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    01 April 2019, Volume 52 Issue 7
    SPECIAL FOCUS ON GRAPE RESEARCH
    Effects of Different Sugar Sources on Protein Kinase Gene Expression in Grape Plantlets
    LIANG GuoPing,LI WenFang,CHEN BaiHong,ZUO CunWu,MA LiJuan,HE HongHong,WAN Peng,AN ZeShan,MAO Juan
    Scientia Agricultura Sinica. 2019, 52(7):  1119-1135.  doi:10.3864/j.issn.0578-1752.2019.07.001
    Abstract ( 550 )   HTML ( 65 )   PDF (4515KB) ( 363 )   Save
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    【Objective】 To explore the effects of different exogenous sugars on the growth and development of grape plantlets and the regulation of protein kinase gene transcription, the candidate genes were tapped in the process of protein phosphorylation by using transcription, which made a foundation for the verification of grape protein kinase-related gene function.【Method】 Sucrose (2%), glucose (2%) and fructose (2%) were added to the basic medium, and the free-sugar treatment was as control, which were named as S20, G20, F20 and CK, respectively. After 37 days of culture, the fresh weight of the leaf-stem and root under different treatments was determined. Transcriptome sequencing of each treated foliages was performed by using Illumina HiSeq TM 2000, and a series of protein kinases related genes were screened by integrated bioinformatics analysis, including reference genomic alignment, differentially expressed gene (DEGs) screening, COG (Cluster of Orthologous Groups of proteins) annotation, GO (Gene Ontology) annotation, etc., and the expression characteristic of these genes were further analyzed by qRT-PCR. 【Result】 Compared with CK, ‘Red Globe’ grape plantlets under F20, G20 and S20 treatments exhibited significant differences in the fresh weight of leaf-stem, and the highest was obtained by F20 treatment, while the weight of fresh root under G20 was the highest. The SNP statistics found that the Transition was the main type of mutation, the second was Transversion. The highest number of SNPs that occurred in the Intergenic, and the next was the Upstream. Splice_Site_Donor and Synonymous_Stop events occurred with the least number of genes and equal. A total of 2 633 deferentially expressed genes were obtained in the 4 samples. The Venn diagram showed that there were a total of 180 differential genes under the 3 treatments compared with CK, and these genes were clustered into 3 groups. In the first group, 127 genes were only highly expressed under CK. The 19 genes of the second group were only highly expressed under G20, while the expression patterns of the 34 genes in third group were different under three treatments. The common 180 differential genes were annotated with 26 genes in the COG database to 11 functional categories, and these DEGs were mainly enriched in general functional categories. In the annotation of GO, the common genes were annotated in 14, 22 and 13 functional categories of molecular function, biological process and cellular component, respectively. Seven kinds of protein kinases were screened by this sequencing, including Glucokinase (GK), Mitogen-activated protein kinases (MAPKs), Calcineurin protein kinase (CBL), Protein phosphatase 2 (PP2A), Hexokinase (HXK), Histidine protein kinase (HPK) and Tyrosine kinase (TK), and these different protein kinases genes showed their own expression patterns among different treatments. By qRT-PCR analysis, 17 out of 20 screened genes expression were consistent with the transcriptome sequencing results. 【Conclusion】 Compared with glucose and sucrose, fructose was the best sugar during grape culture process. The sequencing results showed that 180 DEGs all responded to three different sugars. In the COG annotation, these genes were mainly enriched in membrane ester transport and metabolism, the synthesis, transport and decomposition of secondary metabolites and carbohydrates. In the GO databases, the most of common DEGs were annotated in the activities of protein kinases and oxidoreductases. Seven protein kinases were identified, which were selectively in responses to different exogenous sugars in quantity, functional, category and metabolic pathways, and had their own choice of expression specificity.

    The Accumulation of Monoterpenes and the Expression of Its Biosynthesis Related Genes in ‘Aishen Meigui’ Grape Berries Cultivated in Different Trellis Systems During Ripening Stage
    WANG HuiLing,WANG XiaoYue,YAN AiLing,SUN Lei,ZHANG GuoJun,REN JianCheng,XU HaiYing
    Scientia Agricultura Sinica. 2019, 52(7):  1136-1149.  doi:10.3864/j.issn.0578-1752.2019.07.002
    Abstract ( 510 )   HTML ( 34 )   PDF (2177KB) ( 300 )   Save
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    【Objective】 The effects of two trellis systems on the synthesis of monoterpenes and the expression of its biosynthesis related genes during the maturity of grape berries were studied, in order to reveal the relationship between gene transcription and monoterpenoids accumulation and provide a theoretical basis for trellis selection in production and improvement of aroma quality of grape fruits. 【Method】 The grape berries of Aishen Meigui cultivated in the T and V trellis were used as materials. The berry samples were collected from the beginning of the color change period until the fruit ripened completely. Total soluble solid and titratable acid content in fruits were detected. The changes of monoterpene components and contents in fruits were determined by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (SPEME-GC-MS) in two consecutive years. Additionally, the expression of key enzyme genes in monoterpene biosynthesis pathway including 1-deoxy-D-xylulose 5-phosphate synthase genes (DXS1 and DXS3), 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (DXR), isopentenyl pyrophosphate reductase gene (HDR), linalool synthase gene (Liner syn) and terpineol synthase gene (Terp syn) were analyzed by real-time fluorescence quantitative analysis (qPCR).【Result】During grape berries maturity, the total soluble solids content increased gradually, while titratable acid content decreased gradually. The total soluble solids content of the Aishen Meigui grape cultivated in T trellis was significantly higher than that of grape cultivated in V trellis at ripening stage, and there was no significant difference in titratable acid content. 27 and 28 monoterpene compounds were detected in the grape berries of Aishen Meigui in 2016 and 2017, respectively. The main monoterpene components changed during berry maturity and were different in grape berries cultivated in T and V trellis. As the results shown, linalool, limonene, α-Terpineol, β-cis-Ocimene and geraniol were the main monoterpenes in grape berries cultivated in T trellis, while abundant linalool, α-Terpineol, limonene, nerol oxide and β-cis-Ocimene were detected in the berries cultivated in V trellis, and the content of linalool was the highest among them. At the maturity stage, total monoterpene content of T trellis grape berries reached 108.18 μg?L -1, which was 1.9 times higher than that of V trellis berries in 2016. While in 2017, total monoterpene content of T trellis grape berries reached 403.24 μg?L -1, which was 1.5 times higher than that of V trellis berries. The content of most monoterpene compounds in T trellis fruits were significantly higher than that in V trellis fruits at maturity. During grape berry development, the changes of monoterpenes accumulation in grape berries cultivated in both two trellis systems exhibited two patterns. Most of the monoterpenes including linalool, geraniol, nerol and α-Terpineol followed the first pattern: the content of monoterpenes reached the peak at maturity. But differences were observed between trellises. In the T trellis fruits, the content of compounds, such as (E,Z)-Allo-Ocimene, β-cis-Ocimene, limonene and α-terpineol, firstly decreased, and then increased sharply at 57 days after flowering, followed by a reduction during the later part of ripening stage (76 days after flowering). However, in V trellis berries, the content of these compounds increased gradually during fruit ripening, peaked at 48 days after flowering, and then decreased to the lowest level. The expression of monoterpene synthesis pathway genes (DXS1, DXS3, DXR, DHR, Liner syn and Terp syn) increased with berry ripening, of them, the changes of DXS3, HDR, Liner syn and Terp syn expression were corresponding well to the total monoterpenes accumulation patterns in grape berries cultivated in distinct trellis systems during ripening. The expression of each gene in T-trellis grape berries was significantly higher than that in V-trellis berries, which was consistent with the accumulation pattern of different monoterpenes. 【Conclusion】More accumulation of monoterpenes were detected in Aishen Meigui grape berries cultivated in T trellis. The synthesis of monoterpenes was highly correlated with the expression level of several key genes in the synthesis pathway.

