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    16 June 2021, Volume 54 Issue 12
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Gene Mapping and Candidate Gene Analysis of Grain Width Mutant gw87 in Rice
    ZHANG XiangYu,GUO Jia,WANG San,CHEN CongPing,SUN ChangHui,DENG XiaoJian,WANG PingRong
    Scientia Agricultura Sinica. 2021, 54(12):  2487-2498.  doi:10.3864/j.issn.0578-1752.2021.12.001
    Abstract ( 549 )   HTML ( 83 )   PDF (3182KB) ( 433 )   Save
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    【Objective】Phenotypic identification, genetic analysis, gene mapping and candidate gene analysis of grain width mutant gw87 (grain width87) were performed in rice, so as to lay a good foundation for further understanding molecular mechanism and utilization potentiality of this gene regulating rice grain size.【Method】Through ethyl methanesulfonate mutagenesis, we isolated gw87 mutant showing significant increase of grain width and 1000-grain weight from indica restorer line material 676R. To identify phenotypic and physiological characteristics of the mutant, we performed observation of phenotypes, investigation of agronomic traits, sensitivity analysis of exogenous brassinolide (BL), determination of chlorophyll contents and photosynthetic parameters by using gw87 and its wild type. To analyze its genetic behavior, we investigated F1 phenotypes and F2 segregation of the crossing combination between gw87 and its wild-type parent 676R. To find candidate gene of gw87, we conducted high-throughput sequencing and MutMap analysis of 30 mutant plants from the F2 population of gw87×676R, and molecular mapping using the F2 population crossing gw87 and japonica cultivar Nipponbare. Meanwhile, we confirmed the candidate gene by sequencing its DNA and cDNA sequences. In addition, we detected expression levels of brassinolide synthesis pathway genes OsDWARF4, D11 and D2 using qRT-PCR analysis.【Result】Compared with the wild-type parent 676R, the gw87 mutant showed significant reduction of productive panicle number per plant, main panicle length and seed setting rate, but significant increase of grain width and 1000-grain weight. Meanwhile, the mutant displayed decreased plant height and shortened internodes, in which the first internode was distorted and its length is shortened the most. Besides, leaf length of gw87 was decreased but the width was increased. Sensitivity assays of exogenous BL suggested that the sensitivity of gw87 seedlings to exogenous BL was reduced. Determination of photosynthetic pigments and photosynthetic parameters showed that chlorophyll content and net photosynthetic rate were decreased. Genetic analysis revealed that the mutant phenotype of gw87 was governed by a single recessive nuclear gene. High-throughput sequencing and MutMap analysis suggested that the gw87 locus was located in the middle of chromosome 5, and only one base mutation causing amino acid change of the encoded protein occurred in this chromosome region. Molecular marker linkage analysis demonstrated that the gw87 gene was mapped to a 101 kb genomic region between InDel markers X2 and X3. The MutMap and molecular mapping revealed that the candidate gene was LOC_Os05g32270 that encodes a transcription factor containing the AP2/EREBP DNA-binding domain. Subsequently, we confirmed the candidate gene by sequencing its DNA and cDNA. In the gw87 mutant, a single nucleotide G-to-A substitution occurred at position 1041 in its DNA sequence, and the 76-bp intron sequence adjacent to this mutated nucleotide was spliced into exon in its cDNA, which caused shift of the reading frame and then premature termination of the protein translation. In addition, qRT-PCR analysis showed that the expression of BR synthetic genes were significantly up-regulated in gw87, indicating that BR signal was weakened in the mutant.【Conclusion】gw87 is a new allelic mutant of smos1, shb, rla1, and ngr5. However, unlike these mutants, gw87 exhibited significant increase of grain width and 1000-grain weight relative to its wild type. The reason for the differentiation could be that different mutation sites in the LOC_Os05g32270 gene, resulting in different functional activities of the encoded protein.

    Genetic Diversity and Comprehensive Evaluation of Phenotypic Traits in Sea-Island Cotton Germplasm Resources
    YANG Tao,HUANG YaJie,LI ShengMei,REN Dan,CUI JinXin,PANG Bo,YU Shuang,GAO WenWei
    Scientia Agricultura Sinica. 2021, 54(12):  2499-2509.  doi:10.3864/j.issn.0578-1752.2021.12.002
    Abstract ( 394 )   HTML ( 44 )   PDF (883KB) ( 315 )   Save
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    【Objective】The study is an attempt to the genetic diversity relationship of phenotypic traits of Sea-island cotton germplasm and screen Sea-island cotton germplasm with excellent traits, which provides a theoretical basis for in depth research on quality traits of Sea-island cotton.【Method】Genetic diversity analysis and comprehensive evaluation were carried out with 12 phenotypic traits of 175 Sea-island cotton. Principal component analysis, weights and the use of membership function to generate comprehensive evaluation value D for comprehensive evaluation of Sea-island cotton germplasm resources.【Result】The results (showed that) Sea-island cotton have rich types and consistent with the range of phenotypic traits was 6.40%-28.10%,which the resource genetic diversity index is 1.97 to 2.05, the diversity analysis significant differences in genetic diversity of Sea-island cotton resources between Xinjiang and outside Xinjiang (P<0.05); 3 comprehensive factors were converted from principal component analysis 12 traits, which the contribution rates are: 49.34%, 18.03% and 10.63%, with a total contribution rate of 78%; The larger load of the first principal component is the number of envoys, the height of envoys, the number of effective branches, the number of bolls, the number of effective bolls, the weight of seed cotton per plant and the weight of lint per plant, representing growth and effective yield factors; The larger load of the second component is lint, single boll seed cotton weight and single boll lint weight, which represents the single boll yield factor; The third component load is the plant height and the number of boll shedding, representing plant height and boll shedding factor; The statistical analysis of Sea-island cotton resources evident that the difference was not significant in extreme germplasm at domestic , and there are more materials with medium-performing Sea-island cotton resources in China. In China, the proportion of extreme germplasm outside Xinjiang is relatively high, and the difference was not significant between Xinjiang and foreign intermediate germplasm; Cluster analysis 175 Sea-island cotton resources then divided into 4 groups. Group I has discrepancy germplasm with short stalk of gravity, low yield; Group II has high lint percent and extensive yield of lint yield per plant, and has higher potential yielding germplasm; Group III has perferable comprehensive traits and excellent germplasm; group IV is short-stalked, high-lint, and extremely particular germplasm; comprehensive evaluation screens out 2 excellent comprehensive traits varieties XH30 and 270; By stepwise regression screening Sea-island cotton germplasm screening phenotype of five key indicators (plant height first node height, number of fruit branches, lint weight per boll and cotton weight per seed). 【Conclusion】The genetic diversity of the tested Sea-island cotton germplasm resources is relatively high, but the genetic diversity is low. The plant height, fruit branch number and lint yield per plant are normally distributed among the genotypes, those left traits are skewed. The tested germplasm divided into 4 categories; Plant height, height of the first node, fruit branch number, yield of single boll Lint yield, seed cotton yield per plant, the research provides a theoretical reference to selection of excellent germplasm for sea-island cotton germplasm resources that 5 indicators can be used as comprehensive indicators for the evaluation of core germplasm.

