANG Quangang

2022, 51(12): 10-18. DOI: 10.15933/j.cnki.1004-3268.2022.12.002

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Soil heavy metal pollution，as one of the urgent ecological environment issues needing to be solved，is imminent to remediate and control. Phytoremediation，as an efficient，economical and environmental friendly green restoration technology，is widely recognized and has become an important remediation technology in contaminated soils. This paper reviewed the plant species and remediation modes used in phytoremediation technology，analyzed the existing problems of phytoremediation technology in heavy metal polluted farmland soil，and prospected the research of phytoremediation technology in the treatment of heavy metal pollution in farmland soil as well，which would provide a theoretical basis for the green treatment of heavy metal polluted farmland.

Distribution and Combination Features of Nine Blast Resistant Genes in 291 Rice Germplasm Resources

SANG Shifei, WANG Ya’nan, WANG Junyi, CAO Mengyu, SUN Xiaohan, JI Shengdong

2022, 51(12): 19-27. DOI: 10.15933/j.cnki.1004-3268.2022.12.003

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The molecular markers of eight blast resistant genes（Pi9，Pi‑k^h，Pit，Pi5，Pita，Pb1，Pi2 and Pikm）were collected，and the Pib molecular marker was developed by comparing the sequences of rice blast resistant and susceptible genotypes of Pib.Based on these molecular markers，we analyzed the distribution and combination features of these nine rice blast resistant genes in 291 rice germplasm resources（mainly from Huang‑Huai‑Hai region），so as to provide a theoretical basis for molecular marker assisted selection breeding for rice blast resistance in the future.The results showed that the molecular marker of Pib gene was successfully developed.The number of rice blast resistance genes in a rice germplasm resource was 0—6，and the number of germplasm resources carrying three rice blast resistance genes was the most，accounting for 40.9%，followed by the number of germplasm resources carrying two（20.3%） and four（19.2%） rice blast resistance genes，and the germplasm resources carrying six rice blast resistance genes were the least（0.4%）.The frequency of nine rice blast resistance genes in rice germplasm resources was obviously different，ranging from 7.2% to 59.1%.The frequency of Pi9 gene was the highest（59.1%），followed by Pi‑k^h（50.8%），Pib（41.2%），Pi5（36.4%），Pita（29.2%），Pb1（28.8%），Pi2（19.5%）and Pikm（17.1%），and the frequency of Pit gene was the lowest（7.2%）.There were 93 rice blast resistance gene combinations in 291 rice germplasm resources.Pi9+Pi‑k^h+Pita combination number was the most in rice germplasm resources（18），followed by Pi9+Pi‑k^h+Pib combination（17），Pi9+Pb1combination（11）.The number of Pi9+Pi5+Pb1，Pi9+Pi‑k^h and Pi‑k^h+Pitacombinations was the same（9），and the number of the other combinations was less than eight.

Genetic Differences and Anthocyanin Contents of 15 Black or Purple Wheat Germplasm Resources

YANG Lijuan, FU Liang, REN Xingxu, ZHU Kun, JIANG Zhikai

2022, 51(12): 28-36. DOI: 10.15933/j.cnki.1004-3268.2022.12.004

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crossbreeding，the genetic differences of 15 black or purple wheat germplasm resources were analyzed on the basis of 15K SNP chip data，and HPLC method was used to test the contents of six anthocyanins in seeds of 15 germplasm resources.The results showed that a total of 7 116 effective single nucleotide polymorphism（SNP）loci were obtained from SNP chip data，and 77.12% of the SNP loci were polymorphic among germplasm resources. There were 128—682 SNP loci on each chromosome，which were unevenly distributed in A，B and D genomes.The genome distribution order of SNP loci number was B>A>D，chromosome 3B had the most SNP loci number，and chromosome 4D had the least SNP loci number.The total anthocyanin content in the seeds of black and purple wheat germplasm resources was significantly higher than that of common wheats，and there were big differences in seed anthocyanin contents among 15 wheat germplasm resources. The seed of Zisexiaomai，Weihuiheixiaomai and Zhouheimai 1 contained more kinds of anthocyanin and higher total anthocyanin content than the other germplasm resources.The seed of Shuzimai 1801，Tesexiaomai 1，Tesexiaomai 2，Luozhen 1，Yuzhouheimai 1 and Yuzhouheimai 2 contained lower total anthocyanin content.The 15 wheat germplasm resources could be classified into three groups，marked as Ⅰ，Ⅱ and Ⅲ.The anthocyanin contents of the three classes were very different，showing Ⅱ>Ⅲ>Ⅰ，and the anthocyanin contents of the germplasm resources with great genetic differences were very different.In conclusion，Zisexiaomai，Weihuiheixiaomai and Zhouheimai 1 had high anthocyanin contents，and could hybridize with other germplasm resources with low genetic similarities for breeding.

