Table of Content

01 March 2016, Volume 49 Issue 5

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Establishment and Identification of a Normalized Full-Length cDNA Library of Upland Cotton Ji228

CHI Ji-na, CAI Xiao, ZHANG Jian-hong, ZHEN Jun-bo, LIU Lin-lin, TIAN Hai-yan, TANG Li-yuan, LIU Cun-jing, CUI Rui-min, ZHANG Xiang-yun

Scientia Agricultura Sinica. 2016, 49(5):  813-824.  doi:10.3864/j.issn.0578-1752.2016.05.001

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【Objective】 To improve the efficiency of finding random sequence and rare genes that related to fiber development and provide abundant ESTs of upland cotton, we constructed a normalized fiber cDNA library of elite Gossypium hirsutum cv. Ji228 by decreasing copy numbers of the genes of high degree. 【Method】 To construct the original full-length cDNA library, full-length cDNA of 8 to 40 DPA (days post anther) fibers of elite upland cotton Ji228 were recombined with Gateway donor vector pDONR222. A normalized full-length cDNA library of fiber was established by normalization method, and a large number of ESTs were obtained by sequencing. For bioinformatics analysis, alignment, assembling, COG functional annotation and GO annotation were done. 【Result】 A normalized full-length cDNA library of fiber from elite upland cotton Ji228 was established. The capacity was 1.06×10⁷. The titer of primary library was 3.56×10⁶ cfu·mL^-1, and the average insertion size was 1.2 kb. Results of qRT-PCR demonstrated that normalization produced about 1 000 fold average reduction of two high abundant cotton genes. Random selected 2 384 clones were sequenced. 2 169 high-quality ESTs were obtained and assembled into 1 745 unigenes. Homologous alignments showed that approximately 70% of the unigenes had high homology with known genes. COG function classification revealed that COG function annotation mainly involved ‘translation, ribosomal structure and biogenesis’, ‘carbohydrate transport and metabolism’ and ‘posttranslational modification, protein turnover, chaperones’. According to the results of gene GO annotation, the genes that were involved in cellularity, constitution of organelle and cytomembrane had the highest proportion, moreover, the ratios were higher in cellular and metabolic processes, and genes that function in combining and catalysis also had higher proportions. These genes may play an important role in cotton fiber development. 【Conclusion】 In this study, we established a normalized full-length cDNA library of fiber from elite G. hirsutum cv. Ji228. Results of the library quality testing, the degree of homogenization testing and random cloning sequencing showed that the representation of the library and the integrity of the recombinant fragments met the requirements of library construction. The library had high redundancy. The results of one-by-one comparison of some unigenes demonstrated that the clones obtained from our cDNA library constructed in this study contained genes which not only had high homology with known sequences but also with unique or other sequences in Ji228. These results improved the efficiency of finding random sequence and rare genes that related to fiber development and provided abundant ESTs of upland cotton.

Effects of UV-B Radiation on Pollen Tube Growth in Picea wilsonii

LIU Ai-jing, LI Ni, MA Min, HE Jun-min

Scientia Agricultura Sinica. 2016, 49(5):  825-831.  doi:10.3864/j.issn.0578-1752.2016.05.002

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【Objective】 The effects and mechanisms of UV-B on pollen tube growth and cell wall construction were investigated in the gymnosperm, Picea wilsonii.【Method】To determine the effect and suitable radiation density of UV-B on pollen tube growth of gymnosperms Picea wilsonii, the pollen tube length and tube tip width were measured when pollen grains were incubated for 24 h under visible light with different densities of UV-B radiation. To determine the effect of UV-B on the pollen tube cell wall constructions, pollen grains were incubated for 24 h under visible light alone or in conjunction with suitable UV-B radiation density, and the distribution of cellulose and callose stained with calcofluor and aniline blue, respectively in the pollen tube cell walls were observed under fluorescence microscopy. The distribution of acidic and esterified pectins in the pollen tube cell walls were observed under laser scanning confocal microscopy using the immunolabeled monoclonal antibodies LM19 and LM20, respectively. 【Result】 When pollen grains were incubated under visible light with 0, 0.2, 0.4, 0.6 and 0.8 W·m^-2 UV-B radiation for 24 h, the pollen tubes were gradually shortened and many of them exhibited swollen tips with increasing density of UV-B radiation. This indicated that UV-B inhibits pollen tube growth in a density-dependent manner. When UV-B radiation density was increased to 0.4 W·m^-2, the pollen tube growth was significantly inhibited, suggesting that 0.4 W·m^-2 is a sufficient UV-B radiation density to inhibit pollen tube growth of Picea wilsonii. Under visible light alone, cellulose was distributed uniformly in the wall of pollen tubes, callose was uniformly distributed along the tube shank with a very faint distribution in the apical region of pollen tubes. Acidic pectins were deposited along the tube except in the apical region, while esterified pectins were present only at the tube tips. Under conditions of visible light with 0.4 W·m^-2 UV-B radiation, the distributions of cellulose and callose in the apical region of pollen tubes were significantly higher than that in the tube shank. Acidic pectins were mainly deposited in the apical and subapical regions of pollen tubes, while esterified pectins were deposited throughout the wall of pollen tubes.【Conclusion】UV-B radiation inhibits the polarized growth of Picea wilsonii pollen tubes via affecting the normal orientation distribution of construction components of the pollen tube cell wall, such as cellulose, callose, acidic and esterified pectins.

Analysis of the Role of the PMRP Gene in the Accumulation of Chloroplast Starch and Cold Resistance in Arabidopsis thaliana

GAO Meng-zhu, ZHAO Ya-lin, YAN Qing-di, ZHANG Hai-li, WANG Feng-ru, DONG Jin-gao

Scientia Agricultura Sinica. 2016, 49(5):  832-839.  doi:10.3864/j.issn.0578-1752.2016.05.003

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【Objective】The aim of this study was 1) to analyze the role of PMRP (Putative Membrane Related Protein) in the progress of chloroplast development in Arabidopsis, 2) to illuminate the effect of PMRP on the photosynthetic capacity and cold resistance of Arabidopsis, 3) to provide a theoretical basis for improving the photosynthetic performance and enhancement of plant cold-resistance.【Method】PMRP RNAi and over expression vector were constructed and transformed into Agrobacterium tumefaciens EHA105, and then infected the buds of wildtype Arabidopsis(Columbia, Col-0), so we obtained the PMRP RNAi and overexpressing transgenic Arabidopsis plants; with wild type, PMRP RNAi and over-expressing transgenic Arabidopsis thaliana as materials, cell biology method was used to observe the chloroplast structure and starch grain accumulation; The CO₂ infrared analytical method was used to analyze the photosynthetic rate with different PMRP expression in Arabidopsis transgenic plants; Wild-type (Col-0), overexpression PMRP transgenic Arabidopsis and PMRP-RNAi transgenic Arabidopsis were grown at 21℃ with a 16-h-light/8-h-dark cycle. After 21 days, they were transferred to 4℃cold light incubator for 7 d and then cultivated for 1.5 h at 8℃, then were grown at 21℃ with a 16-h-light/8-h-dark cycle again for 7 d, then we analyzed the chilling resistance of PMRP transgenic Arabidopsis; Wild-type (Col-0), overexpression PMRP transgenic Arabidopsis and PMRP-RNAi transgenic Arabidopsis were grown for 14 days and there rosette leaves were used to determine the conductivity of cellular exudate.【Result】PMRP RNAi and overexpressing transgenic Arabidopsis plants were obtained. The photosynthetic rate of rosette leaves in wild-type (Col-0) was 7.3 μmol·m^-2·s^-1, but the photosynthetic rate in the 3 lines of PMRP-RNAi transgenic Arabidopsis plants was 8.8, 7.8 and 8.5 μmol·m^-2·s^-1 respectively, this results showed that the photosynthetic rate was increased with the decreased PMRP expression. Under cold stress condition, the green leaves rate of wild type (Col-0) was 48%, the green leaves rate of the 3 overexpression PMRP transgenic Arabidopsis lines was 48.6%, 47.8%, 49.2% respectively, the green leaves rate of the 3 PMRP-RNAi transgenic Arabidopsis lines was 65.9%, 67.4%, 68.3% respectively. These data indicate that the decreasing expression of PMRP enhanced the tolerance of Arabidopsis to cold stress. After 30 mins treatment in -8℃ condition, the cellular exudate conductivity of the rosette leaves in wild type Arabidopsis was 70.67 μS·cm^-1, the cellular exudate conductivity of the rosette leaves in the 3 lines of PMRP RNAi transgenic Arabidopsis plants was 48.57, 45.40 and 52.10 μS·cm^-1 respectively, this indicated that the decrease of PMRP expression decreased the damage of cell membrane significantly. We analyzed the ultrastructure of the fully expanded rosette leaf of wild-type and PMRP-RNAi transgenic Arabidopsis, imaging results showed that chloroplasts are ellipse in wild type (Col-0) and was approximately circular shape in PMRP-RNAi transgenic Arabidopsis. Under light condition, the starch grains accumulation obviously in both PMRP transgenic Arabidopsis and wild type; but in the dark environment, the accumulation of starch grains in chloroplasts of PMRP-RNAi transgenic Arabidopsis was more than that of wild type Arabidopsis.【Conclusion】The decreased expression of PMRP changes the chloroplast shape from ellipse to round, increased the starch grains significantly, and enhanced the cold resistance of Arabidopsis thaliana.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Effects of Phosphorus on Winter Wheat Population Characteristics, Canopy Intercepted Photosynthetically Active Radiation and Yield Under Certain Nitrogen-Potassium Rates