    Precise Evaluation of 48 Maize Inbred Lines to Major Diseases
    ZHAO ZiQi,ZHAO YaQi,LIN ChangPeng,ZHAO YongZe,YU YuXiao,MENG QingLi,ZENG GuangYing,XUE JiQuan,YANG Qin
    Scientia Agricultura Sinica. 2021, 54(12):  2510-2522.  doi:10.3864/j.issn.0578-1752.2021.12.003
    Abstract ( 587 )   HTML ( 37 )   PDF (871KB) ( 800 )   Save
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    【Objective】 In order to provide valuable resources to the maize community for enhancing disease resistance, we selected 30 core maize inbred lines from Shaan A and Shaan B heterotic groups, and 18 major maize germplasms worldwide for disease resistance evaluation and resistance gene identification.【Method】Gibberella stalk rot, Gibberella ear rot, northern leaf blight (NLB) and southern leaf blight (SLB) were artificially inoculated, while gray leaf spot (GLS) was naturally infected in replicated field trials at different locations in 2019 and 2020. A randomized complete block design was adopted with two to three replicates being evaluated at each location. Sorghum grain inoculum was artificially inoculated to the whorl of each plant at the seedling stage for NLB and SLB. Gibberella stalk rot was artificially inoculated using corn grain inoculum buried next to the root of each plant at heading time, while Gibberella ear rot was artificially inoculated by injecting conidial suspension through the silk channel and the husk separately. The best linear unbiased predictions (BLUPs) were estimated and correlation coefficients were calculated between each pair of the disease resistance. Disease resistance genes were genotyped using functional markers. 【Result】Nine highly resistant (HR) lines for NLB, two HR lines for SLB, ten resistant (R) lines for GLS, five HR lines for stalk rot, and five R lines for ear rot resistance were identified out of the 48 maize germplasms. Eight inbred lines showed multiple disease resistance to the three foliar diseases, such as 1145, CML170, and so on. We identified seven elite inbred lines showing good performance for all the five diseases, including 1145, CML170, KA105, KB020, X178, Shen137, and Zheng58. NLB, SLB, GLS, and Gibberella stalk rot resistance were significantly positively correlated, while Gibberella ear rot was not correlated with the other four diseases. 1145, KA081, and Shen137 contain the resistant qRfg1 allele. KB109 harbors the resistant Rcg1 allele. Many inbred lines carry the resistant Htn1 and ZmCCoAOMT2 alleles.【Conclusion】1145, CML170, KA105, KB020, X178, Shen137, and Zheng58 could be selected for multiple disease resistance improvement in breeding program. Shen137 is the best line for marker-assisted selection for qRfg1, Htn1, and ZmCCoAOMT2 loci.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Identification of 14-3-3 Client Proteins in Rice Grains and Their Response to Exogenous Hormones During the Grain Filling Stage
    ZHANG ZhiXing,MIN XiuMei,SONG Guo,CHEN Hua,XU HaiLong,LIN WenXiong
    Scientia Agricultura Sinica. 2021, 54(12):  2523-2537.  doi:10.3864/j.issn.0578-1752.2021.12.004
    Abstract ( 366 )   HTML ( 35 )   PDF (1605KB) ( 311 )   Save
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    【Objective】Grain yield and quality of rice mainly depend on grain filling. The family of 14-3-3 proteins is the important regulator of signaling in plant, and plays an important role in plant growth and development. This study was performed to assess the gene expression pattern and to identify client proteins, which would result in important information toward understanding the functional mechanism of 14-3-3 protein during rice grain filling. 【Method】 The expression pattern of each 14-3-3 genes at different grain filling stages were examined by the Real-time RT-PCR (qRT-PCR), and the family members GF14b and GF14e were used for subsequent functional analysis. The function motif of GF14b and GF14e protein were analyzed by using the KEGG database. The amplified open reading frames of GF14b and GF14e were cloned into the PGEX-6P-1 expression vector for production of the N-terminal-tagged fusion proteins GST-GF14b and GST-GF14e. The client proteins of GF14b and GF14e were captured with the affinity chromatography approach and identified by the LC-MS/MS. The protein interaction between client proteins and GF14b and GF14e were validated by the GST Pull-down in vitro. Phosphorylation sites and functions of all client proteins were analyzed using the KinasePhos online website and MapMan software (Version 3.60), respectively. The exogenous hormones (25×10 -6mol·L-1 ABA,10×10-6mol·L-1 IAA,100×10-6mol·L-1 GA,50×10-6mol·L-1 ZR and 2×10-4mol·L-1 BR ) were sprayed on rice grains at 15 days after flowering to investigate the effects of exogenous hormones treatment on the expression of GF14b, GF14e and their interacting genes. 【Result】 Except for the GF14h, the remaining seven 14-3-3 family genes were expressed in rice grains, and the expression of GF14b and GF14e was high and highly variable during grain filling. Two same protein motifs and three differential motifs between GF14b and GF14e were identified with the protein sequence analysis. In total, 59 GF14b- and 72 GF14e-client proteins in rice grains were captured by affinity chromatography on a glutathione-agarose affinity column. Two randomly selected proteins from these client proteins, namely SUS3 and PSA, were further validated by GST Pull-down in vitro, and the results showed that the SUS3 interacted with GF14b and GF14e, whereas PSA specially interacted with GF14e, which confirmed the accuracy of the affinity chromatography assay. Among these client proteins, fourth-three were commonly affinity-identified by the GF14b and GF14e and were predicted to be implicated in the sucrose conversion, starch synthesis, glycolysis and TCA cycle. Meanwhile, it was found GF14b specifically bounded to the nucleotide metabolism and transporters, whilst GF14e tended to interact with the proteins involving C1 metabolism. In addition, most of the identified client proteins possess the phosphorylation sites of Ser or Thr. The present results also showed that the expression of GF14b and GF14e were up-regulated and most of the starch synthesis-related interacting genes (SUS2, AGPS, AGPL, PPDK2, SBE) were down-regulated after exogenous hormones treatment. 【Conclusion】 The magnitude of change in gene expression was greater in GF14b and GF14e than the other 14-3-3 gene family members during grain filling. The GF14b and GF14e could respond the change of hormones concentration and act as a regulator to negatively regulate the expression of starch synthesis related genes through protein-protein interaction, in turn, play an important role in starch synthesis during the grain filling stage.

