Scientia Agricultura Sinica

Variations of Wheat Quality in China From 2006 to 2015

HU Xue-xu, SUN Li-juan, ZHOU Gui-ying, WU Li-na, LU Wei, LI Wei-xi, WANG Shuang, YANG Xiu-lan, SONG Jing-ke, WANG Bu-jun

Scientia Agricultura Sinica. 2016, 49(16): 3063-3072. doi:10.3864/j.issn.0578-1752.2016.16.001

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【Objective】In this paper, the authors intended to give an overview of wheat quality with strong gluten, medium- strong gluten, medium gluten and weak gluten in wheat producing regions of China in recently 10 years, and to discuss the problems and solutions for wheat quality. 【Method】 A total of 7 561 samples of 742 wheat cultivars were collected from wheat producing regions of China in 2006–2015, and the variety structure was summed up, and testing weight, protein content, wet gluten content, sedimentation volume, and dough rheological properties were determined. 【Result】The testing results showed that wheat variety structure had some improvement which the proportion of wheat cultivars with strong gluten (12.6%), medium-strong gluten (16.7%), medium gluten (68.7%) and weak gluten wheat(2.0%) remained stable in years, and the proportion of each type of wheat samples reached 21.0%, 16.9%, 49.4% and 2.0%, respectively, while the proportion of wheat samples with each type reaching the standards was less (4.8%, 4.6%, 25.0%, and 0.4%, respectively). Each type of wheat samples varied among years, i.e., the proportion of strong gluten wheat and weak gluten wheat declined, while the medium gluten wheat and medium-strong gluten wheat increased in recent years. The medium gluten wheat is qualified as the relatively high protein quantity and low protein quality, but the sedimentation volume and dough rheological properties decreased slightly in years. The quality of wheat cultivars with strong gluten, medium- strong gluten and medium gluten remained stable, which the trends of sedimentation volume and dough rheological properties were similar, while the weak gluten wheat declined in quality due to the mean value of protein content, wet gluten content and sedimentation volume increased. There were distinct differences among the four types in quality, which the mean value of testing weight declined from medium strong gluten to strong gluten, and to medium gluten and weak gluten, protein content and wet gluten content declined from strong gluten to medium gluten, and to medium-strong gluten and weak gluten, sedimentation volume and dough rheological properties declined from strong gluten to medium-strong gluten, and to medium gluten and weak gluten. The change in average of quality traits among the four types was also different, of which the change range of sedimentation volume and dough rheological properties declined from strong gluten to medium-strong gluten, and to medium gluten and weak gluten. 【Conclusion】 Chinese wheat variety structure had some improvement, and the proportion of different types reaching the standards was less, which indicated there was much room for improvement of quality. In order to improve the quality of wheat by optimizing the structure of variety, it should increase the quality of breeding high-yielding wheat varieties and promotion efforts, optimize the regional distribution of approved varieties，and intensify policy support and so on.

Mapping QTL of Flowering Time and Their Genetic Relationships with Seed Weight in Brassica napus

HUANG Ji-xiang, XIONG Hua-xin, PAN Bing, NI Xi-yuan, ZHANG Xiao-yu, ZHAO Jian-yi

Scientia Agricultura Sinica. 2016, 49(16): 3073-3083. doi:10.3864/j.issn.0578-1752.2016.16.002

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【Objective】The present research aimed to explore the major QTL controlling the flowering time in European and Chinese rapeseed materials, to analyze the genetic influence of flowering time on QTL for 1000-seeds weight, and thus to provide available information for breeding purpose.【Method】The doubled haploid (DH) Sollux/Gaoyou population with 282 lines was used. The data set of flowering time was obtained from nine environments and over seven years. QTL identification of flowering time was performed using WinQTLCart 2.5. The candidate genes underlining QTLs were screened out by transcriptome analysis using RNA-Seq and alignment between QTL regions and Arabidopsis. Further, conditional QTL estimation was adopted to dissect the genetic relationships between flowering time and seed weight. Finally, using selected DH lines with extreme phenotypes of flowering time, an association evaluation between marker genotypes and phenotypes of flowering time was performed. 【Result】 Seven major QTLs were detected, which showing significant at least in three environments. Their additive effects ranged from 0.58-3.85 days and together accounted for around 84% of the phenotypic variation in population. The sum of eight pairs of epistatic loci (additive × additive) accounted for 41.8% of the total additive effects. QTL by environmental interactions were significant only in few environments with small amount of genetic effects. Four critical orthologous genes of Arabidopsis thaliana for flowering time were mapped in the peak positions of three most significant QTL regions (qFTA2, qFTC2, and qFTC6). It provides valuable information to anchor candidate genes underling QTL. The conditional QTL analysis revealed large impact of flowering time on seed weight in four QTLs (qSWA2, qSWA3, qSWA4, and qSWC2). This partly explained the significant negative correlation between flowering time and 1000-seed weight. While the most important two (qSWA7 and qSWC8) showed independent without being interfered. Six markers linked with three major QTLs showed good fitness between marker genotypes and trait phenotypes (70%-100%), indicating their potentials for breeding purpose. The results demonstrated that the combination of early flowering alleles from qFTA2, qFTC2 and qFTC6 by marker assistant selection of ZAAS548, DNAPL, ZAAS619sa, ZAAS616s, ZAAS846a and C6SGFLO-22 induced not only early flowering but also significantly increased 1000-seed weight, while the oil content and seeds per silique between two extreme flowering time groups showed almost the same.【Conclusion】All seven QTLs of flowering time showed Chinese parent Gaoyou induced early flowering. Four important candidate genes homologous to Arabidopsis controlling flowering time (FT, FLC, AP1, and FY) were physically aligned and mapped underlining the peak positions of the three major QTL qFTA2, qFTC2 and qFTC6. The results indicated that the four loci corresponding to seed weight were genetically influenced by flowering time, however, the most important two (qSWA7 and qSWC8) were independent. Six markers linked to the 3 major QTL were of potentials in the practical breeding program.

Differential Proteomic Research of Three Varieties of Tobacco in China

XU Ying, YAN Guo-quan, ZHANG Yang, YU Hong-xiu

Scientia Agricultura Sinica. 2016, 49(16): 3084-3097. doi:10.3864/j.issn.0578-1752.2016.16.003

