Table of Content

16 July 2015, Volume 48 Issue 14

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Organization Changes of Actin Cytoskeleton and Callose with the Expression Analysis of Related Genes in Physiological Male Sterile Wheat

ZHANG Jiao, ZHU Qi-di, JU Lan, ZHANG Gai-sheng, YU Yong-ang, NIU Na, WANG Jun-wei, MA Shou-cai

Scientia Agricultura Sinica. 2015, 48(14):  2687-2696.  doi:10.3864/j.issn.0578-1752.2015.14.001

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【Objective】The organization of actin filaments and callose and the expression of related genes in physiological male sterility wheat pollen cells were studied to reveal the relationship between actin filaments and callose and pollen abortion in physiological male sterility wheat. The results of the study will provide a theoretical basis for further study on the mechanism of male sterility induced by chemical hybridizing agents SQ-1 in wheat. 【Method】The physiological male sterility line ms(A)-Xinong1376 and corresponding normal fertile wheat (A)-xinong1376 were used as test materials. The TRITC-phalloidin was used to stain actin filaments and Aniline blue was used to stain callose. QRT-PCR was used to analyze the expression of TaADF (Actin depolymerizing factor) and TaGSL (Glucan synthase-like).【Result】At prophase I, metaphase I, and anaphase I, there were no significant differences between physiological male sterility and male fertility. At prophase I, actin filaments distributed in cytoplasm of the cell and there was also distribution of some actin filaments in nuclear zone. At metaphase I, actin filaments distributed in cytoplasm. They were stained deeper in the spindle position and formed actin spindle. At anaphase I, actin filaments between the poles of the chromosomes were stained deeper, so there were more actin filaments distributed there. At early telophase I, we observed that there were no sharp actin filaments at leading edges of phragmoplasts and the overlapped actin filaments at midline were obscure in physiological male sterility. At late telophase I, there was a cell plate at the midline of phragmoplasts, it was linear and smooth, but in the physiological male sterile line, the linear cell plate was not seen, the midzone of dyads was hollow. At the same time, the deposition of callose on the cell plate was insufficient and the cell plate was wrinkled and cleft. At tetrad, actin filaments were obscure and had no silky feeling and callose fluorescence was weaker in the physiological male sterile. Then qRT-PCR technique was performed to analyze the expression patterns of the TaADF gene and TaGSL gene in sterile and fertile anthers during meiosis. The results indicated that the transcript of TaADF in the treated anthers was 4.28 times higher than that of the control. It indicated that because of the increased expression of TaADF, the rate of dissociation of actin filaments was increased, so the microfilaments were obscure. The transcript of TaGSL in the treated anthers was 0.83 times lower than that of the control. Callose deposition was influenced by SQ-1. 【Conclusion】The increased expression level of TaADF in the physiological male sterility line destroyed the normal structure of actin filaments. The actin filaments could not execute their functions. Then it may influence certain metabolic pathways associated with fertility in anther development. At the same time, abnormal actin filaments caused abnormal cell plate formation which may have an effect on callose deposition. Therefore, it was estimated that it has a close relationship among the changes of actin filaments and callose and the physiological male sterility induced by SQ-1.

B Chromosome Polymorphisms in Maize (Zea mays L.) Landrace Populations from Southwest China

YAO Qi-lun, CHEN Fa-bo, LIU Hong-fang, FANG Ping, ZHAO Cai-fang

Scientia Agricultura Sinica. 2015, 48(14):  2697-2704.  doi:10.3864/j.issn.0578-1752.2015.14.002

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【Objective】 The objective of this study is to analyze B chromosome polymorphisms in maize landrace populations from Southwest China, to evaluate their genetic diversity at cellular level and provide evidence for the introduction path of maize in China. 【Method】 Thirty maize landrace populations from Sichuan Province, Chongqing city, Yunan Province, and Guizhou Province in southwestern China were used. Fifty individuals per landrace population were sampled, and ten cells from a single plant were scored for the presence of Bs under a microscope. A total of 15 000 cells (30 × 50 × 10) in the mitotic metaphase were analyzed. Ten slides, from ten individuals per landrace population, were C-banded using conventional Giemsa staining with modifications.【Result】 Besides 10 pairs of standard chromosomes, the landrace populations contain a metacentric B chromosome (B_M) morphologically similar to the first chromosome of the karyotype, a subtelocentric B (B_ST) identical in size to B_M, and a spot-shape microchromosome (B_S) without centromere. Seven maize landrace populations from Sichuan possessed three forms of Bs. In the populations of maize landraces from Chongqing both B_M and B_ST forms were found, whereas B_ST and B_S were identified in the populations of maize landraces from Yunnan. Nine populations from Guizhou were the only one (B_ST) where B_M and B_S were exclusive. The mean length of B_M, B_ST, and B_S was 2.82 µm, 2.78 µm, and 0.9 µm, respectively. In comparison with the metacentric pair of the standard karyotype, measurements conducted on the cells with Bs estimated a relative length of 34%-52% for the B_M, 32%-58% for B_ST, and 13%-18% for the B_S, and relative lengths for B_M, B_ST, and B_S were on the average 43%, 45%, and 15%, respectively. The mean arm ratios for B_M, B_ST, and B_S were 1.19, 1.98, and 1.00, respectively. Of the eighteen populations with B chromosomes, seven, five, four, and two populations originated geographically from Sichuan, Chongqing, Yunnan, and Guizhou, respectively. Eighteen of the thirty populations sampled exhibited numerical polymorphisms and a total of 487 Bs were identified in 421 individuals. The frequency of B chromosomes per individual varied from 0 to 90% with an average of 29.6%. The populations from Sichuan possess a higher frequency of B chromosomes in both population and individual levels. The highest number of B chromosomes in a cell was 2 with the predominant dose being 0. The mean frequency of B chromosomes for the 0B, 1B, and 2B cell were 97.04%, 2.81%, and 0.15%, respectively. B_ST is the predominant form accounting for 67% of the Bs in the populations, followed in turn by B_M and B_S corresponding to 19% and 14%, respectively. It might be hypothesized that B_ST repres­ented an ancestor B chromosome in maize, whereas the B_ST and B_S were variants of the B_M, implying that the evoluation of B_M and B_S have occurred in a smaller time and space scale. The Giemsa staining exhibited that the B_M and B_ST forms, easily distinguished from A chromosomes, shared some homologous DNA sequences and were both heterochromatic as well as GC-rich. The mean C-band number varied from 11.0 to 20.6 in the 0B cell, 10.8 to 19.4 in the 1B cell, and 10.5 to 18.6 in the 2B cell, indicating a negative correlation between B chromosomes and C-bands in the A chromosome set. 【Conclusion】 Cytological observations revealed that maize landrace populations from southwest China had a higher level of genetic diversity. For the B chromosome in them, there existed morphological and numerical polymorphisms. Sichuan is the geographical centre of maize landraces in this region based on geographical distributions of the B chromosome. This supported the theory on a maize introduction path in China.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Characteristics of Lodging Resistance of Super-Hybrid Indica Rice and Its Response to Nitrogen

WU Xiao-ran, ZHANG Wu-jun, WU Long-mei, WENG Fei, LI Gang-hua, LIU Zheng-hui, TANG She, DING Cheng-qiang, WANG Shao-hua, DING Yan-feng

Scientia Agricultura Sinica. 2015, 48(14):  2705-2717.  doi:10.3864/j.issn.0578-1752.2015.14.003