    Effects of Increasing Planting Density on Grain Yield, Leaf Area Index and Photosynthetic Rate of Maize in China
    HOU JiaMin,LUO Ning,WANG Su,MENG QingFeng,WANG Pu
    Scientia Agricultura Sinica. 2021, 54(12):  2538-2546.  doi:10.3864/j.issn.0578-1752.2021.12.005
    Abstract ( 658 )   HTML ( 59 )   PDF (1419KB) ( 519 )   Save
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    【Objective】This study aimed to explore the change of the above-ground canopy structure and function together with increasing planting density of maize in China, so as to provide a theoretical basis for reasonable density in maize production.【Method】In this paper, a total of 82 publicly published academic articles were collected, and a total of 1 338 sets of yield-density data were obtained, including 1 200 sets of maximum leaf area index (LAImax) -density data, and 475 sets of maximum leaf photosynthesis rate (Pnmax) -density data. According to the date of sowing, the total samples were divided into two groups: spring and summer maize. Comprehensive analysis of the relationship between maize yield, planting density and photosynthetic characteristics was carried out by using boundary line analysis and various curve fitting methods. 【Result】(1) The agronomic planting density in China was 100 000 plants/hm2 for a maximum yield of 11.5 t·hm-2. Although the optimal planting density of spring and summer maize was similar, the grain yield of spring maize was 13.0% higher than summer maize. (2) When the planting density reached 110 000 plants/hm2, LAImax no longer increased with planting density increasing. When LAImax reached 6.4, the highest yield could be obtained. The platform value of LAImax for spring maize was 17.6% higher than that for summer maize. The logarithmic function was used to analyze the relationship between LAImax and density, as well as Pnmax and LAImax, and the results showed Pnmax decreased with increasing density and LAImax. (3) Grain yield, LAImax and Pnmax were all significantly improved through analyzing the data of different varieties in recent decades. 【Conclusion】Increasing planting density was one of important ways to increase maize yield. When the density was out the rational range, the photosynthetic characteristics of leaf was altered, which limited yield improvement. Comprehensive analysis of quantitative relationships among maize yield, planting density, and photosynthetic characteristics of ear leaf was important to guide high-yield and high-efficiency maize system with high density.

    PLANT PROTECTION
    Mating Type and Fertility of Cochliobolus heterostrophus Causing Southern Corn Leaf Blight in Sichuan Province
    SUN XiaoFang,LIU Min,PAN TingMin,GONG GuoShu
    Scientia Agricultura Sinica. 2021, 54(12):  2547-2558.  doi:10.3864/j.issn.0578-1752.2021.12.006
    Abstract ( 252 )   HTML ( 24 )   PDF (1199KB) ( 235 )   Save
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    【Objective】The objective of this study is to clarify the distribution of mating types and fertility of Cochliobolus heterostrophus (anamorph: Bipolaris maydis), and to elucidate the possibility of the sexual reproduction of C. heterostrophus isolates in natural fields.【Method】A total of 544 C. heterostrophus isolates were collected from different geographical regions in Sichuan and Yunnan provinces during 2013-2018. The mating type of these isolates was detected by a multiple PCR method, and two standard tested isolates with high combining ability were screened by cross of MAT1-1 and MAT1-2 isolates. Then the 544 C. heterostrophus isolates were tested for their mating type and fertility with two standard test isolates. The fertility was determined by the development of pseudothecia and asci.【Result】Among the 544 isolates, 286 ones were identified as MAT1-1 and 258 as MAT1-2, accounting for 52.57% and 47.43%, respectively. Chi-square test gave a good fit of 1﹕1 ratio (χ2=1.441, P=0.230), indicating that these two mating types distributed in balance in natural fields. In addition, the investigation also showed that two mating types were present in all sampling locations and they had a basically same population structure in different years. After crossing tested isolates with standard isolates MSRS-2-3 (MAT1-1) and DY-12-1-2 (MAT1-2), the percentage of fertile and sterile isolates was 88.79% and 11.21%, respectively, and an obvious fertility differentiation was observed, which were characterized by 12.32% of high fertile, 27.39% of medium fertile, and 49.08% of low fertile isolates, respectively. Fertility structures of isolates varied in different geographical locations and in different collection years. The frequencies of fertile isolates ranged from 77.88% to 94.87% in 2013-2018. The percentage of fertile isolates in Southern Yunnan was highest up to 100%, followed by those in Northern Sichuan, Central Sichuan, Southern Sichuan and Eastern Sichuan regions, accounting for 93.25%, 89.87%, 83.33% and 79.31%, respectively. The percentage of fertile isolates in Western Sichuan region was lowest (69.23%).【Conclusion】MAT1-1 and MAT1-2 achieved an equilibrium in quantity among C. heterostrophus field populations, and the cross combinations between MAT1-1 and MAT1-2 isolates were generally fertile. Although sexual generation was rarely reported in C. heterostrophus population in natural condition, the current results indicated that cryptic sexual cycle might contribute to the genetic diversity of C. heterostrophus.

    Identification of the Antifungal Active Compounds from Bacillus amyloliquefaciens Strain HMB33604 and Its Control Efficacy Against Potato Black Scurf
    LI YangFan,SHAO MeiQi,LIU CHANG,GUO QingGang,WANG PeiPei,CHEN XiuYe,SU ZhenHe,MA Ping
    Scientia Agricultura Sinica. 2021, 54(12):  2559-2569.  doi:10.3864/j.issn.0578-1752.2021.12.007
    Abstract ( 420 )   HTML ( 35 )   PDF (1948KB) ( 371 )   Save
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    【Objective】Potato black scurf caused by Rhizoctonia solani AG-3 is one of the most important potato diseases in China, resulting in considerable economic losses. Microbial fungicides are confirmed as practical and environmental friendly way to suppress plant soil-borne diseases. To develop microbial fungicide for potato black scurf, bacterium with potential biocontrol ability for potato black scurf was obtained and the antifungal mechanism was identified for the biocontrol agent. Results of this study will be useful for optimizing the fermentation of biocontrol agent, as well as application of microbial fungicide developed for the bacterium.【Method】Potential biocontrol agents were screened by dual culture in vitro as well as suppressing potato black scurf in vivo. Bacteria were identified by Biolog Microbiological Identification System, 16S rDNA sequence, as well as phylogenetic tree inferred from the alignment sequence of gyrA, gyrB, rpoB and rpoC. The fermentation broth, cell free supernatant and bacterial cell suspension of biocontrol agent were evaluated for suppressing potato black scurf under greenhouse condition. The lipopeptide produced by the biocontrol agent was isolated by high performance liquid chromatography (HPLC) and the antifungal active compounds were identified by UPLC-Triple TOF-MS/MS. The DNA copies of R. solani in the potato rhizosphere was calculated by real-time PCR.【Result】Three bacterial strains with potential biocontrol abilities for potato black scurf were selected from 2 106 antagonistic strains, and Bacillus amyloliquefaciens strain HMB33604 showed the best suppressing ability with 52.9% biocontrol efficacy for potato black scurf. Both the fermentation broth and cell free supernatant showed significant biocontrol abilities against potato black scurf with 52.2% and 66.4% biocontrol efficacy, respectively. However, only 16.9% biocontrol efficacy was achieved by the cell suspension, so the antifungal compounds produced by HMB33604 played an important role in suppressing potato black scurf. The lipopeptides fengycin, iturin A, and surfactin were separated and purified by HPLC, and only the fengycin and iturin A showed strong inhibitory abilities against the growth of R. solani, as well as causing abnormal hyphal growth in vitro. The number of R. solani was significantly reduced by 60.3% and 64.0% of the fermentation broth and cell free supernatant treatments, respectively, when compared to the control. Meanwhile, R. solani number was only reduced by 10.3% of the cell suspension treatment.【Conclusion】B. amyloliquefaciens strain HMB33604 was successfully selected as a promising biocontrol agent for potato black scurf, and the antifungal active compounds produced by strain HMB33604 played an important role in suppressing potato black scurf. The lipopeptides fengycin and iturinA were the main antifungal active compounds in strain HMB33604 and caused abnormal hyphal growth and decreased the number of R. solani in the potato rhizosphere.