YUE Jun-qin, ZHANG De-qi, LI Xiang-dong, WANG Han-fang, SHAO Yun-hui, FANG Bao-ting, Lü Feng-rong, MA Fu-ju, QIN Feng, YANG Cheng

Scientia Agricultura Sinica. 2016, 49(5):  840-851.  doi:10.3864/j.issn.0578-1752.2016.05.004

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【Objective】The objectives of this study were to investigate the effect of phosphate (P) fertilizer on winter wheat population development, canopy intercepted photosynthetically active radiation (IPAR) and yield under certain nitrogen and potassic (NK) fertilizer levels and to study the relationship between IPAR and leaf area index (LAI) and provide a theoretical foundation for P fertilizer application.【Method】 Zhengmai7698 was used in this study. Four NK fertilizer combinations were designed: Low nitrogen and low potassium (N₁K₁, N 225 kg·hm^-2+K₂O 150 kg·hm^-2), low nitrogen and high potassium (N₁K₂, N 225 kg·hm^-2+K₂O 225 kg·hm^-2), high nitrogen and low potassium (N₂K₁, N 300 kg·hm^-2+K₂O 150 kg·hm^-2), high nitrogen and high potassium (N₂K₂, N 300 kg·hm^-2+K₂O 225 kg·hm^-2). Five P fertilizer application levels were used under each NK fertilizer combinations: P₀ (P₂O₅ 0 kg·hm^-2), P₁ (P₂O₅ 150 kg·hm^-2), P₂ (P₂O₅ 225 kg·hm^-2), P₃ (P₂O₅ 300 kg·hm^-2) and P₄ (P₂O₅ 375 kg·hm^-2). The dynamics of wheat population, LAI, dry matter accumulation after flowering, IPAR and wheat grain yield were measured and analyzed using Data Processing System (DPS) software.【Result】(1)Under the four NK fertilizer combinations, total tillers per stem, dry matter accumulation after flowering and LAI increased with the P (P₂O₅) fertilizer rate changed from 0 kg·hm^-2 to 225 kg·hm^-2_, peaked at 225 kg·hm^-2_, and decreased thereafter. (2) The response of IPAR to different P fertilizer under certain NK fertilizer combination showed the order as P₂＞P₁＞P₃＞P₄＞P₀. The highest IPAR was found in N₁K₁ among all the NK fertilizer combinations. (3) IPAR was positively related to LAI under the four NK fertilizer combinations. The fitting index between IPAR and LAI were 0.8492, 0.8363, 0.7321, 0.8081 under N₁K₁, N₁K₂, N₂K₁, and N₂K₂ combinations. Correlation analysis showed that there existed a quadratic polynomical relation between yield and LAI. The fitting index was 0.7145. (4)Wheat grain yield benefited from P fertilizer (P₂O₅) application when its rate increased from 0 to 225 kg·hm^-2. N₁K₁ had the highest grain yield. Compared to all other P fertilizer rates, P₂ (P₂O₅ 225 kg·hm^-2) demonstrated the highest grain yield and rate of grain increase.【Conclusion】 N₁K₁P₂ (N 225 kg·hm^-2+ K₂O 150 kg·hm^-2 + P₂O₅ 225 kg·hm^-2) was the best NPK combination, which resulted the best canopy structure, optimal LAI, highest dry matter accumulation after flowering, enhanced IPAR and the highest grain yield.

Regulations of Reduced Chemical Nitrogen, Potassium Fertilizer Application and Organic Manure Substitution on Potato Water- Fertilizer Utilization and Biomass Assimilation Under Whole Field Plastics Mulching and Ridge-Furrow Planting System on Semi-Arid Area

ZHANG Xu-cheng, YU Xian-feng, WANG Hong-li, HOU Hui-zhi, FANG Yan-jie, MA Yi-fan

Scientia Agricultura Sinica. 2016, 49(5):  852-864.  doi:10.3864/j.issn.0578-1752.2016.05.005

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【Objective】Chemical nitrogen fertilizer reduction, potassium fertilizer application and organic manure substitution are useful methods to increase crop resource use efficiency, enhance agro-environment, improve quality of agricultural products and decrease the disease risk in China. The understanding on the effects of reduced chemical nitrogen fertilizer dressing, organic manure substitution and potassium fertilizer application on potato dry matter assimilation and water utilization, are helpful to apply the efficient managing strategy of water and nutrient resource.【Method】A 4-year field experiment was carried out from 2011 to 2014 with three treatments: (1) traditional chemical fertilizer application (PM), (2) chemical nitrogen fertilizer reduced by 25% and dressing at flowering stage, and potassium fertilizer application (PMN), and (3) chemical nitrogen fertilizer reduced by 50% and organic manure substitution, and potassium fertilizer application (PMO). The soil moisture, potato biomass and yield had been investigated, crop water consumption, potato growth rate, water use efficiency (WUE) and partial factor productivity from applied fertilizer (PFP) were calculated to understand the effects of different nutrient management methods on potato dry matter assimilation and water-nutrient use efficiency, as well as its regulations on potato water use process on semiarid rain-fed area.【Result】The potato water consumption decreased by 17.4, 28.7, 26.8, 34.2 mm in pre-flowering stage, but increased by 31.1, 34.7, 36.7, 49.2 mm in post-flowering stage in PMN treatment, compared with PM. PMO increased the potato water consumption by 17.8, 24.3, 11.2, 10.3 mm than PM in post-flowering stage, but had no significant effect in the pre-flowering stage. Compared with PM, PMN increased potato above- and under- ground biomass, as well as the growth rate after early blooming, caused 2 595.1 kg·hm^-2 tuber yield increment from 2012 to 2013 averagely; the water use efficiency (WUE) increased by 14.4% and 6.3% in 2013 and 2014, the significant difference observed in these years. PMO significantly increased potato above- and under- ground biomass, and the growth rate, resulted in 2 945 kg·hm^-2 tuber yield increment in 4 experimental years on average. Its WUE was significantly higher than PM from 2012 to 2014. PMN and PMO increased potato fertilizer partial factor productivity from applied fertilizer (PFP_T), chemical fertilizer partial factor productivity from applied fertilizer (PFP_C), nitrogen partial factor productivity from applied fertilizer (PFP_TN) and chemical nitrogen partial factor productivity from applied fertilizer (PFP_CN) as compared with PM. It was indicated that PMN and PMO increased nutrient and water use efficiency synergistically, both realized the objective to regulate water utilization by fertilizer application, and improve nutrient utilization by water management. In 2011 (dry year), PMN and PMO regulated the water consumption between pre- and post- flowering stage, increased above- and under- ground biomass, growth rate and PFP, but the potato tuber yield and WUE didn’t increase significantly.【Conclusion】Both PMN and PMO regulated potato water consumption between pre- and post- flowering stage significantly, and increased potato biomass and growth rate, resulted in increased tuber yield, WUE and PFP. However, PMO showed more significant effects than PMN on the increment of tuber yield, PFP and WUE, so it was the more efficient nutrition management model for higher resource use efficiency and yield under whole field plastics mulching and ridge-furrow planting system on semi-arid areas.

