【Objective】Salt stress is one of the main environmental stresses that restrict rice production. Studying the physiological characteristics under salt stress and analysis the allelic variation and expression of salt-tolerance genes provide key gene resources and genetic materials for breeding salt-tolerance rice varieties. 【Method】This study first evaluated the salt-tolerance ability of the Nangeng series high-quality rice varieties/lines during the seedling stage, using survival rate as an indicator for screening salt-tolerance varieties, which physiological changes under salt stress were analyzed, including chlorophyll, Na+, K+, MDA, H2O2 and soluble sugar. The variation types and expression levels of salt-tolerance genes in rice varieties with resistance to high salt concentration were also analyzed to explaining their molecular mechanisms in response to salt stress. 【Result】Under the condition of treating with 140 mmol·L-1 NaCl for 6 days, the survival rates of NG9108, NG5718, and NGY1 were greater than 60%, with the highest survival rate among the tested varieties. Compared with Nipponbare, the seedlings of NG9108, NG5718, and NGY1 under salt stress had higher chlorophyll content and lower MDA content, indicating that salt stress caused less cell damage to the three varieties. The Na+/K+ values in the roots of NG9108, NG5718, and NGY1 were significantly higher than those in Nipponbare, while the Na+/K+ values in the aerial parts were significantly lower than those in Nipponbare, implying that the three varieties absorb or store more Na+ in roots, but transport less Na+ upwards, which is beneficial for maintaining cell ion balance and causing less ion toxicity and osmotic stress in aerial parts of the seedlings. The three salt-tolerance varieties have 94 SNPs or InDel sites, distributing in exons, introns, 5′UTR, and 3′UTR of the 23 salt-tolerance genes. 24 variation sites of 11 genes occur in the exons, including 7 genes with frameshift mutations or missense mutations which distributed in Os02g0813500 (OsGR2), Os05g0343400 (OsWRKY53), Os06g0685700 (OsRST1), Os07g0685700 (OsEIL2), Os10g0431000 (OsPQT3), Os11g044600 (OsRSS3), Os12g0150200 (P450). Salt stress significantly induces expression of OsSKC1, OsBAG4, OsGPX1, OsCCX2, OsGR3, OsDREB2a, OsRAB21, OsP5CS, OsbZIP23, OsAPX37 and OsLEA3, which help to enhance salt tolerance and reduce the adverse effects of salt damage on rice growth. 【Conclusion】NG9108, NG5718 and NGY1 showed strong salt tolerance phenotype during the seedling growth stage, which is closely related to the balance of sodium and potassium ions under salt stress, allelic variations of multiple salt tolerance genes, and gene expression levels. NG9108, NG5718 and NGY1 have pyramided multiple salt tolerant and high-quality genes, which can be used as backbone parents for genetic improvement and breeding.
【Objective】Cotton bollworm (Helicoverpa armigera) and field weeds are major constraints to high-yield cotton production. Existing varieties with single traits (insect resistance or herbicide tolerance) fail to meet the demands of efficient cultivation. Developing transgenic cotton varieties with combined insect resistance and glyphosate tolerance will provide high-efficiency germplasm resources for stress-resistant cotton breeding.【Method】The insect-resistant fusion gene cry1Ac-vip3Da and glyphosate-tolerant gene g10-epsps were introduced into cotton R15 through Agrobacterium-mediated method, regenerated transgenic plants were screened via PCR, positive lines underwent multi-generation self-pollination to achieve homozygosity, and stable lines with superior resistance were selected. The expression of target genes in different tissues of transgenic lines was analyzed using qRT-PCR and ELISA. Bioactivity assays and glyphosate tolerance tests were conducted to evaluate the genetic stability of insect resistance and herbicide tolerance across generations (T4-T6). Agronomic traits of transgenic lines were comprehensively assessed. 【Result】Eight positive transgenic lines with dual resistance were identified through PCR screening, and CA-6, CA-7 and CA-17 lines exhibited higher resistance. qRT-PCR revealed high expression of cry1Ac-vip3Da and g10-epsps in all tissues of these lines, and expression levels varied significantly among tissues. ELISA analysis demonstrated significant differences in Cry1Ac-Vip3Da and G10-EPSPS protein content across tissues of the three transgenic lines, with the highest levels observed in leaves. Protein accumulation gradually decreased during the developmental stages (from the four-leaf stage to boll-opening stage), but remained stable across T4-T6 generations. Bioactivity assays and glyphosate tolerance tests demonstrated that three transgenic cotton lines (T4-T6 generations) exhibited corrected mortality rates of 65.12%-82.75%, tolerated glyphosate at over four times the recommended dosage, and showed no attenuation of resistance across generations. There were no significant differences in plant height, number of fruit branches, number of bells per plant, bell weight, lint percentage, seed cotton yield, and lint cotton yield between transgenic lines and R15.【Conclusion】The exogenous genes cry1Ac-vip3Da and g10-epsps were stably inherited across generations in transgenic lines CA-6, CA-7, and CA-17, conferring dual insect resistance and glyphosate tolerance without compromising agronomic performance.
