The Colletotrichum fructicola (C. fructicola) is a hemibiotrophic fungus, which causes devastating anthracnose in strawberry. At present, the resistance mechanism to C. fructicola remains poorly understood. Here, we used RNA-sequencing and liquid chromatography-mass spectrometry (LC-MS) metabolomics to excavate the molecular mechanism of strawberry resistance to C. fructicola. The differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) were screened at different stages after C. fructicola infection in the susceptible ‘Benihoppe’ and resistant cultivar ‘Sweet Charlie’. The core common DEGs with high association of common DAMs were identified by multi-omics integration analysis, and showed convergence and divergence in the two strawberry cultivars. Strikingly, the phenylpropanoids biosynthesis was simultaneously enriched in a multi-level omics at different stages after C. fructicola infection in the resistant (R) and susceptible (S) strawberries. Furthermore, we constructed the DEGs-DAMs map of phenylpropanoid biosynthesis. More importantly, we showed that chloroplasts and starch and sugar metabolism related genes, such as chlorophyII A-B binding genes, glycosyl hydrolase (GH) family genes and so on, were differentially expressed. Taken together, our study revealed major changes in genes and metabolites expression associated with C. fructicola resistance, and identified the multi-level regulatory network based on phenylpropanoid biosynthesis, useful for further mechanistic excavation of resistance to C. fructicola in strawberries.

Strawberry anthracnose, caused by Colletotrichum spp., is a devastating disease that significantly reduces strawberry yield and quality. This study aimed to develop a simple diagnostic method to detect infection by the Colletotrichum gloeosporioides species complex (CGSC), the most predominant and virulent Colletotrichum species complex causing strawberry anthracnose in China. In this study, a Cas12aVIP diagnostic method was developed for the rapid detection of the CGSC in strawberry seedlings. This method targets the beta-tubulin gene and combines recombinase polymerase amplification (RPA), the CRISPR/Cas12a system, and a cationic-conjugated polythiophene derivative [poly(3-(3 '-N,N,N-triethylamino-1 '-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT)] mixed with single-stranded DNA. This method shows high sensitivity (10 copies per reaction) and no cross-reactivity against related pathogens. The entire procedure, from sample to result, can be completed within 50 min, including simplified DNA extraction (15 min), RPA reaction (37 degrees C for 20 min), CRISPR/Cas12a detection (37 degrees C for 10 min), and visual detection by the naked eye (1 to 2 min). Furthermore, the Cas12aVIP assay successfully detected the CGSC in naturally infected strawberry seedling samples in field conditions. Asymptomatic infected plants and plant residues have been identified as primary inoculum sources for the CGSC. This method enables visible detection without the need for expensive equipment or specialized technical skills, thereby offering an efficient and straightforward approach for detecting the CGSC in strawberries. The newly developed detection method can be used to promote healthier strawberry production.

Aroma is an important indicator of fruit flavor, but mechanisms of aroma formation in strawberries (Fragaria spp.) during natural ripening are still not clear. In this study, aroma compounds in strawberry cultivars were analyzed using gas chromatography-mass spectrometry (GC-MS). Richly creamy strawberry cultivars in particular expressed high levels of vanillin acetate and coumarin (up-regulated by 12.6- and 9.8-fold, respectively), while the aroma-free cultivars were dominated by differential changes in terpenes and alcohols. Further research using liquid chromatography-mass spectrometry (LC-MS) and RNA-Seq indicated that the activation of the phenylpropanoid biosynthesis and alpha-linolenic acid metabolic pathways constituted the key to formation of aroma compounds in creamy strawberry cultivars. The results of this study not only provide a well-defined database to detect aroma compounds in different strawberry cultivars but also explore the underlying mechanisms of creamy aroma formation in strawberries.