    A New Integrated Soil Disinfection Machine Improves the Uniformity of Dazomet in Soil
    FANG WenSheng,CAO AoCheng,WANG QiuXia,YAN DongDong,LI Yuan,JIN Xi,ZHAO QiLong,QIU YaoKang,ZHAO HongMing
    Scientia Agricultura Sinica. 2021, 54(12):  2570-2580.  doi:10.3864/j.issn.0578-1752.2021.12.008
    Abstract ( 379 )   HTML ( 32 )   PDF (3691KB) ( 243 )   Save
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    【Objective】Dazomet is the granular soil fumigant with the largest applied area and used amount in China, which has excellent control effect on soil-borne pathogens, nematode, underground insects and weeds. However, dazomet application through hand can easily lead to drug drift and uneven soil distribution, which significantly reducing the fumigation effects. Therefore, it is high time to find an efficient and safe application technology of dazomet. In this study, an integrated soil disinfection machine was applied to increase the depth of pesticide application and improve the distribution uniformity of soil layer of dazomet.【Method】The field fumigation was combined with indoor culture experiments to evaluate the distribution uniformity of the fumigant in different depth layers (0-40 cm) as well as the control effects on Fusarium and Phytophthora following dazomet fumigation by a new integrated soil disinfection machine (NIM). The headspace sampling and GC-MS were used to investigate the vertical distribution characteristics of gas methyl isothiocyanate (MITC) and residual MITC in soil at different depths of 0-40 cm. The specific culture medium was used to isolate the soil-borne pathogens of Fusarium and Phytophthora in soil, and then the occurrence of pathogenic bacteria at different depths of 0-40 cm were analyzed.【Result】The fumigation tests in Anhui, Shanxi and Hebei all showed that, under the model of machine application, fumigant dazomet was evenly distributed in 0-40 cm deep soil layer, the concentration of MITC in gas and in soil was 1.46-3.02 and 18.67-26.27 μg/g soil, respectively, and there was no significant difference in MITC solubility among different deep soil layers. However, in the hand-sprinkling mode, dazomet was mainly distributed in 0-10 cm deep soil layer, and no MITC was detected in 20-40 cm deep soil layer. In addition, MITC solubility in 20-40 cm deep soil layer could not be increased by increasing dazomet application dosage under the hand-sprinkling mode, but MITC solubility in 0-10 cm deep soil layer under the hand-sprinkling mode and 0-40 cm deep soil layer under the machine application mode was significantly increased. Furthermore, the results showed that the decrease rate of Fusarium and Phytophthora at 0-40 cm depth was 90%-100% under machine application mode, while the decrease rate at 20-40 cm depth was less than 43% under hand-sprinkling mode. Even though the decrease rate of Fusarium and Phytophthora was as much as 90%-100% at 0-10 cm depth under hand-sprinkling mode. There was no significant difference on Phytophthora control effect between 40 and 60 g·m-2 dazoment application dosage.【Conclusion】The new soil disinfection machine in this study can effectively improve dazomst soil distribution uniformity and increase the prevention and control effects of soil pathogens in the deep soil layer. The pesticide content at each depth soil layer was significantly increased under the machine application mode when dazoment application was increased.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Varying Synthetic Phosphorus Varieties Lead to Different Fractions in Calcareous Soil
    JI BingJie,LI WenHai,XU MengYang,NIU JinCan,ZHANG ShuLan,YANG XueYun
    Scientia Agricultura Sinica. 2021, 54(12):  2581-2594.  doi:10.3864/j.issn.0578-1752.2021.12.009
    Abstract ( 521 )   HTML ( 31 )   PDF (622KB) ( 866 )   Save
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    【Objective】Synthetic phosphorus fertilizers may vary in their behavior when applied to soil because of their inherent differences in nature. In order that the phosphate fertilizers could well match the crops’ P requirement, the selection of appropriate types of P sources are of paramount importance in practice. To achieve this, it is necessary to make a thorough investigation on phosphorus distribution among different soil P fractions and their bioavailability when P applied to soil in various varieties of synthetic phosphate fertilizers. 【Method】We conducted a pot experiment on a calcareous tier soil with a lower Olsen P level, which was collected from a plot without P addition for 20 years. Eight treatments were established: (1) no phosphate fertilizer (Control); (2) superphosphate (SSP); (3) calcium magnesium phosphate (CaMg P); (4) mono-ammonium phosphate (MAP); (5) di-ammonium phosphate (DAP); (6) ammonium poly-phosphate (Poly P); (7) Urea phosphate (Urea P); (8) superphosphate plus ammonium sulfate (SSP+ASA). A maize variety ‘Zhengdan 958’ was used as a test crop. We analyzed the correlations between P uptake in above-ground biomass of maize, soil Olsen P and the content of soil P fractions, which were determined with a P fractionation method modified by Jiang & Gu. 【Result】Results showed that the contents of soil available P (Olsen P) and its dynamics varying with treatments. The average Olsen P contents followed an order of DAP>Urea P≥Poly P>MAP>SSP+ASA>SSP>CaMg P>Control based on their statistical significance during the experimental period of two months. Compared with the control treatment, all treatments receiving P significantly increased the above-ground biomass, phosphorus uptake and phosphorus use efficiency of maize by 64.8%-221.3%, 114.1%-593.0% and 2.1%-11.0%, respectively. The largest increase was observed in treatments receiving DAP and poly P. Both the phosphorus uptake and above-ground biomass of maize were positively and significantly correlated with soil Olsen P. Phosphorus fractions of Ca2-P, Ca8-P, Al-P and Fe-P were enhanced by 36.9%-610.0%, 21.7%-85.5%, 57.2%-83.0%, 28.5%-77.8% in all phosphorus application treatments except O-P and Ca10-P, which remained unchanged except in CaMg P treatment. Of which, the greatest increases in Ca2-P and Ca8-P were observed in treatments given MAP, DAP, poly P and Urea P; the largest increases in Al-P and Fe-P were found in SSP+ASA and SSP treatments; significant increases in O-P and Ca10-P were only obtained in CaMg P treatment. The Poly P treatment had the significantly greater Ca2-P than MAP and Urea P treatments, only lower that DAP treatment, but it contained a significantly lower Ca8-P relative to DAP, MAP and Urea P treatments. Compared with SSP, SSP+ASA significantly increased the contents of Ca2-P and Al-P by 24.9% and 11.9%, respectively, and the available phosphorus content increased by 11.4%. The inorganic P fractions of Ca2-P, Ca8-P and Al-P showed a significant and positive correlations with soil available phosphorus and phosphorus uptake by plants. 【Conclusion】Under the tested soil conditions, DAP had a lower phosphorus fixation and thus a greater available phosphorus pool. Poly P inhibited the transformation of Ca2-P to Ca8-P and therefore reduced the precipitation of phosphorus. The results suggested that Poly P is equivalent to DAP in bioavailability, and these two varieties of phosphate fertilizers could be used where there is an urgent need to improve and maintain soil Olsen P. The application of physiological acidic fertilizer ammonium sulfate combined with SSP in calcareous soil not only increased the content of available phosphorus, but also reduced the fixation of phosphorus.