    Effects of Two Trellis Systems on Viticultural Characteristics and Fruit Quality of Three Table Grape Cultivars
    WANG XiaoYue,ZHANG GuoJun,SUN Lei,ZHAO Yin,YAN AiLing,WANG HuiLing,REN JianCheng,XU HaiYing
    Scientia Agricultura Sinica. 2019, 52(7):  1150-1163.  doi:10.3864/j.issn.0578-1752.2019.07.003
    Abstract ( 465 )   HTML ( 91 )   PDF (1299KB) ( 438 )   Save
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    【Objective】The effects of two different trellis systems on viticultural characteristics, fruit quality and disease incidence of three table grape cultivars were studied deeply to provide theoretical basis for the selection of appropriate trellis system for Northern China alluvial plain.【Method】Vitis vinifera L. were used as plant materials, including Ruiducuixia, Ruiduwuheyi and Ruiduhongyu, which were planted with “T” shaped and “V” shaped trellis systems, respectively. Phenological stages, fruiting habits, photosynthetic characteristics, vine structure, labor intensity, fruit quality indexes including cluster weight, berry weight, berry width, berry length, total soluble solids (TSS), titratable acidity (TA), total anthocyanins, flavonoid and proanthocyanidin of three cultivars in both trellis systems were measured.【Result】There was no significant difference in phenological stages or fruiting habits. In most cases, percentage of germination showed higher value with T-trellis, while other fruiting habits showed no significant differences. T-trellis showed considerable advantages in management operations of the vineyard, in terms of the lateral shoot management, topping of primary shoot, fruit branch binding and winter pruning. Both Soil-bury and untying hours in the fall and out-of-soil and tying hours in the spring showed no statistically difference between the two trellis systems. Ruiducuixia showed higher net photosynthetic rate (Pn) with V-trellis in 2017, Ruiduwuheyi showed higher Pn with V-trellis in 2016, in other cases, T-trellis showed significantly higher Pn. Ruiduwuheyi showed higher stomatal conductance with T-trellis for the three years but the other two cultivars showed inconsistent results. Ruiducuixia had a higher transpiration rate with V-trellis; Ruiduxiangyu had the same trend in 2016. In most cases, there was no difference in intercellular CO2 concentration between the two trellis systems. The shoot width of three cultivars showed trivial difference between the two trellis systems in 2017. However, after the vigor of the vine had been trained to a moderate level, the standard deviation of shoot width of T-trellis had decreased to 2.3, 2.6 and 2.3 for the three cultivars, lower than that of V-trellis (2.7, 2.75 and 3.0), showing higher shoot consistency. Three cultivars showed different results of canopy height and canopy length, while the grapevines grown with V-trellis had larger canopy volume. Leaf area index of Ruiducuixia and Ruiduxiangyu were much higher with V-trellis than T-trellis, while Ruiduwuheyi showed the opposite. The leaf chlorophyll between the two trellis systems showed no statistical difference. As for leaf disease, V-trellis showed much higher disease index and incidence of downy mildew than T-trellis, while no difference was found on powdery mildew. There was no significant difference in cluster weight, single berry weight, vertical diameter, horizontal diameter or total soluble solids. With T-trellis, TSS/TA was in a better range which made the berries taste more balanced and desirable. Ruiducuixia and Ruiduwuheyi showed higher total anthocyanins with T-trellis; all three cultivars had higher level of flavonoid with T-trellis; besides, Ruiduwuheyi showed higher value of proanthocyanidin with T-trellis system, while the other two cultivars showed no significant difference. 【Conclusion】 Based on the three-year study, for soil-bury grape growing areas in Northern China alluvial plain, T-trellis system showed higher photosynthetic use efficiency, less disease incidence, better shoot consistency, higher fruit quality and less labor intensity for vineyard management, manifesting an favorable trellis system to be promoted and practiced in commercial vineyards.