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【Objective】 Experiments were conducted with two super-hybrid rice varieties which have a big difference in lodging resistance. The main objective of this study was to reveal the mechanisms of the divergence of lodging resistance and effects of nitrogen on rice lodging resistance between super-hybrid rice varieties. Results of the study will provide a theoretical basis for breeding and nitrogen management for high yield of rice. 【Method】 Experiments were conducted with super-hybrid rice Yliangyou2 which is strong in lodging resistance and Ⅱyou084 which is weak in lodging resistance to compare their difference in lodging resistance between the two varieties and study the effects of nitrogen on rice morphological, mechanical and physiological traits. In 2012, 0, 150, and 300 kg N·hm^-2 were applied. In 2013, 135, 270, and 405 kg N·hm^-2 were arranged. 【Result】 The yield of Yliangyou2 reached 11.7 t·hm^-2, 9.45% higher than IIyou084. This was mainly attributed to the higher number of spikelets per panicle and spikelets per hectare, which were 28.0% and 31.8% higher than IIyou084, relatively. The lodging index of Yliangyou2 reduced significantly at mature stage, 19.0% higher than IIyou084. This was because the disadvantage of the culm diameter of Yliangyou2 was compensated by the decreased length of the basal internode, thickened stem wall and significant increased dry weight per unit length of leaf sheath. For Yliangyou2, although there was no significant advantage in dry weight per unit length of culm, high structural carbohydrate content lead to the significant increasing of the lodging resistance. With the nitrogen increasing, the lodging index of the super-hybrid indica rice increased significantly as the reduction of the bending moment at breaking. The quality of the stem at heading stage was deteriorated with the excessive nitrogen fertilizer, as a result, the mechanical strength of stem dropped with the dry weight per unit length of basal leaf sheath. In addition, the lignin content of basal stem significantly decreased, which increased the risk of lodging. The breaking resistance of IIyou084 decreased greatly at the lowest nitrogen level, even lower than that of Yliangyou2 at highest nitrogen level, which caused significant reduction of lodging index. With the nitrogen increasing, although the apparent lodging rate of IIyou084 had the tendency of increase, but not significant, the yield decreased obviously.【Conclusion】For the purpose of increasing the lodging resistance of super-hybrid indica rice varieties, it would be favorable to compensate the decrease in culm diameter with increasing wall thickness and shortening the length of basal internode, enhance the protection and support to the culm with increasing the filling degree of leaf sheath. Nitrogen reduced the lodging resistance of super-hybrid indica rice mainly through decreasing the dry weight per unit length of leaf sheath and the content of the structural carbohydrates, especially the content of lignin, in the basal internode.

Yield Components and Population and Individual Characteristics of Growth and Development of Winter Wheat over 10 000 kg·hm^-2 in Hebei Province

WANG Hong-guang, LI Dong-xiao, LI Yan-ming, LI Rui-qi

Scientia Agricultura Sinica. 2015, 48(14):  2718-2729.  doi:10.3864/j.issn.0578-1752.2015.14.004

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【Objective】The purpose of this study is to explore the pathway that the super high yield level of wheat tops 10 000 kg·hm^-2 from 9 000 kg·hm^-2 in Hebei Province, and to definite yield component characters and population and individual features at various growth stages of super high yielding wheat over 10 000 kg·hm^-2, as well as the appropriate ecological conditions, so as to provide a theoretical basis for studying super highly-yielding cultivation technique system of wheat that can achieve the yield over 10 000 kg·hm^-2 stably in Hebei Province.【Method】The super high-yielding research was conducted by adopting different varieties and nitrogen application treatments in high-yielding field during 2010-2014 growing seasons of wheat, and combining with other super high-yielding cultivation technique measures. The yield results were divided into three levels, i.e., 9 000-9 500 kg·hm^-2, 9 500-10 000 kg·hm^-2 and ＞10 000 kg·hm^-2, and then the changes in yield components and population and individual features at various growth stages from 9 000 kg·hm^-2 to 10 000 kg·hm^-2 were analyzed. Meanwhile the appropriate ecological conditions benefiting high yield over 10 000 kg·hm^-2 was confirmed by combining with soil fertility and meteorological data.【Result】The results of comparative analysis on treatments of three yield levels showed that, the change of spike number per hectare was smaller when the grain yield of wheat in Hebei Province increased from 9 000 kg·hm^-2 to ＞10 000 kg·hm^-2, and kernels per spike mainly floated from thirty to thirty five, however, the grain weight increased significantly. The dry matter accumulation amount increased significantly when yield level increased from 9 000-9 500 kg·hm^-2 to 9 500-10 000 kg·hm^-2, and the harvest index also increased when yield level went on increasing to ＞10 000 kg·hm^-2. The relatively ideal yield components and targeting parameters for super high-yielding wheat of ＞10 000 kg·hm^-2 in Hebei Province are that, spikes 800×10⁴ hm², 30-35 kernels per spike, 1 000-grain weight 43 g, dry matter weight at maturity 22 000 kg·hm^-2, and harvest index 0.46, respectively. In treatments with the yield ＞10 000 kg·hm^-2, the area of flag leaf and penultimate leaf was smaller than 20 cm² respectively, the leaf area index at booting stage was 7.69-8.24, which were all lower compared with treatments of 9 000-9 500 kg·hm^-2 yield level. However, in treatments with the yield ＞10 000 kg·hm^-2, the leaf area index on 20 and 30th day after anthesis of wheat were larger than 4 and 2, respectively, which were larger than those in treatments with the yield 9 000-9 500 kg·hm^-2. Accompanied with the yield increased from 9 000 kg·hm^-2 to ＞10 000 kg·hm^-2, there were little change in basal fertility of soil and fertilizing amount, the precipitation and irrigation amount during growing season of wheat was not increased too, while the accumulated temperature and sunshine duration from anthesis to maturity and the whole growing season of wheat were higher or longer.【Conclusion】To break through the bottleneck that grain yield of wheat from 9 000 kg·hm^-2 to ＞10 000 kg·hm^-2 in Hebei Province, the effect of increasing spikes per hectare will be little, increasing the kernels per spike and grain weight should be regarded as the main direction. The high quality population with a modest size and senescing slowly is the guarantee of super high yield over 10 000 kg·hm^-2. Meanwhile, the higher basal fertility, suitable accumulated temperature and longer sunshine duration are the basis to achieve super high yield over 10 000 kg·hm^-2.

Long-Term Simulation of Winter Wheat Yield and Soil Water Response to Conservation Tillage Rotation in Weibei Highland

ZHANG Yu-jiao, LI Jun, GUO Zheng, YUE Zhi-fang

Scientia Agricultura Sinica. 2015, 48(14):  2730-2746.  doi:10.3864/j.issn.0578-1752.2015.14.005

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【Objective】Weibei Highland is a typical rainfed agricultural zone in Northern China, so water shortage is the main factor limiting grain production. Along with the shortage and uneven seasonal distribution of annual rainfall, the growth and development of winter wheat constrains that cause low and unstable yield. The aim of this article is to explore the effects of different rotation tillage patterns on soil water conservation and yield increase in continuous winter wheat region under the long-period in Weibei Highland.【Method】Based on model validation, the WinEPIC model was used to simulate crop yield and soil moisture effects of continuous planting winter wheat field under different tillage rotation treatments (CT (continuous conventional tillage), NT/ST (no tillage/sub-soiling rotation), CT/ST (conventional tillage/sub-soiling rotation), NT/NT/ST (no tillage/no tillage/sub-soiling rotation), NT/CT/ST (no tillage/conventional tillage/sub-soiling rotation)) on Weibei Highland during 1980-2009.【Result】As the gradual decrease of annual precipitations during the simulation period, the simulated wheat yield, WUE (water use efficiency) and water consumption in growth season of winter wheat decreased with fluctuation. The average wheat yield of NT/ST, CT/ST, NT/NT/ST and NT/CT/ST treatments compared to CT treatment increased by 14.4%, 12.2%, 2.4% and 3.2%, respectively, the average wheat yield of no tillage/sub-soiling rotation treatment was the highest, which was 3.53 t×hm^-2. Compared to CT, WUE of NT/ST, CT/ST, NT/NT/ST and NT/CT/ST treatments treatment increased by 6.36%, 6.13%, 6.40% and 6.41%, WUE of NT/NT/ST treatment and NT/CT/ST treatment was better than others, which was 8.68 kg×hm^-2×mm^-1. Compared with CT, the water consumption in the winter wheat growth season of NT/ST, CT/ST, NT/NT/ST and NT/CT/ST treatments increased obviously in the dry year; the difference among them was not significant in the normal rainfall and rainy years. The water consumption in growing season of NT/ST treatment was higher than the other rotation tillage treatments. The monthly available soil water amount in 0-3 m soil layers of winter wheat field showed a seasonal decreasing trend during the simulation period and fluctuated obviously with the seasons, the average available soil water of NT/ST, CT/ST, NT/NT/ST and NT/CT/ST treatments was 28.7%, 27.2%, 26.8% and 26.7% higher than CT treatment, respectively. The water conservation effect of NT/ST treatment was the best, average available soil water in 0-3 m soil layers was 107.1 mm, CT/ST treatment and NT/CT/ST treatment were the second, the last one was continuous conventional tillage treatment. The soil moisture in 0-1 m soil layers changed with the fluctuation of precipitation in wheat field, it also changed in 1-1.5 m in rainy or dry year which was more stable in 1.5-3 m, the difference among different tillage rotation treatments was not significant, and the effect of NT/ST treatment was the best. 【Conclusion】Compared with CT, the yield and effect of water preservation of CT/ST treatment were higher, but the WUE was slightly lower. The WUE and effect of water preservation of NT/NT/ST and NT/CT/ST treatment were higher, but the yield was lower. Considering the yield, WUE and effect of water preservation of the winter wheat field, NT/ST rotation treatment is the most reasonable conservation rotation in long-term continuous winter wheat cropping system on Weibei Highland.