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【Objective】 In order to strengthen the basic scientific research and cultivation of tobacco, differential proteomics is used to select specific proteins and elucidate metabolic pathways in tobacco. 【Method】 Leaf proteins of three varieties of tobacco in China including Honghuadajinyuan, K326 and Yunyan 87 were extracted by phenol. After digestion the protein profiles of three varieties of tobacco were investigated by using tandem mass tag(TMT) coupled with two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS). The proteins and peptides from tobacco were identified with MASCOT search engine after spectra extraction in Proteome Discoverer Software. Then, bioinformatics analysis including data correlation analysis, hierarchical cluster analysis and principal component analysis (PCA) were conducted. Meanwhile, proteins with change ratio of more than 2 fold were defined as differentially expressed tobacco proteins, and they were screened out by volcano plot analysis. Finally, the distribution and function of important proteins in tobacco were analyzed using KEGG pathway analysis. 【Result】 A total of 3 079 proteins in Honghuadajinyuan, K326 and Yunyan 87 were identified and the number of protein ID was 10 343. Protein expression profiles of hierarchical cluster analysis and principal component analysis indicated that K326 and Yunyan 87 were relatively similar, and Honghuadajinyuan was significantly different from the other two. The subsequent protein screening also verified the results. Screening of differentially expressed proteins suggested that there were only 29 proteins differentially expressed between K326 and Yunyan 87. 13 proteins of them covered 8 metabolic pathways including flavonoids biosynthesis. Chalcone synthetase participating in the flavonoids synthesis was significantly higher in K326 than in Yunyan 87. Honghuadajinyuan and Yunyan 87 had 160 differentially expressed proteins. 103 proteins of them covered 42 metabolic pathways including glutathione metabolism. Three kinds of enzymes related to glutathione metabolic pathway including glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase and glutathione S-transferase were significantly lower in Honghuadajinyuan than in Yunyan 87. Honghuadajinyuan and K326 had 119 differentially expressed proteins. 89 proteins of them covered metabolic pathways such as metabolism of xenobiotics by cytochrome P450. It was indicated that several proteins related to stress resistance such as proteinase inhibitor was much less in Yunyan 87 than in K326 and Honghuadajinyuan, and superoxide dismutase in Honghuadajinyuan was the lowest among the three varieties of tobacco. 【Conclusion】Results of the study revealed that the TMT coupled with 2D LC-MS/MS is a powerful method for isolating and identifying differentially expressed proteins in various tobacco varieties. Most of them are involved in photosynthesis, metabolism or stress resistance.

Effects of Five Fungal Chaff Pretreatment Methods on Substrate Properties and Growth of Rice Seedlings

LIU Bin, HAN Ya-nan, YUAN Xu-feng, ZHU Wan-bin, WANG Xiao-fen, CUI Zong-jun

Scientia Agricultura Sinica. 2016, 49(16): 3098-3107. doi:10.3864/j.issn.0578-1752.2016.16.004

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【Objective】 Fungal chaff is a practical choice for rice seedling substrate technology. However, it can cause poor growth, seedling blight, and prevent maturation without pretreatment. Therefore, this study used five different pretreatments to explore optimal growth substrate properties and seedling growth to utilize agricultural wastes as rice seedling substrates.【Method】An experiment for raising rice seedlings carried out in a green house, pretreated substrates with 5methods were used a mix of raw fungal chaff material (T1), fermented compost (T2), adding 10% volume pig manure to ferment compost (T3), steam sterilization (T4), and hot-air sterilization (T5). Soil was used as a control group and spring climate conditions of northeast China were simulated. A comprehensive evaluation was carried out on the bulk density of the substrate, porosity (total porosity, air filled porosity, and water holding porosity), nutrients (total nitrogen content, total phosphorus content, total potassium content, total organic matter content, available K content, available P content and alkaline soluble N content), severity of rice seedling blight (blight areas and spots in weaning stage), and growth of seedlings for each pretreatment (after 30 days, agronomic traits of rice seedlings included leaf age, roots, stem diameters, SPAD values, weights of hundred seedlings), and a calculation method was applied per volume of nutritive element contents rather than a traditional weight ratio, in order to compare differences of treatments.【Result】The results after the pretreatment showed that the bulk densities were within a reasonable range. T1, T2, and T3 significantly improved total porosity and water-holding porosity, whereas T4 and T5 decreased these aspects compared to those of T1. The highest total nitrogen content per volume of nutritive element contents was detected in T3 (3.0×10^-3 g·cm^-3), and the other treatments were 1.6×10^-3–1.8×10^-3 g·cm^-3; total phosphorus content was 4.0×10-4–6.0×10^-4 g·cm^-3. The highest total potassium content was detected in T2 (1.4×10^-3 g·cm^-3), and the other treatments were 7.0×10^-4–9.0×10^-4 g·cm^-3. Total organic matter content was 6.6×10^-2–8.0×10^-2 g·cm^-3. The highest alkaline soluble N content was observed in T3 (2.1×10^-4 g·cm^-3), and the other treatments were 0.9×10^-4–1.2×10^-4 g·cm^-3. Available P content was 3.3×10^-5–5.0×10^-5 g·cm^-3, and available K content was 0.6×10^-4–1.2×10^-4 g·cm^-3. In addition, by calculating with C/N ratio of treatments, results showed that only T3 could reach a value below 20. The incidence rates of rice seedling blight in T1, T5, T2, and T4 were 30.53%, 3.27%, 1.09% and 1.09%, whereas none of the seedlings exposed to T3 developed blight during stage of weaning. Plant heights were 12–14 cm, stem diameters were 0.21–0.23 cm, and total SPAD values at the three-leaf stage were 25–35 in 30-day seedlings, and T3 resulted in the best values for these parameters. Fresh weight per 100 seedlings was 14.50–16.00 g, and dry weight per 100 seedlings was 3.15–3.75 g; the maximum values were observed in seedlings exposed to T2 and T3; the maximum root-top ratio was 0.30 (T2 and T3), the minimum value was 0.22 (T5), fresh-dry ratio of whole plant in all treatments was 0.20-0.23. 【Conclusion】The pretreatments did not significantly affect the nutrients in fungal chaff, as their content was mainly determined by the original composition of the substrate material. Fermenting compost resulted in better substrate properties and seedling growth, suggesting that this could be an effective way to control rice seedling blight, particularly if 10% volume pig manure is co-composted. This method could be developed to use agricultural wastes as a rice seedling substrate.

Effects of Water Management and Controlled-Release Fertilizer on Seed Yield and Seed Quality of Maize (Zea mays L.)

REN Li-sha, GU Ri-liang, JIA Guang-yao, TIAN Kai-xin, SHI Xiu-de, WANG Jian-hua

Scientia Agricultura Sinica. 2016, 49(16): 3108-3118. doi:10.3864/j.issn.0578-1752.2016.16.005

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【Objective】The objective of this study was to investigate the effects of water management and controlled-release fertilizer on seed yield and seed quality and to determine the optimal harvest time for maize hybrid JK968. These results will provide theoretical and technical supports for high quality maize seed production in China.【Method】Maize hybrid Jingke968 was grown in Zhangye city, Gansu province, in 2014 and 2015. Field experiments were designed under split-plot. The main plot treatment was two distinct water managements of normal water irrigation and controlled water irrigation (no water irrigation after grain filling stage that is about 35 days after pollination, DAP). The subplot treatments was two distinct fertilizer treatments of traditional fertilizer (urea and diammonium phosphate) and controlled-release fertilizer (N : P : K ration of 24 : 12 : 10). Seed samples were harvested from 44 to 68 DAP with an interval of 3 for investigating seed yield traits (seed yield, seed number per hectare and 100-seed weight) and seed quality traits (seed moisture, standard germination and accelerated aging germination, and electric conductivity.【Result】Fertilization treatment and irrigation management significantly affected seed yield and seed number per hectare, but none of any effects on seed moisture content, 100-seed weight and seed vigor. Compared to traditional fertilizer, control-released fertilizers increased seed yield by 3.10% and seed number per hectare by 2.13%. Economic benefit by control-released fertilizers also increased by RMB 4 400- 21 700 Yuan and 1 200-13 500 Yuan per hectare under normal water condition and controlled water condition, respectively. Controlled water management decreased seed yield and seed number by 2.00% and 2.49%, respectively, and decreased economic benefit from 1 100 to 17 800 Yuan RMB compared to normal water management. Harvest time significantly affected seed yield, seed moisture, 100-seed weight and seed vigor, but showed no effects on seed number per hectare. Harvest time from 44 to 68 DAP, grain filling and 100-seed weight increased, while seed moisture decreased. Until 68 DAP, seed weight still increased that suggested that the seed have not met their full maturity at this stage. In both years of 2014 and 2015, the vigor of seed harvested from 56-65 DAP showed the highest standard germination rate and accelerated aging germination rate, and the lowest electric conductivity. These results indicated that from 56 to 65 DAP was the best harvest time for maize hybrid JK968, in which the seed vigor was maintained at high level and the damage from cold weather could be avoided. 【Conclusion】The optimal harvest time was from 56 to 65 DAP for Jingke968 seed product, since seed vigor and seed harvest number reached the highest levels during this growth stage. Controlled-release fertilizer improved seed yield, seed harvest number and economic benefit without affecting seed quality. But it decreased grain yield without water irrigation after grain filling stage. Thus controlled-release fertilizer and normal irrigation could save labor and increase economic benefits for seed production.

