Sponsor(s): Chinese Academy of Agricultural Sciences；Chinese Association of Agricultural Science Societies
24 issues per year
Current Issue: Issue 06, 2018
Scientia Agricultura Sinica, the 1st in Comprehensive Agricultural Science, is supervised by Ministry of Agriculture of PRC, and sponsored by Chinese Academy of Agricultural Sciences; Chinese Association of Agricultural Science Societies. Scientia Agricultura Sinica, launched in 1960, is a leading peer-reviewed and mufti-disciplinary journal and published semi-monthly in Chinese with English title, abstract, figures, tables and references. It aims to publish those papers that are influential and will significantly advance scientific understanding in agriculture fields worldwide. The scope covers Crop Genetics, Breeding, Germplasm Resources; Physiology, Biochemistry, Cultivation, Tillage Plant Protection; Soil & Fertilization, Agro-Ecology & Environment, Bio-energy; Animal Science, Veterinary Science, Agricultural Information Science; Food Science; Agricultural Economics and Management; Agricultural Sustainability. The journal is included in JST, CA and CSCD.
Editor-in-Chief Wan Jianmin Associate Editor-in-Chief Zou Ruicang Tang HuaJun Wu Kongming Guo YuYuan Geng Xu Sun Tan Executive Editor Lu Wenru
Induced Immunity Effect and Mechanism of the Weak Pathogenicity Isolate of Verticillium dahliae Vd171 Against Verticillium Wilt in Cotton
Scientia Agricultura Sinica,2018,Vol 51,No. 06
[Objective] The objective of this study is to clarify the induced immunity effect and mechanism of the weak pathogenicity isolate of Verticillium dahliae Vd171 against Verticillium wilt in cotton, and to provide a basis for exploring new ways of biological control of Verticillium wilt in cotton. [Method] The weak pathogenicity isolate of V. dahliae Vd171 was used to evaluate the induced immunity effects of different inoculation methods, inoculation periods, inoculation times and inoculum types in greenhouse. Different inoculation methods included root-dipping, root-irrigation, stem-injection, foliar-spraying and seed-soaking by Vd171 spore suspension with 1 × 10 7 conidia/mL. Totally 10 mL spore suspension for each pot was used in root-dipping, root-irrigation and foliar-spraying and 0.2 mL for each plant in stem-injection. The seeds were soaked for 12 h with spore suspension. Seven treatments were set to confirm the inoculation periods of Vd171 which included 2, 4, 6, 8 days before the inoculation of Vd080 (a high pathogenicity isolate of V. dahliae) and 1, 2, 4 days after the inoculation of Vd080. The inoculation times of Vd171 was one or two times before the inoculation of Vd080. The inoculum types included spore suspension and filtrate of Czapek of Vd171. The cotton seedlings were cultivated by hydroponics and inoculated by Vd080 GFP 4 days after Vd171. Then, the roots were collected in different time periods and the spores of Vd080 GFP were washed in sterile water. The colonization of Vd080 GFP on cotton root was analyzed by counting the spores of Vd080 GFP under microscope. Furthermore, in order to analyze the extension of Vd080 in cotton stem, the small hypocotyl pieces was cultivated on PDA plates on the 7th day after inoculated by Vd080 GFP. The transcript levels of 4CL, CHI, POD, PPO and PAL were analyzed by qPCR. The enzyme activities of POD, PPO, PAL and CHI were measured. The sedimentation of lignin was observed by phloroglucinol staining and the reactive oxygen species (ROS) of leaves was observed by DAB staining. [Result] The highest control efficacy was obtained when Vd171 was inoculated 4 days prior to inoculation by Vd080 (control efficacy of 89.4%). The control efficacy had significant difference among the five inoculation methods. Root-dipping treatment provided a control efficacy of 70.0%, followed by foliar-spraying (54.3%), seed-soaking (45.0%), root-irrigation (39.0%), and stem-injection (2.2%). There was no statistically significant difference between the control efficacy against Vd080 of pre-inoculated by Vd171 for one and two times (85.6% and 81.4%). Particularly, both cotton pre-inoculated with conidiospore and culture filtrate of Vd171 treatments could reduce disease indices of Verticillium wilt, with the control efficacies of 85.6% and 81.1%. There was no significant difference between the two treatments. Cotton plants pre-inoculated with Vd171 could reduce the colonization of Vd080 in cotton root and prevent its expansion in cotton stem. In addition, the transcript levels of Gh PAL, Gh4CL and Gh CHI increased significantly by induction of Vd171, which were 2.2, 8.5 and 2.6 times of the control treatment, respectively. The activities of PPO, PAL, POD and CHI in hypocotyl increased by 31.9%, 131.0%, 57.1% and 102.1% compared with that of control treatment, respectively, and the activities of PAL, CHI and POD in cotyledon also remarkably increased by 22.1%, 39.6% and 7.1% compred with that of control treatment, respectively. Meanwhile, sedimentation of lignin and ROS was induced by Vd171. [Conclusion] The weak virulent isolate Vd171 can effectively induce cotton resistant to Verticillium wilt and has a good application prospect in the control of Verticillium wilt in cotton.