Genetic Analysis of Main Mineral Element Content in Grain of Pubing 3228

WANG Jiansheng, WANG Erwei, MA Aichu, HOU Guiling

2022, 51(12): 37-44. DOI: 10.15933/j.cnki.1004-3268.2022.12.005

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In order to dissect the genetic characteristics of major mineral element content in the new germplasm of wheat‑Agropyron cristatum derivative Pubing 3228，the contents of Ca，Mg and Zn in grains of Pubing 3228，Jing 4839 and their F_2:3 population were assessed，and the genetic characteristics of Ca，Mg and Zn contents of Pubing 3228 were analyzed.The results showed that the contents of Ca，Mg and Zn in grains of Pubing 3228，Jing 4839 and their F_2:3 population were different；the contents of Ca，Mg and Zn in F_2:3 population were 81.76—999.55 mg/kg，337.24—1 380.86 mg/kg and 5.21—148.11 mg/kg，respectively；the content of Zn had the largest variation.The Ca，Mg and Zn contents had similar genetic characteristics，the optimal genetic model was two major genes+polygene model，and the inheritance of the two major genes had the effect of additive‑dominant‑epistasis.The effects of additive，dominance，additive×additive，additive×dominance and dominance×additive of major genes of Ca content were positive，but the effect of dominance×dominance was negative.The effects of additive，dominance，additive×additive and dominance×additive of major genes of Mg content were positive，but the effects of additive×dominance and dominance×dominance were negative. For Zn content，the effects of additive，dominance，additive×additive and dominance×additive of its major genes were positive，but the effects of dominance×additive and dominance×dominance were negative. The main gene heritability of Ca，Mg and Zn contents was greatly different，the main gene heritability of Zn content was the highest，followed by Mg content，Ca content was the lowest.

Quality Evaluation and Construction of Seedling Index of Tea Seedlings

WANG Xueping, GAO Shiwei, YE Fei, TENG Jing, GUI Anhui, ZHENG Pengcheng

2022, 51(12): 45-52. DOI: 10.15933/j.cnki.1004-3268.2022.12.006

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In order to scientifically and conveniently evaluate the quality of tea seedlings，300 container seedlings of 200‑day old Echa No.1 were taken as materials，the comprehensive evaluation index of tea seedlings was constructed by weighted fuzzy comprehensive evaluation method，the seedling index was constructed by principal component analysis method and correlation analysis method，and three tea varieties（Echa No.1，Zhongcha 108 and Jinguanyin）were used to evaluate the stability of the screened seedling index.The results showed that the comprehensive evaluation index of 300 tea seedlings ranged from 0.401 to 0.968.The 17 individual indices of tea container seedlings were classified into three principal components，including underground part indicators，aboveground part indicators and pigment indicators.Twenty‑seven seedling indexes were combined by selecting three indices with great contribution to each principal component.Four seedling indexes were screened out by correlation analysis due to high correlation with the comprehensive evaluation index.Among them，dry weight of aboveground part×dry weight of underground part×chlorophyll a content had the highest correlation coefficient for different varieties of tea seedlings，which could be used as seedling index to evaluate the quality of tea container seedlings.According to the comprehensive evaluation index and seedling index，the quality of tea seedlings were classified into three grades：high‑quality seedlings with seedling index of 0.242—1.089，qualified seedlings with seedling index of 0.064—0.239，and weak seedlings with seedling index of 0.011—0.063. There was significant difference among the above three grades.