    The Effects of Different Training Systems and Shoot Spacing on the Fruit Quality of ‘Kyoho’ Grape
    JI XiaoHao,LIU FengZhi,SHI XiangBin,WANG BaoLiang,LIU PeiPei,WANG HaiBo
    Scientia Agricultura Sinica. 2019, 52(7):  1164-1172.  doi:10.3864/j.issn.0578-1752.2019.07.004
    Abstract ( 501 )   HTML ( 12 )   PDF (365KB) ( 378 )   Save
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    【Objective】The aim of this study was to ascertain the effects of different training systems and shoot spacing on the fruit quality of Kyoho grape and provide the theoretical guidance for the canopy management of Kyoho grape. 【Method】 The experimental materials were six-year-old vines of Kyoho grape using Beta grape as rootstock. Two different training systems, including horizontal shoot positioning and Y-shaped training system, were applied. The distance between the shoots was 10 cm, 15 cm and 20 cm controlled by the rope, respectively. Fruit quality indexes, such as berry weight, soluble solids, titratable acid, vitamin C, anthocyanin and aroma, were determined at maturity stage. 【Result】 Berry weight, soluble solids, titratable acid and vitamin C contents were significantly correlated with the training systems, but not significantly correlated with the shoot spacing. The anthocyanin content was significantly correlated with training systems and shoot spacing. The total aroma content was significantly correlated with the training systems, but not significantly correlated with the shoot spacing. Among them, the content of aldehydes, alcohols and terpenes was consistent with the total aroma content, while the content of esters was only correlated with the shoot spacing. The Topsis comprehensive evaluation analysis was conducted for 9 indicators including berry weight, soluble solids, titratable acid, vitamin C, anthocyanin, ester, aldehyde, terpene and alcohol contents, and the result showed that horizontal shoot positioning training system combining with 15 cm shoot spacing scored the highest. 【Conclusion】 These data strongly suggested that horizontal shoot positioning training system combining with 15 cm shoot spacing was the most suitable for Kyoho grape production.

    Effects of Local Root Zone Salinity on Grapevine Injury, Na + Accumulation and Allocation of Carbon and Nitrogen
    SUN Hong,JIANG YiWen,YU Xin,XIANG GuangQing,YAO YuXin
    Scientia Agricultura Sinica. 2019, 52(7):  1173-1182.  doi:10.3864/j.issn.0578-1752.2019.07.005
    Abstract ( 393 )   HTML ( 23 )   PDF (736KB) ( 382 )   Save
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    【Objective】 Salt stress seriously affects yield and fruit quality of fruit crops. Soil salinity is often heterogeneous in saline fields, and within the different root zones of single plant the salinity of the soil solution might vary widely. This paper was aimed to determine the injury extent of grapevine under the non-uniform salt treatment, and to disclose the corresponding mechanism through the determination of Na + flux and allocation of carbon and nitrogen in grapevine. 【Method】 Saline environment of vine roots was controlled through split-root system and five treatments with different NaCl concentration (mmol·L -1) were set: 0/0, 0/50, 50/50, 0/100, and 100/100. Grapevine injury was evaluated via determining content of chlorophyll and malondialdehyde (MDA) as well as the changes of chlorophyll fluorescence parameters. Na + transport was analyzed by the determination of Na + content, Na + flux and electrical conductivity of culture medium around roots. Nitrogen utilization efficiency and distribution rate of carbon and nitrogen were used to detect the changes of carbon and nitrogen in different tissues under different treatments.【Result】The uniform salt treatment of bilateral roots significantly reduced the content of chlorophyll and enhanced the MDA levels in roots and leaves at 15 and 30 days after treatment. In contrast, salt treatment of local roots alleviated the chlorophyll decrease and the MDA accumulation. Additionally, the determination of chlorophyll fluorescence parameters, such as Fv/Fm and ERT, showed the similar results. Therefore, the roots in the non-saline side could alleviate the grapevine injury in comparison to the uniform salt treatments. All of salt treatments increased Na + content in roots and leaves to varying extents at 15 days after treatment; particularly, the Na + content of the roots in the non-saline side was also enhanced; additionally, local root zone salinity significantly decreased the Na + content in leaves, and local treatment of 100 mmol·L -1NaCl significantly reduced the Na + content in saline side roots, compared to the uniform NaCl treatment. The Na + efflux was observed in non-treated roots, however, the Na + flux was reversed to influx in the non-saline side roots under non-uniform salt treatment. Additionally, the electrical conductivity of the culture medium around the roots in the non-saline side was significantly enhanced. Therefore, the Na + absorbed from the salt-treated side could be transported to the non-saline side roots and thereby expelled out of the roots. Nitrogen utilization efficiency was significantly reduced by the uniform salt treatment and the decline was associated with salt treatment concentration. In contrast, the non-uniform salt treatment alleviated the declines in nitrogen utilization efficiency and particularly, which was significantly enhanced in the non-saline side roots under the 0/100 mmol·L -1treatment. The uniform salt treatments and particularly 100 mmol·L -1NaCl decreased the distribution rate of nitrogen in roots and leaves and increased the values in the two-year-old shoots, favoring the storage of nitrogen. In contrast, the non-saline side roots alleviated the declines of nitrogen distribution rate in roots and leaves. The uniform salt treatment decreased carbon distribution rate in leaves and roots; in contrast, the non-saline side roots not only alleviated the declines of carbon distribution rate in leaves but also elevated carbon distribution rate in roots. It was noteworthy that 50 and 100 mmol·L -1 NaCl treatments imparted different effects on carbon distribution in new shoots and two-year-old shoots, i.e., the uniform and non-uniform treatments of 50 mmol·L -1 NaCl enhanced carbon distribution in the two-year-old shoots while the treatments of 100 mmol·L -1 NaCl produced the contrary results.【Conclusion】Compared with the uniform salt treatment, NaCl treatment of local roots produced the lesser injury for grapevines. Na + absorbed from the salt-treated side was transported to the non-treated side, expelled them from the roots, and thereby reduced Na + accumulation in leaves. The non-saline side roots alleviated the declines in carbon and nitrogen distribution rate of leaves and roots.