果生炭疽菌Colletotrichum fructicola侵染引起的草莓炭疽病是草莓生产中的重要病害.前期研究发现一个在果生炭疽菌侵染阶段高表达的含LysM结构域的候选效应子CfLysM2.为进一步研究CfLysM2的致病功能,运用同源重组方法构建CfLysM2缺失突变株△CfLysM2和回补菌株CfLysM2c.致病力测定结果表明,接种后15 d,ΔCfLysM2接种的草莓叶片病情指数显著下降.荧光定量PCR分析表明CfLysM2基因在果生炭疽菌接种草莓叶片后24 h相对表达量最高.利用转录组技术对野生型菌株(wild-type strain,WT)和△CfLysM2接种后24 h的草莓叶片样品进行比较分析,获得差异表达基因109个.CfLysM2的缺失导致宿主代谢途径尤其是次生代谢物生物合成通路的激活,说明CfLysM2可能在果生炭疽菌侵染草莓过程中通过靶向黄酮醇合成酶等代谢相关基因,抑制植物次生代谢产物合成及其介导的抗真菌免疫,保证其顺利侵染.该研究为揭示CfLysM2机理及对果生炭疽菌和草莓互作研究奠定了理论基础.

The cultivated octoploid strawberry (Fragaria × ananassa) is an economically important fruit that is planted worldwide. The lysin motif (LysM) protein family is composed of the major class of plant pattern recognition receptors, which play important roles in sensing pathogen-associated molecular patterns (PAMPs), and subsequently triggers downstream plant immunity. In the present study, a comprehensive, genome-wide analysis of F. × ananassa LysM (FaLysM) genes was performed to investigate gene structures, phylogenic relationships, chromosome location, collinear relationships, transcription factor binding sites, and protein model analysis. We aimed to identify the LysM genes involved in the defense against plant pathogens. A total of 14 FaLysM genes were identified in the F. × ananassa genome and divided into 2 subgroups (LYP and LYK) on the basis of the phylogenetic analysis. The Ka/Ks ratio for the duplicated pair of most FaLysM genes was less than 1, which indicates that the selection pressure was mostly subject to the purifying selection during evolution. The protein model analysis revealed that FaLysM2-10 contain conserved mode of chitin binding, which suggest the potential role of FaLysM2-10 in pathogen perception and plant immunity. The RNA-Seq results showed the differential regulation of 14 FaLysM genes in response to Colletotrichum fructicola infection, implying the complex interaction between C. fructicola and strawberry. Knockout of candidate effector gene CfLysM2, which was previously proved to be highly expressed during C. fructicola infection, resulted in the up-regulation of six FaLysM genes (FaLysM1, FaLysM2, FaLysM3, FaLysM7, FaLysM8, and FaLysM12), indicating the competitive relations between CfLysM2 and FaLysM genes. Overall, this study provides fundamental information on the roles of LysM proteins in octoploid strawberry and its interaction with C. fructicola, laying useful information for further investigation on the C. fructicola-strawberry interaction and strawberry resistance breeding.

Aroma is an important indicator of the quality of fruit flavor, but the mechanism of aroma formation in strawberries (Fragaria spp.) during natural ripening merit further research. In this study, the aroma compounds in strawberry cultivars of varying types of aromas in different developmental stages were analyzed using gas chromatography-mass spectrometry. Richly creamy strawberry cultivars in particular expressed high levels of volatile phenol esters (mainly vanillin acetate and coumarin), while the aroma-free cultivars were dominated by differential changes in terpenes. Further research using liquid chromatography-mass spectrometry and RNA-Seq indicated that the activation of the phenylpropanoid biosynthesis and α-linolenic acid metabolic pathways, which used phenylalanine and α-linolenic acid as precursors and phenylalanine ammonia lyase and lipoxygenase as reactive enzymes, constituted the key to formation of aroma compounds in creamy strawberry cultivars. This study provides a new technical basis to understand the types and mechanisms of formation of aroma compounds in strawberries.