Effects of Cattle Manure and Wheat Straw Compost instead of 20% Chemical Fertilizer on Soil Total Nutrients in Plough Layer during Wheat‑Corn Rotation System

NIE Shengwei, ZHANG Qiaoping, PAN Xiuyan, WANG Jianchao, ZHENG Nian, XU Jidong, ZHANG Yuting, CHEN Zhaoxia

2022, 51(12): 53-62. DOI: 10.15933/j.cnki.1004-3268.2022.12.007

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The effects of compost consisting of cattle manure and wheat straw，which was applied at 6 000（M1‑20%F）and 9 000（M2‑20%F）kg/ha，respectively instead of 20% chemical fertilizer in wheat and corn growing seasons on soil total nutrients concentration during two continuous years（2018—2019 was the first year and 2019—2020 was the second year）were studied. Single applying chemical fertilizer treatment was used as CK‑F. The results showed that both M1‑20%F and M2‑20%F treatments could increase wheat（by 0.52% and 13.90%，respectively）and corn（by 17.65% and 50.28%，respectively）grain yields in the first wheat‑corn rotation（WCR）year. However，wheat grain yield in the treatments of compost instead was reduced in the second WCR year，and maize grain yield in M1‑20%F treatment was slightly reduced，but in M2‑20%F treatment increased by 8.21%.Compared with CK‑F treatment，soil total nitrogen（TN）and total phosphorus（TP）contents in compost instead treatments were increased，and significantly increased under M2‑20%F treatment at corn maturing stage. Soil total potassium（TK） content in compost instead treatments had increasing trends.Soil organic matter（SOM）content increased generally at corn maturing stage，while it increased at the first wheat maturing stage and decreased slightly at the second wheat maturing stage，respectively.Compared with total soil nutrients content of the treatment itself in plough layer at the local planting season，TN content was reduced at the first and increased at the second wheat maturing stage，respectively；while the TN content in M1‑20%F treatment was reduced and in M2‑20%F treatment was increased at the corn maturing stage，respectively.Moreover，TP，TK，and SOM contents were increased at the first wheat maturing stage and decreased at the second wheat maturing stage，respectively.The concentration of soil nutrients at the second corn maturing stage was increased. Compared with total soil nutrients content of the treatment itself at the wheat planting stage，the total nitrogen content of each treatment was reduced at the first WCR year，and the reduction content in instead treatments was lower than that in CK‑F treatment，with the order of CK‑F（0.321 t/ha）>M1‑20%F（0.096 t/ha）>M2‑20%F（0.058 t/ha）.However，the total nitrogen content of each treatment was increased at the second WCR year. TP content of each treatment was increased at the first rotation year；TP contents of CK‑F and M2‑20%F treatments at the second rotation year were increased by 0.450 and 0. 499 t/ha，respectively，while that of M1‑20%F treatment was reduced by 0.079 t/ha. TK and SOM contents of each treatment were all increased.The TK contents for two years of CK‑F，M1‑20%F，and M2‑20%F treatments were increased by 30. 292，31. 392，31. 605 t/ha，respectively，the SOM contents were increased by 16.145，25.983，21.371 t/ha respectively，and the total increase rates content of compost instead treatments were all higher than that of CK.In conclusion，compost of cattle manure and wheat straw instead of 20% chemical fertilizer could supply rational nutrients and obtain reasonable grain yields in wheat‑corn rotation area.

Effects of Straw Deep Burial Combined with Nitrogen Fertilizer on Organic Carbon Components and Nutrients in Black Soils

XU Sisi , LÜ Xuemei, ZHOU Meng, SUI Yueyu, JIAO Xiaoguang

2022, 51(12): 63-72. DOI: 10.15933/j.cnki.1004-3268.2022.12.008

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With farmland black soils in Northeast China as the research object，the effects of straw deep burial and nitrogen fertilizer on soil organic carbon components and nutrients were investigated.Four nitrogen fertilizer（pure N）levels（no nitrogen：0 kg/ha；low nitrogen：135 kg/ha；medium nitrogen：180 kg/ha；high nitrogen：225 kg/ha） were set up for treatments（no straw and straw deep burial）.The changes of soil nutrients and organic carbon components were systematically analyzed.The results showed that the contents of total nitrogen（TN），total phosphorus（TP）and total potassium（TK）in soil were not significantly affected by straw deep burial（P>0.05）.The contents of soil organic carbon（SOC），light group organic carbon（LFOC） and soluble organic carbon（DOC） were significantly increased（P<0.05）by 2.45%—10.96%，8.76%—70.50% and 47.53%—64.21%，respectively.There were no significant effects on soil easily oxidized organic carbon（ROC）and particulate organic carbon（POC）（P>0.05）.Under the condition of straw deep burial，nitrogen application had no significant effect on soil DOC and POC（P>0.05）.However，it significantly increased the SOC content by 5.56%—11.96%（P<0.05）.The contents of LFOC，ROC and TP were significantly reduced by 19.71%—31.10%，4.75%—8.75% and 3.14%—6.40% respectively（P<0.05）.The SOC and DOC contents were the highest under straw deep burial with medium nitrogen.The contents of LFOC and ROC in soil were the highest under straw deep burial with no nitrogen. The soil TN content was the highest under straw deep burial with high nitrogen. Furthermore，straw deep burial increased the correlation between SOC and DOC，and reduced the correlation between SOC and other organic carbon components，TN，TP and TK.By overall consideration，straw deep burial with 180 kg/ha of pure N is more conducive to the improvement of soil fertility.