    Effects of Nitrogen Reduction and Film Mulching on Wheat Yield and Nutrient Absorption and Utilization in Loess Plateau
    LIU Kai,XIE YingHe,LI TingLiang,MA HongMei,ZHANG QiRu,JIANG LiWei,CAO Jing,SHAO JingLin
    Scientia Agricultura Sinica. 2021, 54(12):  2595-2607.  doi:10.3864/j.issn.0578-1752.2021.12.010
    Abstract ( 328 )   HTML ( 44 )   PDF (588KB) ( 260 )   Save
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    【Objective】The Loess Plateau is an important wheat growing area in China, however the phenomenon of unreasonable fertilization and low yield is widespread. The aim of this study was to clarify the yield variation of winter wheat and the absorption and utilization of soil nutrients under long-term nitrogen reduction and quantitative monitoring of fertilization and mulching, so as to provide a recommendation for the scientific and rational fertilization of winter wheat and the increase of regional wheat production in the loess tableland. 【Method】 Based on the long-term positioning of the experimental land in the winter wheat growing area in the dry land of south Shanxi Province, three treatments were set up from 2012 to 2019, including farmer pattern (FP) , monitoring fertilization (MF) and monitoring fertilization plus ridge mulching-furrow planting (RFSF), to study the yield and composition of winter wheat, the absorption and utilization efficiency of nitrogen(N), phosphorus (P) and potassium (K) fertilizer, the transfer and absorption of nutrients before and after flowering and the N residue in the soil layer of 0-2 m .【Result】Compared with the FP, the total nitrogen application of MF decreased by 33.5% and balanced addition of phosphate and potassium fertilizer, while there was no significant difference in grain yield, biological yield and yield composition between FP and MP; the nitrogen uptake rate under MF was increased by 8.9%, and the apparent recovery rate of nitrogen fertilizer, phosphate fertilizer and nitrogen partial productivity under MF were increased by 7.6%, 2.7% and 55.0%, respectively; the transplanting amount of vegetative organs before flower under MF was 24.2% higher than that under FM, and the nitrogen uptake after flower was reduced. The total residual nitrate nitrogen in the soil layer of 0-2 m was 34.4% lower than that under the farmer model. Mulch was carried out on the basis of quantitative monitoring and fertilization of nitrogen reduction, grain yield and biological yield under RFSF increased by 24.3% and 25.5% than that under no mulch, respectively. The N, P and K uptake of grains increased by 20.9%, 35.0% and 33.1%, respectively. The apparent recovery and partial productivity of N, P and K fertilizer increased by 3.7%, 2.7%, 4.8% and 22.8%, 22.8%, 22.0%, respectively; nitrogen and phosphorus uptake increased significantly by 25.4% and 57.5% before flowering, respectively; the total residue of nitrate nitrogen in the 0-2 m soil layer was reduced by 25.1%.【Conclusion】On the premise of achieving stable winter wheat yield, the utilization efficiency of fertilizer could be improved and the residual amount of nitrogen in soil could be reduced by reducing the amount of nitrogen fertilizer and balanced application of phosphate fertilizer. Mulching on the basis of quantitative monitoring and fertilization of nitrogen reduction could increase the yield of winter wheat, further improve the utilization efficiency of fertilizer, and had a more significant effect on reducing environmental risks caused by nitrate nitrogen accumulation.

    Water Use Characteristics of Maize-Green Manure Intercropping Under Different Nitrogen Application Levels in the Oasis Irrigation Area
    LI HanTing,CHAI Qiang,WANG QiMing,HU FaLong,YU AiZhong,ZHAO Cai,YIN Wen,FAN ZhiLong,FAN Hong
    Scientia Agricultura Sinica. 2021, 54(12):  2608-2618.  doi:10.3864/j.issn.0578-1752.2021.12.011
    Abstract ( 365 )   HTML ( 40 )   PDF (1468KB) ( 304 )   Save
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    【Objective】 The excessive reliance on chemical fertilizers and lower water productivity have constrained maize production in continuous maize cropping. This study explored the characteristics of water consumption through intercropping maize with green manure and reducing of N fertilizer rate. The aim was to investigate the characteristics of water utilization of maize production due to intercropping green manure, so as to provide a theoretical and practical basis for the optimization of nitrogen reduction and water use efficiency (WUE) in maize production. 【Method】 The field experiment was conducted from 2018 to 2019 in Hexi oasis irrigation region. There were two cropping patterns, i.e. maize-green manure intercropping and sole maize (M). Two N fertilizer rates, i.e. 25% N fertilizer reduction (N1) and traditional N fertilizer application (N2), were managed. The green manure included common vetch (V) and rape (R). Water consumption characteristics and yield performance of various treatments were studied. 【Result】 Intercropping green manure did not reduce grain yield of maize even with lower N application, as no significant difference of grain yield between N1 and N2. Intercropping green manure couple with 25% N fertilizer reduction could increase soil water content at the green manure clipping and maize harvest, and the increase range of M/VN1 was the largest, which was 17.2%-18.9% and 28.6%-31.3% higher than that of sole maize under traditional N fertilizer application (MN2). Intercropping green manure couple with 25% N fertilizer reduction significantly reduced the crop water consumption. Compared with MN2, the water consumption of M/VN1 was decreased by 38.8-48.8 mm, 32.9-53.9 mm and 80.9-92.7 mm, respectively, at before green manure planting to clipping, green manure clipping to maize harvest, and the entire growing period. Intercropping green manure also improved the WUE of maize; and M/V treatment was the most prominent, which was 14.9%-15.6% higher than M treatment. Compared with N2, N1 treatments of M/R and M/V increased WUE by 3.4%-5.2% and 5.7%-6.5%, respectively; while M treatment decreased WUE by 4.0%-5.5%. Intercropping maize with green manure integrated 25% N fertilizer reduction had greater WUE, the WUE of M/VN1 was increased by 14.8%-16.8% compared with MN2. Thus, intercropping maize with common vetch integrated 25% N fertilizer reduction could effectively improve soil water content, reduce water consumption of the system, increase WUE of maize, and have high water productivity. 【Conclusion】 The results showed that intercropping green manure to substitute partial chemical fertilizer N in water limited oasis irrigation region had high water supply potential. Intercropping maize with common vetch integrated 25% N fertilizer reduction was the optimum practice.