Triplex PCR Detection for Corynespora cassiicola,Colletotrichum orbiculare and Pseudomonas syringae pv. lachrymans

GAO Shi-gang, ZENG Rong, XU Li-hui, LUO Jin-yan, CHEN Lei, DAI Fu-ming

Scientia Agricultura Sinica. 2016, 49(16): 3119-3129. doi:10.3864/j.issn.0578-1752.2016.16.006

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【Objective】The objective of this study is to develop a rapid triplex PCR detection method of Corynespora cassiicola,Colletotrichum orbiculare and Pseudomonas syringae pv. lachrymans. 【Method】 Specific primers for the three pathogens were designed based on ITS sequences of C. cassiicola andC. orbiculare and 16S rDNA sequence of P. syringae pv. lachrymans, which were confirmed by amplifying specific fragments. Three pairs of primers specific to C. cassiicola, C. orbiculare or P. syringae pv. lachrymans were suitable for triplex PCR and selected for triplex PCR. Primer concentration, annealing temperature, extension time and cycle number were optimized to develop the triplex PCR detection system. 【Result】Five, seven and six pairs of primers were designed specific to C. cassiicola, C. orbiculare and P. syringae pv. lachrymans, respectively. CC4F/CC4R and CC5F/CC5R primers could specifically amplify ITS sequence of C. cassiicola; CL1F/CL1R, CL2F/CL2R, CL3F/CL3R, CL3F/CL4R, CL3F/CL5R, CL3F/CL6R and CL3F/CL7R could specifically amplify ITS sequence of C. orbiculare; PS3F/PS4R and PS4F/PS4R primers could specifically amplify 16S rDNA sequence of P. syringae pv. lachrymans. Fragments in lengths of 370, 275 and 698 bp were amplified by CC5F/CC5R, CL3F/5R and PS3F/4R, respectively, which were separated fully by 3% agarose gel electrophoresis, and the three pairs of primers were used for triplex PCR. Three target fragments were amplified effectively with 0.16 μmol·L^-1 of CC5F/CC5R, 0.4 μmol·L^-1 of CL3F/5R and 0.16 μmol·L^-1 of PS3F/4R in 25 μL of triplex PCR system. Part target fragments were not amplified effectively at >65℃ of annealing temperature. Finally, the triplex PCR system suitable for the detection of C. cassiicola, C. orbiculare and P. syringae pv. lachrymans were developed and confirmed. This PCR system contained 12.5 μL of 2×Hiff^TM PCR Master Mix (With Dye), 0.16 μmol·L^-1 of CC5F/CC5R, 0.4 μmol·L^-1 of CL3F/CL5R and 0.16 μmol·L^-1 of PS3F/PS4R. The PCR program was as follows: an initial denaturation at 95℃ for 3 min followed by 35 cycles of denaturation (95℃ for 30 s), annealing (65℃ for 30 s) and extension (72℃ for 2 min), and a final extension at 72℃ for 10 min. 【Conclusion】The detection method could rapidly detect C. cassiicola,C. orbiculare and P. syringae pv. lachrymans from leaves infected by the three pathogens, the sensitivity of which was 0.4 pg·μL^-1.

Analysis, Expression and Identification of the Common Structural Domain of Bacillus thuringiensis (Bt) Cry1 Toxins

LIU Bei-bei, ZHANG Xiao, XIE Ya-jing, JIAO Ling-xia, LIU Yuan, ZHANG Cun-zheng, ZHAO Yan-yan, WU Ai-hua, LIU Xian-jin

Scientia Agricultura Sinica. 2016, 49(16): 3130-3139. doi:10.3864/j.issn.0578-1752.2016.16.007

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【Objective】The objective of this study is to express the optimal common structural domain through analyzing and locating the common structure of five Bacillus thuringiensis Cry1 toxins. This research will lay a foundation for producing the generic antibody and developing the detection method for Cry1 toxins. 【Method】Through bioinformatics, molecular simulation technique and homology modeling to build the three-dimensional structure models of five Cry1 toxins. The structures were evaluated using three programmes, Ramchandran plot, Verify3D and ERRAT. Domain I was identified as the common structure domain of five Cry1 toxins finally. In order to construct the pET-26b-Domain I vector, primers were designed according to the Cry1Ac gene of Bacillus thuringiensis ssp. kurstaki. As well as insert with Nco I and Not I digestion sites. When it was identified by PCR, restriction enzyme digestion and sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) which was induced with 1 mmol·L-1 IPTG, 20℃ for 16 h. The supernatant and precipitate were collected and verified by SDS-PAGE after E. coli BL21 (DE3) cells were collected and crushed by ultrasonic wave. The soluble fusion protein was purified by His-Trap HP nickel affinity column and verified by SDS-PAGE, Western blot and ELISA. 【Result】 According to the analysis of amino acid sequences and three- dimensional structures of the five Cry1 toxins, the sequences of Domain I were the highest identity part and its three-dimensional structure was very similar and then the Domain I was chosen as the common structure domain of the five Cry1 toxins. The expression vector pET-26b-Domain I was constructed successfully, and soluble Domain I protein was expressed and purified. The molecular weight of the fusion protein was confirmed to be 33.4 kD by SDS-PAGE and Western blot, which also showed specific activity to anti-6×His monoclonal antibody. The ELISA assay showed that the Domain I protein had a good sensitivity with the specific antibodies of the five Cry1 toxins, and the epitope prediction results showed that both the Domain I protein and the complete Cry protein existed multiple potential epitopes, and the percentage of their antigenic peptides were 48.4% and 63.6%, respectively. These results indicate that the Domain I protein has good immunogenicity and immune response. 【Conclusion】Based on molecular simulation and molecular cloning technology, the conserved structural domain protein was successfully expressed and purified. This study has established a foundation for the following studies and the common structural domain protein would as target molecule for the production of the generic antibody of Cry1 toxins in further research.