PLANT PROTECTION

Construction and Application of a Yeast Two-Hybrid cDNA Library from Rice Spikelets Infected with Ustilaginoidea virens

WANG Yu-qiu, LI Guo-bang, YANG Juan, LI Liang, ZHAO Zhi-xue, FAN Jing, WANG Wen-ming

Scientia Agricultura Sinica. 2016, 49(5):  865-873.  doi:10.3864/j.issn.0578-1752.2016.05.006

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【Objective】The occurrence of rice false smut disease is increasing in rice production worldwide, but the underlying mechanism of Ustilaginoidea virens-rice interaction is still unclear. The objective of this study is to construct a yeast two-hybrid library from rice spikelets infected with U. virens, and apply it for screening interacting proteins of a U. virens effector Uv_1261, so as to provide valuable resource for studying the mechanism of U. virens-rice interaction. 【Method】A susceptible indica rice Chuannong H2S was challenged with the inoculum of a GFP-tagged U. virens strain P4. Infected rice spikelets were collected after confirmation by a confocal laser scanning miscroscope at 13 days post inoculation (dpi), when pathogen hyphae extended into inner floral organs of rice spikelets. The total RNA was isolated from U. virens-infected spikelets, and used for synthesizing the first-strand cDNA by Oligo (dT) primer. ds cDNA was amplified by PCR and purified by agarose gel electrophoresis before mixed with vector pGADT7-Rec. The yeast two-hybrid cDNA library was constructed by homologous recombination technology in yeast line Y187. Then Uv_1261 bait vector was constructed and subjected to self-activation verification. Then the library was screened, and the blue colonies that grew well in medium containing SD/-Ade/-His/-Leu/-Trp/X-α-Gal were selected as candidate interacting proteins of Uv_1261. After sequencing confirmation, these candidates were annotated and classified by Blast and gene ontology (GO) analyses. 【Result】The cDNA library titer was 5.7×10⁸ cfu/mL and the average length of the insertions was 750 bp, indicating that this library is of high quality. Yeast transformation analysis showed that Uv_1261 had no self-activation. Screening for the library with Uv_1261 revealed 56 candidate interacting proteins including 28 proteins in U.virens, 16 protiens in rice and 12 unknown proteins. The candidate interacting proteins from U. virens were involved in 17 biological processes including translation, metabolic process, oxidation-reduction process, cellular amino acid biosynthetic process, etc; or possessed molecular functions such as metal ion binding, hydrolase activity, ATP binding, etc; or were associated with cell components like ribosome, cytoplasm, mitochondrion, etc. Uv_1261-interacting proteins from rice were involved in 11 biological processes including protein dephosphorylation, carbohydrate metabolic process, translation, etc; or possessed molecular functions such as metal ion binding, nucleotide binding, transferase activity; or were associated with cell components like ribosome, membrane, nucleus, etc. 【Conclusion】The cDNA library has good quality, and is suitable for identifying interacting proteins of U. virens effectors. This work can provide a valuable resource for studying molecular interaction between U. virens and rice.

Effects of Fresh and Composted Ageratina adenophora on Physiology of Three Solanaceae Vegetables and Yield and Quality of Pepper

JIAO Yu-jie, SANG Yu-jie, YANG Lei, WANG Ya-qi, WU Ye-kuan, DU Ru-wan, YUAN Ling

Scientia Agricultura Sinica. 2016, 49(5):  874-884.  doi:10.3864/j.issn.0578-1752.2016.05.007

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【Objective】 Ageratina adenophora is a noxious invasive weed and widely spread in southwest China. This weed can’t be put into filed directly because of their strong vitality and reproduction by roots, stems, and seeds. The objective of this study is to research the effects of fresh and composted A. adenophora on physiology of three Solanaceae vegetables (Capsicum annuum, Solanum lycopersicom and Solanum melongena), and yield and quality of pepper in order to provide technical support for harmless treatment and resource utilization. 【Method】 A microbial inoculum made from Pseudomonas putita sp. and Clostridium thermocellum sp., the bacteria insensitive to the toxicity of A. adenophora, was used to compost this invasive plant materials in fields. Then pure culture experiment was conducted to study the effects of the extracts from both fresh and decomposed A. adenophora on seed germination, young seedling growth, seed inclusions (protein, free amino acid, starch, soluble sugar, total phosphorus, and soluble phosphorous), and some physiological indexes (nitrate reductase activity, chlorophyll concentration, and root activity) of young seedlings. In addition, a field experiment was carried out to investigate the yield and quality of pepper in the composted A. adenophora (OF, organic fertilizer) and inorganic fertilizer treatments (CK, without fertilizer; CF, chemical fertilizer; CF+OF, chemical fertilizer plus organic fertilizer).【Result】In the pure culture experiment, the inhibition of seed germination and seedling growth were intensified as the concentration of the extract from fresh A. adenophora (EFA) increased in contrast to the extract from composted A. adenophora (EDA) which behaved otherwise. The seed germination rate was increased by 2.48%-17.78% and seedling height by 51.28%-105.02% following seed treatment with EDA compared with sterile water. Simultaneously, the concentrations of protein and starch in seeds increased with EFA concentrations increased in seed soaking treatments, despite of no significant changes in total phosphorus. In the same case, however, the concentrations of free amino acid, soluble sugar, and soluble phosphorus decreased. Opposite effects was found on free amino acid, soluble sugar, and soluble phosphorous in seeds with EDA soaking treatment. Nitrate reductase activity was decreased by 66.67%-73.17%, chlorophyll by 17.79%-80.57%, and root activity by 41.52%-61.15% in the young seedlings which came from the seeds treated with 100 mg·L^-1 EFA compared with the sterile water. Providing same concentration of EDA to treat the seeds significantly increased these three physiological index values, with nitrate reductase activity showing 88.89% of maximum increment, followed chlorophyll concentration (73.61%), and root activity with 95.82%, respectively. Therefore, EFA inhibited significantly the hydrolysis of protein, starch and inositol phosphates in endosperm, resulting in low seed germination rate. The depression of chlorophyll concentration, root activity and nitrate reductase activity in seedlings by EFA could be unbeneficial to photosynthesis, nutrient uptake, and nitrogen assimilation, which might lead to the inhibition of seedling growth. On the other hand, EDA promoted the hydrolysis of high molecular metabolites in endosperm, by which increased soluble inclusions in seeds, and also enhanced chlorophyll concentration, root activity and nitrate reductase activity in young seedlings. In the field experiment, the fruit yield of pepper changed in the sequence: CF+OF > CF ≈ OF > CK. The fruit yield of pepper received CF+OF was increased by 14.42% compared to CF. The best fruit quality was also obtained by supplying the pepper with CF+OF because of the highest free amino acids and vitamin C but lower nitrate. 【Conclusion】The composting A. adenophora with microbial inoculum eliminated the physiological inhibition of seed germination and young seedling growth produced by EFA. Application of composted A. adenophora in combination with chemical fertilizer increased pepper yield and improved the fruit quality. Composting A. adenophora realized harmless treatment and resource utilization, which provided an organic fertilizer resource for the local vegetables cultivation.

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

Characteristics of Nitrogen Pools and Nitrogen Balance in Soil Profile in Typical Spring Maize Planted Regions in Northeast China

WANG Na, FAN Mei-rong, LIU Shuang-quan, ZHAO Shi-cheng, ZHAO Ying, QIU Shao-jun, HE Ping, ZHOU Wei

Scientia Agricultura Sinica. 2016, 49(5):  885-895.  doi:10.3864/j.issn.0578-1752.2016.05.008

