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16 July 2020, Volume 53 Issue 14

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SPECIAL FOCUS: SORGHUM BREEDING AND CULTIVATION

New Research Progress on Sorghum Breeding and Cultivation Techniques

ZOU JianQiu

Scientia Agricultura Sinica. 2020, 53(14):  2769-2773.  doi:10.3864/j.issn.0578-1752.2020.14.001

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Sorghum has become increasingly important in China’s agricultural structure adjustment due to its excellent stress tolerance and wide adaptability. To meet the new needs of specialization and mechanization of modern agricultural production, many new achievements have been made in sorghum research. Six articles published in this issue of “Scientia Agricultura Sinica”,introduced the recent research progress of China Agriculture Research System on Millet and Sorghum from the aspects of sorghum genetic selection theory, breeding methods, nutritional quality characteristics and cultivation physiological basis. So as to be helpful to the development of sorghum industry in China.

Formation Regulating and Micro-Structure of Sorghum Starch with Different Types of Endosperm

KE FuLai,ZHU Kai,LI ZhiHua,SHI YongShun,ZOU JianQiu,WANG YanQiu

Scientia Agricultura Sinica. 2020, 53(14):  2774-2785.  doi:10.3864/j.issn.0578-1752.2020.14.002

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【Objective】 Sorghum is the main material for liquor-making and brewing well-aged vinegar, and its starch composition and structure are the main factors that influence the brewing quality of sorghum. In this paper, the Dynamic Changes of Key Enzymes Involved in Starch Synthesis are analyzed, the ultrastructure of starch is observed, for confirming the regulatory effect of related enzymes on sorghum starch synthesis, and revealing the ultrastructure characteristics of sorghum starch.【Method】Three sorghum cultivars（Liaonian3, Liaoza19, Liaoza10） with different endosperm types were used as experimental materials. The changes of key enzymes involved in starch synthesis were detected with enzymological technique. Scanning electron microscopy was used to observe the morphology features of starch granules. The regulation of related enzymes in starch synthesis was studied by correlation analysis. 【Result】 The activity of UDPG, ADPG, SSS, SBE, DBE enzymes of sorghum with different endosperm types showed a single peak curve during starch accumulation. The activity of GBSS enzyme of sorghum with waxy endosperm showed a single peak curve, while other types sorghum showed bimodal curves. Activities of UDPG and ADPG, SSS enzyme were significantly correlated with amylose and amylopectin accumulation rate. Activities of SBE, DBE enzyme were positively correlated with amylopectin accumulation rate. Activities of GBSS enzyme was positively and significantly correlated with amylopectin accumulation rate in non-waxy sorghum and semi-waxy sorghum, it showed a positively correlation trend, but not significant in waxy sorghum. For sorghum with different endosperms, the formation process of starch grains showed a similar tendency. The starch accumulation rate was higher during 14-35 d after flowering. The starch grains of waxy sorghum are the smallest, with a diameter within 10 μm, and carved with round-hole or wedge-shaped cavity inside. The starch grains of nonwaxy sorghum are the largest, irregularly globular, with scarcely cavity inside. The size distribution of sorghum starch granules with semi-waxy endosperm ranges widely, most of them with wedge-shaped or star-shaped cavities inside, only a few with no cave. 【Conclusion】 UDPG, ADPG, SSS play important adjustive roles in the accumulation of amylopectin and amylose. GBSS, SBE and DBE are the key enzymes to regulate the ratio of amylose/amylopectin. Higher activities of SBE and DBE are the main reason that makes waxy sorghum contains higher amylopectin. The ratio of amylose/amylopectin may be the main factor affecting the starch grain structure of sorghum.

Heterosis Prediction of Sweet Sorghum Based on Combining Ability and Genetic Distance

WANG LiMing,YAN HongDong,JIAO ShaoJie,JIANG YanXi,SU DeFeng,SUN GuangQuan

Scientia Agricultura Sinica. 2020, 53(14):  2786-2794.  doi:10.3864/j.issn.0578-1752.2020.14.003

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【Objective】 The heterosis, general combining ability (GCA) and special combining ability (SCA) of main agronomical traits of sweet sorghum were analyzed. Meanwhile, the feasibility of heterosis prediction based on combining ability, phenotypic genetic distance (PGD) and molecular genetic distance (MGD) was estimated. The result will provide theoretical reference for germplasm improvement and hybrid breeding of sweet sorghum.【Method】 Sixty-four hybridized combinations were generated with 8 sterile lines as females and 8 restorer lines as males in accordance with a North Carolina Design II mating scheme. Twelve agronomical traits including days to flowering, growth duration, plant height, panicle length, stem diameter, tillers, panicle weight, 1000-grain weight, grain yield per plot, biomass per plant, biomass per plot and sugar content of hybrids and their parent lines were investigated in two years. The heterosis, GCA, SCA, PGD and MGD were analyzed, as well as the correlation between combining ability, genetic distance and heterosis. 【Result】 Mid-parent heterosis (MPH) of traits from high to low was: biomass per plant, grain yield per plot, panicle weight, biomass per plot, plant height, panicle length, 1000-grain weight, stem diameter, growth duration, days to flowering, tillers and sugar content. Among which, heterosis of growth duration, days to flowering, tillers and sugar content was negative. The sequence of MPH for all traits was almost the same as that of better parent heterosis (BPH). Combining ability analysis showed that the GCA of different parents varied greatly in each trait. Meanwhile, the SCA of different combinations was also significantly different. Most combinations with high SCA also showed high GCA in their parent lines. Heterosis of biomass per plant, grain yield per plot, panicle weight, biomass per plot, panicle length, 1000-grain weight, tillers and sugar content was positively and highly significantly correlated with GCA and SCA of their parent lines simultaneously. Heterosis of growth duration was positively and highly significantly correlated with SCA. Meanwhile, heterosis of days to flowering was positively and significantly correlated with SCA. The PGD of parent lines was 2.86-6.82, and MGD was 0.50-0.96. The correlation between heterosis and MGD was greater than that of PGD in biomass per plant, grain yield per plot, panicle weight, biomass per plot, plant height, panicle length, stem diameter and sugar content. Among which, heterosis of biomass per plot, biomass per plant, panicle length and stem diameter was positively and highly significantly correlated with MGD. 【Conclusion】 Heterosis of yield related traits was higher, and that of sugar content and tillers was lower among all traits. In the prediction of heterosis, combining ability was more effective than genetic distance, therefore, can be used to predict heterosis. MGD was more effective in heterosis prediction compared with PGD.

