Scientia Agricultura Sinica

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Scientia Agricultura Sinica. 2018, 51(19): 0-0.

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The Establishment and Application of Rice Protein Sample Library RiceS-A300

ZHANG JianShuo, MA JinJiao, ZHANG Tong, CHEN Yue, WEI Jian, ZHANG Liu, SHI JiaNan, XU Shan, YAN GaoWei, DU TieMin, DOU ShiJuan, LI LiYun, LIU LiJuan, LIU GuoZhen

Scientia Agricultura Sinica. 2018, 51(19): 3625-3638. doi:10.3864/j.issn.0578-1752.2018.19.001

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【Objective】 The aim of the study is establish a protein sample library namely RiceS-A300, which was collected from rice seedlings treated by abiotic stressed conditions and investigate the expression patterns of the reference protein HSP82 by Western blot analysis using the library of RiceS-A300. 【Method】The seeds of japonica rice variety TP309 were soaked in water for 3 days at 30℃, and then cultured either in soil mixture (soil and vermiculite 1:1) or aquaculture condition at 30℃ with photoperiod L12 h/D12 h for 5 days. Then the seedlings were treated by cold (4℃), heat (44℃ and 48℃), submerge, constant light, constant dark, PEG 6000 (20%) and NaCl (0.2 mol·L^-1), while the seedlings cultured at 30℃ with photoperiod L12 h/D12 h was used as control. Rice seedlings were monitored, photographed and samples were collected at different time points, the plant height and fresh weight were measured under different stressed conditions. To establish a library of protein samples total proteins were extracted from rice seedlings. SDS-PAGE separated proteins were stained with Coomassie blue to evaluate the quality of protein sample. Western blot (WB) was carried out to investigate the expression patterns of reference protein HSP82. 【Result】The phenotype, plant height, fresh weight and total protein content of rice seedlings at 9 time points under various stressed conditions were investigated. It was found that cold (4℃), hot (44℃ and 48℃) stresses demonstrated the inhibitory effect of the elongation of plant height in 2 d, while the submerge treatment promotes the elongation of plant height. Constant dark and constant light inhibited the elongation of plant height, the seedlings became yellow under the constant dark treatment. PEG and NaCl treatments had an inhibitory effect on the plant height also, and the leaf tip was dry under NaCl stress at 5 d. The results for fresh weight investigation showed that cold (4℃), hot (44℃ and 48℃), PEG and NaCl stresses reduced the fresh weight of the seedlings, while the submerge treatment had little effect on the fresh weight, constant dark treatment reduced the fresh weight but constant light treatment had no obvious effect. The three kinds of temperature stresses increased the total protein content, and constant dark stress decreased the total protein content, the other stresses had little effect on the total protein content. Collectively, a total of approximately 300 protein samples are collected，the rice protein sample library (RiceS-A300) was established. The total volume of each sample was 2 mL, enough for about 200 WBs. As standard operational protocol was used for sample collection and protein isolation, the scale of sample can be enlarged. It is expecting that repeatable results can be achieved from different laboratories using similar treatment conditions. RiceS-A300 was used to investigate the expression patterns of HSP82 protein, the results showed that heat stress increased the expression of HSP82 significantly, constant light, PEG and NaCl stresses increased the expression of HSP82 slightly, cold stress, submerge and constant dark had little effect on the abundance of HSP82 protein.【Conclusion】This study proposed a process for the construction and evaluation of the protein sample library, and the first version of the library (RiceS-A300) derived from rice seedlings under different stressed conditions was established. On the basis of RiceS-A300, the expression patterns of reference protein HSP82 was investigated using WB and it was found that HSP82 was specifically induced under heat stress.

Arabinoxylan Feruloyl Transferase Gene Cloning and Development of Functional Markers in Common Wheat

ZHAN ShuaiShuai, BAI Lu, XIE Lei, XIA XianChun, REN Yi, Lü WenJuan, QU YanYing, GENG HongWei

Scientia Agricultura Sinica. 2018, 51(19): 3639-3650. doi:10.3864/j.issn.0578-1752.2018.19.002

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【Objective】 The arabinoxylan feruloyl transferase (AFT) gene was cloned from wheat, and the functional markers linked to the content of FAX were developed to improve the accuracy of predicting the content of FAX , in order to provide the basis for the improvement of wheat processing quality. 【Method】 The complete gDNA sequence of wheat FAX gene was obtained by homologous cloning method，using FR846233 as a probe. The sequence differences between high and low FAX content varieties were compared by the DNAMAN software; based on sequence difference, specific primers were designed with Primer5.0 software to develop functional markers closely linked to FAX content and a set of Chinese spring Nulli-tetrasomic lines and ditelosomic line 3AS, and 3AL were used for chromosome physical mapping; and the practicability of functional markers were verified by using 253 wheat varieties (lines) from the main winter wheat regions in China, combined with the method of PCR verification. IBM SPSS statistics 19.0 software was used to analyze the correlation between FAX content and genotypes. 【Result】Two pairs of specific primers B1 and B2 finally amplified fragments of 800 bp and 710 bp, respectively. And 80 bp overlaps of PCR fragments amplified by B1 and B2 were spliced to obtain the AFT gene that is located on chromosome 3A. The TaBahd-A1 sequence is consists of 1429 base pairs and allelic variants TaBahd-A1a and TaBahd-A1b are obtained. The two allelic variants possess a 1266 bp open reading frame, two exons and one intron. The introns conformed to the typical GT-AG structure. The similarity between the alleles was 98.08%, with 24 SNPs and 3 InDels, which could encode 421 amino acid residues and the predicted molecular weight was 45.2 kDa. Two complementary dominant markers AFTA2 and AFTB2 were developed based on the 107 bp SNP. AFTA2 was able to amplify a 692 bp fragment in the TaBahd-A1a material, which was associated with a high FAX content, but not in the material with TaBahd-A1b allelic variation. AFTB2 could only amplify a 438 bp fragment in TaBahd-A1b type material and correlate with low FAX content, but not in TaBahd-A1a type material, and AFTA2 and AFTB2 were located in 3AL chromosome of wheat by a set of Chinese spring Nulli-tetrasomic lines. Using the functional markers AFTA2 and AFTB2 to detect 253 Chinese winter wheat materials, the results showed that the difference in FAX content of different genotypes reached a significant level (P < 0.05), and there was no significant difference in FAX content in Northern China Plain Winter Wheat Region; but in the Yellow & Huai River Valley Winter Wheat Region, The FAX content of the TaBahd-A1a-containing variety was significantly higher than that of the TaBahd-A1b- containing variety (P<0.05). Therefore, the complementary dominant markers AFTA2 and AFTB2 are related to FAX content and can be effectively used for genetic improvement of FAX content. The frequency of TaBahd-A1 allele variation indicates that TaBahd-A1a is an excellent allelic variant associated with high FAX content, and the frequency of TaBahd-A1a in the Northern China Plain Winter Wheat Region (71.3%) is significantly higher than that in the Yellow & Huai River Valley Winter Wheat Region (60.2%). 【Conclusion】 The results suggested that these two STS markers are closely related to FAX content related gene and could be used for the improvement of wheat processing quality for wheat-based products.