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【Objective】The characteristics of nitrogen pools in 0-90 cm soil depth and the nitrogen balance was elucidated to make clear the potential of nitrogen immobilization under the current farmers’ fertilizer practices in typical spring maize planted in northeastern China.【Method】Seventeen sites in each of the provinces of Heilongjiang, Jilin, and Liaoning, adding up to 51 sites, were observed on the farmer’s agricultural systems during the whole growth period of spring maize in the year of 2012. The soil total nitrogen (TN), mineral nitrogen (NO₃-N, NH₄-N), particulate organic nitrogen (PON), microbial biomass nitrogen (SMBN), and dissolved organic nitrogen (DON) in 0-30, 30-60, 60-90 cm soil depth and spring maize yield were determined, and nitrogen balance between before the maize was sown and after the maize was harvested was calculated.【Result】The TN stock of 0-90 cm soil depth in Heilongjiang, Jilin, and Liaoning was 357.9, 286.9, and 218.1 kg·hm^-2 respectively in typical spring maize planted in northeastern China. The mean of TN stock in each province was significant (P＜0.05). In 0-30 cm soil depth, the mean of TN concentration in Heilongjiang, Jilin, and Liaoning province was 1.4, 1.0, 0.7 g·kg^-1, respectively; moreover, the mean of TN concentration in each province was significant (P＜0.05). In 30-60 cm or 60-90 cm soil depth, the mean of TN concentration in Heilongjiang, Jilin, and Liaoning provinces were 0.9, 0.6, 0.4 g·kg^-1and 0.6, 0.4, 0.3 g·kg^-1, respectively, and in either soil depth, the mean of TN concentration in Heilongjiang was significantly (P＜0.05) higher than that in Liaoning. In 0-30 soil depth, the difference of the mean of PON concentration, PON/TN, SMBN/TN among Heilongjiang, Jilin, and Liaoning provinces increased and SMBN concentration decreased as latitude decreased, and the difference of the mean of PON concentration, PON/TN, or SMBN concentrations at each province was significant (P＜0.05) respectively. The difference of the mean of SMBN/TN between Heilongjiang and Liaoning provinces was significant (P＜0.05). In 30-60 cm soil depth, the mean of PON/TN among Heilongjiang, Jilin, and Liaoning provinces increased as latitude decreased, and the difference of the mean of PON/TN at each province was significant (P＜0.05). The mean of PON concentration in Heilongjiang province was significantly (P＜0.05) lower than that in Jilin or Liaoning province, and the mean of DON concentration in Heilongjiang province was significantly (P＜0.05) higher than that in the other two provinces. The mean of DON/TN in Jilin province was significantly (P＜0.05) lower than that in Liaoning province. In 60-90 cm soil depth, the mean of SMBN or SMBN/TN in Jilin province was significantly (P＜0.05) higher than that in Heilongjiang and Liaoning provinces, and the mean of DON/TN in Heilongjiang was significantly (P＜0.05) lower than that in Jilin or Liaoning provinces. On the whole, total nitrogen and soil active nitrogen pools at each province decreased as the soil depth increased. The mean of NO₃-N in Liaoning province in 0-30 cm soil depth was significantly lower than that in Heilongjiang or Jilin province, and the mean of NH₄-N in Heilongjiang and Jilin was significant (P＜0.05). The mean of NO₃-N in Jilin province in 30-60 cm or 60-90 cm soil depth was significantly (P＜0.05) higher than that in Heilongjiang or Liaoning province. Because of the highest nitrogen rate in Jilin province among the three provinces, nitrogen balance in Jilin province was significantly (P＜0.05) higher than the other two provinces, and Jilin province showed N surplus, while Heilongjiang and Liaoning province showed a balanced N on the whole. In the year of 2012, the mean of the yield of maize in Heilongjiang, Jilin, and Liaoning provinces was at 11.9, 11.3 and 10.8 t·hm^-2 respectively, and the average yield in Heilongjiang province was significantly higher than the other two provinces.【Conclusion】 There wasn’t completely consistency in the increased or decreased trend between total nitrogen and soil active nitrogen pools among Heilongjiang, Jilin, and Liaoning provinces; the yield maintained 11.0 t·hm^-2 in northeastern China on the whole, and the negative environmental effect of nitrogen in the spring maize planted region in Jilin province was relatively higher than the other two provinces.

An Investigation on Optimal Irrigation and Nitrogen Rates of Greenhouse Tomato Based on Spatial Analysis for High Yield and Quality

YANG Hui, CAO Hong-xia, LI Hong-zheng, GUO Li-jie, DU Ya-dan

Scientia Agricultura Sinica. 2016, 49(5):  896-905.  doi:10.3864/j.issn.0578-1752.2016.05.009

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【Objective】The objectives of the study were to investigate the effects of different water and nitrogen supply rates on fruit yield and quality of tomato (M6088), irrigation water and nitrogen use efficiency，and to obtain the best combination of irrigation and nitrogen for maximum tomato production when a single indicator was considered and the optimal range of water and nitrogen rates for high yield and quality tomato production. 【Method】A pot experiment was conducted in greenhouse at the Key Laboratory of Agricultural Soil and Water Engineering in Arid and Semiarid Area of Ministry of Education in Northwest A&F University in 2013. The treatments comprised three irrigation upper limitations (W1, 70%θ_f; W2, 80%θ_f; W3, 90%θ_f, θ_f is the field capacity), and three nitrogen rates (N1, N 0.24 g·kg^-1; N2, N 0.36 g·kg^-1; N3, N 0.48 g·kg^-1). And the treatments were replicated fifteen times in random complete block designs. The multivariate regression analysis was used to obtain the best combination of irrigation and nitrogen for maximum tomato production when a single indicator was considered. The spatial analysis method was further used to comprehensively evaluate the tomato yield and quality to seek optimal range of water and nitrogen rates.【Result】The amount of irrigation and rate of nitrogen significantly affected the fruit yield, fruit quality, irrigation water productivity (IWP, in kg·m^-3, kg fruit produced per cubic metre water irrigated) and partial factor productivity of nitrogen (PNP). The fruit yield per plant increased first and then decreased with the irrigation and nitrogen rates were increased (except for N1, W1 treatments). Increasing irrigation amount remedied production reduction caused by nitrogen deficiency under N1 condition; increasing nitrogen rate relieved restrain from drought on fruit yield under W1 condition. Yield and the water and nitrogen supply exhibited a binary quadratic regression relationship. The maximum yield (1 599.4 g/plant) was achieved when the irrigation and nitrogen rates were 62.3 L/plant and 0.3864 g·kg^-1 soil, respectively. When the irrigation and nitrogen rates increased constantly, the tomato yield decreased. The IWP increased with the decreasing irrigation amount and increasing nitrogen rate, while the PNP was in contrast. The rates of water and nitrogen significantly influenced fruit quality parameters. With the increasing of nitrogen rate, soluble solids and organic acid content increased while vitamin C, lycopene and soluble sugar content and sugar acid ratio first increased and then decreased. Soluble solids and vitamin C content decreased with increasing irrigation amount (excepted for N2 treatment). Lycopene and soluble sugar content and sugar acid ratio first increased and then decreased with increasing irrigation amount (except for N1 treatment). The entropy weight method was used to determine the subjective weight of single tomato quality attributes. Results indicated that the attributes were ranked based on their importance by lycopene＞soluble sugar＞sugar-acid ratio＞organic acid＞Vc＞soluble solids. 【Conclusion】Through the multivariate regression analysis and the spatial analysis methods, the results showed that acceptable yield and quality of tomato (≥95% of corresponding maximum values) were obtained within the range of irrigation upper limitation 80% θ_f and nitrogen rate 0.34-0.44 g·kg^-1 soil.

Optimized Fertilization Effects and Environmental Benefits Evaluation of Nitrogen and Phosphorus in the Paddy Soil

ZHANG Zhi, LI Xiao-kun, CONG Ri-huan, REN Tao, HUANG Tie-ping, LU Yan-hong

Scientia Agricultura Sinica. 2016, 49(5):  906-915.  doi:10.3864/j.issn.0578-1752.2016.05.010