    Effects of Different Light Intensity and Temperature on PSII Photochemical Activity in ‘Cabernet Sauvignon’ Grape Leaves Under Ozone Stress
    GENG QingWei,XING Hao,ZHAI Heng,JIANG EnShun,DU YuanPeng
    Scientia Agricultura Sinica. 2019, 52(7):  1183-1191.  doi:10.3864/j.issn.0578-1752.2019.07.006
    Abstract ( 403 )   HTML ( 25 )   PDF (417KB) ( 290 )   Save
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    【Objective】Ozone stress, high light and high temperature usually exist simultaneously in nature, but little is known about the effect of the combination of three stress factors on plants. So the combined effects of different temperature (26, 40℃), light (800, 1 600 μmol?m -2?s -1) and ozone treatments on the operation of photosynthetic apparatus were investigated to reveal the mechanism of inhibition of photosynthesis, which provided a theoretical basis for reducing ozone injury by regulating the light and temperature. 【Method】 Potted Cabernet Sauvignon grapes were used as materials to be treated with different temperatures (26, 40℃), two kinds of light intensities (800, 1 600 μmol?m -2?s -1) combined with (120±20) nL?L -1 ozone. Chlorophyll a fluorescence parameters were analyzed to investigate the effects of different treatments on the PSII function. 【Result】 40℃ and 1 600 μmol·m -2·s -1 both significantly decreased the maximal photochemical efficiency (Fv/Fm), linear electron transport rate (ETR), the unit area number of reactive centers (RC/CSm) and photochemical quenching coefficient (qP) of leaf under ozone stress. At the same time, the decreased excitation energy partition coefficient (α) of PSI and increased excitation energy partition coefficient (β) of PSII induced a serious imbalance of excitation energy distribution between the two photosystems and caused the damage of photosystem II, and the influence of strong light intensity (1 600 μmol·m -2·s -1) was more significant. The maximal photochemical efficiency (Fv/Fm) under T2 and T3 treatment decreased 10.3% and 38.8% than that under T1 treatment, Fv/Fm under T4 treatment decreased most for 54.8%. PSII quantum efficiency Fv/Fo under T2, T3 and T4 treatment decreased 30.4%, 69.6% and 80.3% than that of T1 treatment, respectively. The number of reaction centers per unit area (RC/CSm) under T2, T3 and T4 treatment decreased 30.4%, 69.6% and 80.3% than that of T1 treatment, respectively, and the probability of that a trapped exciton the moves an electron further than QA by trapped exciton (Ψo) under T2, T3 and T4 treatment decreased 11.2%, 21.6% and 40.8% than that of T1 treatment, respectively. The effective photochemical efficiency Fv'/Fm' under T2, T3 and T4 treatment decreased 7.9%, 22.1% and 42.3% than that under T1 treatment, respectively. Linear electron transport rate (ETR) of T4 decreased most (decreased 62.5% than that under T1 treatment), and ETR under T2 and T3 treatment decreased 17.6% and 37.5% than that of T1 treatment, respectively. The photochemical quenching coefficient (qP) under T2, T3 and T4 treatment decreased 10.7%, 19.8% and 39.5% than that of T1 treatment, respectively. The photochemical reactions share under T2, T3 and T4 treatment decreased 18.8%, 38.8% and 62.0% than that of T1 treatment, respectively. While the antennal heat dissipation share under T2, T3 and T4 treatment increased 15.9%, 36.2% and 60.5% than that of T1 treatment, respectively, and the excess light energy share of T2, T3 and T4 treatment increased 12.5%, 19.1% and 25.2% than that of T1 treatment, respectively, the excitation energy partition coefficient (α) of PSI under T2, T3 and T4 treatment decreased 6.6%, 12.8% and 25.1% than that under T1 treatment, respectively, and the excitation energy partition coefficient (β) of PSII under T2, T3 and T4 treatment increased 4.5%, 8.7% and 17.1% than that under T1 treatment, respectively, and the change trend of imbalance of partitioning of excitation energy between PSⅠand PSⅡ(β/α-1) was consistent with the change of the excitation energy partition coefficient, β/α-1 under T2, T3 and T4 increased 37.7%, 78.3% and 187.8% than that under T1 treatment.【Conclusion】Temperature and light stress increased the sensitivity of PSII activity to ozone stress, and the influence of light stress was more significant, while high temperature and high light stress caused serious damage to photosystem II.

    Effects of 1-MCP and Na2S2O5 Composite Preservative on Postharvest Physiology and Storage Quality of Red Globe Grapes
    CHEN Hao,ZHANG RunGuang,FU LuYing,ZHANG YouLin
    Scientia Agricultura Sinica. 2019, 52(7):  1192-1204.  doi:10.3864/j.issn.0578-1752.2019.07.007
    Abstract ( 404 )   HTML ( 16 )   PDF (1157KB) ( 463 )   Save
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    【Objective】 Red Globe grapes easily appears dehydration, threshing, decay and dry stem during storage. The objective of this study was to provide theoretical basis and technical parameters for prolonging the storage period of Red Globe grapes by studying the effects of 1-MCP and Na2S2O5 on the postharvest physiology and storage quality.【Method】 Shaanxi Heyang Red Globe grape was selected as the test material and sealed with 0.04 mm perforated polyethylene (PE) film bag. The Red Globe grapes were stored under the condition of low temperature (-1±0.5)℃and relative humidity of 90%-95%. The Red Globe grapes were treated with no preservative agent as the control. And then, fruit respiration intensity, weight loss rate, titratable acid content, fruit hardness, malondialdehyde content, soluble solid content, fruit cell membrane relative permeability, protopectin, soluble pectin, reducing sugar content, antioxidant enzyme activities of superoxide dismutase (SOD), polyphenol oxidase (PPO), polygalacturonase (PG), peroxidase (POD) and catalase (CAT) were used as measurement indexes to study the effects of respiratory types of Red Globe grapes and 1-MCP, Na2S2O5 and 1-MCP+Na2S2O5 three preservative treatments on the postharvest physiological and related physicochemical indexes of Red Globe grapes after harvest and during storage. Commercial fruit percentage were calculated during storage, including sensory characters such as color, aroma, taste and shape of Red Globe grapes, which were comprehensively evaluated when stored for 180 days.【Result】 The spike-stalk of Red Globe grapes was respiratory climacteric, and ear and fruit grain were non-respiratory climacteric type. Three kinds of preservatives had different effects on the quality of Red Globe grapes. 1-MCP and Na2S2O5 combined treatment could inhibit activities of PPO, POD, SOD and PG, enhance the activity of CAT, delay the increase of malondialdehyde, soluble pectin content, fruit cell membrane relative permeability and weight loss rate, slow down fruit hardness, protopectin and respiration intensity, prevent Red Globe grapes decay and deterioration effectively, and maintain the contents of soluble solid, titratable acid and reducing sugar. Sensory evaluation at 180 days after storage showed that the indexes under 1-MCP and Na2S2O5 combined treatment of Red Globe grapes were higher than that under other treatments, the commercial fruit rate under this treatment was 90.4%, and the sensory evaluation was 90.2 points.【Conclusion】 Postharvest Red Globe grapes were treated with 3 mg·kg -1 FW 1-MCP and 4 g·kg -1 FW Na2S2O5, which could slow down the change rate of fruit softening, protopectin and respiratory intensity, maintain high content of soluble solids, titratable acid and reducing sugar, and keep good quality of fruits.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Evaluation of Crop Rotation Yield Effect in Cold and Arid Regions of Northwest Hebei Province