Effects of Biochar on Fertility and Nitrogen Utilization Efficiency in Fluvo‑Aquic Soil

ZHANG Keke, SONG Xiao, GUO Doudou, HUANG Shaomin, YUE Ke, GUO Tengfei, ZHANG Shuiqing , YUE Yanjun

2022, 51(12): 73-80. DOI: 10.15933/j.cnki.1004-3268.2022.12.009

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This study aimed to investigate the effect of biochar on soil nutrients，crop yield and nitrogen use efficiency in fluvo‑aquic soil. Five treatments［no fertilization and no biochar（CK），conventional fertilization（B0），and low amount of biochar（B7.5，7 500 kg/ha），medium amount of biochar（B15，15 000 kg/ha）and high amount of biochar（B22. 5，22 500 kg/ha）］were set up to study the effects of biochar on wheat yield and nutrient uptake in fluvo‑aquic soil，and soil nutrient content in 0—15 and 15—30 cm soil layers. The results showed that，biochar had significant effect on soil nutrient content，wheat yield and nitrogen use efficiency.The content of organic carbon，total nitrogen and available potassium in the 0—15 and 15—30 cm soil layers increased with the increase of biochar dosage；the content of soil available phosphorus in the 0—15 and 15—30 cm soil layers reached the maximum under B15 treatment，which were increased by 29.35% and 19.92% compared to the B0 treatment，respectively.With the increase of biochar dosage，wheat yield，1 000‑grain weight and number of grains per ear increased first and then decreased，and the B15 treatment reached the maximum. Compared with the B0 treatment，the average yield and 1 000‑grain weight in 2018—2019 increased by 31.05% and 6.44%，respectively，and the number of grains per ear increased by 3.55.At the same time，nitrogen agronomic efficiency and nitrogen use efficiency also increased first and then decreased with the increase of biochar dosage，the nitrogen agronomic efficiency increased by 6.88 kg/kg and nitrogen use efficiency increased by 21.77 percentage points respectively compared with B0 treatment. In summary，appropriate amount of biochar can increase soil carbon and nitrogen storage and available nutrients content. In order to achieve high yield of wheat，the optimum biochar application rate was 15 000 kg/ha.

Screening，Identification and Effect Evaluation of Three Plant Growth‑Promoting Rhizobacteria from Salvia miltiorrhiza

WANG Fei, LI Xuemeng, YANG Jin, WEN Yi, ZHAO Chenchen, ZHAO Ying, LIU Yuxia, GAO Suxia, QI Wenping, QIN Yanhong, LU Chuantao

2022, 51(12): 81-89. DOI: 10.15933/j.cnki.1004-3268.2022.12.010

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In order to obtain plant growth‑promoting rhizobacteria（PGPR）to promote growth and control root rot（Fusarium proliferatum）of Salvia miltiorrhiza，we isolated bacteria from the rhizosphere soil of Salvia miltiorrhiza by dilution coating plate method.PGPR isolates were screened by confront culturing and indoleacetic acid（IAA）assay.Then the screened strains were identified and evaluated according to their biocontrol and growth‑promoting effects.The results showed that three PGPR strains（K03‑4，Pp03‑41 and Pp146）of 657 isolates from the rhizosphere soil of S.miltiorrhiza were screened.And the three PGPR strains were all characterized as Paenibacillus polymyxa by morphological identification and 16S rRNA gene sequence analysis.The antagonistic effects of K03‑4，Pp03‑41 and Pp146 on F.proliferatum were 66.15%，61.03% and 48.21%，respectively.At the same time，the three strains had the ability to secrete IAA，dissolve inorganic phosphorus，fix nitrogen and produce siderophore.The pot experiments showed that the control effects of three PGPR strains on root rot of S.miltiorrhiza were 83.46%，49.02% and 68.31%.Meanwhile，the height of S.miltiorrhiza was increased by 29.77%，32.60% and 27.64%，the fresh weight of stem and leaf was increased by 18.76%，30.57% and 28.42%，the root number was increased by 89.12%，76.53% and 112.59%，and the fresh weight of root was increased by 88.82%，122.49% and 144.78%，respectively.In conclusion，the three PGPR strains have the function of biocontrol of root rot（F.proliferatum）of S.miltiorrhiza and growth promotion.

