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    01 May 2021, Volume 54 Issue 9
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Mechanism of DA-6 Treatment Regulating Wheat Seed Vigor After Anthesis
    XU Chen,WANG WenJing,CAO Shan,LI RuXue,ZHANG BeiBei,SUN AiQing,ZHANG ChunQing
    Scientia Agricultura Sinica. 2021, 54(9):  1821-1834.  doi:10.3864/j.issn.0578-1752.2021.09.001
    Abstract ( 529 )   HTML ( 79 )   PDF (707KB) ( 346 )   Save
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    【Background】High-vigor seeds germinate quickly and efficiently, and they are highly stress resistant and advantageous during crop production. Diethyl aminoethyl hexanoate (DA-6), as a tertiary amine plant growth regulator, increases plant photosynthetic rates, regulates carbon and nitrogen metabolism and improves crop quality and yield. However, there are limited studies on its effects on seed vigor. 【Objective】This study analyzed the effects of spraying DA-6 post-flowering on the compounds accumulated in grains, expression levels of key genes associated with metabolism and stress-resistance, 1000-grain weights, chemical compositions and seed vigor to determine DA-6’s mechanism for regulating wheat seed vigor, which has important theoretical and practical significance in the production of high-vigor wheat seeds. 【Method】Wheat varieties, Liangxing 77 and Shannong 23, grown in fields of the Mazhuang Experimental Base of Shandong Agricultural University in 2017-2018 and 2018-2019 (117°E, 36°N) were sprayed with DA-6. Two treatment concentrations (0 and 6 g·L-1 DA-6), two treatment periods (2 and 6 days after anthesis) and three zone groups, each having an area of 6 m × 3 m, were used. Fresh samples of grains, stems below spikes and flag leaves were collected at the early and middle stages of grain filling (12 and 22 days after anthesis, respectively), and the expression levels of key metabolism-related enzymes were analyzed. Samples were taken on different days of grain development (25, 30, 35 and 40 days after anthesis), and the 1000-grain weights, protein contents and starch contents were determined after natural drying and threshing. For wheat spikes harvested at the maturity stage, the germination and vigor indices of the whole ear, different spike positions (upper, middle and lower) and different grain positions (lower and upper grain) were measured. 【Result】Spraying 6 g·L-1 DA-6 at 6 days after anthesis significantly increased the 1000-grain weights, seed protein contents, and plant dry weights of the upper spikes and upper grains in both wheat varieties, and it significantly improved the seed vigor indices. DA-6 spraying at 6 days after anthesis effectively reduced the seed vigor difference between different spike and grain positions, and it improved the seed vigor of different spike and grain positions in wheat as a whole, especially those of upper and middle spikes, as well as the upper grain. However, DA-6 spraying at 2 days after anthesis significantly reduced seed vigor, 1000-grain weights and seed protein contents. The 1000-grain weights of Liangxing 77 and Shannong 23 increased first and then decreased as the grain-filling period was extended. However, relative and absolute protein contents increased with time. Additionally, both varieties showed the highest 1000-grain weights and protein contents after spraying 6 g·L-1 DA-6 at 6 days after anthesis. While the 1000-grain weight of Liangxing 77 was not significantly different from that of the control group, the relative and absolute protein contents were significantly greater. The 1000-grain weight and absolute protein content of Shannong 23 significantly increased after spraying 6 g·L-1 DA-6 at 6 days after anthesis. DA-6 spraying at 2 days after anthesis had no significant effects on 1000-grain weights, relative and absolute protein contents or starch contents. Real fluorescence quantitative PCR showed that DA-6 spraying at 6 days after anthesis significantly increased the expression levels of protein phosphatase and sucrose phosphate synthase in flag leaves. The expression levels of class I heat shock protein (hsp) and hsp70 in the stem, like those of protein disulfide isomerase and hsp82 in grains, increased significantly. The α-amylase activity during seed germination was measured in samples sprayed with DA-6 at 6 days after anthesis. The activity gradually increased along with germination time in both control and treated Shannong 23. DA-6 spraying at 6 days after anthesis significantly increased the α-amylase activity levels in seeds of both varieties after germinating for 72 h. 【Conclusion】Spraying DA-6 at 6 days after anthesis promoted the expression levels of proteins, key enzyme-encoding genes of sucrose metabolism and hsps. It also facilitated the synthesis of seed storage substances, increased the protein contents of seeds and significantly increased the 1000-grain weights. The dry weight of individual plants during germination and seed vigor of upper, middle spikes and the upper grain of harvested seeds in two varieties significantly improved after spraying DA-6. The seed vigor difference between different spike positions decreased, and the α-amylase activity increased during seed germination. Thus, spraying DA-6 after anthesis promoted the accumulation of seedling dry matter and improved material transformation capacities during seed germination. Additionally, seed vigor improved. Our studies suggest that spraying 6 g·L-1 DA-6 on wheat at 6 days after anthesis is an effective way to improve seed vigor.

    Fusarium verticillioides Resistant Maize Inbred Line Development Using Host-Induced Gene Silencing Technology
    HE KeWei,CHEN JiaFa,ZHOU ZiJian,WU JianYu
    Scientia Agricultura Sinica. 2021, 54(9):  1835-1845.  doi:10.3864/j.issn.0578-1752.2021.09.002
    Abstract ( 375 )   HTML ( 35 )   PDF (2384KB) ( 218 )   Save
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    【Objective】Fusarium verticillioides (F. verticillioides) is a common pathogen, which can cause ear rot, stalk rot, seedling blight, and seed rot in maize. These diseases caused by Fusarium verticillioides not only affected the yield and quality of maize, but also seriously threatened to the safety of human and livestock by a variety of fungal toxins such as fumonisin which produced during the metabolic process of the pathogen. So far, there is no report about the major resistance gene cloned and utilized for Fusarium verticillioides in maize. Using host-induced gene silencing technology provides a new strategy for resistance breeding in maize. 【Method】Key genes associated with the Fusarium verticillioides development were cloned using homologous gene sequence method, and the dsRNA were produced by in-vitro transcription (IVT) assay. The dsRNA for different genes was premixed with suspension spores of Fusarium verticillioides used for RNA silencing experiment in vitro. For investigate the degree of disease, the seeds of the susceptible inbred line Xi502 were sterilized and inoculated, and then were cultured in a petri dish at 28℃ in the dark for 48 h. For investigate the incidence of the seeds after inoculation, glucose was added to the spore suspension mixed with dsRNA, then spore germination and mycelia growth were observed under the microscope after 25℃ culture for 24 h. The Xi502 seedlings of the trifoliate stage were transferred to the spore suspension with premixed dsRNA for culture, and the incidence of seedlings blight was observed after 7 days. In order to select the target gene for HIGS, combine the seed morphological observation result after inoculation and seedling inoculation result. Then, the silent vector about these key target genes were constructed and transferred into the susceptible inbred line Xi502. The transgenic seeds were evaluated by artificial inoculation. The total RNA of the transgenic seeds after inoculation was extracted, and the relative expression of target genes in F. verticillioide was analyzed by qRT-PCR to determine the silencing effect of HIGS line. 【Result】Eighteen candidate genes related to growth were cloned by homologous cloning method in Fusarium verticillioides. It was found that the disease level of seeds was significantly reduced after 11 candidate genes silencing by seed inoculation experiments. Furthermore, six of the 11 candidate target genes, deo, Ras2, Dpdc, Hsp90, Frp1, and Atg15, were found that response to the spore germination and mycelium growth after gene silencing. Finally, based on the results of seedling inoculation, 3 silencing target genes deo, Atg15 and Frp1 with significant inhibitory effect in vitro were selected. Then the silencing vector was constructed by combine three specific segments from the three target genes, transgenic plants were obtained. It was found that the resistance level was highly increased in T2-generation seeds compared to the none-transgenic plants. As well as the expression levels of all the three target genes were significantly decreased in Fusarium verticillioides. 【Conclusion】Three genes, deo, Atg15 and Frp1, are important for development of Fusarium verticillioides. By constructing transgenic HIGS plants for target gene deo, Atg15 and Frp1, the increase the resistance to Fusarium verticillioides in maize.