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【Objective】 Environmental issue caused by unreasonable fertilization has been widely noticed in the paddy field. Yield increase potential and nutrient surface balance in the plant-soil system were analyzed, aiming to achieve scientific fertilizer principle and management strategy from the view of yield productivity and environmental benefit. 【Method】 A total of 735 site-year fertilization experiments were conducted on early rice, middle rice and late rice in Hunan province under the Project of Soil Testing and Fertilizer Recommendation during the period of 2005-2010. Three treatments were selected, control (i.e., CK), farmer’s practice (i.e., FP), and optimum fertilization (i.e., OPT). The yield increment and partial factor productivity (PFP) of nitrogen (N), phosphorus (P) and potassium (K) between the OPT and FP treatments for early, middle and late rice were analyzed. Three groups (i.e. high-yield, high-efficiency and high-yield & efficiency) were divided through screening the 75-100th percent of the yield and/or PFP_N for the OPT treatment. Evaluation of N saving and emission reduction in the paddy soil were combined the empirical model between N rates and N losses (i.e., nitrous oxide emission, N leaching and runoff, and ammonia volatilization). Apparent balance of P for early, middle and late rice were calculated to analyze the P residuals. In addition, the planting areas of early, middle and late rice in Hunan province were applied to assess the regional environmental benefit of N and P. 【Result】 Compared with the CK treatment, rice (average of early, middle and late rice) yield increment of FP and OPT treatments were 41.9% and 52.0%, respectively. And yield increase rate reached 7.0%-8.3% for the OPT treatment as compared with FP treatment for early, middle and late rice. Compared with the FP treatment, the optimum fertilization improved the PFP of N and P fertilizers by 16.8%-19.6% and 5.5%-37.3%, respectively, and slightly decreased (6.4%) in PFP_K since K rates were increased in the OPT treatment. In general, the high-yield group had high yield but also high N rate, and the high- efficiency group got low N rate but also low yield. The high-yield & efficiency group was proper to estimate the potential of yield and efficiency. Under the group of high-yield & efficiency, N fertilizers would be saved by 81 kg N·hm^-2 annually (32 kg N·hm^-2 for late rice), nitrogen loss decreased by 15 kg N·hm^-2, and nitrous oxide emission reduced by 20%. Total N saving potential would be 12.5×10⁴ t, mainly contributing by early rice and late rice, with the emission reduction potential of 2.35×10⁴ t. Optimum fertilization improved the total P uptake of rice from 59-66 kg·hm^-2 (FP treatment) to 63-71 kg·hm^-2 (OPT treatment). However, total P uptake of rice was not improved by increasing P rate. Clearly, when phosphorus rate meets the requirement of rice, surplus P would be residual in soil. There were 46%, 44% and 15% of the samples appeared to P residual for early, middle and late rice, respectively. The ratio of P residual for OPT treatment was obviously lower than that of the FP treatment. Phosphorus residual amounts of early, middle, and late rice for OPT treatment can be dropped to 1.36-5.30 kg P₂O₅·hm^-2, which were 33.7%-48.5% lower than the FP treatment. For the whole province, the total phosphorus residual of early, middle, and late rice would be decreased by 18.14×10³, 3.59×10³ and 5.30×10³ t. In the other hand, no P application was still exist in the farmer practice. Balance fertilization is very important for crop growth and soil nutrient maintaining. 【Conclusion】 Optimized fertilization is an important guarantee for food and environment security. Applying lower nitrogen fertilizer would improve nitrogen use efficiency, decrease nitrogen loss and produce less greenhouse gases. Controlling total phosphorus fertilization rate would reduce the pollution risk of soil phosphorus residual.

HORTICULTURE

Overcoming Continuous Cropping Obstacles—The Difficult Problem

LI Tian-lai, YANG Li-juan

Scientia Agricultura Sinica. 2016, 49(5):  916-918.  doi:10.3864/j.issn.0578-1752.2016.05.011

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Differential Protein Analysis of Tomato Leaves in Tomato-Potato Onion Intercropping System

LIU Shu-qin, ZHOU Xin-gang, WU Feng-zhi, LIU Shou-wei

Scientia Agricultura Sinica. 2016, 49(5):  919-932.  doi:10.3864/j.issn.0578-1752.2016.05.012

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【Objective】The objective of this study was to better understand the soil sickness mechanism of the tomato intercropped with four different allelopathic potential potato onions which has high yield and less plant diseases, obtain the differential expressed proteins of tomato leaves in tomato-potato onion intercropping system, and improve the foundation and extend of tomato cultivation mode.【Method】 A comparative proteome analysis was conducted to study the impact of tomatoes under normal monoculture and intercropping with four varieties of potato onion with different allelopathic potential (high allelopathic potential cultivars: ‘Suihua’, ‘Wuchanghongqishe’, and low allelopathic potential cultivars: ‘Ninganhongcheng’, ‘Qitaihe’). Total proteins were extracted from these tomato leaves, and subjected to two-dimensional electrophoresis, followed by MALDI-TOF-MS mass spectrometry. Types and functions of proteins differently expressed in the leaves were analyzed.【Result】The results showed that 39 proteins differently expressed in the leaves of intercropped tomato. These proteins were divided into main 7 categories, including proteins related to photosynthesis, metabolism, energy metabolism, plant resistance, protein synthesis, nucleic acid synthesis and unknown protein. The proteins showing a higher abundance were primarily involved in 11 photosynthesis proteins, 10 metabolic proteins and 8 plant resistance proteins discovered. The photosynthesis of intercropped tomato enhanced and resistance also increased. The expression levels of heat shock-related protein, thioredoxin peroxidase, polyphenoloxidase, sedoheptulose-1,7- bisphosphatase, S-adenosylmethionine synthase, ATP-binding protein increased in the leaves of intercropped tomato. Interestingly, the expression levels of these proteins from the tomato intercropped with potato onions with high allelopathic potentials were significantly higher than that intercropped with potato onions with low allelopathic potentials.【Conclusion】Our results showed that potato onion with different allelopathic potentials intercropped have different influences on tomato at the protein expression. The tomato intercropped with potato onions with high allelopathic potentials were stronger than tomato intercropped with potato onions with low allelopathic potentials, and is an effective way for promoting tomatoes healthy growth and better plant resistance．

Effects of Garlic Straw Application on Controlling Tomato Root-Knot Nematode Disease and Rhizospheric Microecology

GONG Biao, ZHANG Li-li, SUI Shen-li, WANG Xiu-feng, WEI Min, SHI Qing-hua, YANG Feng-juan, LI Yan

Scientia Agricultura Sinica. 2016, 49(5):  933-941.  doi:10.3864/j.issn.0578-1752.2016.05.013

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【Objective】 The effects of garlic straw application on controlling tomato root-knot nematode disease and rhizospheric microecology have been studied with the purpose of showing comprehensive effects among plants, root-knot nematode disease and rhizospheric microecology. This study will provide the theoretical and technical basis for the exploitation of garlic straw. 【Method】 This study was performed with five treatments including 0 (CK), 1% (T1), 2% (T2), 4% (T3) and 8% (T4) garlic straw application (w/w) in pots (both the diameter and depth are 20 cm) with 5 kg garden soil. Then, we planted one tomato seedling in each pot and cultivated them in a greenhouse (28-32℃/15-23℃). Seven days later, five holes were dug around the tomato seedlings and inoculated about 5000 root-knot nematode J2 per seedling. There are 30 pots in each treatment. Tomato root and rhizospheric soil by dithering at the 10, 30, and 50th days after treatment were collected and then we determined the biomass of each tomato plant; disease index including numbers of root-knot nematode, numbers and ratios of female, numbers of egg masses, eggs index, reproduction index, galling index and relative control efficiency; activities of soil enzymes including urease, phosphatise, sucrose, cellulose, catalase and polyhenoloxidase; microbial population of bacteria, fungi and antinomyces, numbers of different nematode trophic groups including phytophagous nematodes, bacterial-feeding nematode, fungal-feeding nematodes and omnivorous-predator nematodes. 【Result】 With the increasing consumption of garlic straw, the biomass of tomato plant firstly increased and then decreased, among which, T1 treatment was little higher than CK and T2 treatment was lower than CK. However, the relative control efficiency was increased with the increasing consumption of garlic straw, reaching 13.6%, 50.0%, 72.7% and 81.8% separately. The ratio of females was decreased by applying garlic straw, which decreased the numbers of egg masses, egg index and reproduction index. The activities of urease and phosphatise were decreased in garlic straw treatments. However, the activities of cellulose, invertase, catalase and polyphenoloxidase in rhizospheric soil were increased. In addition, application of garlic straw significantly increased the numbers of bacterium, fungi and actinomycetes. The numbers of bacterium firstly increased and then decreased, and the maximum value could be observed in T2 treatments. However, numbers of fungi and actinomycetes were increased with the increasing consumption of garlic straw. Application of garlic straw significantly decreased the numbers of phytophagous nematode, but increased the numbers of bacterial-feeding and omnivorous-predator nematode. The increase of the fungal-feeding nematode was not significant. 【Conclusion】 Comprehensive consideration of the growth of tomato plants, the protection of root-knot nematode disease and rhizospheric microecology, this study indicated that T2 garlic straw application was better than other treatments.