Enhanced Effect of Two Years Plastic Film Mulching with Reduced Tillage on Grain Yield Formation of Wheat Rotation Under Reduced Irrigation and N Application

FAN ZhiLong, ZHAO Cai, LIU Chang, YU AiZhong, YIN Wen, HU FaLong, CHAI Qiang

Scientia Agricultura Sinica. 2018, 51(19): 3651-3662. doi:10.3864/j.issn.0578-1752.2018.19.003

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【Objective】Continuous cropping is normally employed for maize cultivation with plastic film mulching in arid oasis irrigation regions. Lower resources use efficiency and residual film pollution usually result from unreasonable management of irrigation/N application and single planting pattern. The purpose of this study was to determine the enhanced effect of two years plastic film mulching with reduced tillage on grain formation of wheat with reduced amount of irrigation and N application, so as to provide the theoretical basis for exploiting of planting pattern with lower resources input and greater resources use efficiency.【Method】A field experiment was conducted at Zhuwangpu county, Hexi Corridor of Gansu province in 2016 and 2017. Two tillage patterns were designed as: wheat planted with two years used plastic film mulch and no-tillage in the preceding fall (RT), wheat strip planted with conventional tillage and without mulching (CT). Wheat growth under two irrigation levels (the reduced 20% amount of traditional irrigation (I₁), the amount of traditional irrigation (I₂)) and three nitrogen levels (the reduced 40% amount of traditional nitrogen application (N₁), the reduced 20% amount of traditional nitrogen application (N₂), and the amount of traditional nitrogen application (N₃)). 【Result】 The significant difference of growth and yield was not observed between 2016 and 2017. Growth rate of wheat was enhanced by RT as 8.5% at jointing, as 9.0% from booting to flowering compared with that of CT; The growth rate of wheat was not significant difference between different irrigation levels or nitrogen application levels, respectively, in each growth stage. Average leaf area index (LAI) of RT was greater as 13.9% than that of CT. However, the LAI was decreased under reduced irrigation, the average LAI of the I₁ was decreased by 12.2% than that of I₂. LAI of wheat was also affected by nitrogen application level, the LAI of N₁ was decreased by 13.3% than that of N₂, and LAI of N₂ was decreased by 13.3% than that of N₃. Under I₁ irrigation level, RT had the greater improving effect on grain yield than conventional tillage. RT enhanced grain yield of wheat by 9.1% in comparison to CT. Two years used plastic film mulch achieved the greatest grain yield in I₁ irrigation level and N₂ nitrogen application level, which was 7 168 kg·hm^-2 in 2016 and 7 537 kg·hm^-2 in 2017, and grain yield of RTI₁N₂ was equaled to that of traditional irrigation and nitrogen application levels. While biomass yield was decreased and harvest index was increased under reduced irrigation and nitrogen application. The harvest index of I₁ was increased by 10.7% than that of I₂. The N₁ and N₂ had higher the harvest index by 9.2% and 7.1% than that of N₃, respectively. Under I₁ irrigation level, the transfer amount from photosynthetic product for grain after flowering was increased by 26.2% under RT, compared with CT, meanwhile the contribution was increased as 4.9%. The significant difference on the transfer amount from photosynthetic product for grain between the reduced 20% and traditional nitrogen was not observed. Yield component factors and the path analysis indicated that, improving kernel weight via greater leaf area index support photosynthetic product and translation to grain was useful for achieving high yield. 【Conclusion】 RT had improved wheat grain yield with the I₁ irrigation level and N₂ nitrogen application. It was therefore suggested that, in arid oasis irrigation regions, the treatment of RTI₁N₂ was one of effective cultivation patterns for the integrated measures with the reduced amount of water, nitrogen and plastic film resources.

Effects of Silicon on Plant Growth and Photosynthetic Characteristics of Soybean Seedlings Under Artificial Shade Stress

LI ShuXian, LIU WeiGuo, GAO Yang, LIU Ting, ZHOU Tao, DU YongLi, YANG Huan, ZHANG Hao, LIU JunDou, YANG WenYu

Scientia Agricultura Sinica. 2018, 51(19): 3663-3672. doi:10.3864/j.issn.0578-1752.2018.19.004

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【Objective】 This experiment mainly studied the effects of silicon on the growth and photosynthetic characteristics of soybean seedlings under shade stress, in order to provide reference for cultivating strong seedlings of soybean intercropping. 【Method】 In 2017, in the experimental base of Sichuan Agricultural University, two soybean varieties Nandou12 (strong shade-tolerance) and Nan 032-4 (weak shade-tolerance) were used as experimental materials, and the black sun-shade net (50% light transmittance) was used to simulate the shade environment conditions of maize-soybean intercropping. The outdoor pot experiment was carried out, and 4 treatments were set up, including CK (Normal, spraying distilled water), S0 (50% shade, spraying distilled water), S1 (50% shade, spraying 100 mg·kg^-1 Na₂SiO₃·9H₂O water solution), and S2 (50% shade, spraying 300 mg·kg^-1 Na₂SiO₃·9H₂O aqueous solution), spraying leaf surface of soybean at the seedling stage. The content of silicon, photosynthetic parameters, chlorophyll content, stem diameter and flexural strength of stem, root morphology and root/shoot ratio, dry matter accumulation, and soluble sugar content were measured. Finally, the effects of silicon on the shade tolerance of soybean seedlings were analyzed. 【Result】 The total dry matter accumulation of two soybean varieties, silicon content and soluble sugar content of each organ decreased. At the same time, stem diameter, stem resistance, as well as root length, root surface area, root volume and root/shoot ratio of root traits, were reduced. The sun-shade net reduced the radiation of sunlight to soybean, resulting in the decrease of net photosynthetic rate (Pn) and stomatal conductance (Gs) and transpiration rate (Tr) of soybean plants. While intercellular CO₂concentration (Ci) and chlorophyll content increased with the decrease of light. Soybean varieties with different shade tolerance had different degrees of response to the same shading environment. Nandou12 had strong adaptability under shade stress, therefore, which had higher photosynthetic capacity (net photosynthetic rate, intercellular carbon dioxide concentration, transpiration rate, stomatal conductance, chlorophyll content), stem resistant ability (stem diameter, stalk resistance), and root growth ability (root length, root surface area, root volume, root dry weight). As compared with Nan032-4, Nandou12 had stronger shade tolerance. After S1 treatment under shading conditions, the plants of two soybean varieties had more dry matter accumulation, thicker stem, and stronger stem resistance. In addition, the root volume, root surface area and root volume of the soybean seedlings increased with the increase of Si concentration. And net photosynthetic rate, stomatal conductance and transpiration rate of leaves were higher than that of shade treatment (S0). The intercellular carbon dioxide concentration decreased and chlorophyll content increased. The soluble sugar content of stem and leaf increased. Eventually, seedlings became stronger. However, the sensitivity of different shade tolerant soybean varieties to different concentrations was different. Inside, under the S2 treatment, the net photosynthetic rate, soluble sugar content of leaves and stems of Nandou12 increased by 20.3%, 12.9% and 4.3%, respectively compared with S0 treatment, however, those of Nan032-4 increased by 20.1%, 94.3% and 72.6%, respectively. The intercellular CO₂concentration of Nandou12 and Nan032-4 decreased by 10% and 45.2%, respectively.【Conclusion】Silicon could effectively improve the growth of soybean seedlings under shade stress, enhance photosynthesis ability, stem resistant ability and root absorptive capacity, so as to improve the shade tolerance of soybean.

