Citrus yellow vein clearing virus (CYVCV) is a new citrus virus that has become an important factor restricting the development of China’s citrus industry, and the CYVCV coat protein (CP) is associated with viral pathogenicity.  In this study, the Eureka lemon zinc finger protein (ZFP) ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.  Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid (SA)-related and hypersensitive response marker genes, and triggered a reactive oxygen species burst, ion leakage necrosis, and the accumulation of free SA.  Furthermore, the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4% that in control plants 6 mon after inoculation, with only mild leaf chlorotic spots observed in those transgenic plants.  Taken together, the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.  This is the first report that ZFP is involved in the immune response of a citrus viral disease, which provides a basis for further study of the molecular mechanism of CYVCV infection.

Canopy temperature strongly influences crop yield formation and is closely related to plant physiological traits.  However, the effects of nitrogen treatment on canopy temperature and rice growth have yet to be comprehensively examined.  We conducted a two-year field experiment with three rice varieties (HD-5, NJ-9108, and YJ-805) and three nitrogen treatments (zero-N control (CK), 200 kg ha^–1 (MN), and 300 kg ha^–1 (HN)).  We measured canopy temperature using a drone equipped with a high-precision camera at the six stages of the growth period.  Generally, canopy temperature was significantly higher for CK than for MN and HN during the tillering, jointing, booting, and heading stages.  The temperature was not significantly different among the nitrogen treatments between the milky and waxy stages.  The canopy temperature of different rice varieties was found to follow the order: HD-5>NJ-9108>YJ-805, but the difference was not significant.  The canopy temperature of rice was mainly related to plant traits, such as shoot fresh weight (correlation coefficient r=–0.895), plant water content (–0.912), net photosynthesis (–0.84), stomatal conductance (–0.91), transpiration rate (–0.90), and leaf stomatal area (–0.83).  A structural equation model (SEM) showed that nitrogen fertilizer was an important factor affecting the rice canopy temperature.  Our study revealed: (1) A suite of plant traits was associated with the nitrogen effects on canopy temperature, (2) the heading stage was the best time to observe rice canopy temperature, and (3) at that stage, canopy temperature was negatively correlated with rice yield, panicle number, and grain number per panicle.  This study suggests that canopy temperature can be a convenient and accurate indicator of rice growth and yield prediction.

Fruit development and ripening is a complex procedure (Malus×domestica Borkh.) and can be caused by various factors such as cell structure, cell wall components, and cell wall hydrolytic enzymes.  In our study, we focused on the variations in fruit firmness, cell wall morphology and components, the activity of cell wall hydrolytic enzymes and the expression patterns of associated genes during fruit development in two different types of apple cultivars, the hard-crisp cultivar and the loose-crisp cultivar.  In this paper, the aim was to find out the causes of the texture variations between the different type cultivars.  Cell wall materials (CWMs), hemicellulose and cellulose content were strongly associated with variations in fruit firmness during the fruit development.  The content of water soluble pectin (WSP) and chelator soluble pectin (CSP) gradually increased, while the content of ionic soluble pectin (ISP) showed inconsistent trends in the four cultivars.  The activities of polygalacturonase (PG), β-galactosidase (β-gal), cellulase (CEL), and pectate lyase (PL) gradually increased in four cultivars.  And the activities of PG, β-gal, and CEL were higher in ‘Fuji’ and ‘Honeycrisp’ fruit with the fruit development, while the activity of PL of ‘Fuji’ and ‘Honeycrisp’ was lower than that of ‘ENVY’ and ‘Modi’.  Both four cultivars of fruit cells progressively became bigger as the fruit expanded, with looser cell arrangements and larger cell gaps.  According to the qRT-PCR, the relative expression levels of MdACO and Mdβ-gal were notably enhanced.  Our study showed that there were large differences in the content of ISP and hemicellulose, the activity of PL and the relative expression of Mdβ-gal between two different types of apple cultivars, and these differences might be responsible for the variations in the texture of the four cultivars.

Nutrition poverty alleviation is an effective measure to improve the nutritional status of economically disadvantaged individuals, fundamentally improving their health and reducing poverty.  Based on the Entitlement Theory and using the China Health and Nutrition Survey (CHNS) data, this paper examines the relationship between farmers’ nutritional intake, production structure and regional market conditions.  Results show that farmers with diversified production have better nutritional intake than those who specialize.  Furthermore, the correlation between regional market conditions and nutritional intake varies between economically disadvantaged and non-economically disadvantaged households.  Market conditions significantly influence the carbohydrate and fat intake of economically disadvantaged households and are positively associated with the dietary structure and nutritional intake of non-economically disadvantaged ones.  Moreover, income is positively correlated with the nutritional intake of non-economically disadvantaged households but not with economically disadvantaged ones.  