    HORTICULTURE
    Effects of Dark and Light Treatments on Wound Healing of Potato Tubers
    WEI YaNan,PENG Hui,YANG Qian,ZHENG XiaoYuan,TANG ZhongQi,ZHU Yan,XIE JianMing,BI Yang
    Scientia Agricultura Sinica. 2021, 54(12):  2619-2629.  doi:10.3864/j.issn.0578-1752.2021.12.012
    Abstract ( 378 )   HTML ( 24 )   PDF (1385KB) ( 218 )   Save
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    【Objective】 Wound healing is a typical reaction in damaged tuber tissue of potato, which is greatly affected by external environmental factors. The effects of dark and light treatments on the wound healing of potato tubers were compared to explore its partial mechanisms. 【Method】In this study, the "Atlantic" potato seeds were selected as the test material. After being cut in half and disinfected, the tubers were healed under light (11 000 lx) and dark at ambient condition ((20±3)℃, RH (95±5)%). The determination was conducted on the basis of weight loss and disease index of tubers after inoculated with Fusarium sulphureum on 7 d during healing to evaluate the efficacy of the wound healing. The accumulation of suberin and lignin, and the chroma at wound sites of the tubers were observed. The activity of the key enzymes of phenylpropanoid metabolism and the activities of peroxidase, polyphenol oxidase and hydrogen peroxide were analyzed in order to explore the partial mechanism of light and dark at the wounded sites of potato tubers. 【Result】During healing, the weight loss of tubers under dark and light was increased gradually. Dark treatment significantly decreased the weight loss of wounded tubers and disease index of inoculated tubers, which were 54.3% and 59.0% lower than that under light after 7 d of healing, respectively. Accumulation of suberin poly phenolic, suberin poly aliphatic and lignin of tubers under dark and light were increased gradually during healing. The dark treatment comparatively improved the accumulation of suberin poly phenolic, suberin poly aliphatic and lignin at the wounded sites of potato tubers. The thickness of suberin poly phenolic, suberin poly aliphatic and lignified cell layers was increased by 38.2 %, 36.8 % and 14.7 % respectively than that under light treatment after 14 d of healing. During healing, L* and b* values of wounded tuber surface under dark and light was decreased first and then increased. The dark treatment reduced the L* and a* and elevated the b* value of the wound surface of tubers. The L* and a* values were lower than that of light by 13.51% and 32.74% on the 5th day of wound healing, respectively. The b* value was 42.11% higher than that of light on day 5 of wound healing. During healing, PAL activity at the wounded sites of tubers in dark treatment increased rapidly, while in light treatment increased slowly, C4H activity at the wounded sites of tubers under both treatments first increased and then stabilized, and 4CL activity in two treatments first increased rapidly and then decreased continuously at the wounded sites. The dark treatment significantly increased the activity of phenylalanine ammonia-lyase, cinnamic acid-4-hydroxylase and 4-coumaryl coenzyme A ligase at the wounded site of tubers, which were 79.6%, 10.7% and 72.5% higher respectively than the ones under light treatment after 7 d of healing. Moreover, the content of H2O2 in tuber wound under dark and light increased rapidly in the first week at the wounded sites of tubers and then flatten out. The peroxidase activity and hydrogen peroxide content at the wounded sites of tubers under dark were 49.8% and 49.9% higher than that under the light treatment after 5 d of healing, respectively. In addition, polyphenol oxidase activity was increased obviously at wound sites of tubers treated with dark on the 3rd of healing, which was 12.93% higher than that of light.【Conclusion】Dark improved the wound healing of potato tubers, while the light showed a certain extent inhibition. The improvement mechanism of the dark on wound healing involved in eliciting phenylpropanoid metabolism and the acceleration of polyphenol oxidase and peroxidase activities to promote the accumulation of suberin and lignin at wounded sites of tubers.

    Influence of Plastic Film Covering of Tree Canopy on Fruit Postharvest Storage Performance in Shatangju Tangerine
    ZHU ZhiFeng,LIU Ping,XU RangWei,CHEN ChuanWu,DENG ChongLing,NIU Ying,ZHU Yi,WANG PengWei,DENG XiuXin,CHENG YunJiang
    Scientia Agricultura Sinica. 2021, 54(12):  2630-2643.  doi:10.3864/j.issn.0578-1752.2021.12.013
    Abstract ( 307 )   HTML ( 60 )   PDF (2764KB) ( 303 )   Save
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    【Objective】This study aimed to provide a theoretical basis for the postharvest storage and production of Shatangju tangerine (Citrus reticulate) under the plastic film covering of the tree canopy.【Method】 Shatangju tangerine was used as the research subject under two treatments, including canopy covering with plastic film and no canopy covering (control). The samples were collected in seven periods. Then, the color index, total soluble solids (TSS), titratable acid (TA), primary metabolites, respiration rate, hormone levels, fruit surface wax components, expression levels of wax synthesis-related genes, weight loss rate and decay rate were measured.【Result】Compared with the control, canopy covering with plastic film resulted in a slower decline rate of glossiness on fruit surface, redder peel color, 1.37%-3.37% increases in total soluble solid contents, 1.87-8.12 increases in solid-acid ratio, 1.93%-16.45% decreases in respiration rate, 13.48-15.04 ng·g-1 increases in endogenous ABA content, 1.14-3.47 μg·cm-2 increases in wax, 22.24%-29.27% reduction of weight loss, and 1.06%-17.65% reduction of decay rate; besides, the expression levels of most genes related to wax synthesis, such as CER1 and CER26, were higher than those of the control. With the delay of the harvest time under plastic film covering of the canopy, the glossiness and solid-acid ratio of the fruit showed continuous decreasing from the end of December to February 28, followed by a slight increase in March; in addition, the peel color gradually changed from bright red to yellow and then turned to red in March. The soluble solid content and weight loss rate showed a continuous rising trend. The postharvest decay rate increased gradually, and the samples on February 3, February 28 and March 16 showed decay rates of 19.26%, 32.06% and 30%, respectively, after 25 days of storage. Furthermore, the flowering time of Shatangju tangerine in the Guilin production area was the late March. Hence, too late harvest might affect the flowering and fruit setting of the tree in next season.【Conclusion】The results showed that plastic film covering of canopy could significantly enhance the ABA content and surface total wax of Shatangju, reduce postharvest decay and loss, and improve freshness preservation, but too late harvest would affect the freshness preservation performance of the fruit. It was suggested that the reasonable harvest period of Shatangju tangerine with plastic film covering of canopy in Guilin Guangxi should be before late February.

    Analysis of Key Genes About Flower Color Variation in Iris hollandica
    LIN Bing,CHEN YiQuan,ZHONG HuaiQin,YE XiuXian,FAN RongHui
    Scientia Agricultura Sinica. 2021, 54(12):  2644-2652.  doi:10.3864/j.issn.0578-1752.2021.12.014
    Abstract ( 300 )   HTML ( 29 )   PDF (1211KB) ( 304 )   Save
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    【Objective】 Flower color variation is of great significance for enriching color of ornamental plants, but it is difficult to clarify variation mechanism due to uncertainty of flower color variation. Dutch iris (Iris hollandica) is an important bulbous ornamental plant. In this study, the blue-purple wild type ‘Zhanchi’ and white mutant strain 'Yufei' of Dutch iris were investigated to explore molecular mechanism and difference of pigment accumulation, so as to provide a basis for mechanism of flower color variation.【Method】In this study, using inner tepals of ‘Zhanchi’ and ‘Yufei’ from Dutch Iris as materials, UHPLC-QTOF-MS method was used to determine types and contents of anthocyanins and flavonols from two varieties of Dutch Iris, and the different expression genes related to anthocyanin synthesis were screened by transcriptome sequencing. Using different flowers in developmental stages of two varieties as materials, the different expression genes were verified by qRT-PCR.【Result】Results of metabolomics analysis revealed that delphinidin, cyanidin and its derivatives were accumulated in blue-purple flowers, which were almost no accumulation in white flowers, while the flavonol contents were increased in white ‘Yufei’. Results of RNA-seq analysis revealed that a total of 46 485 unigenes were obtained, and 27 073 unigenes of them were functionally annotated by public databases, accounting for 41.85% of the total. And 701 differentially expressed genes were obtained, 485 genes of which were up-regulated and 216 genes were down-regulated in white ‘Yufei’. Two dihydroflavonol-4-reductase genes and one flavonol synthase gene involving in anthocyanin biosynthetic pathway had different expression, named IhDFR1, IhDFR2 and IhFLS1. Down-regulated expression of IhDFR1 and IhDFR2 as well as up-regulated expression of IhFLS1 in white ‘Yufei’ led to significant decrease of anthocyanins and accumulation of flavonols, which caused metabolic flow from anthocyanin to flavonol. The qRT-PCR results of three genes showed that expression levels of IhDFR1 and IhDFR2 increased in blue-purple flowers during flower developmental stage, but low expression in white flowers, and expression level of IhFLS1 increased in white flowers during flower developmental stage, but low expression in blue-purple flowers, which was consistent with RNA-seq results.【Conclusion】Low expression of IhDFR1 and IhDFR2 as well as high expression of IhFLS1 in white ‘Yufei’ blocked accumulation of anthocyanins, and some of metabolic flow changed from anthocyanins to flavonols, resulting in the change of flower color from blue violet to white.