Real-Time Pixel-Wise Classification of Agricultural Images Based on Depth-Wise Separable Convolution

LIU QingFei, ZHANG HongLi, WANG YanLing

Scientia Agricultura Sinica. 2018, 51(19): 3673-3682. doi:10.3864/j.issn.0578-1752.2018.19.005

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【Objective】In order to improve the accuracy and real time recognition of crops and weeds, the field color image of seedling beet was taken as the research object, and a pixel-wise classification method based on deep separable convolution was proposed.【Method】In this paper, the field color image of the seedling beet was used, the pixels in the color image were tagged into three categories of crops, weeds and soil by the manual pixel marking method, and the single classification information was placed in three different image channels, which was used for training and testing. First, a deep separable convolution neural network model based on encoder and decoder was set up. The encoder part and decoder part were merged in multi scale. The pixel location was determined by the encoder part, and the decoder part got the pixel classification. In order to solve the problem of the unbalance of the coverage rate of the classification category, the single channel standard was used. In order to control the size of the network parameters, the number of the point convolution kernel was controlled by the width multiplier and the network was used under the different resolution input conditions to control the network parameters. The model was further tested to discuss the real-time performance of the network model. Finally, we used random data enhancement technology to expand data sets, 80% of the data sets were used for training network parameters, and 20% of them were used to test network performance. 【Result】 (1) Compared with the existing pixel-wise classification method, the proposed method achieved higher classification accuracy. The average accuracy rate of the SegNet method was 90.06%, the average accuracy of the U-Net method was 92.06%, the average accuracy rate of the three channel marking training was 92.70%, and the average network accuracy of the single channel marking training was 94.99%. (2)The advantages of the single channel annotation information training method in dealing with the unbalance of classified category coverage and the less training samples were demonstrated by calculating the indexes of the single category by pixel classification by different methods. The accuracy rate of weeds pixel-wise classification SegNet method was 18.39%, U-Net method was 18.33%, the network of three channel marking training was 22.87%, and the network accuracy of single channel marking training was 41.94%. (3) The parameter size of the network model could be effectively controlled by the width multiplier. When the width multiplier was 1, the parameter size was 6.768 million, and the parameter size was reduced to 77.2 thousand when the width multiplier was 0.1. It was 1.14% of the original network parameter scale, and the accuracy rate for the classification of soil, weeds and crops was only 2.81%, 2.78% and 3.7%, respectively. According to the accuracy requirement, the scale of parameters could be further reduced. (4) Under the combined action of input resolution and width multiplier, the real-time processing capability of the network was discussed. Using GPU hardware acceleration, the rate of simultaneous recognition of three classes could reach 20 fps, and the rate of single class recognition was 60 fps. It could satisfy the real-time operation of agricultural weeding system and crop monitoring system. 【Conclusion】The pixel-wise classification method based on deep separable convolution proposed in this paper could effectively classify the soil, weeds and crops in agricultural images. At the same time, this method could deal with a single category by pixel classification in real time to meet the needs of the actual system.

Composition and quantitative analysis of Fusarium species in maize rhizosphere soil

GE Bo, WANG BaoBao, GUO Cheng, SUN SuLi, CHEN GuoKang, WANG XiaoMing, ZHU ZhenDong, DUAN CanXing

Scientia Agricultura Sinica. 2018, 51(19): 3683-3693. doi:10.3864/j.issn.0578-1752.2018.19.006

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【Objective】The objective of this study is to understand the population structure and relative content of Fusarium spp. in maize rhizosphere soil in China, and to provide an early warning of the occurrence of soil-borne maize diseases.【Method】Using dilution plate method, a total of 58 Fusarium isolates were obtained from 47 maize soil samples of 17 provinces (autonomous region, municipality). Based on morphological characteristic, specific PCR amplification, and ITS and TEF-1α gene sequences analysis, these Fusarium spp. were further determined. The reference strain sequences were downloaded from GenBank and Fusarium MLST. Multi-gene loci phylogenetic tree was constructed by neighbor-joining method (NJ) using MEGA 6.0 software. Some representative strains were selected for pathogenicity test by maize seedlings, seeds and in vitro leaves infection. The content of Fusarium spp. and some species in maize rhizosphere soil samples was detected using RT-qPCR determination system. Statistical analysis was carried out for different maize producing areas.【Result】 A total of 9 Fusarium species were identified. Among them, F. verticillioides (22.41%), F. quisete (20.69%) and F. graminearum species complex (18.97%) were isolated more frequently. The rest of 6 species were F. oxysporum species complex, F. proliferatum, F. dimerum, F. acuminarum, F. solani and F. commune, with the isolation frequency of 12.07%, 8.62%, 5.17%, 5.17%, 3.40%, and 3.40%, respectively. The results showed that the same Fusarium spp. and the reference strain were in the same branch, and the diversity of Fusarium spp. existed in different regions. All Fusarium species were pathogenic except F. dimerum. F. commune was isolated from maize rhizosphere soil and confirmed to be pathogenic to maize seed, seedling, and detached leaf for the first time. The pathogenicity of different strains of the same Fusarium species was not the same. Using RT-qPCR detection system, the total of Fusarium, F. verticillioides and F. graminearum species complex in maize soil samples were quantitatively assayed, with the content range of 2.91-169.90, 0.08-5.34 and 0.76-78.37 pg·g^-1, respectively. The content of Fusarium species in different maize producing areas was different, and the content of F. graminearum was higher than that of F. verticillioides. 【Conclusion】There are multiple species of Fusarium in maize rhizosphere soil and F. verticillioides, F. equiseti, and F. graminearum species complex are dominant species. The content of Fusarium spp. differs in different areas. Among them, the content of Fusarium spp. is the highest in Southwest mountainous area of maize, and the F. graminearum content in the Huang-huai-hai summer sowing maize area is higher than that in other regions.

Function analysis ofγ-glutamyl phosphate reductase-encoded gene SsGPR1 in Sclerotinia sclerotiorum

DU Jiao, WANG YaBo, LI XueHua, HUANG ZhiQiang, YANG YuHeng, BI ChaoWei, YU Yang

Scientia Agricultura Sinica. 2018, 51(19): 3694-3703. doi:10.3864/j.issn.0578-1752.2018.19.007

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【Objective】The gamma-glutamyl phosphate reductase (GPR) is a key enzyme in fungal proline synthesis pathway. The objective of this study is to silence a GPR-encoded gene SsGPR1 in Sclerotinia sclerotiorum via the RNA interference strategy, research the mycelial growth, sclerotial formation and pathogenicity of the gene-silencing transformants, so as to lay a foundation for revealing the growth, development and pathogenicity of S. sclerotiorum. It also provides important clues for the green prevention and control of Sclerotinia disease.【Method】Homology analysis and phylogenetic tree construction were performed through the BLAST search and MEGA 5.0 software. The real-time RT-PCR was used to detect the expression pattern of SsGPR1 at the different stages of mycelial growth, sclerotial development and germination, and infection processes. The gene silencing vector of SsGPR1 was constructed based on the principle of RNA interference, and the vector was used to transform wild-type strain 1980 by PEG-mediated transformation of protoplasts methods. The gene-silenced strains were identified by real-time RT-PCR. The mycelial morphology, growth rate and sclerotial formation of the gene-silenced strains were observed and the hyphal growth rate under the oxidative stress was measured. The gene-silenced strains were inoculated on Brassica napus leaves and Arabidopsis thaliana plants, and lesion size was observed and measured. The free proline of gene-silenced strains was assayed using the acid ninhydrin method. 【Result】 SsGPR1 of S. sclerotiorum is 1 454 bp in length and encodes 449 amino acids. SsGPR1 protein contains a GPR domain at amino acid H¹⁰-N⁴²⁶. SsGPR1 showed high sequence similarity with Botrytis cinerea BC1G_13183 (95% identities) and Sclerotinia borealis SBOR_2215 protein (94% identities). The three proteins clustered into a small branch according to the result of phylogenetic tree. SsGPR1 showed high expression level during hyphae growth. The expression level was similar during the different development stages of sclerotia, but it significantly decreased compared with that during the hyphal growth period. The expression level of SsGPR1 increased gradually during the pathogenic period, and it reached the highest at 9 h post inoculation. The SsGPR1 silencing vector, pSIGPR1 was transformed into the wild-type strain of S. sclerotiorum, and the expression level of SsGPR1 in different transformants was detected by real-time RT-PCR. The results showed that SiGPR1-104 and SiGPR1-149 were SsGPR1 gene-silenced transformants. When cultured on PDA medium, SsGPR1 gene-silenced strains had no significant difference with wild-type strain on the number and average dry weight of sclerotia, which can germinate to form apothecium. However, the gene-silenced strains produced denser hyphae and showed a significantly reduce in growth rate. The hyphal growth of SsGPR1 gene-silenced strains was inhibited more strongly when cultured on medium containing H₂O₂, indicating that the gene-silenced strains were more sensitive to oxidative stress. SsGPR1 gene-silenced strains led to small lesions on B. napus leaves and A. thaliana plants, indicating that the pathogenicity of the gene-silenced strains was impaired. The content of proline produced by SsGPR1 gene-silenced strains had no significant difference with wild-type strain.【Conclusion】SsGPR1 is related to hyphal growth and mycelial morphology, and involved in the oxidative stress resistance and pathogenicity of S. sclerotiorum.

