Table of Content

01 May 2019, Volume 52 Issue 9

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Genetic Diversity and Population Structure of Broomcorn Millet in China Based on Fluorescently Labeled SSR

KOU ShuJun, HUO AHong, FU GuoQing, JI JunJian, WANG Yao, ZUO ZhenXing, LIU MinXuan, LU Ping

Scientia Agricultura Sinica. 2019, 52(9):  1475-1477.  doi:10.3864/j.issn.0578-1752.2019.09.001

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【Objective】Genetic diversity and population genetic structure of 131 broomcorn millet accessions in China were analyzed based on fluorescently labeled SSR markers for variety improvement and germplasm innovation as well as effective utilization of broomcorn millet germplasm.【Method】Six broomcorn millet accessions with phenotypical diversity from different geographical origins were used to select SSR markers with highly polymorphism by denaturing polyacrylamide gel electrophoresis. The polymorphic markers were labeled with four fluorescent dyes: 6-FAM, HEX, ROX and TAMRA at the 5' end of forward primers which used in subsequent analyses. The size of each allele locus was determined by DNA Analyzer to estimate the genetic diversity and genetic population structure of the tested accessions. 【Result】 Twenty-two polymorphic and stably amplifying markers were selected for both traditional denaturing PAGE electrophoresis detection and fluorescent SSR labeling-automatic analysis technology. A total of 128 alleles were detected with an average of 5.82 alleles per locus, the gene diversity index, polymorphism information content and Shannon's information index ranged from 0.3572 to 0.8132, 0.2934 to 0.8150 and 0.5427 to 1.7681, respectively, with an average of 0.6284, 0.5874 and 1.2062 respectively. Genetic distance and genetic identity of accessions from different ecotypes were 0.0764-0.7251 (average value is 0.3121) and 0.4843-0.9265 (average value is 0.7465), respectively, the smallest genetic distance was found between NSP ecotype and LPSS ecotype. UPGMA cluster analysis showed that NSP ecotype and LPSS ecotype were clustered into one group. The clustering result of the cultivars and landraces from NES ecotype was consistent, the cultivars from LPSS ecotype were divided into different groups. By plotting the relationship between K and ΔK, ΔK is the largest when K=4. Based on K and △K values, 131 broomcorn millet accessions were grouped four genotypes by the population genetic structure analysis. Group Ⅰ is composed of NSP ecotype. Group Ⅱ consist of NES ecotype. Most accessions of group Ⅲ are from NSP ecotype. Group Ⅳ mainly contain LPSS ecotype. Most of the accessions in same group have same genetic component while only a few accessions contained genetic component of other groups. The analysis result of population genetic structure was consistent with the UPGMA clustering, indicating that the genetic diversity is related to geographical region. 【Conclusion】 There have more abundant genetic diversity in NSP and LPSS ecotype than other regions. The cultivars of NES ecotype were bred mainly from the landraces. The cultivars of LPSS ecotype were introduced from a wide range of resources during the breeding process, harboring genetic exchanges with other ecological regions.

Correlation Between Genetic Distance of Parents and Heterosis in Upland Cotton

QU YuJie, SUN JunLing, GENG XiaoLi, WANG Xiao, Zareen Sarfraz, JIA YinHua, PAN ZhaoE, HE ShouPu, GONG WenFang, WANG LiRu, PANG BaoYin, DU XiongMing

Scientia Agricultura Sinica. 2019, 52(9):  1488-1501.  doi:10.3864/j.issn.0578-1752.2019.09.002

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【Objective】The correlation between heterosis and genetic distance (GD) of quantitative traits between parents was analyzed by 1500 hybrid combinations in upland cotton, and the possibility of using GD between parents of large-scale combinations to improve the efficiency of hybrid vigour prediction of upland cotton was discussed in order to provide theoretical guidance for cotton hybrid breeding and utilization of heterosis.【Method】305 upland cotton core collections from 15 countries and 23 provinces (municipalities) of China were selected as parents, and 1500 cross combinations were produced by L×T (Line×Tester) cross design. From 2012 to 2013, ten yield and fiber quality related traits, including plant height (PH), boll weight (BW), boll number per plant (BN), lint percentage (LP), fiber length (FL), fiber strength (FS), fiber elongation (FE), fiber length uniformity (FU), micronaire (MIC) and spinning consistent index (SCI), were investigated in 13 ecological conditions in north and south China. F₁ hybrids mid-parent heterosis (MPH), heterobeltiosis (HB), GD between parents and population structure were analyzed. The correlation between GD and hybrid vigour was calculated by four schemes (Cor1-Cor4). 【Result】The mean values of MPH of the ten traits ranged from 1.70% to 7.40%, with an average of 4.36%, and F₁ hybrids were divided into 5 groups (A-E) according to different male parents, the mean values of MPH: A>E>B>C>D. The mean values of HB ranged from -4.17% to 1.87%, with an average of -0.17%, and the average values of group A, B, and E were positive. In 5 groups, except for MIC of group D and E, other 9 traits had obvious MPH, among them, MPH of BW and FL were mainly positive (more than 80%) in the 5 groups, the maximum MPH values were 34.01% and 9.83% respectively, and the corresponding HB values were 24.25% and 5.80% respectively. The significant difference analysis between F₁ hybrids and their parents indicated that BW, PH, FL, FE, and FU showed some HB. The GDs between male parents (testers) and 300 female parents ranged from 2.280 to 61.430, with an average of 21.550. The mean GDs between 5 testers and female parents: D>C>E>A>B, in which the nearest value was 11.721, and the farthest value was 33.271. According to “Ward” clustering method, 305 upland cotton parents were divided into two groups, including five subgroups. The results of four correlation analysis methods between GD and heterosis showed that the consequences varied with the sample size, the range of GD, and the male parent, the correlation increased with the sample size. Cor1 was the overall embodiment of Cor2 results; compared with Cor1 and Cor2, Cor3 had different correlations between MPH and GD in some traits; Cor4 had the weakest correlations. To sum up, the genetic distance was positively correlated with the MPH of LP, FS, FU, and SCI, the correlation between GD and MPH of other traits was different due to the different analysis schemes. In the four schemes, except for FU, the relationship between GD and HB was negatively correlated on the whole, and there was a strong correlation between genetic distance and HB of MIC, FL and LP. 【Conclusion】There is a linear relationship between GD of quantitative traits and hybrid vigour in upland cotton. The correlations are positive or negative, strong or weak due to different traits, and the larger the sample size, the stronger the correlation. Thus, the large-scale hybrid combinations are used to well study the relationship between GD and heterosis in upland cotton.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effects of Different Sowing and Harvest Dates of Winter Wheat- Summer Maize Under Double Cropping System on the Annual Climate Resource Distribution and Utilization