Legume cultivars affect N uptake, component crop growth, and soil physical and chemical characteristics in maize–legume intercropping systems.  However, how belowground interactions mediate root growth, N fixation, and nodulation of different legumes to affect N uptake is still unclear.  Hence, a two-year experiment was conducted with five planting patterns, i.e., maize–soybean strip intercropping (IMS), maize–peanut strip intercropping (IMP), and corresponding monocultures (monoculture maize (MM), monoculture soybean (MS), and monoculture peanut (MP)), and two N application rates, i.e., no N fertilizer (N–) and conventional N fertilizer (N+), to examine relationships between N uptake and root distribution of crops, legume nodulation and soil N availability.  Results showed that the averaged N uptake per unit area of intercrops was significantly lower than the corresponding monocultures.  Compared with the monoculture system, the N uptake of the intercropping systems increased by 31.7–45.4% in IMS and by 7.4–12.2% in IMP, respectively.  The N uptake per plant of intercropped maize and soybean significantly increased by 61.6 and 31.8%, and that of intercropped peanuts significantly decreased by 46.6% compared with the corresponding monocultures.  Maize and soybean showed asymmetrical distribution of roots in strip intercropping systems.  The root length density (RLD) and root surface area density (RSAD) of intercropped maize and soybean were significantly greater than that of the corresponding monocultures.  The roots of intercropped peanuts were confined, which resulted in decreased RLD and RSAD compared with the monoculture.  The nodule number and nodule fresh weight of soybean were significantly greater in IMS than in MS, and those of peanut were significantly lower in IMP than in MP.  The soil protease, urease, and nitrate reductase activities of maize and soybean were significantly greater in IMS and IMP than in the corresponding monoculture, while the enzyme activities of peanut were significantly lower in IMP than in MP.  The soil available N of maize and soybean was significantly greater increased in IMS and IMP than in the corresponding monocultures, while that of IMP was significantly lower than in MP.  In summary, the IMS system was more beneficial to N uptake than the IMP system.  The intercropping of maize and legumes can promote the N uptake of maize, thus reducing the need for N application and improving agricultural sustainability.

In the past decade, there has been extensive global surveillance for highly pathogenic avian influenza (HPAI) infection in both animals and humans, however, few studies on epidemiology of avian influenza in Democratic People’s Republic of Korea (DPRK) were published.  During the period 2013–2014, HPAI H5N1 viruses were detected with outbreaks in domestic poultry in DPRK.  Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to clade 2.3.2.1c with high homology.  The HPAI H5N1 virus found in ducks at the Tudan Duck Farm in 2013 was might introduced by migratory birds and then led to the outbreaks on neighboring chicken farms in 2014.  These data provide direct evidence for the transmission of avian influenza viruses from wild birds to waterfowl to terrestrial birds.  Therefore, the monitoring and control of influenza virus in ducks must be given top priority, which are essential components to prevent and control HPAI.

The GDSL esterase/lipase family contains many functional genes that perform important biological functions in growth and development, morphogenesis, seed oil synthesis, and defense responses in plants.  The expression of GDSL esterase/lipase genes can respond to biotic and abiotic stresses.  Although GDSL esterase/lipase family genes have been identified and studied in other plants, they have not been identified and their functions remain unclear in tomato.  This study is the first to identify 80 GDSL esterase/lipase family genes in tomato, which were named SlGELP1–80.  These genes were mapped to their positions on the chromosomes and their physical and chemical properties, gene structure, phylogenetic relationships, collinear relationships, and cis-acting elements were analyzed.  The spatiotemporal expression characteristics of the SlGELP genes in tomato were diverse.  In addition, RNA-seq analysis indicated that the expression patterns of the SlGELP genes in tomato differed before and after inoculation with Stemphylium lycopersici.  qRT-PCR was used to analyze the expression of five SlGELP genes after treatments with S. lycopersici, salicylic acid and jasmonic acid.  Finally, this study was the first to identify and analyze GDSL esterase/lipase family genes in tomato via bioinformatics approaches, and these findings provide new insights for improving the study of plant disease resistance.