Research on the Occurrence in Fields and Detection of Tobacco Viruses in Henan Province

BAI Jingke, WU Yanhui, NIU Longlong, HAO Haohao, WANG Xuefen, ZHANG Xiaoli, LI Shujun

2022, 51(12): 90-96. DOI: 10.15933/j.cnki.1004-3268.2022.12.011

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Tobacco virus disease is an important disease affecting tobacco growth，which occurs periodically and regionally.To clarify the occurrence in fields and variation of tobacco viruses in Henan Province in recent years，we systematically investigated the dynamics of tobacco viruses in the southern，central and western regions of Henan respectively，and detected tobacco viruses samples by ELISA simultaneously. The results indicated that the dynamic curve of tobacco virus disease in field was S‑shaped，with a initial stage appearing at 25—30 days after transplanting，then a peak emerging after 60—70 days，and at last reaching a stable stage when plants were ripe.It also showed that the main virus species were tobacco mosaic virus（TMV），cucumber mosaic virus（CMV），tobacco etch virus（TEV），tobacco vein banding mosaic virus（TVBMV）and potato virus Y（PVY）from 2017 to 2021.

Transcriptome Analysis of Pleurotus ostreatus at Different Growth Stages in Response to IAA

CUI Xiao, ZHANG Yuting, KONG Weili, HU Sujuan, LIU Qin, WANG Yanpo, WU Jie, SHI Ziwen

2022, 51(12): 97-109. DOI: 10.15933/j.cnki.1004-3268.2022.12.012

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To investigate the mechanism of Pleurotus ostreatus at different growth stages in response to IAA，transcriptome sequencing of mycelium（7 d，15 d）and fruiting body stage（25 d，27 d，31 d）under two concentrations of IAA（high concentration：1×10^‑3 mol/L；low concentration：1×10^‑8 mol/L） was performed to screen the differentially expressed genes（DEGs），with no treatment（0 mol/L IAA）as the control.Through Venn analysis，cluster analysis，functional enrichment analysis，and the construction of co‑expression network，the molecular mechanism of P.ostreatus at different growth stages in response to IAA concentration was revealed.Results indicated that 217 DEGs，6 735 co‑expression pairs at the mycelium stage and 10 DEGs，32 co‑expression pairs at the fruiting body stage were screened under the high concentration of IAA，while 81 DEGs and 87 DEGs，and 766 co‑expression pairs and 1 565 co‑expression pairs were dug out under the low concentration of IAA at the mycelium and fruiting body stage，respectively.The GO and KEGG results showed that the low concentration of IAA could regulate ribosome synthesis of endogenous plant hormones through TRINITY_DN2732_c0_g1，while promote the metabolism of the fruiting body stage through TRINITY_DN530_c0_g1 and TRINITY_DN3832_c0_g1.High concentration of IAA could regulate glycerophospholipid metabolism through TRINITY_DN41591_c0_g1，regulate chitin metabolism through TRINITY_DN14069_c0_g1，and regulate oxidoreductase activity through TRINITY_DN35150_c0_g1，TRINITY_DN5314_c0_g1 and TRINITY_DN43009_c0_g1，while at the fruiting body stage，high concentration of IAA regulated ATP synthesis via TRINITY_DN21430_c0_g1.The above results indicate that low concentration of IAA affects the phytohormone synthesis in the mycelium stage，and the metabolic process in the fruit body stage of P.ostreatus through DEGs；high concentration of IAA affects the oxidative stress mechanism in the mycelium stage，and the ATP synthesis in the fruit body stage of P.ostreatus through DEGs.