ZHOU BaoYuan, MA Wei, SUN XueFang, GAO ZhuoHan, DING ZaiSong, LI CongFeng, ZHAO Ming

Scientia Agricultura Sinica. 2019, 52(9):  1501-1517.  doi:10.3864/j.issn.0578-1752.2019.09.003

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【Objective】 The study was carried out to optimize the inter-season climatic resource distribution of traditional winter wheat-summer maize cropping system and explore the optimal two-season climatic resource distribution model, so as to further increase the annual yield potential and resource utilization efficiency of Huang-Huai-Hai region. 【Method】 In this study, five sowing dates of winter wheat and corresponding harvest dates of summer maize were set from early October to early December, and the field experiment was conducted at Xinxiang county from 2015 to 2017. Based on the field experiments, The annual yield, climate resources distribution and resources use efficiency were studied. 【Result】 With the sowing/harvest dates delayed, days of wheat growth period and amount of radiation, temperature, and precipitation resources gradually reduced, more growth time and resources were transferred to maize season, and the resources distribution rate between two seasons changed from treatmentⅠ (46%:54%, 60%:40%, 42%:58%) to treatment V (34%:66%, 49%:51%, 34%:66%), which resulted in decrease of wheat grain yield. However, due to greater number of ears and grains of ZM66, no significant difference was found in wheat yield between treatmentⅠ and treatment V. Maize grain weight increased by 13.1% and 15.5% due to 15 d, 143.8 and 120.7 MJ·m ^-2, 290.5 and 281.6℃, 12.4 and 25.7 mm increasing in 2016 and 2017, respectively, in maize growth duration, radiation, accumulated temperature, and precipitation, eventually the annual grain yield of treatment V increased by 7.9% and 6.7% compared than that of treatmentⅠ, respectively. In addition, the grain water content decreased to 14.4%-17.3% due to 15 d, 322.5 and 336.3 MJ·m ^-2, 509.6 and 497.8℃, 56.7 and 14.1 mm increasing in maize growth duration, radiation, accumulated temperature, and precipitation in 2016 and 2017, respectively. At the same time, because of radiation and temperature resources in wheat season of treatment V decreased significantly, especially the irrigation water reduced 150 mm, the radiation, temperature and water production efficiency of wheat for treatment V increased by 12.5% and 15.8%, 10.9% and 7.7%, 39.6% and 59.3% in 2016 and 2017, respectively, compared than treatmentⅠ. During maize growth season, radiation and temperature production efficiency under treatment V decreased, but water production efficiency increased significantly than that under treatmentⅠ, so the annual radiation, temperature and water production efficiency of treatment V increased by 7.3% and 9.1%, 5.6% and 5.1%, 17.3% and 29.3% in 2016 and 2017, respectively, compared than treatmentⅠ, respectively. 【Conclusion】 It is of great significance for promoting the sustainable development of winter wheat-summer maize double cropping system in the Huang-Huai-Hai plain by changing sowing and harvest dates (Wheat was sown in early December and maize was harvested in mid-November) to optimize the distribution of resources between two seasons for winter wheat-summer maize double cropping system without any input.

Spectral Diagnosis of Leaf Area Density of Maize at Heading Stage Under Lodging Stress

ZHOU LongFei, GU XiaoHe, CHENG Shu, YANG GuiJun, SUN Qian, SHU MeiYan

Scientia Agricultura Sinica. 2019, 52(9):  1518-1528.  doi:10.3864/j.issn.0578-1752.2019.09.004

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【Objective】 Leaf area density (LAD) reflects the difference of the total leaf area per volume in vertical direction and the distribution of the leaf area in the canopy with the change of height. The purpose of this study was to explore the characterization ability of maize leaf area density and its spectral response to lodging stress intensity. 【Method】 Taking lodging summer maize at heading stage as the research object, the multi-stage of LAD and canopy spectral data after lodging were obtained. The first-order differential and wavelet transform of the canopy spectrum of lodging maize were processed. Based on the correlation analysis between LAD, the first-order differential and wavelet decomposition coefficients of canopy spectrum, the sensitive bands of LAD and the optimal wavelet decomposition scale were screened. Partial least squares (PLS) method was used to construct the LAD spectral diagnosis model of lodging maize, and the accuracy of the model was verified by the measured samples.【Result】The LAD of maize increased with the increase of lodging stress, and LAD could effectively characterize the intensity of lodging stress and recovery ability of maize. After lodging, the canopy structure of maize changed greatly. The spectral reflectance of lodging maize canopy was higher than that of normal maize. The increase of near infrared band was higher than that of visible band. The stronger lodging intensity was, the higher spectral reflectance was. The sensitive bands of LAD were mainly distributed in the blue band 354-442 nm and 472-495 nm, the red band 649-829 nm, and the near infrared band 903-1 195 nm and 1 564-1 581 nm. Comparing with the first-order differential, the validation R ² of LAD diagnostic model of maize lodging based on continuous wavelet transform increased by 6.08%-9.11%, and RMSE decreased by 23.08%-31.63%. The scale of wavelet decomposition had a certain influence on the diagnostic accuracy of LAD. The accuracy of the low- and medium-scale model was better than that of the high-scale model, and the model constructed by the fifth scale had the best fitting effect on LAD (R ²=0.898, RMSE=1.016). 【Conclusion】 The application of continuous wavelet transform to analyze the maize canopy hyperspectral could effectively diagnose maize leaf area density under lodging stress. It could provide necessary prior knowledge for remote sensing monitoring of maize lodging stress disaster.