Effects of Fertilization on Winter Wheat-Summer Maize Rotation System in Different Soil Types

HUANG Xiao-ting, ZHAO Ya-li, YANG Yan, ZHANG Xing, ZHANG Mou-biao, LI Chao-hai

Scientia Agricultura Sinica. 2016, 49(16): 3140-3151. doi:10.3864/j.issn.0578-1752.2016.16.008

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【Objective】The objective of the experiment was to study the nutrient absorption characteristics and the effects of fertilization on crop yield of winter wheat and summer maize in different soil types, the results of the study can provide a basis for fertilization and high efficient utilization of resources.【Method】The experiment with four kinds of soil types (clay fluvo-aquic soil, sandy fluvo-aquic soil, black soil and yellow-cinnamon soil) and three fertilizer levels (no fertilizer, middle fertilizer and high fertilizer) was conducted by a pool experiment to investigate the soil nutrient supply characteristics, the effects of fertilization on production and fertilizer use efficiency in different soil types.【Result】The results showed that under no fertilizer application, the nitrogen, phosphorus and potassium accumulation and yields of winter wheat and summer maize decreased in the four kinds of soil in the order: clay fluvo-aquic soil＞black soil＞yellow-cinnamon soil＞sandy fluvo-aquic soil, which indicated a significant difference among different soil types in soil nutrient supplying capacity and productivity. Fertilization significantly increased crop nutrient accumulation and yield in four soil types. Compared with no fertilizer treatment, the nitrogen accumulation in whole year increased by 37.7%-98.7%, phosphorus accumulation increased by 23.6%-75.3%, potassium accumulation increased by 29.3%-76.1%, and yields of winter wheat and summer maize increased by 26.5%-64.4%. The increases of nitrogen, phosphorus and potassium accumulation and yield of winter wheat and summer maize in four soil types were in the order of sandy fluvo-aquic soil ＞yellow-cinnamon soil＞black soil＞clay fluvo-aquic soil, which showed that the higher of soil productivity, the bigger of nutrient supply ability, the lower effect of fertilization on crop yield. Agronomic efficiency of fertilizer of winter wheat and summer maize in four soil types were characterized by: sandy fluvo-aquic soil＞yellow-cinnamon soil＞black soil＞clay fluvo-aquic soil, which means that the lower of productivity of soil, the higher the fertilizer use efficiency. The nutrient accumulations and yields of winter wheat and summer maize increased significantly under high fertilizer in sandy fluvo-aquic soil and yellow-cinnamon soil. However, there was no significant increase in clay fluvo-aquic soil and black soil.【Conclusion】The higher nutrient supply ability, lower effect of fertilization on crop yield and lower fertilizer use efficiency were found in the higher productivity soil types. While there was a contrary rule in the lower productivity soil types. Therefore, attention should be made to increase soil nutrient and decrease fertilizer application in the clay fluvo-aquic soil and black soil with high fertility, while increase fertilizer application in the sandy fluvo-aquic soil and yellow-cinnamon soil with low fertility to increase soil productivity constantly.

Influences of Actinomycete Strain JXJ-0136 on the Growth of Brassica chinensis and Vigna unguiculata and Its Phosphate Solubilization

ZHANG Bing-huo, LI Han-quan, LUO Juan-yan, YANG Jian-yuan, SHI Hong-qiu, SUN Feng-zhen

Scientia Agricultura Sinica. 2016, 49(16): 3152-3161. doi:10.3864/j.issn.0578-1752.2016.16.009

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【Objective】The objective of this study is to determine the taxonomic status of an actinomycete strain JXJ-0136, investigate its ability of dissolving insoluble phosphorus, the colonization in the rhizospheric soil of crops, and its influences on the seed germination, seedling growth and yield of vegetables, and to evaluate the application value of strain JXJ-0136 in developing microbial fertilizer.【Method】Taxonomic status of strain JXJ-0136 was determined on the basis of the cultural and morphological characteristics, and the phylogenetic analysis of 16S rRNA gene sequence. Influences of strain JXJ-0136 on the seed germination and seedling growth were studied using agar plate. The field cultivation tests were carried out to investigate the influences of strain JXJ-0136 on the growth and yield of vegetables. The total contents of phosphorus in the soil before and after the field trial were measured to investigate the influence of strain JXJ-0136 on the utilization of phosphorus in the soil by crops. The colonization of strain JXJ-0136 in the rhizospheric soil of the plants was investigated by isolation of the microorganisms in rhizosphere soil. The efficiencies of strain JXJ-0136 to dissolve insoluble inorganic and organic phosphorus were investigated using liquid pure culture. The model vegetables of the study were Brassica chinensis and Vigna unguiculata. 【Result】 Growth of actinomycete strain JXJ-0136 was observed at 6-45℃, pH 4.0-13.0 and 0-4% (w/v) NaCl, with optimal growth at 28℃, pH 8.0 and 1% (w/v) NaCl. Strain JXJ-0136 developed well-branched aerial mycelia on ISP 2 medium. The aerial mycelia was off-white in color. Its spore chains were fascicular with elliptical spores. The 16S rRNA gene sequence was closest to Streptomyces violascens, S. somaliensis, S. hydrogenans, S. albidoflavus and S. daghestanicus with the similarities of 97.98%, 97.71%, 97.30%, 97.23% and 97.03%, respectively. However, strain JXJ-0136 formed different clades on phylogenetic tree. The culture broth of strain JXJ-0136 enhanced the seed germination and the seedling growth significantly. After addition of 0.2%-0.8% broth culture of strain JXJ-0136, the seed germination rate, plant height and root length of B. chinensis were 3.55%-12.61%, 13.91%-53.03% and 7.37%-51.92% higher than those of the controls, respectively. The seed germination rate, plant height and root length of V. unguiculata were 4.71%-21.18%, 3.60%-22.33% and 2.37%-20.08% higher than these of the controls, respectively. The field cultivation tests indicated that strain JXJ-0136 could colonize in the rhizospheric soil of the plants, and promoted crops to utilize phosphorus in the soil, and enhanced the yields of the crops. After inoculating with 5 mL broth culture of strain JXJ-0136 to each plant, the soil total phosphorus contents of B. chinensis and V. unguiculata decreased by (23.56±2.65)% and (37.10±1.98)%, respectively, at the end of the tests, which were (1.77±0.29) and (2.70±0.15) times of the controls (P＜0.01). The yields of B. chinensis and V. unguiculata increased by (27.59±6.15)% and (70.29±5.15)% (P＜0.01) than the controls, respectively. After inoculating strain JXJ-0136 and culturing for 5 days under liquid pure culture condition, the pH values of inorganic and organic phosphorus cultures decreased to 5.0 and 6.0 initially from pH 7.0, respectively, and available phosphorus in the cultures of inorganic and organic phosphorus increased by (73.94±0.94) and (7.12±0.28) mg (P＜0.01), respectively. 【Conclusion】Actinomycete JXJ-0136 is a member of the genus Streptomyces. With good properties including increasing the seed germination, plant height and root length of seedling, colonizing in rhizospheric soil, increasing the content of the available phosphorus in the soil, enhancing the crops to utilize the phosphorus in the soil and promoting the growth and yields of crops, strain JXJ-0136 has a potential application value in developing microbial fertilizer.

