Scientia Agricultura Sinica

Genetic Diversity Analysis and Comprehensive Evaluation of Sorghum Breeding Materials Based on Phenotypic Traits

ZHANG YiZhong, ZHANG XiaoJuan, LIANG Du, GUO Qi, FAN XinQi, NIE MengEn, WANG HuiYan, ZHAO WenBo, DU WeiJun, LIU QingShan

Scientia Agricultura Sinica. 2023, 56(15): 2837-2853. doi:10.3864/j.issn.0578-1752.2023.15.001

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【Objective】 The present study analyzed the genetic variation of phenotypic traits and genetic diversity of sorghum breeding materials. Additionally, the study explored a comprehensive method for the evaluation of germplasm materials and screening of excellent sorghum germplasm to provide an important basis for sorghum germplasm innovation and variety selection.【Method】 In total, 263 sorghum germplasms from different sources were used as the test materials, and 17 phenotypic traits were identified under different environments for two years. Genetic diversity of the phenotypic traits was calculated based on the Shannon-Wiener information diversity index. The sorghum germplasms were comprehensively evaluated using the correlation analysis, principal component analysis, cluster analysis, and stepwise regression. Excellent sorghum germplasms were screened according to the phenotypic comprehensive evaluation value (F value) and target traits.【Result】 Sorghum breeding materials exhibited high genetic diversity. The diversity index distribution of different traits ranged from 0.497 to 2.075, with the diversity index of spike shape being the smallest and that of spike stalk length being the largest. The coefficient of variation of seven plant height, stem diameter, panicle length, panicle stalk length, grain weight per spike, thousand grain weight, period of duration varied in different years; the smallest variation was observed in the period of duration, followed by the panicle length, whereas the largest variation was observed in grain weight per spike, followed by stem diameter. A comprehensive evaluation of the breeding materials showed that when the cumulative contribution percentage was >80%, the number of the total principal components was 11. F value of the sorghum breeding materials was calculated using the membership function method. The average F value was found to be 0.464, with the restorer line L28 having the highest F value (0.581) and the maintainer line 72B/DORADO having the lowest the F value (0.330). Through stepwise regression, a regression equation was established, with 12 traits (main vein color, ear type, ear shape, awn character, glume coating degree, grain shape, plant type, stem diameter, ear length, grain weight per ear, 1000-grain weight, and growth period) as independent variables. The equation could be used for a comprehensive evaluation of the phenotypic traits of breeding materials of sorghum breeding materials. Based on F value clustering, 263 materials were divided into six groups. Among these, 33 materials in group Ⅳ exhibited excellent agronomic characteristics and high F value, which could be used as parent materials for material innovation and cross breeding.【Conclusion】 Sorghum phenotypic traits exhibit rich genetic variation and high genetic diversity. A total of 33 excellent germplasms were obtained. Using multivariate statistical analysis is a feasible approach to comprehensively evaluate sorghum germplasm.

Status, Existing Problems and Strategy Discussion on Northward Expansion of Winter Rapeseed in China

LIU ZiGang, WEI JiaPing, CUI JunMei, WU ZeFeng, FANG Yan, DONG XiaoYun, ZHENG GuoQiang

Scientia Agricultura Sinica. 2023, 56(15): 2854-2862. doi:10.3864/j.issn.0578-1752.2023.15.002

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Since the 1950s, with the introduction and application of winter rapeseed (Brassica napus) in China, it has led to the rapid transformation, i.e., replacing winter turnip rape by winter oil rape, in the Yangtze River Basin being the main production areas of winter rapeseed in China. In the late 1980s, with the continuous breakthroughs in cold tolerance breeding, the planting area of winter oil rapeseed continued to extend northward. Winter oil rapeseed had replaced winter turnip rapeseed in the main production areas in China, such as the HuangHuai River Basin, Weihe River Basin, and Weibei Dry Plateau. In recent years, strong cold resistant cabbage type winter oil glycerol 4 and other varieties have been developed, which can replace winter turnip rapeseed in arid and cold regions of northern China, achieving doubled yield of winter rapeseed, “double low” quality, and suitable machine harvesting for lodging resistance. The essence of the successful northward migration of winter rapeseed was the northward migration of winter oil rapeseed in China, namely cold resistant varieties of winter oil rapeseed replaced winter turnip rapeseed in the original production area, which have greatly promoted the development of winter rapeseed industry. Nevertheless, the northward migration of winter turnip rapeseed faces completely different difficulties. Since 1955, the northward migration trial of winter turnip rapeseed has been terminated by the introduction of spring oil rapeseed into the planting regions of spring turnip rapeseed at the same time. In the subsequent trial, there is a deviation in the research direction on techlonogies of the northward migration, i.e., solely paying close attention to the cold resistance, while ignoring drought tolerace of varieties which have been migrated into the northward migration area with further reduction in precipitation. There was resulting in technology output not meeting the actual needs of the industry. As a result, the northward migration practice has been carried out for decades and has not yet formed a stable winter rapeseed planting area outside of traditional production areas. In recent years, the original planting area has also continued to shrink, and the northward migration of winter turnip rapeseed has had little effect. In practical condition, the main challenge faced by the northward migration of winter turnip rapeseed is water limitation and the combination of drought and cold stress. The low comparative efficiency is the leading factor in the predicament of industrial development. In recent years, the strong cold-tolerant varieties of winter oil rapeseed developed can stably overwinter in northern cold and arid regions, replacing the varieties of winter turnip rapeseed, significantly improving yield, quality, planting efficiency of winter rapeseed, etc., which is the hope for winter rapeseed in strong winter regions to break through industrial difficulties. We have reviewed the history of winter rape northward-extension in China, achievements and existing problems. The reasons for the dilemma of northward extension were analyzes, and suggestions were made. The revolution of replacing winter turnip rape with winter oilseed rape should be promoted to meet the challenges in winter rape industry development in north China.

Effect of Nitrogen Application Rate and Planting Density on the Lodging Resistance and Grain Yield of Two Winter Wheat Varieties

MU HaiMeng, SUN LiFang, WANG ZhuangZhuang, WANG Yu, SONG YiFan, ZHANG Rong, DUAN JianZhao, XIE YingXin, KANG GuoZhang, WANG YongHua, GUO TianCai

Scientia Agricultura Sinica. 2023, 56(15): 2863-2879. doi:10.3864/j.issn.0578-1752.2023.15.003