Antibody Preparation and Subcelluar Localization of dsRNA Degrading Enzyme in Locusta migratoria

SONG HuiFang, ZHANG JianQin, FAN YunHe, LI Tao, MA EnBo, ZHANG JianZhen

Scientia Agricultura Sinica. 2018, 51(19): 3704-3713. doi:10.3864/j.issn.0578-1752.2018.19.008

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【Objective】The dsRNA degrading enzyme (dsRNase) is an important obstacle of using RNAi technology for pest control. The objective of this study is to obtain specific antigens of LmdsRNase2 and LmdsRNase3 by using prokaryotic expression system, and then to prepare antibodies, and to detect the protein expression level of midgut and subcelluar localization. The results will provide strong evidence from protein level to further analysis of functional differentiation of LmdsRNase2 and LmdsRNase3 in the midgut of Locusta migratoria.【Method】The specific antigen sequences (R2’ and R3’) of LmdsRNase2 and LmdsRNase3 (GenBank: ARW74135.1, ARW74134.1) were chosen by alignment analysis. The primers with BamHI, HindIII restriction sites were designed according to the complete cDNA sequences. The target sequences were amplified by PCR and ligated to pET-32a vector through double enzyme digestion. The recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells, and then the cells were induced with 0.5 mmol·L^-1 IPTG for 4 hours at 37℃. SDS-PAGE electrophoresis was performed to examine the target proteins. After that, large amounts of E. coli cells were cultured for protein extraction. The target proteins R2’ and R3’ were purified through Ni-NTA agarose chromatography, and the protein concentration was determined according to the method of Bradford. The anti-LmdsRNase2 and 3 polyclonal antibodies were obtained after immunizing New Zealand white rabbit. The specificity of antibody was detected by western blot analysis, the recombinant LmdsRNase2 and LmdsRNase3 proteins were used as antigens. The titers of two antibodies were determined by ELISA. Furthermore, the total proteins were extracted from L. migratoria midgut and midgut fluid on day 3 of 5th instar nymph, and western blot was used to test the expression level of LmdsRNase2 and LmdsRNase3. Finally, the paraffin sections of the midgut on day 3 of 5th instar nymph were prepared, the subcellular localization of LmdsRNase2 and LmdsRNase3 proteins in the midgut of L. migratoria was conducted by immunofluorescence.【Result】LmdsRNase2 and LmdsRNase3 showed 37% sequence identity by full-length amino acid sequence alignment, and the variant sequence regions (named as R2’ and R3’ for LmdsRNase2 and LmdsRNase3, respectively) were selected for PCR primer design. The length of R2’ and R3’ is 157 and 153 a.a., respectively, and the calculated molecular mass of them is 17.0 and 16.8 kD, respectively. The protein expression was induced with 0.5 mmol·L^-1 IPTG for 4 hours at 37℃, finally the expressed proteins were only detected in inclusion bodies. The recombinant proteins R2’ and R3’ were purified using Ni-NTA agarose chromatography, and the purity of R2’ is 85%, it is suitable to directly immune New Zealand white rabbit. But the purity of R3’ is under 85%. To further obtain highly purified R3’, R3’was loaded on a gel and the expected band was cut for protein extraction and following immunization. After 36 days, ELISA results indicated that the titer of LmdsRNase2 and LmdsRNase3 antibodies was 1﹕102 400. Western blot demonstrated that anti-LmsRNase2 and 3 polyclonal antibodies could specifically detect LmdsRNase2 or LmdsRNase3 proteins, respectively, no cross hybridization was observed. The molecular weight was consistent with the predicted size. LmdsRNase2 protein was highly expressed in L. migratoria midgut by western blot, nevertheless, there was no detectable signal in midgut fluid. However, LmdsRNase3 could not be detected in both midgut and midgut fluid. Immunofluorescence results showed that both LmdsRNase2 and LmdsRNase3 were expressed in cytoplasm of L. migratoria midgut cells, but the expression level of LmdsRNase3 protein was much lower than that of LmdsRNase2.【Conclusion】Anti-LmdsRNase2 and 3 specific polyclonal antibodies were successfully prepared, western blot and immunofluorescence showed that LmdsRNase2 protein was highly expressed in midgut, whereas the expression of LmdsRNase3 was very low. These results provide evidence of protein level for the functional differentiation of LmdsRNases in the midgut of L. migratoria.

Impact of Land Use Type on Soil Organic Carbon Fractionation and Turnover in Southeastern Tibet

XU Meng, LI XiaoLiang, CAI XiaoBu, LI XiaoLin, ZHANG XuBo, ZHANG JunLing

Scientia Agricultura Sinica. 2018, 51(19): 3714-3725. doi:10.3864/j.issn.0578-1752.2018.19.009

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【Objective】 Land use conversion from native vegetation to cropland has a great impact on soil organic carbon (SOC) storage. Recently, the area of croplands that converted from native forests or grasslands increases gradually in the southeastern part of Tibetan Plateau, yet its influence on SOC fractionation and turnover remains unknown. It is therefore in great need to understand the extent and mechanisms that difference in land use type has exerted on SOC storage, which will provide scientific basis for sustainable management of agricultural soils in southeastern Tibet. 【Method】In the present study, soil samples were collected from cropland that had been cultivated for more than 50 years, as well as adjacent native forest and grassland. A combination of physical and chemical method was conducted to partition the SOC into different fractions. For each SOC fractions, the present in soil mass, SOC content and stable carbon isotope composition (δ¹³C) were analyzed to investigate the differences in SOC fractionation and turnover under different land use types. 【Result】 The SOC that stored in top 20 cm soil of cropland was (39.4±2.0) Mg C·hm^-2, which was 52% and 45% lower than native forest ((81.5±8.5) Mg C·hm^-2) and grassland ((71.4±7.3) Mg C·hm^-2), respectively. Compared to native forests and grasslands, long-term cultivation led to a decrease of 63.4%-70.8% in the mass of coarse particulate organic matter (cPOM), whereas the mass of microaggregate (μagg) and easily dispersed silt and clay (dSilt+Clay) increased 10.0%—25.9% and 65.7%-86.2%, respectively. The C content in each SOC fraction was significantly lower in cropland soils than that in forest and grassland soils. SOC content in cropland soils was 51.7%-58.1% lower than that in forest and grassland soils. The SOC contents of unprotected C, physically protected C and biochemically protected C pool reduced 79.8%-86.3%, 72.4%-73.1% and 32.4%-39.8% in cropland soils, respectively, and were positively correlated with changes in total SOC content following land use conversion. The SOC content in chemically protected C pool, however, was not affected by land use conversion. The C/N ratio and δ13C differed among different SOC fractions and three land use types. The C/N ratio of cPOM in cropland soils (10.0±0.5) was significantly lower than forest soils (13.5±0.4), whereas its δ¹³C value ((-21.6±0.5) ‰) was significantly higher than forest soils (-23.6±0.4) ‰. The δ¹³C values of microaggregate-protected particulate organic matter (iPOM) and non-hydrolyzable fraction (NH-dSilt+Clay and NH-μSilt+Clay) were comparably lower (-25.3‰- -27.2‰) than other SOC fractions, and had significantly lower C/N ratio in cropland soils (8.4-9.4) compared to forest soils (13.5-15.9). 【Conclusion】Results of the present study indicated that long-term cultivation had resulted in c.a. 50% reduce in SOC stock compared to native vegetation in southeastern Tibet. Agricultural cultivation strongly promoted the turnover of unprotected particulate organic matter, and suppressed the formation of more stabilized SOC, such as microaggregate-protected SOC fraction, which contributed great proportions to the dramatic decrease in SOC storage. Therefore, implement of no-tillage or other protective management would be necessary to reduce the negative influence of agricultural land use on SOC storage and to maintain the sustainable utilization of natural soil sources in southeastern Tibet.