Variation Characteristics of Microtopography of Ridge Tillage of Purple Soil Under Different Rainfall Patterns

LUO Jian, YIN Zhong, ZHENG Zi-cheng, HE Shu-qin, LI Ting-xuan

Scientia Agricultura Sinica. 2016, 49(16): 3162-3173. doi:10.3864/j.issn.0578-1752.2016.16.010

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【Objective】Soil surface microtopography is one of the important factors that affects processes such as surface depression storage, penetration rate and surface runoff. Conversely, some of these processes also alter microtopography due to erosion and deposition. The objective of this paper was to investigate the dynamic variation characteristics of microtopography under different rainfall patterns, which to further understand water erosion mechanism on sloping farmland, and to provide a scientific basis for optimizing the configuration of tillage measures and the prediction of soil erosion.【Method】This paper took sloping of ridge tillage in Hilly Area of Central Sichuan basin as the research object and the linear slope was used as control. From the perspective of soil roughness, methods involving artificial rainfall and GIS were adopted to analyze the variation characteristics of microtopography of purple soil.【Result】The range of relative elevation was from -180 mm to 80 mm on the sloping of ridge tillage under different rainfall patterns, which mainly focused on -20 mm-20 mm, the proportion of soil surface elevation reduction was relatively higher. However, the range of relative elevation was from -10 mm to 10 mm on the linear slope, which mainly focused on -5 mm-5 mm. The soil erosion area of increased rainfall series was higher than that of decreased rainfall series for different tillage measures. The value of soil roughness was from 57.47 mm to 65.32 mm on the sloping of ridge tillage under different rainfall patterns, and the value of soil roughness showed that upslope＞downslope＞midslope under different slope positions. The value of soil roughness was from 5.71 mm to 6.28 mm on the linear slope under different rainfall patterns, but soil roughness of different slope positions had obvious variability affected by the random roughness. Grid number of micro-slope of the sloping of ridge tillage showed a decreasing-increasing-decreasing with the increase of slope under different rainfall patterns, and the slopes were mainly concentrated at 0°-5°and 30°-35°. Grid number of micro-slope of the linear slope decreased gradually with the increase of slope, and the slope was mainly concentrated at 0°-10°. Grid number of micro-aspect of the sloping of ridge tillage mainly focused on the northern slope and the southern slope, and distributions of the other micro-aspect were relatively even, but there was an obvious difference in the linear slope under different rainfall patterns. 【Conclusion】The variation of elevation values and soil roughness reflected the vertical variability of microtopography, while the terrain factors could reflect the spatial distribution of microtopography in the process of water erosion. The combination of them reflected variation characteristics of microtopography of the sloping of ridge tillage well. The increased rainfall pattern was similar to the major erosion rainfall pattern in purple soil region in summer which resulted in relatively severe soil erosion on sloping farmland, and it was the main rainfall pattern of soil erosion prevention and control in the study area. This study could provide a new ideas for quantification of microtopography and reveal the essence of soil erosion on sloping farmland.

Content and Analysis of Biosynthesis-Related Genes of Flavonoid Among Four Strains of Malus sieversii f. neidzwetzkyana F₁ Population

XU Hai-feng, WANG Nan, JIANG Sheng-hui, WANG Yi-cheng, LIU Jing-xuan, QU Chang-zhi, WANG De-yun, ZUO Wei-fang, ZHANG Jing, JI Xiao-hao, ZHANG Zong-ying, MAO Zhi-quan, CHEN Xue-sen

Scientia Agricultura Sinica. 2016, 49(16): 3174-3187. doi:10.3864/j.issn.0578-1752.2016.16.011

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【Objective】In order to develop the theory and breeding technology of functional apple, the molecular mechanism of the differences of flavonoid biosynthesis in several cross progenies of Malus sieversii f.neidzwetzkyana and M. domestica cv. Fuji was studied. 【Method】 Four apple stains (Zihong NO.2, Hongcui NO.1, Hongcui NO.2 and Hongcui NO.4) with significant difference in red-flesh degree during the latter growth period were used as materials. The type of MYB10 promoter was identified, and the components and contents of flavonoids and the relative expressions of related genes were determined.【Result】The type of MYB10 promoter in Hongcui NO.1, Hongcui NO.2, and Hongcui NO.4 was R6R1, and that of Zihong NO.2 was R6R6. The contents of flavonoids in the mature period between Hongcui NO.1 (3.0 mg.g^-1) and Zihong NO.2 (3.1 mg·g^-1) were equivalent, while the anthocyanin content in Zihong NO.2 (23.9 U·g^-1 FW) was twice as that in Hongcui NO.1 (12.2 U·g^-1FW), and other contents of flavonoid of anthocyanin in Zihong NO.2 (1 635.3 mg·kg-1) was only 69% of that in Hongcui NO.1 (2 355.0 mg·kg^-1). The transcription factors and anthocyanin biosynthesis genes such as MYB10 and UFGT in Zihong NO.2 had higher expression during the latter growth period (110-125 d). The expression of MYB10 in Hongcui NO.4 during the latter growth period (110-125 d) was higher, but the expression of bHLH3, TTG1, ANS and UFGT were lower. The contents of flavonoid components among Hongcui NO.1 (2 355.0 mg·kg^-1), Hongcui NO.2 (1 247.5 mg·kg^-1) and Hongcui NO.4 (1 337.5 mg·kg^-1) indicated significant differences. In Hongcui NO.1, the MYB12, FLS, LAR and ANR showed higher expression, while the expression of MYB16 and MYB111 were lower. The MYB12, FLS, LAR and ANR in Hongcui NO.2 and Hongcui NO.4 showed lower expression, while the expression of MYB16 and MYB111 were higher.【Conclusion】The transcription factors, such as MYB10, BHLH3, TTG1, and the structure genes which were associated with anthocyanin biosynthesis including ANS, UFGT were obviously up-regulated during the latter growth period, and it might be the main reason that caused high anthocyanin content in Zihong NO.2 flesh in the mature period. Meanwhile, the transcription factors, for example, MYB12, MYB16, MYB111, and the structure genes that relative to flavonoid biosynthesis such as DFR, FLS, LAR, ANR had different expressions, and it might be the main reason that led to the difference in the components and contents of flavonoid among the 3 strains of Hongcui NO.1, Hongcui NO.2 and Hongcui NO.4.