    Influence of Anthocyanin Biosynthesis on Leaf and Fiber Color of Gossypium hirsutum L.
    YUAN JingLi,ZHENG HongLi,LIANG XianLi,MEI Jun,YU DongLiang,SUN YuQiang,KE LiPing
    Scientia Agricultura Sinica. 2021, 54(9):  1846-1855.  doi:10.3864/j.issn.0578-1752.2021.09.003
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    【Objective】Cotton is an important economic and oil crop. Both its leaves and fibers can accumulate pigments and present different colors. Studies have confirmed that chlorophyll, carotenoids, and anthocyanin are the main pigments in cotton leaves and their relative proportion changes leaf colors. While proanthocyanidins and their derivatives, which are oligomeric and polymeric products from anthocyanidins, are thought to be responsible for the color formation of brown fibers. This article intends to explore the relationship between the color of leaves and fibers in upland cotton through analyzing the anthocyanidin content and gene expression level in the anthocyanin biosynthesis pathway in different leaf color mutants. The result will help to lay the foundation for the utilization of leaf color mutants and the improvement of the color of colored cotton fibers. 【Method】In this experiment, the anthocyanidin contents in leaves of 21 upland cotton leaf color mutants were detected. According to the leaf and fiber color as well as the anthocyanidin content level, 6 cotton leaf color mutants were selected as materials to measure the anthocyanidin level in leaves and fibers at 15 days post anthesis to analyze the relationship between anthocyanidin content and the leaf or fiber color. Then the expression levels of GhCHS, GhLAR and GhANR in leaves and fibers at different developmental stages (5, 10, 15, 20 DPA) were measured to analyze the influence of target genes on the color formation of leaves and fibers. 【Result】The anthocyanidin content in the leaves of 21 leaf color mutants of G. hirsutum was significantly different, and the purple or fuchsia leaves had higher anthocyanidin content. In the selected six leaf color mutants, the anthocyanidin content in leaves was significantly higher than that in fibers, and brown fibers accumulated more anthocyanidins than white fibers. Compared to GhCHS, GhANR and GhLAR expressed at a very low level in leaves, and no significant correlation was found between leaf color and their expression level. While in fibers, the expressions of GhANR and GhLAR were obviously higher in brown fibers than in white fibers, especially in fibers of 5 DPA to 15 DPA. 【Conclusion】Anthocyanins played important roles in color formation of both leaves and fibers in upland cotton. Purple-red or purple leaves and brown fibers accumulated more anthocyanidins, while the formation of fiber color did not directly correlate with leaf color. In fibers, the contents of anthocyanidins directly related to the expression levels of GhANR and GhLAR, indicating that coloration mechanism of cotton leaves and fibers was not exactly the same, and proanthocyanidins mainly accumulated in fibers.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Mixed-Cropping Improved on Grain Filling Characteristics and Yield of Maize Under High Planting Densities
    HU DanDan,LI RongFa,LIU Peng,DONG ShuTing,ZHAO Bin,ZHANG JiWang,REN BaiZhao
    Scientia Agricultura Sinica. 2021, 54(9):  1856-1868.  doi:10.3864/j.issn.0578-1752.2021.09.004
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    【Objective】The aim of this study was to evaluate the effects of mixed planting of maize varieties on grain filling characteristics and yield under close planting conditions. 【Method】Zhengdan 958 (ZD958) and Denghai 605 (DH605) were used as experimental materials. Three planting densities (D1, 67 500 plants/hm2; D2, 82 500 plants/hm2; D3, 97 500 plants/hm2), and two different mixed-cropping planting patterns (mixture (M), random sowing after mixing seeds of the two varieties in the same proportion; I, one row of ZD958 and one row of DH605) were arranged, with the same density of Zhengdan 958 (SZD958) and Denghai 605 (SDH605) as controls. Effects of mixed planting of maize varieties on the dry matter accumulation and translocation, grain filling characteristics and yield of summer maize were investigated under close planting conditions. 【Result】With the increase of planting density, the dry matter accumulation of different planting methods after anthesis increased, while the dry matter accumulation per plant at maturity and the grain filling parameters decreased. Although the 1000-grain weight decreased, the population yield increased significantly. There were no significant differences in the grain yields among the different treatments at D1 density. The grain yields obtained under the M and I treatments were higher than those of the monoculture treatments at D2 and D3 densities. Under D2 density, the 2-year average data showed that the grain yields obtained under the M and I treatments increased by 8.70% and 8.09% than that of SZD958, and 6.92% and 6.32% than that of SDH605, respectively. At D3 density, the grain yields obtained under the M and I treatments increased by 7.24% and 7.55% than that of SZD958, and 4.98% and 5.28% than that of SDH605, respectively. At D2 and D3 densities, the Gmax (maximum grain-filling rate), Wmax (kernel weight at the maximum grain filling rate) and grain weight were increased under the M and I treatments. And the 100-kernel weight was extremely significantly positively correlated with the days needed for reaching the maximum grain-filling rate (Tmax), Wmax, Gmax, and active grain filling period (P) at P<0.01. At D2 density, the average Wmax for two years under M and I treatments increased significantly by 11.61% and 11.12% than that of SZD958, and 5.86% and 5.38% than that of SDH605, respectively. The average Wmax at M and I treatments at D3 density increased significantly by 10.32% and 9.75% than that of SZD958, and 5.63% and 5.08% than that of SDH605, respectively. The dry matter accumulation per plant at maturity, dry matter accumulation after anthesis, the transfer amount and translocation efficiency of dry matter for M and I treatments increased than those of SZD958 and SDH605. The 2-year average data showed that dry matter accumulation after anthesis obtained under the M and 1:1 treatments increased by 4.43% and 7.56% than that of SZD958, and 5.25% and 8.36% than that of SDH605 at D2 density, respectively. The dry matter accumulation after anthesis obtained under the M and I treatments increased by 3.85% and 4.68% than that of SZD958, and by 4.52% and 5.36% than that of SDH605 at D3 density, respectively. 【Conclusion】There were no significant differences in the grain yields among the different treatments at low density. Under 82 500 plant/hm2 and 97 500 plant/hm2 density, the mixed cropping significantly increased dry matter accumulation and transport after anthesis, improved the maximum grain filling rate of summer maize and weight of maximum grain filling rate, promoted grain filling, and finally increased the yield significantly.

    The Degradation Characteristics of Different Plastic Films and Their Effects on Maize Yield in Semi-Arid Area in Western Liaoning
    FENG Chen,FENG LiangShan,LIU Qi,LI HaoRu,ZHENG JiaMing,YANG Ning,BAI Wei,ZHANG Zhe,SUN ZhanXiang
    Scientia Agricultura Sinica. 2021, 54(9):  1869-1880.  doi:10.3864/j.issn.0578-1752.2021.09.005
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    【Objective】This study clarified the degradation characteristics of degradable mulching films and their effects on maize yield in semi-arid area in western Liaoning, which may provide a basis for regional screening and application of degradable plastic films. 【Method】A field study combined with laboratory analysis was conducted in National Agricultural Experimental Station for Agricultural Environment in Fuxin, with three treatments, including conventional plastic film mulching (T1), additive degradation film mulching (T2) and biodegradable film mulching (T3). Through 2 years of field experiment, the regular field observation and sampling analysis were carried out to determine the maize yield under different mulching treatments. By using scanning electron microscope (SEM) and fourier transform infrared spectroscopy (FTIR), the surface morphology, mechanical properties and chemical structure of the mulching film were measured. The degradation process and degree of different types of mulching film in semi-arid area in western Liaoning were systematically analyzed. 【Result】There was no significant difference in maize yield among different treatments, and T2 and T3 had similar degradation process, in which T2 and T3 all showed cracks from the 38th day after mulching and significant degradation from the 58th day after mulching. The degradation process under T3 treatment was generally faster than that under T2 treatment, while the conventional plastic film showed almost no degradation. As plastic film degraded, the water vapor transmittance of degradable plastic film increased significantly, while the mechanical properties (tensile load, tensile strength and elongation at break) decreased significantly. The micro-structure and chemical structure of plastic film surface changed remarkably, while these indexes of conventional plastic film did not change significantly. The water vapor transmission capacity of different plastic film was in the order of T3>>T2>T1, while the mechanical properties was in order of T1>T2>T3, and the change of micro-structure of plastic film on the 98th day after mulching was in the order of T3>T2>T1, which were consistent with the field observation results and the corresponding physical indexes of mulching film. The degradation rates under T2 and T3 were 37.4% and 47.8%, respectively. The small and medium sizes of 4 cm2 and 4-25 cm2 were predominant. 【Conclusion】Biodegradable plastic film mulching could guarantee maize yield, achieve self-degradation and reduce plastic film residue. In terms of yield, degradation characteristics and residual rate, PBAT biodegradable plastic film was more feasible to replace common plastic film for maize mulching cultivation in semi-arid areas in western Liaoning.