Responses of Soil Carbon Emission and Carbon Balance of Maize Field to Plastic Film Mulching Pattern and Row Space

YU AiZhong, CHAI Qiang, YIN Wen, HU FaLong, FAN ZhiLong, ZHAO Cai

Scientia Agricultura Sinica. 2018, 51(19): 3726-3735. doi:10.3864/j.issn.0578-1752.2018.19.010

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&【Objective】Field experiment was carried out to analyze and evaluate the effects of plastic film mulching pattern and row space on soil carbon emission and carbon balance of maize field in arid inland river irrigated area. The experimental results provided a reference for evaluating ecological effect of typical cropping systems and its key cultivating techniques in this area.【Method】Full-film mulching with uniform row (50 cm), full-film mulching with paired row (80 cm+40 cm), half-film mulching with uniform row (50 cm), half-film mulching with paired row (80 cm+40 cm) were layout in the year of 2013 and 2014. Dynamics of soil respiration rate and CO₂-C emission during maize growth stage were to be measured by using portable Li-8100A (LI-COR). Carbon emission efficiency (CEE) and net ecology productivity (NEP) were used as indexes to estimate carbon sequestration intensity.【Result】Soil respiration rate was significantly affected by different film mulching pattern and row space, and it showed a single peak curve during the entire growth stage of maize. Full-film mulching treatments increased soil CO₂ emission significantly, compared with half-film mulching treatments. The contribution rate of root CO₂-C emission to soil total CO₂-C emission varied from 18.8% to 77.2% between different film mulching pattern and row space treatments and its average was 49.1%. Half-film mulching with paired row space showed the highest CEE among the four cropping patterns, up to 2.64 kg·kg^-1, which was 37.3% and 26.2% higher than that of full-film mulching with uniform row space and paired row space cropping pattern, respectively. Half-film mulching with paired row space showed the highest NEP in four cropping patterns, up to 7 446.2 kg C·hm^-2, which was significantly higher than that of other three cropping patterns. 【Conclusion】Soil CO₂ emission of maize cropland was affected significantly by different film mulching patterns and row spaces, and the four main maize crop systems were sinks of atmospheric CO₂ in arid inland river irrigation area. Half-film mulching with paired row space showed the lowest carbon emission and the highest carbon sequestration intensity.

The Degradation of Typical Antibiotics and Their Effects on Soil Bacterial Diversity in Spinach Soil

ZHANG Cheng, FENG Yao, LIU YuanWang, CHENG DengMiao, ZHENG Yu, LI ZhaoJun

Scientia Agricultura Sinica. 2018, 51(19): 3736-3749. doi:10.3864/j.issn.0578-1752.2018.19.011

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【Objective】 To provide an efficiently and eco-friendly solution for elimination of antibiotics in the soils and find a foundation for the evaluation of antibiotics contaminated land, this study was carried out about the residue of antibiotics in soils and their impacts on the abundance, diversity and composition of soil bacterial community. 【Method】 In this study, two types of antibiotics (gentamicin and tylosin) and two antibiotic-degrading microbe (a fungus named Aspergillus Terreus FZC3 to degrading gentamicin, a bacterium named Achromobacter to degrading tylosin) were selected to mix in spinach-growing soil. There were 7 treatments in total, including gentamicin (CG), gentamicin and FZC3 (CGF), tylosin (CT), tylosin and Achromobacter (CTW), two antibiotics (CM), two antibiotics and their degrading microbe (CMM), and blank control (CC). Solid phase extraction and HPLC-MS/MS technology were employed to study the impacts of degrading-microbe on antibiotics. The interactive influence of antibiotics and their degrading-microbe on soil bacterial community were studied by Illumina high-throughput sequencing technologies. 【Result】 Antibiotic-degrading microbe could improve the removal of the two antibiotics in the soil, but as time went by, the differences in the residual rates of antibiotics among all the treatments gradually minimized. During the entire test period, the differences of antibiotics residual rates between treatments were most significant in the first week. Under the CMM treatment, which spiked with two antibiotics and their degrading microbe, the residual rate of gentamicin was lowest (53.93%). Under the CTW treatment, which only spiked tylosin and Achromobacter, the residual rate of tylosin was as low as 3.92%. In the first week, the residual rate of gentamicin under the CGF treatment (spiked with gentamicin and FZC3) decreased about 3.3% compared with the CG treatment (spiked with gentamicin), and the residual rate of tylosin under the CTW treatment decreased about 4.1% compared with the CT treatment (spiked with tylosin). According to the results of high-throughput sequencing, there were significant differences (P＜0.05) between the bacterial richness and diversity in the three treatments of CG, CTW and CMM, compared with CC. Comparing the samples of the first week, the relative abundances of Massilia, Bacillus and etc. under the CG treatment were significantly lower than those in CC treatment; the relative abundance of Massilia in CT treatment was significantly lower than that in the CC treatment; but the decrease of inhibitory effects of antibiotics on these bacteria in the second week, indicating that gentamicin and tylosin in the early period of pot experiment had a greater impacts on soil bacterial communities. The bacterial community structure under the CGF treatment was similar to that under the CC treatment within the fortnight, indicating that gentamicin-degrading fungus FZC3 could alleviate the inhibitory effects of gentamicin on bacteria. In contrast with CT treatment, the relative abundance of Achromobacter was significantly increased while other genus decreased under the CTW treatment, indicating that the tylosin-degrading bacteria had a greater impact on the bacterial community structure. There was no significant difference in bacterial richness, diversity and community structure within fortnight between CC treatment and CM treatment (spiked with two antibiotics), indicating that there might be an antagonistic effect in soil bacteria when gentamicin and tylosin were added simultaneously. 【Conclusion】 Residues of gentamicin and tylosin in soil had different degrees of influence on bacterial abundance, diversity, and structure in soils. The addition of exogenous antibiotic-degrading microbe would not only speed up the removal efficiency of the two antibiotics, but also alleviate the effect of antibiotics on soil bacterial communities to some extent.