PLANT PROTECTION

Establishment and Application of Real-Time PCR for Quantitatively Detecting Plasmopara viticola in Vitis vinifera

LI WenXue, XIAO RuiGang, LÜ MiaoMiao, DING Ning, SHI HuaRong, GU PeiWen

Scientia Agricultura Sinica. 2019, 52(9):  1529-1540.  doi:10.3864/j.issn.0578-1752.2019.09.005

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【Objective】Grape downy mildew caused by Plasmopara viticola is one of the most important monoetic diseases on Vitis vinifera. The objective of this study is to establish a real-time PCR detection system of P. viticola based on the pathogen sequence information, and to provide a scientific basis for early diagnosis and prediction of grape downy mildew.【Method】According to the cox2 gene sequence of P. viticola in GenBank, a pair of specific primers, F-cox-Pv/R-Pv, was designed to establish and optimize the conventional PCR and real-time PCR reaction system. The mycelium DNA of 14 species of grape and other crop pathogens and antagonistic fungus (including P. viticola, Colletotrichum gloeosporioides, Uncinula necator, Botrytis cinerea, Coniella diplodiella, Botryosphaeria dothidea, Alternaria brassicae, C. capsica, Fusarium solani, Sphaerotheca fuliginea, Sclerotinia sclerotiorum, F. equiseti, F. oxysporum, Trichoderma harzianum) were used to detect the specificity of conventional PCR and real-time PCR. The sensitivity and repeatability of the system were also evaluated. The pathogen DNA in the grape leaves during the latent infection period of artificial inoculation with P. viticola was quantitatively detected by the established real-time PCR system. The relationship between inoculation time and latent infection amount of P. viticola in the grape leaves was analyzed by SPSS 19.0 software. 【Result】 The primers designed in this study had high specificity. A 139 bp fragment of genome DNA was only amplified from P. viticola by conventional PCR. The detection results of real-time PCR assays showed that the primers had only one absorption peak for P. viticola, and no product absorption peak was detected for other test strains. The sensitivity of conventional PCR was 10 pg·μL ^-1 genomic DNA, while the sensitivity of real-time PCR was 0.1 pg·μL ^-1, which was 100 times higher than that of the conventional PCR. The linear relationship between real-time PCR cycle threshold (Ct) and template concentration was constructed by using recombinant plasmid containing cox2 gene fragment as standard material. The standard curve was y=42.27-3.36x, with the correlation coefficient of 0.997 and amplification efficiency of 98.50%. The linear range was up to 7 orders of magnitude, showing a good linear relationship at 2.4×10 ³-2.4×10 ⁹ copies/μL. The real-time PCR system was used to detect the pathogen DNA in the grape leaves during the latent infection period of artificial inoculation with P. viticola. The results showed that the latent infection of P. viticola in the grape leaves increased exponentially with the change of inoculation time, the curve equation was y=6.34×10 ⁴·e ^{0.084 x}, with the correlation coefficient of 0.936. The real-time PCR system could detect the P. viticola DNA 6 h after inoculation, and the DNA content was 5.68×10 ⁴copies/μL.【Conclusion】The sensitivity of the established real-time PCR system for detection of grape downy mildew is much higher than that of conventional PCR, and the specificity and reproducibility of this real-time PCR system are good. There was a good linear relationship between Ct value and template concentration, and the amplification efficiency is high. This method can be used to quantitatively detect the latent infection of P. viticola.

Ecological Safety Evaluation of Different Bt Proteins on the Predator Chrysopa pallens

ZHAO Man, TANG JinRong, NIU LinLin, CHEN Lin, LIANG GeMei

Scientia Agricultura Sinica. 2019, 52(9):  1541-1552.  doi:10.3864/j.issn.0578-1752.2019.09.006

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【Objective】The objective of this study is to evaluate the potential effects of Cry1Ac, Cry1F and Cry2Ab proteins, which have been transformed into Bt cotton, on Chrysopa pallens, the main predatory enemy in Bt cotton fields of China. 【Method】The effects of Cry1Ac, Cry1F and Cry2Ab proteins on the important life table parameters (weight of 4th instar larvae, developmental period of larvae, pupation rate, pupa weight, developmental period of pupae, eclosion rate, adult weight and total fecundity) of C. pallens were evaluated by the method of adding the high-dose Bt proteins in the artificial diet of C. pallens, and the pure artificial diet of C. pallens treated without insecticidal compound was used as negative control while the artificial diet of C. pallens treated with PA (KH₂AsO₄) was used as positive control. Meanwhile, the concentrations of Bt protein in larvae, pupae and adults of C. pallens after feeding with Bt protein-treated diet were measured by double-antibody sandwich enzyme-link immunosorbent assay (DAS-ELISA) method. The stability and bioactivity of Bt protein in the fresh prepared diet or diet that had been exposed to C. pallens for 2 days were measured by ELISA and sensitive-insect bioassay, respectively. In addition, the main digestive enzymes (total protease, tryptase, chymotrypsin, and aminopeptidase), detoxifying enzymes (α-naphthyl acetate esterase, carboxylesterase, glutathione-S-transferase, acetylcholinesterase, acid phosphatase, and alkaline phosphatase) and antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) of C. pallens after feeding on control artificial diet, Bt protein-treated diet or PA-treated diet were compared by using enzyme activity measurement method. 【Result】 The results of life table evaluation showed that there was no significant adverse effect on the weight of 4th instar larvae, developmental period of larvae, pupation rate, pupa weight, developmental period of pupae, eclosion rate, adult weight and total fecundity of C. pallens by adding Bt protein or Bt protein mixture to the artificial diet, but the addition of PA in the artificial diet extremely significant prolonged the developmental period of larvae and pupae, and extremely significant decreased the weight of 4th instar larvae, pupation rate, pupa weight, eclosion rate, adult weight and total fecundity of C. pallens. The measurement results of Bt protein concentration in C. pallens showed that there was a certain amount of Bt protein in the larvae, pupae and adults of C. pallens after feeding on Bt protein-treated diet. The ELISA and sensitive-insect bioassay measurement results indicated that both the fresh prepared diet and diet that had been exposed to C. pallens for 2 days contained high concentration of Bt protein with biological activity. Thus, C. pallens was exposed to high concentration of Bt protein with biological activity in the whole process of bioassay. Furthermore, ingestion of Bt protein-treated diet had no significant effect on the activities of digestive enzymes, detoxifying enzymes and antioxidant enzymes in C. pallens larvae and adults, but ingestion of PA-treated diet remarkably decreased the activity of aminopeptidase in C. pallens larvae and adults and increased the activities of other testing enzymes of C. pallens. 【Conclusion】 C. pallens is insensitive to the three tested Bt proteins (Cry1Ac, Cry1F and Cry2Ab). Ingestion of the tested Bt proteins has no obviously detrimental effect on the development and activities of main digestive enzymes, detoxifying enzymes and antioxidant enzymes of C. pallens. The biological evaluation system established in this study can also be used to evaluate the ecological safety of other insecticidal proteins from new insect-resistant genetically modified crops to C. pallens.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Effect of Straw Addition on the Formation of Aggregates and Accumulation of Organic Carbon in Dryland Soil