    DU YuQiong,ZHANG LiFeng,YAO ZhaoLei,ZHANG JiZong,LIU YuHua
    Scientia Agricultura Sinica. 2019, 52(7):  1205-1214.  doi:10.3864/j.issn.0578-1752.2019.07.008
    Abstract ( 413 )   HTML ( 28 )   PDF (372KB) ( 319 )   Save
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    【Objective】The study was carried out to promote the rational and balanced utilization of water and nutrients in crop production in cold and arid areas of Northwest Hebei province, where water, temperature and soil resources were scarce, and to protect the farmland ecological environment. 【Method】A field experiment of rotation and continuous cropping with five main crop combinations was designed in the meadow chestnut soil and sandy chestnut soil farmland by using the cross planting method. The yield effect of rotation of main crop in the region was studied by repeating the experiment between years. 【Result】Compared with continuous cropping, the yield of potato, beet and oat under rotation conditions in meadow chestnut soil farmland increased by 58%-94%, 28%-53% and 16%-70%, respectively; The yield of faba bean increased by 4%-43% rotated with other crops except for beet; The yield of potato and faba bean stubble forage maize increased by 12% and 5%, respectively, compared with continuous cropping, and the yield of beet and oat stubble forage maize was only 81% and 93% of that of continuous cropping, respectively. In sandy chestnut soil farmland, the yield of potatoes and beets in different Preceding crop fields increased by 5%-36%, 13%-36%, respectively, compared with continuous cropping; The yield of beet stubble forage millet decreased, and it increased by 5%-30% rotating with other crops compared with continuous cropping; The yield of forage millet stubble oats decreased, while that of other stubbles increased by 9%-15% compared with continuous cropping. The yield of potato and oat stubble flax was 5% and 8% higher than that of continuous cropping, respectively, and the yield of forage millet and beet stubble flax was only 94% and 99% of continuous cropping, respectively. 【Conclusion】The rotation of potato and oat, potato and beet in meadow chestnut soil was mutually beneficial, forage maize stubble potato, oat stubble beet, faba bean stubble beet, forage maize stubble beet and faba bean stubble potato showed commensalism rotation. In sandy chestnut soil field, oats stubble potato, potato stubble beet showed commensalism rotation, and this could be applied in production.

    Physiological Response Mechanism of Drought Stress in Different Drought-Tolerance Genotypes of Sesame During Flowering Period
    SUN Jian,YAN XiaoWen,LE MeiWang,RAO YueLiang,YAN TingXian,YE YanYing,ZHOU HongYing
    Scientia Agricultura Sinica. 2019, 52(7):  1215-1226.  doi:10.3864/j.issn.0578-1752.2019.07.009
    Abstract ( 571 )   HTML ( 30 )   PDF (420KB) ( 357 )   Save
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    【Objective】The aims of this study were to study the effects of drought stress on the physiological and biochemical characteristics of leaves and roots of different drought-tolerant sesame varieties, to analyze the physiological responses of sesame different genotypes under drought stress during flowering period, thus providing a theoretical reference for the studies of sesame drought-tolerant and its improvement. 【Method】This experiment adopted the method of potted to drought-tolerant varieties “Jinhuangma” and drought-sensitive varieties “Zhushanbai” as the experimental materials, and treatment with mild (T1), moderate (T2) and severe (T3) drought stress at the flowering period, with normal irrigation as the control (CK). The contents of malondialdehyde (MDA), H2O2, proline (Pro), soluble sugar (SS), soluble protein (SP), free amino acids (AA), reduced ascobate (AsA), and reduced glutathione (GSH), as well as activities of superoxide dismutase(SOD), peroxidase (POD), catalase (CAT), phenylalanine ammonialyase (PAL) in plant leaves and roots were determined, respectively. And then the differences of physiological response mechanism between two genotypes under drought stress were analyzed. 【Result】 After drought stress, the contents of MDA, H2O2, Pro, SS, SP, and GSH with the activities of SOD, POD and PAL in sesame leaves, as well as contents of MDA, SS, SP, AA, GSH, AsA and CAT and PAL activity in roots showed an increasing trend with the aggravation of stress degree, and AA, AsA content and CAT activity in leaves and contents of H2O2, Pro and SOD, POD activity in roots showed a trend of first rising and then falling with the aggravation of stress degree. The results of correlation analysis showed that the measured value of physiological and biochemical indexes in leaves were all positively correlated with them in roots, and the drought-tolerance of varieties was negatively correlated with the content of membrane lipid peroxidation damage substances (MDA and H2O2), and positively correlated with partial osmotic regulatory substances, defense system substances and protective enzyme activities. 【Conclusion】Under drought stress during the flowering period of sesame, 12 physiological and biochemical indexes of two different genotypes of sesame all showed different degree of increase, with great difference in response, and the effect on the leaves was greater than that of the roots. Drought-tolerant varieties showed less cell membrane lipid peroxidation damage, more accumulation of osmotic regulatory substances, stronger activity of protective enzymes (SOD and CAT) and higher content of antioxidant substances (GSH and AsA) than that in drought-sensitive varieties. Drought-tolerance of sesame showed comprehensive defense in many factors on physiological and biochemical indexes.