The objective of this study was to evaluate the effects on chemical and microbiological properties of paddy soil of short-term biochar, straw, and chemical fertilizers compared with chemical fertilization alone.  Five soil fertilization treatments were evaluated: regular chemical fertilizers (RF), straw+regular chemical fertilizers (SRF), straw biochar+regular chemical fertilizers (SCRF), bamboo biochar (BC)+regular chemical fertilizers (BCRF), and straw biochar+70% regular chemical fertilizers (SC+70%RF).  Their effects were investigated after approximately 1.5 years.  The soil pH and cation exchange capacity (CEC) were significantly higher in biochar-treated soils.  The soil phosphorous (P) and potassium (K) contents increased with biochar application.  The soil Colwell P content was significantly increased with the addition of straw biochar in the treatments of SCRF and SC+70%RF.  The oxygen (O):carbon (C) ratio doubled in BC picked from the soil.  This indicated that BC underwent a significant oxidation process in the soil.  The denaturing gradient gel electrophoresis (DGGE) fingerprints of microbial communities differed among the treatments.  Soils with added biochar had higher Shannon diversity and species richness indices than soils without biochars.  The results suggest that biochar can improve soil fertility.

Understanding the waxy maize starch physicochemical properties response to heat stress during grain filling could improve starch quality. The effects of heat stress during early (1–15 days after pollination, DAP) and late (16–30 DAP) grain filling stages on the starch physicochemical properties of four waxy maize varieties were evaluated. Crystallinity only increased in Suyunuo 5 after exposure to high temperature at late grain filling stage. The effects of heat stress on digestibility and swelling power were dependent on varieties and stages. Generally, swelling power was increased by heat stress at early grain development stage and digestibility was increased by high temperature at late grain filling stage, respectively. The results of correlation analysis indicated the starch with large granule size could swell well and easy digest. Peak, trough, final, and breakdown viscosities in response to heat stress were dependent on stages and varieties. In general, peak, trough and final viscosities were decreased and increased by heat stress at early grain formation and late grain filling stages, respectively; whereas the breakdown and setback viscosities were similar among the three treatments. Heat stress increased the gelatinization temperatures and retrogradation percentage. Gelatinization range decreased under heat stress at 1–15 DAP but remained constant under heat stress at 16–30 DAP in all varieties. The starch exposed to high temperature at 16–30 DAP presented higher digestibility and peak viscosity and lower retrogradation percentage than those at 1–15 DAP. Therefore, heat stress at early grain formation stage severely affects the physicochemical properties of starch.

     Heterosis is an important biological phenomenon, and it has been used to increase grain yield, quality and resistance to abiotic and biotic stresses in many crops. However, the genetic mechanism of heterosis remains unclear up to now. In this study, a set of 184 chromosome segment substitution lines (CSSLs) population, which derived from two inbred lines lx9801 (the recurrent parent) and Chang 72 (the donor parent), were used as basal material to construct two test populations with the inbred lines Zheng 58 and Xun 9058. The two test populations were evaluated in two locations over two years, and the heterotic loci for plant height and ear height were identified by comparing the performance of each test hybrid with the corresponding CK at P<0.05 significant level using one-way ANOVA analysis and Duncan’s multiple comparisons. There were 24 and 29 different heterotic loci (HL) identified for plant height and ear height in the two populations at two locations over two years. Three HL (hlPH4a, hlPH7c, hlPH1b) for plant height and three (hlEH1d, hlEH6b, hlEH1b) for ear height were identified in the CSSLs×Zheng 58 and CSSLs×Xun 9058 populations as contributing highly to heterosis performance of plant height and ear height across four environments. Among the 29 HL identified for ear height, 12 HL (41.4%) shared the same chromosomal region associated with the HL (50.0%) identified for plant height in the same test population and environment.

    The objective of this study was to isolate lactic acid bacteria (LAB) strains from different origins and to select the best strains for ensiling Robinia pseudoacacia (RB) and Morus alba L. (MB) leaves. The LAB strains were inoculated into the extracted liquid obtained from RB and MB leaves to evaluate the fermentation products. 11 LAB strains were selected for further experiments based on the highest products of lactic or acetic acid, including 1 strain of Weissella confusa, 2 of Lactobacillus reuteri and 8 of Lactobacillus plantarum. The API 50 CH fermentation experiment indicated that all of the selected 11 LAB strains utilised most of the carbohydrates. All the strains grew at temperatures between 10 and 45°C and at a pH of 3.5 to 4.5; however, L. reuteri F7 and F8 tolerated a pH as low as 3.0. All 11 LAB strains showed antibacterial activity against Listeria monocytogens, Escherichia coil, Salmonella sp. and Acetobacter pasteurianus; however, after excluding the effect of organic acids, only F7 and F8 still exhibited antibacterial activity. The present study indicated that the selected 11 LAB strains could be used to prepare silages of RB and MB leaves, especially L. reuteri F7 and F8.