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【Objective】 To investigate the interactions between genotype, nitrogen application rate and planting density on the regulation of wheat lodging resistance and grain yield, so as to identify the optimal combination of nitrogen-density that matches the biological characteristics of varieties. The results provide theoretical basis and technical support for stable and abundant winter wheat yield and resistant strain cultivation. 【Method】 A split-split plot field experiment was conducted in Jiaozuo, Henan Province, China, for two consecutive years from 2020 to 2022. Two wheat varieties Xinhuamai 818 and Xinmai 26 with different lodging resistance were selected in the main plots. The nitrogen fertilizer application rates were used as split-plots, and five levels were set: no N application (N0), 180 kg·hm^-2 (N1), 240 kg·hm^-2 (N2), 300 kg·hm^-2 (N3) and 360 kg·hm^-2 (N4), the planting densities were used as split-split plots, and three levels were set: 2.25 million plants/hm² (D1), 3.75 million plants/hm² (D2) and 5.25 million plants/hm² (D3). The study focused on analyzing the effects of the three-factor combination of variety, nitrogen application and planting density on the anatomical structure of wheat culms, field lodging rate and yield. 【Result】 The results showed that nitrogen application rate and planting density significantly regulated the vascular bundle structure of both wheat varieties. The number and area of big vascular bundles and the ratio of number and area of big and small vascular bundles were significantly and positively correlated with culm wall thickness and culm breaking strength, while the area of small vascular bundles was significantly and negatively correlated with culm wall thickness. Compared with Xinmai 26, Xinhuamai 818 had more big vascular bundles and larger area, while the number of small vascular bundles was equal and the area was smaller. This may be the anatomical basis for the superiority of Xinhua 818 over Xinmai 26 in terms of lodging resistance. Under the same planting density, the number and area of big vascular bundles of both wheat varieties showed a trend of increasing and then decreasing with the increase of nitrogen application rate, with the largest number and area of big vascular bundles in N3 treatment. The average increase of number and area of big vascular bundles of Xinhuamai 818 and Xinmai 26 under N3 treatment compared with the minimum treatment were 14.61%, 15.80% and 16.18%, 20.10% respectively. The number and area of small vascular bundles showed similar changes. Under the same level of nitrogen application rate, the number and area of big vascular bundles of both varieties were the largest in the low density D1 treatment. Compared with the minimum value of high density D3, the average increase in the number and area of big vascular bundles of Xinhuamai 818 and Xinmai 26 under D1 treatment were 6.14%, 5.20% and 8.95%, 11.42%, respectively.【Conclusion】 Nitrogen-density control combination D1N2 with 240 kg·hm^-2 and planting density of 2.25 million plants/hm² can optimize the vascular bundle structure, coordinate the development of big and small vascular bundles. Specifically, the number and area of big vascular bundles and the number ratio and area ratio of two vascular bundles were increased in this treatment. The combination can also increase the thickness of the culm wall between the basal nodes and improve the breaking strength of the plant. These changes realize the synchronous improvement of lodging resistance and yield of wheat. We think this treatment can be used as a suitable nitrogen-density combination pattern for high-yielding and efficient cultivation of winter wheat in high-yielding irrigation areas in northern Henan.

Effects of Tassel Sizes on Post-Flowering Dry Matter Accumulation and Yield of Different Maize Varieties Under High Temperature Stress During Pollination

MU XinYuan, LÜ ShanShan, LU LiangTao, LIU TianXue, LI ShuYan, XUE ChangYing, WANG HongWei, ZHAO Xia, XIA LaiKun, TANG BaoJun

Scientia Agricultura Sinica. 2023, 56(15): 2880-2894. doi:10.3864/j.issn.0578-1752.2023.15.004

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【Objective】 High temperature stress is one of the most critical meteorological disaster factors that restrict the high and stable yield of maize. This study explored the effect of tassel sizes on yield of different maize varieties under high temperature stress during pollination, so as to provide the theoretical basis and reference for stress-resistant cultivation and high temperature resistance breeding of maize. 【Method】 This study was conducted by plot experiment in a greenhouse with two maize varieties as the experimental materials, Xundan 20 (XD20) and Nonghua 101 (NH101), from 2020 to 2021. The effect of tassel sizes on dry matter accumulation, distribution and yield of maize under high temperature stress during pollination was investigated by setting the tassel branch removal treatment at tasseling stage. 【Result】 The results of two years showed that high temperature stress during pollination had little effect on tassel length, branch number, spikelet number and flowering dynamics of male and female. However, high temperature stress resulted in the decrease of dry matter accumulation capacity and distribution ratio to ear, which affected the growth and development of ear, resulting in the significant decrease of cob length and diameter, the significant decrease of grain number per ear, the decrease of proportion of matter accumulation to grain after anthesis, and the significant decrease of yield. Under high temperature stress, the decrease of ear length of NH101 was less than that of XD20, but the decrease of grain number per row, grain number per ear and the proportion of matter accumulation to grain after anthesis was higher than that of XD20, resulting in the decrease of yield of NH101 more than that of XD20, and the yield of XD20 and NH101 decreased by 12.32% and 25.00% respectively. XD20 is more resistant to heat than NH101. The tassel branch removal treatment significantly reduced the number of tassel branch and spikelet of XD20 and NH101 by 58.57%, 42.91% and 57.30%, 41.34%, respectively, but had little effect on the flowering dynamics of male and female. Under the two temperature conditions, the tassel branch removal treatment promoted the growth of ear, increased the grain number per ear, increased the proportion of matter accumulation to grain after anthesis, and thus increased the yield. Among them, the yield increase of XD20 under high temperature conditions was the largest. Compared with normal tassel branch treatment, tassel branch removal treatment increased the yield of XD20 and NH101 by 2.76% and 4.37% under normal temperature conditions, while increased by 12.47% and 5.75% under high temperature conditions, respectively. 【Conclusion】 High temperature stress during pollination has little effect on the growth and development of tassel, but it caused irreversible damage to the growth and development of ear, reduced the number of grains per ear, limited the distribution of photosynthate to grains after anthesis, and significantly reduced the yield. Under high temperature conditions, properly reducing the number of branches in tassel can promote the growth and development of ear, increase the number of grains per ear, promote the accumulation of matter to grains after anthesis, and increase the yield. In addition, the yield increase of the large tassel-type variety XD20 was higher than that of the small tassel-type variety NH101.

Yield-Increasing Effects Under Plastic Film Mulching of Potato in China Based on Meta-Analysis

XU JuZhen, ZHANG MengLu, HE WenQing, SUI Peng, CHEN YuanQuan, CUI JiXiao

Scientia Agricultura Sinica. 2023, 56(15): 2895-2906. doi:10.3864/j.issn.0578-1752.2023.15.005

