绿色荧光蛋白(GFP)标记杧果细菌性角斑病病原菌及其定殖规律

为研究杧果细菌性角斑病病菌在杧果不同器官组织中的定殖规律。利用电击转化法将质粒pBBR1MCS2-Tac-EGFP导入野油菜黄单胞菌杧果致病变种(Xanthomonas citri pv.mangiferaeindicae)菌株Xcm003中,通过转化子生长表型和外源基因的PCR鉴定,成功获得带绿色荧光蛋白(GFP)标记的转化子Xcm003-EGFP,采用室内刺伤和盆栽苗灌根接种试验,结合组织学观察法,追踪该病原菌在不同杧果器官组织中的定殖规律。结果表明,病原菌通过伤口侵入杧果果实果皮外层细胞层,随着侵染时间推移,向内层果皮细胞层垂直扩散并定殖,后期病原菌在伤口处大量聚集,出现隆起开裂状病斑。在杧果叶片中,病原菌从伤口侵入叶片上表皮细胞层,随后迁移到叶肉细胞间隙,侵染后期,病原菌在气孔和伤口定殖,造成气孔和伤口堵塞,导致接种点出现黑褐色水浸病斑。盆栽苗灌根后发现,该病原菌可在土壤中定殖,入侵杧果根部,但并不扩散至其他杧果组织,不会为害整株幼苗。说明杧果细菌性角斑病病菌可在不同杧果组织中定殖,但仅表现为局部侵染特性。

化橘红挥发油对罗汉果斑枯病菌的抑制作用及机理

采用平板抑菌法结合盆栽防治试验测定化橘红(Citri grandis Exocarpium)挥发油对罗汉果(Siraitia grosvenorii)斑枯病菌(Stagonosporopsis cucurbitacearum)的抑制活性。通过显微观察病原菌菌丝体的形态及测定挥发油处理病原菌菌丝体后处理液的电导率及可溶性蛋白质含量变化情况,探究化橘红挥发油的抑菌机理。平板抑菌结果表明,化橘红挥发油稀释≤1 000倍对罗汉果斑枯病病原菌菌丝生长的抑制率均达100.0%,显著高于80%多菌灵可湿性粉剂稀释1 000倍的抑制率(88.7%)。盆栽试验结果表明,化橘红挥发油稀释1 000倍,45%戊唑·咪鲜胺乳油和10%苯醚甲环唑乳油对罗汉果斑枯病病斑扩展的抑制率均在80.0%以上,且均显著高于80%多菌灵可湿性粉剂的抑制率(70.68%)。显微镜下观察发现,经化橘红挥发油处理7 d的病原菌菌丝呈念珠状膨大并有断裂现象。菌丝体与化橘红挥发油共培养结果表明,混合液的电导率及可溶性蛋白质含量均随着化橘红挥发油处理浓度的加大、互作时间的延长而提高;浓度为2.0 g/L的化橘红挥发油与病原菌菌丝共培养48 h,培养液的电导率为279.208μS/cm,为各处理最大值;挥发油终质量分数0.2%、作用60 h后,培养液中的可溶性蛋白质含量达90.6μg/mL,为各处理的最大值。

Activation of autophagy by Citri Reticulatae Semen extract ameliorates amyloid-beta-induced cell death and cognition deficits in Alzheimer’s disease

Amyloid-beta-induced neuronal cell death contributes to cognitive decline in Alzheimer’s disease.Citri Reticulatae Semen has diverse beneficial effects on neurodegenerative diseases,including Parkinson’s and Huntington’s diseases,however,the effect of Citri Reticulatae Semen on Alzheimer’s disease remains unelucidated.In the current study,the anti-apoptotic and autophagic roles of Citri Reticulatae Semen extract on amyloid-beta-induced apoptosis in PC12 cells were first investigated.Citri Reticulatae Semen extract protected PC12 cells from amyloid-beta-induced apoptosis by attenuating the Bax/Bcl-2 ratio via activation of autophagy.In addition,Citri Reticulatae Semen extract was confirmed to bind amyloid-beta as revealed by biolayer interferometry in vitro,and suppress amyloid-beta-induced pathology such as paralysis,in a transgenic Caenorhabditis elegans in vivo model.Moreover,genetically defective Caenorhabditis elegans further confirmed that the neuroprotective effect of Citri Reticulatae Semen extract was autophagy-dependent.Most importantly,Citri Reticulatae Semen extract was confirmed to improve cognitive impairment,neuronal injury and amyloid-beta burden in 3×Tg Alzheimer’s disease mice.As revealed by both in vitro and in vivo models,these results suggest that Citri Reticulatae Semen extract is a potential natural therapeutic agent for Alzheimer’s disease via its neuroprotective autophagic effects.