【Objective】Aflatoxin contamination is one of the important factors that hinders sustainable development of the peanut industry. Precise evaluation of germplasm resources from China and abroad for resistance to A. flavus infection and creation of new resistant germplasms will facilitate the development of resistant cultivars. 【Method】The A. flavus infection index of 322 peanut germplasm lines were characterized following in-vitro inoculation of seeds harvested from 3 different “environments” (CA2020, CS2020, CS2021). Aspergillus flavus strain As 3.4408, known for its strong infectivity and high toxin production, was used as the inoculation strain. The botanical type, plant type and nutritional quality of kernels were measured and analyzed. Accessions exhibiting resistance with novel traits were comprehensively evaluated and screened. 【Result】Thirteen accessions with stable resistance were identified, accounting for 4.04% of the total germplasm lines evaluated, most of which belonged to var. hypogaea, including two with stable and high resistance (C203 and C206), while no accession was observed to be immune to Aspergillus flavus infection. The frequency distribution of infection index of 322 accessions exhibited continuous variation, with the broad-sense heritability exceeding 0.8, indicating that the A. flavus-resistance of kernels was significantly influenced by genotypes and “environments”, and the phenotypic variation was primarily controlled by genetic factors. Correlation analysis revealed significant positive correlation of infection index of accessions among the different “environments” (P<0.001), and the phenotype of each accession harvested from various “environments” was relatively consistent. Additionally, no significant correlation was found between nutritional quality and infection index. Comparative analysis of infection index among peanut accessions of different botanical and plant types revealed that var. hypogaea/prostrate-type peanuts were more likely to exhibit resistance to A. flavus infection within the existing peanut germplasm resources. 【Conclusion】The phenotypes of peanut germplasms harvested from different “environments” in response to A. flavus infection were relatively stable. Variation of kernels resistance to A. flavus infection was primarily controlled by genotype. Accessions C203 and C206, exhibiting stable and high resistance, can serve as excellent resistant parents for the mining of aflatoxin resistance genes and for the improvement of peanut varieties resistant to aflatoxin contamination.