Sugar and Organic Acid Components in Fruits of Plum Cultivar Resources of Genus Prunus

LIU Shuo, LIU You-chun, LIU Ning, ZHANG Yu-ping, ZHANG Qiu-ping, XU Ming, ZHANG Yu-jun, LIU Wei-sheng

Scientia Agricultura Sinica. 2016, 49(16): 3188-3198. doi:10.3864/j.issn.0578-1752.2016.16.012

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【Objective】 The aim of this study is to provide a basis for the evaluation and utilization of plum cultivar resources by measuring the components of sugars and organic acids in fruits of Prunus species.【Method】The sugar and organic acid contents in 57 representative accessions of plum, including eight Prunus species, four wild types of European plum and three plum × apricot hybrids, were detected using the high performance liquid chromatography (HPLC) method. 【Result】 The highest content of the total soluble sugar, including sucrose, fructose, glucose and sorbitol, existed in Prunus simonii Carr, followed by P. salicina Lindl., P. ussuriensis Kov.et Kost. and P. domestica L., while the lowest existed in P. cerasifera Enrhart. A significant difference in sucrose contents was observed among plum species. No sucrose was detected in P. cerasifera, P. americana Marsh, P. spinosa L. and wild type of P. domestica L., but a middle concentrations in P. domestica, P. nigra Ait. and plum×apricot hybrids, and a high concentrations in P. simonii, P. salicina and P. ussuriensis were detected. The sugar components were similar in P. domestica, wild type of P. domestica and P. spinosa, mainly containing glucose and sorbitol. P. cerasifera contained the highest total organic acid content, followed by wild type of P. domestica and P. spinosa, while P. ussuriensis contained the lowest. The main organic acids in P. domestica were quinic and malic acids, accounting for 93.12% of the total acids, while malic acid was main organic acid, accounting for more than 60% of total acids in other species. A high content of quinic acid was also detected in P. cerasifera, P. americana, P. nigra, P. spinosa and wild type of P. domestica in comparison with malic acids. The first three PCs accounted for 71.26% of total variance, indicating the sugar and acid component profile in Prunus species. 【Conclusion】 In the cultivated species of plum, except P. domestica, sucrose is the main sugar, followed by glucose and fructose, and malic acid is main organic acid components, while in wild Prunus species, fructose and glucose are main sugar components, followed by sorbitol, and malic and quinic acids are main organic acid components. A high glucose and sorbitol in sugar components and a high quinic and malic acids in organic acid components presented in cultivated P. domestica, while the main sugar and organic acid components are sucrose and malic acid in P. salicina, which contributed to the special flavors of P. domestica and P. salicina.

The Mechanism of Myosin Phosphorylation Regulating Actomyosin Dissociation of Skeletal Muscle During Postmortem

GAO Xing, LI Xin, LI Zheng, DU Man-ting, ZHANG Cai-xia, ZHANG De-quan, DING Wu

Scientia Agricultura Sinica. 2016, 49(16): 3199-3207. doi:10.3864/j.issn.0578-1752.2016.16.013

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【Objective】The aim of this study was to investigate the effect of myosin phosphorylation modification on actomyosin dissociation by analyzing the correlation between myosin phosphorylation and actomyosin dissociation, and then reveal the function of myosin phosphorylation to sarcomere length and tenderness during postmortem.【Method】Samples of ovine longissimus dorsi after storage at 4℃ for 6, 24, 48, 72 h were collected for myosin phosphorylation and actomyosin dissociation by SDS-PAGE, Pro-Q Diamond staining and western blotting, actomyosin ATPase activity. And sarcomere length was measured by transmission electron microscope.【Result】The phosphorylation level of myosin light chain 2 decreased sharply from 0.5-48 h (P＜0.05), then it increased from 48-72 h, and the final value was lower than the initial phosphorylation value. While to the dissociation of actomyosin, it displayed a gradual decrease from 0.5-6 h and then increased from 6-48 h (P＜0.05), finally kept stable during 48-72 h postmortem, and the final dissociation degree was significantly higher than 0.5 h. The actomyosin ATPase activity increased to the highest value at 24 h, followed by gradual decrement from 24 to 72 h. On the contrary, the sarcomere length decreased to the shortest value at 24 h, followed by gradual sarcomere relaxation from 24 to 72 h.【Conclusion】The phosphorylation level of myosin light chain 2 had a great influence on myosin and actin interaction, in addition, the sarcomere contraction (myosin and actin interaction force) and the dissociation of actomyosin (myosin and actin interaction amount) happened out of sync during postmortem. Phosphorylation modification of myosin light chain 2 caused the sarcomere shrinkage and relaxation by negative regulating actomyosin dissociation and actomyosin ATPase activity negatively, which could control the final meat tenderness.

The Odor of Rabbit Meat Extracted by Supercritical Carbon Dioxide Fluid Extraction

XIE Yue-jie, HE Zhi-fei, LI Hong-jun

Scientia Agricultura Sinica. 2016, 49(16): 3208-3218. doi:10.3864/j.issn.0578-1752.2016.16.014

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【Objective】The aim of this study was to explore the conditions for extracting the odor of rabbit meat by supercritical carbon dioxide fluid extraction (SFE-CO₂), to determine the species, components and contents of rabbit meat odor and to provide the database for further deodorization and processing industry. 【Method】 SFE-CO₂ technology was employed to extract the odorants in rabbit meat with Box-Behnken response surface analysis based on single factor experiments, using extraction yield as a target. The odorants of rabbit meat were detected by gas chromatograph in tandem with mass spectrum, with 2, 4, 6-trimethylpyridine (TMP) as an internal standard. Calculating odor active value (OAV) of the detected matters, and combining with gas chromatography olfactory (GC-O) and sensory evaluation, the main contents of rabbit meat odor was determined.【Result】The results of single factor experiment indicated that the extraction yield could reach 97.91% and the peak areas were 1.58×10⁹ when the extraction temperature was 40℃. When the extraction time was 4 hours, the extraction yield and peak areas were 97.83% and 2.42×10⁹, respectively. The extraction rate reached 97.78% with peak areas of 3.78×10⁸ when the extraction pressure was 25 MPa. Among the three factors, the extraction temperature and extraction pressure not only affected the solute diffusion coefficient, but also the density of CO₂ fluid. The diffusion coefficient increased when the extraction temperature was raised, but the extraction yield decreased due to the decreased CO₂ fluid density. Although the density of CO₂ fluid was larger at higher extraction pressure, the compressibility decreased. Hence, the extraction yield decreased due to the reduction of diffusion coefficient and solubility. When the extraction time was too long, the proportion of the odorants was reduced, for some non-volatile compounds were extracted, with the total amount increased. According to the response surface analysis, it was concluded that the optimal extraction temperature, extraction pressure and extraction time were 40.67℃, 25.67 MPa and 3.13 h, respectively, with extraction yield reached 98.01%. And the actual operation showed that the extraction temperature, extraction pressure and extraction time were 40℃, 25 MPa and 3 h, respectively. Under these conditions, the extraction yield was 98.11%. Through quantitatively adding internal standard substance TMP, GC - MS quantitatively analyzed for rabbit meat volatile flavour compounds of five classes, including aldehydes, acids, esters, heterocyclic compounds and hydrocarbons, a total of 38 kinds of flavor compounds. Comparing the five types of matter content: acids ((1 394.25±3.45) μg·kg^-1) > esters ((569.26±1.23) μg·kg^-1) > hydrocarbon ((471.82±1.11) μg·kg^-1) > aldehyde ((168.46±0.97) μg·kg^-1) > heterocyclic ((86.71±0.64) μg·kg^-1). The key odorants of rabbit meat odor were pentanal, hexanal, hexanoic acid and 2-pentyl furan via calculating OAV. Those four materials with liver off-flavor, grass odor, muttony odor and beany odor respectively were regarded as the main bodies of odor in rabbit meat by GC-O. In addition, the extractant showed rabbit meat odor obviously through sensory evaluation.【Conclusion】The method of SFE-CO₂ which provides a new extraction way for researching rabbit meat odor is feasible. The optimal extraction temperature, extraction time and extraction pressure was 40℃, 3 h, 25 MPa. Pentanal, hexanal, hexanoic acid and 2-pentyl furan were preliminarily regarded as the main bodies of rabbit meat odor.