Analysis of Soil Microbial Community Structure and Diversity in Cucumber Continuous Cropping Nutrition Medium by Biolog-ECO

ZOU Chun-jiao, QI Ming-fang, MA Jian, WU Chun-cheng, LI Tian-lai

Scientia Agricultura Sinica. 2016, 49(5):  942-951.  doi:10.3864/j.issn.0578-1752.2016.05.014

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【Objective】 As an effective way to alleviate a continuous cropping obstacle, nutrition medium cultivation has been widely used. However, cropping obstacles still happen in nutrition medium cultivation after years of cultivation. Because of insufficient study on the mechanism and repair methods, replacement of the nutrition medium is the main method to solve the problem of a continuous cropping obstacle, resulting in the wasting of resources. The objectives of this study were to analyze the soil microbial community structure and the diversity of the nutrition medium, in order to explore the mechanism of a continuous cropping obstacle and find a reasonable method of repairing a continuous cropping obstacle of nutrition medium.【Method】 An experiment of continuous cropping of cucumbers in a nutrition medium (made from straw, soil, and chicken manure) was conducted in a greenhouse in order to study the microbial community structure and diversity of the nutrition medium microenvironment. The Shannon-Wiener diversity index, the Simpson dominance index and the McIntosh index were calculated by the biology-ECO method to reveal the microbial community structure and diversity. In addition, the changes of the microbial community structure and diversity in different rotation nutrition mediums were analyzed by the principal component analysis method. 【Result】 With the increase of rotation, the average yield of cucumber reached a peak in continuous cropping stubble 5, and that in continuous cropping stubble 11 was significantly lower than in stubble 1. As for the pH value of a continuous cropping cucumber nutrition medium, it was gradually reduced with the increase of rotation, and it was significantly lower in continuous cropping stubble 13 than in stubble 1. The content of organic carbon, available nitrogen, phosphorus, and potassium of the continuous cropping cucumber nutrition medium reached the highest level at stubble 5. The available nitrogen and available potassium content in stubble 11 were significantly lower than in stubble 1, and the organic carbon and the available phosphorus content in stubble 13 was significantly lower than in stubble 1. The principal component analysis of the microbial community carbon metabolism showed that microbial carbon source utilization was significantly different after continuous cropping stubble 7. The Shannon-Wiener index, McIntosh index, and Richness index all reached peak in continuous cropping stubble 5. The Shannon-Wiener index and Richness index in continuous cropping stubble 11 began to be significantly lower than in stubble 1, The McIntosh index in stubble 13 was significantly lower than in stubble 1. Simultaneously, with the increase of rotation, the microorganism metabolic activity decreased significantly when L-Arginine, D-Galactonic acid latone, Tween 40, Tween 80, Pyruvic acid methyl ester, Methyl-D-glucoside, and N-Acetyl-D-glucosamine were used as the carbon source. On the contrary, the activity increased significantly when L-Asparagine, 2-Hydroxy benzoic acid, 4-Hydroxy benzoic acid, Putrescine, and D-Mannitol was the carbon source. 【Conclusion】 The results showed that in continuous cropping stubble 5, the average yield of cucumbers, microbial carbon metabolism, indicators of microbial diversity, and high utilization of single carbon source number were all highest. After continuous cropping stubble 11, the ability of microbial carbon metabolism declined significantly, microbial diversities were significantly reduced, microorganisms of carboxylic acid type of carbon source utilization improved significantly, so it is stated that the microbial population preferred carboxylic acid as a carbon source to get enrichment and microbial community structure appeared simplified.

Effect of Wheat Residues on Growth and Rhizosphere Microorganisms of Continuously Monocropped Watermelon

HAN Zhe, XU Li-hong, LIU Cong, KONG Ling-kun, WU Feng-zhi, PAN Kai

Scientia Agricultura Sinica. 2016, 49(5):  952-960.  doi:10.3864/j.issn.0578-1752.2016.05.015

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【Objective】The microbial community structure was the important element of soil environmental quality in soil. The purpose of this study was to compare the influence of D123 and D125 wheat residues on the growth and rhizosphere microorganisms of watermelon in continuous cropping soil, the relation of Fusarium oxysporum f. sp. niveum and rhizosphere microorganism in soil, to provide a reference for ecological control about watermelon continuous obstacles and watermelon wilt disease. 【Method】The D123 and D125 wheat residues were smashed and mixed in the continuous cucurbites cropping soil. The growth index and yield of watermelon have been measured by conventional methods. Rhizosphere microorganism numbers were determined with a dilution plate method. Abundances of Bacillus and Fusarium oxysporum f. sp. niveum were assessed by real-time PCR. 【Result】The results showed that the vine length of watermelon was promoted after D123 and D125 wheat residues treatment, the fresh weight of over ground root and plant were increased significantly in the later growth stage (P＜0.05), and D125 was higher than D123. The yield per plant was increased after the stubble treatment but was non-significant. Stubble treatment increased the yield per plant of watermelon, but this difference was insignificant. With the statistics of the rhizosphere soil microorganism number, the bacterial population of D125 was remarkably higher than the D123 and control (CK) at 20 d after transplanting (P＜0.05), but the discrepancy was not significant between D123 and CK. D125 treatment bacterial population was remarkably higher than CK at 30 d after transplanting (P＜0.05), there was a non-significant difference between D123 and the CK, D125 and D123 (P＜0.05). The fungus amount researches show that D125＞D123＞CK each period, this rule shows a significant difference at 30 d after transplanting (P＜0.05). The research of Fusarium oxysporum f. sp. niveum number show that D125＞D123＞CK, D125 treatment is significantly higher than D123 and the CK at 30 d after transplanting (P＜0.05), but D123 treatment and CK had no remarkable difference. The B/F specific value had a declining curve along with the sampling period, the multiply speed of bacterial population was lower than fungus and Fusarium oxysporum. On 20 d after transplanting, the B/F specific value of D125 treatment was remarkably higher than D123 treatment and CK in rhizosphere soil (P＜0.05), D123 treatment had non-significant difference with CK. The amount of Fusarium oxysporum f. sp. niveum had a growth trend with the plant growth of watermelon, Fusarium oxysporum assume CK＞D123＞D125 in all periods, the data show that wheat residues significant reduce the amount of Fusarium oxysporum f. sp. niveum at 20 d and 40 d after transplanting (P＜0.05). The Bacillus data indicate that the amount of Bacillus reaching the maximum value at 30 d after transplanting, but it was lower in other periods. Wheat residues increase the number of Bacillus in rhizophere soil contemporaneity, D125 treatment outstanding higher than CK in each period (P＜0.05), and D123 treatment significantly greater than CK at 20 d and 30 d after transplanting (P＜0.05), D125 treatment was salience higher than D123 treatment on 20 d and 40 d after transplanting (P＜0.05), and it showed an adverse result at 30 d after transplanting. 【Conclusion】Wheat residues can promote watermelon growth, increase microbial numbers and the size of the beneficial microflora, and reduce the number of pathogens. Wheat residues can repair soils continuously cropping of watermelon.

Effect of Main Phenolic Acids of the Apple Replanted Soil on the Roots of Malus hupehensis Rehd. Seedlings

YIN Cheng-miao, HU Yan-li, WANG Gong-shuai, ZHANG Xian-fu, ZHOU Hui, SHEN Xiang, CHEN Xue-sen, MAO Zhi-quan

Scientia Agricultura Sinica. 2016, 49(5):  961-969.  doi:10.3864/j.issn.0578-1752.2016.05.016