Phenotypic Diversity Analysis of Different Cultivars Groups of Herbaceous Peony Germplasm Resources

WANG Huijuan, WANG Erqiang, FU Zhenzhu, LI Yanmin, WANG Xiaohui, WANG Zhanying, YUAN Xin, GAO Jie, WANG Limin, ZHANG Hechen

2022, 51(12): 110-121. DOI: 10.15933/j.cnki.1004-3268.2022.12.013

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109 herbaceous peony germplasm resources from different cultivar groups were used to study the diversity of 38 phenotypic traits（4 quantitative traits，27 qualitative polymorphism traits and 7 qualitative binary traits）by the analysis of variance，coefficient of variation analysis，correlation analysis，principal component analysis and cluster analysis.The results showed that the phenotypic traits of 109 herbaceous peony germplasm resources had a high degree of variation and rich genetic diversity.Among them，the average variation coefficient of quantitative traits was 17.59%，with the largest variation coefficient for plant height（24.71%）.The average Shannon‑Wiener index of quantitative traits was 2.00，and plant height had the maximum value（2.10）.The average of Shannon‑wiener index in qualitative indexes（1.06）was less than that in quantitative traits，and the Shannon‑Wiener index of flower color was the highest（1.82），followed by that of flower type（1.67）.The results of correlation and principal component analysis showed that there were 67 pairs of extremely significant correlation and 53 pairs of significant correlation among the characters. The cumulative contribution rate of first 14 principal components was 69.905%.Plant height，plant type，stigma size，flowering time and stem state were the main traits affecting the phenotypic differences of herbaceous peony. Hierarchical clustering divided herbaceous peony germplasm resources into four groups.Phenotypic traits such as plant height，plant type，flower color，and leaf margin sawtooth were closely related to variety clustering. Groups 1—3 were mainly traditional peony varieties of China.The herbaceous peony germplasm resources introduced from abroad belonged to three cultivars groups and clustered into one group alone，which had quite different phenotypic traits（thick stems and half‑opened plant types） from the traditional varieties of China.This clustering result reflected the difference of provenance and long‑term breeding objectives to a certain extent.

Effects of Cadmium and Lead Combined Stress on Seed Germination and Seedling Growth of Herbaceous Flowers

LI Jiewei, CHI Yongqing, WU Tianfang, ZHU Yangchun, DING Fenghua

2022, 51(12): 122-130. DOI: 10.15933/j.cnki.1004-3268.2022.12.014

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In order to explore the tolerance to cadmium and lead combined stress in the germination stage of herbaceous flowers，the seeds of seven species of herbaceous flowers were chosen as experimental materials to study the effects of cadmium and lead combined stress on seed germination and seedling growth.The results showed that under cadmium and lead stress，5 mg/L+50 mg/L mass concentration could promote germination index and vigor index of Zinnia elegans，Lupinus micranthus and Orychophragmus violaceus. The dry weight of Cosmos bipinnatus and Mirabilis jalapa was higher under cadmium and lead concentration of 10 mg/L+250 mg/L than that of the control with water.The root length of seven species of herbaceous flowers was significantly inhibited with cadmium and lead concentration of 40 mg/L+1 000 mg/L. 80 mg/L+2 000 mg/L cadmium and lead stress completely inhibited the seed germination and seedling growth.The growth inhibition of root was greater than that of seedling.According to the D value of weighted membership function，the cadmium and lead tolerance of herbaceous flower seeds was in the order of Mirabilis jalapa>Gomphrena globose>Cosmos bipinnatus>Lupinus micranthus>Calendula officinalis>Zinnia elegans>Orychophragmus violaceus.The cluster analysis of the membership function integrated values showed that the test herbaceous flower seeds were divided into three categories including high resistance（Mirabilis jalapa），medium resistance（Gomphrena globose，Cosmos bipinnatus，Lupinus micranthus）and low resistance（Calendula officinalis，Zinnia elegans，Orychophragmus violaceus）

Development and Application of SYBR Green Ⅰ‑Based Real‑time PCR Assay Method for Detection of Fowl Adenovirus Serotype 4

LU Qingxia, JIN Qianyue, FENG Lili, CHAI Yongxiao, GUO Zhenhua, XING Guangxu, ZHANG Gaiping

2022, 51(12): 131-138. DOI: 10.15933/j.cnki.1004-3268.2022.12.015

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To develop an assay method for the clinical detection of fowl adenovirus serotype 4（FAdV4），a pair of primers was designed according to the Hexon gene sequence of FAdV4 in GenBank.The amplified Hexon gene was ligated with the vector pEASY‑Blunt to construct recombinant plasmid pEASY‑Blunt‑Hexon，which then served as the template.Real‑time PCR assay method for the detection of FAdV4 was established using primers amplifying the conserved region of Hexon gene.The amplified product was 95 bp in length and the limit of detection reached 7.1×10² copies/μL.The assay had no cross‑reaction with other poultry viruses.The coefficient of variation was less than 2% within and between the groups，which showed good specificity and repeatability.The detection result of the proliferation of FAdV4 on leghorn male hepatocellular（LMH）cells exhibited good associations with the viral titers by measuring TCID₅₀.In conclusion，the real‑time PCR assay method developed in this study is applicable for the early detection of FAdV4 clinically.