PLANT PROTECTION

Construction of a Rapid Screening System of Pathogenicity-Related Mutants in Verticillium dahliae

WANG Xin-yan, ZHANG Dan-dan, GUI Yue-jing, LI Nan-yang, XU Ming, CHEN Jie-yin, DAI Xiao-feng

Scientia Agricultura Sinica. 2015, 48(14):  2747-2756.  doi:10.3864/j.issn.0578-1752.2015.14.006

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【Objective】 The objective of this study is to construct a rapid screening system of pathogenicity-related mutants in Verticillium dahliae, which will provide a technical support to identification of T-DNA mutant library.【Method】The cotton roots were inoculated with V. dahliae conidial suspension by root-dipping method for 30 min, including five concentrationsof 5.0×10⁴, 5.0×10⁵, 5.0×10⁶, 5.0×10⁷and 5.0×10⁸ conidia/mL, to determine the suitable concentration range of causing the obvious Verticillium wilt. The culture flasks were used for the V. dahliae culture, and 15, 25, 35 and 45 mL of sterilized water were added into the flasks to elute the conidial. The concentration of conidial was calculated by the hemocytometer to detect the effect of sterilized water volume on the conidial concentration. Based on the unit of mutants in 96-well plates, the single spore of mutants was isolated on the petri dish and expanding cultured in the flasks, then the sterilized water at suitable volume was directly added into the flasks to elute the conidial and the cotton seedling roots were dipped into the conidial suspension in the flasks for 30 min, subsequently the infected cotton seedlings were cultured for 14 d and the disease index was investigated. A rapid screening system of pathogenicity-related mutantsin V. dahliae was constructed after optimization of cotton planting, mutants single spore isolation, expanding culture, inoculation, and results investigation. The quantitative inoculation method was used for validation of the reliability results of pathogenicity-related mutants, 30 cotton seedlings with three replicates for each mutant, the concentration of suspension was 5.0×10⁶ conidia/mL, 5 mL suspension for each seedling, and inoculated for 30 min, finally the disease indexes of pathogenicity-related mutants were investigated on 5, 8, 11 and 14 d after inoculation.【Result】The conidial concentration up to 5.0×10⁵ conidia/mL could obviously infect the cotton and cause the Verticillium wilt symptom. A standard culture method of V. dahliae was constructed which includes single spore isolation for 5 days and expanding culture in standard culture flasks for 9 days. The appropriate volume of sterilized water used for eluting the conidial suspension was 25 mL. The test results showed that the concentration for eluting the conidial suspension of 20 mutants were（2.55±0.58)×10⁶-(1.72±0.25)×10⁷ conidia/mL. Subsequently, the inoculation steps were optimized for construction of the rapid process of V. dahliae pathogenicity identification, which includes single spore isolation, expanding culture, conidial suspension preparation, inoculation, and results investigation. Moreover, a rapid screening system of pathogenicity-related mutants in V. dahliae was constructed after coordinative design. The test results showed that one people could finish 1 334 mutants within 54 days by running one cycle which includes seven processes, and the work load is only 21 man-days. The validation of target mutants by quantitative inoculation method showed that the pathogenicity of these mutants was significantly reduced, consistent with the results of the rapid screening system, which indicated that the rapid screening system is appropriate for the screening of pathogenicity-related mutants in V. dahliae.【Conclusion】A rapid screening system ofpathogenicity-related mutants in V. dahliae was constructed after optimization and standardization of cotton planting, mutants single spore isolation, expanding culture, preparation of the conidial suspension, inoculation, and results investigation, which will provide a technical support for the pathogenicity-related gene research in future.

Determination of Spore Viability of Fusarium solani Based on Dual Fluorescence Assay

CHAI A-li, HAN Yun, WU Jun, SHI Yan-xia, XIE Xue-wen, LI Bao-ju

Scientia Agricultura Sinica. 2015, 48(14):  2757-2766.  doi:10.3864/j.issn.0578-1752.2015.14.007

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【Objective】Quantitative determination of spore viability of Fusarium solani is the foundation for effective control of the diseases. The traditional spore germination assay is complicated and time consuming, and often with poor sensitivity. Rapid and effective determination methods are needed. The objective of this study is to develop a dual fluorescence assay by combining fluorescein diacetate (FDA) with propidium iodide (PI) for quantitative detection of spore viability of F. solani sensitively and accurately. 【Method】The optimal incubation time, optimal concentrations of FDA and PI were determined, and then the FDA-PI dual fluorescence assay were established. To evaluate the accuracy of FDA-PI assay, pre-defined percentages of dead F. solani spores (0, 25%, 50%, 75% and 100% dead) were detected by FDA-PI assay. The practical mortalities (via FDA-PI assay) were compared with theoretical mortalities. Moreover, spores of F. solani were subjected to physical, chemical and fungicide stress. Relationship between spore mortality (via FDA-PI dual fluorescence assay) and rate of spore germination (via spore germination assay) were analyzed. 【Result】A FDA-PI dual fluorescence assay was developed to assess the activity of F. solani spores. The optimal working concentration of FDA was 100 µg?mL^-1, incubated for 20 min at 25℃. The optimal working concentration of PI was 3 µg?mL^-1, and incubated for 10 min at 4℃. For pre-defined percentages of dead spores, a correlation existed between practical (via FDA-PI assay) and theoretical mortality of spores (R²=0.99, P＜0.05). The result demonstrated that FDA-PI assay allowed for quantification of spores viability of F. solani. Physical treatments (pressurised heat, dry heat and UV light) and chemical treatments (ethanol and hydrogen peroxide) induced gross changes in the activity of the spores of F. solani. There was a negative correlation between spore mortality (via FDA-PI assay) and rate of spore germination (via spore germination assay) (R²=0.99, P＜0.05). For fungicide (fludioxonil, carbendazim and calcium cyanamide) treatment, spore mortalities of F. solani raised as the concentration of fungicide increased. As to spores of F. solani treated by calcium cyanamide, the spore mortality detected by FDA-PI assay was consistent with the result detected by spore germination assay. While the results of FDA-PI assay was below than spore germination assay after fludioxonil and carbendazim treatment. 【Conclusion】A new method was developed for rapid, high-throughput and automatic detection of spore viability of F. solani based on FDA-PI dual fluorescence assay and flow cytometry. This method would replace the traditional spore germination assay, and could shorten the detection time and improve the efficiency greatly.