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【Objective】Effect of soil measured concentrations of phenolic acids and their mixture on the biomass and root system related indexes of Malus hupehensis Rehd. seedlings was studied to provide theoretical basis for the research of the mitigation measures of apple continuous cropping obstacle and the guidance of the old orchard. 【Method】 The test includes the control (CK), T1 (phloretin), T2 (mixture of phenolic acids), T3 (vanillic aldehyde), T4 (phlorizin), T5 (salicylic acid), and T6 (benzoic acid), a total of 7 treatments. Effects of soil concentration of five phenolic acids and their mixture on the biomass, root mitochondria-related indicators, root activity, root antioxidant related enzymes (SOD, POD, CAT), and changes of the tricarboxylic acid cycle (TCA) related aconitase enzymes on seedlings of Malus hupehensis Rehd. 【Result】 Five phenolic acids and their mixture reduced the root and stem fresh weight of the Malus hupehensis Rehd. seedlings and the mixture of phenolic acids (T2) had the most inhibitory effect, which fresh weight were 27.3% and 51.7% as control, respectively. Compared to a single phenolic acid treatment, the largest damage treatment was phloridzin (T4), and the root and stem fresh weight were 27.3% and 51.7% as control, respectively. Treatments that caused damage to the biomass of Malus hupehensis Rehd. seedlings were sorted as: Mixed phenolic acids＞Phlorizin＞Phloretin＞Vanillic aldehyde＞Salicylic acid＞Benzoic acid＞Control. Different phenolic acids treatments reduced the root activity of the Malus hupehensis Rehd. seedlings. With the extension of treatment time, the root activity decreased more significantly. Root activity of T2 reduced the most significant after treatment of 1, 3, 5 d, which were 73.0%, 71.7% and 76.1% as control, respectively. Root activity of T4 reduced the most significant after treatment of 10, 15 d, which reduced from 85.5% to 54.4% as control. Different phenolic acids treatments also reduced the root activity of SOD, POD and CAT. Different phenolic acids treatments could significantly increase the content of malondialdehyde (MDA). With the extension of treatment time, the content of MDA increased more significantly. The MDA of T2 increased the most significant after treatment of 1, 3, 5 d, which were 7.71, 1.66, and 1.66 times as control, respectively. The content of hydrogen peroxide (H₂O₂) increased first and then decreased. The H₂O₂ of T1, T2, T3, T4, T5, T6 increased the most significant after treatment of 3 d, which were 2.50, 2.36, 2.58, 2.59, 2.40 and 2.58 times as control, respectively. Different phenolic acids treatments inhibited the TCA cycle related activity of aconitase and isocitrate dehydrogenase, which consistent declined in root activity. However, different phenolic acids treatments had no effect on fumaric acid enzyme activity. The phenolic acids increased the opening of mitochondrial permeability transition pores, decreased the membrane electric potential and cytochrome c/a. The cytochrome c/a of T2 reduced the most significant after treatment of 1, 3, 5, 10, 15 d, which were 76.0%, 76.9%, 69.1%, 66.4%, 64.2% as control, respectively. Followed by T4 treatment, the cytochrome c/a of T4 at 15 d varied from low to high: Mixed phenolic acids＞Phlorizin＞Phloretin＞Vanillic aldehyde＞Salicylic acid＞Benzoic acid＞Control.【Conclusion】Phenolic acids mixtures, phlorizin and phloretin had greater inhibition effect on the growthof Malus hupehensis Rehd. seedlings. The results showed that phlorizin and phloretin were the main phenolic acids in the replanted apple orchards. When updating the old orchard, a variety of measures can be used to degrade phlorizin and phloretin primarily to ease harm caused by phenolic acids in the apple replant disease.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

The Expression Efficiency of Human IFNα-2b Regulated by β-Casein Gene Promoters from Different Sources

LI Hui, LIU Qing-you, SHI De-shun

Scientia Agricultura Sinica. 2016, 49(5):  970-978.  doi:10.3864/j.issn.0578-1752.2016.05.017

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【Objective】In order to obtain a high efficiency mammary gland specific promoter, in the present study, promote efficiency of β-casein gene promoters derived from Holstein cow, Jersey cow and water buffalo will be comparatively studied.【Method】β-casein gene promoter sequences of Holstein cow, Jersey cow and water buffalo in Genbank were comparatively analyzed, primers of promoter sequences and 3′ployA sequences were designed according to the conservative fragment respectively, primers of the human IFN-2b gene and EGFP-Neo selective marker fragment were also designed. To facilitate the constructing expression vectors, appropriate restriction enzyme digestion sites were introduced into all the primers. Genome DNA was isolated from the leukocytes in venous blood samples. β-casein gene promoter fragments and 3′ployA fragments were amplified by PCR using the genome DNA as templates. Human IFNα-2b gene and EGFP-Neo selective marker fragments were also amplified by PCR using the plasmids conserved in our lab. Sequencing analyses were carried out; making sure that all the fragments were correct. Then, all fragments were inserted into the pMD18-T backbone in the designed order. Finally, 3 mammary gland expression vectors, pHSTBCNp-IFN, pJSBCNp-IFN and pSNBCNp-IFN were constructed. Transgenic cell lines were generated by transfected 3 recombinant plasmids into Bacp-37 cells and G418 selected. Transgenic cell lines were induced by combined hormones including Insulin (1 mg·L^-1), Transferrin factor (1 mg·L^-1), Hydrocortisone (1 mg·L^-1) and PRL (250 IU·L^-1). IFNα-2b expression levels were analyzed at mRNA and protein level by PCR, Western blot, QRT-PCR and ELISA.【Result】β-casein gene promoter and 3′ployA fragments of Holstein cow, Jersey cow and water buffalo were obtained by PCR. The fragments lengths were 5 219, 5 244 and 5 216 bp respectively. In the promoter fragments, the first exon, first intron and the partial second exon were included. 51 nucleotides in the partial second exon encoding 17 amino acid signal peptide. For the 3′ployA fragments were all 1 166 bp. After 3 transgenic expression vectors were generated, and transfected into Bcap-37 cells. After G418 screening, 3 positive transgenic cell lines were obtained successfully. PCR, Western blot, QRT-PCR and ELISA results showed that IFNα-2b was successfully expressed in these 3 transgenic cell lines when hormone induced. Transgenic cell lines contain pJSBCNp-IFN expression vector showed a significantly higher expression level than the other two at mRNA and protein levels (P＜0.05).【Conclusion】Jersey cow β-casein gene promoter possesses a higher efficiency in regulating foreign gene expression, and it is a considerable mammary gland specific promoter in transgenic research. Expression vectors constructed in the present study laid a foundation for constructing IFNα-2b transgenic mammary gland bioreactor.

Effects of 2-methylbutyrate on Digestive Enzymes Activities and Gene Expression of Glucose Cotransporter of Small Intestine in Calves

ZHANG Hang, LIU Qiang, WANG Cong, ZHANG Yan-li, PEI Cai-xia, WANG Yong-xin, GUO Gang, HUO Wen-jie, ZHANG Shuan-lin

Scientia Agricultura Sinica. 2016, 49(5):  979-987.  doi:10.3864/j.issn.0578-1752.2016.05.018

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【Objective】 There are obvious changes observed in the development of the gastrointestine and its function of absorption and metabolism in pre- and post-weaning dairy calves. Especially, the development of the gastrointestine and even its future production performance which are influenced by the feeding and management of calves during the pre-weaning period. 2-methyl butyric acid is a kind of branched-chain short volatile fatty acids, mainly from the degradation of branched chain amino acids in the rumen, can be used as a control agent of ruminant animal gastrointestinal development. The aim of this study was to reveal the 2-methyl butyric acid on development mechanism of the small intestine by observing the effects of 2-methyl butyrate supplementation on the small intestine digestive enzymes activities and expression of growth hormone receptor (GHR) and sodium-glucose cotransporter (SGLT1) mRNA of small intestinal mucosa in dairy calves. 【Method】Thirty-two Holstein male calves (15-day-old) with similar body weight (44.7±0.3) kg were chosen and divided into four groups randomly. Calves in the control were fed milk in pre-weaning and calf-concentrate and alfalfa hay in the post-weaning period. Calves in treatments were supplemented 2-methylbutyrate at 3, 6 and 9 g per calf per day, respectively. All calves were weaned at 45 days of age. At 30 and 90 days of age, four calves from each group were selected and slaughtered before the morning feeding. Digesta and mucosa of duodenal, proximal jejunal, distal jejunal and ileal were collected to determine the activities of lactase, amylase, trypsase and lipase, and mRNA expression of GHR and SGLT-1, respectively. 【Result】The results showed that lactase enzyme activity and mRNA expression of mucosal GHR and SGLT1 in every part of small intestine in post-weaning calves were lower than that in pre-weaning calves, while activities of amylase, lipase and trypsin in every part of the small intestine in post-weaning calves were higher than that of pre-weaning calves. Lactase activities of proximal jejunal, distal jejunal and ileal in pre-weaning calves supplemented with 2-methyl butyrate at 6 and 9 g·d^-1 were higher than that of the control group and the 3 g·d^-1 2-methyl butyrate group (P＜0.05). Lactase activities of duodenal and ileal in post-weaning calves in the group supplemented with 2-methyl butyrate at 6 and 9 g·d^-1 were higher than that of the control group and the 3 g·d^-1 2-methyl butyrate group (P＜0.05). Activities of trypsase and lipase in the duodenal, proximal jejunal, distal jejunal and ileal, and GHR mRNA expression of the small intestine in pre- and post-weaning calves supplemented with 2-methyl butyrate at 6 and 9 g·d^-1 were higher than that of the control group and the 3 g·d^-1 2-methyl butyrate group (P＜0.05). The SGLT-1 mRNA expression of the duodenal in pre- and post-weaning calves supplemented with 2-methyl butyrate at 6 and 9 g·d^-1 were higher than that of the control group (P＜0.05). The SGLT-1 mRNA expression of the proximal jejunal, distal jejunal and ileal in pre- and post-weaning calves supplemented with 2-methyl butyrate at 6 and 9 g·d^-1 were higher than that of the control and 3 g·d^-1 2-methyl butyrate group (P＜0.05).【Conclusion】Results indicated that digestive enzymes activities in the small intestinal digesta and mRNA expression of GHR and SGLT-1 in the small intestinal mucosa were improved by supplemental 2-methyl butyrate, and the optimum dose of 2-methyl butyric acid was 6 g·d^-1.