Effect of Nitrogen Fertilizer on Dry Matter Accumulation, Transportation and Nitrogen Metabolism in Functional Leaves of Broomcorn Millet at Late Growth Stage
Scientia Agricultura Sinica,2018,Vol 51,No. 06
[Objective] The purpose of this study was to investigate the basis of nutritional requirement and supply for high quality of broomcorn millet through studying structure characteristics of dry matter and regularity of nitrogen metabolism in functional leaves of broomcorn millet at late growth stage under different nitrogen levels. [Method] Broomcorn millet cultivar of Yumi2 was selected as the material in a field experiment from 2015 to 2016. Dry matter accumulation, distribution and transportation were analyzed at heading stage, flowering stage, grain-filling stage and mature stage under different nitrogen levels of 60 kg·hm −2 (N1), 105 kg·hm −2 (N2), 150 kg·hm −2 (N3), 195 kg·hm −2 (N4). Gglutamine synthetase (GS) activity, nitrate reductase (NR) activity, free amino acid content and soluble protein content in functional leaves were detected. Yield and yield components were analyzed. The correlation of the dry matter accumulation, transportation of broomcorn millet and nitrogen metabolism of the leaves with yield was summarized. [Result] The results showed that the accumulation of dry matter of aboveground organs increased with increasing N application rates up to 150 kg·hm −2, and then decreased when N application rate was 195 kg·hm −2. The stem dry weight, leaf dry weight, sheath dry weight and spike dry weight under N3 treatment increased by 51.2%, 40.8%, 64.2% and 41.3%, respectively, compared with the non-fertilization treatment at the flowering stage. The transportation of dry matter in different organs was improved by applying nitrogen fertilizer. The contribution rates of aboveground organs to grain were increased after heading, which increased by 9.6% under N3 treatment, compared with the non-fertilization treatment. The GS activity, NR activity, free amino acid and soluble protein content in different leaves first increased and then decreased under nitrogen treatment. The nitrogen metabolism activity was in the descending order: the flag leaves > the second leaves > the third leaves at the same period, among which, N3 treatment reached the maximum value, but the overall change of nitrogen metabolism was not affected in functional leaves. Compared with no nitrogen application, the nitrogen contents of grain under N1, N2, N3 and N4 treatments increased by 4.0%, 6.0%, 7.8% and 8.9%, respectively, and the protein contents of grain by nitrogen fertilizers were significantly increased by 3.89%, 5.75%, 7.54% and 8.59%, respectively. The panicle length, stem diameter, the number of panicles per plant, the 1 000-grain weight and the yield were also significantly increased by nitrogen fertilizer. Compared with no nitrogen application, in 2015, the yields of broomcorn millet under N1, N2, N3 and N4 treatments were significantly increased by 10.09%, 29.71%, 44.73% and 35.99%, respectively, and in 2016, they were significantly increased by 19.08%, 30.60%, 65.85% and 39.14%, respectively. The biggest increase of yield was found in N3 treatment in two years. Therefore, N3 treatment had the best effect on increasing yield. [Conclusion] The dry matter accumulation, transportation and nitrogen metabolism in functional leaves of broomcorn millet could be promoted by nitrogen fertilizer at late growth stage, and yield and yield components could be improved. Under this experimental condition, N3 treatment (150 kg·hm −2) was the suitable amount of nitrogen in Yulin, Shaanxi.