Localization of Melanin Biosynthesis Enzyme Genes in the Genome and Expression Pattern Analysis of Setosphaeria turcica

JIA Hui, MENG Qing-jiang, LI Zhi-yong, GONG Xiao-dong, ZANG Jin-ping, HAO Zhi-min, CAO Zhi-yan, DONG Jin-gao

Scientia Agricultura Sinica. 2015, 48(14):  2767-2776.  doi:10.3864/j.issn.0578-1752.2015.14.008

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【Objective】The objectives of this study are to determine the location in the genome, and to investigate the structure of melanin biosynthesis genes StPKS, StSCD, St3HNR, St4HNR, StLAC1 and StLAC2 of Setosphaeria turcica. Furthermore, to elucidate the relationship between melanin biosynthesis enzyme genes and morphogenesis and pathogenicity of S. turcica viaanalyzing the expression pattern of six genes during infection progress from conidial germination to penetration and the mycelia growth period.【Method】 Based on the S. turcica genome database and genome searching with Blastp, the locations of six genes were identified and genes linkage were analyzed. Primers were designed according to the six melanin synthesis enzyme gene sequences. qRT-PCR system was used to detect the six genes expressions of S. turcica, in which total RNAs from different infectious morphogenesis stages including conidial germination to penetration were extracted and used as templates and β-tubulin as the reference gene, and the expression patterns between vegetative and reproductive growth stages were compared.【Result】In genome database, the StPKS and St3HNR were located on the positive and negative strand of scaffold_12, respectively. St4HNR and StLAC2 were on the negative strand scaffold_11. StSCD and StLAC1 located on the positive of strand scaffold_1 and scaffold_7, respectively. Furthermore, StPKS and St3HNR were clustered within the 26.9 kb chromosomal region. Relative expression of six genes had two expression profiles, which was up-down- up regulation or up- down- up- down regulation at five stages from conidial germination to penetration. The StLAC2 had the highest expression in conidia, and there was a significant difference in expression. However, St3HNR and StSCD had a higher expression level than other genes in mycelia.【Conclusion】StPKS and St3HNR in melanin biosynthesis were clustered within a 26.9 kb chromosomal region. Six genes were whole expressed at five stages from conidial germination to penetration. They had a similar expression pattern. Unfortunately, they had a significant difference in expression levels. The results showed that six genes of melanin biosynthesis enzyme participated in the infection process and played an important role in the pathogenic of S. turcica. In addition, St3HNR and StSCD played an important role at mycelial stage. StLAC2 had the highest expression at conidial stage.

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

Dynamic Kinetic Characteristics of Different Ratios of Ammonium and Nitrate Absorbed by Banana Seedlings

ZHANG Chao-yi, FAN Xiao-lin

Scientia Agricultura Sinica. 2015, 48(14):  2777-2784.  doi:10.3864/j.issn.0578-1752.2015.14.009

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【Objective】Ammonium and nitrate and their interaction effect on N absorption by banana seedlings were studied to screen the optimum ratio of ammonium to nitrate for banana growth and efficient utilization of nitrogen. 【Method】 The NH₄⁺-N, NO₃^--N and total nitrogen kinetics of N uptake by banana seedlings were studied by modified depletion method based on the principle of nutrient absorption kinetics in the solution culture with different ratios of ammonium to nitrate. 【Result】 Michaelis-Menten enzyme kinetic model or equation could be applied to describe the patterns of absorption of NH₄⁺-N, NO₃^--N and total N by banana seedlings in all the treatments and the co-relationship coefficient r was at a significant level. When the rate of NH₄⁺-N was at 10% and 70%, NH₄⁺-N absorption rate increased with the NO₃^--N addition gradually. The maximum absorption rate (Vmax) of NH₄⁺-N uptake reached the maximum of 55.56 μmol·g^-1·h^-1, when the NH₄⁺-N ratio increased to 70%. The NH₄⁺-N absorption rate decreased again when the NH₄⁺-N ratio was more than 70%. However, the rate of nitrate absorption of banana declined with the increase of NH₄⁺-N. When the proportion of NH₄⁺-N increased from 10% to 90%, the Vmax of NO₃^--N decreased by 2.62 times. Therefore, adding NH₄⁺-N would significantly inhibit the banana seedlings to absorb NO₃^--N. There was no obvious effect of the ratio of ammonium to nitrate on affinity of banana root to NH₄⁺-N and NO₃^--N. When ammonium to nitrate ratio was 3﹕7, the total nitrogen absorption rate was 83.33 μmol·g^-1·h^-1, being the maximum rate and in favor of banana to take up nitrogen. 【Conclusion】 When the proportion of NH₄⁺-N was lower than 70%, increase of NO₃^--N could enhance the banana seedlings to take up NH₄⁺-N. When the ammonium ratio was more than 70%, increase of NO₃^--N would inhibit the banana seedlings to take up NH₄⁺-N. Increasing ratio of NH₄⁺-N could significantly inhibit the banana seedlings to absorb NO₃^--N. The optimum ratio of ammonium to nitrate being favor of nitrogen absorption was 3﹕7.

Nitrogen Release of Returned Maize Straw and Its Effects on Loess N Supply and Nitrogen Uptake by Winter Wheat in Guanzhong Plain

HUANG Ting-miao, ZHENG Xian-feng, WANG Zhao-hui

Scientia Agricultura Sinica. 2015, 48(14):  2785-2795.  doi:10.3864/j.issn.0578-1752.2015.14.010

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【Objective】 In order to optimize the nitrogen nutrient management under straw returning to soil condition in the winter wheat-summer maize rotation region of Shaanxi Guanzhong Plain, a field experiment was carried out to study the N release characteristics of maize straw and the effects of returning straw to soil on loess N supply and nitrogen uptake by winter wheat. 【Method】The field experiment was initiated in October 2012 and ended in May 2013 at Zhongnan town, Zhouzhi county in Shaanxi Province. Some little nylon net bags containing the air dried maize straw were buried between the rows after local winter wheat sowing. The experiment was conducted by a combination of net bag buried in the field and ¹⁵N isotope cross labeled maize straw and N fertilizer with four replications. The changes of residual N from the straw and N content in different organs of wheat at harvest were measured for studying the N release of maize straw during the wheat growing season, the fate of straw N and fertilizer N, and their contributions to N uptake in the above-ground part of winter wheat by using two treatments, which were no nitrogen and 200 kg N·hm^-2 fertilization under returning straw to soil condition. 【Result】 The amount of total N remained in the straw was decreased from seeding to the period before the winter dormant stage of wheat, then increased until reviving stage and declined gradually later. From sowing to harvest of wheat, the amounts of net N release to soil were 9.4 and 7.2 kg·hm^-2 by the returned straw under no and 200 kg N·hm^-2 fertilization, respectively, and they were the balance of the N release of 19.7 and 18.3 kg N·hm^-2 from the straw, and the N capture of 10.4 and 7.5 kg·hm^-2 from the soil and 3.6 kg N·hm^-2 from fertilizer into the straw.At harvest, it was found that 65.1% and 67.7% of the straw N were still left in the returned straw, 31.5% and 30.4% were released into the soil or lost, and only a few, 3.4% and 1.9% were absorbed and used by winter wheat, at respectively no N and 200 kg N·ha^-1 fertilization. Also, 25.0%, 73.2% and 1.8% of fertilizer N were observed respectively to be absorbed and used by winter wheat, left in the soil or lost, and captured by the returned maize straw under straw returning condition, when the N application rate was 200 kg N·hm^-2. Under straw returning condition, soil N was shown to contribute the most to the above-ground part N uptake of wheat, and then was the fertilizer N, while the straw N contributed the least, of which they were 98.3% and 69.2%, 0 and 30.1%, 1.7% and 0.6%, at no N and 200 kg N·hm^-2 fertilization, respectively. In the above-ground part of wheat, N derived from soil, fertilizer and straw were mainly distributed in the grain of wheat, and they were respectively 98.1%, 0 and 1.9% at no N fertilization, and 68.8%, 30.5% and 0.7% as N fertilizer was applied into the soil.【Conclusion】In the winter wheat-summer maize rotation region of Shaanxi Guanzhong Plain, more than 65% in straw N was still left in the straw after one winter wheat growing season, also as high as 70% of fertilizer N was mainly immobilized by soil or lost under maize straw returning condition. And soil N contributed the most to the above-ground part N uptake of wheat, which approximated 70%.