Resistance Monitoring of Field Populations of Panonychus citri to Three Pesticides

[Objectives]The paper was to investigate the development of Panonychus citri resistance to commonly used pesticides in major citrus producing areas in China,and to screen out new green and efficient agents for its control and prevention.[Methods]The resistance changes of field populations of P.citri to abamectin,pyridaben,and bifenazate in 12 locations across five provinces in China were compared using the leaf disc impregnation method.[Results]P.citri in the tested areas exhibited the most severe resistance to abamectin,with approximately 91.7%of the field populations showing high levels of resistance to abamectin(112.1-560.5 times);50%of P.citri populations exhibited high levels of resistance to pyridaben(123.0-202.7 times),while the remaining populations showed intermediate levels of resistance(25.6-80.3 times);except for the Zhejiang Xiangshan 2019 and Jiangxi Yudu 2019 populations,which exhibited a medium level of resistance to bifenazate,the remaining 10 monitored populations demonstrated a high level of resistance to bifenazate(140.4-686.1 times).[Conclusions]It is advisable to discontinue the use of abamectin and bifenazate due to significant resistance observed in populations of P.citri monitored in 12 locations across 5 provinces.It is recommended to reduce the frequency of pyridaben use and alternate with other acaricides that have different mechanisms of action due to the varying degrees of resistance developed.

A Network Pharmacology Study on Active Components and Targets of Citri Reticulatae Pericarpium for Treating Keloids

[Objectives]To investigate the mechanisms and pharmacologic effects of Citri Reticulatae Pericarpium against keloids by network pharmacology systematically.[Methods]TCMSP,Uniprot and BATMAN-TCM databases were used to obtain the active constituents and targets of Citri Reticulatae Pericarpium."Keloid"was used as key word to search for related therapeutic targets from Drug Bank,OMIM,TTD,and GEO databases.The Chinese medicine compound-target network was constructed by Cytoscape software.Besides,gene ontology(GO)and Kyoto Encyclopedia of genes and genome enrichment analysis were also performed.Afterward,Discovery Studio software was used to assess the interaction of key components and genes.[Results]Five active components of Citri Reticulatae Pericarpium,773 compound targets and 676 keloid treatment targets were obtained in the databases.After the intersection,there are 47 targets of Citri Reticulatae Pericarpium for treating keloids.Hub genes were identified such as MMP9,IL6,TNF,TP53,and VEGFA,which were enriched in tumor necrosis factor-α,nuclear factor kappa-B,and other signaling pathways.The molecular docking stimulation confirmed the interaction between the MMP9 and three components of Citri Reticulatae Pericarpium.[Conclusions]Citri Reticulatae Pericarpium may play an important role in treating keloids through modulating genes and signaling pathways.The present study sheds light on the mechanisms of active compounds of Citri Reticulatae Pericarpium for the treatment of keloids.

环境和气象因素对柑橘黄龙病重要传播媒介——柑橘木虱发生规律的影响

研究了柑橘木虱(Diaphorina citri)对环境和气象因素的敏感性,以及其与柑橘黄龙病(Huanglongbing,HLB)传播的关系。总结了柑橘木虱(Diaphorina citri)的基本情况,并探究了环境因素(如海拔、纬度、土壤、田间管理、寄主和生物因素)和气象因素(如温度、湿度、降雨、光照和气压)对其发生规律的影响。这些信息有助于预测柑橘木虱的发生情况和制定有效的防治策略,从而减少柑橘黄龙病的传播风险。

Transcriptomic analysis reveals the defense mechanisms of citrus infested with Diaphorina citri