Study on Microbial Diversity of Water in Rice‑crayfish‑eel Coculture System

ZHANG Yuanyuan, ZHAO Liangjie, LI Hong, ZHU Wenjin

2022, 51(12): 139-146. DOI: 10.15933/j.cnki.1004-3268.2022.12.016

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To study the microbial diversity in different spatial regions of rice‑crayfish‑eel coculture system，Illumina MiSeq was used to analyze the microbial diversity of water samples inside the cages （T1），outside the cages（T2）and different positions in circular groove（T3，T4）.The results showed that a total of 3 737 OTUs were obtained from 16S rRNA gene in water samples from the system of rice‑crayfish‑eel，and a total of 47 phyla，137 classes，309 orders，509 families，929 genera and 1 697 species were identified.Proteobacteria，Cyanobacteria and Actinobacteria were the dominant groups in different sampling sites. Eel cage‑culture did not significantly affect the microbial diversity index in different areas of water，but significantly changed the microbial community structure. With the development of culture，the difference of bacterial composition among different sampling sites was more and more obvious. Redundancy analysis（RDA） results showed that NO₂^-‑N，TN and NH₄⁺‑N were highly correlated with the microbial community composition of the system，and CODMn had the least correlation with the microbial community composition.Cluster of orthologous group（COG）functional prediction analysis showed that the bacteria associated with amino acid metabolism and energy conversion had the highest abundance.

Molecular Identification of Giardia duodenalis and Cryptosporidium spp.from Sheep

SUI Panbo, XU Feifei, LIANG Guanda, DU Haili, LANG Jiashu, LI Junqiang

2022, 51(12): 147-152. DOI: 10.15933/j.cnki.1004-3268.2022.12.017

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To investigate the molecular characteristics of Giardia and Cryptosporidium from sheep in Anyang area，Henan Province，microscopy positive fecal samples based on Lugol’s iodine staining techniques and Sheather’s sugar flotation were obtained，followed by DNA extraction from sixteen Giardia positive samples and three Cryptosporidium positive samples.The nested PCR amplification based on 16S rRNA locus and 18S rRNA locus was performed，respectively.The positive amplicons were sequenced，followed by phylogenetic and evolutionary analysis.The results indicated that the specific gene loci of Giardia and Cryptosporidium from sheep were successfully amplified by PCR.Molecular characteristics analysis of the specific loci of the two intestinal protozoans showed that the Giardia strains were identified as G.duodenalis assemblage E（93.75%，15/16）and assemblage A（6.25%，1/16）.And the Cryptosporidium strains were identified as C.ubiquium（66.67%，2/3）and C.suis（33.33%，1/3）.

Research on Maize Pest Detection Based on Instance Segmentation

ZHAO Kangdi, SHAN Yugang, YUAN Jie, ZHAO Yuanlong

2022, 51(12): 153-161. DOI: 10.15933/j.cnki.1004-3268.2022.12.018

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In order to achieve accurate and rapid identification of maize pests，this paper proposes a pest detection method using convolutional neural network combined with transfer learning based on instance segmentation.Taking eggs，larvae and adults of Spodoptera frugiperda as detection objects，the image data was expanded by data enhancement，and the pre‑training weights of Yolact++ model on COCO data set were migrated to the detection of Spodoptera frugiperda.Using the idea of focal loss to solve the imbalance of difficult and easy samples，the loss function in the model was optimized. The Detnet model was used to improve the Resnet trunk model in the Yolact++ model to improve the effect of small target detection.In the training process，the convolution layer was first frozen and then thawed，and the local and global training methods were combined to obtain the optimal weight model，and the model was tested. The test results showed that compared with the detection algorithms of YoloV3+migration learning，SSD+migration learning，Yolact+migration learning and Yolact++，this method had better accuracy and missed detection rate for complex background image detection. The accuracy of the test reached 96.32%，the missed detection rate was 5.51%，and the false detection rate was 5.33%.