Molecular Aided Breeding System of Photosensitive Forage Sorghum Based on SSR

NIU Hao,PING JunAi,WANG YuBin,ZHANG FuYao,LÜ Xin,LI HuiMing,CHU JianQiang

Scientia Agricultura Sinica. 2020, 53(14):  2795-2803.  doi:10.3864/j.issn.0578-1752.2020.14.004

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【Objective】 The aim of this study is to use molecular marker to construct a breeding system to improve the traditional breeding method,thereby reducing breeding costs and improving breeding efficiency. At the same time, it lays a theoretical foundation for the formation of a new breeding system. 【Method】 The F₂ population was constructed by crossing the photosensitive(non-heading) forage sorghum variety Jinguang 1R as female and the photo insensitive(heading) sorghum variety BMRC-3-2 as male. According to heading and non-heading phenotypes, F₂ population was divided into two groups. Thirty plants were selected from each group and DNA was extracted from leaves, SSR and BSA were used to map photosensitive genes in F₂ population of Jinguang 1R/BMRC-3-2 and screen for specific SSR primers. The specific primers were used to identify the photosensitivity trait in F₁ generation（F₁ Hybrids breeded from high generation stable restorer line with Jinguang 1R as parent and matched with other male sterile lines）, and the final target primers were determined by comparing with the phenotype in the field, so as to construct a new photosensitive breeding system for forage sorghum. 【Result】 After SNP index analysis, the region of about 250 Kb before and after the highest point of 99% threshold line was selected as the candidate region for trait correlation. The total length of the region was 500 kb, with 400 SNP loci, and six of them were non-synonymous mutations or stop gain or stop loss SNP loci. Finally, two candidate genes were identified to be related to photosensitivity, which were located on chromosome 7. A pair of specific primers 70.2-3 was developed by SSR. The primers can distinguish between heading and non-heading sorghum plants to a great extent. The results showed that according to the "single peak" at 251bp, the accuracy of primers 70.2-3 in the identification and selection of 50 non-photosensitive F₁ hybrids reached 100%, and all the materials showed a peak value near 251bp. Based on the "double peaks" in the vicinity of 214 bp and 251 bp, the accuracy rate of the primer for the identification and selection of the other 50 photosensitive F₁ hybrid species reached 90%. Three materials, No. F₁-69, F₁-70 and F₁-71, showed "double peaks" near 214 bp and 262 bp. Material F₁-81 has a "single peak" at 251bp, while material No. 86 has a "double peak" near 251 bp and 232 bp.【Conclusion】 In this study, we revealed that the candidate genes controlling heading date of sorghum were LOC8068537 and LOC8068548, which were between 810000 and 1310000 bp on chromosome 7 of sorghum. The acquisition of specific primers 70.2-3 improved the traditional breeding methods. The primer validation in laboratory could replace the field crossing observation, saving the breeding cost and improving the breeding efficiency.

Effects of Harvesting at Different Growth Stage on Agronomic and Nutritional Quality Related Traits of Sweet Sorghum

WANG HaiLian,WANG RunFeng,LIU Bin,ZHANG HuaWen

Scientia Agricultura Sinica. 2020, 53(14):  2804-2813.  doi:10.3864/j.issn.0578-1752.2020.14.005

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【Objective】 Sweet sorghum (Sorghum bicolor (L.) Moench) is one of the most important forage crops and is widely cultivated. Studies on biological yield, nutritional quality and forage value of sweet sorghum at different growth stages could provide theoretical guidance for determining the suitable harvesting time of sweet sorghum as forage. 【Method】 Sweet sorghum hybrid, Jitianza No.2, was used as an experiment material, planted in Licheng and Jiyang District in Jinan, and Dongying Cities, and mowed at booting, flowering, milky, dough and physiologic maturity stages. Agronomic and nutritional quality related traits were analyzed, and relative feed values (RFV) at five growth stages were evaluated. 【Result】 The largest value of plant height of Jitianza No.2 were 440.0 cm and 390.0 cm at dough stage in Licheng District and Dongying City, and 445.9 cm at physiologic maturity stage in Jiyang District. Stem is the main component of total fresh weight (TFW) and occupied 83.8%, 83.3%, 78.9%, 78.4% and 78.5% of TFW at five growth stages, respectively. With development of plant, TFW was gradually increased from boot stage to dough stage and decreased at physiologic maturity stage in Licheng District and Dongying City. The maximum TFW were 1 970.5 g/plant and 1 977.5 g/plant. TFW was increased from boot stage to physiologic maturity stage with the maximum TFW of 2 389.4 g/plant in Jiyang District. Total dry weight (TDW) showed the same change trend as TFW, and the maximum TDW of 487.2 g/plant and 469.0 g/plant were reached at dough stage in Licheng District and Dongying City, and 573.5 g/plant at physiologic maturity stage in Jiyang District. Significant effects by environment, growth stage and interaction between environment and growth stage were identified in plant height, leaf fresh weight, stem fresh weight, panicle fresh weight, TFW and TDW. The content of neutral detergent fiber (NDF) was gradually increased with development of plant and the minimum were 45.27% and 46.33% at booting stage, respectively, in Jiyang District and Dongying City. Acid detergent fiber (ADF) content had a similar trend with NDF and the minimum of 29.06% and 32.07% were found at booting stage in Jiyang District and Dongying City. Crude protein (CP) content varied largely in each growth period, with the highest values of 6.29% at booting stage in Jiyang District and 6.83% at flowering stage in Dongying City. Soluble carbohydrate (SC) content was increased significantly at each growth period and reached the maximum of 14.09% and 15.69% at physiologic maturity stage, respectively, in Jiyang District and Dongying City. Ash content was gradually increased with development of plant and the maximum of 8.53% and 5.36% were reached at physiologic maturity stage in Jiyang District and dough stage in Dongying City. Effects by environment, growth stage and interaction between environment and growth stage were significant in NDF, ADF, CP, SC and ash content. With growth of plant, dry matter intake (DMI) at five growth stages was gradually decreased, and the maximum were 2.65% and 2.59% at booting stage in two environments, respectively. Digestible dry matter (DDM) had the same change trend as DMI and the maximum were 66.26% and 63.92% in Jiyang District and Dongying City. Similarly, the maximum RFV of 136.17 and 128.35 were found at booting stage. Significant effects by the environment, growth stage and interaction between environment and growth stage were found in DMI, DDM and RFV.【Conclusion】Biomass, nutritional quality and RFV of sweet sorghum were significantly affected by environment, harvesting stage and environment-harvesting stage interaction. The largest biomass could be obtained at about dough stage, and the highest DMI, DDM and RFV could be reached at booting stage. However, considering the optimal combination of biomass, nutrient quality and silage quality, the optimum harvesting stage was between milk and dough stage.