HORTICULTURE

Study on Screening of Taste Evaluation Indexes for Apple

ZHENG Li-jing，NIE Ji-yun，LI Ming-qiang，KANG Yan-ling，KUANG Li-xue，YE Meng-liang

Scientia Agricultura Sinica. 2015, 48(14):  2796-2805.  doi:10.3864/j.issn.0578-1752.2015.14.011

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【Objective】 The aim of this study is to screen scientific evaluation indexes for apple taste. 【Method】 Six indexes of 132 apple samples were determined, including pH determined by acidity meter, total soluble solid (TSS) determined by brix meter, soluble sugar (SS) determined with the fehling reagent titration, titratable acidity (TA) determined with the indicator titration method, the taste of each sample was determined by tasting, the ratio of total soluble solid to titratable acidity (RTT), and the ratio of soluble sugar to titratable acidity (RST). Statistical methods (such as level analysis，regression analysis, cluster analysis, and factor analysis) were used to clarify the level of each index and its distribution, to clarify the variation tendency between indexes and the quantitative relationships between/among indexes, and to screen appropriate indexes for evaluating and classifing apple taste. 【Result】 The variances of three indexes (pH, TSS and SS) were all small, with the variable coefficient of 8.5%, 10.7% and 10.6%, respectively, while the variances of other three indexes (TA, RTT, and RST) were all big, with the variable coefficient of 45.1%, 64.9% and 66.5%. Both TSS and SS distributed normally, with the probability values of 0.6241 and 0.6298. pH, TA, RTT and RST distributed unnormally. pH, RTT and RST all had a significant negative correlation with TA, with the correlation coefficient of -0.8810, -0.8117 and -0.8116. Both RTT and RST had a significant positive correlation with pH, so did SS with TSS, and RST with RTT. pH had a significant logarithmic-function concerning TA with a determination coefficient of 0.8522. Between TSS and SS, as well as between RTT and RST, there was a significant linear function. RTT as well as RST had a significant power function concerning TA. Each of the six indexes mentioned above, except TA (with low fitting precision and low prediction accuracy), had a multivariate linear model concerning the other five indexes with the mean fitting error of 1.88%-2.74% and the mean prediction error of 1.83%-2.59%. From the six indexes studied, TA, TSS and RTT were screened as evaluation indexes of apple taste, which represented sour index, sweet index and comprehensive taste index, respectively. With use of these indexes, 132 apple samples can be divided into five classes that were sweet, sour-sweet, sweet-sour moderately, sweet-sour, and sour. 【Conclusion】 Apple taste can be scientifically evaluated and classified by three indexes, including TSS, TA and RTT.

Differential Expression and Functional Analysis of Glutathione Peroxidase Gene from jujube (ZjGPX ) Under Drought and Salt Stresses

XIAO Rong, LUO Hui-zhen, ZHANG Xiao-juan, DENG Shu, ZHANG Chun-fen, REN Ying, MENG Yu-ping, CAO Qiu-fen, NIE Yuan-Jun

Scientia Agricultura Sinica. 2015, 48(14):  2806-2817.  doi:10.3864/j.issn.0578-1752.2015.14.012

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【Objective】As a foundation for further studying on the application of fruit stress tolerance genetic engineering, this study analyzed the function of a cDNA encoding GPX, which was isolated from the fruit-bearing shoot of Ziziphus jujuba Mill. Hupingzao and designated as ZjGPX (GenBank accession No. AB608053). 【Method】 The ZjGPX sequence was analyzed and expression functions were forecasted by using bioinformatics analysis tool first. The differential expressions of ZjGPX on drought and salt stresses were tested by real-time quantitative PCR, and a vector named PEZR(K)-LNY-ZjGPX was constructed and introduced into Arabidopsis by floral dip method. Transgenic and wild Arabidopsis plants were tested under NaCl and drought stresses to verify ZjGPX’s function.【Result】Sequence analysis indicated that ZjGPX was 510 bp in length, encoding 169 amino acids. Blast analysis exhibited that ZjGPX was highly homologous to other GPXs (>80%). Real-time quantitative PCR analysis showed that ZjGPX expression could be rapidly induced by drought and salt stresses just in 15 minutes in Z. jujuba Mill. Lajiaozao test tube seedling. After agrobacterium-mediated transformation, 10 ZjGPX transgenic Arabidopsis lines were obtained. Results of the drought resistance and salt tolerance tests pointed out that in the stress treatments, the transgenic plants exhibited higher seed germination rates than the untransformed control plants. After 15 days of stress treatment, the untransformed control plants showed signs of death, but the transgenic plants maintained normal growth.【Conclusion】The results showed that ZjGPX was likely related to drought and salt resistances and over-expression of ZjGPX gene increased the drought resistance and salt tolerance of the transgenic Arabidopsis.

STORAGE·FRESH-KEEPING·PROCESSING

Effects of Calcium Salts Based on Vacuum Infiltration on Quality of Postharvest Agaricus bisporus

CHEN Li-juan, WANG Zhao-gai, YANG Hui, ZHANG Le, WANG Xiao-min, SHI Guan-ying

Scientia Agricultura Sinica. 2015, 48(14):  2818-2826.  doi:10.3864/j.issn.0578-1752.2015.14.013

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【Objective】This study is aimed at investigating the effect of concentration of calcium lactate solution on the physiological and biochemical quality during postharvest of Agaricus bisporus by using the technology of vacuum infiltration and its optimization, so as to provide the guidance for storage conditions, a theoretical basis for postharvest loss and application of techniques for storage of Agaricus bisporus.【Method】 The mushroom of A. bisporus variety ‘4607’ were stored at (2±1)℃ and the effect of concentration of calcium lactate solution (0.016, 0.032, 0.048 and 0.064 mol·L^-1), vacuum degree (0.01, 0.03, 0.05, 0.07 and 0.09 MPa) and processing time (0.5, 1, 2, 3 and 5 min) were determined as a single factor on the effect of browning degree. At the same time, processing conditions were optimized for orthogonality, and physiological and biochemical quality changes were obtained during storage of A. bisporus by using chromatic meter, CO₂ analyzer and other equipments.【Result】Under the condition of a single factor, the appropriate concentration of calcium lactate solution was 0.032 mol·L^-1 and 0.048 mol·L^-1, the vacuum degree was 0.05 MPa, and the processing time was 1-2 min. The best condition of orthogonal optimization was 0.048 mol·L^-1 calcium lactate solution, 0.05 MPa vacuum degree and 2 min vacuum processing time. During storage of orthogonal optimization, compared with the control group, A. bisporus in the calcium lactate treatment group and calcium lactate vacuum infiltration group could be effectively alleviated in physiological and biochemical index change and kept in good quality. However, for some indexes, calcium lactate vacuum infiltration group was significantly better than that of calcium lactate processing group. Compared with calcium lactate treatment group, during storage of 0-9 d, there was no significant difference in decrease of whiteness values between two groups (P＞0.05), and effect of calcium lactate vacuum infiltration treatment was obviously better than that of calcium lactate treatment group from 9 to 15 d (P＜0.05). Both the treatment groups of A. bisporus reached respiratory peak in 6 d, the breath peak was postponed for 3 d, and calcium lactate vacuum infiltration treatment was lower than that of calcium lactate group (P＜0.05). Weight loss rate of calcium lactate vacuum infiltration group was lower than that of calcium lactate treatment group, but there was no significant difference (P＞0.05). Cap opening rate of both the treatment groups increased significantly and began to open respectively, delayed cap opening rate during storage of 6 d. During 6-15 d, calcium lactate vacuum infiltration treatment group was significantly lower than that of calcium lactate treatment group (P＜0.05). Both the treatment groups could effectively restrain the increase of membrane permeability, and calcium lactate vacuum infiltration treatment group remained better than that of calcium lactate during 3-15 d (P＜0.05). Malondialdehyde accumulation of calcium lactate vacuum infiltration treatment group was at a lower level, and also was lower than that of calcium lactate treatment group (P＜0.05). There was no significant difference in PPO activity between the two treatment groups (P＞0.05) from 0 to 6 d, and significantly different during 6-15 d (P＜0.05).【Conclusion】Calcium lactate vacuum infiltration treatment could retain whiteness better, inhibit respiration intensity, open umbrella rate and weight loss rate, and reduce the cell membrane permeability, MDA accumulation and PPO activity. Vacuum infiltration technology can be used as an auxiliary method in A. bisporus preservation, and can better keep the storage quality of agaricus bisporus.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Study on the Developmental Changes of Meat and Antioxidant Capacity of Grazing and House-Feeding Goats

HUANG Jin-yu, JIAO Jin-zhen, RAN Tao, TANG Shao-xun, KANG Jin-he, TAN Zhi-liang

Scientia Agricultura Sinica. 2015, 48(14):  2827-2838.  doi:10.3864/j.issn.0578-1752.2015.14.014