【Objective】Nitrogen panicle fertilizer is one of the key factors affecting rice yield and quality. Studying its impact on the yield, quality, and aroma of aromatic japonica rice in southern China could provide a theoretical basis for high-yield and high-quality cultivation of southern japonica rice. 【Method】 Conducted from 2022 to 2023, this study used Nanjing 9108, a representative variety of aromatic japonica rice in southern China, as the material, and three nitrogen application modes were set up: no nitrogen fertilizer (N0), no panicle fertilizer (N1), and conventional application of panicle fertilizer (N2, with 70% base and tillering fertilizer + 30% panicle fertilizer). In addition, the experiment of applying ear fertilizer at different leaf age stages, including the top sixth leaf, fifth leaf, fourth leaf, third leaf, second leaf and first leaf just after emerging from the sheath (designated as L6, L5, L4, L3, L2, and L1), was conducted to study the synergistic regulation mechanism of nitrogen panicle fertilizer on yield, quality, and aroma of Nanjing 9108.【Result】Compared with no nitrogen fertilizer application and no panicle fertilizer application, the application of panicle fertilizer could significantly increase the effective panicle number per unit area and grains per panicle of aromatic japonica rice, thereby enhancing its yield. As the period of panicle fertilizer application was delayed, the yield first increased and then decreased, reaching a maximum at the treatment of applying fertilizer at the fourth leaf from the top (counted downwards from the flag leaf). The application of panicle fertilizer improved rice processing quality, appearance quality, and aroma quality. The period of panicle fertilizer application had an impact on these qualities of aromatic japonica rice. With the delay in the period of panicle fertilizer application, the milled rice rate of Nanjing 9108 showed an increasing trend, but the chalkiness degree increased, leading to a deterioration in appearance quality. Simultaneously, the amylose content decreased while the protein content increased, resulting in a decline in taste value and eating quality. The content of 2-acetyl-1-pyrroline (2-AP), as the main component of aroma, also decreased with the delay in the period of panicle fertilizer application. The application of panicle fertilizer significantly increased the proline content and proline dehydrogenase activity in grains. Advancing the period of panicle fertilizer application had a significant promoting effect on proline accumulation during the rice filling stage, and proline dehydrogenase activity also increased, which was conducive to maintaining higher proline content and proline dehydrogenase activity in grains during the maturity stage, thereby promoting the synthesis of 2-AP in rice grains. Based on a comprehensive evaluation of the effects of panicle fertilizer application period using indicators, such as actual yield, milled rice rate, chalkiness degree, taste value, and grain 2-AP content, it was found that the treatment of applying fertilizer at the fourth leaf from the top had the highest comprehensive score. 【Conclusion】Under the experimental conditions of this study, the application of panicle fertilizer contributed to the synergistic improvement of yield and quality. On the basis of ensuring stable yield, the application of panicle fertilizer at the fourth leaf from the top achieved the best overall benefits in terms of yield, taste, and aroma.
【Objective】This study aimed to explore the effects of multiple cropping green manure combined with different levels of nitrogen fertilizer on the yield and grain quality of spring wheat, so as to provide the theoretical guidance for the construction of chemical fertilizer reduction and high-quality production mode based on green manure in Qinghai province.【Method】The split plot experiment was carried out in the experimental site of the Academy of Agricultural and Forestry Sciences of Qinghai University from 2023 to 2024. Two planting patterns were set up in the main area: multiple cropping green manure after wheat (W-G) and leisure after wheat (W), and three nitrogen fertilizer levels in the sub-area included: no nitrogen application (N0), nitrogen fertilizer reduction by 30% (N1, 157.5 kg N·hm-2), and the local custom of nitrogen application (N2, 225 kg N·hm-2). Wheat yield and grain quality (grain protein content, sedimentation value, formation time, stability time, wet gluten, etc.) were determined.【Result】The grain yield of spring wheat under W-GN1 treatment was 5.5% and 13.4% higher than that under WN1 in 2023 and 2024, and 2.0% and 5.3% higher than that under WN2 treatment, respectively; the biological yield under W-GN1 in 2023 and 2024 was 5.1% and 10.6% higher than that under WN1, and 1.5% and 4.6% higher than that under WN2, respectively. W-G could obtain higher harvest index than W. The compensation effect of multiple cropping green manure was negative under no nitrogen application level, and it was between 2.0%-14.0% under nitrogen application level. The contribution of multiple cropping green manure combined with 30% reduction of nitrogen fertilizer to increasing crop yield was the best. At the same time, W-GN1 could improve grain quality by increasing grain protein content, sedimentation value, formation time and stabilization time. The grain protein content under W-GN1 treatment was 10.62% and 9.48% higher than that under WN1, respectively. The grain sedimentation value increased by 25.05% and 18.13%, respectively. The grain formation time increased by 34.70% and 8.66%, respectively. The grain stability time increased by 41.30% and 13.68%, respectively. Through principal component analysis, it demonstrated that the multiple cropping of green manure after wheat had a more significant promoting effect in grain protein content, sedimentation value, formation time and stability time.【Conclusion】The grain yield and quality of spring wheat were significantly improved by 30% reduction of nitrogen fertilizer (N1,157.5 kg N·hm-2) combined with green manure after wheat harvest, which could be used as a suitable planting mode and nitrogen application level for improving quality and stable yield of wheat under the condition of reducing chemical fertilizer in Qinghai Province.