    FOOD SCIENCE AND ENGINEERING
    Extraction Technology, Preliminary Structure and α-glucosidase Inhibition of Polysaccharide with Alkaline-Extracted from Sugarcane Peel
    WANG XuanXuan,LIU ChunYu,XIE BeiYu,ZHANG ShuShu,WANG DanYang,ZHU ZhenYuan
    Scientia Agricultura Sinica. 2021, 54(12):  2653-2665.  doi:10.3864/j.issn.0578-1752.2021.12.015
    Abstract ( 271 )   HTML ( 31 )   PDF (3133KB) ( 323 )   Save
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    【Objective】 The objectives of this research were to establish the optimized technology, further explore the structural characteristics and evaluate α-glucosidase inhibitory effect of polysaccharide with alkaline-extracted from sugarcane peel.【Method】Single factor experiment and response surface methodology (RSM) were used to optimize the extraction process of alkali-extracted sugarcane peel polysaccharide (SPAP). The method of phenol sulfuric acid was used to determine the content of polysaccharide. After deproteinization by Sevag method and depigmentation by AB-8, the structure of SPAP was characterized. The molecular weight distribution, monosaccharide compositions and characteristic functional groups of SPAP were detected by HPLC, GC-MS and FT-IR, respectively. Finally, the method of 4-nitrophenol-2-β-D-glucopyranoside (PNPG) was used to determine the α-glucosidase inhibitory effect of polysaccharide extract. 【Result】 The optimum extraction conditions were as follows: extraction temperature 37℃, sodium hydroxide concentration 5%, ratio of material to solvent 1﹕46 (g·mL-1), and extraction times 4 times. Under these conditions, the yield of SPAP reached 10.84%. After removing protein and pigment, the content of SPAP reached 86.54%. Furthermore, SPAP was mainly composed of arabinose, xylose, glucose and galactose, with molecular weight of 3.03×103 kD. It might be a pyran hetero-polysaccharide with α or β configuration. In addition, SPAP showed good inhibitory effect on α-glucosidase. The inhibition rate of SPAP reached 78.31%.【Conclusion】 The process optimization of alkali-extracted of polysaccharides from sugarcane peel could effectively utilize raw materials and improve the yield and efficiency. The structural characteristics of SPAP were preliminarily described by HPLC, FT-IR and GC-MS. At the same time, SPAP showed good inhibitory effect on α-glucosidase and potential hypoglycemic activity. These results provided a theoretical basis for further study on the structure-activity relationship of SPAP.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    Advances of Biosynthesis and Toxicity of Cereulide Produced by Emetic Bacillus cereus
    CUI YiFang,ZHENG Min,DING ShuangYang,ZHU Kui
    Scientia Agricultura Sinica. 2021, 54(12):  2666-2674.  doi:10.3864/j.issn.0578-1752.2021.12.016
    Abstract ( 599 )   HTML ( 34 )   PDF (2807KB) ( 405 )   Save
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    Bacillus cereus (B. cereus) is a gram-positive facultative anaerobe that can produce spores to survive adverse environments. And it is widely present in soil, water, air and a variety of foods. Pathogenic B. cereus is one of the most common food-borne pathogens, and the toxins produced by B. cereus are the main cause of food poisoning. Cereulide is a major toxin produced by pathogenic B. cereus, which is a small molecule lipophilic cyclic dodecadepsipeptide with stable structural properties. Cereulide can cause mild food poisoning with emetic symptoms, such as nausea and vomiting, and it may induce severe fatal diseases such as hepatic encephalopathy or acute liver failure. Current researches believed that cereulide caused vomiting by stimulating the vagus nerve, and induced the loss of mitochondrial membrane potential by acting as a potassium ionophore, which ultimately led to cell death. However, the toxic mechanism of hepatic encephalopathy or acute liver failure caused by cereulide remains unclear. Cereulide is encoded by the cereulide synthetase gene cluster (ces) and is synthesized by the non-ribosomal peptide synthetase (NRPS) system. Cereulide is composed by two hydroxy acids and two amino acid residues [-D-HIC-D-Ala-L-HIV-L-Val-], which forms a trimer phenolphthalein after three iterations and shows a structural specificity and representativeness. However, isocereulides may be produced due to the flexibility of the NRPS system. Therefore, the toxicity of cereulide is closely related to its biosynthesis process. Based on previous studies, this review summarized and proposed the biosynthesis mechanism of cereulide. Firstly, the CesA and CesB domained in ces recognize D-α-ketocarboxylic acid, L-alanine, L-α-ketoisovalerate and L-valine, respectively, which formed the main synthetic unit dipeptide of cereulide by covalent bonding. Secondly, a tetrapeptide was synthesized by repeating the above process. Thirdly, the second tetrapeptide was synthesized through repeated reactions, and the two tetrapeptides formed an octapeptide through esterification. Fourthly, the above reaction was repeated to form a ternary complex product peptide. Lastly, because the surface structure of the active center of the thioesterase domain in ces-NRPS prevented external water molecules from entering, it induced an internal nucleophilic attack reaction and finally released a circular cereulide. The risk of food poisoning caused by cereulide producing B. cereus was underestimated. In addition, our previous studies have found that some probiotic Bacillus products were contaminated with cereulide-producing B. cereus strains. This posed a potential risk to food safety and public health. This review briefly summarized the characteristics and toxic mechanisms of cereulide, which would provide a scientific basis for the prevention of cereulide. This review also summarized and proposed the biosynthesis process of cereulide. Functions of two domains in the synthesis process need to be focused. The main function of ketoreductase (KR) domain was that it could catalyze the formation of esters of keto acid at the beginning of the biosynthesis processes. The important role of thioesterase (TE) domain was to form repeating units and the cyclic peptide in the last link of synthesis. These could serve as a model for other cyclic peptides synthesized by the non-ribosomal peptide synthetase system.