Dwarfing Effect and Molecular Mechanism of An Elite Sorghum Male Sterile Line 01-26A in Its Hybrids

ZOU JianQiu,WANG YanQiu,LI JinHong,ZHU Kai

Scientia Agricultura Sinica. 2020, 53(14):  2814-2827.  doi:10.3864/j.issn.0578-1752.2020.14.006

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【Objective】The mechanized production of sorghum is the inevitable direction of future development, and the ideal plant type is the basis and key factor for mechanized production. The dwarf male sterile line 01-26A was found a unique effect in reducing the plant height in F₁ generation when crossed with available grain sorghum restorer lines. Therefore, a study was conducted to determine the genetic mechanism and regulation mechanism of plant height dwarfing by its genetic effects and regulatory gene loci. 【Method】This study used sorghum male sterile line 01-26A (A₁ cytoplasm) with dwarf plant height effect and sorghum male sterile line 7050A (A₂ cytoplasm) without dwarf plant height effect as the test material, focusing on 7 restorer lines, including 6 grain restorer lines and 1 sweet sorghum restorer line, and their cross F₁ generation hybrids, the genetic effects of plant height, number of nodes, the total internode length under peduncle, the peduncle length and the head length were analyzed, the gene loci of Dw₁ to Dw₃ related with the plant height were also measured and analyzed. Dw₄ was not included because it had not been cloned.【Result】the male sterile line 01-26A had a significant dwarf effect on the plant height of grain sorghum, and its hybrids is 15.8% lower than that of the 7050A, generally, the absolute value of plant height did not exceed 160 cm. The plant height of F₁ generation derived from 01-26A with sweet sorghum restorer line had not been obviously reduced, so it had not dwarf effect on sweet sorghum hybrid. The genetic dwarf effect of 01-26A was mainly manifested in the shortening of the internode length under peduncle, and the internode length under peduncle has more correlated with the plant height variation. The peduncle length reduction of F₁ crossed by 01-26A with restorer lines is another reason of plant becoming shorter, but the effect was less than that of the internode length under peduncle. While the head length had much little effect on plant height variation. The dwarf genotype (Dw₁ to Dw₃) of 01-26A was determined by PCR and sequencing of Dw₁ to Dw₃ genes. And combined the analysis of plant height genetic data of multiple cross combinations, the plant height genotype of 01-26A was deduced to be dw₁dw₁Dw₂ Dw₂dw₃dw₃dw₄dw_4, a 3-dwarf sorghum sterile line. In addition, by the analysis of plant height regulation genes, we found that the dw1 and dw3 of 01-26A may had a greater effect on the plant height of grain sorghum, while the presence of DW₂ was the immanent cause for not dwarfing on sweet sorghum F₁.【Conclusion】 01-26A was likely to be a 3-dwarf sorghum male sterile line with the genotype of dw₁dw₁Dw₂Dw₂dw₃dw₃dw₄dw₄. It could achieve dwarf regulation of its F₁ by reducing the internode length (main effect) and the peduncle length (secondary effect). However, 01-26A, had not been found obvious dwarfing effect when crossed with sweet sorghum, which may be due to the presence of Dw₂.

Genetic Analysis on Growth Period and Plant Height Traits of Early-maturing Dwarf Sorghum Male-Sterile Line P03A

DUAN YouHou,LU Feng

Scientia Agricultura Sinica. 2020, 53(14):  2828-2839.  doi:10.3864/j.issn.0578-1752.2020.14.007

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【Objective】In order to identify the genetic effect of early-maturing and dwarf traits on sorghum male-sterile line P03A, here we, provide a theoretical basis for sorghum early-maturing and dwarf breeding improvement by genetic analysis on growth period and plant height. 【Method】In 2016, sorghum male-sterile line P03A,L025A, L080A, L081 and P02A were used as female parent lines, and restoring line L242, L2381, LNK1, L280, L237, and L298 were used as male parent lines the F₁ hybrid seeds were obtained by NCⅡ crossing method. And in the winter of 2016, F₂ seeds were harvested from F₁ selfing plantlets in Hainan province. During 2017-2018, Combining ability analysis on growth period and plant height traits were performed, together with 4 generation conjoint analysis from hybrid F₁ and F₂ population using mixed major gene plus poly-gene inheritance model. P03A and L237 were selected as female and male parent respectively. 【Result】 Hybrid combination P03A/L237 show the characters of short growth period and short plant height through interaction of the 2 parent lines. P03A contribute to shortening the growth period and plant height in hybrid. Compared with the rest 4 sterile lines, the hybrid combinations with P03A show shorter growth period and plant height, and heritability on the 2 traits was identified. 4-generation analysis of P03A/L237 on growth period and plant height were performed using major gene plus poly-gene inheritance model, which indicate that growth period and plant height traits were both controlled by two major genes with additive-dominate-epistatic effects and poly-genes. Analysis result of growth period suggest that additive effect is higher than epistatic and dominate effects. Heritability of major genes is 81.13%, and heritability of poly-genes is 10.36% respectively. 91.49% phenotypic variation is conducted by major genes plus poly-genes, and 8.51% phenotypic variation is conducted by environmental factors. Analysis of plant height indicate that additive effects and dominant effects of the first major gene are all stronger than the second major gene, and the dominant effect is much more important. Heritability of major genes and poly-genes is 84.80%, and 6.89% respectively. 8.31% phenotypic variation is conducted by environmental factors. 【Conclusion】Genetic effects of growth period and plant height of sorghum sterile line P03A were analyzed in this study. It was identified that the heritability of the two traits mentioned above are relatively high, not easily affected by environmental factors, and with stable hereditary characters. Thus, P03A can be utilized due to its early maturing and dwarf genes in sorghum hybrid breeding, which can meet the requirement of sorghum mechanized production.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effects of Subsoiling and No-Tillage Frequencies on Soil Aggregates and Carbon Pools in the Loess Plateau

ZHANG Qi, WANG ShuLan, WANG Hao, LIU PengZhao, WANG XuMin, ZHANG YuanHong, LI HaoYu, WANG Rui, WANG XiaoLi, LI Jun

Scientia Agricultura Sinica. 2020, 53(14):  2840-2851.  doi:10.3864/j.issn.0578-1752.2020.14.008

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【Objective】This study was aimed to clarify the effects of reducing the frequencies of subsoiling on the soil structures and soil carbon pool in spring maize fields on the Loess Plateau. 【Method】A long-term positioning experiment of conservation tillage with different frequencies of subsoiling and no-tillage was carried out in spring maize fields on Weibei dryland from 2007 to 2019, with continuous subsoiling (S) as the contrast, which set up combination tillage modes of no-tillage and subsoiling to reduce the frequency of subsoiling: subsoiling once two years (NS) and subsoiling once three years (NNS). The effects of reducing the frequency of subsoiling on soil aggregates, carbon preservation capacity of soil aggregates, soil carbon pool composition and carbon pool management index under different tillage treatments were observed after 12 year continuous experiment. 【Result】(1) Decreasing the frequency of subsoiling improved the content of macroaggregates (R_0.25), the content of 0-40 cm soil layer mechanical-stability aggregates (DR_0.25) increased by 3.8% and water-stable aggregates (WR_0.25) increased by 38.9% under NNS treatment, respectively. NS treatment increased the WR_0.25 by 41.8%. Meanwhile, NNS decreased the destruction rate (PAD) and unstable aggregate index (E_LT) of aggregates, with the mean weight diameter (MWD) and geometrical mean diameter (GMD) significantly increased by 13.3% and 16.6%. (2) The total carbon preservation capacity (TOPC) of the aggregates under NNS and NS was significantly increased. The average carbon sequestration capacity of aggregates in 0-40 cm soil layer under NNS was increased by 10.8%, whereas it was decreased in 20-30 cm soil layer. The carbon preservation ability of aggregates in different sizes indicated that the carbon preservation ability of aggregates with particle size of <0.25 mm was stronger. (3) NNS treatment had no significant effect on the total organic carbon (TOC) content in 0-40 cm soil layer, but increased the TOC content in the 10-20 cm soil layer, reduced stratification effect of surface soil organic carbon, and decreased the TOC content in 30-40 cm soil layer and caused the effect of organic carbon layering in deep soil. (4) The content of active organic carbon (EOC) in the 0-40 cm soil layer under NNS treatment obviously increased by 24.9%. Furthermore, the increase of EOC content lead to higher EOC/TOC ratio, carbon pool activity (A) and carbon pool activity index (AI), with the carbon pool management index (CPIM) increased by 39.8% compared to S. 【Conclusion】Long-term continuous subsoiling was not conducive to the formation of soil aggregates and the cycle of soil carbon pools. While subsoiling once three years tillage mode helped to reduce the degree of damage about soil aggregates, improve the carbon pool management index and adjust the renewal and cycling of soil carbon pool, which was a suitable tillage model for the region.