 

Effects of Different Feeding Levels on Energy Metabolism Balance of Meat Ewes During Non-Pregnancy and Lactation

LOU Can, DENG Kai-dong, JIANG Cheng-gang, MA Tao, JI Shou-kun, CHEN Dan-dan, ZHANG Nai-feng, TU Yan, DIAO Qi-yu

Scientia Agricultura Sinica. 2016, 49(5):  988-997.  doi:10.3864/j.issn.0578-1752.2016.05.019

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【Objective】 Nutrition supply in different physiological periods has great impact on reproduction of ewes, which determines the conception rate, birth weight, survival rate and potential growth performance of lambs. The nutrition after weaning compensate for the body reserve which was consumed during pregnancy and lactation, and is necessary for the ewes to prepare for the next reproduction cycle. The energy metabolism is essential in the study of nutrition of mutton sheep, especially during early growth period. The lack of study in related area limited the development of mutton sheep industry in China. This experiment was conducted to investigate the effect of different feeding levels on energy and gas metabolism in Dorper × Thin-tailed Han crossbred ewes during non-pregnancy and lactation periods. 【Method】 Non-experimental factors should be limited in the feeding trial, which is a common method in the research of nutrient requirement. Combined with the digestibility and gas metabolism trials, the energy balance of ewes in different physiological periods could be systemically investigated. Fifteen healthy ewes treated for estrus synchronization were assigned into three feeding levels according to the randomized block design: ad libitum, 80% of ad libitum, and 60% of ad libitum. Another nine non-pregnant ewes of the same age and species were used as a blank group. Digestibility and respirometry trials were conducted on 20, 50 and 80 d of lactation. 【Result】The apparent digestibility of carbon (C) and energy of non-pregnant and lactating ewes increased significantly with decreasing levels of feed intake (P＜0.05). Milk C decreased significantly with increasing lactation periods (P＜0.05). Fecal and urinary C decreased significantly with decreasing feeding levels (P＜0.05), and it was higher in lactation period than in non-pregnant period (P＜0.05). The metabolic rates of digestible energy increased with the decreasing feeding levels (P＜0.05), and were 78.55%-82.93%, 79.53%-85.89%, 79.40%-83.49% and 80.99%-85.33% in the period of non-pregnancy, early, middle and late lactation, respectively. The respiration quotients decreased significantly with the decreasing feeding levels (P＜0.05). However, they were similar between non-pregnancy and lactation periods, ranging from 0.83 to 0.96. The methane (CH₄) production and the ratio of CH₄ to DMI (kg) or metabolic weight (W^0.75) decreased significantly with the decreasing feeding levels (P＜0.05). 【Conclusion】 Feed intake significantly influenced the C and energy apparent digestibility of Dorper × Thin-tailed Han ewes during non-pregnency and lactation periods. There were correlations between the O₂, CO₂, CH₄, RQ and feed intake (P＜0.05). Lactation significantly improved the digestion and utilization of C and energy.

Transcriptome Analysis of Ovary Tissue in Early Laying Period and Egg Laying Peak Period of Shanma Ducks

ZHU Zhi-ming, CHEN Hong-ping, LIN Ru-long, MIAO Zhong-wei, XIN Qing-wu, LI Li, ZHANG Dan-qing, ZHENG Nen-zhu

Scientia Agricultura Sinica. 2016, 49(5):  998-1007.  doi:10.3864/j.issn.0578-1752.2016.05.020

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【Objective】The objective of this study was to find the transcriptome differences between ovary tissue in early laying period and egg laying peak period of Shanma ducks, and enrich the laying duck transcriptome data.【Method】Selecting 6 Shanma duck ovarian tissues as a sample (early laying period and peak period 3 samples respectively), then the transcriptome libraries of ovary tissue in the early laying period and the egg laying peak period of Shanma ducks were constructed by using an Illumina HiSeq^TM 2000 sequencing technique, and the bioinformatics methods were analyzed subsequently, such as sequencing assess and gene function annotation. 【Result】 The results showed that the base ratios with quality value higher than 20 in reads (Q20) were more than 93%. 43 489 798 reads and 438 084 706 bp transcriptome data were obtained in the early laying period, and 42 782 676 reads, 4 307 499 083 bp transcriptome data were obtained in the egg laying peak period. Respectively, 70.92% and 72.70% reads could be compared to the duck reference genome sequence. Comparison of the transcriptome of ovary tissue in the early laying period and the egg laying peak period, 26 808 expressed genes were found in the early laying period, 27 013 expressed genes were found in the egg laying peak period. As the early laying period for reference, 1 929 differentially expressed genes were found in the egg laying peak period, including 989 up-regulated genes and 940 down-regulated genes. Annotation analysis indicated that 1 423 genes were annotated in Nr data, including 695 up-regulated genes and 728 down-regulated genes. Through COG analysis, there were 663 functional annotations of these differentially expressed genes, involving 25 functional classifications. With GO function annotation classifications, a total of 1 122 genes were divided into 61 function categories, in which many functional categories were mainly involved, such as molecular binding, catalytic activity, cell process, biological regulation, which including 406 related annotation genes involved development and reproduction biological process. KEGG analysis showed a total of 425 genes were annotated to 160 metabolic pathways, and GnRH signaling pathway, GPI-anchor biosynthesis, TGF-beta signaling pathway, ribosome biosynthesis pathway were significantly enriched. The GnRH and TGF-β signaling pathway played an important role in the ovarian physiology and reproduction activities, but the role of GPI-anchor and ribosome biosynthesis was not clear in ovarian physiology and reproductive activity.【Conclusion】The transcriptome study of the ovary tissue in the early laying period and the egg laying peak period of Shanma ducks by using high-throughput sequencing technology revealed the number of differentially expressed genes under different physiological status of the ovarian tissue in Shanma ducks, obtained the function, classification and metabolic pathway of differentially expressed genes, which enriched the ovary transcriptome information of laying ducks, and laid a foundation for the study on related genes with reproductive traits and molecular mechanisms in laying ducks.

RESEARCH NOTES

Spatial Distribution and Content of Soil Calcium in Farmland: A Case Study in Dazhuang Town, Wugong County

SHI Hong-ping, WANG Yi-quan, SHI Zong-lin, LIANG Hua-xue, WANG Jia-xu

Scientia Agricultura Sinica. 2016, 49(5):  1008-1016.  doi:10.3864/j.issn.0578-1752.2016.05.021

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【Objective】 To reveal the effects of intensive land use, fertilization and tillage system changes on soil health under the condition of modern agricultural production, the history of the development and evolution of soil in Guanzhong region was studied. 【Method】 Soil samples within 600 m to villages of the same natural conditions and land use types were analyzed for Ca change. Calcium content of samples in radiation lines was compared, and the data of soil survey made in 1982 were used as reference, so the methods of horizontal reference standard and vertical (time) standard were employed for study of the evolution of soil Ca. 【Result】 The results indicated that the variation of soil calcium carbonate and exchangeable calcium in 0-40 cm layer were not obvious in the horizontal scales in the range of 0-400 m from the village, but increased obviously in soils from 400-600 m to the villages. The content of soil water soluble calcium was gradually decreased in the range of 0-600 m. In the range of the same soil layer, the content of calcium carbonate in 0-20 cm layer was lower than that in the 20-40 cm layer. Exchangeable calcium and water soluble calcium was higher than that in the 20-40 cm layer. In 0-40 cm, the content of calcium carbonate 50 m away from the village was lower than that 500 m from the village, and the content in the same layer was lower than that in 1982. The content of calcium carbonate was significantly increased under 40 cm layer. The total storage capacity of soil calcium at 50 m away from the village was significantly higher than that at 500 m in 0-100 cm layer. 【Conclusion】 Therefore, the modern intensive land use and technology measures employed actuated and accelerated the process of soil calcium leaching and deposition in the limited level of space, resulting in the spatial variability of calcium in soils and the concealed degradation process of soil calcium in farmland. Cautions should be paid to the influences of modern agricultural technology measures on soil health.