This study investigated effects of dietary fibre and grit on growth performance, gastrointestinal tract (GIT) development, serum indexes and grit retention of Sichuan white goslings in China. The experiment was a 3×2 factorial design consisting of dietary crude fibre (CF) at 2.0, 4.0 and 6.0% levels by adding grass meal and with or without grit supplementation. A total of 720 1-d-old male goslings were randomly allocated into 36 pens of 20 goslings/pen (n=6 pens/treatment). At 21 d of age, GIT, blood samples and grit in gizzards were collected. Body weight (BW), empty BW (without GIT), average daily gain (ADG) and average daily feed intake (ADFI) were affected by CF levels and the medium (4.0%) had the highest value. Adding grit improved BW, empty BW, ADG and ADFI. CF affected relative weight or length of proventriculus, gizzard and duodenum. Increasing CF levels decreased jejunum villus height and affected caecal crypt depth and villus height to crypt depth ratio. Higher CF levels decreased serum triglyceride. Adding grit increased relative weight of abdominal fat. Grit addition increased grit weight in gizzard and the ratio of grit in 0.45-1 and 1-2 mm while decreasing the ratio of grit <0.45 and 2-3 mm. In conclusion, moderate CF level and grit addition increased performance of goslings without interaction. Dietary CF levels affected the development of upper GIT and serum triglyceride. Grit needed by goslings should be less than 2 mm. Goslings’ gizzard would accumulate grit from feed when other grit could not be acquired.

Breeding of male-sterile lines has become the mainstream for the heterosis utilization in foxtail millet, but the genetic basis of most male-sterile lines used for the hybrid is still an area to be elucidated. In this study, a highly male-sterile line Gao146A was investigated. Genetic analysis indicated that the highly male-sterile phenotype was controlled by a single recessive gene a single recessive gene. Using F2 population derived from cross Gao146A/K103, one gene controlling the highly male- sterility, tentatively named as ms1, which linked to SSR marker b234 with genetic distance of 16.7 cM, was mapped on the chromosome VI. These results not only laid the foundation for fine mapping of this highly male-sterile gene, but also helped to accelerate the improvement of highly male-sterile lines by using molecular marker assisted breeding method.

CRISPR-Cas9 emerged as a powerful tool for gene editing, which has been widely used in plant functional genomics research and crop genetic breeding (Chen et al. 2019). The target specificity of CRISPR-Cas9 relies on the 20-base-pair single guide RNA (sgRNA), making it relatively quick and straightforward to create plant-specific mutant libraries through large-scale synthesis of sgRNAs targeting multiple genes or even the whole genome. Several CRISPR-Cas9 mutant libraries have been developed for crops such as rice (Lu et al. 2017; Meng et al. 2017), soya bean (Bai et al. 2020), Brassica napus (He et al. 2023), and cotton (Sun et al. 2023). However, no CRISPR-Cas9 mutant library has yet been generated in woody crop plants. Grape (Vitis vinifera L.) is one of the oldest and most economically valuable fruit crops worldwide. The MYB family is one of the most abundant and versatile transcription factor families in plant (Wu et al. 2022). Here, we described a strategy for generating a collection of MYB mutant lines in grape using a sgRNA library.

We obtained 138 grape MYB transcription factor sequences from the Plant Transcription Factor Database (PlantTFDB, http://planttfdb.gao-lab.org/family.php?sp=Vvi&fam=MYB) (Appendix A). Since genes with similar sequences often share similar functions, a phylogenetic tree was constructed and divided all MYBs into 30 sets based on the sequence similarity (Fig. 1-A and Appendix B-a). A total of 127 sgRNAs were designed, each targeting conserved regions shared by two or more MYB transcription factors with fewer than three base-pair differences. This approach aimed to simultaneously mutate multiple MYB members within a cluster using a single sgRNA, addressing the challenge of genetic redundancy (Appendix B-b). These shared target sites were designated as Target1 (T1). In addition, specific sgRNAs targeting individual MYB transcription factors were designed using the  CRISPR-P 2.0 online tool (http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR) (Liu et al. 2017). The design criteria included selecting target sites within exons near the start of open reading frames (ORFs), a GC content of at least 40%, and an off-target efficiency below 0.4. Finally, 138 sgRNAs targeting specific sites for each MYB transcription factor were designed, referred to as Target2 (T2). In total, a comprehensive sgRNA library comprising 265 sgRNA was developed to target 138 MYBs, with an average  of 1.92 sgRNA per MYB (Fig. 1-A and Appendix C).