Effect of Rapeseed Rotation on the Yield of Next-Stubble Crops

ZHANG ShunTao, LU JianWei, CONG RiHuan, REN Tao, LI XiaoKun, LIAO ShiPeng, ZHANG YueQiang, GUO ShiWei, ZHOU MingHua, HUANG YiGuo, CHENG Hui

Scientia Agricultura Sinica. 2020, 53(14):  2852-2858.  doi:10.3864/j.issn.0578-1752.2020.14.009

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【Objective】 The aim of this study was to clarify the impact of rapeseed rotation on the yield of the next-stubble crops in the multiple cropping rotation area of the Yangtze River Basin, and to verify that it was a common phenomenon that rapeseed cultivation increased crop yield in the subsequent season, so as to provide a basis for rapeseed as an alternate husbandry crops to promote both yield of grain and oil, and yield stability. 【Method】 Field experiments with different crop rotation patterns were carried out in different areas of the Yangtze River Basin: in the upper Yangtze River, rapeseed-rice and wheat-rice rotation in Beibei (Chongqing), and rapeseed-maize and wheat-maize rotation in Yanting (Sichuan province) were selected; in the middle Yangtze River, rapeseed-rice and wheat-rice rotations, rapeseed-maize and wheat-maize rotations in Shayang (Hubei province), rapeseed-early rice and winter fallow-early rice-late rice rotations in Hengyang (Hunan province), rapeseed-rice and wheat-rice rotations in Xinyang (Henan province) were selected; in the lower Yangtze River, rapeseed-rice and wheat-rice rotations in Rugao (Jiangsu province) were selected. The differences in yield, yield components and nutrient uptake of rice or maize in the subsequent season of winter crop wheat (or winter fallow) and rapeseed at the same fertilization level were analyzed. 【Result】 Compared with that in wheat-rice rotation, the rice yield of rapeseed-rice rotation in Beibei, Shayang, Xinyang and Rugao increased by 323, 483, 1 569 and 569 kg·hm^-2, respectively, with increase rate of 4.6%, 6.6%, 17.3% and 6.0%, respectively. Compared with that in wheat-maize rotation, the maize yield of rapeseed-maize rotation in Yanting and Shayang increased by 487 and 579 kg·hm^-2, respectively, with increase rate of 7.0% and 14.8%, respectively. Compared with that in winter fallow-rice-rice rotation, the early rice and late rice yields of rapeseed-rice-rice rotation in Hengyang increased by 718 and 726 kg·hm^-2, respectively, with increase rate of 11.1% and 10.5%, respectively. Compared with the wheat-rice rotation, the rice panicle number and grains per panicle of rapeseed-rice rotation in Shayang and Xinyang increased by 7.0×10⁴, 27.7×10⁴ spikes/hm² and 18.1, 20.2 grains. Compared with the wheat-rice rotation, the rice biomass of the rapeseed-rice rotation in Shayang and Beibei increased by 1 711 and 2 625 kg·hm^-2, respectively, and the nitrogen accumulation increased by 23.9 and 23.2 kg·hm^-2, respectively. 【Conclusion】 In different planting areas in the Yangtze River Basin, rapeseed rotation could increase the yield and nutrient accumulation of next-stubble crops in different rotation patterns in varying planting areas in Yangtze River Basin, being a good alternate husbandry crop in a rotation.

The Methodology and Application of Agricultural Monitoring and Early Warning Model Cluster

XU ShiWei, DI JiaYing, LI GanQiong, ZHUANG JiaYu

Scientia Agricultura Sinica. 2020, 53(14):  2859-2871.  doi:10.3864/j.issn.0578-1752.2020.14.010

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【Objective】The accurate prediction and evaluation of agricultural product supply and demand is an important manifestation for the improvement of agricultural monitoring and early warning capabilities. A multi-variety multi-link model cluster to construct that can efficiently solve analytical technical problems which are difficult to solve with single links or single models.【Method】 The methodology characterized by "factor classification decoupling, parameter conversion adaptation" for multi-variety agricultural products was proposed to build a multi-temporal dimension monitoring and early warning model cluster, which took into account the important factors of agricultural products supply and demand, namely production, consumption, trade volume, price, etc., the strong linkage among commodities, the entangled complex natural, social and economic factors, and the multivariate strong coupling, non-linear, time-varying characteristics of parameters in the model development.【Result】 The model clusters were developed covering production, consumption, trade and price for different agricultural products, based on the "factor classification decoupling, parameter conversion adaptation" methodology. These model clusters could be used to analyze and project the supply and demand situation of major agricultural products including rice, corn, wheat and meat in different spatial and temporal dimensions, and to support the generation of major agricultural products balance sheets in the China Agricultural Outlook Report. The 6-year average forecast accuracy was higher than 97%.【Conclusion】The methodology of agricultural monitoring and early warning model cluster proposed in the paper has effectively improved the solution efficiency and accuracy of agricultural product multi-variety model clusters, enhanced the systematic and intelligent analysis and projection of agricultural supply and demand. The research provided a new technical method for systematically revealing the complex characteristics of supply and demand of agricultural products in time and space, and promoting the scientific and predictable regulation of agricultural products market.