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【Objective】 Plastic mulching film (PMF) has various effects, including improving soil temperature, moisture retention, and weed inhibition. It is an efficient and simple technical measure to alleviate the limitations of potato production. Based on publication data, this study quantified the effect of PMF on potato yield and water use efficiency (WUE), and further analyzed the influencing factors to provide a reference for sustainable potato production. 【Method】 Based on a meta-analysis of 291 field experiments on PMF of potato production in China from 1981 to 2021, published in Web of Science and CNKI databases, including four regions, Northern single farming area (NSFA), Southwest mixed farming area (SWMFA), South winter farming area (SWFA), and Central plains second farming area (CPSFA). Meta-analysis was used to quantify the effects of PMF on potato yield and WUE. The study examined the effects of PFM on potato yield and WUE from various perspective, including the regions, natural conditions (average annual precipitation, soil bulk density, and soil organic matter content), and different management conditions (potassium fertilizer application rate, planting density, mulching color, and mulching method). 【Result】 Compared with no mulching, PMF increased potato yield and WUE by 24.9% and 28.3% respectively. The effects of PMF on yield and WUE varied among different regions: NSFA (27.2%), SWFA (23.6%), SWMFA (18.1%) and CPSFA (10.1%). However, WUE was only significantly improved in the NSFA (29.1%). The response of yield and WUE to PMF varied among different regions and was influenced by natural conditions and management conditions. The response of yield to PMF varied among different regions under different natural condition. Planting density and mulching method had consistent effect in all regions, with the best yield increase achieved with low planting density and ridge. In the NSFA, PMF significantly improved WUE under low average annual precipitation, relatively low soil organic matter content, low soil bulk density and fertilizer application rate, medium planting density, black mulching color, and ridge. 【Conclusion】 PMF had been widely used in China and the results showed it increased potato yield. The yield increase varied among different regions and was in the following order: NSFA, SWFA, SWMFA, and CPSFA. PMF only improved WUE in the NSFA. PMF performed better on yield increase under natural condition of low average annual precipitation, poor soil fertility, and loose soil, as well as under management conditions of relatively low fertilization level, relatively low planting density, black mulching, and ridge. In the NSFA, the conditions required for achieving optimal yield increase and WUE increase using PFM were comparable.

Effect of Humidity on Sporulation and Release of Corynespora cassiicola and Control Technology

CHAI ALi, YANG HongMin, WANG ShaoHua, ZHAO Kun, GAO Wei, SHI YanXia, XIE XueWen, LI Lei, FAN TengFei, LI BaoJu

Scientia Agricultura Sinica. 2023, 56(15): 2907-2918. doi:10.3864/j.issn.0578-1752.2023.15.006

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【Objective】 Cucumber target leaf spot, caused by Corynespora cassiicola, has brought great economic losses to the cucumber industry. Sporulation and release of C. cassiicola spores play a significant role in the epidemiology of the disease. In this study, the regularity and the effect of humidity on sporulation and release of C. cassiicola, and the optimal application method and time for control of cucumber target leaf spot were evaluated.【Method】 The sporulation regularity of C. cassiicola was evaluated by quantifying the spore concentration on lesions of diseased cucumber leaves at 0: 00, 3: 00, 6: 00, 9: 00, 12: 00, 15: 00, 18: 00, and 21: 00, respectively. The release regularity of C. cassiicola was evaluated in different seasons of spring, summer, autumn and winter, air samples were collected from naturally infested cucumber greenhouse at 0: 00, 3: 00, 6: 00, 9: 00, 12: 00, 15: 00, 18: 00, and 21: 00, respectively, and the concentrations of C. cassiicola in the air were evaluated. The effect of relative humidity on sporulation and release of C. cassiicola was also assessed in artificial climate exposure chambers and plastic greenhouses at four different humidity conditions of continuous high humidity (RH>90%, 24 h), continuous low humidity (RH<60%, 24 h), high humidity for 12 h followed by low humidity for 12 h, and low humidity for 12 h followed by high humidity for 12 h. The control efficiency of 60% carbendazim·diethofencarb wettable powder (WP) and 500 million spores/g Pseudomonas fluorescens WP on cucumber target leaf spot disease was compared, by using powder spraying and water spraying at different application times in the field.【Result】 The study on the daily variation regularity of sporulation and release of C. cassiicola showed significant differences in the quantity of spores on diseased leaves and in the greenhouse air at different times of the day. There was a complementary relationship between the number of spores on diseased leaves and in the greenhouse air at the same time. After 18: 00, as the duration of high humidity (RH>90%) prolonged, the number of spores on diseased leaves increased, reaching a peak of 1 344 spores/cm² at 6: 00 the next day. Then, the humidity decreased gradually to RH<60% after opening the air vent of the greenhouse, and spores were released into the greenhouse space. At 12: 00, the spore concentrations in the greenhouse air reached a peak of 12 445-110 697 spores/m³. In different seasons of spring, summer, autumn, and winter, the daily variation regularity of sporulation and release is consistent, showing that C. cassiicola produced a large amount of spores under high humidity (RH>90%) at night, and released to greenhouses under low humidity (RH<60%) during the day. In artificial climate exposure chambers and plastic greenhouses, the highest quantity of C. cassiicola spores was detected under alternating wet and dry conditions, which was significantly higher than that under continuous high humidity or continuous low humidity condition. By powder spraying at 19: 00, 60% carbendazim·diethofencarb WP and 500 million spores/g P. fluorescens WP gave the best control efficiency of 80.60% and 75.08%, respectively, and the spore inhibition efficiency was higher than 84%.【Conclusion】 Humidity is a key environmental factor affecting the spore reproduction and release of C. cassiicola. The alternating day-dry and night-wet environment in the greenhouse promotes the reproduction and diffusion of C. cassiicola, and accelerates the spread of cucumber target leaf spot. Powder spraying method is better than water spraying method for disease control, and the best application time is the evening before spore reproduction. The results of this study will contribute to the development of new strategies for the effective alleviation and control of cucumber target leaf spot.

Function of Transcription Factor NbERF RAP2-1 in Cucumber Green Mottle Mosaic Virus Infection

YU LianWei, JIANG XingLin, YANG LingLing, WANG He, ZHANG YuYang, XIE LiNa, XIA ZiHao, LI HongLian, YANG Xue, SHI Yan

Scientia Agricultura Sinica. 2023, 56(15): 2919-2928. doi:10.3864/j.issn.0578-1752.2023.15.007