WANG BiSheng,YU WeiShui,WU XuePing,GAO LiLi,LI Jing,LI ShengPing,SONG XiaoJun,LIU CaiCai,LI Qian,LIANG GuoPeng,CAI DianXiong,ZHANG JiZong

Scientia Agricultura Sinica. 2019, 52(9):  1553-1563.  doi:10.3864/j.issn.0578-1752.2019.09.007

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【Objective】To understand the regularity of aggregation and organic carbon changes after adding straw in long-term conventional tillage soil and to explore the main reason of improved soil organic carbon, the effects of straw incorporation on soil aggregates and the organic carbon content in aggregate were determined, so as to provide theoretical basis for carbon fixation in dryland agriculture.【Method】 An in-lab incubation experiment was conducted for 180 days in a constant temperature incubator at 25℃ with the soil collected from conventional tillage and no-tillage field plot. Four treatments were set up, namely conventional soil without straw (CT), no-tillage soil without straw (NT), conventional tillage soil with straw (CTS) and no-till soil with straw (NTS). Each treatment was sampled 15 replicates periodically for aggregate and organic carbon determinations. The straw was the aboveground parts of maize collected from conventional tillage, and the dosage was 5% dry soil weight.【Result】 (1) Aggregate in CT and NT were dominated by 250-53 μm fraction, accounting for 52%-66% of total aggregates, while in CTS and NTS, the aggregates were dominated by 2 000-250 μm fraction, accounting for 41%-50% of total aggregates. CTS and NTS improved 2 000-250 μm aggregate by 230%-302% and 92%-134% relative to CT and NT, respectively. (2) Straw incorporation significantly increased the mean weight diameter (MWD), geometric mean diameter (GMD) and macro-aggregate content (R_0.25) of water-table aggregates. In the 180th day, CTS increased 133%, 130%, and 235%, respectively; compared with CT, NTS increased 53%, 75% and 87%, respectively, compared with NT. (3) In the 180th day, compared with CT, CTS increased the organic carbon content in aggregate of 250-53 μm and <53 μm by 70% and 54%, respectively, and compared with NT, NTS increased in the same aggregates by 30% and 25%, respectively. (4) The contribution of organic carbon in 2 000-250 μm aggregate to soil organic carbon was significantly increased by CTS and NTS, with improvement of 49%-61% and 50%-60%, respectively, which was more affected by aggregate composition. 【Conclusion】 The addition of straw could effectively increase the content of macroaggregates (>250 μm) in the dryland, enhance its stability and increase the contribution of macroaggregates to soil organic carbon, and had a greater effect on the conventional tillage soil.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Decomposition Characteristics and Driving Factors of Organic Materials in Typical Farmland Soils in China

MA Xiang, XU MingGang, ZHAO HuiLi, DUAN YingHua

Scientia Agricultura Sinica. 2019, 52(9):  1564-1573.  doi:10.3864/j.issn.0578-1752.2019.09.008

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【Objective】Understanding of the dynamics and mechanisms of organic materials in soil are essential for improving the utilization of organic wastes and developing nutrient management strategies in many intensive farming regions around the world. The objective of this study was to elucidate the decomposition characteristics of straw and manure in typical farmland soils in China.【Method】The field experiment was conducted in three soils (Red soil, Aquic soil, and Black soil) at the “National Soil Fertility and Fertilizer Effects Long-term Monitoring Network” experimental station. Four organic materials (wheat straw (WS), maize straw (MS), pig manure (PM) and cattle manure (CM)) were dried and then packed separately into 48 μm-mesh size nylon net bag. Each bag contained 20 g organic materials was cut into 2 mm pieces. Based on the temperature difference, each treatment was sampled 6 times within 49-360 days after landfill. We analyzed the change of organic carbon content with soil accumulated temperature and residual rate during decomposition to study the relative contribution of climate factors, organic material properties and soil nutrients in the decomposition rate of different organic materials.【Result】Humification coefficient of straw and manure were 11%-39% and 50%-57%, respectively, suggesting faster decomposition of straw than manure. The organic labile decomposable carbon pool accounted for 76% and 43% at straw and manure, respectively, while recalcitrant carbon pool accounted for 17% and 53%, respectively. The decomposition rates constants (k) of labile decomposable carbon pool were similar in straw and manure, with an accumulated turnover temperature (1/k) of 1 400-2 000℃. VPA (variance decomposition analysis) analysis showed that the nature of the organic material, contributing 28% to the variance, was the main influencing factor in its decomposition. Individually, the greatest contributor during the decomposition of straw was the combined interaction of climate, organic material properties and soil (42.3%). In contrast, the decomposition of manure was mainly controlled by the climate (38.3%).【Conclusion】The decomposition rate and proportion of labile decomposable carbon pool in straw were higher than that of manure. The decomposition of straw was influenced by the synergistic effect of climate, soil and material properties, while manure was influenced mainly by climate factors. It was critical to determine the returning time and returning amount of straw in the field in combination with the local hydrothermal conditions. Manure was recommended to be piled up along with proper timing of its application before returning to the field.