PLANT PROTECTION

Cloning and Functional Analysis of VdHP1 in Verticillium dahliae from Cotton

SUN Qi,HE Fang,SHAO ShengNan,LIU Zheng,HUANG JiaFeng

Scientia Agricultura Sinica. 2020, 53(14):  2872-2884.  doi:10.3864/j.issn.0578-1752.2020.14.011

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【Objective】The objective of this study is to determine the function of a novel gene (VdHP1) in Verticillium dahliae causing cotton verticillium wilt, and to provide a basis for analyzing the pathogenic mechanism of V. dahliae and the prevention and treatment of cotton verticillium wilt.【Method】The full length of VdHP1 was cloned and sequenced from V. dahliae wild-type strain V592 genomic DNA and cDNA. The relative expression of VdHP1 in V592 strain induced by cotton roots for different times and from different tissues of V592 was measured by reverse transcription quantitative real-time PCR (RT-qPCR). VdHP1 gene knockout vector, complementary vector and overexpressed vector were constructed to produce VdHP1 gene knockout strains, complementary strains and overexpressed strains by Agrobacterium tumefaciens-mediated transformation, respectively. Taking wild-type strain V592 as the control, colony growth on PDA and hypha morphology were observed, microsclerotia production, conidial production and pathogenicity to cotton of VdHP1 gene knockout mutants and complementary strains were measured. The relative expression of other genes involved in pathogenicity in VdHP1 knockout mutants and overexpressed strains was measured by RT-qPCR.【Result】The full length of VdHP1 was determined to be 862 bp and deduced protein contained 268 amino acids, which shared no significant sequence similarity to any known annotated gene in GenBank. The transcriptional expression of VdHP1 was significantly up-regulated when V592 strain induced by cotton roots for 6-12 h, indicating that VdHP1 plays a role at the early stage of the infection. The transcriptional expression of VdHP1 in conidia was significantly higher than that in mycelia and microsclerotia, indicating that VdHP1 is differentially expressed in different tissues of V. dahliae. Compared with wild-type strain V592, VdHP1 gene knockout mutants showed significantly decreased conidia and conidiophores, branching hyphae were spirally shaped, and the pathogenicity to cotton was significantly decreased. The relative expression of genes involved in penetration peg formation (VdCrz1, VdNoxB, VdPls1), delivery of secretory protein (VdSep5) and conidial production (Vdpf, VdSge1, VGB, VdPLP, VdCYC8, VdNLP1, VdNLP2) was significantly down-regulated in VdHP1 knockout mutants, but was up-regulated in the overexpressed strains; whereas, the relative expression of genes involved in melanin synthesis (VdCmr1, VdSho1, VdLAC, VdPKS1) was significantly up-regulated in VdHP1 knockout mutants, and was down-regulated in the overexpressed strains. 【Conclusion】VdHP1 is participated in the production of conidia and conidiophores, and is involved in pathogenicity in V. dahliae. VdHP1 positively regulates the transcriptional expression of genes involved in penetration peg formation, delivery of secretory protein and conidial production, and negatively regulates the transcriptional expression of genes involved in melanin synthesis.

Preparation of A Novel Silver Nanoparticle and Its Antifungal Mechanism Against Alternaria alternata

XIANG ShunYu,WANG Jing,XIE ZhongYu,SHI Huan,CAO Zhe,JIANG Long,MA XiaoZhou,WANG DaiBin,ZHANG Shuai,HUANG Jin,SUN XianChao

Scientia Agricultura Sinica. 2020, 53(14):  2885-2896.  doi:10.3864/j.issn.0578-1752.2020.14.012

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【Objective】In order to explore the agricultural application prospect of biological polysaccharide synthesis of silver nanoparticles and provide a theoretical basis for the development of safe and efficient antifungal agent, the silver nanoparticle based on the polysaccharide was prepared and its inhibitory activity and mechanism against Alternaria alternata was analyzed.【Method】The sodium alginate was used as reductant and surfactant to synthesize the silver nanoparticles in water bath (65℃). The transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), ultraviolet spectrophotometer (Uv-vis), Zeta potential-particle size analyzer and X-ray diffraction energy spectrum (XPS) were used to characterize and analyze the particle size, dispersion, stability and chemical composition of S-AgNPs, respectively. The Nano Measure software was used to measure the mean particle size of S-AgNPS in TEM and AFM images. Then, the S-AgNPs inhibition ability against mycelial growth was tested in the potato dextrose agar medium (PDA) with different S-AgNPs concentrations (0.0625, 0.125, 0.25, 0.5, 1.0 μg·mL^-1). After A. alternata cultivated in the liquid PDA medium (PDB) with S-AgNPs at the concentration of 1.0 μg·mL^-1, the effects of S-AgNPs on the mycelium weight, morphology, the cell membrane permeability and the soluble protein content were investigated by testing the fresh and dry weights of mycelia, SEM observation, conductivity measurement and coomassie brilliant blue G-250 solution stain. Then, the control efficacy of S-AgNPs on tobacco brown spot was tested in vitro leaves by the spore suspension injection method. Finally, crucian carp was used to evaluate the biosafety of S-AgNPs.【Result】The TEM, AFM and SEM images showed that the strategy could be used to well control the size of the S-AgNPs (average particle size of 9.83 nm), and the prepared S-AgNPs showed a high stability and dispersibility in aqueous solvent. The results of bioassay showed that the inhibition rate of S-AgNPs on the growth of A. alternata reached 83.9% at 1.0 μg·mL^-1. The fresh weight and dry weight of mycelium in S-AgNPs treatment group were significantly lower than those of the control group. The SEM image of mycelium showed that S-AgNPs could obviously destroy mycelium surface structure. Further investigation on the antifungal mechanism of S-AgNPs showed that S-AgNPs could destroy the biofilm structure by inhibiting the synthesis of soluble total protein in A. alternata, reducing the water retention capacity of biofilm, rapidly destroying the membrane permeability of mycelium and causing a large amount of cytoplasmic leakage to inhibit the growth and development of mycelium. Finally, in vitro experiments confirmed that S-AgNPs could effectively inhibit A. alternata infecting tobacco leaves. Moreover, S-AgNPs in the effective antifungal concentration had no toxic effect on crucian carp and did not affect its normal life.【Conclusion】The method established in this study can stably synthesize silver nanoparticles with good dispersion, uniform particle size and strong stability. In addition, the silver nanoparticle has a strong inhibitory ability against A. alternata and can effectively control tobacco brown spot, which suggests that S-AgNPs has a potential application prospect.

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

Characteristics of Soil Enzyme Activities and CO₂ and CH₄ Emissions from Natural Wetland and Paddy Field in Karst Areas

YUAN Wu,JIN ZhenJiang,CHENG YueYang,JIA YuanHang,LIANG JinTao,QIU JiangMei,PAN FuJing,LIU DeShen

Scientia Agricultura Sinica. 2020, 53(14):  2897-2906.  doi:10.3864/j.issn.0578-1752.2020.14.013

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【Objective】 The purpose of this study was to explore the impacts of land-use change on the relationship between soil enzyme activities and greenhouse gas emissions of wetlands. 【Method】 Huixian karst wetland and the top soil (0-20 cm) from a lake wetland and its adjacent paddy fields were used as a research site and research objects, respectively. The soil enzyme activities and CO₂ and CH₄ emissions were detected with colorimetric method and static-chamber method, respectively.【Result】The results showed that the activities of β-glucosidase, cellulase, invertase, chitinase, urease and alkaline phosphatase in the paddy soil were all higher than those in the lake wetland, with a range of 11.8%-32.7%. The fluxes of CO₂ and CH₄ from the paddy field were 255.9-789.7 mg·m^-2·h^-1and -0.41-1.74 mg·m^-2·h^-1, respectively. The average values of CO₂ and CH₄ fluxes from the paddy field were 445.8 and 0.42 mg·m^-2·h^-1, respectively, which were lower than those in the lake wetland. Compared with the lake wetland, the amount of CO₂ and CH₄ emissions from the paddy field were decreased by 22.3% and 83.3%, respectively, while the GHGs (including N₂O) was decreased by 29.6%. Correlation analysis showed that the CO₂ flux was significantly negatively correlated with the activities of β-glucosidase, cellulase, invertase and chitinase, while the CH₄ flux was significantly negatively correlated with 6 soil enzyme activities (P<0.05).【Conclusion】The above results indicated that the conversion of a natural lake wetland into a paddy field had significantly increased soil enzyme activities and decreased the emissions of CO₂ and CH₄ at the same time, which was conductive to the improvement of microbial carbon-use-efficiency and the sequestration of soil carbon in Huixian karst wetland.