    PLANT PROTECTION
    Construction and Analysis of Broad-Spectrum Resistance Gene Combination Pattern for Japonica Rice in Lower Region of the Yangtze River, China
    WU YunYu,XIAO Ning,YU Ling,CAI Yue,PAN CunHong,LI YuHong,ZHANG XiaoXiang,HUANG NianSheng,JI HongJuan,DAI ZhengYuan,LI AiHong
    Scientia Agricultura Sinica. 2021, 54(9):  1881-1893.  doi:10.3864/j.issn.0578-1752.2021.09.006
    Abstract ( 360 )   HTML ( 27 )   PDF (3784KB) ( 309 )   Save
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    【Objective】Gene pyramiding is one of the most effective ways to achieve broad-spectrum resistance against Magnaporthe oryzae. The objective of this study is to construct a set of polygene pyramiding lines (PPLs) under the background of japonica rice, to evaluate their resistance performances and analysis the components of their resistance effects using M. oryzae strains collected from lower region of the Yangtze River, China, thus providing broad-spectrum resistance gene combination pattern and germplasm resources for japonica rice resistance breeding in lower region of the Yangtze River, China. 【Method】Monogenic lines with multiple alleles of the Piz locus (Pigm, Pi40, Pi9, Pi2, Pizt and Piz) with the background of japonica rice 07GY31 as the backbone, crossed with other broad-spectrum resistance gene Pi1, Pi54 and Pi33, respectively using the incomplete NCII mating design. A total of 18 different PPLs were constructed using marker-assisted selection (MAS) and agronomic traits screening. Artificial inoculation assays at seedling and heading stage with 109 representative M. oryzae strains collected from lower region of the Yangtze River, combined with natural induction identification under multiple field environments were conducted to evaluate the resistance performances of different PPLs, and to analyze the component factors of the resistance effects of the PPLs. 【Result】Genotyping by sequencing (GBS) analysis shows that the constructed PPLs all have a high background recovery rate, which was ranging from 97.08% (PPL Piz/Pi33) to 99.08% (PPLPigm/Pi1), indicated that the genetic background of all PPLs was almost fully identical to that of the recurrent parent 07GY31. The seedling blast and panicle blast resistance levels of most PPLs were significantly higher than those of monogenic lines under artificial inoculation conditions, the PPLs with better resistance to seedling blast are PPLPigm/Pi1, PPLPigm/Pi54, PPLPigm/Pi33, PPLPi9/Pi33, PPLPi9/Pi54, PPLPi40/Pi54, PPLPi40/Pi33, PPLPi40/Pi1 and PPLPi9/Pi1, respectively, and the PPLs with outstanding performance in panicle blast are PPLPigm/Pi1, PPLPigm/Pi54, PPLPigm/Pi33, PPLPi40/Pi33, PLPi40/Pi54, PPLPi40/Pi1 and PPLPizt/Pi33, respectively. Different resistance gene combinations produced different effects after pyramiding. High complementary effect and which could be fully expressed is the key factor for the improvement of the resistance level of seedling blast and panicle blast of the PPLs. In addition, PPLPigm/Pi1, PPLPigm/Pi54 and PPLPigm/Pi33 displayed broad-spectrum resistance in artificial inoculation at seedling and heading stage, and showed stable broad-spectrum resistance under different disease nurseries. Besides, agronomic traits evaluation also showed PPLs with these three gene combinations were at par to the recurrent parent. Therefore, Pigm/Pi1, Pigm/Pi54 and Pigm/Pi33 are broad-spectrum resistance gene combination patterns suitable for japonica rice resistance breeding in lower region of the Yangtze River, China. 【Conclusion】The combination pattern of resistance genes affects the resistance level of the PPLs, and high complementary effect and which could be fully expressed is the key factor for the improvement of the resistance level of the PPLs in japonica background. In addition, the development of PPLs and component factors analysis in this study provides valuable theoretical support and innovative germplasm resources for the precise breeding broad-spectrum japonica varieties in lower region of the Yangtze River, China.

    Functional Analysis of the Nucleoporin Gene FgNup42 in Fusarium graminearium
    ZHANG ChengQi,LIAO LuLu,QI YongXia,DING KeJian,CHEN Li
    Scientia Agricultura Sinica. 2021, 54(9):  1894-1903.  doi:10.3864/j.issn.0578-1752.2021.09.007
    Abstract ( 373 )   HTML ( 42 )   PDF (4741KB) ( 226 )   Save
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    【Objective】The nucleoporin Nup42 plays important roles in gene expression regulation, nuclear mRNA processing and export. The objective of this study is to analyze the function of the nucleoporin gene FgNup42 in growth and development, stress, pathogenicity and mycotoxins production of Fusarium graminearum. 【Method】Targeted gene deletion and mutant complementation constructs were generated using the double-joint PCR and yeast gap repair methods, respectively. Then the gene deletion mutant ΔFgNup42 and complemented strain ΔFgNup42-C were obtained by PEG-mediated protoplast transformation. The vegetative growth, asexual and sexual reproduction of the gene disrupted mutant ΔFgNup42 were examined. Moreover, the sensitivity of ΔFgNup42 to osmotic stress, fungicides and cell wall damaging agent were investigated. Pathogenicity of the mutant ΔFgNup42 was assayed by point-inoculated the flowering wheat heads and incubation on maize silks. Mycotoxins produced by ΔFgNup42 were quantified using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The relative transcription levels of 7 TRIs involved in trichothecene mycotoxins biosynthesis were determined by qRT-PCR between PH-1 and ΔFgNup42. 【Result】Phenotype analysis showed that the growth rate of ΔFgNup42 was 50% of that of wild type PH-1, but more dense in hyphal branching. Microscopic examination revealed that the conidiation of ΔFgNup42 mutant decreased by 85.45% compared with the wild type PH-1, and percentage of conidia with septum range 0 to 2 was significantly increased. The ΔFgNup42 mutant produced more perithecia than the wild type strain in mating cultures suggesting improved sexual reproduction. The mutant ΔFgNup42 displayed increased tolerance to osmotic stress NaCl and KCl, fungicides tebuconazole and phenamacril, also cell wall damaging agent Congo red. Pathogenicity assays exhibited that disruption of gene FgNup42 caused a dramatic reduction in pathogenicity by inoculated the flowering wheat heads and maize silks. Furthermore, the amount of mycotoxins DON, 3ADON and 15ADON produced by ΔFgNup42 were significantly lower than that produced by wild type. 【Conclusion】The nucleoporin gene FgNup42 plays a key role in fungal growth and development, pathogenesis, mycotoxins production and responses to various environmental stresses in F. graminearum.