    PLANT PROTECTION
    Effects of Oxytetracycline Treatment on the Control of Sweet Orange Huanglongbing and the Expression of PP2 Gene
    YAO TingShan,ZHOU Yan,DIANN ACHOR,WANG Nian,ZHOU ChangYong
    Scientia Agricultura Sinica. 2019, 52(7):  1227-1236.  doi:10.3864/j.issn.0578-1752.2019.07.010
    Abstract ( 629 )   HTML ( 9 )   PDF (2256KB) ( 429 )   Save
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    【Objective】The objective of this study is to evaluate the control efficacy of oxytetracycline (OTC) on sweet orange Huanglongbing (HLB), detect the expression of phloem protein 2 (PP2) gene, and to provide a scientific basis for the effective prevention and control of HLB as well as a reference for OTC mechanism research. 【Method】OTC, which can effectively inhibit and kill pathogenic bacteria, was injected with 0.1 g/tree of 4-year-old Valencia sweet orange with different severity of HLB (newly infected, mild infected and severely infected plants), and then the contents of Cadidatus Liberibacter asiaticus (Las), starch and PP2 gene of leaves were evaluated at 7, 30, 60, 90 days post injection, respectively. Furthermore, 90 days after injection, the young and mature tissues of the plants were observed by using starch staining light microscopy (LM). The extraction of tender leaves was investigated 80 days after OTC injection. 【Result】Trunk injection of OTC 0.1 g/tree had the most obvious therapeutic effect on newly infected Valencia sweet orange. Seven days after injection, Las could not be detected and it maintained for 90 days. The starch content in mature leaves decreased obviously, but starch grains were still enriched in stems. The Las content of mild infected Valencia leaves decreased from (1.68×10 6±858884) cells/g leaf to (7.21×10 4±30981) cells/g leaf, the descending range was obvious, and the starch content in mature leaves showed a decreasing trend within 90 days. The Las content of severely infected Valencia leaves decreased from (4.10×10 8±3.04×10 8) cells/g leaf to (2.80×10 7±2.70×10 7) cells/g leaf, but the starch content in mature tissues of severely infected plants was still high, except the newly autumn shoots. The results indicated that 0.1 g/tree OTC was not enough to cure 4-year-old severely infected Valencia. The expression of PP2 gene in Valencia significantly decreased 30 days after OTC injection, and remained stable in the subsequent 90 days, which was consistent with the expression level at 30 days after injection.【Conclusion】OTC can be used in the prevention and control of HLB, and has better therapeutic effect on newly infected Valencia (Las content is less than 9.00×10 5 cells/g leaf) and mild infected Valencia (Las content is 9.00×10 5-9.00×10 7 cells/g leaf). It was not suitable for the treatment of severely infected plants (Las content is more than 9.00×10 7 cells/g leaf). After injection of OTC, the expression of PP2 gene decreased obviously, suggesting that OTC can effectively reduce the stress of phloem pathogens.

    Potential Functions of Nilaparvata lugens GSK-3 in Regulating Glycogen and Trehalose Metabolism
    DING YanJuan,LIU YongKang,LUO YuJia,DENG YingMei,XU HongXing,TANG Bin,XU CaiDi
    Scientia Agricultura Sinica. 2019, 52(7):  1237-1246.  doi:10.3864/j.issn.0578-1752.2019.07.011
    Abstract ( 531 )   HTML ( 20 )   PDF (470KB) ( 247 )   Save
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    【Objective】The insect insulin signaling pathway can mediate glycogen synthase kinase 3 (GSK-3 or GSK3) to regulate glucose metabolism in the body, such as glycogen and trehalose, thereby controlling different life activities of insects. The objective of this study is to explore the regulation of glycogen synthase kinase on the metabolism of glycogen and trehalose in Nilaparvata lugens.【Method】 Firstly, based on the cDNA coding sequence of GSK-3, the GSK-3 amino acid sequence was translated using the ExPASy tool to predict the molecular weight and isoelectric point (pI) of the protein, and then the signal peptide was analyzed by SignalaIP4.1Server. Secondly, the N. lugens raised in the author’s laboratory was collected every 12 hours from the 4th instar to 48-h-old adult. The total RNA of N. lugens was extracted by Trizol method. The first strand DNA was synthesized according to the reverse transcription kit, and 18S was used as the internal reference gene. The relative expression of GSK-3 in different ages at mRNA level was detected by quantitative real-time PCR (qRT-PCR). Then, double-stranded RNA (dsRNA) was microinjected into N. lugens with RNAi technology to inhibit the GSK-3, and N. lugens of dsGFP was injected as a control group. The expression of GSK-3 was detected by qRT-PCR 48 h after injection to determine the inhibitory effect. In addition, the N. lugens was taken 48 h after injection, and the change of trehalose, glucose, glycogen content and trehalase (TRE) activity in N. lugens was determined. Finally, the relative expression of related genes in insulin signaling pathway (including insulin receptor (InR), insulin-like peptides (Ilps)) and trehalose metabolism pathway (TRE, trehalose-6-phophate synthase (TPS), glycogen phosphorylase (GP), glycogen synthase (GS)) was detected by qRT-PCR to analyze the regulation of GSK-3 in insulin signaling pathway and trehalose metabolic pathway.【Result】The open reading frame of N. lugens GSK-3 is 1 914 bp, encoding 637 amino acids; the predicted molecular weight of the protein is 69.25 kD, and the isoelectric point is 9.15. It is a basic protein with no signal peptide structure and the sequence is highly conserved. The results of developmental expression pattern showed that the expression of GSK-3 was inconsistent at different developmental stages, and the expression was low before and after molting of 5th instar nymph. At 48 h after the dsRNA injection of GSK-3, the expression of GSK-3 decreased significantly compared with the dsGFP of the control group, indicating that the RNA interference effect was obvious. Glycogen content and two types of trehalase activity decreased significantly, while trehalose content increased significantly. It is speculated that glycogen and glucose are converted to trehalose as an energy source for their physiological activities. The results of qRT-PCR showed that the expression of TRE1-2 significantly decreased 48 h after the inhibition of GSK-3 expression, while the expression of TRE1-1 and TRE2 extremely significant decreased. In addition, the expression of two TPS genes, GS and GP genes all extremely significant decreased; the expression of two InR genes and four Ilps genes in the insulin signaling pathway were also inhibited, indirectly indicating that InR can regulate the expression of GSK-3. 【Conclusion】 The low expression of GSK-3 in N. lugens can regulate glycogen and trehalose metabolism by regulating insulin signaling pathway and trehalose metabolism pathway related gene expression. The relevant research results will help to explore more comprehensive molecular mechanisms for the regulation of the balance of trehalose and carbohydrates by insect glycogen synthase kinases such as N. lugens.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Characteristics of Soil Water Utilization in Spring Wheat Field with Different Straw Retention Approaches in Dry Inland Irrigation Areas
    YIN Wen,CHAI Qiang,HU FaLong,FAN ZhiLong,FAN Hong,YU AiZhong,ZHAO Cai
    Scientia Agricultura Sinica. 2019, 52(7):  1247-1259.  doi:10.3864/j.issn.0578-1752.2019.07.012
    Abstract ( 373 )   HTML ( 19 )   PDF (465KB) ( 403 )   Save