Measurement of Skin Thickness Along the Whole Body Sites and Association Study with Candidate SNPs on Sus Scrofa Chromosome 7 in Bamaxiang Pigs

HUANG Tao, HUANG Xiao-chang, QIU Heng-qing, YAN Guo-rong, HUANG Yi-zhong, ZHANG Yi-feng, JIANG Jia-cheng, ZHOU Li-sheng, REN Jun, MA Jun-wu, XIAO Shi-jun, HUANG Lu-sheng, YANG Bin, AI Hua-shui

Scientia Agricultura Sinica. 2016, 49(16): 3219-3228. doi:10.3864/j.issn.0578-1752.2016.16.015

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【Objective】Skin thickness at 9 typical sites of the whole pig body were measured in Bamaxiang pig breed, one of the Chinese local miniature pig breeds. There are 46 candidate SNPs located in a major QTL affecting skin thickness on sus scrofa chromosome 7 (SSC7) were genotyped in the Bamaxiang pig population. One aim of this study is to make clear variation rule of skin thickness at different body sites in Bamaxiang pigs. The other aim is to verify the major QTL affecting skin thickness on SSC7 in Bamaxiang pig population by association study in a specific genomic region. Result of this study will contribute to further revealing the genetic mechanism of swine skin thickness in a larger pig population at whole genome level, and increase our understanding about pig skin. 【Method】A totle of 50 Bamaxiang pigs, including 27 females and 23 castrated males, were randomly selected from a Bamaxiang pig population consisting of 319 adult pigs, which were slaughtered at 300 days. The skin with 5 cm² area were cut from 9 body sites of cheek, shoulder, back, loin, rump, chest, belly, armpit and trotter. Skin thickness at these 9 different body sites were accurately measured using digital caliper. The difference of skin thickness between pairwise body sites and the difference between females and castrated males were tested using the basic statistic package of R language. Pairwise Pearson's correlation coefficients were calculated between skin thickness at different body sites, meanwhile their significant tests were done. A total of 46 SNPs located in the region from 34.5 to 36.2 Mb on SSC7 were selected and designed for genotyping on a SEQUENOM MassARRAY SNP Genotyping platform. Combined with the above phenotypes of skin thickness, association studies on the target candidate region were performed by a general mixed linear model using SNPasscoc package of R language. Based on the association results and biological function of genes, positional candidate genes were determined. 【Result】One-way analysis of variance showed that an extreme significant difference with a 2.95×10^-117 probability existed among skin thickness at 9 body sites in Bamaxiang pigs. The thickest body sites were loin and back with skin thickness of (5.15 ± 0.92) and (4.97 ± 0.85) mm, respectively, while the thinnest sites were belly and armpit with skin thickness of (1.77 ± 0.36) and (1.97 ± 0.68) mm, respectively. The body sites with skin thickness from thick to thin in turn were loin, back, shoulder, cheek, rump, trotter, chest, armpit and belly. Skin thickness of castrated male pig at armpit was significantly thinner than female pig with a 0.021 probability. When compared between other pairwise body sites, skin thickness had no significant differences. There was no significant correlation between skin thickness at belly and back, between belly and shoulder, and between belly and cheek. Other pairwise combinations had a significant positive correlation. Association analysis between skin thickness at 9 different body sites and the above candidate SNPs suggested that the major QTL on SSC7 affecting skin thickness was also identified in Bamaxiang pig population. Three most promising SNPs were worthy of being further focused on. SNP (chr7:34856565) showed most significant association with skin thickness at loin (P_cor = 5.15×10^-6), which was also the most significant site associated with skin thickness at shoulder (P_cor = 5.75×10^-6). SNP (chr7:35543837) was the most significant site associated with skin thickness at armpit (P_cor = 3.05×10^-5), at rump (P_cor = 0.010), at chest (P_cor = 0.013) and cheek (P_cor = 0.025), also was the second most significant site with shoulder. SNP (chr7:35573869) was the top significant site associated with back skin thickness (P_cor = 1.17×10^-5), and followed by SNPs (chr7:35543837 and chr7: 34856565). Based on nearest genes of top SNPs and their biological function, ANKS1A and HMGA1 were preliminarily suggested to be positional candidate genes possibly affecting skin thickness in pigs. 【Conclusion】It was the first time to measure skin thickness at 9 different body sites and report the variation rule of skin thickness along the whole body in Bamaxiang pig breed, one of the Chinese local miniature pig breed. Meanwhile the major QTL affecting skin thickness on SSC7 was verified in Bamaxiang pig population, it provided an important clue and base to reveal the genetic mechanism of pig skin thickness in future. ANKS1A and HMGA1 might be positional causal genes affecting skin thickness in pigs, and it needs further verification of biological functional test.

Priority Approaches, Techniques and Models to Sustainably Tap the Grassland Productivity Potential

HOU Xiang-yang

Scientia Agricultura Sinica. 2016, 49(16): 3229-3238. doi:10.3864/j.issn.0578-1752.2016.16.016

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Grassland which covers 41.7% of the land area is the biggest terrestrial ecosystem of China, with an area at about 0.4 billion hm². Grassland is not only the key resources for modern agriculture and animal husbandry, but also the critical defense for country’s ecosystem safety. However, because of the longtime unreasonable exploitation and utilization, the grassland are facing serious problems such as degrading ecological system, decreasing productivity, prominent conflicts between grass and livestock, low efficiency in animal production, etc. Because of the decreasing of the ecological and productive functions, the grassland area has become one of the limited areas for sustainable development strategy of China. Exploiting the potential of grassland production systematically and sustainably and improving the ecological and productive function of the grassland effectively are the key guarantees for reinforcing ecological safety and ecological civilization construction, accelerating the transformation and development of modern animal husbandry industry. There are three approaches to exploit the potential of grassland productivity. First, reconstruction of the degraded grasslands and improvement of the productivity of natural grasslands. Second, construction of the dry land artificial pasture. Third, development of high efficiency grass industry. Methods such as natural restoration, optimized utilization, and artificially development et al can be taken according to the degrading condition of the grasslands. As a result, these methods can accelerate the process of grassland restoration and improve the grassland productivity by 20% to 30%, even up to 40%-50%. Concerning the limitation of water and soil resources, in semiarid areas, the emphases should be put on changing crop land into forage land and developing dry cultivated pasture; breeding good quality forage varieties with the characteristics of drought resistance, cold hardiness, and grazing tolerance by exploiting the potential of native forage genetic resources. Improvement of the usage efficiency of water and soil resources and dry land productive efficiency by developing and using dryland cultivating technology, harvesting and processing technology, soil conservation technology, are all effective technologies. Through these development methods, grassland productivity can be increased by two to three times. In areas with sufficient and balanced water and heat resources, high efficiency water-saving irrigated artificial pasture should be constructed appropriately according to the local hydrological conditions. Assuming that if the productivity of this high efficient irrigated pasture can be improved by 10 to 20 times, the annual forage yield will be increased by 15 to 30 million tons, which is about the yield of 2 to 4 million ha natural grassland. Suggestions were put forward on guarantee of tapping the grassland potential continuous. First, taking full advantage of the driving function of grassland eco-compensation policy. It needs pushing on the transformation of the development mode to encourage the optimized management of grassland with the category of grass-animal balance. Second, carrying out the research and development actions to tap grassland productive potential sustainably. The key research and develop actions including the integration and demonstration of critical techniques to increase natural grassland productivity, the model and demonstration of high efficiency grass-animal husbandry industry techniques in semi farm semi herd areas, the technique choke points in forage production and procession to improve the usage efficiency of forages. Third, constructing modern grass industry demonstration in different areas. By constructing regional demonstration plot according to the grassland characters and areas, and building whole industrial chain, achieve the integration and optimal allocation of all kinds of resources. Forth, cultivating new business entities, improving and perfecting the market system, boosting the development of grass economics.