【Objective】The current post-planting film-covering technology in the Yellow River Basin cotton-growing areas relies on manual seedling release and thinning, which hinders the development of full-scale mechanization. This study explored the adaptability of the dry sowing and wet emergence technology (used in northwestern inland cotton regions) to the Yellow River Basin. By comparing the effects of different sowing methods on seed germination environment, cotton emergence rate, and seedling growth, this study aimed to identify key environmental constraints and provide the theoretical support for optimizing mechanized planting techniques. 【Method】From 2023 to 2024, using the cotton cultivar Ji863 as the experimental material, four treatments were implemented: single-seed seeding without mulching (T1), single-seed post-planting film covering (T2), dry sowing and wet emergence with single-seed sowing (T3), and dry sowing and wet emergence with double-seed sowing (T4). The study was conducted in Quzhou County, Hebei Province, and the effects of these treatments on soil environment, cotton emergence, and growth of above-ground and root systems were evaluated. 【Result】Compared with T1, T2 and T3 significantly increased soil temperature at 5 cm, soil moisture content, emergence rate, plant height, above-ground dry matter weight, root length, root surface area, root volume, and root vigor, while significantly reduced soil compaction, emergence time, and root diameter. Compared with T2, in 2023 and 2024, T3 reduced the daily temperature range at 5 cm soil depth by 3.67 and 1.58 ℃ within 30 days after sowing, and reduced soil compaction by 9.36% and 27.06% within 10 days after sowing, respectively, which decreased emergence days in 2024 by 0.6 days, and increased root length, surface area, volume, and root activity in both years. Compared with T4, single-grain sowing in 2023 and 2024 significantly increased emergence time and decreased emergence rate but increased aboveground dry matter weight by 13.98% and 55.00%. The structural equation model showed that different sowing methods affected cotton seedling emergence rate by altering soil temperature at 5 cm depth, daily temperature range, soil moisture content, and soil compaction, while seedling dry matter weight was mainly affected by soil moisture content, plant height, and soil compaction. 【Conclusion】In the Yellow River Basin cotton areas, the dry-sowing and wet-emergence improved emergence speed and rate by reducing the diurnal temperature fluctuation at 5 cm and soil compaction, thereby promoting uniform and robust seedlings, and sowing one seed per hole was the optimal strategy.
【Objective】Plum cultivation is an important industry in Liaoning region, while the large-scale epidemic of bacterial shot hole has seriously affected the sustainable development of plum cultivation. The purpose of this paper is to clarify the pathogenic bacteria species of plum bacterial shot hole in Liaoning, and to explore the toxicity of different agents against the pathogenic bacteria, so as to provide a theoretical basis for the prevention and control of the disease.【Method】From 2023 to 2024, the occurrence of plum bacterial shot hole was investigated in 10 plum production areas of Liaoning Province. By collecting 20 disease samples of leaves, fruits, and twigs with typical symptoms, a total of 30 pathogenic bacteria were isolated, purified and preserved. The characteristics of these colonies including morphology, color, size and edge morphology were observed and recorded. Transmission electron microscopy was employed to further observe their morphology and the Gram staining was performed. The bacterial 16S rDNA universal primer 27F/1492R and multiple gene loci were used for amplification. The sequencing result was compared by BLASTn analysis in the NCBI database and the reference sequences of the corresponding genera were downloaded. Then, a multi-gene joint phylogenetic tree was constructed using the maximum likelihood method on the website of GIPRES Science Gateway. Subsequently, the leaves of ‘Qiu Ji’ plum were inoculated with pathogenic bacteria by the stab inoculation of bacterial suspension, and then cultivated at 28 ℃ under high humidity. The disease occurrence of the leaves was regularly observed and recorded. Furthermore, the pathogenic bacteria were re-isolated from the disease-health junction of the leaves to complete the verification of the whole Koch’s postulates. The sensitivities of pathogenic bacteria to 0.15% tetramycin, 80% ethylicin, 3% benziothiazolinone, 1.8% octylamine, 3% zhongshengmycin and 6% kasugamycin were determined by the inhibition zone method, respectively.