Huanglongbing(HLB) is a devastating disease that has led to an acute crisis for growers of citrus, one of the world's most important fruit crops. The phloem-feeding Asian citrus psyllid(ACP), Diaphorina citri, is the main pest at the new shoot stage and is the only natural vector of HLB pathogenic bacteria. Little is known about how plants perceive and defend themselves from this destructive pest. Here, we characterized changes in the expression of various genes in citrus plants that were continuously infested by D. citri for different durations(12, 24, and 48 h). A total of 5 219 differentially expressed genes(DEGs) and 643 common DEGs were identified across all time points. Several pathways related to defense were activated, such as peroxisome, alpha-linolenic acid metabolism, and phenylpropanoid and terpenoid biosynthesis, and some pathways related to growth and signal transduction were suppressed in response to D. citri infestation. The expression of genes including kinases(CML44, CIPK6, and XTH6), phytohormones(SAMT, LOX6, and NPR3), transcription factors(bHLH162, WRKY70, and WRKY40), and secondary metabolite synthesis-related genes(PAL, 4CL2, UGT74B1 and CYP82G1) was significantly altered in response to D. citri infestation. The findings of this study greatly enhance our understanding of the mechanisms underlying the defense response of citrus plants to D. citri infestation at the molecular level. Functional characterization of the candidate defense-related genes identified in this study will aid the molecular breeding of insect-resistant citrus varieties.

Occurrence of Diaphorina citri in Guangxi and Integrated Control Technique

In order to promote the healthy and sustainable development of citrus industry in Guangxi and reduce the harm of Diaphorina citri and citrus Huanglongbing on citrus industry,the paper summarizes the occurrence of D.citri in Guangxi,and put forward comprehensive control technique,so as to provide the reference for guiding prevention and control of major pests in citrus industry.

Overview of Occurrence,Prevention and Control of Panonychus citri in China

In order to further promote the prevention and control research of Panonychus citri and improve its control effect,this paper summarizes the main influencing factors of the outbreak,main control technology and drug resistance of P.citri,and puts forward the research focus and control strategy.

几种杀虫剂对柑橘木虱的毒力及防效研究

在室内条件下，采用浸虫法测定了10种杀虫剂对柑橘木虱的毒力，结果表明，高效氯氟氰菊酯和溴氰菊酯对柑橘木虱成虫具有较高的毒力，其24 h的 LC50值分别为2.116、3.030 mg ·L -1，而吡蚜酮、螺虫乙酯和吡虫啉毒力较差，尤其以吡蚜酮的毒力最差，其24 h的LC50值为2025.489 mg · L -1；采用灌根施药法测定了4种内吸性杀虫剂对柑橘木虱成虫的防效，4种供试验药剂各处理对柑橘木虱成虫的短期效果较差，但10％烯啶虫胺2000倍液、70％吡虫啉14000倍液及7000倍液处理灌根施药后14～28 d对柑橘木虱成虫的防效均保持在87％以上，至药后28 d防效高达97％以上，持效期长。

柑橘皮多糖抗氧化性研究

[目的]研究柑橘皮多糖的抗氧化活性。[方法]利用水提醇沉法,提取柑橘皮多糖并进行初步纯化,采用苯酚-硫酸法测定柑橘皮多糖的提取率并采用水杨酸法研究柑橘皮多糖清除.OH的效果。[结果]柑橘皮中多糖的含量为6.32%;不同浓度的柑橘皮多糖对H2O2/Fe2+体系通过Fenton法反应产生的.OH都具有清除作用,且随浓度的增加,清除率逐渐升高,即清除率与多糖的用量存在一定的量效关系。[结论]柑橘皮多糖具有明显地抗氧化性,对.OH具有清除作用。

赣南脐橙园柑橘红蜘蛛空间分布格局

通过对赣南脐橙园柑橘红蜘蛛(Panonychus citri)的田间调查,发现柑橘红蜘蛛在叶片、树冠和植株3个空间尺度的分布型均为聚集分布;柑橘红蜘蛛在脐橙园的分布常存在中心株,且树冠的各个方位分布无显著差异;卵多产在叶的主脉两侧,尤以叶基部主脉两侧居多。根据赣南脐橙园柑橘红蜘蛛分布规律,提出了相应的防治对策。