    Detection of Citrus Leaf Blotch Virus by Reverse Transcription- Recombinase Polymerase Amplification (RT-RPA)
    DUAN Yu,XU JianJian,MA ZhiMin,BIN Yu,ZHOU ChangYong,SONG Zhen
    Scientia Agricultura Sinica. 2021, 54(9):  1904-1912.  doi:10.3864/j.issn.0578-1752.2021.09.008
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    【Objective】The objective of this study is to establish a rapid reverse transcription-recombinase polymerase amplification (RT-RPA) detection system for citrus leaf blotch virus (CLBV), and to provide an efficient and simple detection method for CLBV. 【Method】RT-RPA primers for CLBV detection were designed on conserved sequence of the ORF1, and their specificity was verified. The reaction temperature and reaction time were then optimized to establish RT-RPA system for CLBV detection. Nucleic acids from samples infected with citrus tristeza virus (CTV), citrus yellow vein clearing virus (CYVCV), citrus exocortis viroid (CEVd), citrus tatter leaf virus (CTLV), citrus psorosis virus (CPsV), satsuma dwarf virus (SDV), Xanthomonas citri subsp. citri (Xcc) and Candidatus Liberibacter asiaticus (CLas), respectively, were used to evaluate the specificity of the established RT-RPA method. The 10-fold dilution of CLBV positive samples was used to compare the detection sensitivity of RT-RPA, RT-PCR and RT-qPCR. In order to evaluate the applicability of the RT-RPA, 72 citrus samples were parallelly detected by the above three methods. 【Result】In order to establish a RT-RPA method for CLBV detection, a series of experiments have been approached. Firstly, RCLBV-F/RCLBV-R2 that selected out of three designed primer pairs could specifically amplify the ORF1 fragments of CLBV with the aim size of 144 bp by RT-RPA, whereas no specific band could be obtained from samples infected with CTV, CYVCV, CEVd, CTLV, CPsV, SDV, Xcc or CLas, showing the strong specificity of the RT-RPA method. Secondly, through gradient test of 6 temperatures and 5 reaction times, the optimal reaction temperature and time of the RT-RPA system were determined to be 40℃ and 30 min, respectively. Thirdly, the sensitivity of the RT-RPA was compared to that of RT-PCR and RT-qPCR, which is similar to the former but less than the latter. The RT-RPA established in this study was addressed to detect 72 citrus samples, 11 samples were detected to be CLBV positive with positive rate of 15.28%, which was consistent with that of RT-PCR and RT-qPCR. 【Conclusion】A RT-RPA system for CLBV detection was established and optimized, which has advantages of short time consuming and easy operation with potential usage for a relatively large scale of virus monitoring in field.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Effects of No-Tillage and Straw Returning on Soil C-Cycling Enzyme Activities in China: Meta-Analysis
    MA LiXiao,LI Jing,ZOU ZhiChao,CAI AnDong,ZHANG AiPing,LI GuiChun,DU ZhangLiu
    Scientia Agricultura Sinica. 2021, 54(9):  1913-1925.  doi:10.3864/j.issn.0578-1752.2021.09.009
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    【Objective】The objectives of this study were to assess the effects of no-tillage and straw returning on C-cycling enzyme activities in China, so as to provide some insights into organic matter transformation and soil health improvement. 【Method】Based on 56 peer-reviewed papers in China, the database related to soil C-cycling enzyme activities (i.e., invertase, cellulase, β-glucosidase and polyphenol oxidase) under conventional tillage (CT, 507 sets), conventional tillage + straw returning (SR, 305 sets), no-tillage (NT, 291 sets) and no-tillage + straw returning (NTS, 122 sets) were constructed. By using meta-analysis and boosted regression tree (BRT) model, the effects of tillage and residues management practices on the soil C-cycling enzyme activities, and quantified the relative contribution of some variables (i.e., climate, soil properties, planting systems and duration) regulating the invertase activities were analyzed. 【Result】Compared with CT, the overall C-cycling enzyme activities under SR, NT and NTS soils were enhanced by 28.0%, 13.7%, and 23.2%, respectively. Specifically, the invertase activity was increased by 25.3% in SR and 16.2% under NT and 22.5% under NTS relative to CT. In addition, the cellulase activity in SR soil was higher by 36.6% than that in CT soil. In the soils with lower organic carbon concentration (SOC<10 g·kg-1), SR, NT and NTS enhanced invertase activity by 26.7%, 24.2% and 37.9%, respectively. In the soils with higher soil pH (>7.5), the invertase activity was higher by 22.3% under SR and 28.7% under NTS, respectively. Considering the soil texture, the invertase activity in soils with lower clay content (i.e., <20%) was increased by 21.5% under SR and 22.3% under NT. Instead, this invertase activity in soils with moderate clay content (20%-30%) under the SR, NT and NTS was higher by 26.1%, 16.1% and 25.3%, respectively, relative to CT. In the regions with higher aridity index (2-3.5 and >3.5), the enhanced invertase activity was observed in the SR (29.1% and 20.5%), NT (13.4% and 17.0%) and NTS (9.0% and 36.9%) treatments. The application of SR and NTS in the crop rotation systems enhanced invertase activity by 24.0% and 29.7%, respectively, relative to CT, whereas only SR practice in continuous cropping system enhanced the invertase activity by 29.4%. The improved invertase activity by NTS varied with experimental duration showing long-term duration (>10 years; 39.9%) > medium-term duration (5-10 years; 31.7%)>short-term duration (<5 years; 17.6%). Moreover, SR increased invertase activity by 22.0% in short-term duration and 27.3% in medium-term duration. The interaction of no-tillage and straw returning on the invertase activity was limited in the soils with lower SOC concentrations (<10 g·kg-1), higher soil pH (>7.5), lower clay content (<20%), higher aridity index (>3.5), crop rotation system and longer duration (>10 years). The BRT model indicated that clay content and soil pH played the most important roles on the invertase activities in the SR incorporated soils, while SOC concentration and drought index dominated in the no-tillage soils (NT and NTS) in controlling invertase activities. 【Conclusion】The application of no-tillage and straw returning had great significance to enhance invertase activity, especially in soils with lower SOC concentrations, lower clay content and higher drought index.