Potential Analysis of Reducing Chemical Nitrogen Inputs While Increasing Efficiency by Organic-Inorganic Fertilization in Winter Rapeseed Producing Areas of the Middle and Lower Reaches of the Yangtze River

JIANG QianHong,LU ZhiFeng,ZHAO HaiYan,GUO JunJie,LIU WenBo,LING Ning,GUO ShiWei

Scientia Agricultura Sinica. 2020, 53(14):  2907-2918.  doi:10.3864/j.issn.0578-1752.2020.14.014

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【Objective】In order to provide a scientific basis for reducing chemical nitrogen (N) inputs and improving N efficiency in the rapeseed production, the reduction potential of chemical fertilizer and regional suitability was evaluated in winter rapeseed producing areas along the middle and lower reaches of the Yangtze River. 【Method】To analyze the effects of organic and inorganic fertilizer on yield and chemical N fertilizer use efficiency of rapeseed, the experiments were set up in Gaochun, Anren, Shayang, Xiuning and Dangtu across in 4 provinces in 2018, which consisted of two factors, including manure dosages (0, and 2 250 kg·hm^-2) and nitrogen application rates (0, 90, 135, 180, 225, and 270 kg·hm^-2). In total, 12 treatments were contained in all field experiments. Meanwhile, the optimal chemical nitrogen rates for winter rapeseed were evaluated in different regions under the optimal yield and fertilization benefit. 【Result】Compared with only application of chemical fertilizer, applying organic fertilizer could significantly increase the yield of rapeseed by 7.7% to 43.3%. With the target to the highest yield under the application of organic fertilizer, the recommended chemical N fertilizers rates were: 195, 199, 195, 179 and 185 kg·hm^-2 in Gaochun, Anren, Shayang, Xiuning and Dangtu, respectively. 26.7%-45.9% of chemical N could be replaced by organic fertilizer to maintain the same highest yield at each site. Moreover, as the soil fertility was better, the substitution rate on chemical N by organic fertilizer got higher. In addition, the N use efficiency decreased with the increase of chemical N rate under different dosage of organic fertilizer, while the combined application of manure and mineral fertilizer could significantly increase the nitrogen fertilizer partial productivity by 24.4%-53.0% and agronomic utilization efficiency by 26.3%-89.9% in rapeseed production. In Anren, Xiuning and Dangtu, the income increases were the highest under 180 kg N·hm^-2 input combined with organic fertilizer, and the corresponding values were 8 915, 1 0358 and 6 569 yuan/hm², respectively. However, in Gaochun and Shayang, the highest income increases were under only application of chemical fertilizer (225 kg N·hm^-2 input), and were 11 252 and 8 500 yuan/hm², respectively. 【Conclusion】In the experimental sites along middle and lower reaches of the Yangtze River, combined application of chemical and organic fertilizer could reduce 26.7%-45.9% chemical N fertilizer input, increase grain yield, chemical nitrogen use efficiency and income increases for winter rapeseed production, and achieve the goal of reducing chemical N input with increased efficiency.

HORTICULTURE

Response Characteristics of Green Onion (Allium fistulosum L.) to LED Light Quality Under Artificial Climate Chamber

GAO Song,LIU XueNa,LIU Ying,CAO BiLi,CHEN ZiJing,XU Kun

Scientia Agricultura Sinica. 2020, 53(14):  2919-2928.  doi:10.3864/j.issn.0578-1752.2020.14.015

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【Objective】The purpose was to explore the response characteristics of green onion growth, product quality and photosynthetic characteristics of green onions to different light qualities, and to provide technical parameters for optimizing the regulation of light environment in industrial production of green onions and improve the production efficiency of green onions based on fresh green leaves. 【Method】 Under the condition of LED light source in artificial climate chamber, two different varieties of green onion ‘Zhangqiu’ and ‘Tianguang’ were used as experimental materials. The seedlings of green onion with about 15 cm in seedling height and 2-3 pieces of true leaves were cultured under five different light qualities, including blue light (B), red light (R), green light (G), yellow light (Y) and white light (W). The light intensity was controlled at (301.6 ± 12.7) μmol·m^-2·s^-1, the light time was 12 h/d, and the day/night temperature was controlled at 25℃/18℃, respectively. Samples were taken at 0, 10, 20, 30 and 40 days after treatment, and the photosynthetic parameters of green onions treated with different light quality were measured, as well as the growth and product quality of green onion at 40 days of culture. 【Result】The growth, product quality, leaf pigment content, photosynthetic rate (Pn), apparent quantum efficiency (AQY) and RuBP maximum regeneration rate of green onion under W were significantly better than those under monochromatic light. After 40 days of culture, the fresh weight (FW) per plant of green onion treated with W was 25.21 g, which was 7.83%, 20.28%, 35.68% and 60.78% higher than B, R, G and Y, respectively. The Pn under W was 7.63 μmol·m^-2·s^-1, which was 11.39%, 24.07%, 39.23% and 59.62% higher than B, R, G and Y, respectively. The photo-saturated photosynthetic rate (Pmax) under W reached 13.29 μmol·m^-2·s^-1, which was 5.39%, 9.47%, 15.57% and 21.48% higher than B, R, G and Y, respectively. There was no significant difference in light saturation point (LSP) among other monochromatic light, while the light compensation point (LCP) was higher in Y, followed by G and R, and lower in B and W. There were also significant differences among different monochromatic light, the FW per plant of B was significantly higher than Y, which reached 24.22 g and 16.52 g, respectively, G and R were in the middle. Pn, AQY, Pmax, carboxylation efficiency (CE) and maximum regeneration rate of RuBP of green onion leaves treated with B were also significantly higher than those of other monochromatic light. The quality indexes in green onion, such as soluble sugar, crude cellulose, pyruvate, soluble protein, free amino acid and dry matter in W, were significantly higher than those in monochromatic light. However, the B was higher among the monochromatic light, and the others were red light, green light, and yellow light in order. 【Conclusion】 The W was the most beneficial to the growth of green onions, which showed that the photosynthetic efficiency of leaves was higher and the product quality was better, and the effect of each monochromatic light treatment was higher than B, followed by R, Y and G. It reflected that green onions had strong utilization ability to W and B, which laid a foundation for further study on the formation of yield and quality of green onions by compound light.