Introduction Blueberries have a high antioxidant content and are produced as healthy food worldwide. Long non-coding RNAs (lncRNAs) are a type of regulatory RNAs that play a variety of roles in plants. Nonetheless, information on lncRNAs and their functions during blueberry fruit development is scarce in public databases. Methods In the present study, we performed genome-wide identification of lncRNAs in a southern highbush blueberry using strand-specific RNA sequencing (ssRNA-Seq). Differentially expressed lncRNAs (DE-lncRNAs) and their potential target genes were analyzed at four stages of fruit development. Cis-regulatory DE-lncRNAs were predicted using co-localization analysis. Results These findings included a total of 25,036 lncRNAs from 17,801 loci. Blueberry lncRNAs had shorter transcript lengths, smaller open reading frame (ORF) sizes, fewer exons, and fewer isoforms than protein-coding RNAs, as well as lower expression levels and higher stage-specificity during fruit development. A total of 105 DE-lncRNAs were identified among the comparison group of PAD vs. CUP, 443 DE-lncRNAs were detected when comparing CUP with PINK fruits, and 285 DE-lncRNAs were revealed when comparing PINK and BLUE fruits. According to Kyoto Encyclopedia of Genes and Genomes annotation, target genes of DE-lncRNAs were primarily enriched in the “Autophagy-other”, “DNA replication”, “Endocytosis”, ‘photosynthesis’ and ‘chlorophyll metabolism’ pathways, suggesting that lncRNAs may pay potential roles in fruit expansion and ripening. Moreover, several lncRNAs have been proposed as cis-regulators of the key genes involved in flavonoid biosynthesis. MSTRG.107242.6, and its putative target gene, BTB/POZ and TAZ domain-containing protein, might play critical roles in anthocyanin accumulation in blueberries. Discussion These findings highlight the regulatory function of lncRNAs and aid in elucidating the molecular mechanism underlying blueberry fruit growth.

为明确引起上海地区桃果实腐烂的病原菌种类,于2019年6—7月在奉贤区、浦东新区和金山区等上海桃产区采集桃烂果,进行组织分离、培养,并对病原菌进行形态学分析、柯赫氏法则验证及分子鉴定.结果表明:上海地区引起桃烂果的病原菌有5种,分别是美澳型核果褐腐病菌(Monilinia fructicola)、层出镰孢菌(Fusarium proliferatum)、白地霉(Galactomyces candidum)、葡萄座腔菌(Botryosphaeria dothidea)和灰霉菌(Botrytis cinerea),其中M.fructicola的分离率为75.00％,其次是F.proliferatum(8.33％)和G.candidum(8.33％).本研究对上海地区桃采前及采后腐烂病害的综合防治具有一定指导意义.

In this study, we used virus-mediated gene silencing technology and found that the HSP17.4 gene-silenced cultivar Sweet Charlie plants were more susceptible to Colletotrichum gloeosporioides than the wild-type Sweet Charlie, and the level of infection was even higher than that of the susceptible cultivar Benihopp. The results of differential quantitative proteomics showed that after infection with the pathogen, the expression of the downstream response genes NPR1, TGA, and PR-1 of the salicylic acid (SA) signalling pathway was fully up-regulated in the wild-type Sweet Charlie, and the expression of the core transcription factor MYC2 of the jasmonic acid (JA) pathway was significantly down-regulated. The expression of the proteins encoded by these genes did not change significantly in the HSP17.4-silenced Sweet Charlie, indicating that the expression of HSP17.4 activated the up-regulation of downstream signals of SA and inhibited the JA signal pathway. The experiments that used SA, methyl jasmonate, and their inhibitors to treat plants provide additional evidence that the antagonism between SA and JA regulates the resistance of strawberry plants to C. gloeosporioides.

为明确上海地区'红美人'柑橘的病毒种类,2020年4月在崇明、金山、奉贤、嘉定、闵行和浦东采集疑似病毒感染的叶片样品11份,采用小RNA深度测序技术结合RT-PCR对不同来源的混合样品进行病毒种类鉴定.结果 表明,样品中含有柑橘衰退病毒Citrus tristeza virus (CTV)、柑橘黄化脉明病毒Citrus yellow vein clearing virus(CYVCV)、柑橘树皮裂纹类病毒Citrus bark cracking viroid(CBCVd)和柑橘类病毒Ⅴ Citrus viroid Ⅴ(CVd-Ⅴ).同时发现,上海地区'红美人'柑橘病毒复合侵染现象普遍.基于CTV和CYVCV的CP全长基因及CBCVd和CVd-Ⅴ全基因组序列系统进化分析结果表明:除了CYVCV的分离物与地理来源具有明显的相关性外,其余病毒及类病毒均没有严格的地域相关性.研究结果为开展'红美人'柑橘种苗病毒检测及病毒病的防治奠定基础.