    Analysis of Chemical Fertilizer Application Reduction Potential for Paddy Rice in Liaoning Province
    GONG Liang,JIN DanDan,NIU ShiWei,WANG Na,XU JiaYi,SUI ShiJiang
    Scientia Agricultura Sinica. 2021, 54(9):  1926-1936.  doi:10.3864/j.issn.0578-1752.2021.09.010
    Abstract ( 340 )   HTML ( 32 )   PDF (1467KB) ( 278 )   Save
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    【Objective】The rice planting area in Liaoning Province is approximately 500 000 hm2, decreasing by one fifth from the size it was 10 years ago. However, due to the continuous increase in unit production, the annual output has been stable at over 4 million tons. As the main producing area with the highest unit yield for japonica rice in Liaoning Province, the potential and space remains to be further studied in the context of zero growth of chemical fertilizer, at the same time, both soil fertility and rice yield should be taken into consideration for chemical fertilizer application reduction. 【Method】Three major rice producing areas were investigated in 2020, including the Liaohe Delta, the Southeastern Area along the Yellow Sea, and the North-central Area of Liaoning. In each rice planting area, the representative towns with high, medium and low yields were selected as research spots. In each research spot, two to five villages (ten households per village) were selected to carry out the investigation, counting up to 590 questionnaires. The recommended amount of nitrogen (N) fertilizer for each rice growing area was determined on the basis of previous studies. According to crop nutrient requirement and soil nutrient status in paddy fields, the suitable amount of phosphate (P) and potassium (K) fertilizer was estimated by the measurement and monitoring method of P and K. Based on this analysis, the potential of chemical fertilizer reduction for paddy rice in Liaoning Province was calculated. 【Result】Average yields in the Liao River Delta, the southeastern area along the Yellow Sea and the north-central area of Liaoning were 10.4, 7.7 and 8.7 t·hm-2, respectively. The difference within the same rice planting area was about 4 t·hm-2, while the difference between different rice planting areas was about 2.7 t·hm-2. The average amount of N, P2O5 and K2O for the different paddy rice fields in Liaoning Province were 229.4, 102.8 and 91.1 kg·hm-2, respectively. Compared with the amount of fertilizer application before 2004-2017 year, the N fertilizer was reduced by 12%-32% and the P and K fertilizer was increased to some extent. The ratio of N, P and K fertilizer was 2.2﹕1﹕1. The average amounts of N and P fertilizer application varied greatly. The difference of the average amount between the maximum and the minimum is 79.5 kg·hm-2for nitrogen and 35.4 kg·hm-2for P2O5. The difference between the amounts of pure N application by farmers was about 4-fold. About 2.5% of the farmers did not apply K fertilizer, and some farmers did not apply P fertilizer. The maximum application of P2O5 and K2O were 294.5 and 225.0 kg·hm-2, respectively. The phenomenon of unscientific fertilization still existed in these regions. Taking the average yield increase of 5% in the last three years as the target yield, the recommended fertilizer amount was calculated. The target yield in the rice planting area of the Liaohe Delta was 10 000 kg·hm-2. The recommended application of N, P2O5 and K2O were 234, 111 and 101 kg·hm-2, respectively. About 82.1% of the farmers had low N fertilizer efficiency. This excessive N fertilizer application was a serious problem. About 21.7% of the farmers could potentially reduce N fertilization by 13.7%, about 9.6% of farmers could potentially reduce P fertilizer by 5.1%, and K fertilizer needed to be supplemented appropriately. Target yield in the Southeastern Area along the Yellow Sea was 7 500 kg·hm-2. The recommended application of N, P2O5 and K2O were 179, 83 and 76 kg·hm-2, respectively. About 68.0% of the farmers had low P fertilizer efficiency with a generally excessive P fertilizer application phenomenon. 28.0% of the farmers could potentially reduce the amount of P fertilizer by 18.6%. About 20% of farmers fell in the low-yield and low-efficiency levels of N and K fertilizer. The application reduction potential was around 7%. The target yield in the North-central Area of Liaoning was 9 000 kg·hm-2. The recommended application of N, P2O5 and K2O were 210, 80 and 91 kg·hm-2, respectively. About 30% of farmers had the potential to reduce the application of N, P and K fertilizers. The application reduction potential for N, P and K fertilizer was approximately 1%, 1% and 4%, respectively. 【Conclusion】After the implementation of a five year ‘zero growth in fertilizer use’ policy, the situation of fertilizer application in the paddy fields of Liaoning Province has be drastically improved, but the phenomenon of unscientific fertilization by farmers still exists. In the Liaohe Delta, the excessive application of N fertilizer was prominent. About 21.7% of the farmers could reduce the application of N fertilizer by 13.7%. In the Southeastern Area along the Yellow Sea, the phenomenon of excessive application of P fertilizer is common. About 28.0% of the farmers could reduce P fertilizer input by 18.6%. In the north-central area of Liaoning Province, about 30% of farmers in rice planting areas have the potential to reduce the application of chemical fertilizers. The percentage for potential application reduction of N and P fertilizer was about 1%, while that of K fertilizer was about 4%.

    Effects of Continuous Straw Returning with Chemical Fertilizer on Annual Runoff Loss of Nitrogen and Phosphorus in Rice-Rape Rotation
    JIN YuTing,LIU YunFeng,HU HongXiang,MU Jing,GAO MengYao,LI XianFan,XUE ZhongJun,GONG JingJing
    Scientia Agricultura Sinica. 2021, 54(9):  1937-1951.  doi:10.3864/j.issn.0578-1752.2021.09.00
    Abstract ( 350 )   HTML ( 37 )   PDF (2467KB) ( 292 )   Save
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    【Objective】To explore the effects of straw returning on the loss of nitrogen, phosphorus in rape rice double cropping system in Chaohu Area, so as to provide scientific basis for source control of non-point source pollution in Chaohu Lake Basin. 【Method】Four treatments no straw + no fertilization (CK), conventional fertilization (F), straw returning + conventional fertilization (SF) and straw returning + conventional fertilization reduced by 15% (SDF) of field plot experiment were carried out for three consecutive years (2016-2019). By measuring the concentrations and losses of nitrogen, phosphorus in farmland surface runoff under rape rice rotation, soil nutrients and crop nitrogen and phosphorus nutrient absorption and yield during rape and rice harvest, the effects of straw returning on nutrient loss and soil nutrient content in farmland runoff were discussed. 【Result】The results showed that straw returning combined with chemical fertilizer decreased nitrogen concentration in farmland runoff and increased phosphorus concentration. In rape and rice season, compared with F treatment, the average mass concentration of total nitrogen (TN) decreased by 15.6% and 26.0%, total phosphorus (TP) increased by 12.5% and 8.0%. SF, SDF treatment reduced the loss of nitrogen and phosphorus. In 2017-2019, the runoff loss of TN, TP of rape and rice season were 11.9-26.7 kg·hm-2, 1.3-2.8 kg·hm-2 and 15.6-27.0 kg·hm-2, 0.8-2.0 kg·hm-2 under F treatment. Compared with F, TN of SF treatment in rape and rice season were significantly decreased by 18.4%-29.7% and 21.9%-28.1%, TP loss decreased by 1.3%-4.0% and 1.0%-6.6%. Straw returning can increase soil organic matter and other nutrients, and reduce soil pH value in a short time. Compared with F, the average contents of organic matter, total nitrogen, total phosphorus, available phosphorus and alkali hydrolyzable nitrogen under SF treatment were respectively increased by 6.2%, 8.4%, 27.3%, 19.5%, 5.0% in rape season and 7.0%, 10.9%, 17.7%, 7.5%, 5.1% in rice season. The results showed that straw returning combined with chemical fertilizer could improve the nitrogen and phosphorus accumulation of crop aboveground. The nitrogen and phosphorus accumulation of above ground crops of F treatment in rape and rice seasons were 105.0 kg·hm-2, 20.4 kg·hm-2 and 134.3 kg·hm-2, 36.7 kg·hm-2, respectively. Compared with F treatment, SF increased nitrogen accumulation by 28.9% and 7.8%, phosphorus accumulation by 12.1% and 5.9% in rape and rice season. Compared with F treatment, SF significantly increased the annual yield of rape rice rotation by 7.8% (2017) and 6.4% (2019). 【Conclusion】Under the rape rice rotation system, straw returning combined with chemical fertilizer can not only ensure crop yield, but also increase soil nutrient content and reduce nitrogen and phosphorus loss load.