有机柑橘种植中五种溃疡病防治药剂作用评价

通过田间药效试验筛选防治柑橘溃疡病(Xanthomonas axonopodis pv.citri)的有效有机药剂,以清水为对照,工农-16型背负手摇喷雾器喷雾,以咪鲜.松、代森铵和叶青双作比较,对比研究了硫酸铜钙、噻菌铜、四霉素、春雷霉素与王铜混配剂、水胆矾和两水硫酸钙结合体等5种杀菌剂对柑橘溃疡病的防治效果。结果表明,硫酸铜钙、噻菌铜防治效果较好,与咪鲜.松相当,四霉素、春雷霉素与王铜混配剂(加瑞农)、水胆矾和二水硫酸钙结合体防治效果较差,低于代森铵和叶青双;首次对上述药剂的抗雨水能力进行了评价,结果表明,加瑞农、咪鲜.松抗雨水能力最强,其次是硫酸铜钙、水胆矾和两水硫酸钙结合体,噻菌铜、四霉素效果较差,代森铵、叶青双最差。试验中各处理均无药害产生。

橘全爪螨种群动态与环境因素的研究

通过田间试验和数理统计对橘全爪螨(Panonychus citri Me Gregor)田间动态及环境因素对其的影响进行探讨。橘全爪螨在湖北温州蜜柑产区一年有2次虫口高峰,分别出现在5～6月和9～10月。温度是影响橘全爪螨生长发育的主要因素,螨与卵的田间增长在旬平均气温15～20℃时最快。虫口高峰一般出现在22～27℃、相对湿度为75%～83%时。长期阴雨不利于其种群发展,旬雨量超过100 mm引起种群密度急剧下降。如果环境条件适合则种群增殖力随其成螨密度增加而增强。田间天敌混合种群与害螨田间动态符合Leslie模型,当天敌量达到每叶0.05～0.08头时,可控制害螨种群发展。

橘全爪螨种群空间分布与危害

通过理论分布的适合性检验、空间格局指标分析,对橘全爪螨(Panonychus citri McG)在温州蜜柑上的空间分布特征、动态与危害进行了系统研究。结果表明,橘全爪螨田间种群分布符合负二项分布,低密度下偶尔符合Neyman A分布。空间分布的基本成分为疏松的个体群,个体群内成员间的分布为随机分布,个体群间呈聚集分布,聚集强度随种群密度的变化而变化,种群低密度下的聚集主要由环境因素引起,高密度下的聚集与该螨自身生物学特性有关。当田间种群数量达到某一危害级别时,相应级别的危害空间容积不超过总体空间的30%,相应的田间药剂防治面积也不应超过总面积的30%。

柑桔酸腐病菌的分离鉴定及系统发育树的构建

从来自浙江省多个柑桔种植区的58份具有酸腐病状的柑桔果实样本上，分离得到6株致病真菌。致病力检测结果表明，G50菌株致病力最强，对桩柑果实有伤接种茵悬液后6天即致全果腐烂。6株真菌的ITS序列已在GenBank中登录，登录号为JQ425846-JQ425851，其中5株菌与GenBank中的AF411060茵株的相似度〉94％。基于核糖体DNA-ITS的系统发育分析表明，G10与G27距离最近，6株分离菌株与GenBank中的AF411060、HM999937聚在同一分支上，均为酸腐菌Geotrichumcirri—aurantii。

广西金柑黑点病病原菌形态学及分子生物学鉴定

近年广西金柑黑点病发生呈逐年上升趋势,为鉴定广西金柑黑点病的病原,从广西阳朔县和融安县10年生金柑上采集黑点病组织样品,按柯赫法则获取病原,采用形态学方法对病原进行初步鉴定,然后采用rDNA-ITS系列分析技术及系统发育分析方法对其进一步鉴定。结果表明,金柑黑点病分离病原真菌为柑桔间座壳菌Diaporthe citri(无性态为Phomopsis citri)。

Establishment of HPLC Fingerprint, Cluster Analysis and Principle Component Analysis of Citri Reticulatae Pericarpium Viride