To investigate the mechanisms of cytoplasmic male sterility (CMS) in pepper (Capsicum annuum L.), cytological observation and metabolomics analysis of different pollen development processes were carried out. The pollen development processes were divided into five stages. Resin semi-thin section analysis indicated an abnormality of the sterile anthers started at the tetrad stage (S2); the cell of the tapetal layer was over-vacuolated. The tapetal began to degrade and lose its function. The tetrad could not get essential energy and materials which resulted in pollen abortion. Anthers of S2, and uninucleate pollen stage (S3), were selected and analyzed by non-target metabolomics. A total of 454 metabolites in S2 and S3 stages of maintainer line were detected, and 201 were annotated. For the CMS line, 459 metabolites in two stages were detected and 211 were annotated. Alanine, arginine, serine, and tryptophan were found in the two stages of maintainer line. A marked decrease of glutamine and pyruvic acid during the pollen abortion process were especially found in the pepper CMS line. The decrease of pyruvate concentration further leads to the abnormal synthesis of various kinds of amino acids and ATP which may ultimately contribute to the phenotype of the CMS line.

Plants, via physiological and molecular processes, respond to unsuitable environmental conditions, resulting in stress tolerance. Most previous studies have focused on plant responses to a single abiotic stress, but the effects of combined water deficit and high temperature stresses are more severe and complex than those due to a single stress. Therefore, our study aimed to explore the differences in the damage caused by combined vs. single stresses. Grapevines were subjected to water deficit, high temperature, and water deficit plus high temperature treatments. The transcript levels of heat- and drought-stress genes, activities of photosystem II (PS II) and antioxidant enzymes (superoxide dismutase, catalase, and peroxidase), and changes in abscisic acid (ABA) biosynthesis were evaluated. The activities of PS II and antioxidant enzymes were lower under the water deficit plus high temperature treatment than under the heat treatment alone. The concentration of ABA and the transcript levels of ABA biosynthesis-related genes increased under both types of stress. The enhanced thermo-tolerance observed under drought stress could be attributed to increased PS II efficiency, as well as to changes in antioxidant pathways, mediated by a common regulatory system or including a substantial cross talk between heat- and drought-stress signaling.

Liquid chromatography and mass spectrometry based label-free proteomics and non-target metabolomics technology were used to study the proteome and metabolome change of disease-resistant grape cultivar ‘Shenfeng’ infected with Botrytis cinerea. There were 1 374 proteins and 33 metabolites showing more than 1.5-fold changes in ‘Shenfeng’ leaves infected with B. cinerea, respectively. The differentially expressed proteins and metabolites were analyzed by gene ontology annotation and bioinformatics. The results showed that B. cinerea infection changed the expression level of chloroplast proteins, and mainly focused on plant and pathogen interaction, synthesis pathways of plant hormones and alkaloids. Multi-omics analysis further showed that there was a consistent increase in the expression levels of chorismic acid, salicylic acid, isochorismic pyruvate lyase pchB, transcription factor TGA and pathogenesis-related protein PR-1 in the salicylic acid-mediated disease-resistant signal transduction pathway. The full activation of salicylic acid-mediated disease resistance signaling pathway is an effective means for grape leaves to resist B. cinerea infection. The result is of great benefit to further deeply reveal the molecular mechanisms of plant-pathogen interactions and the breeding of pathogen-resistant grape varieties.

Label-free based phosphoproteomics technology was used to study the phosphoproteome change of disease-susceptible strawberry cultivar ‘Benihopp’ and resistant cultivar ‘Sweet Charlie’ infected with Colletotrichum gloeosporioides. There were 154 and 173 phosphoproteins showing more than 1.5-fold change in ‘Benihopp’ and ‘Sweet Charlie’, respectively. The differentially expressed phosphoproteins were analyzed by gene ontology annotation and bioinformatics, and we found that, most differentially expressed phosphoproteins were involved in macromolecule complexe formation, and the biological processes of structural localization, stimulus response and signal transduction. Compared with ‘Benihopp’, ‘Sweet Charlie’ may specifically own two motif types of S*Y and T*F of differentially expressed phosphorylated peptides, and its plant hormone signal transduction pathway and carbon fixation pathway had higher levels of phosphorylation. The result is of great benefit to further deeply reveal the molecular mechanisms of plant-pathogen interactions and the breeding of pathogen-resistant strawberry cultivars.