    HORTICULTURE
    Functional Identification of Grape Potassium Ion Transporter VviHKT1;7 Under Salt Stress
    LIU Chuang,GAO Zhen,YAO YuXin,DU YuanPeng
    Scientia Agricultura Sinica. 2021, 54(9):  1952-1963.  doi:10.3864/j.issn.0578-1752.2021.09.012
    Abstract ( 291 )   HTML ( 32 )   PDF (4302KB) ( 229 )   Save
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    【Objective】The aim of this study was to explore the role of VviHKT1;7 in the salt tolerance mechanism of grapes, so as to provide a theoretical reference for the subsequent cultivation of new salt-tolerant varieties. 【Method】DANMAN and MEGA software were used to analyze the biological information of VviHKT. The strongly salt resistant rootstocks SA15, SA17 and the commonly used rootstock 1103P tissue cultured seedlings were used as materials. Seedlings were treated under 100 mmol·L-1 NaCl for 0, 3, 6, 12, 24, 48 h, and the corresponding time of water treatment were taken as control. Real-time quantitative PCR (qRT-PCR) was used to detect the relative expression of HKT1 in the roots of grapes. VviHKT1;7 was cloned from SA17 cDNA and then linked with pRI101-AN-GFP, and the inflorescence of Arabidopsis thaliana was infected by Agrobacterium tumefaciens. Subsequently, T3 homozygous lines were screened out from resistant MS plates. Wild-type and transgenic Arabidopsis seeds were sowed on MS plates and MS plates (150 mmol·L-1NaCl added), their germination and growth were observed, and the root length and fresh weight were counted. The SA17 transgenic grape roots were obtained by Agrobacterium rhizogenes technology. After being treated with 100 mmol·L-1NaCl for 24 hours, the NMT in vivo physiological detector based on non-damaging micro-measurement technology was used to detect the net flow of Na+ and K+ instantaneous flow under salt stress in the roots of wild-type and transgenic grapes. 【Result】Multiple sequence alignment and phylogenetic tree analysis showed that VviHKT had high homology, among which the VviHKT1;7 open reading frame sequence length was 1 380 bp and it was the closest to VviHKT1;6. Salt stress significantly induced the expression of HKT1 gene in three varieties of grapes. Among them, the relative expression of VviHKT1;7 was up-regulated, which was still increased after long-term stress. The relative expression of VviHKT1;7 reached the peak at 6 or 12 h under salt stress, and its relative expression in SA17 and SA15 was significantly higher than 1103P. Results of germination and growth experiments in Arabidopsis showed that there was no significant difference between wild-type and transgenic Arabidopsis under normal conditions, but the germination rate, root length and fresh weight of transgenic Arabidopsis were significantly higher than those of wild type under salt stress. Fluorescence detection experiments showed that green fluorescence could be seen in the transgenic grape roots under fluorescence, rather than in the wild-type roots. Further, qRT-PCR results also showed that the relative expression of VviHKT1;7 in the transgenic grape roots was 20-folds higher than that in the wild-type roots. The results of ion flow rate detection showed that the net flow of Na + both in wild-type and transgenic roots showed efflux under normal conditions. Besides, no significant difference was found between wild-type and transgenic roots (208 and 205 pmol·cm-2·s-1) and the fluctuation range of ion flow rate in each time period was small. After salt stress, the Na+ net fluxes of them increased significantly, and the fluctuations in each time period also increased; the average net fluxes of wild-type and transgenic roots were 1 053 and 1 340 pmol·cm-2·s-1, respectively. Under normal conditions, the K+ absorption and efflux of the two roots were in a dynamic equilibrium. Salt stress significantly induced K+ efflux, and the efflux of K+in transgenic roots was significantly smaller than that in the wild type, which were 406 and 952 pmol·cm-2·s-1, respectively. The results indicated that the ability of removing Na+ and keeping K+ of transgenic roots was significantly greater than that of wild type. 【Conclusion】VviHKT1;7 played an important role in the response of grapes to salt stress, and the overexpression of this gene could improve the adaptability of Arabidopsis and grape roots under salt stress.

    Characterization of Volatiles Changes in Chinese Dwarf Cherry Fruit During Its Development
    LI XiaoYing, WU JunKai, WANG HaiJing, LI MengYuan, SHEN YanHong, LIU JianZhen, ZHANG LiBin
    Scientia Agricultura Sinica. 2021, 54(9):  1964-1980.  doi:10.3864/j.issn.0578-1752.2021.09.013
    Abstract ( 319 )   HTML ( 22 )   PDF (717KB) ( 220 )   Save
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    【Objective】The component and content of volatiles in Chinese dwarf cherry fruit during its development were studied, and the characterization of odor types transform and possible involved metabolic pathways were revealed, in order to provide the data support for the metabolic mechanism and quality breeding of fruit aroma. 【Method】The volatiles of Yanshan 1-Chinese dwarf cherry fruit were analyzed by headspace solid phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC- MS), and the AMDIS, NIST11 and RI were applied to volatiles confirmation, and internal standard method to contents calculation. Then the odor-activity values (OAVs) and qualities were evaluated to define the odor types transform feature and the main involved metabolic pathways. 【Result】Total of 140 volatiles were detected, and the volatiles types was much more at young fruit stage than that at other stages; the volatiles contents increased considerably at green fruit stage, and esters and terpenoids were the main volatiles, up to 90% of content. The volatiles of green-type had rich content at young fruit and coloring stage, then significantly decreased at commercial ripen stage, floral-type was notably enhanced during the full ripen stage. OAVs indicated that floral fragrance was the main characteristic at young fruit and green ripen stage, then fruity feature increased and equivalent with floral feature which formed the typical mature fruit aroma at full ripen stage. The volatiles generated relied on the substrates and key enzymes participated, terpenoids derived from two parallel pathways, the mevalonate (MVA) pathway, and the methylerythritol 4-phosphate (MEP) pathway, aldehydes, alcohols, esters were via fatty acid and amino acid pathway. It showed complicated pathways maybe involved at young fruit stage, with more volatiles generated; the amino acids pathway related to esters and terpenoids were dramatically reduced, the unsaturated fatty acid pathway were obviously enhanced during green ripen stage to commercial ripen stage; then the saturated fatty acid and amino acid participate in ester synthesis, and β-oxidation and cleavage of carotenoids in monoterpene pathways enhanced at following full ripen stage, with large of branch and aromatic esters and ring structure monoterpene formed. 【Conclusion】More pathways involved at early development stage, related to fruit development and environmental adaptation; the pathways of hexyl and hexene esters derived from linoleic and linolenic acid were strengthen and accumulated during green ripening stage; with the fruit growth rapidly, the volatiles synthesis decreased and the pathways transformed during coloring stage; then the pathways of branch and aromatic esters as cyclic monoterpenes of typical mature fruits aroma were markedly enhanced at following stages. Thus, the aroma mature maybe involved by three stages, refer to the young fruit stage, green fruit stage to commercial ripen stage, and full ripen stage; the mainly typical aroma substances were ripen at full ripen stage, and which was later than the physiological mature.

    Quality Analysis of Frozen Pear Based on Color, Aroma, Taste and Texture
    WANG Yang,WANG WenHui,TONG Wei,JIA XiaoHui,DU YanMin
    Scientia Agricultura Sinica. 2021, 54(9):  1981-1992.  doi:10.3864/j.issn.0578-1752.2021.09.014
    Abstract ( 385 )   HTML ( 26 )   PDF (690KB) ( 228 )   Save
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    【Objective】Three suitable frozen pear varieties (Nanguoli, Jianbali and Huagaili) were studied to analyze the color, aroma, taste and texture of fresh pear and frozen pear. 【Method】Color values (L*, a*, b*, C, h, ∆E) were determined by chromatic meter, aroma component and content were tested by gas chromatograph-mass spectrometer, sugar component and content were measured by ion chromatograph, acid component and content were determined by liquid chromatograph, and texture include hardness, adhesiveness, cohesiveness, springiness, gumminess and chewiness were tested by physical property analyzer of peel and flesh of three fresh and frozen pears, respectively. 【Result】The peel color of frozen pear was black brown, and its flesh was yellow brown which changed obviously. The L* of peel and flesh of frozen pear decreased. The color of frozen pear was darker and color values fell near the achromatic region. Compared to flesh, the color of peel changed obviously. Twenty-eight kinds of aroma components were detected in fresh Nanguoli peel, while twenty-seven kinds in frozen Nanguoli peel. Among them, twenty kinds of aroma components were retained in frozen Nanguoli peel, but fifteen kinds of aroma components were significantly different (P<0.05). Twenty-two kinds of aroma components were measured in fresh Nanguoli flesh, while twenty-six kinds of aroma components were detected in frozen Nanguoli flesh. Among them, there were nineteen common aroma components. The contents of eighteen kinds of aroma components were significantly different (P<0.05) except for methyl butyrate. Twenty-five kinds of aroma components were determined in Jianbali peel, and twenty-three kinds in frozen Jianbali peel. Among them, twenty kinds of aroma components did not change. The contents of other sixteen kinds of aroma components were significantly different (P<0.05) except for ethyl propionate, isoamyl acetate, ethyl 3-hydroxybutyrate and reverse 2-octyl aldehyde. Thirteen kinds of aroma components were tested in fresh Jianbali flesh, and sixteen kinds in frozen Jianbali flesh. Among them, eleven kinds of aroma components were retained in frozen Jianbali flesh. The contents of other eight kinds of aroma components were significantly different (P<0.05) except for ethyl propionate, ethyl caproate and ethyl 3-hydroxycaproate. Eleven kinds of aroma components were detected in Huagaili peel, and seventeen kinds in frozen Huagaili peel. Among them, nine kinds of aroma components were always present. The contents of other eight kinds of aroma components were significantly different (P<0.05) except for methyl butyrate. Seven kinds of aroma components were detected in Huagaili flesh, and sixteen kinds in frozen Huagaili flesh. Among them, six kinds of aroma components did not change and were significantly different (P<0.05). Compared with fresh pear, the glucose content of frozen pear increased and the total sugar content decreased. The sorbitol and fructose contents of frozen Huagaili decreased significantly (P<0.05), and no sucrose content was detected in the frozen Nanguoli and Huagaili. Furthermore, the contents of malic acid, shikimic acid and total acid in frozen pear decreased, both of the content of fumaric acid in frozen Nanguoli and citric acid in frozen Huagaili decreased significantly (P<0.05). 【Conclusion】The color, aroma, taste and texture of fresh and frozen pear were analyzed in this study, and the result showed that compared to fresh pears, the frozen pears peel color were changed to black and brown significantly. Three frozen pear varieties showed different aroma, but esters with fruit flavor were detected in frozen pear peel and flesh. The content of total sugar and total acid decreased. The texture of frozen pears were soft, in addition the flesh were chewed easily and could be sucked.