Functional Analysis of Burkholderia pyrrocinia WY6-5 on Phosphate Solubilizing, Antifungal and Growth-Promoting Activity of Maize

GONG AnDong, ZHU ZiYu, LU YaNan, WAN HaiYan, WU NanNan, Cheelo Dimuna, GONG ShuangJun, WEN ShuTing, HOU Xiao

Scientia Agricultura Sinica. 2019, 52(9):  1574-1586.  doi:10.3864/j.issn.0578-1752.2019.09.009

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【Objective】 The study was carried out to screen microbe with phosphate solubilizing (P-solubilizing) and antagonistic activity, to evaluate their efficacies for P-solubilizing, fungal inhibition and plant growth promoting, and to identify antifungal compounds, so as to provide new resources for the development of microbial fertilizers. 【Method】 Rhizosphere soil samples of tea trees were collected from Cheyun mountain factory in Xinyang, Henan, China. Each sample was diluted, and spread onto the surface of insoluble organic and inorganic media. The diameter of P-solubilizing zone was measured after 5 days of incubation. The strain WY6-5 was chosen for further studies because it showed the highest P-solubilizing activity on insoluble phosphate. Additionally, the strain WY6-5 was inoculated in liquid medium and soil for 20 days to test P-solubilizing activity, and inoculated in maize grown soil to test plant growth promoting effects. Moreover, strain WY6-5 was co-cultured with eight different fungi to determine antagonistic activity by using a two dish face-to-face cultural method. The volatiles were characterized and identified with gas chromatography-tandem mass spectrometry (GC-MS/MS). 【Result】 Three strains with P-solubilizing activity were isolated from rhizosphere soils of tea trees, and which were capable of dissolving insoluble organic and inorganic phosphorus medium. The strain WY6-5 demonstrated the highest P-solubilizing activity with the solubilizing halo up to 2.3 cm on insoluble organic medium, and 3.6 cm on insoluble and inorganic phosphorus medium. The ratios of the P-solubilizing halo diameter to the colony diameter on both media were 4.6 and 7.2, respectively. Based on morphological characteristics, physiological, biochemical and phylogenetic analyses, the strain WY6-5 was identified to be Burkholderia pyrrocinia. P-solubilizing activity of WY6-5 was also observed in the liquid medium or in soil after 20 days of incubation. The concentration of soluble phosphate in liquid medium was up to 520.4 mg·L ^-1, which was 176 times higher than that in control treatment. During 3-20 d, the phosphate concentrations in soil under WY6-5 treatments were constantly higher than that under control treatment. In addition, the strain WY6-5 significantly promoted the growth of maize seedling in terms of the number, length, width and area of leaf as well as plant height and fresh weight. Moreover, the volatile compounds produced from the WY6-5 inhibited the growth of all eight different fungi, with the mycelium inhibition rate up to 100%. The antifungal volatile was subsequently identified as dimethyl disulfide through GC-MS/MS. 【Conclusion】 The Burkholderia pyrrocinia strain WY6-5 isolated from rhizosphere soils of a tea tree was found to be able to dissolve insoluble phosphate in both liquid medium and soil, to promote the growth of maize seedlings, and to produce volatile dimethyl disulfide with broad antifungal activity, implying an important biological functions.

HORTICULTURE

Chemical Compositions and Gene Mapping of Wax Powder on Watermelon Fruit Epidermis

GONG ChengSheng, ZHAO ShengJie, LU XuQiang, HE Nan, ZHU HongJu, DOU JunLing, YUAN PingLi, LI BingBing, LIU WenGe

Scientia Agricultura Sinica. 2019, 52(9):  1587-1600.  doi:10.3864/j.issn.0578-1752.2019.09.010

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【Objective】Wax powder is the first protective barrier for plants to resist external stress. In order to find out the physiological and biochemical mechanism of this trait and to get candidate genes, the structure, chemical compositions and inheritance of wax powder on watermelon fruit epidermis were investigated in this research, and the candidate genes were predicted. 【Method】Six-generation populations (P₁, P₂, F₁, F₂, BC₁P₁, and BC₁P₂) were constructed by crossing the inbred line ‘Meijiaxuanhei’ (in glossy) and ‘FH’ (in waxy). Scanning electron microscopy (SEM) was used to observe the structure of wax powder on mature watermelon fruit epidermis. The composition of wax powder was determined by gas chromatography-mass spectrometry (GC-MS), and the contents of different chemical components were quantitatively calculated based on the peak area. The wax powder trait was preliminary mapped by BSA-seq, and BLAST software was used to annotate the coding genes in the localization interval in many databases. By analysis of detailed gene annotation information and mutation site, candidate genes were quickly screened. 【Result】The color of wax powder on ‘Meijiaxuanhei’ fruit was gray-white, and the structure was compact plate shaped with about 5 μm in length, while ‘FH’ fruit was found smooth epidermis and no similar structure. 24 kinds of aliphatic compounds were detected in the wax powder, which were classified as hydrocarbons, alcohols, esters, acids, phenols and aldehydes. There were 10 kinds of hydrocarbons were found, which accounted for 77.72% of the total effective chemical extracts of epidermal wax powder and chain length ranged from C17 to C36. Among them, most of the saturated n-alkanes were C27, C28, C29, C32, C33, C34 and C36, 5 kinds of alcohols accounted for 12.60%, 5 kinds of esters accounted for 0.43%, 2 kinds of acids accounted for 0.53%, 1 kind of phenol accounted for 0.80%, and 1 kind of aldehydes accounted for 3.99%, respectively. The top five chemical components in watermelon wax powder were n-34, n-29, 1,30-triacontanediol, n-33 and n-octacosane. The fruit of F1 and BC₁P₂ progenies had wax, the segregation of waxy and glossy in F₂ progenies corresponded to the Mendelian ratio of 3:1, and the segregation ratio in BC₁P₁ progenies was 1:1, so it could be concluded that the hereditary mode of waxy was the dominant heredity of single gene, and the waxy type was the dominant character. By SNP and InDel correlation analysis, 3.16-4.84Mb association region of chromosome 1 was obtained, which contained 144 genes, 138 genes in them were functionally annotated, including 10 non-synonymous mutations and 1 frameshift mutation. Combining with the literature reports, there were five non-synonymous mutation genes might be related to the formation of watermelon epidermal wax powder: Cla002367 was an enoyl ACP-reductase (ECR) gene, which was essential for the synthesis of extra-long chain fatty acids. Cla011514, Cla002337 and Cla002342 were cytochrome P450 (CYP) family genes, and some proteins encoded by the CYP family genes could catalyze the formation of aliphatic compounds through alkyl hydroxylation and other reactions; Cla002353 gene annotation information was ABC transporter, and ABC transporters were closely related to the transport of waxy molecules.【Conclusion】Wax powder on watermelon fruit epidermis was a plate structure, which was mainly composed of aliphatic compounds derived from long chain fatty acids. The trait conformed to the single gene inheritance model, and wax powder was dominant. The association region of chromosome 1 in the range of 1.68Mb was obtained by BSA correlation analysis. 5 non-synonymous mutation genes Cla002367, Cla011514, Cla002337, Cla002342 and Cla002353 were predicted as candidate genes for wax powder on watermelon fruit epidermis.