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【Background】 Cucumber green mottle mosaic virus (CGMMV) is an important quarantine plant virus in China, which has caused serious economic losses to vegetable and melon industry. The ERF transcription factor is involved in biotic and abiotic stress and the defense response against a variety of plant pathogens. In previous study, it was demonstrated that the host transcription factor NbERF RAP2-1 was significantly down-regulated after CGMMV infection.【Objective】 The objective of this study is to clarify the function of the ERF transcription factor family members in CGMMV infection, and to provide a theoretical basis for disease control caused by CGMMV.【Method】 MEGA7.0 was used to construct a phylogenetic tree to analyze the amino acid sequence of NbERF RAP2-1. The expression vector NbERF RAP2-1-GFP was constructed to observe the subcellular localization of NbERF RAP2-1. The transcript level of NbERF RAP2-1 at different stages of CGMMV infection was analyzed by qRT-PCR. VIGS and transient overexpression of NbERF RAP2-1 were conducted to analyze the function of NbERF RAP2-1 during CGMMV infection.【Result】 Phylogenetic tree analysis showed that NbERF RAP2-1 was highly homologous to ERF transcription factors in tobacco, and was far from ERF transcription factors in Arabidopsis thaliana. The results of subcellular localization showed that NbERF RAP2-1 was localized in the nucleus and acted as a transcription factor. The effect of CGMMV infection on the transcript level of the NbERF RAP2-1 showed that the expression level of NbERF RAP2-1 did not significantly change at the 6, 9, and 12 d after inoculation, but at the 15 and 18 d after inoculation, the expression level of NbERF RAP2-1 was significantly down-regulated. Tobacco rattle virus (TRV) mediated VIGS was used to silence the NbERF RAP2-1, and CGMMV was inoculated in TRV: NbERF RAP2-1 and TRV: 00 plants. At 8 d after inoculation, the leaves of TRV: 00 plants showed mottling and curling, while the TRV: NbERF RAP2-1 plants showed no symptoms. At the same time, the detection results of CGMMV RNA and protein levels showed that TRV: NbERF RAP2-1 could effectively inhibit the accumulation of CGMMV. Similarly, transient overexpression of NbERF RAP2-1 inhibited the accumulation of CGMMV at 24, 48, and 72 h, respectively.【Conclusion】 NbERF RAP2-1 effectively inhibit the initial CGMMV replication, i.e. the viral RNA replication stage. With the invasion of CGMMV, i.e. the intercellular and systemic movements of CGMMV, CGMMV recognizes and inhibits the transcript level of NbERF RAP2-1. When NbERF RAP2-1 is knocked down, that may inhibit the transcription of downstream proteins, thereby inhibiting the accumulation of CGMMV at a later stage. NbERF RAP2-1 plays an important role during the CGMMV infection.

Effects of Diversified Cropping on the Soil Aggregate Composition and Organic Carbon and Total Nitrogen Content

SUN Tao, FENG XiaoMin, GAO XinHao, DENG AiXing, ZHENG ChengYan, SONG ZhenWei, ZHANG WeiJian

Scientia Agricultura Sinica. 2023, 56(15): 2929-2940. doi:10.3864/j.issn.0578-1752.2023.15.008

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【Objective】 In this study, the composition as well as carbon and nitrogen distribution characteristics of soil aggregates under diversified cropping system of poaceae and legumes in the black soil region of Northeast China were elucidated, which could provide the theoretical basis and technical guidance for promoting the optimization of cropping system of combined use and cultivation in black soil. 【Method】 Field experiment was conducted from 2016 to 2020 in Gongzhuling Experimental Station, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Four diversified cropping systems were considered, including maize-soybean intercropping (M/S), maize-soybean rotation (M-S), maize-peanut intercropping (M/P), and maize-peanut rotation (M-P), while the maize continuous cropping (CM) system was used as control. Soil samples of 0-20 cm and 20-40 cm layers were collected after harvesting in October 2020. Then, soil aggregates and their soil organic carbon (SOC) and total nitrogen (TN) content under diversified cropping system were analyzed. 【Result】 Diversified cropping was beneficial to increase the content of water-stable macro-aggregates in 0-20 cm and 20-40 cm soil (>0.25 mm), and to reduce the content of silt and clay (<0.053 mm), the mean weight diameter (MWD) and geometric mean diameter (GMD) of soil aggregates under four diversified cropping systems were significantly higher than those of maize continuous cropping system (P<0.05). Soil aggregates ratios of >0.25 mm under M/S and M-S treatments were 17.5% and 13.4% higher than that under CM treatment in 0-20 cm soil layer, respectively. Soil aggregates ratios of >0.25 mm under M/S, M-S and M-P treatments was 10.4%, 8.3% and 10.5% higher than that under CM treatment in the 20-40 cm soil layer, respectively. Diversified cropping increased the SOC and TN content of soil aggregates. In 0-20 cm soil layer, the SOC of >2 mm soil aggregate under M/S, M-S, M/P and M-P treatments was 20.7%, 24.3%, 18.8% and 17.8% higher than that under CM treatment, respectively; the TN of >2 mm soil aggregate under M-S, M/P and M-P treatments was 13.0%, 16.8% and 14.8% higher than that under CM treatment, respectively. When compared with CM treatment, the contribution rates of >2 mm soil aggregate to SOC and TN under M-S and M/P treatments were higher in 0-20 cm soil layer, while the contribution rate of <0.053 mm soil aggregate to SOC and TN under M/S and M-S treatments were lower in 0-20 cm soil layer. 【Conclusion】 Diversified cropping increased the content of soil macroaggregates, decreased the content of clay particles, improved the soil aggregate stability, SOC and TN of soil aggregates, which was conducive to promoting the SOC and TN sequestration in black soil region of Northeast China.

Effects of Phosphorus Reduction on Soil Phosphorus Pool Composition and Phosphorus Solubilizing Microorganisms

SHEN KaiQin, LIU Qian, YANG GuoTao, CHEN Hong, LIANG Cheng, LAI Peng, LI Chong, WANG XueChun, HU YunGao

Scientia Agricultura Sinica. 2023, 56(15): 2941-2953. doi:10.3864/j.issn.0578-1752.2023.15.009

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【Objective】 The effects of continuous reduction of phosphorus fertilizer application on soil phosphorus components and phosphorus-solubilizing microorganisms were studied, and the transformation law of soil phosphorus was explored to provide a theoretical basis for rational application of phosphorus fertilizer and efficient utilization of phosphorus in soil. 【Method】 In 2014, the phosphorus reduction positioning test was carried out in Longmen Town, Mianyang, Sichuan province. The test set 4 treatments, namely P0 (no phosphorus fertilizer), P1 (1/2 reduction, 45 kg·hm^-2), P2 (1/3 reduction, 60 kg·hm^-2), and P3 (normal fertilization, 90 kg·hm^-2), and 0-20 cm soil samples were collected to measure and analyze soil total phosphorus (TP), available phosphorus (AP), and phosphorus activation coefficient (PAC), phosphorus fractions and the change characteristics of phosphorus-dissolving microorganisms. 【Result】 Reducing the application of phosphorus fertilizer significantly reduced the TP content of the soil; compared with 2014, after 3 years of continuous treatment, the TP content under P0 and P1 treatments decreased by 7.2% and 0.9%, respectively, however, which under P2 and P3 treatments increased by 2.6% and 7.3%, respectively; after 6 years of continuous treatment, the TP difference between treatments was further expanded: compared with 6 years ago, the TP under P0 and P1 treatments decreased by 15.2% and 5.7%, respectively, which under the P2 and P3 treatments increased by 7.8% and 21.6%, respectively. The variation trend of AP content was similar to that of TP. After continuous treatment for 3 years, the content of AP under P0 treatment decreased by 18.1%, while that under P1, P2, and P3 treatments increased by 21.2%, 72.2%, and 132.1%, respectively; compared with 6 years ago, the AP variation of each treatment expanded to -24.6%-201.6% after continuous treatment for 6 years. The PAC was determined by the content of AP and TP, and its variation trend was generally consistent with that of AP. Reducing the application of phosphorus fertilizer mainly caused a significant decrease in the content of H₂O-P, NaHCO₃-Pi and NaOH-Pi, but had no significant effect on Residual-P. Among them, NaOH-Pi was the main phosphorus form that caused the change of soil TP content, and NaHCO₃-Pi was the most critical phosphorus fraction in the process of soil phosphorus activation. There were differences in the abundance of phosphorus-solubilizing microorganisms in different phosphorus fertilizer treatments. Redundancy analysis results showed that Nocardioides, Mycobacterium, Bacillus, Hyphomicrobium and Rhizobium were positively correlated with each form of phosphorus, among which, the genus Hyphthora was highly correlated with NaHCO₃-Pi and NaOH-Pi. 【Conclusion】 The reduction of phosphorus fertilizer significantly changed the composition of soil phosphorus pool, and the changes of soil AP and TP were mainly affected by the changes of NaHCO₃-Pi and NaOH-Pi. There were significant differences in the abundance of soil phosphorus-dissolving microorganisms after 6 years of continuous reduction of phosphorus fertilizer, and the genus Hyphomicrobium was the key microorganism involved in the transformation of phosphorus in the experimental area. Reducing the application of 1/3 phosphorus fertilizer for 6 consecutive years could effectively reduce the accumulation of slow-release phosphorus in the soil in this experimental area while maintaining a high level of available phosphorus.