Effects of Annual Continuous Tillage on Soil Water Conservation and Crop Yield in Lime Concretion Black Soil Farmland

JIN Hai-yang, XIE Ying-xin, LI Meng-da, LIU Yu-juan, HE De-xian, FENG Wei, WANG Chen-yang, GUO Tian-cai

Scientia Agricultura Sinica. 2016, 49(16): 3239-3250. doi:10.3864/j.issn.0578-1752.2016.16.017

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【Objective】 The aim of this study is to select the appropriate tillage practices for improving soil water conservation and increasing crop yield of lime concretion black soil farmland in the summer maize and winter wheat rotation system. 【Method】Under straw returning conditions, the effects of five summer maize-winter wheat annual tillage practices (no tillage-rotary tillage, no tillage-deep tillage, subsoiling tillage-rotary tillage, subsoiling tillage-no tillage, no tillage-no tillage) on soil bulk density, soil water content and grain yield were studied through field location experiment in summer maize and winter wheat rotation system of Huanghuai region, China. 【Result】 Compared with no tillage-rotary tillage, no tillage-deep tillage significantly decreased soil bulk density in 15-25 cm soil layer during the maize harvest period and in 20-35 cm soil layer during the wheat harvest period. Subsoiling tillage-rotary tillage and subsoiling tillage-no tillage significantly decreased soil bulk density in 15-40 cm soil layer during the maize harvest period and in 20-25 cm soil layer during the wheat harvest period. No tillage-no tillage significantly increases soil bulk density in 0-10 cm soil layer during the maize harvest period and in 5-20 cm soil layer during the wheat harvest period. No tillage-deep tillage, subsoiling tillage-rotary tillage and subsoiling tillage-no tillage significantly increased soil water storage in 0-40 cm layer during the maize harvest period, but subsoiling tillage-rotary tillage and no tillage-no tillage significantly decreased soil water storage in 0-40 cm layer during the wheat harvest period. Compared with no tillage-rotary tillage, subsoiling tillage-no tillage increased soil water content in 20-40 cm soil layer during the whole growth period, no tillage-no tillage increased soil water content in 40-160 cm soil layer during the crop harvest period, but subsoiling tillage-rotary tillage decreased soil water content in 40-160 cm soil layer during the wheat harvest period. Compared with no tillage-rotary tillage, subsoiling tillage-rotary tillage and subsoiling tillage-no tillage significantly increased annual grain yield by 7.67% and 10.21%, respectively. No tillage-deep tillage decreased maize yield but increased wheat yield that finally resulted in decrease of the annual grain yield by 0.44%. The grain yield of summer maize and winter wheat decreased by no tillage-no tillage, the annual yield decreased by 2.19%. 【Conclusion】 Subsoiling tillage-rotary tillage and subsoiling tillage-no tillage could decrease soil bulk density, increase grain yield of summer maize and winter wheat. Especially, subsoiling tillage-no tillage could improve the potential of soil water conservation, and also could have the optimum effects on increasing grain yield and economic benefit. Therefore, subsoiling tillage-no tillage can be selected as an appropriate tillage practice in lime concretion black soil farmland.

Development of a Freeze-Dried Kit for Isothermal Amplification Assay of Mycoplasma bovis

WU Tong, LIU Xu, LI Jia-he, YAN Xin-bo, ZHANG Ning, WU Wen-xue

Scientia Agricultura Sinica. 2016, 49(16): 3251-3260. doi:10.3864/j.issn.0578-1752.2016.16.018

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【Objective】Mycoplasma bovis (M. bovis) is a pathogen related to a variety of syndromes of infected cattle, and it spreads widely in the world. For the survey of the epidemiology of M. bovis in China, simple and sensitive kits are needed.【Method】A recombinant plasmid with the uvrC gene of M. bovis was constructed and transfected into TOP10 competent cell, by which a strain of recombinant Escherichia coli was selected. The recombinant plasmid rP-uvrC was extracted, purified and diluted to the concentration of 10⁴copies/μL, which was used as the positive control. Betaine solution and color developing solution (mixture of SYBR Green I and HNB) were prepared to solve the freeze-dried amplification reagent and visualize the result of the amplification reaction, respectively, according to the published reports. Three kinds of freeze-drying protective agent which did not inhibit the amplification reaction were selected from 8 agents generally used to produce vaccine, then they were combined by different ratios to form 27 combinations, and the best combination was selected according to the physical characters. The isothermal amplification reagent made with the best freeze-drying protective agents were transported to the lyophilizer to detect the eutectic point, and the best heating-up time of the first drying, the first and the second drying times were determined. The best lyophilizing curve was selected to lyophilized the isothermal amplification reagent by testing of the physical characters, vacuum and residual water content. The lyophilized isothermal amplification kit consists of one tube of lyophilized isothermal amplification agent, betaine solution, positive control and color developing solution. The sensitivity of the kit was determined with a series of rP-uvrC solutions (10⁰-10⁵ copies), and the specificity was determined with 10⁴ CCU·mL^-1 solutions of M.bovis strains PG-45, HB-1, SD-2, 10⁸ CCU·mL^-1solutions of M. bovirhinis and M. agalactiae, and 10⁸CFU·mL^-1 Pasteurella multocida and Tuberculosis mycobacteria, respectively. The steady of the kit was also evaluated under different temperatures.【Result】Among the 8 freeze-drying protective agents, only trehalose, mannitol and bovine serum albumin did not interfere the isothermal amplification reaction, and the best freeze-drying protective agents composition is 5% trehalose +1.25% mannitol +1.25% bovine serum albumin. The eutectic point was -16℃, and the best heating-up time of the first drying was 3h, the first and the second drying times are 6h and 4h, respectively. The detection limit of the freeze-dried kit is 10 copies, 10⁴ CCU·mL^-1 solutions of M.bovis strains PG45, HB-1 and SD-2 showed positive results, 10⁸ CCU·mL^-1 solutions of M. bovirhinis and M. agalactiae, 10⁸CFU·mL^-1 Pasteurella multocida and Tuberculosis mycobacteria showed negative results. The sensitivity of the kit didn't change even it was stored for 6 months under 20℃ or 10 d under 37℃. So, the shelf life of the freeze-dried kit could be about 24 months.【Conclusion】The above results suggest that the freeze-dried kit has higher sensitivity, specificity and stability, could be used as a point-of-care (POC) test product since it is convenient for storage and very easy for the veterinarians to use in the farms.

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