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    【Objective】In oasis irrigated agricultural region, water resources scarcity is one of the most prominent constraints for crop production, which also leads to the unstable yield and the lower water use efficiency of crop production with conventional tillage. In this study, the characteristics of soil water utilization in spring wheat field with different straw retention approaches were investigated in the areas, so as to optimize the farming practices and to improve the water use efficiency. 【Method】A field experiment was carried out in a typical oasis irrigation region, Wuwei, Gansu Province, from 2014 to 2016, to determine the effects of treatments of straw retention patterns on soil water utilization of spring wheat field. The treatments included reduced tillage with 25 to 30 cm high straw standing (NTSS), reduced tillage with 25 to 30 cm high straw covering (NTS), conventional tillage with 25 to 30 cm high straw incorporation (TS), and conventional tillage without straw retention (CT, the control). 【Result】Reduced tillage with straw retention could decrease evapotranspiration of spring wheat field, furthermore NTSS and NTS treatments decreased evapotranspiration by 3.1% to 7.8%, 3.7% to 7.7%, compared to CT treatment, respectively. NTSS and NTS treatments decreased evapotranspiration of wheat before early-filling stage but increased it afterwards, so this created a more optimal balance between early- and late-stage water demand of spring wheat. NTSS and NTS treatments could enhance the effectiveness of water by inhibiting soil evaporation and reducing the proportion of evaporation to evapotranspiration (E/ET) for the spring wheat field. NTSS and NTS treatments reduced soil evaporation by 9.3% to 17.4% and 10.8% to 23.3% over CT treatment, and reduced by 4.0% to 5.8% and 5.6% to 11.4% over TS treatment, respectively. Among the two reduced tillage with straw retention treatments, NTS had the best effect on inhibiting soil evaporation, thus this treatment reduced E/ET by 6.9% to 21.3%. The grain yield of NTSS, NTS, TS was 16.6% to 24.9%, 18.6% to 27.3%, 10.2% to 18.7% greater than that of CT treatment, respectively, among the three straw retention treatments, NTSS and NTS had greater grain yield by 5.2% to 5.9% and 7.2% to 9.5% than that of TS treatment, respectively. Thus, straw retention treatments had greater water use efficiency (WUE), compared to CT treatment, NTSS, NTS, and TS treatments improved WUE by 21.1% to 28.3%, 26.6% to 30.6%, 13.1% to 20.3%, respectively. Across the three straw retention treatments, NTSS and NTS treatments improved WUE by 6.7% to 11.9%, 8.6% to 13.7%, in comparison to TS treatment, respectively. 【Conclusion】 Our results showed that reduced tillage in combination with 25 to 30 cm high straw standing and covering was the feasible technology for realizing high yield, stable yield and efficient utilization of irrigation water of spring wheat production in the oasis irrigation region.

    Short-Term Effect of Biochar Amendments on Total Bacteria and Ammonia Oxidizers Communities in Different Type Soils
    ZHANG MengYang,XIA Hao,LÜ Bo,CONG Ming,SONG WenQun,JIANG CunCang
    Scientia Agricultura Sinica. 2019, 52(7):  1260-1271.  doi:10.3864/j.issn.0578-1752.2019.07.013
    Abstract ( 476 )   HTML ( 25 )   PDF (470KB) ( 408 )   Save
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    【Objective】Ammonia oxidation is the first step in nitrification and the rate-limiting step in nitrification. It is a key link in the global nitrogen cycle. The purpose of this experiment was to study the effects of peanut shell biochar application on bacteria and ammonia oxidation in different type soils in China, so as to provide a theoretical basis for the promotion and use of biochar.【Method】Yellow-brown soil, fluvo-aquic soil and black soil were utilized as the tested soil. Through short-term culture experiments, 16SrRNA sequencing was used to study the effects of biochar on ammonia-oxidizing microorganisms, bacterial community structure and related enzyme gene expression in different type soils. Four treatments for each soil included CK (no fertilizer and biochar), F (single fertilization), C (single 2% peanut shell biochar), and FC (application of fertilizer + 2% peanut shell biochar).【Result】Acid soil pH increased significantly by 0.5-1.0 units after application of biochar (C, FC), but alkaline soil pH decreased significantly by 0.5-0.6 units; the microbial abundance and diversity of yellow-brown soil increased significantly caused by single application of biochar (C). The fluvo-aquic only significantly increased the microbial diversity index of the soil when it was applied to biochar alone (C). Biochar and chemical fertilizers did not significantly change soil abundance and diversity in black soil; the abundance of ammonia-oxidizing bacteria in three soils was higher than that of ammonia-oxidizing archaea. The measured OTU abundance of ammonia-oxidizing bacteria was about 8.1 times that of ammonia-oxidizing archaea. Biochar and chemical fertilizers did not significantly alter the OTU abundance in the thaumarchaeota, but had a significant effect on the OTU abundance in the beta and gamma proteobacteria. The ammonia-oxidizing bacteria of the three soils were mainly β-proteobacteria, accounting for about 60%. In addition, the application of biochar (C, FC) significantly changed the microbial community structure of yellow-brown soil on PC1 (40.4%), and significantly changed the microbial community structure of fluvo-aquic soil on both PC1 (42.3%) and PC2 (21.3%). After application of biochar (C, FC), the expression of ammonia synthesis related enzyme gene in fluvo-aquic soil decreased significantly by 14.7%-39.9% in a short period of time, and the ammoxidation archaea abundance decreased by 70.5% and 48.7% under C treatment and FC treatment, respectively.【Conclusion】After application of biochar, the microbial community structure of yellow-brown soil and fluvo-aquic soil was significantly changed, and the ammoxidation of fluvo-aquic soil was obviously inhibited in a short period of time.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    The Effect of Krüppel-Like Factor 3 (KLF3) Gene on Bovine Fat Deposition
    GUO HongFang,NING Yue,CHENG Gong,ZAN LinSen
    Scientia Agricultura Sinica. 2019, 52(7):  1272-1281.  doi:10.3864/j.issn.0578-1752.2019.07.014
    Abstract ( 453 )   HTML ( 31 )   PDF (648KB) ( 457 )   Save
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    【Objective】This study aimed to investigate the effects of bovine Krüppel-like factor 3 (KLF3) gene on bovine adipocytes differentiation and fatty acid metabolism, and to explore the effect of KLF3 gene on lipid deposition. 【Method】This research synthesized interference RNA (siRNA) of KLF3, and then transfected KLF3 gene SiRNA and negative to the Qinchuan cattle preadipocytes when the cells grew to a confluence of 70%-90%. The QPCR method was used to determine the expression of adipocyte differentiation marker genes PPARγ and C/EBPα as well the key genes FAS, ACCα and FABP4 in fatty acid metabolism at adipocytes induced differentiation 0 and 4 days when the expression levels of KLF3 gene interfered. At the same time, on the 4th day of inducing adipocyte differentiation after interfering with KLF3 gene treatment, oil red O staining was used to observe the difference of lipid droplets between the KLF3 gene treatment group and the control group, and the enzyme-linked immunosorbent assay (ELISA) method was used to determine the triglyceride content to further determine the effect of the KLF3 gene on bovine adipocyte differentiation and lipid metabolism. 【Result】The results indicated that KLF3-Si2 had the highest interference efficiency, which was 73%. After transfected with KLF3-Si2, on the 0th and 4th day of bovine adipocytes induced differentiation, the expression of PPARγ was extremely significant (P<0.01) down-regulated by 58% and 37%, respectively; the expression of C/EBPα was also extremely significant (P<0.01) down-regulated by 64% and 41%, respectively; the expression level of FABP4, a key gene for lipid metabolism, was down-regulated by 89% and 60%, respectively; while the expression of ACCα interfered with the KLF3 treatment group and the control group was down-regulated by 50% and 37%, respectively; the expression of FAS was down-regulated by 73% and 19%, respectively, and all the difference was extremely significant (P<0.01). On the 4th day of induced differentiation, oil red O staining and triglyceride content determination also showed that the lipid droplets were decreased and the triglyceride content was significantly decreased in the silence KLF3 gene treated group compared with the control group (P<0.05). 【Conclusion】 Interfering with bovine KLF3 gene could inhibit bovine adipocytes differentiation and fatty acid synthesis key genes expression, thus affecting fat deposition.