Nitrogen and Phosphorus Surplus and Soil Nitrate Nitrogen Accumulation in Typical Rice-Vegetable Rotation and Banana Garden in Hainan

ZHAO YongJian, ZHANG BoFei, ZHANG Chong, JU XiaoTang

Scientia Agricultura Sinica. 2023, 56(15): 2954-2965. doi:10.3864/j.issn.0578-1752.2023.15.010

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【Objective】 Hainan is the province with a large area of rice-vegetable rotation and banana cultivation in China, but the nitrogen (N) and phosphorus (P) fertilizers input by farmers far exceed the nutrient requirements of crops, which may have a negative impact on Hainan's ecological environment. The aim of this study was to investigate the N and P surpluses, and soil nitrate accumulation in typical cropping systems in Hainan, thus to provide scientific basis for evaluating nutrient losses and their impacts and achieving sustainable nutrient management. 【Method】 In 2021-2022, a typical area for rice-vegetable rotation and banana cultivation in Chengmai, Hainan, was selected as the research area and 20 rice-vegetable rotation plots and 15 banana orchards were identified. The information of chemical and organic fertilizer application, straw returning method and amount of above fields were obtained by real-time record of farmers' agricultural activities, crop biomass and the nutrient content were determined at crop harvest, and other nutrient input include nutrient deposition and biological N fixation were obtained by literature survey. Five banana orchards were selected and soil was collected by soil auger method and nitrate N accumulation was measured in the 0-400 cm soil profile.【Result】 The N and P fertilizer inputs to the rice-vegetable rotation were 1 308 kg N·hm^-2 (975 kg N·hm^-2 of chemical and 333 kg N·hm^-2 of organic fertilizer) and 515 kg P·hm^-2 (385 kg P·hm^-2 of chemical and 130 kg P·hm^-2 of organic fertilizer); the aboveground N and P uptake of the crop were 248 kg N·hm^-2 and 48 kg P·hm^-2; the surplus of N and P in rice and vegetable rotation was 1 196 kg N·hm^-2 and 484 kg P·hm^-2. The N and P fertilizer inputs to banana orchards were 1 340 kg N·hm^-2 (1 293 kg N·hm^-2 of chemical and 47 kg N·hm^-2 of organic fertilizer) and 447 kg P·hm^-2 (442 kg P·hm^-2 of chemical and 5 kg P·hm^-2 of organic fertilizer); the aboveground N and P uptake were 242 kg N·hm^-2 and 23 kg P·hm^-2; the banana N and P surpluses were 1 271 kg N·hm^-2 and 435 kg P·hm^-2. The nitrate-N accumulation in the 0-400 cm soil profile of banana orchards was 1 131 kg N·hm^-2. 【Conclusion】 Excessive application of N and P fertilizers has led to the large nutrient surplus in typical soil-crop systems in Hainan, and large amount of nitrate-N has accumulated in banana orchard in the deep soil layer. Hainan produces typical high-value fruit and vegetables at the cost of large nutrient losses and negative environmental impacts, optimized nutrient management should be implemented to ensure its environmental safety.

Development of Ogura CMS Restorers of Broccoli via Genetic Transformation of Rfo

XING MiaoMiao, XU YuanYuan, LU YuYu, YAN JiYong, ZENG AiSong

Scientia Agricultura Sinica. 2023, 56(15): 2966-2976. doi:10.3864/j.issn.0578-1752.2023.15.011

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【Objective】 The broccoli restorers were developed by transforming the matching fertility restorer gene of Rfo from radish into the leading cultivar Ogura CMS line of Nai Han You Xiu (named as SFB45), so as to efficaciously use Ogura cytoplasmic male sterility (CMS) hybrids in breeding and to provide resources for genetic improvement of germplasm.【Method】 The sequence of CDS with its preceding promoter of Rfo were synthesized and the pRfo::Rfo plant expression vector was constructed to infect the cotyledon with petiole, and hypocotyl explants of the SFB45 through Agrobacterium-mediated transformation method. After regeneration and screening of herbicide resistant seedlings, transgenic plants were identified by detection of the Bar resistant marker. The fertility of transgenic plants were observed at flowering stage. The pollen viability of SFB45 and the transgenic plants were analyzed using the alexander stain. Total RNA of <3 mm and >3 mm flower buds from SFB45 and the transgenic plants were isolated and transcribed into cDNA. The Rfo specific primers were designed, and RT-PCR was performed to analyze the expression levels of Rfo and also the genes related to tapetum and pollen wall development in flower buds of SFB45 and its restorer lines.【Result】 A total of 10 transgenic plants were obtained by genetic transformation. Among which, the fertilities of 8 were restored to varying degrees with the average pollen viability ranging from 84.2% to 90.4%. RT-PCR analysis showed that Rfo was expressed in flower buds of fertility restored plants. The key regulators of tapetum development (DYT1 and TDF1) and the essential gene (ACOS5) for the synthesis of sporopollenin, a major component of the pollen wall were up-regulated in <3 mm flower buds of Ogura CMS restore lines. The tapetum degradation related gene AMS and tetrad callose wall and pollen outer wall development related genes CalS5 and CYP703 were up-regulated in >3 mm flower buds of the restore lines. The analysis of the self- and hybrid-crossing progenies of the positive transgenic lines R-1, R-3 and R-6 showed that the introduced Rfo could be stably inherited, which was consistent with the Mendelian inheritance.【Conclusion】 The fertility restorer lines of Ogura CMS SFB45 were obtained by genetic transformation of Rfo, and the integration of Rfo into the genome of SFB45 recovered the expression of genes associated with tapetum and pollen wall development.