【Result】Based on morphological observation and molecular biology identification, the pathogenic bacteria were identified as Xanthomonas arboricola pv. pruni (Xap) (36.67%, 11 strains) and Pantoea vagans (63.33%, 19 strains). The sensitivity results demonstrated that the best inhibitory activities were achieved by 0.15% tetramycin and 80% ethylicin, with the EC50 of 0.15% tetramycin being 0.026 and 0.502 μg·mL-1 for Xap and P. vagans, respectively, and that of 80% ethylicin being 1.162 and 25.643 μg·mL-1, respectively. The second was 3% benziothiazolinone, and its EC50 values against Xap and P. vagans were correspondingly 5.200 and 96.075 μg·mL-1, followed by EC50 of 1.8% octylamine against Xap and P. vagans of 176.008, 273.072 μg·mL-1, and 3% zhongshengmycin against Xap and P. vagans of 621.697 and 72.270 μg·mL-1. As for 6% kasugamycin, it had an EC50 of 886.467 μg·mL-1 for P. vagans and was less effective in inhibiting Xap.【Conclusion】The pathogens causing plum bacterial shot hole in Liaoning were identified as Xap and P. vagans. Moreover, the 0.15% tetramycin and 80% ethylicin were screened and selected as exerting better inhibitory effects on these two pathogens. The results of the study will provide a theoretical basis for the precise prevention and control of plum bacterial shot hole in the field.
【Objective】The study aims to establish a novel visual detection technique for prunus necrotic ringspot virus (PNRSV) by combining reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR/Cas12a system (RT-RAA-CRISPR/ Cas12a).【Method】Primers with high amplification efficiency and strong specificity were designed and selected based on the conserved regions of the coat protein (CP) gene of PNRSV. The detection conditions, including primer, probe concentration, temperature, and reaction time were optimized to develop a visual detection method for PNRSV by RT-RAA-CRISPR/Cas12a technology. The specificity of this method was evaluated by detecting PNRSV and common Prunus viruses, including plum pox virus (PPV), apple mosaic virus (ApMV), cucumber mosaic virus (CMV), potato virus X (PVX), and potato virus Y (PVY). The total RNAs from PNRSV-infected fruit were diluted in 10-fold gradients, then RT-PCR, RT-RAA and RT-RAA-CRISPR/Cas12a were performed to compare the sensitivity of the three methods. The RT-RAA-CRISPR/Cas12a and RT-PCR methods were used to detect 31 peach fruit test samples suspected to be infected with the virus collected at the port to verify the practicability of the visual detection method.【Result】The RT-RAA-CRISPR/Cas12a-based visual detection method for PNRSV was successfully established. The optimized working concentrations were as follows: RT-RAA-PNRSV-F2/R2 primers at 0.4 μmol·L-1, fluorescent reporter (FQ) at 800 nmol·L-1, CRISPR-Cas12a at 200 nmol·L-1, and PNRSV-crRNA (CRISPR RNA) at 240 nmol·L-1, the reaction conditions were performed at 41 ℃ for 45 min. This method showed high specificity for PNRSV and had no cross-reaction with other common Prunus viruses. The limit of detection for PNRSV RNA in peach fruit samples reached 3.06 pg·μL-1 and 306 fg·μL-1 using RT-RAA and RT-RAA-CRISPR/Cas12a methods, respectively, showing the sensitivity of RT-RAA-CRISPR/Cas12a was 10 times higher than that of RT-RAA and RT-PCR. Among the 31 tested peach fruit samples at the port, 14 positive samples were identified by RT-PCR, while 15 positive samples were found by RT-RAA-CRISPR/Cas12a, indicating a high level of consistency between the two methods.【Conclusion】The RT-RAA-CRISPR/Cas12a visual detection method for PNRSV has been established. It is characterized by simplicity, rapidity, high sensitivity, high specificity, and visual readability, making it well-suited for rapid on-site detection of PNRSV.