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【Objective】The objective of this experiment is to explore developmental changes of meat quality and antioxidant capacity of grazing and house-feeding goats. 【Method】Thirty-six kid goats were collected from 25 Xiangdong black female goats with estrus synchronization. All postnatal goats were fed on goat milk in 20 days. At 0, 7, and 14 d, 4 kid goats were slaughtered, respectively. And then the rest of these kids were assigned into two groups of 12 per group based on different feeding systems-grazing and house-feeding. At 21 d, the grazing group started to feed goat milk and forage, and the house-feeding group was fed on goat milk and a diet of forage supplemented with concentrate starter. All kids were weaned at 40 days. At 42, 56, and 70 d, 4 kids were slaughtered, respectively, in each group. The longissimus dorsi muscle from all 36 kids were collected and crude protein, intramuscular fat, amino acids, fatty acids contents and antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, reduced glutathione and total antioxidant capacity ) activities were determined.【Result】The live weight and carcass weight of kids increased significantly with age (P＜0.05). When compared with house-feeding kids, grazing kids had lower (P＞0.05) live weight, but higher (P＞0.05) carcass weight. The content of crude protein in dry matter showed a linear increasing trend in two groups (P＜0.05), while intramuscular fat (IMF) showed an increasing trend (P＞0.05).Grazing kids presented lower contents of crude protein (P＞0.05) and higher IMF than house-feeding kids. The level of total amino acids (TAA), adult essential amino acids (AEAA) and baby amino acids (BEAA) were not affected significantly by age, whereas both TAA (P＞0.05) and flavor amino acids (FAA) (P＜0.05) showed an increasing trend. Likewise, TAA, AEAA and BEAA were not affected remarkably by feeding system; grazing kids had lower TAA, AEAA and FAA, but higher BEAA than house-feeding kids (P＞0.05). (4) Age did not affect saturated fatty acids (SFA) in grazing kid, but had a quadratic effect on SFA in rearing kids; monounsaturated fatty acids (MUFA) gradually decreased (P＜0.05), while polyunsaturated fatty acids (PUFA) and essential fatty acids (EFA) remarkably increased (P＜0.05) with age. Compared to house-feeding group, the grazing group showed lower levels of SFA, PUFA and EFA (P＞0.05), but higher level of MUFA (P＜0.05). Age did not have an effect on the active levels of catalase (CAT) and superoxide dismutase (SOD), however, had a quadratic effect on glutathione peroxidase (GSH-Px) (P＜0.05), and an increasing effect on total antioxidant capacity (T-AOC) (P＜0.05); reduced glutathione (GSH) in grazing group showed a linear decreasing trend (P＜0.05), but no significant difference in house-feeding group (P＞0.05).【Conclusion】Whatever the feeding system, kid goats at different ages had an increasing live weight and carcass weight, and in longissimus dorsi muscle, crude protein gradually increased, intramuscular fat showed an increasing trend, and amino acids and fatty acids contents fluctuated with age. Meanwhile, the grazing kids presented a relatively lower level of meat quality while higher antioxidant capacity than the house-feeding kids. Consequently, for producing the desirable chevon and achieving higher profit, an adaptive feeding system should be selected according to the natural environment, feedlot condition and the development profile of the muscle.

Difference in Susceptibility to Mycoplasma Pneumonia Among Various Pig Breeds and Its Molecular Genetic Basis

FANG Xiao-min, ZHAO Wei-min, FU Yan-feng, TU Feng, LI Bi-xia, WANG Xue-min, ZHAO Fang, REN Shou-wen

Scientia Agricultura Sinica. 2015, 48(14):  2839-2847.  doi:10.3864/j.issn.0578-1752.2015.14.015

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【Objective】The objective of study is to detect susceptibility differences to M. hyopneumoniae among Chinese native pigs, western pigs and their crossbreed pigs, screen differentially expressed genes and explore the molecular genetic basis of M. hyopneumoniae infection in swine. It wouldThe results of the study will provide a basis and possibility for researchers to investigate the molecular mechanisms of M. hyopneumoniae infection in swine. 【Method】 In this study, Meishan pigs (sensitive to M. hyopneumonia, n=20 ), Landrace (excellent tolerance to M. hyopneumonia, n=20) and Suzhong pigs (cultivated breed derived from crossbred progeny of Meishan and Landrace pigs, n=20) were selected for artificial infection with M. hyopneumoniae. And the pigs were allocated randomly into the treatment group (15 pigs of each breed) or control group (5 pigs of each breed). The pigs of treatment group were administered with M. hyopneumoniae strain JS. Meanwhile, the control pigs were injected with physiological saline solution. The treatment and control groups were isolated and fed an antibiotic-free diet throughout the experimental period. And then, clinical symptoms were observed daily, and average daily weight gain, antibodies level, X-ray transmission and M. hyopneumoniae pathogen were detected at 18 and 28 days post M. hyopneumoniae challenge, respectively. Twenty-eight days post challenge, the pigs were sacrificed and inspected for hepatization of the lung lobes as an indicator of Mycoplasma pneumonia. Then, 2 infected individuals and 2 non-infected individuals of each breed pigs by M. hyopneumoniae were selected to perform Agilent Pig 4x44K Gene Expression Microarray analysis, and the double comparison method of inter- and intra-breed was used to investigate differential expression genes. Furthermore, Gene Ontology (http://www. geneont ology. org) and KEGG pathway (http://www. genome.jp/kegg/) were applied to analyze the roles of these differentially expressed genes involved in signaling pathway and regulatory network to discuss the molecular genetic basis in the process of M. hyopneumoniae infection in swine.【Result】 The results showed that, from day 1 to day 18 of experiment period, the average daily weight gain of the treated pigs was significantly lower than that of the control pigs (0.01≤P＜0.05), especially for Meishan pigs. From day 19 to day 28, negative growth (an average daily weight gain of less than 0.00 kg) and a drop in average daily weight gain were recorded for pigs in the treatment group. And the average daily weight gain of treated pigs was significantly lower than that of the control pigs (P＜0.01). Eighteen days post M. hyopneumoniae challenge, the antibody level of Landrace pigs and Suzhong pigs had little change and were low with the negative s/p ratio. But the s/p ratios of Meishan pigs were rapidly up to positive level, and were significantly higher than that of Landrace and Suzhong pigs (0.01≤P＜0.05), respectively. Twenty-eight days after treatment, the antibody level of Meishan pigs was up to 0.97±0.26, which was significantly higher than those of Landrace (0.15±0.10) and Suzhong pigs (0.46±0.20) (P＜0.01, respectively). At 18 days after treatment, clinical symptoms of mycoplasma pneumonia appeared in 11 Meishan pigs in the treatment group. Only two Landrace pigs showed signs of a slight cough. Five Suzhong pigs had early symptoms of mycoplasma pneumonia, such as coughing and lethargy. Twenty-three days into the experiment, one Meishan pig from the treatment group died after infection of M. hyopneumoniae. At the end of study, day 28, all Meishan pigs in the treatment group showed typical clinical symptoms and signs of mycoplasma pneumonia. Seven Landrace pigs from the treatment group had intermittent cough, lethargy and decreased appetite. Five Suzhong pigs had typical clinical symptoms of mycoplasma pneumonia, and further more six pigs had a slight cough. The results of M. hyopneumoniae pathogen detection were generally in accordance with description above. Moreover, on day 18 after treatment administration, the results of X-ray transmission detection revealed that 15 Meishan pigs in the treatment group had cloudy flocculent shadow, and amongst them, six animals had typical clinical symptoms and signs of mycoplasma pneumonia. While, only one of the Landrace pigs had a cloudy flocculent shadow and showed increased lung markings in the lung field, and four pigs had a small cloudy flocculent shadow. The number and degree of M. hyopneumoniae infection of Suzhong pigs appeared to be between that of Meishan and Landrace pigs. On day 28, all Meishan pigs in the treatment group had typical clinical symptoms and signs of mycoplasma pneumonia. Seven Landrace pigs and thirteen Suzhong pigs had clinical pathological evidence of mycoplasma pneumonia. At the end of experiment period, pathological examination of infected pigs indicated that the lung lesion scores of Meishan pigs (mean 23.20±2.54) were significantly higher than that of Landrace pigs  (mean 10.27±8.18) (P＜0.01) and Suzhong pigs (mean 18.71±4.78) (0.01≤P＜0.05). Profile microarray results showed that 119 differential genes were identified in Mycoplasma pneumonia pigs, including 49 up-regulated and 70 down-regulated genes, compared with the control group. These genes were involved in 18 signal pathways, such as B cell receptor signaling pathway, complement and coagulation cascades, steroid hormone biosynthesis and so on.【Conclusion】The obvious sensitivity differences do exist in the occurrence of swine mycoplasma pneumonia among various pig breeds. The primary immunodeficiency, Toll-like receptor signaling, and steroid metabolism pathways play important roles in the regulation of inflammatory response to M. hyopneumoniae infection in swine.