The Mechanism of Resistance to Fusarium oxysporum f. sp. niveum Race 1 in Tetraploid Watermelon

JI WanLi, ZHU HongJu, LU XuQiang, ZHAO ShengJie, HE Nan, GENG LiHua, LIU WenGe

Scientia Agricultura Sinica. 2018, 51(19): 3750-3765. doi:10.3864/j.issn.0578-1752.2018.19.012

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【Objective】The Fusarium wilt disease caused by the continuous cropping obstacles is one of the main factors which limits the production of watermelon in recent years. Field observation shows that tetraploid watermelon is more resistant to Fusarium wilt disease than autodiploid watermelon. The objective of this study is to reveal the mechanism of resistance to Fusarium wilt in tetraploid watermelon by comparing the resistant differences of diploid and tetraploid watermelon, and to provide a theoretical basis for polyploidy breeding and disease resistance breeding of watermelon.【Method】The diploid and autotetraploid watermelon seedlings of Zhengzhou No. 3 were used as materials in this research, the differences of the resistance to Fusarium wilt of different ploidy watermelon seedlings were compared after inoculating with Fusarium oxysporum f. sp. niveum race 1 (Fon 1) at one leaf stage, the Fon 1 with green fluorescent protein (GFP) was used to observe the infection process in different ploidy watermelon seedlings, then the activity/content of metabolites related to disease resistance such as peroxidase (POD), phenylalanine ammonialyase (PAL), malondialdehyde (MDA), total phenols, flavonoid and the expression levels of PR3, MPK7, PAL and MYB at different stages of diploid and tetraploid watermelon roots were determined.【Result】After inoculation with Fon 1, for the diploid watermelon seedlings, the Fusarium wilt was observed at the 4th day, and most of the plants died at the 10th day, while the disease was observed at the 7th day, and most of them died at the 13th day in the tetraploid watermelon seedlings. The wilt symptom delayed 3 days in tetraploid watermelon seedlings, tetraploid watermelon seedlings were more tolerant to Fusarium wilt than diploid ones. The infection pathway of Fon 1 was the same in both diploid and tetraploid watermelons. In addition, there was no significant difference in the germination rate of conidia between diploid and tetraploid watermelon roots. However, with the infection of pathogens, the infection speed of Fon 1 in tetraploid seedlings was obviously slower and the intercellular hyphae were less than those in diploid ones. The colonization rate of Fon 1 in the xylem of tetraploid watermelon seedlings was lower than that in diploid plants, which showed significant differences in the roots and extremely significant differences in the petioles of the seedlings, the expansion of the pathogen in the stems and leaves of tetraploid plants was limited to a certain extent. The infection process of Fon 1 in tetraploid watermelon apparently lagged, which coincided with the disease symptoms. After inoculation with Fon 1, the activity of POD and PAL in tetraploid watermelon seedlings increased, the POD activity of tetraploid watermelon seedlings increased more than that of diploid watermelon seedlings. Tetraploid roots produced more POD and PAL to enhance the plant disease resistance and protect cells from damage. The increasing of total phenols and flavonoid contents in tetraploid watermelon roots were higher than those in diploid ones. The advantages of these secondary metabolites made tetraploid plants more resistant to the invasion of Fon 1. In addition, the content of MDA in the root of tetraploid watermelon was obviously lower than that in diploid at the same time, which indicated that the damage degree of root cell membrane of tetraploid watermelon seedlings was lower after inoculation with Fon 1. Analysis of the expression of resistant genes showed that the expression level of PR3 tetraploid watermelon roots increased continuously after inoculation with Fon 1, and reached the highest level at the 10th day, which was 10 times of that of diploid at the same time. At the late stage of inoculation, tetraploid roots exhibited higher expression of MPK7 to transmit resistance signals, and the expression of disease resistance genes could be promoted, the damage of Fon 1 on watermelon seedlings would be reduced. The expression level of PAL increased firstly and then decreased, and the expression level was higher than that of diploid at the same period, the maximum expression level was 6 times of that of autodiploid. The expressions of MYB in tetraploid watermelon roots was continuously higher than that of autodiploid, and the expression level was 80 and 35 times of that of diploid on the 7th and 10th day after inoculation with Fon 1.【Conclusion】Through inoculation and identification of Fon 1 at seedling stage, observation of pathogen infection process, changes of metabolic substance content and gene expression level research, the results show that tetraploid watermelon seedlings are more resistant to Fusarium wilt than autodiploid watermelon seedlings.

The Genetic Diversity and Population Structure Analysis on Malus baccata (L.) Borkh from 7 Sources

GAO Yuan, WANG Kun, WANG DaJiang, ZHAO JiRong, ZHANG CaiXia, CONG PeiHua, LIU LiJun, LI LianWen, PIAO JiCheng

Scientia Agricultura Sinica. 2018, 51(19): 3766-3777. doi:10.3864/j.issn.0578-1752.2018.19.013

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【Objective】Genetic diversity and population structure of Malus baccata from 7 newly collected sources were analyzed using fluorescent SSR molecular markers. The identification of genetic diversity and structure within and among populations can provide references for germplasm collection and preservation of Malus and the study of the phylogenetic evolution of species. 【Method】19 pairs of polymorphic SSR primers were screened to detect the polymorphism of Malus baccata from 7 sources. GenAlEx 6.501 was used to calculate the index of genetic diversity and analyze the molecular variation (AMOVA) among populations. The genetic differentiation among populations were analyzed by GenepopV4 and Fstat293. Based on the Nei genetic distance DA, the Neighbour-Joining (NJ) evolutionary tree of 269 accessions was constructed using POPULATION 1.2. The Bayesian cluster was carried out using STRUCTURE 2.3.4 to analyze the genetic structure of populations.【Result】392 polymorphic alleles were detected by 19 pairs of SSR primers, with an average allele number of 20.6 and effective allele number of 9.070. The average values of heterozygosity and expected heterozygosity were 0.628 and 0.855 respectively, and the Shannon index was 2.392. Dividing populations according to their sources, the highest number of observed alleles in Heilongjiangpopulation was 15.684. The genetic diversity in Russian population was the lowest, and the highest genetic diversity was in Hebei population. The coefficient of genetic differentiation Fst between every two populations was from 0.019 to 0.111. The gene of Hebei population communicate frequently with other populations, andGansu population was the most stable among 7 populations. The genetic differentiation and gene exchange among populations were not completely related to the geographical location far or near. The cluster analysis based on Nei genetic distance could divided 269 accessions into 7 groups at 0.7444. Most of groups were not related to geographical location, among which group Ⅰ and Ⅱ were far away from other groups, and the cluster of groupⅡ and Ⅲ was mixed, the sources of group Ⅵ was the most complex, groupⅣ and Ⅴ were relatively pure, and finally 99% of all accessions in group Ⅶ were from Heilongjiang. The population structure analysis divided 269 accessions into 3 groups with 3 possible genetic sources. The accessions from different sources were distributed into every group, and there was no clear correlation with the geographical location. Only most part ofHeilongjiang, Shanxi and Gansu population, as well as some of accessions from Russia and Hebei belonged to a relatively simple group. This result was similar to the results of clustering. In 269 accessions, the Q value of 232 accessions were higher than 0.6, and most of them had relatively single genetic background. 【Conclusion】19 pairs of SSR primers were highly polymorphic and could be used for the evaluation of genetic diversity and population structure. The genetic diversity of Malus baccata from 7 regions was high with the highest genetic diversity of thosefrom Hebei, and genetic variation mainly occured within populations and individuals. There was genetic communication among populations with the most frequent communication between Hebei and other groups, but at the same time, it also resisted genetic differentiation among populations caused by gene drift. Genetic differentiation and gene exchange among populations were not completely related to geographical location.