The QTL Analysis of Single Fruit Weight Associated Traits in Melon Based on CAPS Markers

LIU XiangYu, ZHANG YuShu, LIU Liu, LIU Shi, GAO Peng, WANG Di, WANG XueZheng

Scientia Agricultura Sinica. 2019, 52(9):  1601-1613.  doi:10.3864/j.issn.0578-1752.2019.09.011

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【Objective】 In our study, based on melon genome-wide resequencing technology, SNP loci were explored and CAPS markers were developed to construct a genetic linkage map. The preliminary QTL analysis of traits related to single fruit weight in melon was carried out, which laid a theoretical foundation for the detection of genes related to single fruit weight traits in melon.【Method】F_2:3 population materials were constructed from X207 and M4-130. The cultivated melon accession M4-130 was selected as female parent and wild melon accession X207 as male parent. Correlation analysis was carried out for fruit traits, including single fruit weight, fruit length, fruit width, pulp thickness, fruit shape index and soluble solids content in melon fruit. Parents were resequenced at 20× depth. SNP loci between parents were extracted in the whole genome by the use of BWA, SAMTools, VCFTools and other software. CAPS markers were developed by using SNP2CAPS software and combined with restriction endonuclease digestion sites in melon genome for genetic linkage map construction. Finally, QTLs of single fruit weight, fruit length, fruit width, pulp thickness, fruit shape index and soluble solids content in melon fruit were analyzed by composite interval mapping.【Result】In this experiment, single fruit weight significantly correlated with fruit length, fruit width and pulp thickness. 185 CAPS markers were developed and subsequently used for genetic linkage map construction. The genetic linkage map contained 12 linkage groups covering a total length of 1 600.45 cM and average distance of 8.65 cM. 7 QTLs (FW3.1, FW4.1, FW5.1, FW6.1, FW8.1, FW8.2 and FW11.1) were related to the single fruit weight. 7 QTLs (FL2.1, FL3.1, FL4.1, FL5.1, FL6.1, FL8.1 and FL11.1) were related to fruit length. 5 QTLs (FWID3.1, FWID4.1, FWID8.1, FWID10.1 and FWID11.1) were related to fruit width. 2 QTLs (FT6.1 and FT11.1) were related to pulp thickness. 1 QTLs (FS2.1) was related to fruit shape index. 2 QTLs (SS6.1 and SS12.1) were related to soluble solids content.【Conclusion】Twenty-three QTLs in total were identified. A major QTL FW8.1 was identified with phenotypic variance contribution rate of 25.8774% and the LOD value was 16.8746. It was also found that single fruit weight was closely related to fruit length, fruit width and pulp thickness. Location intervals were same or adjacent, closely concentrated on chromosomes 3, 4, 5, 6, 8 and 11.

FOOD SCIENCE AND ENGINEERING

Lethal Effect of Meat-Borne Pseudomonas fluorescens to Acidic Electrolyzed Water

CAI LinLin, HU HaiJing, YI XiaoKun, WANG HuHu, XU XingLian, PENG Bin

Scientia Agricultura Sinica. 2019, 52(9):  1614-1623.  doi:10.3864/j.issn.0578-1752.2019.09.012

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【Objective】The objective of this study was to investigate whether the acidic electrolyzed water (AEW) could effectively control meat-borne Pseudomonas fluorescens and to investigate the changes induced in bacteria, which was to provide a reference for the development of novel disinfection approaches.【Method】In this paper, the meat-borne P. fluorescens was treated by AEW with available chlorine concentrations of 20, 40, and 60 mg?L ^-1 for 5, 10, 15, 20, 25 and 30 minutes. The lethal effect of AEW on meat-borne P. fluorescens was determined by plate counting, and the characteristic morphology of meat-borne P. fluorescens after 5 min treatment of 20, 40, and 60 mg?L ^-1 AEW was observed under scanning electronic microscopy (SEM) images. In addition, multiple fluorescent probes were used to measure the effects of AEW on cell membrane integrity, intracellular pH, intracellular ATP concentration and membrane potential. The extracellular polymeric substances (EPS) was also extracted to preliminarily investigate the interaction between EPS and AEW. 【Result】 All treatments of AEW with researched concentrations of 20, 40, and 60 mg?L ^-1could reduce the viable cell number of meat-borne P. fluorescens by 7 log CFU/mL in 5 min, and the increasing concentration of AEW above 40 mg?L ^-1 played no significant role in the survival of bacterial cell (P>0.05). No viable cells were detected after 10 min treatment of AEW. SEM observations showed that meat-borne P. fluorescens cells exposed to all concentrations of AEW treatment exhibited surface indentations and cell distortions, which indicated the loss of typical structure. AEW significantly decreased the bacterial membrane integrity, membrane potential, intracellular ATP concentrations, intracellular pH and EPS content (P<0.05). Under treatments of 20, 40 and 60 mg?L ^-1 AEW, the membrane integrity decreased to 2.26%, 1.87% and 1.20%, respectively; the membrane potential declined by 40%, 50% and 50%, respectively; the intracellular pH dropped from 7.50 to 6.53, 5.90 and 5.83, respectively. The intracellular ATP concentration was decreased nearly under detection. The decrease significantly depended on the increasing concentration of AEW, however, when the concentration of AEW was above 40 mg?L ^-1, there were no significant changes in membrane potential, intracellular ATP concentrations and intracellular pH. After treatment with AEW, the contents of protein and carbohydrate in EPS were significantly decreased (P<0.05). The protein and carbohydrate of L-EPS were decreased by 71.08% and 62.23%, respectively, which those of B-EPS were decreased by 99.32% and 40.62%, respectively. When added to AEW, EPS decreased the ORP of AEW significantly (P<0.05), but played no significant role in AEW’s pH and ACC (P>0.05).【Conclusion】AEW had good bactericidal effect against meat-borne P. fluorescens and it impaired the cell membrane, destroying cell homeostasis. EPS played an important role in hindering the lethal effect of AEW on bacteria.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