Analysis of the Interaction Between VvGAI1 and VvJAZ9 Proteins in Grape and Its Expression Pattern Under Low Temperature

LIU DeShuai, FENG Mei, SUN YuTong, WANG Ye, CHI JingNan, YAO WenKong

Scientia Agricultura Sinica. 2023, 56(15): 2977-2994. doi:10.3864/j.issn.0578-1752.2023.15.012

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【Objective】 DELLA protein belongs to plant-specific GRAS protein family, which is a significant regulatory factor in GA signal transduction pathway and plays important roles in plant growth, development and resistance to different stresses. In this study, the European grapevine VvGAI1 gene was cloned and the analysis of subcellular localization, protein interaction and expression were performed, so as to lay the foundation for the further study of the function of DELLA protein in response to cold stress in grapevine.【Method】 The VvGAI1 gene sequence was obtained by homologous cloning from the leaves of Vitis vinifera cv. Chardonnay. The VvGAI1 sequence was analyzed by bioinformatics method, and the multiple sequence alignment and phylogenetic trees were performed by DNAMAN and MEGA7.0, respectively. The location of VvGAI1 protein in cells was determined by subcellular localization, and the transcriptional activation activity of the VvGAI1 protein was confirmed by a yeast assay. The interaction between VvGAI1 protein and VvJAZ9 protein was verified by yeast two-hybrid and BiFC assays. The VvGAI1 protein polyclonal antibodies from rabbit was prepared by VvGAI1 protein purified from prokaryotic expression. The VvGAI1 protein expression at low temperature was detected by Western blot method. The effect of exogenous MeJA and GA₃ on the cold resistance in grape was analyzed by relative electrical conductivity.【Result】 The VvGAI1 gene was cloned from Chardonnay leaves, with carrying an ORF of 1 773 bp, encoding 590 amino acids, locating on chromosome 1, and containing only one exon. The VvGAI1 protein had a molecular weight of 64.87 kDa and pI of 5.31, which was an acidic unstable hydrophilic protein. The VvGAI1 belonged to GRAS family and had the conserved DELLA and GRAS domains. Protein clustering analysis showed that VvGAI1 was closely related to Arabidopsis GAI and tobacco GAI1. The results of subcellular location and transcriptional activation showed that VvGAI1 was a transcription factor localized in the nucleus and had transcriptional self-activation activity. The interaction between VvGAI1 and VvJAZ9 was confirmed by yeast two-hybrid and BiFC assays. The VvGAI1 gene sequence was cloned into the prokaryotic expression vector to form a pET28b-VvGAI1 recombinant vector, and the Escherichia coli BL21 carrying pET28b-VvGAI1 recombinant vector was incubated in culture medium with 1.0 mmol∙L^-1 isopropyl-β-d-thiogalactoside (IPTG) at 16 ℃ to obtain VvGAI1-His fusion protein. The anti-VvGAI1 polyclonal antibody (rabbit-derived) was prepared by antigen immunization and serum purification and used to specifically detect VvGAI1 protein in Chardonnay grapes. Western blot results showed that the VvGAI1 protein in grape protoplasts under low temperature treatment showed an increasing first and then decreasing trend, which indicated the expression of VvGAI1 protein was induced at a low temperature. For 50 μmol·L^-1 MeJA and 50 μmol·L^-1 GA₃ treatments, compared with the control group exogenous MeJA treatment could improve the cold resistance of grape, whereas GA₃ treatment made grapes more sensitive to cold.a【Conclusion】 Grape VvGAI1 protein was a transcription factor and interacts with VvJAZ9. The VvGAI1 responded to low temperature stress, and exogenous MeJA was able to positively regulate the cold stress, while GA₃ negatively regulated the cold response.

Background Selection and Comparison of Marker Superiority and Inferiority of Aphid-Resistant Seedlings in an Interspecific Cross Peach Population

LIU SuNing, BIE HangLing, WANG JunXiu, CHEN XueJia, WANG XinWei, WANG LiRong, CAO Ke

Scientia Agricultura Sinica. 2023, 56(15): 2995-3005. doi:10.3864/j.issn.0578-1752.2023.15.013

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【Objective】 To establish a background selection system in peach, the seedlings contained aphid-resistance locus and high female parent recovery rate were screened from an F₂ population crossed by Xiang Pi You Tao peach (big fruit and susceptible to aphid) and Zhou Xing Shan Tao peach (small fruit and resistant to aphid). 【Method】 Firstly, three methods were used to select background markers, including the high polymorphic single nucleotide polymorphism (SNP) obtained from the previous study (Pre-work SNP), SNP randomly selected in the whole genome (Random SNP), and functional SNP affecting the start and stop codon (Functional SNP). The number of final SNP selected by the above methods were 775. Then, using these SNPs, the parents recovery rate for all 121 individuals of the F₂ population were calculated, respectively. The repeatability of the selection methods was evaluated by comparing whether the top 10 seedlings with different selection markers were coincident or not. After completing the evaluation of aphid resistance, single fruit weight, and soluble solids content of F₂ population, 10 seedlings with extreme phenotypes for the single fruit weight and soluble solids content were selected, respectively. And the superiority and inferiority of different selection methods were estimated by comparing the significance of the differences in Xiang Pi You Tao recovery rates between the two types of phenotypes. Finally, the SNPs in the aphid-resistant location area were used as the foreground markers to screen the elite seedlings with high maternal genetic background and aphid resistance. 【Result】 The background recovery rates of the F₂ seedlings which calculated by the three methods were 36.34%-71.99%, 31.75%-74.92%, and 4.51%-66.53%, respectively. Among the top 10 seedlings with high Xiang Pi You Tao recovery rates screened by the three background markers, Pre-work SNP and Random SNP had two duplicate single plants, and so do Pre-work SNP and Functional SNP, and there were 6 repetitive single plants in Random SNP and Functional SNP. This result indicated that the repeatability between the Random SNP and Functional SNP was the highest among all comparisons. When single fruit weight was selected as the breeding target, among the extreme phenotypic monocots, the three background markers, such as Pre-work SNP, Random SNP, and Functional SNP, had a significant Xiang Pi You Tao background recovery rate of 0.069, 0.26, and 0.092, respectively, which meant high relativity was found between the background recovery rate calculated by Pre-work SNP and their fruit weight, followed by Functional SNP, and Random SNP difference was not significant. When soluble solids content was selected as the target, the Xiang Pi You Tao background recovery rates among extreme phenotypic monocots were significant at 0.77, 0.65 and 0.31, respectively, and the differences among the three background markers were not significant. Finally, two individuals with high recovery rate of Xiang Pi You Tao peach were screened, including N20 and N36. Among them, N20 comprised the aphid-resistant markers, and this individual showed aphid resistance with an average fruit weight of 34.42 g and soluble solids content of 16.1%, which was considered to be the superior single strain of this population. 【Conclusion】 In this study population, Pre-work SNP showed a stronger correlation between single fruit weight and Xiang Pi You Tao background recovery rates than Functional SNP and Random SNP, confirming the superiority of this background marker selection method, and the superior performance of N20 plants, which selected with this background marker in the target traits also supported this result. This study provided an idea of background selection and a method to judge the superiority and inferiority of different background markers in the study population, which could effectively improve the efficiency of resistance breeding in fruit crops.