Molecular Cloning and Functional Characterization of Purple Acid Phosphatase Related Gene MdPAP10 of Apple
Scientia Agricultura Sinica,2018,Vol 51,No. 06
[Objective] This study is aiming at cloning apple purple acid phosphatase gene MdPAP10 and studying its expression pattern and low phosphorus response process. The mechanism of action of MdPAP10 under low phosphorus condition was further studied. This study laid the foundation for further study of the molecular mechanism of MdPAP10 affecting phosphorus uptake in woody fruit trees. [Method] MdPAP10 gene was cloned by homology sequence alignment and PCR technique from Malus × domestica ‘Royal Gala’ apples. The protein structure of MdPAP10 was analyzed by NCBI, and the PAP10 amino acid sequences of 10 species such as white pear, peach and strawberry were obtained. The phylogenetic tree was constructed by MEGA5.0. The induced expression and tissue-specific expression profiles of MdPAP10 gene in the apple with low phosphorus stress were detected by real-time fluorescent quantitative PCR (qRT-PCR). MdPAP10 was ligated into the plant overexpression vector pBI121. The resultant construct was transformed into Agrobacterium tumefaciens LBA4404 for the infection of apple calli. The MdPAP10 transgenic calli were obtained by screening on resistant medium and PCR identification. The acid phosphatase accumulation, the tolerance to low phosphorus stress, and phosphorus content were detected by culturing MdPAP10 transgenic calli on low phosphorus medium. Finally, qRT-PCR was used to detect the expression of phosphorus-related genes in MdPAP10 transgenic calli.[Result] An apple purple acid phosphatase gene MdPAP10 (MDP0000272096) was cloned from Malus × domestica ‘Royal Gala’. The sequence analysis showed that the open reading frame (ORF) of MdPAP10 was 1 332 bp, which encoded 443 amino acids. The protein structure analysis showed that MdPAP10 contained a signal peptide and a phosphatase domain. The gene structure analysis showed that MdPAP10 contained five exons and four introns. A phylogenetic tree indicated that the apple MdPAP10 exhibited the highest sequence similarity to Pyrus bretschneideri PbPAP10. The expression analysis showed that MdPAP10 was expressed in roots, stems, leaves, flowers, and fruits, and the expression level in roots was the highest. MdPAP10 had a significant response to low phosphorus conditions, and the expression level in roots increased gradually, reached the maximum at 6 h and then decreased gradually. The expression level in leaves was always lower than that in the control group. The MdPAP10 transgenic calli were obtained by A. tumefaciens infection and verified by PCR and qRT-PCR. The MdPAP10 transgenic calli could significantly advance the secretion of acid phosphatase in low phosphorus conditions. Overexpression of MdPAP10 in transgenic calli under low phosphorus conditions improved the tolerance of calli to low phosphorus stress and increased the uptake of phosphorus. The result of qRT-PCR showed that overexpression of MdPAP10 could significantly promote the expression of apple phosphorus-related genes. [Conclusion] MdPAP10 can respond significantly to low phosphorus stress and promote phosphorus uptake and acid phosphatase secretion under low phosphorus conditions. MdPAP10 plays an important regulatory role in response to low-phosphorus stress.
An Electrochemical Aptasensor for Detection of Salmonella typhimurium with Methylene Blue as Hybridization Indicator
Scientia Agricultura Sinica,2018,Vol 51,No. 06
[Objective] A novel assay of electrochemical aptasensor for quantitative detection of Salmonella typhimurium with better practicability was constructed and investigated in order to overcome the shortcomings of traditional Salmonella detection methods in terms of time consumption, sensitivity, simplicity, etc. [Method] The prepared graphene oxide (GO) solution was dropped onto the glassy carbon electrode (GCE) surface and was reduced in PBS buffer by electrochemical reduction method to obtain reduced graphene oxide (rGO), and then Au nanoparticles (AuNPs) were electrochemically deposited onto the electrode by submersion in HAuCl 4. The complementary strands (S) of the aptamers of Salmonella typhimurium were attached to the surface of rGO/AuNPs GCE by Au-S bond, and then the electrode surface was blocked with MCH. Subsequently, the aptamers (Apt) of Salmonella typhimurium were dripped onto the modified electrode to make Apt bind with S. The modified electrode was immersed into the mixture containing Salmonella typhimurium and exonuclease I ( Exo I) at 37 °C. In terms of the characteristics of Exo I that could amplify electrical signals and the aptamers that could exclusively bind with Salmonella typhimurium, the aptamers were taken away from S circularly. Then, the modified electrode was immersed in methylene blue (MB) solution for a while. Finally, the conditions of the incubation time in bacteria liquid and the Exo I concentration were optimized and the electrical signals of the electrode surface were monitored to construct the aptasensor. This electrochemical aptasensor was used to test Escherichia coli, Staphylococcus aureus, Shigella, Listeria monocytogenes and Vibrio parahaemolyticus to ensure the electrochemical aptasensor’s specificity. The electrochemical aptasensor was used to detect 2 × 10 2–2 × 10 7 cfu/mL Salmonella typhimurium to ensure the electrochemical aptasensor’s sensitivity. Then this electrochemical aptasensor was used to detect the pork to evaluate the practical use of electrochemical aptasensor. [Result] The optimization of the electrochemical aptasensor incubation time in bacterial liquid and the Exo I concentration were studied in detail, and the optimal conditions were 40 min and 0.8 U·μL −1. The developed aptasensor was specific to Salmonella typhimurium and did not react with non-target bacteria. The electrochemical aptasensor could successfully detect the Salmonella typhimurium target down to 67 cfu/mL. A good recovery of Salmonella typhimurium in the range of 97.3%–106.7% was obtained in pork by the electrochemical aptasensor assays developed. [Conclusion] This electrochemical aptasensor can detect Salmonella typhimurium with a high sensitivity, a high specificity, an easy operation, a rapid detection and a low cost, which provides a good application prospect in the field of rapid quantitative detection of Salmonella typhimurium.