CEBPα and p53 Regulate Kiss1 Gene Expression in Porcine Ovary Granulosa Cells

XIN XiaoPing, WANG JiaYing, ZHANG AiLing, ZHONG YuYi, HE YingTing, CHEN ZanMou, ZHANG Zhe, ZHANG Hao, LI JiaQi, YUAN XiaoLong

Scientia Agricultura Sinica. 2019, 52(9):  1624-1634.  doi:10.3864/j.issn.0578-1752.2019.09.013

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【Objective】The objectives of this study were to predict the potential transcription factors in the upstream region of Kiss1, and then to verify the regulatory role of these transcription factors on the expression of Kiss1 in the ovarian granulosa cells of pigs. This study could provide the basic data for the molecular mechanism of Kiss1 in granulosa cells of pigs. 【Method】 By using the bioinformatic software, we predicted the potential binding site of transcription factors in the upstream region of Kiss1 in NCBI database. In addition, we reviewed a lot of relevant papers and references, and then CEBPα and p53 were selected as potential binding transcriptional factors in the upstream region of Kiss1 gene. The primers were designed near the potential binding sites in the upstream region of Kiss1 genes, and verified the binding of transcriptional factors CEBPα and p53 to the upstream region of Kiss1 gene by ChIP. According to the mRNA sequences of CEBPα and p53 in NCBI database, Primer Premier 5 software was used to design primers, and CDS regions of CEBPα (containing KpnI and XhoI sites) and p53 (containing KpnI and Hind III sites) were amplified by PCR and identified by sequencing. Then the CDS regions were connected to the eukaryotic expression vector pcDNA3.1, and the constructed plasmids were extracted with endotoxin-freely, named pcDNA3.1-CEBPα and pcDNA3.1-p53. The interfering siRNA fragments of CEBPα and p53 were synthesized through the chemical method. Pig ovaries were collected from the slaughterhouse, and the ovarian granulosa cells were isolated and cultured. The eukaryotic expression vector or siRNA fragment was transfected into ovarian granulosa cells by cationic liposome, and the effects of p53 and CEBPα on the expression of Kiss1 were verified by the real-time fluorescence quantitative PCR and Western Blotting, respectively. 【Result】 The bioinformatic prediction indicated that there were putative binding sites of p53 (tumor protein p53, p53), CEBP (CCAAT/enhancer binding protein, CEBP), Stat4 (signal transducer and activator of transcription 4, Stat4) and other potential of transcription factor binding sites in the upstream (-850 -+221) of Kiss1. And the putative binding site of CEBPα (GenBank Gene ID: 397307, XM_003127015.4) were found in -744—-733 bp region and the putative binding site of p53 (GenBank Gene ID: 397276, NM_213824.3) were found in -533—-523 bp region. The results of ChIP showed that p53 and CEBPα were bounded at -533—-523 and -744—-733 of Kiss1, respectively. After overexpression of p53 or CEBPα, both mRNA and protein expression level of Kiss1 significantly decreased (P<0.05). Furthermore, both mRNA and the expression level of Kiss1 increased significantly (P<0.05) by interfering p53 or CEBPα. 【Conclusion】 In pigs, p53 and CEBPα could bind at the upstream region of Kiss1 to inhibit its expression in ovarian granulosa cells.

Research Progress and Prospect of Life Cycle Assessment in Animal Husbandry

JIANG MingHong, LIU XinChao, TANG HuaJun, XIN XiaoPing, CHEN JiQuan, DONG Gang, WU RuQun, SHAO ChangLiang

Scientia Agricultura Sinica. 2019, 52(9):  1635-1645.  doi:10.3864/j.issn.0578-1752.2019.09.014

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Life Cycle Assessment (LCA) is a generally accepted method to evaluate the overall environmental burden and production efficiency during the entire life cycle of a product, which is widely used for production management structure optimization in the enterprises and policy formulation in governments. In recent years, for food safety and environmental protection considerations, more and more studies emphasize that the LCA method should be used in animal husbandry to comprehensively assess the environmental burden and resources consumption during the animal-source food production. In China, LCA research and application of animal husbandry are rare, especially in the northern grassland pastoral area, which are the main livestock produce region, and no LCA research on the local livestock production system has been carried out. This paper reviewed the framework and research status of livestock production LCA methods on the main sections and processes, including (1) the goal and scope definition of livestock husbandry LCA; (2) life cycle inventory (LCI); and (3) life cycle impact assessment (LCIA). Through review the domestic and foreign LCA literatures on animal husbandry production, we noticed that the frame and methodology of animal husbandry LCA had been constructed well in developed country. It had an importance instructing significance for the research and practical application of Chinese animal husbandry LCA. However, we also needed to recognize that due to the differences in the specific situations of the livestock industry between domestic and foreign country, foreign LCA experience should not be applied directly in China. First of all, in the northern pastoral areas of China, family farm is the main production system mode of local livestock production, unclear boundaries between herdsmen's life needs and livestock production inputs exist in this system, therefore, it is difficult to apply the foreign experiences directly. Secondly, it is really a challenge to collect the data required for livestock husbandry LCA, which is the biggest restrictive factor of LCA application in livestock production in China. Third, compared with the animal husbandry developed countries, such as European countries, the United States and Australia, due to the gaps in livestock production technology and management methods, the livestock production in the northern pastoral areas of China is affected more by natural factors, such as precipitation, and various marketing factors, including the market supplies and demands. This is also an important issue that must be considered in the research and application of animal husbandry LCA in China. In order to widely practice Chinese livestock husbandry LCA, the following aspects should be improved: (1) the investigation about material input and output of livestock husbandry production in the northern pastoral areas of China should be carried out to collect necessary data; (2) the Chinese animal husbandry LCA database should be established in order to improve the accuracy of livestock husbandry LCA; and (3) we should strengthen the interdisciplinary study to clarify the natural and social driving factors in the development of animal husbandry, and to provide data reference and technology support for the sustainable development of animal husbandry in the northern pastoral areas of China.