为了改善‘沪培1号’葡萄穗肩部和穗尖部果实着色不一致问题,采取转色前对葡萄进行套袋处理,分析成熟期果皮颜色和果实糖分积累情况.结果 表明,不同套袋处理间果实可溶性固形物、葡萄糖和果糖含量无明显差异,但均显著低于不套袋处理,说明套袋对果实糖分积累有负面影响,可在成熟前2周提前脱袋;套袋处理能显著改善‘沪培1号’着色不一致现象,在生产中可推广使用.

在花期,对葡萄新梢摘心是平衡营养生长和生殖生长的重要栽培技术之一.本试验在花前7 d、初花期和盛花期,对3个四倍体鲜食葡萄品种'申丰''信浓乐''巨玫瑰'开展摘心处理,结果发现'申丰'和'信浓乐'适宜在初花期至盛花期摘心,试验中观察到花前7 d摘心更早出现卷须,消耗植株养分,对坐果不利,影响生殖生长和营养生长的平衡.在果实成熟期检测粒质量、可溶性固形物和可滴定酸含量,结果表明果实品质与摘叶处理间无相关性.

Simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) markers were applied to analyze genetic diversity among 43 strawberry (Fragaria×ananassa Duch.) cultivars and effectivenesses of these two kinds of molecular markers were also compared. The results showed that there were 6.43 polymorphic sites per primer pair of 30 SSR primers, and the average polymorphism information content (PIC) of each site was 0.628 4. While there were 14.30 polymorphic sites per primer pair of 20 SRAP primers, and the average PIC of each site was 0.911 4. The correlation coefficient between clustering results based on SSR markers and SRAP markers was 0.817, which was significant. The correlation coefficients between SSR markers, SRAP markers and SSR+SRAP joint markers were 0.938 and 0.966, respectively, which reached extremely significant levels. Both SSR and SRAP markers can be used to analyze the genetic diversity of strawberry, but the effect of SRAP marker is better than that of SSR marker. The analysis of SSR+SRAP joint markers can better evaluate the genetic diversity and genetic relationship of strawberry germplasm.

以不同抗性的3个葡萄品种‘巨峰’ ‘夏黑’ ‘金手指’的叶片为试材,对灰葡萄孢菌接种温度、湿度和时长等发病条件进行研究,形成了葡萄灰霉病抗性离体叶片快速鉴定方法,并应用该法对‘夏黑’ ‘巨峰’ ‘醉金香’ ‘阳光玫瑰’和申沪系列等在内的18个葡萄品种和杂交株系的灰霉病抗性进行了鉴定和比较.研究结果表明,温度20℃、湿度90％是灰葡萄孢菌的最佳侵染条件,在此条件下接种2d后的病情指标可以较好地区分18个葡萄品种(系)对灰霉病的抗性差异. ‘沪22’对灰葡萄孢菌显示高抗,而‘沪21’和‘沪27’显示高感.

The various post-translational modifications (PTMs) of plant proteins have important regulatory roles in development. We therefore examined various modified proteins from strawberry stigmata and found that succinylation of lysine residues was the most abundant type of modification. We then subjected proteins from strawberry stigmata to an efficient enrichment method for succinylated peptides and identified 200 uniquely succinylated lysines (Suks) in 116 proteins. A bioinformatics analysis revealed that these proteins are involved in important biological processes, including stress responses, vesicular transport, and energy metabolism. Proteomics, combined with immunoprecipitation and immunoblotting, revealed an obvious increase in succinylation of the assembly polypeptide 2 (AP2) and clathrin from 0.5 to 2 h after pollination, suggesting that succinylation is involved in the recognition of pollen-stigma signaling substances and vesicular transport. These results suggest that AP2/clathrin-mediated vesicular transport processes are regulated by lysine succinylation during pollen recognition.