越南地区柑橘黄龙病发生及防治

黄龙病是全球广泛发生的毁灭性病害,柑橘木虱为其主要媒介昆虫。越南最早于上个世纪六十年代有相关报道。通过一些国际合作项目,越南对柑橘木虱和黄龙病进行了一系列研究并积极采取措施有效防控:包括消除病株、筛选抗病虫品种、改变种植密度和时间、化学防治与生物防治、作物间种、喷施矿物油乳剂、施用有机肥、使用无病苗和无病接穗并在运输过程中封闭保护等。其中控制黑臭蚁Dolichoderus thoracicus增殖黄猄蚁Oecophylla smaragdina、交错式间种番石榴以控制柑橘木虱和黄龙病值得中国借鉴和参考。     

防治果园周边柑橘木虱控制黄龙病效果研究

在新种柑橘园中栽种柑橘无毒苗,定期监测和防治果园内柑橘木虱Diaphorina citri Kuwayama,防治时期为各新梢萌芽期,重点防治期为早夏梢期和早秋梢期;与此同时,调查柑橘园周围柑橘木虱寄主植物,每年2月下旬、5月上旬、8月下旬和11月下旬定期喷药,清除柑橘园周围寄主植物上的柑橘木虱。结果表明,果园内柑橘木虱和黄龙病均得到有效的控制,果园内柑橘木虱种群数量控制在很低的水平,最高为225头/公顷,最低为0;黄龙病发病率也很低,2008年为8‰,实施该技术后,发病率逐年下降,2012年降低到01‰。     

不同种类寄主对柑橘黄龙病菌侵染能力及虫媒适应性的影响

柑橘木虱Diaphorina citri是柑橘黄龙病菌“Candidatus Liberibacter asiaticus”的主要媒介昆虫,探明不同植物种类寄主对柑橘黄龙病菌侵染能力及柑橘木虱适应性的影响,在设计黄龙病防控策略及寄主抗性研究中具有参考价值。本文选取蕉柑Citrus reticulata、蜜柚Citrus grandis、黄皮Clausena lansium和九里香Murraya exotica为研究材料,对比其田间植株黄龙病发病率和柑橘木虱数量及带菌情况,并在实验室条件下比较柑橘木虱对不同寄主的适应性（包括发育历期、存活率、携菌能力和传菌能力）。结果发现：（1）田间蕉柑和蜜柚的感染率最高,分别为47%和37%;而黄皮和九里香的带菌率较低,分别为18%和4%,且菌浓度均较轻微;（2）田间九里香上柑橘木虱卵、若虫及成虫数量均最多,蕉柑和黄皮其次,蜜柚最少,但蕉柑和蜜柚上柑橘木虱的携菌率和携菌量最高;（3）4种寄主上柑橘木虱卵和1~3龄若虫的发育历期没有显著性差异,柑橘木虱4龄、5龄若虫和总若虫历期在九里香上最短,而在蜜柚上最长;4种寄主上4龄、5龄若虫存活率没有显著性差异,但蜜柚上木虱卵的孵化率、1~3龄及总若虫存活率最低。（4）蕉柑和蜜柚上带菌木虱的携菌能力及传菌能力最高,其次为黄皮,九里香最低。该研究证明了除了寄主植物本身抗病性差异外,虫媒对寄主植物的适应性差异也是造成田间黄龙病发病差异的重要因素。     

柑橘黄龙病株不同部位内生细菌群落结构的多样性

采用PCC(Pearson correlation coefficient)、系统聚类和多样性指数研究了柑橘黄龙病(citrus huanglongbing,HLB)植株不同部位叶片、枝条及其根部内生细菌的相互关系及其群落结构多样性.结果表明,从供试柑橘中分离得到26株内生细菌(叶片10株,枝条14株,根部2株)隶属于10个属19个种.Nested-PCR结果显示,柑橘植株不同空间叶片中,黄龙病病原的阳性检出率为58.3％.用LSD法比较显示,柑橘内生细菌在不同器官的分布量大小依次为根＞叶＞枝条.上、中、下不同部位叶片和枝条内生细菌分布量差异不显著,而东、西、南、北不同方位的叶片和枝条内生细菌分布量差异显著(P＜5％).柑橘内生细菌之间的PCC分析表明,Bacillus pumilus和Bacillus sp.与柑橘黄龙病病原菌呈显著负相关,Bacillus sp.和Bacillus pumilus与Bacillus subtilis呈显著正相关.Bacillus brevis和Escherichia hermannii与Bacillus vesiculariss存在显著正相关.对柑橘不同器官内生细菌的PCC分析表明,同种器官的内生细菌之间呈正相关,不同器官的内生细菌之间呈负相关.以内生细菌在各器官的分布量为指标分析不同器官之间的相关性,结果表明,叶片与枝条呈正相关,PCC为0.55,叶片与根部、枝条与根部均呈负相关,PCC分别为-0.19和-0.13.16S rDNA序列聚类分析表明,柑橘内生细菌可分为两大类,第Ⅰ大类为革兰氏阳性菌,第Ⅱ大类为革兰氏阴性菌.第Ⅰ大类又可以分为芽孢杆菌属和短小杆菌属两个亚类,第Ⅱ大类也可以分两个亚类.对柑橘黄龙病病原与叶片内生细菌进行聚类分析,可以分为三类,第Ⅰ类特征为内生细菌存在所有部位的叶片;第Ⅱ类特征为内生细菌与黄龙病病原菌存在负相关性;第Ⅲ类特征为内生细菌只存在特定部位叶片.当马氏距离为27.23时,可将柑橘黄龙病内生细菌群落结构聚为3类,第Ⅰ类特点是在不同部位叶片均有分布,为完全分布类型,且分布量较大,第Ⅱ类特点是在各器官中均为不完全分布类型,且分布量不均匀,第Ⅲ类特点是根部分布特性且分布量较大.分析多样性指数表明,SHANNON(H1)指数在叶片最高,根部最低.SIMPSON(D)指数在枝条最高,根部最低.Pielou指数在根部最高,枝条最低.     