Comprehensive Evaluation of Fruit Texture and Taste Quality of Pineapple Based on Multiple Methods

FU Shan, LIANG Ye, XU JiuLiang, RUAN YunZe, LUO Jian, LI TingYu

Scientia Agricultura Sinica. 2023, 56(15): 3006-3019. doi:10.3864/j.issn.0578-1752.2023.15.014

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【Objective】 This study adopted different methodologies to identify the key texture characteristics and physicochemical indexes affecting the total sensory quality of pineapple (Ananas comosus (Linn.) Merr.), and to establish a new comprehensive evaluation method for the precise testing of fruit texture and taste quality.【Method】 In this study, seven different varieties of pineapple were selected for the measurement of sensory attributes, texture characteristics, and physicochemical compositions. Based on the variance and correlation analysis, the key texture properties and physicochemical indexes that affect sensory quality were identified. Further, the principal component regression analysis was performed, with key texture characteristics and physicochemical indexes as independent variables, and the total score of sensory evaluation as dependent variables, to obtain a statistically significant prediction model for the comprehensive evaluation of pineapple quality.【Result】 There were significant differences in some texture properties and physicochemical indexes among different varieties of pineapple, such as hardness, chewiness, maximum shear force, sugar-acid ratio, soluble protein, vitamin C and soluble pectin; the coefficient of variation among varieties was greater than 25%, while the difference in elasticity and cohesiveness was not significant among varieties. The overall satisfaction score of different pineapple varieties from the highest to lowest was Tainong 17>Tainong 16>Tainong 4>MD-2>Tainong 11>Smooth Cayenne>Comte de Paris. Tainong 17 showed the best quality of fruit texture and taste, its total soluble solid content was 16.23%, sugar to acid ratio was 31.82, soluble pectin content was 23.72 mg∙g^-1, hardness was 1 826.55 N, Chewiness was 789.77 mJ, and the maximum shear force was 3 491.37 N. Correlation analysis showed that there were six key indexes affecting the overall satisfaction of sensory significantly, including hardness, chewiness, maximum shear force and physicochemical index of soluble solids, sugar-acid ratio and soluble pectin. The determination coefficient R² of the sensory quality prediction model based on the principal component regression analysis was 0.916, and the standard deviation was 0.11. 【Conclusion】 The texture and taste quality of pineapple vary greatly among different varieties, and a single evaluation method could not accurately evaluate its comprehensive quality. A prediction model was established for pineapple sensory evaluation based on the key texture properties and physicochemical indexes, which could accurately predict the texture and taste quality of pineapple, and made up for the lack of objectivity in artificial sensory analysis.

Screening of Anti-Apoptotic Protein GRP94 Interaction Proteins in Porcine Hepatic Stellate Cells by Immunoprecipitation Combined with LC-MS/MS

WANG XiaoHong, XING MingJie, GU XianHong, HAO Yue

Scientia Agricultura Sinica. 2023, 56(15): 3020-3031. doi:10.3864/j.issn.0578-1752.2023.15.015

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【Background】 Extensive stress reactions often occur in pigs due to poor breeding environment, thick subcutaneous fat and lack of sweat glands in pigs. Endoplasmic reticulum (ER) stress (ERS) may be the earliest stress response, which is closely related to apoptosis and metabolism. Liver is the main metabolic organ of the body. In the process of pig breeding, the artificial operations, such as weaning, feed mildew, and inhalation of harmful gases and temperature changes, will cause ERS in pig liver, which will not only cause liver damage, but also cause liver fat metabolism disorder and extensive inflammatory reaction, affecting animal production performance and reproductive performance. The regulation of ERS in production is helpful to reduce the recessive loss in the pig breeding process. 【Objective】 Immunoprecipitation combined with mass spectrometry was used to screen the cellular proteins interacting with glucose-regulated protein 94 (GRP94) in porcine hepatic stellate cells (HSC) under ERS condition, which could lay a foundation for further study on the protective mechanism of GRP94 on biological function of HSC. 【Method】 Protein complexes bound to GRP94 bait protein were eluted, collected, and verified by SDS-PAGE gel electrophoresis. The successfully verified sample eluent was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the interaction proteins of GRP94 under normal condition and ERS condition were identified. The detected proteins were analyzed by GO, KEGG and interaction network. Vimentin, one of the interacting proteins, was verified by co-immunoprecipitation. 【Result】 In porcine hepatic stellate cells, 146 proteins were interacting with GRP94 under normal condition, 76 proteins under ERS condition, and 44 proteins under both conditions. The results showed that 76 proteins interacting with GRP94 under ERS were mainly involved in the negative regulation of apoptosis process, peptide cross-linking, ubiquitin-dependent ERAD (endoplasmic reticulum associated degradation) pathway and hydrogen peroxide catabolic process, among them, the specific proteins interacting with GRP94 in the negative regulation of apoptosis process were albumin, catalase, filament A, heat shock protein family A member 5, keratin 18, and prohibin 2, indicating that GRP94 might play an anti-apoptotic role with these proteins. Besides, the vimentin protein that made up the intermediate filament was involved in multiple GO enrichment terms, which might have an important interaction with GRP94, which was further confirmed by co-immunoprecipitation test, further demonstrating that there was indeed an interactive relationship between this two. Further analysis showed that some GRP94 interacting proteins (Such as peroxiredoxin, death inducer obliterator 1, catalase, glandular kallikrein, pyruvate kinase and so on) specifically expressed under ERS were closely related to anti-apoptosis. 【Conclusion】 Under ERS, GRP94 interacting proteins were mainly involved in anti-apoptosis, folding of unfolded proteins and maintenance of intracellular homeostasis-related signal pathways. This conclusion laid a foundation for further study on the mechanism of GRP94 involved in liver ERS regulation.