The sgRNA fragments from the same set were mixed as one sgRNA pool, ligated into the pKSE401 vector using Gibson ligation, and subsequently transformed into the Escherichia coli  TOP10 competence cells (Fig. 1-B and Appendix D-a). To evaluate the ligation efficiency of the sgRNA pool with the vector, 90 E. coli clones from sets #1-3 were randomly selected and sequenced. The results demonstrated that the ligation efficiency exceeded 90% and the sgRNA coverage ratio over 80%, confirming the feasibility of this method (Appendix D-b). Using this approach, approximately 1300 (~5×) positive E. coli clones were obtained across the 30 sets (Fig. 1-D). Plasmids extracted from each set were mixed in equal proportions and transformed into Agrobacterium tumefaciens GV3101 competence cells. Finally, all Agrobacterium colonies were collected and verified with next-generation sequencing (NGS). The results revealed that 95.31% of the sequences in the library were accurate, and 178 of 265 sgRNAs were represented by at least one read, targeting 125 (90.58%) MYB transcription factors. Most (83.93%) sgRNA read counts fell within the range of 2⁸-2¹⁵. These results indicated that the sgRNAs library in Agrobacterium exhibited high accuracy and gene coverage, which is usable for grape transformation (Fig.1-E).

Vitis vinifera L. cv. Cabernet Sauvignon is one of the most renowned red wine grape varieties, widely cultivated worldwide. In this study, pro-embryonic masses of ‘Cabernet Sauvignon’ were used as recipient material for Agrobacterium-mediated transformation (Fig. 1-C and Appendix E). A total of 1354 kanamycin-resistant seedlings were obtained, and which 341 were confirmed as transgenic lines (PCR positive). And, all the lines were determined to harbor a single correct sgRNA, representing 13 unique sgRNAs targeting 18 MYB transcription factors. Target site DNA was amplified and sequenced, revealing only 67 gene-edited lines with mutations in 8 MYB transcription factors (Fig. 1F and Appendix F and G). Among these, 56 lines were chimeric mutants, nine were biallelic mutants, one was a homozygous mutant and one was a heterozygous mutant (Appendix H). Five Target1 type transgenic lines were obtained, three of them did not mutate in all of the targeted genes, in which the sgRNA was targeting two or more completely conserved sites. Additionally, gene-edited lines for GSVIVT01032467001-T1 and GSVIVT01014770001-T1 were producted. However, the sgRNA harbored in these transgenic lines only caused the mutations in GSVIVT01032467001 and GSVIVT01014770001, without off-target effects on genes with similar sequences (Appendix I and J). All MYB-edited lines were subsequently transplanted into a greenhouse for observation (Appendix K). Phenotypic analysisrevealed that the GSVIVT01026481001 edited lines exhibited significantly enhanced tolerance to drought stress (Fig. 1-G).

CRISPR-Cas9 has greatly accelerated gene function research and breeding in plants. In this study, we developed a strategy for generating a collection of MYB mutant lines in grape using a CRISPR-Cas9 library. However, the relatively lower transformation efficiency in grape limited the number of mutants obtained. Factors affecting grape transformation efficiency primarily included the regeneration rate of the recipient material and the efficiency of Agrobacterium infection. Numerous studies have demonstrated that plant regeneration efficiency enhanced using developmental regulators such as BABY BOOM (BBM), WUSCHEL (WUS), GROWTH-REGULATING FACTOR (GRF), and REGENERATION FACTOR (REF) (Debernardi et al. 2020; Yang et al. 2022). Additionally, plant transformation and gene editing efficiency improved through optimized genetic transformation methods and gene editing vector designs (Debernardi et al. 2024; Yan et al. 2024). We confirmed that a large number of edited plants could be obtained simultaneously using a sgRNA mixed-pool library, provided that grape transformation efficiency is improved. This strategy holds significant potential for constructing genome-wide mutant libraries in woody crop plants in the future.