[Objectives] This study aimed to establish HPLC fingerprint and conduct cluster analysis and principle component analysis for Citri Reticulatae Pericarpium Viride. [Methods] Using the HPLC method, the determination was performed on XSelect~® HSS T3-C_(18) column with mobile phase of acetonitrile-0.5% acetic acid solution(gradient elution) at the flow rate of 1.0 mL/min. The detection wavelength was 360 nm. The column temperature was 25℃. The sample size was 10 μL. With peak of hesperidin as the reference, HPLC fingerprints of 10 batches of Citri Reticulatae Pericarpium Viride were determined. The similarity of the 10 batches of samples was evaluated by Similarity Evaluation System for Chromatographic Fingerprint of TCM(2012 edition) to determine the common peaks. Cluster analysis and principal component analysis were performed by using SPSS 17.0 statistical software. [Results] The HPLC fingerprints of the 10 batches of medicinal materials had total 11 common peaks, and the similarity was 0.919-1.000, indicating that the chemical composition of the 10 batches of medicinal materials was consistent. There were 11 common components in the 10 batches of medicinal materials, but their contents were different. When the Euclidean distance was 20, the 10 batches of samples were divided into two categories, S4 in the first category, and the others in the second one. When the Euclidean distance was 5, the second category could be further divided into two sub-categories, S1 and S10 in one sub-category, and S2, S3, S5, S6, S7, S8 and S9 in the other one. The principle component analysis showed that cumulative contribution rate of the two main component factors was 92.797%, and the comprehensive score of S7 was the highest with the best quality. [Conclusions] The results of HPLC fingerprinting, cluster analysis and principle component analysis can provide reference for the quality control of Citri Reticulatae Pericarpium Viride.

A survey of FLS2 genes from multiple citrus species identifies candidates for enhancing disease resistance to Xanthomonas citri ssp. citri.

Pathogen-associated molecular patterns(PAMPs)-triggered immunity(PTI)is an important component of plant innate immunity.In a previous study,we showed that the PAMP flg22 from Xanthomonas citri ssp.citri(Xflg22),the causal agent of citrus canker,induced PTI in citrus,which correlated with the observed levels of canker resistance.Here,we identified and sequenced two bacterial flagellin/flg22 receptors(FLS2-1 and FLS2-2)from‘Duncan’grapefruit(Citrus paradisi,CpFLS2-1 and CpFLS2-2)and‘Sun Chu Sha’mandarin(C.reticulata,CrFLS2-1 and CrFLS2-2).We were able to isolate only one FLS2 from‘Nagami’kumquat(Fortunella margarita,FmFLS2-1)and gene flanking sequences suggest a rearrangement event that resulted in the deletion of FLS2-2 from the genome.Phylogenetic analysis,gene structure and presence of critical amino acid domains all indicate we identified the true FLS2 genes in citrus.FLS2-2 was more transcriptionally responsive to Xflg22 than FLS2-1,with induced expression levels higher in canker-resistant citrus than in susceptible ones.Interestingly,‘Nagami’kumquat showed the highest FLS2-1 steady-state expression levels,although it was not induced by Xflg22.We selected FmFLS2-1,CrFLS2-2 and CpFLS2-2 to further evaluate their capacity to enhance bacterial resistance using Agrobacterium-mediated transient expression assays.Both FmFLS2-1 and CrFLS2-2,the two proteins from canker-resistant species,conferred stronger Xflg22 responses and reduced canker symptoms in leaves of the susceptible grapefruit genotype.These two citrus genes will be useful resources to enhance PTI and achieve resistance against canker and possibly other bacterial pathogens in susceptible citrus types.

The small and large subunits of carbamoyl-phosphate synthase exhibit diverse contributions to pathogenicity in Xanthomonas citri subsp.citri

Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citrisubsp. citri, carA and carB encode the small and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that carA did not affect any of the phenotypes, while carB played multiple roles in pathogenicity. The deletion of carB rendered the loss of pathogenicity in host plants and the ability to induce a hyper- sensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type Ill secretion system were suppressed when interacting with citrus plants. The mutation in carB also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracellular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp, citri.