Molecular Marker-Assisted Identification of Yellow/White Flesh Trait for 122 Peach Cultivars (Lines)

LU ZhenHua,SHEN ZhiJun,NIU Liang,PAN Lei,CUI GuoChao,ZENG WenFang,WANG ZhiQiang

Scientia Agricultura Sinica. 2020, 53(14):  2929-2940.  doi:10.3864/j.issn.0578-1752.2020.14.016

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【Objective】It shows that peach flesh color (yellow/white) is controlled by the gene CCD 4 (carotenoid cleavage dioxygenase 4). Based on three types of CCD4 allele variations, molecular markers of indel, SSR-CE, and Sanger sequence for SNP were used to analyze the genotypes of 122 peach cultivars (lines), with the aim to determine the correlation between flesh color and genetic variation. This result could provide information for parental matching and the selection of corresponding molecular markers for phenotyping in their offspring. 【Method】Three types of variations were detected via PCR. LTR transposable element insertion was detected by 1% agarose gel electrophoresis, and SSR repeat numbers were detected using CE-SSR in ABI3730XL. Nucleotide substitution was detected using the Sanger sequence and analyzed with the ContigExpress software. In total, the genotypes of 122 cultivars (lines) were analyzed, and the correlation between phenotype and genetic variation was determined.【Result】After genotyping of the 122 cultivars, it was found that 31 accessions were LTR transposable element insertions with 729-bp amplified fragments, accounting for 25.4% of the total accessions, of which eight accessions (25.8%) were homozygous. Sixty-eight cultivars (lines) were SSR repeat number variations, accounting for 55.7% of the total accessions and including 25 (36.8%) homozygous types with 2-bp insertions. Of the 122 cultivars (lines), only one cultivar (Fertilia Morettini) was caused by nucleotide substitution and SSR repeat number variation, accounting for 0.82%, which was not widely used in the breeding program. LTR transposable element insertion and SSR repeat number variation were the key types affecting flesh color. Of the 122 cultivars (lines), seven yellow-flesh cultivars (lines) were caused by SSR repeat number variation and LTR transposable element insertion, accounting for 5.7%. One homozygous or two heterozygous sequence variations were both responsible for yellow flesh. The results showed that genotypes were identical with phenotypes of the 122 accessions, with 100% accuracy. 【Conclusion】The genotypes of 122 peach cultivars (lines) (white and yellow flesh color) were identified using molecular markers, which could be applied for parental selection, offspring identification in breeding programs, and flesh color selection (white or yellow) using molecular marker-assisted selection.

FOOD SCIENCE AND ENGINEERING

Effects of Xanthan Addition on the Gel Properties and Gel Mechanism of Alkaline-Induced Konjac Glucomannan Gels

LI XiaoFei,LI PeiYuan,LI AnQi,YU WenYan,GUO Chuo,YANG Xi,GUO YuRong

Scientia Agricultura Sinica. 2020, 53(14):  2941-2955.  doi:10.3864/j.issn.0578-1752.2020.14.017

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【Objective】The effects of xanthan addition amounts on the gel properties and gelation mechanism of konjac glucomannan (KGM) gels were investigated, which was expected to provide a theoretical basis and technique references for developing KGM-related gel foods.【Method】The mixed KGM and xanthan systems were prepared by adding different concentrations of xanthan into 2% KGM solution, and then the mixed polysaccharide systems were subjected to heating at 90℃ for 2 h in the presence of 2% Na₂CO₃. After cooling to room temperature, the composite KGM-xanthan gels were prepared. By determining the gel strength, the effects of deionized water immersion and 2% citric acid immersion as well as freeze-thaw treatment on the composites were investigated. Meanwhile, scanning electron microscope was used to visualize the microstructures of the composite gels, and then the effects of different treatment methods and xanthan addition amounts on the gel structures were also revealed. Moreover, based on rheology, X-ray scattering and thermogravimetric analyses, the gel formation process of the mixed KGM and xanthan systems during heating at hot alkaline condition was explored, and the gelation mechanism was also revealed.【Result】During heating at 90℃ for 2 h, it was found that with the increasing of xanthan addition, the gel strength of the composite gels was decreased, indicating that the presence of xanthan negatively affected the formation of KGM gel network in this process. However, at room temperature, with increasing xanthan content, the gel strength of the composite gels was increased. The higher the xanthan content, the greater the gel strength, indicating that xanthan might strengthen the composite gels during cooling. After dezionized water and 2% citric acid immersion, gel strength decreased, and the gel samples with citric acid immersion showed a more pronounced decreased trend. Besides, after freeze-thaw treatment, KGM gels exhibited significant syneresis effect, with an approximately 50% of syneresis rate. However, after adding xanthan, the syneresis rate of the composite gels was significantly decreased. The higher the xanthan content, the lower the syneresis rate. Rheological test results suggested that during heating at 90℃ for 2 h, with the increasing of xanthan content, the gelling rate of the mixed KGM-xanthan system was decreased, indicating that the addition of xanthan decreased the formation of KGM gel network. Subsequent decreasing temperature sweep results showed that when the temperature decreased from 90℃ to 60℃, the elastic modulus of the composite gels exhibited a decreasing trend. When the temperature was continuously decreased to room temperature, the elastic modulus showed an increasing trend, and the transition temperature was corresponding to 60℃, suggesting that xanthan began to synergistically bind with KGM gel network at this temperature.【Conclusion】The addition of xanthan significantly improved the gel strength of alkali-induced KGM gels at room temperature, and also enhanced the freeze-thaw stability of the gels. This result could provide valuable reference to development of KGM gel-related food.

ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT

The Analysis of PI3K-AKT Signal Pathway Based on the Proteomic Results of Sheep Embryonic Skeletal Muscle

WANG XinYue,SHI TianPei,ZHAO ZhiDa,HU WenPing,SHANG MingYu,ZHANG Li

Scientia Agricultura Sinica. 2020, 53(14):  2956-5963.  doi:10.3864/j.issn.0578-1752.2020.14.018