    FOOD SCIENCE AND ENGINEERING
    Effect of Low Voltage Electrostatic Field-Assisted Short-Term Frozen Storage on Quality of Pork
    HU FeiFei,QIAN ShuYi,HUANG Feng,JIANG Wei,QIANG Yu,JIANG Feng,HU HaiMei,LI Xia,ZHANG ChunHui
    Scientia Agricultura Sinica. 2021, 54(9):  1993-2005.  doi:10.3864/j.issn.0578-1752.2021.09.015
    Abstract ( 370 )   HTML ( 23 )   PDF (1318KB) ( 323 )   Save
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    【Objective】The current study was aimed to explore the effects of low voltage electrostatic field (LVEF) on pork quality traits during short-term frozen storage (28 d), and the results might provide a theoretical basis for the development of novel technology in meat preservation. 【Method】In the current study, pork longissimus thoracis et lumborum muscles were used as experimental material. Quality indices of the meat samples frozen at -18, -12 and -6℃ under LVEF (to output a voltage of 2 500 V and a current of 0.2 mA) or in the absence of LVEF (control group) were determined during frozen storage periods (0, 7, 14, 21 and 28 d), which included total bacterial count, total volatile basic nitrogen (TVB-N) content, color, purge loss, cooking loss, shear force, TBARS value and sulfhydryl content. Moreover, the morphology of ice crystals was observed, and the myowater distribution of meat samples was determined by using low-field nuclear magnetic resonance. 【Result】Throughout storage period of samples, there were significantly lower (P<.05) total bacterial counts, TVB-N contents and TBARS values observed in LVEF samples than that of control group. Compared with control group, the meat samples subjected to LVEF groups exhibited higher water holding capacity and fresher color. During freezing, the meat samples under LVEF formed smaller and more uniform ice crystals in muscle, which resulted in less damage to the muscle microstructure. It was noteworthy that at 28 d, no significant differences (P>0.05) were observed among the total bacterial counts (4.50 lg (CFU/g)), TVB-N content (8.73 mg/100g) and TBARS value (0.1691 mg MDA?kg-1) in meat samples subjected to LVEF group at -12℃, and those of control group at -18℃ (4.48 lg (CFU/g), 8.91 mg/100 g and 0.1754 mg MDA?kg-1, respectively). Additionally, the purge loss, shear force and color between two groups showed no significant difference (P>0.05). 【Conclusion】In general, LVEF could effectively alleviate the quality deterioration of pork during frozen storage. However, there were no significant differences observed (P>0.05) in the meat quality traits between LVEF group at -12℃ and control group at -18℃ throughout the short-term storage (28 d), and the similar trend was found between LVEF group at -6℃ and control group at -12℃.

    Implantation and Persistence of Inoculated Active Dry Yeast in Industrial Wine Fermentations
    SUN Yue,YANG HuiMin,HE RongRong,ZHANG JunXiang
    Scientia Agricultura Sinica. 2021, 54(9):  2006-2016.  doi:10.3864/j.issn.0578-1752.2021.09.016
    Abstract ( 232 )   HTML ( 20 )   PDF (615KB) ( 148 )   Save
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    【Objective】The purpose of this study was explore the implantation and persistence of commercial active dry yeast (ADY) during industrial wine fermentations, and their competitive relationship between Chinese indigenous Saccharomyces cerevisiae during fermentation process, so as to provide the theoretical basis for the breeding of indigenous S. cerevisiae strains and provide the reference for the use of ADY in wine production. 【Method】Industrial wine fermentations were carried out at wineries in Eastern Foot of Helan Mountain in Ningxia. Four vats of Cabernet Sauvignon gape must were inoculated with BDX, XR, FR and FX10, respectively. Samples were collected and analyzed at 1 d, 3 d and 5 d after the inoculation. Interdelta and SSR analysis were used to investigate the genotypes of different S. cerevisiae strains. Therefore, the number and proportion of S. cerevisiae strains in different fermentation stages were analyzed, and the colonization ability of commercial ADY was tracked. Genetic diversity parameters were calculated by PopGen32 software. The genetic correlation between commercial yeast and Ningxia indigenous yeast was revealed by NTsys2.10e software. 【Result】Interdelta fingerprint showed 6 kinds of fingerprints, namely 6 genotypes. And XR and FR showed more than one genotype; BDX and FX10 showed one genotype, respectively. SSR analysis showed that there was one genotype in each ADY for the 9 locus. 225 S. cerevisiae isolates were isolated from the 4 inoculated fermentations. Interdelta fingerprint showed 42 genotypes, of which 36 genotypes were indigenous strains. The degree of variability (16%, 42/225) was intermediate. SSR analysis showed 20 genotypes, of which 16 genotypes were indigenous strains. The analyzed 9 microsatellite prime pairs generated a total of 75 polymorphic bands, 8.3333 alleles for per locus. The heterozygosity observed was 0.2000-0.5000. The polymorphism information contents (PIC) of all strains at 9 loci were 0.6339-0.8620, suggesting that the 9 SSR loci were hypervariable. The indigenous genotypes were the most abundant in the fermentation inoculated with BDX (11 Interdelta types and 8 SSR types), followed by FR (11 Interdelta types and 6 SSR types). ADY did not dominate all three stages. Moreover, the genotypes of the dominant strains were also different for different stages in the same fermentation. Interdelta and SSR analysis showed FR was not dominant in the corresponding fermentation. Although BDX existed in the whole fermentation process, it was only dominant at d 3 after the inoculation. In the fermentation inoculated with XR and FX10, Interdelta analysis showed that they were not the dominant strains, while SSR analysis showed that they were the dominant strains in the corresponding fermentations, respectively. Indigenous strains of genotype β (SSR genotype BDX-7), genotype γ (SSR genotype BDX-6), genotype A (SSR genotype XR), genotype a (SSR genotype FX10), genotype b (SSR genotype FX10), genotype bb (SSR genotype FR-2) and genotype ee (SSR genotype FR-4) showed strong competitiveness in the corresponding fermentations. Cluster analysis showed that the genetic diversity among the S. cerevisiae strains isolated from the same fermentation was large. 【Conclusion】The genotypes of indigenous S. cerevisiae strains in the industrial wine fermentations were rich. The inoculated fermentations were completed by both indigenous strains and commercial ADY, and they competed with each other in the same fermentations and showed dynamic succession of different strains.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    Comparison Analysis on Eggshell Quality, Biochemical Index of Calcium Metabolism and Calcium Binding Protein CaBP-D28k mRNA Expression Between Langya Chicken and Its Synthetic Lines
    ZHANG NingBo,HAN ZhaoQing,JIN TaiHua,ZHUANG GuiYu,LI JiongKui,ZHENG QuanSheng,LI YongZhu
    Scientia Agricultura Sinica. 2021, 54(9):  2017-2026.  doi:10.3864/j.issn.0578-1752.2021.09.017
    Abstract ( 241 )   HTML ( 37 )   PDF (552KB) ( 256 )   Save