Analysis and Evaluation on Quality of Not from Concentrate Apple Juices in Different Maturation Period

GOU XiaoJu, TIAN You, GUO YuRong, YANG Xi, HOU YanJie, PING JiaXin, LI Ting

Scientia Agricultura Sinica. 2018, 51(19): 3778-3790. doi:10.3864/j.issn.0578-1752.2018.19.014

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【Objective】The study was to investigate the effects of different maturation periods on the quality of not from concentrate (NFC) apple juices, and to clarify the differences of physicochemical properties in NFC juices, in order to provide theoretical basis for juice industry.【Method】NFC juices of twenty-two varieties from different maturation periods were prepared by squeezing, inactivating enzymes, pasteurization and hot filling. By determining their physicochemical properties including the total soluble solids (TSS) content, pH, titratable acidity (TA), TSS/TA, turbidity, color quality and polyphenol constituents, the property differences among these juices were evaluated by variance analysis. Additionally, principal component analysis (PCA) and liner discriminant analysis (LDA) were applied to classify twenty-two juices so as to illuminate the differences in physicochemical properties of NFC juices.【Result】The total soluble solids content of all NFC juices increased with the extended maturation period, and the pH in early and early-medium and medium mature varieties were relatively lower than those of medium-late and late mature varieties, as suggested by the analysis of physicochemical properties. The results of PCA showed that the NFC juices of early maturity and early-medium and medium maturity could be split in the same category, featuring the low contents of TSS. Additionally, the NFC juices of medium-late and late maturity were intersected on PCA score chart. ‘Yuhuazaofu’ ‘Jonathan’ ‘Delicious’ ‘Starkrimson’ ‘Granny Smith’ ‘Nagafu 2’ and ‘Indo’ constituted the group with the characteristic of low TSS/TA and high content of TA, indicating the sour taste. And ‘Qinhong’ ‘Jiguan’ ‘Newworld’ ‘Qinguan’ and ‘Pink lady’ formed the same group which was characterized by high brightness and turbidity. ‘Huali’ and ‘Hanfu’ were the same group with high TSS content and relatively low pH and TSS/TA. With the similar results of PCA, LDA implied that early maturity and early-medium and part medium maturity NFC juices showed the close physicochemical properties; Nevertheless, no significant difference between medium-late and late maturity was observed. Ultra high performance liquid chromatography (UHPLC) analysis demonstrated that the NFC apple juices predominantly possessed chlorogenic acid, procyanidin B2, epicatechin and epigallocatechin. The NFC juices from 22 varieties had the same individual polyphenol components but different contents (P＜0.05). 【Conclusion】The NFC juices of early maturity and early-medium and medium maturity were different in the physicochemical properties compared with the varieties of medium-late and late maturity. Thus, maturity should be taken into consideration as a primary index when producing NFC apple juices.

Effects of Cold and Heat Stress on Milk Production Traits and Blood Biochemical Parameters of Holstein Cows in Beijing Area

HU LiRong, KANG Ling, WANG ShuHui, LI Wei, YAN XinYi, LUO HanPeng, DONG GangHui, WANG XinYu, WANG YaChun, XU Qing

Scientia Agricultura Sinica. 2018, 51(19): 3791-3799. doi:10.3864/j.issn.0578-1752.2018.19.015

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【Objective】 The experiment was conducted to study the effects of cold and heat stress on milk production traits and blood biochemical parameters of Holstein cows, and to evaluate potential blood biochemical parameters to discriminate the temperature stress response, so as to provide theoretical basis for managing and selecting resistant individuals of Holstein cows in Beijing. 【Method】In current study, Holstein cows in Sanyuan dairy farm were selected as experimental animals, Temperature-humidity index (THI) of cowshed was continuously monitored, and their milk production data and blood samples used for the detection of blood biochemical parameters were collected in August 2014 (heat stress, N=178), November 2014 (non-stress, N=120) and January 2015 (cold stress, N=126), respectively. The changes of 7 milk production traits and 14 blood biochemical parameters induced by cold or heat stress were evaluated by MIXED models of SAS9.2 computer program, and Logistic analysis was used to analyze the accuracy of blood biochemical indicators which significantly changed under heat or cold stress in identification of temperature stress.【Result】Our results showed 1) in August 2014, the average temperature stood at 31.80℃ and the average THI reached 81.57 of Sanyuan dairy farm. It also should be noted that there were 21 days with THI greater than 78 for more than 8 hours in this month. These data indicated Chinese Holstein cows in Beijing suffered danger heat stress during August. In whole November 2014, with respect to conditions of average temperature with 12.76℃ and average THI only in 55.43, it displayed that there was no thermal stress in this month. While in January 2015, the mean of temperature was -6.70℃ and the average THI was 25.63, and a total of 21 days temperature difference were beyond 12℃. It showed that Chinese Holstein cows were under a mild cold stress in January; 2) in response to heat stress, the average milk yield of 7 days (AMY) of Chinese Holstein cows dropped by 1.34 kg, and significant decrease was found in FP, PP, F/P, SP (P＜0.01) and LP (P＜0.05), but significant increase in SCS (P=0.01) in milk. And heat stress significantly increased the concentrations of serum GH, LD, PRL and SOD in blood (P＜0.01), but decreased the concentrations of BUN, CRP, LDH, LPO, NE and K⁺ (P＜0.01). Among above 10 blood biochemical parameters changed under heat stress, the AUCs of PRL, GH and CRP based on ROC curve analysis were greater than 0.80 with the value in 0.91, 0.85 and 0.83, respectively; 3) in response to cold stress, the AMY of Chinese Holstein cows declined by 1.13 kg, and the FP and SP significantly decreased (P＜0.01) as well as F/P (P＜0.05), but the SCS significantly increased (P＜0.01) in milk. A significantly higher concentrations of serum COR, CORT and K⁺ in experimental cows appeared in cold stress than non-stress (P＜0.01), but opposite changes for DA, GH, LDH, PRL and SOD (P＜0.01), and ATCH (P＜0.05). Using ROC curve analysis, the AUC of SOD showed greater than 0.8 with value in 0.84. 【Conclusion】These results revealed that Holstein cows in Beijing were undergoing severe temperature stress in August and January, furthermore, heat and cold stress had damaging effects on milk production traits and physiological condition of Holstein cows in Beijing. The PRL and GH might be used as candidates for evaluation of heat stress and SOD for cold stress in early period of Holstein cows.

Pathogenicity of ME 49 Strain Toxoplasma gondii Tachyzoites in Kunming Mice

LU YaoYao, SU RuiJing, DONG Hui, WANG MengYao, YANG YuRong

Scientia Agricultura Sinica. 2018, 51(19): 3800-3806. doi:10.3864/j.issn.0578-1752.2018.19.016

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【Objective】In order to provide a basis for study the pathogenic characteristic of Toxoplasma gondii (T. gondii), ME 49 strain of T. gondii tachyzoites were explored to understand its virulence.【Method】In this study, the tachyzoites of ME 49 strain T. gondii were diluted with the concentration of ＜10⁰, 10⁰-10⁶ and inoculated to Kunming mice by intraperitoneal injection. The tachyzoites in lung and mesentery lymph nodes of mice were checked, and the cysts of T. gondii in brain were numbered. Besides, the paraffin sections in the lungs were stained with HE and IHC. The results of smear and MAT were used to analyze the infection rate and survival rate of mice.【Result】0 DPI-60 DPI, the result of T. gondii IgG antibody by MAT showed that the seroprevalence of mice with ＜10⁰, 10⁰, 10¹ and ≥10² were 0, 20%, 60% and 100%, respectively. Three mice with 10⁴ tachyzoites died at 12 DPI, 16 DPI and 19 DPI, respectively. Two mice died at 11 DPI and one mouse died at 8 DPI with 10⁵ tachyzoites. Two mice with 10⁶ tachyzoites died at 9 DPI, three mice died at 7 DPI, 8 DPI and 10 DPI, respectively, and the survival rate of mice infected with T. gondii was 62.07% (18/29). The tachyzoites of lungs and mesentery lymph nodes of dead mice were found by microscopy, and the cysts and antigen of T. gondii were observed in the lung by HE and IHC. The cysts in brain of T. gondii infection-positive mice with ＜10⁰, and 10⁰ tachyzoites were not found, the cysts in brain of T. gondii positive mice with 10¹ tachyzoite were 60, 100 and 0, respectively, 10² tachyzoites of that were 20, 40, 60, and 0, respectively, 60 DPI-600 DPI, three mice died with 10³ tachyzoites at 184 DPI, 435 DPI and 569 DPI, respectively, but no cysts of T. gondii were detected in the brains of mice. One mouse died at 188 DPI with the 10⁵ tachyzoites and the cysts was 20, while the survived time of remaining mice was 590 DPI.【Conclusion】In this study, the pathogenicity of ME 49 strain of T. gondii tachyzoites in Kunming mice was explored. 100% infection rate for mice was ≥10² T. gondii tachyzoites, the 100% lethal dose was 10⁶ tachyzoites, the time of death in infected mice was 7 DPI-19 DPI, the brain cysts was 0-53 per mouse, and the longest survival time was 590 DPI. The T. gondii tachyzoites of ME 49 strain had low pathogenicity to Kunming mice, and the cysts could be formed in brain, and only cause death of mice at high concentration of tachyzoites.