Effects of Controlled Atmosphere Storage on the Shelf Quality of Jintao Kiwifruit

ZHANG Sipu, WANG Xinrui, CUI Wei, ZHANG Ke, HU Qingxia, LU Yunfeng, MIAO Jianyin, LIU Cheng, NIU Jiajia

2022, 51(12): 162-171. DOI: 10.15933/j.cnki.1004-3268.2022.12.019

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In order to explore the effects of 1%—4% CO₂ treatment at 5% O₂ on the shelf quality of kiwifruit after air‑conditioned storage，Jintao kiwifruit was used as test material，and the controlled atmosphere parameters were set as 5%O₂+1%CO₂，5%O₂+2%CO₂，5%O₂+3%CO₂，and 5%O₂+4%CO₂，respectively.After storage at（1±0. 5）℃ for 210 days，the decay rate，chilling injury index，weight loss ratio，fruit firmness，total soluble solid（TSS）content，titratable acid（TA）content，ethylene release rate，peroxidase（POD）activity，catalase（CAT）activity，superoxide anion（O₂·-）content and hydrogen peroxide（H₂O₂）content were determined at room temperature（25±1）℃.The results showed that different treatments could reduce decay rate，chilling injury index and ethylene production rate，inhibit the increase of weight loss rate and TSS content，delay the decrease of fruit firmness and TA content，reduce the accumulation of H₂O₂ and O₂·-，and maintain the activities of CAT and POD at higher levels.Among them，5%O₂+3%CO₂ treatment had the best effect. After shelf life of 10 days，the firmness and TA content of fruit were the highest，which were 1.86 kg/cm²and 1.18%，respectively.TSS content was the lowest（14.6%）.CAT and POD activities were the highest，which were 30.0 U（/ min·g）and 245 U（/min·g），respectively.O₂·- content was the lowest as 2.12 μmol/g，and H₂O₂content was 4.5 μmol/g.And the above indexes were significantly different from general refrigerated treatment（P<0.05）.Therefore，it is suggested to use 5%O₂+3%CO₂ treatment to keep Jintao kiwifruit fresh in production，which can effectively improve the shelf quality of fruit. After 210 days of low temperature storage，the shelf life is more than 10 days.

Study on Curing Characteristics and Quality of New Flue‑cured Tobacco Variety Xujin 101

LIU Fang, SONG Xiaolong, ZONG Shengjie, MENG Zhiyong, ZHU Yinfeng, SUN Huan, MA Haobo, ZHANG Guoping

2022, 51(12): 172-180. DOI: 10.15933/j.cnki.1004-3268.2022.12.020

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In order to clarify the curing characteristics and the quality of tobacco leaves of a new self‑bred flue‑cured tobacco variety Xujin 101，with the main variety Zhongyan 100 as a control，the yellowing，water loss，chlorophyll degradation and polyphenol oxidase activity of Xujin 101 tobacco leaves during the dark‑box and curing process were studied，and the differences in appearance quality，sensory evaluation quality，neutral aroma‑causing substances and main chemical components of cured tobacco leaves were analyzed.The results showed that under dark‑box conditions，Xujin 101 took 72 h and 48 h to turn completely yellow in the middle and upper leaves，respectively，and the time for 30% browning was 120 h and 72 h，respectively. The yellowing index and browning index were both higher than those of Zhongyan 100.On the whole it was easy to turn yellow and brown. The water loss balance was 1.16 and 0.47，respectively，and the water loss balance was good. The degradation rate of chlorophyll reached 1.32%/h and 1.37%/h respectively at 72 h，the degradation amount reached 94.88% and 98.71%，and the degradation characteristics were good.The average PPO activities after baking 24—96 h were 0.409 U and 0.420 U，respectively，the middle leaves had poor baking resistance，and the upper leaves had moderate baking resistance. Although the baking characteristics of Xujin 101 were slightly better than Zhongyan 100，but the initial curing plan had a greater impact on the quality of Xujin 101 tobacco leaves after curing，and the overall quality was slightly worse than that of Zhongyan 100.According to comprehensive analysis，Xujin 101 is easy to bake，but has average baking resistance.In order to highlight the characteristics of Xujin 101，it is necessary to set up a baking process according to its baking characteristic. In the early stage of yellowing，the temperature difference between dry and wet bulbs should be appropriately reduced to reduce the moisture loss of tobacco leaves.In the later stage，the temperature difference between dry and wet bulbs should be appropriately increased to improve the dehumidification strength，promote drying and prevent browning，and ensure safe color setting of tobacco leaves to improve its use value and economic value.

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