RESEARCH NOTES

Subcellular Distribution and Chemical-Extraction of Lead in Wheat with Different Characteristics of Lead Absorption

YANG Su-qin, CHENG Hai-kuan, JING Xin-xin, ZHOU Zhi-yun, SUN Xiao-xue, SHI Zhen-ya, ZHANG Biao, ZHAO Peng

Scientia Agricultura Sinica. 2015, 48(14):  2848-2856.  doi:10.3864/j.issn.0578-1752.2015.14.016

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【Objective】The objectives of this study were to examine the subcellular distribution of lead (Pb) and various chemical extraction in wheat cultivars Bainong160 and Yanzhan4110, to provide theoretical support for screening for cultivars of wheat with low Pb accumulation. 【Method】Based on a preliminary investigation, two wheat cultivars with different characteristics of Pb absorption were selected as study materials. A sand-culture experiment was carried out at three levels of Pb stress: 0 (control), 100 and 800 mg·L^-1 Pb as Pb (NO₃)₂. After 4-week Pb stress, the plant was harvested. Biomass, subcellular distribution and chemical extraction of Pb were determined, and the differences in biomass, subcellular distribution and chemical extraction of Pb between two wheat cultivars under Pb stress were analyzed. 【Result】 Pb at 100 mg·L^-1 could improve the growth of wheat. Bainong160 and Yanzhan4110 had a 4.78% and 2.66% increase in biomass, respectively. When the concentration of Pb increased to 800 mg·L^-1, wheat growth was inhibited. There were 25.66% and 38.60% decrease in biomass for Bainong160 and Yanzhan4110, respectively. In addition, there was a difference in root/shoot ratio between the two cultivars. When the concentration of Pb increased from 100 mg·L^-1 to 800 mg·L^-1, Bainong160 had no increase, but there was an increase from 0.71 to 0.81 in Yanzhan4110. After 4-week stress, the Pb contents of stems and leaves, and roots in Bainong160 were lower than those in Yanzhan4110. For subcellular distribution, Pb was mainly observed in the cell wall and cytoplasm, which accounted for 72%-86% of the total, in both wheat cultivars under different levels of Pb stress. When the concentration of Pb increased from 100 mg·L^-1 to 800 mg·L^-1, Yanzhan4110 had a 8% decrease in the proportion of the root cell wall and cytoplasm of Pb, and 4% decrease in this proportion stems and leaves, but the value for Bainong160 increased by 9% for root and 1% for stems and leaves. For chemical forms, poorly-soluble Pb lead (i.e. acetic acid and hydrochloric acid extractable) was mainly detected, which accounted for 67%-85% of the total. NaCl extractable Pb accounted for 6%-18% of the total, and the sum of ethanol extractable, deionized water extractable and residue Pb accounted for 8%-16%. In addition, the contents of active-Pb (i.e. ethanol and deionized water extractable) in roots of Bainong160 were lower than those in Yanzhan4110. The active-Pb in stems and leaves was different from that in roots. At the level of 100 mg·L^-1 Pb, the contents of active-Pb in stems and leaves of Bainong160 were lower than those in Yanzhan4110, when Pb increased to 800 mg L^-1, there was no difference in contents of active-Pb in stems and leaves between two cultivars.【Conclusion】Under Pb stress, root-surface of Bainong160 presented stronger resistance to Pb than Yanzhan4110. The cultivar had Pb fixation on the cell wall, which limits further transport of Pb and reduce the toxicity of Pb to shoots. For chemical forms, the distribution ratio of various forms of Pb is closely related to detoxification mechanism, but performances are not the same for different organs.

Analysis of Genome Recombination and CP Sequence Diversity of ACLSV Apple Isolate from Shandong

LI Ke, SHI Hong-wei, JING Chen-chen, SUN Xian-chao, ZHOU Chang-yong, QING Ling

Scientia Agricultura Sinica. 2015, 48(14):  2857-2867.  doi:10.3864/j.issn.0578-1752.2015.14.017

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【Objective】The objective of this study is to determine the molecular characterization and potential recombination events of genome of Apple chlorotic leaf spot virus (ACLSV) isolated from apple in Shandong Province, and diversity of CP gene of these ACLSV isolates.【Method】The whole genome sequence of ACLSV was obtained by RT-PCR through segment amplification, cloning and gene splicing. The molecular characterization and phylogeny of whole genome of ACLSV were then described and analyzed by comparison with reported sixteen full-length genome or near full-length genome sequences. The CP of ACLSV isolates was amplified for phylogenetic and diversity analysis. 【Result】The whole genome sequence of the ACLSV isolate, QD-13, was cloned and sequenced. The molecular characterization showed that QD-13 (GenBank accession number KJ522693) was 7 557 nt in length containing three ORFs, and shared the highest identity of 85.9% with Japanese apple isolate B6. The phylogenetic tree showed that the isolate QD-13 was grouped into the same cluster with isolate B6. Segmented comparisons of seventeen genome sequences of ACLSV isolates indicated that there were great differences in the sequences of 5′UTR and 3′UTR. ORF3 was relatively conserved and all of the identities of putative amino acid sequences were more than 91.2% except isolates Ta Tao 5 and MS. The ORF1 coding region between 527-665 aa was least conservative and the identities of this region were 15.4%-59.7% among all the seventeen compared sequences. Recombination event was considered as significant when it was simultaneously detected by more than three detection methods with P-Value less than 0.05. The results showed that there were three potential significant recombination events occurred in 6 507-6 247, 6 397-6 497 and 3 851-4 760 nt, respectively. The isolates QD-15, QD-20 and YT-24 were randomly selected for CP amplification. The diversity analysis showed that twenty-two sequences of CP obtained from QD-15 and YT-24 includes three types. Comparison of amino acid sequences of CP revealed that the three types are different combinations of amino acid conserved sites which were Met⁶⁰-Ser⁷³-Ser⁷⁹-Asp⁸²-Asn⁹⁷-Gly⁹⁸, Leu⁵⁹-Met⁸³-Ile¹⁹³ and Ala⁴⁰-Leu⁶⁰-Ala⁷²-Phe⁷⁵-Ile⁸⁶-Arg⁸⁸- Ser¹³⁰-Gly¹³⁷-Met¹⁸⁴, respectively. 【Conclusion】The complete genome sequence of ACLSV apple isolate in China was reported. The molecular characterization of QD-13 was confirmed and three major recombination events occurred in the genome of QD-13. Three types of CP gene were found in the isolates of ACLSV from Shandong Province, which indicated that CP gene of ACLSV has rich diversity.