Pharmacokinetics and Bioavailability of Tildipirosin Solution in Pigs

YAN ChaoQun, LI JianYe, ZHANG Shen, XIE Shun, HU Lang, GU Xin, CAO Ying, HUANG ShiXin, HUANG XianHui

Scientia Agricultura Sinica. 2018, 51(19): 3807-3814. doi:10.3864/j.issn.0578-1752.2018.19.017

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【Objective】The aim of this study was to investigate the pharmacokinetic properties and bioavailability of tildipirosin in pigs after an intravenous or intramuscular administration of 4 mg·kg^-1 body weight. 【Method】 Twenty healthy pigs were selected and randomly divided into 2 groups and received 4 mg·kg^-1of tildipirosin injection by either intravenous or intramuscular administration. The blood was collected at 5 min, 10 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d, 9 d, 10 d, 11 d, 12 d, 13 d, 14 d and 15 d after administration. Phenomenex Luna C18 (150 mm×2 mm, 5 μm) was used. Acetonitrile-0.1% formic acid aqueous solution was used as the mobile phase. The gradient elution procedure was used. The flow rate was 0.25 mL·min^-1, the column temperature was 30℃, and the injection volume was 5.0 μL. The sample was thawed naturally, 0.5 mL of plasma was accurately pipetted into a 5 mL centrifuge tube, 2 mL of acetonitrile was added, vortexed and shaken for 10 min, centrifuged at 8000 r/min for 10 min. And then the supernatant was dried with nitrogen at 35℃ and reconstituted in 1 mL of solution. 0.22 μm microporous membrane, LC-MS/MS detection analysis. Tildipirosin showed a good linearity in the concentration range of 4-1000 ng·mL^-1, with a correlation coefficient of 0.994-0.998, a detection limit of 2 ng·mL^-1, and a limit of quantification of 4 ng·mL^-1. The relative recovery rate of this method was 87.91%-104.93%, which showed a good linear relationship with the correlation coefficient of 0.994—0.998. The coefficient of intra-assay coefficient of variation was 2.12%—12.09%, and the inter-assay coefficient of variation was 3.92%—10.65%. The experimental method had high sensitivity and simple operation, and could be used for the pharmacokinetic study of tildipirosin in pigs. MS conditions of detection method: ESI ion source, positive ion scan, ion spray voltage: 5500V, TEM: 550℃, the pressure of CUR: 25 psi, the pressure of GS2 50L/min, the pressure of collision CAD: 6 psi. the quantitative ion is: tildipirosin m/z→735.1/98.0. Blood samples were collected and detected by high-performance liquid chromatography (HPLC) with tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters were estimated using the WinNonlin5.2.1 software package and SPSS 16.0 analysis of the time and concentration data.【Result】After intravenous injection, AUC_lastand AUC_{inf (pred)} were (18030.30 ±7560.75) h·ng·mL^-1 and (18795.31±7455.23) h·ng·mL^-1, respectively;t_1/2 was (99.42±22.25) h, and MRT was (81.71±12.15) h. After intramuscular injection, C_max was (886.00±155.63) ng·mL^-1; T_maxwas (0.51 ± 0.30) h. AUC_last and AUC_{inf (pred)}were (19702.05±6442.36) h·ng·mL^-1 and (20840.08±6849.76) h·ng·mL^-1, respectively; t_1/2 was (100.83±20.23) h and MRT was (81.80±9.44) h; the absolute bioavailability was 109.27%. 【Conclusion】 After intramuscular injection, the tildipirosin was absorbed quickly in pigs, distributed widely, peaked rapidly, and eliminated slowly.

Effect of Different Intercropping Types on the Growth of Replanted Apple Tree and Soil Environment

MA ZiQing, DUAN YaNan, SHEN Xiang, CHEN XueSen, YIN ChengMiao, MAO ZhiQuan

Scientia Agricultura Sinica. 2018, 51(19): 3815-3822. doi:10.3864/j.issn.0578-1752.2018.19.018

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【Objective】Taking the soil of the old apple orchard as the object, this study is to explore the effects of mixed planting of different crops and replanted young apple trees on replanting plants and soil environment, select the more effective mixed plant to reduce the apple replant disease, and to provide technical support for the renewal of old orchards.【Method】The experiment included 5 treatments, which were CK (the old apple orchard soil), T1 (the methyl bromide fumigation old apple orchard soil), T2 (grafted apple seedlings mixed with Allium fistulosum), T3 (grafted apple seedlings mixed with Triticum aestivum), T4 (grafted apple seedlings mixed with Brassica juncea). the effect of mixed planting of different crops on the aboveground biomass, soil enzymes activity, soil microbial quantity, and soil phenolic acid content was detected. The copy number of the major harmful fungi in soil of old orchard was detected by real-time quantitative PCR technology.【Result】Mixed the three kinds of crops and young apple tree obviously enhanced the young apple tree plant height, ground diameter, fresh weight, dry weight, average fresh shoot length, and total fresh shoot length, and the treatment of mixed planting with A. fistulosum (T2) had the most beneficial effect, which plant height, ground diameter, fresh weight, dry weight, average fresh shoot length, and total fresh shoot length were 1.12, 1.31, 1.71, 1.65, 1.25 and 2.09 times of the control, respectively. mixed planting three crops could increase the number of soil bacteria and reduce the number of soil fungi. Compared with the control, the number of soil bacteria increased by 37.93%, and the number of soil fungi reduced by 41.33% under the treatment of mixed planting with A. fistulosum, the number of soilbacteria increased by 31.03% under the treatment of mixed T. aestivum, the number of soil bacteriaincreased by 13.79% under the treatment of mixed B. juncea. The ratio of bacteria to fungus in the soil increased by 135.11%, 128.55% and 89.66% when mixed A. fistulosum, T. aestivum, B. juncea, respectively. Compared with the control, the gene copy number of Fusarium genus in the mixed three kinds of crops decreased, and showed significant differences. The gene copy number of F. moniliforme, F. oxysporum, f. solani and F. proliferatum declined by 61.01%, 37.11%, 40.97% and 53.11% under the treatment of mixed planting with A. fistulosum, respectively. Mixed the three kinds of crops and young apple tree could increase the activity of main soil enzymes, among which the mixed planting of B. juncea had the obvious promotion effect of urease activity compared with the control, which increased by 44.59%. the mixed planting of T. aestivum increased the activity of phosphatase and sucrose by 59.26% and 50.37%, respectively. The content of phlorzin in mixed planting A. fistulosum, T. aestivumcrops and B. juncea soil decreased by 81.23%, 20.56% and 86.11%, respectively, the total amount of phenolic acid in the soil of the mixed planting of A. fistulosum decreased by 28.27% compared with the control, and the effect was better than other treatments.【Conclusion】Mixed planting of A. fistulosum, T. aestivum and B. juncea has great influence on soil environment and biomass of replanted young trees. All of the three mixed crops can promote the growth of replanted young trees, optimize the structure of microbial community and increase the activity of main enzymes in replanted soil, decrease the content of phlorzin in soil, and reduce the apple replant disease. Among them, the effect of mixed with A. fistulosum is the best.

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