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    【Objective】The aim of this research was to provide a theoretical basis for the conservation of Langya Chicken breed resources and the development of its synthetic lines based on the comparative study on eggshell quality and calcium metabolism of Langya Chicken and its two synthetic lines. 【Method】Each of one hundred and eighty 240-day-old birds of Langya Chicken and its two synthetic lines (Light linen and Dark linen synthetic lines), were randomly divided into 6 repeats, each with 30 replicates. When the chicken were raised to 300 days of age under the same breeding conditions, thirty eggs and six birds were randomly collected from each repeat, blood samples were taken from the veins under the wings, and tissue samples from the left leg tibia and duodenum, egg shell glands, and kidneys were collected after slaughter. Calcium- and phosphorus-related indicators and the expression levels of calcium binding protein CaBP-D28k mRNA were detected. 【Result】The results showed that the egg weight of the light linen synthetic line was significantly lower than that of the dark linen synthetic line (P<0.05), and the eggshell weight was lower than other lines (P>0.05). In addition, the eggshell thickness and eggshell ratio were significantly higher than those of the linen line (P<0.05), the strength of eggshell was significantly higher than that of other lines (P<0.05). The calcium content of egg yolk, eggshell, and tibia in light linen synthetic line was significantly higher than that in dark linen synthetic line (P<0.05). The content of phosphorus in the tibia was significantly higher than that of other lines (P<0.05), and the ash content in eggshells and tibia was the highest in the light linen synthetic line, followed by the Langya Chicken and the dark linen synthetic line (P<0.05). The body weight and tibia length of light linen synthetic line was significantly lower than those of dark linen synthetic line (P<0.05). The contents of plasma calcium and calcitonin in the light linen synthetic line were significantly lower than those in other lines, while the plasma phosphorus, alkaline phosphatase, and parathyroid hormone contents in the light linen synthetic line were significantly higher than those in other lines, and the differences between the lines were significant (P<0.05); the calcium binding protein content of the light linen synthetic line was significantly lower than that of the dark linen synthetic line (P<0.05). While the osteocalcin of the synthetic lines was significantly higher than that of the Langya Chicken (P<0.05). The expression level of calcium binding protein CaBP-D28k mRNA in duodenum of light linen synthetic line was significantly higher than that of other lines (P<0.05), and the expression level in eggshell gland was significantly higher than that of dark linen synthetic line (P<0.05). The expression level of calcium binding protein in kidney was significantly higher than that of Langya Chicken (P<0.05). 【Conclusion】The above results showed that the plasma phosphorus, alkaline phosphatase, parathyroid hormone, osteocalcin and other related active substances and calcium-binding protein CaBP-D28k mRNA expression level in duodenum, kidney, and shell glands of light linen synthetic lines was all higher than other lines during the 240-300 day-old laying period. which could promote the absorption of calcium and phosphorus in the upper small intestine and the release and transport of bone calcium, affect the deposition of minerals in egg yolk and eggshell, and improve the quality of eggshell and tibia.

    Expression Analysis of IGF1-PI3K-Akt-Dependent Pathway Genes in Skeletal Muscle and Liver Tissue of Yellow Feather Broilers
    SHU JingTing,JI GaiGe,SHAN YanJu,ZHANG Ming,JU XiaoJun,LIU YiFan,TU YunJie,SHENG ZhongWei,TANG YanFei,JIANG HuaLian,ZOU JianMin
    Scientia Agricultura Sinica. 2021, 54(9):  2027-2038.  doi:10.3864/j.issn.0578-1752.2021.09.018
    Abstract ( 591 )   HTML ( 39 )   PDF (477KB) ( 289 )   Save
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    【Objective】IGF1-PI3K-Akt signal pathway have been implicated in the regulation of growth and development of chicken. In the present study, the expression of IGF, IGF1R and IRS1genes of IGF1-PI3K-Akt signal pathway was first studied in skeletal muscle and liver tissues of Guangxi partridge chickens with slow growth rate and Huashan partridge chickens with moderate growth rate, which would provide a reference for clarifying the regulation mechanism of growth and development of yellow feather broilers. 【Method】SPSS20.0 software was used to compare the differences of growth traits between Guangxi partridge chickens and Huashan partridge chickens. Expression of IGF1, IGF1R and IRS1 was quantified by RT-PCR in the breast muscle (BM), leg muscle (LM) and liver on days 9, 12, and 16 of embryonic development, as well as at 0, 7, 21, 35, 49, and 63 days post-hatching (PH) in the two chicken breeds, and the correlations between the gene expression level and body weight as well as tissue weight were also analyzed by SPSS20.0 software. 【Result】The body weight, skeletal muscle and liver weight of chicken in the early growth development showed significant breed- and age-specificity. The growth of Huashan partridge chickens had far exceeded that of Guangxi partridge chickens from the late embryonic development. The expression patterns of IGF1, IGF1R and IRS1 showed significant differences in breed-, tissue- and age-specific fashion. Overall, the expression levels of the three genes in skeletal muscles of embryonic development were much higher than those in liver; in contrast, the expression levels in liver were higher than those in skeletal muscle at post-hatching development. As for breed, expression levels of the studied genes in skeletal muscle of Guangxi partridge chickens were higher than those in Huashan partridge chickens; in contrast, liver expression levels were higher in Huashan partridge chickens. IGF1 mRNA could be detected as early as on E9 d in the skeletal muscles of both chicken breeds, and the highest level was appeared at this stage in LM, significantly higher than those on the other studied development stages expect on E12 d, and the lowest level was appeared on 0 d at hatching; while in BM, the highest level was appeared on E12 d and lowest level was appeared on 63 d; however, the level of IGF1 mRNA differed between breeds in liver. For Guangxi partridge chickens, the highest level was observed on 21 d and there was no expression during embryonic development, whereas the highest level was found on 49 d and very low level during embryonic development in Huashan partridge chickens. IGF1R mRNA could be detected at the whole studied stages in skeletal muscles and liver of both chicken breeds, and the highest level was appeared on E9 d in both BM and LM, significantly higher than those on the other studied development stages in Guangxi partridge chickens, and significantly higher than those on the other studied development stages expect on E12 d in Huashan partridge chickens. In liver, the highest expression level of IGF1R was detected on 21 d in Guangxi partridge chickens, significantly higher than those on E9 d, E12 d, E16 d and 0 d; while the highest level was found on 63 d and significantly higher than those on the other studied development stages. IRS1 mRNA could also be detected at the whole studied stages in skeletal muscles and liver of both chicken breeds. In skeletal muscles, expression of IRS1 were higher on E9 d or E12 d than any other age, and then decreased with age and kept relative low level during PH development in both breeds. The liver IRS1 expression pattern was consistent with IGF1R in both breeds. Significant positive relationships were observed for the expression of studied genes in BM, LM and liver tissues of both chicken breeds. Meanwhile, the skeletal muscle expression of these three genes were all showed significant negative relationships with body weight and breast (leg) muscle weight, whereas the liver expression of these three genes were all showed significant positive relationships with body weight, breast (leg) muscle weight and liver weight. 【Conclusion】These results implied that the expression of selected genes that comprise the IGF1-PI3K-Akt pathway existed identical trends, and differential expression between breeds and tissues might be one of the main reasons for the different growth rate of the different types of yellow feather broiler.