    Changes of LncRNA Expression Profile in Spleen of Diarrhea and Non-diarrhea Individuals in F17 of Hu Sheep Lamb
    HUANG SaiNan,JIN ChengYan,BAO JianJun,WANG Yue,CHEN WeiHao,WU TianYi,WANG LiHong,LÜ XiaoYang,GAO Wen,WANG BuZhong,ZHU GuoQiang,DAI GuoJun,SUN Wei
    Scientia Agricultura Sinica. 2019, 52(7):  1282-1294.  doi:10.3864/j.issn.0578-1752.2019.07.015
    Abstract ( 430 )   HTML ( 13 )   PDF (3268KB) ( 283 )   Save
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    【Objective】The objective of this study was to investigate the effect of lncRNA on anti-diarrhea in sheep by screening lncRNA differentially expressed in E. coli F17 fimbriae non-diarrhea and diarrhea sheep spleen. 【Method】 In this study, individuals with non-diarrhea and diarrhea were obtained by oral administration of E. coli F17 strain to Lake Lamb. The success of the challenge was verified by using intestinal counts and pathological sections of the lambs. A cDNA library of spleen from lambs in non-diarrhea group and diarrhea group was constructed and sequenced by using Illumina HiSeq 2500 platform. Functional description and cell pathway analysis of differentially expressed transcripts were performed by Gene Ontology (GO) and KEGG Pathway enrichment analysis by using FPKM method. The expression levels of lncRNA and mRNA transcripts were screened by high-throughput sequencing technology RNA-seq for differential expression of lncRNA in spleens of non-diarrhea and diarrhea individuals; then, Quantitative PCR was used to detect spleen tissues in non-diarrhea and diarrhea lambs. The expression levels of differentially expressed (DE) lncRNA and DE mRNA were used to verify the role of screened DE lncRNA in the non-diarrhea group. 【Result】 After oral administration of E. coli F17 strain, there were two phenotypes of non-diarrhea and diarrhea. The number of bacteria in the intestine of the diarrhea group was significantly higher than that in the non-diarrhea group (P<0.05), and the jejunal mucosa of the diarrhea group appeared different degrees of damage, dull color, part of the small intestine villi off. We used RNA-seq to screen 34 DE lncRNAs and 703 DE mRNAs in non-diarrhea and diarrhea lamb spleens. A total of 12 DE lncRNA and DE mRNA were randomly selected and verified by q-PCR. Relative expression levels in the diarrhea and non-diarrhea lambs were found to be consistent with RNA-seq results. The comparison between DE lncRNA and GO database by GO and KEGG pathway enrichment analysis indicated that a total of 34 lncRNAs were annotated and classified into 302 functional subclasses. There were more than one functional subclass of lncRNA, such as sheep protein binding (GO: 0005515), nuclear (GO: 0005634), poly (A) RNA binding (GO: 0044822), cytoplasm (GO: 0005737), tissue remodeling (GO: 0048771), regulation of endopeptidase activity (GO: 0052548) ), 6-phosphate fructose-2-kinase/fructose-2,6-bisphosphatase complex (GO: 0043540), phosphatidylinositol phosphorylation (GO: 0848654), fructose-2, 6.2-phosphite 2-phosphatase activity (GO: 0004331) and calcium-dependent phospholipase C activity (GO: 0050429), while the remaining functional subclasses had less lncRNA distribution. The alignment of DE lncRNA with the KEGG pathway database indicated that a total of 34 lncRNAs were annotated and classified into 149 KEGG pathways, the sheep thyroid hormone signaling pathway (path: ko04919), Spliceosome (path: ko03040), white blood cell cross Endothelial migration (path: ko04670), neurotrophin signaling pathway (path: ko04722), lysosome (path: ko04142), MAPK signaling pathway-pathway (path: ko04011), sphingolipid signaling pathway (path: ko04071), phagocytosis the body (path: ko04145), oxidative phosphorylation (path: ko00190) and other 9 KEGG pathways had more lncRNAs, while the remaining KEGG pathways had less lncRNA distribution. Through lncRNA-mRNA interaction network analysis, we found six co-expressed genes: MYO1G, TIMM29, CARM1, ADGRB1, SEPT4, and DESI2. 【Conclusion】 This study explored the expression profile of lncRNA in the spleen of non-diarrhea and diarrhea lambs for diarrhea. It was found that lncRNA differentially expressed in the spleen of non-diarrhea and diarrhea lambs, which helped to find out how lambs resist diarrhea and provided a scientific basis for lambs to resist diarrhea.