Investigation on the Antibiotic Resistance of Staphylococcus Methicillin-Resistant MLS_B from Food Animals in Six Provinces of China

LI ShuMin, FANG LiangXing, LI Liang, ZHAO Meng, LU Xiao, GU WeiQi, LIAO XiaoPing, SUN Jian, XIONG YanQiong, LIU YaHong

Scientia Agricultura Sinica. 2019, 52(9):  1646-1656.  doi:10.3864/j.issn.0578-1752.2019.09.015

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【Objective】 The aim of this study was to investigate the prevalence of genes encoding resistance to macrolides, lincosamides and streptogramins (MLS_B) among methicillin-resistant Staphylococcus aureus from food animals in China. 【Method】Staphylococcus aureus were isolated from about 6 500 samples of pig, chicken and duck origins among six different provinces, including Guangdong, Henan, Hebei, Fujian and so on, in China. LA-MRSA was confirmed from staphylococcus aureus through detecting the susceptibility of oxacillin and the presence of mecA and mecC genes. Susceptibility test of LA-MRSA resistant to MLS_B and other common antibiotics were determined by agar dilution method and microdilution broth method. And the MLS_B resistance genes (ermA-C and ereA-B) and other common resistant genes were detected by PCR. 【Result】Four hundred and eighty Staphylococcus aureus isolates and 101 LA-MRSA isolates were found among about 6 500 samples, and the isolation rate of Staphylococcus aureus and the detection rate of LA-MRSA among Staphylococcus aureus isolates were 1.54% (101/6500) and 21.04% (101/480), respectively. Susceptibility test showed high resistance rate of LA-MRSA to MLS_B was found with over 99.00% among LA-MRSA isolates. High resistance rate to other common antibiotics was also found among LA-MRSA, including florfenicol, tetracycline, cefotaxime and gentamycin with the resistance rates of 99.01%, 96.39%, 96.39%, 96.39% and 94.05%, respectively. MLS_B resistance genes detection showed that ermC was found in all LA-MRSA isolates, followed by ereB (79.20%), ermA (45.54%) and ereA (40.59%), while, ermB was present with a low detection rate (11.88%). Other drug-resistant genes test results showed that fexA, tetL, aadA1, aph (4') - Ia detection rate was higher, which was 92.10%, 97.02%, 97.29% and 83.17%, respectively; secondly, tet (M), the detection rate was 71.29%; the detection rate of aac (6') - Ib, aac (3') - Ic, aph (3') -II, aph (3') -IV, optrA, tet (A) and tet (C) was relatively low, which was 49.50%, 46.53%, 39.60%, 37.62%, 33.67%, 29.70% and 20.79%, respectively; the detection rate of cfr, tet (K), lnuA and lnuF was the lowest, which was 17.82%, 14.85%, 5.94% and 3.96%, respectively. The relationship of the number of MLS_B resistance genes and the number of resistance to antibiotics was further analyzed, which indicated the increased number of resistance to antibiotics was corresponding with the increased number of MLS_B resistance genes and high detection rate of other resistant genes among the LA-MRSA isolates. 【Conclusion】In conclusion, in this study, a high resistance to MLS_B was found and it was consistent to the high detection rate of ermC gene among LA-MRSA isolates. The multi-drug resistance of LA-MRSA isolates was serious and this was closely related to the number of MLS_B resistance genes. Therefore, it is urgently needed to take action in the rational use of antibiotics, as to reducing the emergence of multi-drug resistance LA-MRSA.

RESEARCH NOTES

Cloning and Expression Analysis of Jasmonic Acid Carboxyl Methyltransferase Gene from Perilla frutescens

BAI HuiYang, LU Geng, LU JunXing, GUAN Li, TANG Xin, ZHANG Tao

Scientia Agricultura Sinica. 2019, 52(9):  1657-1666.  doi:10.3864/j.issn.0578-1752.2019.09.016

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【Objective】Jasmonic acid carboxyl methyltransferase (JMT) is a key enzyme in the biosynthesis of methyl jasmonate. By cloning the Perilla frutescens JMT and studying its expression patterns under different stresses and different developmental stages of seeds, it provides a theoretical basis for studying the role of JMT in plant defense and seed development.【Method】Primers were designed based on the transcriptome sequencing results of Perilla seeds, and the DNA and cDNA sequences of the JMT were cloned from Perilla, named PfJMT. Analysis of the structure, stability, hydrophilicity, subcellular localization and conserved domain of PfJMT by bioinformatics methods. Analysis of the evolutionary relationship between PfJMT and other species JMT proteins using phylogenetic tree. Tissues such as perilla roots, stems, leaves, and flowers in flowering stage were used for tissue-specific expression analysis. Seeds at 5, 10, 15, 20, 25, and 30 days after flowering were used for the study of JMT expression patterns in different developmental stages of seeds. The perilla seedlings with 4 true leaves were sprayed with 25 μmol·L ^-1 methyl jasmonate (MeJA) and 1 mmol·L ^-1 salicylic acid (SA) and the roots were watered, perilla root and leaf tissues from different individuals were taken after 0, 2, 4, 8, 16, 24, and 48 hours of treatment to study the expression pattern of JMT under different stresses.【Result】PfJMT has an ORF of 1 050 bp and encodes 349 amino acids. Bioinformatics analysis showed that PfJMT is an unstable hydrophilic protein, which is located in the cytoplasm and contains a Methyltransf-7 conserved domain. Compared with the JMT protein sequence of other species, the sequence identity of Perilla JMT and Salvia miltiorrhiza JMT was the highest, which was 80.5%, and the sequence with Oryza sativa Japonica Group was the lowest, which was 36.8%. In the analysis of phylogenetic tree based on JMT protein construction system in many different plants, it was found that the dicotyledonous plants such as Perilla and Arabidopsis thaliana and Salvia miltiorrhiza were closely related, but the relationship with the monocotyledons such as Cymbidium ensifolium and Oryza sativa Japonica Group was far. This indicates that JMT may have large differences in the evolution of monocotyledonous and dicotyledonous plants. The results of real-time PCR showed that the relative expression of PfJMT in roots and stems of Perilla was the lowest, leaves and flowers was slightly higher, 5 days after flowering was the highest, and it was gradually down-regulated with the development of seeds. This indicates that JMT plays an important role in seed development. PfJMT expression was significantly down-regulated in Perilla root and leaf tissues treated with MeJA and SA. This result supports the theory that JMT may not directly participate in defense responses, but indirectly participate in plant defense by regulating JA levels.【Conclusion】PfJMT was successfully cloned, and the expression of PfJMT was gradually down-regulated with the development of seeds, and the expression level was significantly down-regulated under exogenous MeJA and SA stress.