    Protective Effects of Chinese Propolis Extract Against Lipopolysaccharide- Induced Acute Mastitis and Mammary Barrier Functions in Mice
    SONG MeiJie,OU AiQun,XUE XiaoFeng,WU LiMing,SHOU QiYang,WANG Kai
    Scientia Agricultura Sinica. 2021, 54(12):  2675-2688.  doi:10.3864/j.issn.0578-1752.2021.12.017
    Abstract ( 331 )   HTML ( 36 )   PDF (2428KB) ( 354 )   Save
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    【Background】 Dairy cow mastitis is a common and frequent dairy cow disease, which not only threatens the health of dairy cows, but also causes significant economic losses. The traditional medicine for the treatment of dairy cow mastitis is largely dependent on the usage of antibiotics, which easily caused antibiotic resistance/abuse. Therefore, it is with practical significance to develop an alternative approach of antibiotics for the prevention and treatment of mastitis in dairy cows. Propolis is a natural product with good anti-inflammatory and antibacterial activities. Propolis is collected by western honeybees from plant resin and mixing with bees’ maxillary gland and wax gland secretions. Accordingly, propolis has great potential for the prevention and treatment of mastitis in dairy cows. Despite that there have some previous reports on the prevention and treatment of mastitis using propolis, scant information is available on the effects of propolis on the breast blood barrier function. 【Objective】The aim of this study was to evaluate the protective effect of ethanol extract of Chinese propolis, EECP, against bacterial lipopolysaccharide- induced mouse acute mastitis, and we focused on its effect on the expression of tight junction proteins in mammary epithelial cells. This outcome of this study would lay a foundation for the in-depth study for the usage of propolis for prevention and treatment on dairy cow mastitis. 【Method】 The type and content of the main polyphenolic compounds in EECP were determined by ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). Female ICR mice were divided into four groups, including the normal control group, model group (injecting LPS into the mammary gland, 1mg·kg-1 b.w.), EECP group and positive control group (dexamethasone). The female mice were subjected to continuous gavage by EECP or positive control drugs for seven days. The LPS model group, EECP group and positive control group were injected with 1 mg·kg-1 LPS through the fourth and the fifth pair of nipple tubes, to induce acute mastitis in mice. After 24 h, they were killed for collecting the mammary tissue samples. Hematoxylin-Eosin (HE) staining method and Sirius red staining method were used to evaluate pathological changes in mouse mammary tissue. Enzyme-linked immunosorbent assay (ELISA) was used to determine the release of inflammatory factors and quantitative real-time PCR was applied to determine mouse mammary tissue gene expressions of tight junction proteins (Occludin, Cluadin-1, ZO-1). Finally, the immunohistochemical technology was used to study the expression and distribution of mouse mammary tight junction proteins (Occludin and ZO-1). These above indicators were applied to comprehensively evaluate the anti-mastitis activity and its effect on mammary tight junction proteins by EECP. 【Result】 Based on UHPLC-QqQ-MS/MS, an accurate quantitative method was established for the 17 main polyphenolic compounds in EECP. The results showed that the five compounds with the higher content and their contents were: galangin (12.88 ±0.57 μg·mg-1), 3-O-Acetylpinobanksin (12.93±0.59 μg·mg-1), pinocembrin (8.56±0.27 μg·mg-1) and pinobanksin (8.52±0.25 μg·mg-1). Animal study results showed that after a week of regular oral administration of EECP reduced on the infiltrations of inflammatory cells in mammary gland under LPS stimulation, and the structural damages were alleviated by EECP. Moreover, Chinese propolis administration inhibited the overexpression of inflammatory factors (IL-1β, IL-6, IL-10) in mouse mammary gland tissue, and increased the transcription level of tight junction protein genes (Occludin, Claudin-1, ZO-1) mRNA, and enhanced the expression of tight junction protein Occludin, ZO-1. 【Conclusion】EECP showed good preventive effect against lipopolysaccharide-induced mastitis in mice, with potent anti-inflammatory effects. It also increased the expressions of tight junction proteins in the mammary gland, which maintained the integrity of the tight junction structure in the mammary gland and protected the blood-milk barrier. Nevertheless, the molecular mechanism of Chinese propolis on the regulation of tight junctions needed to be clarified in the future research.

    The Effect of Flumethrin on Metabolism of Worker Larvae of Apis mellifera with LC-MS Technique
    YU LongTao,YANG HeYan,SU YuChen,YAN WeiYu,WU XiaoBo
    Scientia Agricultura Sinica. 2021, 54(12):  2689-2698.  doi:10.3864/j.issn.0578-1752.2021.12.018
    Abstract ( 375 )   HTML ( 30 )   PDF (1581KB) ( 375 )   Save
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    【Objective】Flumethrin belongs to the second generation pyrethroid insecticides and acaricides, which is used for the control of honeybee mites. Because of the toxicity of acaricides, it can not only kill mites, but also threat the health of honeybees. The different metabolites of Apis mellifera worker larvae treated with different concentrations of flumethrin were tested using liquid chromatography-mass spectrometry (LC-MS) technology and the metabolic pathways involved of different metabolites were analyzed, so as to explore the toxicological effect of flumethrin on honeybees and provide references for scientific using in beekeeping.【Method】The queen was controlled to lay eggs on an empty worker frame for 12 h, and the spawning area was divided into four groups. From the 5th day, the small larvae of each group were fed with sugar water containing different concentrations of flumethrin (0, 0.5, 5, 50 mg·kg-1), the dose was increased daily from day 5 to day 8 (1.5, 2, 2.5, 3 μL), and lymph fluid from larvae was collected on day 9. The metabolites of A. mellifera larvae were analyzed by LC-MS and the metabolites with significant difference were screened by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), while the metabolic pathways of common differential metabolites in flumethrin treatment groups were analyzed.【Result】Compared with the control group, a total of 190 different metabolites were found and 87 types were identified in 0.5 mg·kg-1 group, and a total of 275 different metabolites were identified and 97 types were identified in 5 mg·kg-1 group, while there were a total of 275 different metabolites and a total of 131 species were identified in 50 mg·kg-1 group. Meanwhile, 29 common differential metabolites in treatment groups were screened, of which 16 metabolites were up-regulated, 12 metabolites were down-regulated, and 1 metabolite was down-regulated in 0.5 and 50 mg·kg-1 groups while it was up-regulated in 5 mg·kg-1 group. These differential metabolites include ribose, purine and its derivatives, fatty acids with their conjugates. After enrichment analysis of metabolic pathways, significant differences (P<0.05) were found in the metabolic pathways, which include amino sugar and nucleotide sugar metabolism, drug metabolism-other enzymes, α-linolenic acid metabolism and other pathways.【Conclusion】The LC-MS technology can effectively analyze the changes of metabolites in the honeybee larvae treated with flumethrin, and flumethrin can cause the contents of UDP-N-acetylglucosamine, azathioprine, traumatic acid, 9-oxononanoic acid and 13(s)-HPODE abnormal in honeybee larvae. The changes of these different metabolites confirm that flumethrin causes various substance metabolism disorders in honeybees. The analysis of these metabolic processes can further explain the mechanism of honeybees metabolizing toxic compounds, and provide a theoretical basis for the stress of acaricides and other toxic compounds on honeybees.