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【Objective】Sheep is an important economic livestock and its skeletal muscle growth and development have a deep bond with meat production traits. The sheep embryonic period is an essential stage for skeletal muscle growth, analyzing and mining the proteome data of sheep embryonic skeletal muscle in this period has a great significance to reveal the muscle development process and screen their key regulation proteins.【Method】The longissimus dorsi of Chinese merino sheep at embryonic age of 85 days, 105 days and 135 days were selected for protein qualification by using tandem mass tag (TMT) and 1316 differential abundance proteins were obtained finally. GO, KEGG and R bioinformatic methods were used to cluster, annotate and analyze the differential abundance proteins. And the candidate proteins were testified by using bioinformatic methods.【Result】Based on the previous results, the cluster analysis on differential abundance proteins illustrated that the cluster 5 proteins were significantly expressed on embryonic age of 105 days with high abundance. GO and KEGG analysis on cluster 5 proteins showed these proteins were significantly involved in protein metabolism biology process and notably enriched in PI3K-AKT signal pathway in which RAC-beta serine/threonine-protein kinase isoform X1(ATK2) has a high abundance. Meanwhile, the results of bioinformatics showed that the AKT2 was composed of 481 amino acids and the theoretical molecular weight was 55.58kD. It consists of 66 positively charged amino acid residues and 72 negatively charged amino acid residues, the theoretical isoelectric point was 6.08, the hydrophilicaverage coefficient was -0.454, 12 N-terminal glycosylation sites and 71 phosphorylation sites were found in AKT2. The homology of AKT2 and protein kinase-like (PK-like) was 99% and it belongs to the family of protein kinases catalytic subunit.【Conclusion】The proteome data analysis of sheep embryonic skeletal muscle showed that embryonic age of 105 days is a key point of sheep embryonic skeletal fiber cell from proliferation and differentiation to hypertrophy. The PI3K-AKT signaling pathway which has function of regulating growth and development of embryonic skeletal muscle fibers was significantly enriched, and ATK2 is a key candidate regulation protein in this pathway. To summarize, the study has a theoretical guiding significance to reveal the growth and development and its molecular regulation mechanism of embryonic skeletal muscle.

Identification and Properties of Lactic Acid Bacteria Isolated from Wild Boar Feces

WANG Yi,LI Miao,LI YongFeng,SUN Yuan,QIU HuaJi

Scientia Agricultura Sinica. 2020, 53(14):  2964-2973.  doi:10.3864/j.issn.0578-1752.2020.14.019

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【Objective】In order to screen safe lactic acid bacteria (LAB) strains with probiotic properties, LAB were isolated in this study for further developing probiotics and providing resources for animal related products such as feed additives. 【Method】 A total of 13 samples of wild boar feces were collected from Greater Khingan Mountains in China, and the samples were quickly returned to the laboratory at 4°C for isolation and purification of LAB. The genomic DNA of the isolated Gram-positive bacteria was extracted by bacterial genomic DNA extraction kit. After 16S rDNA identification, the sequence was compared with information in GenBank database using BLAST, and the classification status of each strain was determined. The tolerance capacity of LAB was evaluated under acidic pH condition (pH 3.0) and bile salt (0.3%). The LAB was cultured overnight, observed and determined at different time, and then the autoaggregation ability was evaluated. The strains cultured overnight were mixed with Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium at room temperature for coaggregation test. In vitro, the adhesion ability of LAB to Caco-2 cells and IPEC-J2 cells was measured and evaluated. The anti-pathogenic activities were detected by measuring the bacterial inhibition rings on plates of LAB against E. coli, S. aureus, and S. typhimurium. Evaluation of the safety of LAB strains by in vivo and in vitro tests. LAB isolates were cultured in MRS medium for 18-24 h at 37℃. Streak plate methods were performed on sheep blood agar plates to analyze hemolytic activity. The absence/presence of virulence factor genes for the isolated strains was performed using PCR amplification with primers. After 21 days of continuous LAB supplementation, the parameters of general health status including body weight gain and organ index were calculated to assess the safety of the LAB in vivo. 【Result】Three LAB strains isolated from wild boar feces were Enterococcus mundtii, Enterococcus durans, and Lactobacillus mucosae, with the excellent tolerance to acid and bile salt. The results showed that these three strains showed strong adhesion and aggregation ability, and the antimicrobial effect of L. mucosae on the three pathogenic bacteria was better than others. The result of safety evaluation demonstrated that these strains were free of hemolytic activity, and no virulence genes could be detected. The percentage of body weight gain of the mice treated with L. mucosae was significantly higher (P<0.01) than that of the control mice on day 21. 【Conclusion】Taken together, the results indicated that three probiotic strains, especially L. mucosae, had good probiotic properties and safety, so this study provided a scientific basis for further development of probiotic preparations.

RESEARCH NOTES

Species Identification and Virulence Determination of Beauveria bassiana Strain BEdy1 from Ergania doriae yunnanus

ZHANG Lei,JIA Qi,WU Wei,ZHAO LuPing,XUE Bing,LIU HuanHuan,SHANG Jing,YONG TaiWen,LI Qing,YANG WenYu

Scientia Agricultura Sinica. 2020, 53(14):  2974-2982.  doi:10.3864/j.issn.0578-1752.2020.14.020

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【Objective】Ergania doriae yunnanus has become one of the main pests in soybean fields in southern China. The objective of this study is to investigate the species and biological characteristics of an entomopathogenic fungus that naturally caused the death of E. d. yunnanus, determine the virulence of this fungal strain to E. d. yunnanus adults, and to provides a new way for biological control of E. d. yunnanus.【Method】The fungus from E. d. yunnanus was isolated and purified, and the fungal DNA was extracted for amplification of rDNA-ITS. Subsequently, BLAST alignment and phylogenetic tree construction were used to identify fungal species that caused the natural death of E. d. yunnanus. The growth rate and sporulation of the fungus were measured by cross method and hemocytometer method at different culture temperatures. The virulence of fungus BEdy1 to E. d. yunnanus was assessed on the lethality of the pests by treatment with different concentrations of spore suspension and estimated by comparison with the commercial fungal agent LH. Finally, the soybean leaves sprayed with BEdy1 spore suspension were used to evaluate the lethal effect after E. d. yunnanus feeding.【Result】The fungus caused the death of E. d. yunnanus under natural conditions was identified as Beauveria bassiana, named BEdy1. BEdy1 had a high growth rate at 22-28℃, and the highest growth rate was 4.34 mm·d^-1 at 26℃. There was a high sporulation at 20-26℃, and the highest sporulation was 4.63×10⁶ spores/mm²at 22℃. The mortality at 10th day of E. d. yunnanus adults treated with 1.0×10⁵, 1.0×10⁶, 1.0×10⁷ and 1.0×10⁸ spores/mL of B. bassiana BEdy1 spore suspension was 49.33%, 77.33%, 88.67% and 98.00%, respectively. The mortality of the control treatment was only 10%. The effect was best under the treatment of 1×10⁸ spores/mL, with the LT₅₀ of (6.79±0.13) d and the cadaver rate of 74.67%. The LC₅₀ and LC₉₅ at 10th day were 4.80×10⁴ and 3.57×10⁷ spores/mL, respectively. Treatment with 1×10⁸ spores/mL of a commercial B. bassiana agent LH, the mortality and cadaver rate at 10th day were only 58.00% and 4.67%, which were significantly lower than those of the same concentration of BEdy1 treatment. After 10 days of E. d. yunnanus feeding on BEdy1 treated soybean leaves, the mortality and cadaver rate were 100% and 63.33%, respectively, and the LT₅₀ was (5.27±0.35) d. 【Conclusion】Under appropriate conditions, the B. bassiana strain BEdy1 has fast growth rate, high sporulation, and high lethality rate to E. d. yunnanus adults, which has great potential for development and application in the future.