Assessment of Genomic Selection Accuracy for Slaughter Traits in Broilers Based on Microarray and Imputed Sequencing Data

YIN Chang, ZHU Mo, CHEN YanRu, TONG ShiFeng, ZHAO GuiPing, LIU Yang

Scientia Agricultura Sinica. 2023, 56(15): 3032-3039. doi:10.3864/j.issn.0578-1752.2023.15.016

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【Background】 In the breeding work of livestock and poultry, the core of which is the accuracy of genomic estimated breeding values. Different levels of genetic marker densities have a great impact on estimated breeding values, and with the development of genotyping technology and the decrease of high-throughput sequencing prices, genomic selection studies based on sequencing data have emerged. Theoretically, higher marker density can obtain higher prediction accuracy. This is because Quantitative Trait Loci (QTL) affecting the target trait are in linkage disequilibrium with at least one of the high-density markers covering the entire genome. A higher density of marker levels theoretically ensures tight linkage between markers and QTL, thus ensuring higher prediction accuracy. However, compared with microarray data, it has also been shown that the accuracy of genomic prediction for imputed sequencing data is not significantly improved. 【Objective】 Using the GBLUP method, we compared the genomic selection accuracy of imputed sequencing data and microarray data for slaughter traits in broiler chickens to provide a theoretical basis for genotyping strategies for broiler genomic selection breeding. 【Method】 In this study, we used SNP array data and imputed whole-genome sequence level (WGS) data to perform genomic prediction for the traits of breast muscle weight, carcass weight and thigh muscle weight in white feather broilers using the GBLUP method, and then we conducted a comparative study on their accuracy in genomic prediction. First, 3 362 chickens were genotyped using the Jingxin No. 1 chicken 55 K SNP chip, and 230 chickens were randomly selected from the ninth batch of generation 7 for whole-genome resequencing, and then the 55 K SNP chip data were imputed to the resequencing data level using Beagle 5.1 software. Considering the effect of chromosome size on the filling accuracy, the larger chromosome 3 and the smaller chromosome 14 were used to calculate the allele correct rate (CR) and genotype correlation coefficient (Cor), and the imputed WGS accuracy was determined by this study. The genomic breeding values of three slaughter traits were predicted using the imputed WGS data, and the accuracy, rank correlation and unbiasedness of the prediction results were evaluated using a 5-fold cross-validation method. 【Result】 The results showed that the average allelic accuracy of the two chromosomes was 0.924 and the average genotype correlation was 0.885, and the imputed WGS accuracy was high enough to be used for genomic prediction studies at a later stage. The accuracy of the predicted genomic breeding values calculated from microarray data ranged from 0.2194 to 0.2629, and the accuracy of the predicted genomic breeding values calculated from imputed sequencing data ranged from 0.2110 to 0.2695. The results show that the difference in the accuracy of the prediction of genomic breeding values from the imputed sequencing data was not significant compared with the 55 K SNP chip results. 【Conclusion】 Compared with the results of 55 K SNP microarray, the improvement in the accuracy of genomic breeding value prediction for three slaughter traits (breast muscle weight, carcass weight and leg muscle) in white feather broiler using imputed genomic level data was not significant, which provides a reference for the selection of data types in livestock genetic breeding work.

The Protective Efficacy of Commercial Vaccines Against H9N2 Avian Influenza Virus of Branch h9.4.2.5 Isolated in China

MA Qi, HE XinWen, WANG Yan, LIU YanJing, PAN ShuXin, HOU YuJie, SHI JianZhong, DENG GuoHua, BAO HongMei, LIU JingLi, GUO XingFu, MAO ShengGang, HU JingLei, LU Tong, YANG Fan, TIAN GuoBin, ZENG XianYing, CHEN HuaLan

Scientia Agricultura Sinica. 2023, 56(15): 3040-3050. doi:10.3864/j.issn.0578-1752.2023.15.017

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【Objective】 There are many commercial inactivated vaccines against the H9 subtype avian influenza approved for use in China, and their protection efficacy and selection have been widely concerned by farmers. In this study, the protection efficacy of main commercial vaccines against H9N2 viruses recently isolated in the field were evaluated, so as to provide the research reference for guiding the prevention and control of H9 subtype AI by immunization in China.【Method】 According to the vaccine batch release of the National Veterinary Drug Basic Information Database in China, 4 commercial vaccines (labeled as A-D) with large batches were selected from 40 kinds of sold H9 commercial vaccines. The four H9N2 subtype AIV, including CK/XJ/S1204/2015, DK/JX/S4512/2017, CK/YN/S1666/2020 and CK/NX/S4590/2020, belonging to h9.4.2.5 branch and isolated at different times and places, were used to evaluate the protection efficacy of selected commercial H9 subtype AI vaccines. The 50% chicken embryo infection (EID₅₀), 50% chicken infection (CID₅₀), and 50% cell infection (TCID₅₀) of the four viruses were tested to determine the challenge dose of the animal experiment and the infection dose of the cell test. 40 3-week-old SPF chickens were injected intramuscularly with 4 kinds of inactivated vaccine, and the groups of 10 similar SPF chickens were inoculated with PBS as control group. 3 weeks post vaccination (p.v.), the serum of all experimental chickens were collected before challenge, the hemagglutination inhibition (HI) and neutralization (NT) antibody titers were detected by HI test and neutralization test; the 40 immunized chickens of each vaccine were randomly divided into four groups, and 10 chickens of each vaccine and 10 control chickens were infected intranasally with the 10 CID₅₀ of each H9N2 virus. Oropharyngeal and cloacal swabs were collected on days 3 and 5 post challenge (p.c.) for virus shedding detection and calculation of the protective rate of each vaccine. 【Result】 The CID₅₀ of the four strains were 10^3.5 EID₅₀/0.1 mL, 10^2.5 EID₅₀/0.1 mL, 10^2.5 EID₅₀/0.1 mL and 10^3.5 EID₅₀/0.1 mL, respectively. At three weeks p.v., the mean HI antibodies titers against the commercial H9 subtype HI test antigen (CK/SH/10/2001) in 4 groups of vaccinated chickens ranged from 9.4 log₂ to 11 log₂, while the mean HI antibodies titers against challenge virus in 4 groups of vaccinated chickens ranged from 4.6 log₂ to 10.8 log₂, and the significant differences of HI antibodies titers were observed among different vaccine groups, for the maximum difference of mean HI antibodies titers were 64-fold among them. The mean NT antibodies titers against challenge virus in 4 groups of vaccinated chickens ranged from 6.7 log₂ to 12.2 log₂, while the maximum difference of mean HI antibodies titers were 32-fold among them, and the HI antibody and NT antibody of the control group were negative. After intranasal infection with different H9 viruses, the immune effects of the four vaccines were quite different. In CK/XJ/S1204/2015 challenged groups, 3 vaccines (B-D) could provide more than 80% protection. In DK/JX/S4512/2017 challenged groups, 1 vaccine (B) could provide more than 80% protection. In CK/YN/S1666/2020 challenged groups, 2 vaccines (A and B) could provide more than 80% protection. In CK/NX/S4590/2020 challenged groups, the protection efficacy of the four vaccines (A-D) were all less than 80%. In the same period, the virus shedding rate of chickens under the control group was more than 8/10. 【Conclusion】 There was a great difference in the immune protection efficacy of the four kinds of commercial vaccines after challenge with recently isolated H9 strains, and the difference of antigenicity between vaccine antigens and isolates was the main reason for the decrease of immune protection efficacy of the commercial vaccines. The titers of HI antibody and NT antibody against H9N2 prevalent strains could be as important data for evaluation of the commercial H9N2 AI vaccines. This study provided the scientific references for selection and usage of the commercial vaccines against H9N2 AI.

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