Comparison and Analysis of Genetic Parameters Estimation of Early Growth Traits of Alpine Merino Sheep by Different Animal Models
Scientia Agricultura Sinica,2018,Vol 51,No. 06
[Objective] This study aims to investigate the effects of different animal models on the estimation of genetic parameters of early growth traits of Alpine Merino Sheep, and to select the best animal model. The best model was used to estimate the genetic parameters of early growth traits, which could provide a theoretical basis for the breeding of Alpine Merino Sheep at the early stage. [Method] Firstly, F-test was performed using ASReml software to determine whether the factors in the fixed effects were significant. And the fixed effects which had significant influences on the early growth traits of Alpine Merino sheep were selected for further analysis. Secondly, four animal models were constructed considering different random effects. And the genetic parameters in each model were estimated by the Average Information Restricted Maximum Likelihood (AIREML) in ASReml software. The individual additive genetic effect was considered as random effect in model 1. Besides individual additive genetic effect, the maternal genetic effect and individual permanent environmental effect were considered as random effects in model 2 and model 3, respectively. Finally, the optimal animal model was selected by comparing the random effects in different models with the Akzo Information Criterion (AIC) Index and Likelihood Ratio Test (LRT). [Result] (1) The results showed that bloodline, gender, birth type, birth year, birth month, mating month and flocks highly affected the early growth traits of Alpine Merino Sheep ( P < 0.001). Except gender and birth type, other fixed effects were significant on gestation length ( P < 0.001). (2) The direct heritabilities were (0.092 4 ± 0.016 0)–(0.207 3 ± 0.022 6), (0.065 1 ± 0.012 6)–(0.102 7 ± 0.015 9), (0.068 1 ± 0.013 0)–(0.100 1 ± 0.106 1), (0.086 5 ± 0.014 8)–(0.093 7 ± 0.014 9), and (0.090 2 ± 0.017 4)–(0.111 9 ± 0.018 9) for birth weight (BWT), weaning weight (WWT), pre-weaning average daily gain (ADG), weaning staple length (WSL), and gestation length (GL), respectively. The maternal heritabilities were 0.162 3 ± 0.011 3, (0.109 7 ± 0.040 7)–(0.109 8 ± 0.011 2), 0.089 8 ± 0.011 2, 0.017 3 ± 0.010 7, and 0.047 7 ± 0.014 6 for BWT, WWT, ADG, WSL, and GL, respectively. (3) The comparative analysis of the random effects in different models by AIC and LRT showed that the early growth traits were significantly affected by the individual additive genetic effects and maternal genetic effects, but the individual permanent environmental effects were negligible. Therefore, model 2 was the best model for the early growth traits of Alpine Merino Sheep. [Conclusion] The early growth traits of Alpine Merino Sheep were affected by the maternal genetic effects more than other random effects. Model 2 was the best animal model for estimating the genetic parameters of early growth traits of Alpine Merino Sheep. Based on the best animal model, the direct heritabilities were 0.092 4 ± 0.016 0, 0.065 1 ± 0.012 6, 0.068 1 ± 0.013 0, 0.086 5 ± 0.0148, and 0.0902 ± 0.017 4 for BWT, WWT, ADG, WSL, and GL, respectively. The maternal heritabilities of these traits were 0.162 3 ± 0.011 3, 0.109 8 ± 0.011 2, 0.089 8 ± 0.011 2, 0.0173 ± 0.0107, and 0.047 7 ± 0.014 6, respectively.