Impacts of MdcyMDH Overexpression on Photosynthesis, Hormone and Growth in Apple

WANG Qing-jie, JIN Zhong-xin, ZHOU Li-jie, HAO Yu-jin, YAO Yu-xin

Scientia Agricultura Sinica. 2015, 48(14):  2868-2875.  doi:10.3864/j.issn.0578-1752.2015.14.018

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【Objective】 The objective of this study is to obtain MdcyMDH-overexpressed apple in vitro shoot cultures, to evaluate the impacts of MdcyMDH overexpression on growth, and to disclose the corresponding mechanism by determining photosynthesis and hormone level. The potential results are expected to lay a basis for further unraveling the mechanism related to growth regulation by MdcyMDH.【Method】The open reading frame of MdcyMDH was amplified by PCR from apple leaves. The PCR products were cloned into the expression vector pBI121. The resultant construct was introduced into the Agrobacterium LBA4404 and transformed into apple leaves by Agrobacterium mediated transformation method. The transgenic plants would be produced by leaf regeneration. The transgenic lines were preliminarily screened by PCR using the specific primer pairs from the sequence of 35S promoter and MdcyMDH. The transgenic lines were further confirmed by semi-quantitative and quantitative RT-PCR. The apple in vitro shoot cultures at 40 and 60 days after subculture were selected to subject to rooting culture. The growth parameters were determined at 30 days after rooting treatment, such as stem height, leaf quantity and fresh root weight. The photosynthesis and chlorophyll content were determined using the leaves from the wild type and transgenic apple trees grown in the field; the chlorophyll content was determined by spectrophotometer. The hormones in the leaves of apple in vitro shoot cultures were detected by LC-MS.【Result】The PCR using DNA template preliminarily identified three transgenic lines, i.e., line 2, line 3 and line 4, which were confirmed byexpression analysis with semi-quantitative and quantitative RT-PCR. It was found that MdcyMDH expression was increased by 12 folds and 5 folds in line 2 and line 4, respectively, compared to the wild types. The expression levels of chloroplast malate dehydrogenase gene (MdchMDH) were also elevated in the transgenic lines. As to the apple in vitro shoot cultures at 30 days after rooting culture, MdcyMDH overexpression did not generate significant impacts on plant height, leaf number, stem diameter and above-ground fresh weight; by contrast, plant height and above-ground weight were increased to a small extent for the transgenic lines. In addition, it was found that the photosynthesis was significantly increased compared with the control, and the increments of 13.2% and 15.1% were found in line 2 and line 4, respectively. By contrast, stomatal conductance and transpiration rate were also significantly increased, while CO₂ concentration was significantly reduced. Besides, MdcyMDH overexpression led to 20.4% and 15.9% increments of total chlorophyll contents in line 2 and line 4, respectively. On the other hand, there was no significant difference in IAA and GA₃ between the control and transgenic lines. By contrast, MdcyMDH overexpression significantly inhibited ABA levels and produced nearly 50% decline of ABA content.【Conclusion】MdcyMDH overexpression promoted growth of the transgenic plants and roots in especial by increasing photosynthesis and decreasing ABA level.

Isolation and Identification of Dermatophytes from Beef Cattle in Ningxia

GE Song, JIANG Wan, HE Sheng-hu, YU Yong-tao, ZHANG Lei-lei, GUO Shu-qiang, WANG Jing

Scientia Agricultura Sinica. 2015, 48(14):  2876-2883.  doi:10.3864/j.issn.0578-1752.2015.14.019

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【Objective】 An experiment was carried out to isolate and identify the pathogenic fungi from the skin scrapings of beef cattle affected by dermatophytes in Ningxia, in order to provide reference for the diagnosis, prevention and control of dermatophytes in cattle population. 【Method】 The samples of scrapings and hairs were collected from beef cattle affected by dermatophytes in Ningxia. Each sample was placed on microslide containing a drop of 10% potassium hydroxide solution and covered with a cover slip, and observed under the light microscope. Each sample was inoculated onto the Sabouraud’s dextrose agar medium（SDA）to isolate and purify the fungi, and the fungal isolates were observed under the light microscope containing lactophenol cotton blue stain, the identification and classification of each isolate were performed preliminarily according to the morphological characteristics. The conidial suspension at a density of 1×10⁸ cfu/mL from the isolates was used to infect the healthy ICR mice’s skin to confirm whether the isolates do has pathogenicity or not and to screen pathogenic fungi. The suspected pathogenic fungi’s genome DNA were extracted, the ITS sequences of which were amplified and sequenced, and finally classified according to the homology comparision and phylogenetic analysis combined with the morphological characteristics. 【Result】  According to the microscopic examination, many fungal hyphae and chlamydospore were observed in chains in the samples, and a lot microcondia inside the hairs. Twenty-three fungi were isolated from the samples, and the isolates were classified into the genera like Trichophyton, Aspergillus, Lichtheimia, Alternaria, Fusarium, Epicoccum, Mucor. Two of Trichophyton NXGY1 and NXGY2 were confirmed to be pathogenic fungi according to the skin infection experiments in mice, and all the infected mice with clinical symptoms like itching, dandruff increasing, scabbing, unhairing. The ITS sequence lengths of NXGY1 and NXGY2 were 660 bp and 662 bp, and the homology compared with Trichophyton verrucosum was up to 99% according to the ITS sequences homology comparison. Phylogenetic analyses showed that NXGY1 and NXGY2 were also highly similar to T. verrucosum. 【Conclusion】 The results indicated that NXGY1, NXGY2 isolated in this research were Trichophyton verrucosum.

Improvement of the Effects of Phytase Application by Lowering the High Level of Copper in Piglets Diets

LU Yang, HU Er-yong, ZI Zheng-hao, SUN Guo-rong, XIA Dong

Scientia Agricultura Sinica. 2015, 48(14):  2884-2890.  doi:10.3864/j.issn.0578-1752.2015.14.020

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【Objective】To promote the growth of piglets and nutrient digestibility, high copper and/or phytase additives are usually added to weaned piglet diets. However, the effects of high dietary copper on the phytase application are not very clear. This study was conducted to determine the effects of supplementation of phytase on nutrient apparent digestibility (AD), growth performance, fecal copper (FCu) and fecal phosphorus (FP) in piglets with high copper diets, and the effects of reducing copper dosage on the phytase application. 【Method】Total of 180 Landrace ´ Yorkshire piglets with initial body weight of 9-10 kg were randomly assigned to three dietary treatments group, 5 replicate pens of 12 piglets in each group. The diets based on corn-soybean meal meet or exceed NRC (1998) nutrient requirements. For the first group (control group, Group 1), 180 mg·kg^-1 copper from copper sulfate addition was added to the diet without phytase. For the second and third group (phytase treatment groups, Group 2 and Group 3), 180 and 120 mg·kg^-1 copper from copper sulfate addition was added to the diets with 500 IU·kg^-1 phytase, respectively. Feed and water were provided ad libitum throughout the three weeks of experimental period, following by three days of adaptation. Feed consumption and diarrhea were checked daily, and body weight was recorded at the beginning and end of the experiment to calculate average daily bodyweight gain (ADG), average daily feed intake (ADFI), feed conversion ratio (F/G) and diarrhea frequency. At the last week, fececs were spot sampled for three consecutive days from each pen. Every 100 gram of feces was immediately added with 10 mL of 10% ethanol and stored at -20°C until required for further analysis. Nutrients apparent digestibilities were calculated using acid-insoluble ash as an index in feed and fecal samples. 【Result】 The results showed that adding phytase to high copper level diet increased phosphorus (P) apparent digestibility by 21.12% (Group 2 to Group 1, P＜0.05), decreased FCu by 4.41% (P=0.09) and diarrhea frequency by 19.22 % (P=0.35). No significant difference was observed in apparent digestibility of dry matter (DM) and crude protein (CP), ADFI, ADG or F/G. Compared with two phytase treatment groups, reducing the copper supplement from 180 mg·kg^-1 to 120 mg·kg^-1 increased the apparent digestibility of calcium (Ca) and P by 13.74% and 9.79% (Group 3 to Group 2, P=0.02 and P=0.01), decreased FP by 18.86% (P＜0.01) and diarrhea frequency by 25.24% (P=0.25). No significant difference was observed in digestibility of DM, CP, ADFI, ADG or F/G. In contrast to high copper diet without phytase addition, lower copper diet with phytase addition showed higher apparent digestibility of DM, CP, Ca and P by 1.80%, 2.84%, 17.20% and 32.98% (Group 3 compare with Group 1, P=0.02, P=0.04，P＜0.01 and P＜0.01) and lower diarrhea frequency by 39.6% (P=0.04). 【Conclusion】Results of the current experiment indicate that reducing copper supplementation in the corn-soybean meal basal diet from 180 mg·kg^-1 to 120 mg?kg^-1 could prevent severely diminishing phytase’s positive effects on Ca and P apparent digestibility and FCu and FP content.