柑橘黄龙病菌内参基因的筛选与评估

【背景】柑橘黄龙病是世界柑橘生产上最具毁灭性的病害之一,主要由候选韧皮部杆菌属亚洲种("Candidatus Liberibacter asiaticus",CLas)引起。CLas全基因组测序已经完成,因而该病原菌的基因表达研究和功能验证得以进行。【目的】筛选CLas内参基因并评估其不同侵染时期和在不同品种植物寄主中的表达稳定性。【方法】基于基因功能类别,利用实时荧光定量PCR技术分析23个CLas的候选内参基因相对表达情况(16Sr RNA基因作为参照基因)。结合Ct值标准差和geNorm、NormFinder、RefFinder软件,评价内参基因的表达稳定性。【结果】在感染黄龙病不同时期和不同品种的植物寄主样本中,14对引物表现出较强的特异性和稳定性。内参基因稳定性排名:ftsZgyrArpoB1ftsAsecAgapzapEgmk2rpoDsecYrpoOftsWgmk1recA,根据geNorm配对变异值Vn/n+1选择稳定性最好的ftsZ和gyrA作为内参基因作进一步评估。以ftsZ+gyrA以及16S rRNA基因分别作为内参基因检测柑橘黄龙病菌致病基因LasΔ5313的表达水平,所得的表达模式相同。【结论】柑橘黄龙病菌中涉及DNA复制和细胞分裂功能的管家基因表达较稳定,在CLas的基因表达研究中可选择ftsZ+gyrA的基因组合作为内参。本研究为后续利用实时荧光定量PCR分析CLas基因表达及研究CLas致病机理奠定基础。     

亚洲柑橘木虱与柑橘黄龙病菌互作的研究进展

亚洲柑橘木虱Diaphorina citri Kuwayama作为柑橘产业重要病害柑橘黄龙病的主要传播媒介,已经成为重点防治对象。该害虫与黄龙病之间的互作一直是相关研究的热点,本文就近年来该领域的研究进展做了一个总结,从亚洲柑橘木虱的获菌与传病机制、病原菌与柑橘木虱之间的互作以及病原菌感染寄主植物后对木虱的影响等方面进行了综述。期望为深入开展黄龙病相关研究、寻找防控新途径提供依据。     

亚洲柑橘木虱传播黄龙病菌的特性与机制研究进展

黄龙病Huanglongbing(HLB)是世界性的重大柑橘病害,在中国主要由亚洲柑橘木虱Diaphorina citri Kuwayama传播。该害虫的传病特性与机制一直是相关研究热点之一。本文综述了近些年该领域的研究成果,包括亚洲柑橘木虱传播黄龙病的方式、传病过程与机制,以及影响传病效率的因素等方面,为该木虱及黄龙病的防控与深入研究提供参考。     

橘园蓟马的种类、发生规律及防治研究

本文以我国柑橘主产区当阳和赣州为试验基地,系统调查了橘园中蓟马为害的严重性及其发生规律;经鉴定发现我国柑橘主产区橘园内蓟马有11种:包括蓟马科中的茶黄蓟马Scirtothrips dorsalis Hood、褐三棕蓟马Lefroyothrips lefroyi(Bagnall)、花蓟马Frankliniella intonsa(Trybom)、八节黄蓟马Thripsflavidulus(Bagnall)、黄胸蓟马T.hawaiiensis(Morgan)、棕榈蓟马T.palmi Karny、色蓟马T.coloratus Schmutz和管蓟马科中的边腹曲管蓟马Rhaebothrips lativentris Karny、华简管蓟马Haplothrips chinensis Priesner、桔简管蓟马H.subtilissimus(Haliday)、狭翅简管蓟马H.tenuipennis Bagnall;并对蓟马进行药剂防治试验,结果表明,吡虫啉、敌百虫和阿维菌素3种药剂都能有效的减少蓟马的虫口数目,在柑橘花期之前和柑橘花期施药可以有效的降低蓟马对果实的为害率。     

基于23S／5SrDNA序列的柑橘黄龙病病原菌亚洲种系统发育研究

根据柑橘黄龙病亚洲种23S/5S的DNA序列设计一对引物对不同地理来源的6个柑橘黄龙病样品DNA进行扩增,扩增片段大小均为1 654 bp包括一个假定细胞壁水解酶假基因(putative cell wall hydrolase pseudogene)和5S rRNA 基因.序列同源性分析结果表明;6个柑橘黄龙病病原菌样品与柑橘黄龙病病原菌亚洲种Sihui样品的同源性为99%,然而与土壤杆菌,布鲁氏菌,根瘤菌,中华根瘤菌,巴通体菌和中慢生根瘤菌的同源性只有89%～95%,说明在23S/5S rDNA序列上黄龙病病原菌亚洲种与α变形菌纲根瘤菌目的其他病原菌相差较大.对黄龙病病原菌亚洲种种内的23S/5S rDNA序列进行比较分析,结果发现黄龙病病原菌亚洲种种内之间putative cell wall hydrolase pseudogene和5S rRNA的基因序列非常保守,但不同地理来源的柑橘黄龙病样品碱基序列间确实存在差异,差异的大小与地理的远近无关.利用简约法对黄龙病病原菌亚洲种及α变形菌纲其它病原菌的23S/5S rDNA序列构建的系统发育树显示黄龙病病原菌亚洲种单独聚为一类,其他细菌聚为另一类,该结果与基于rplJ基因及16S rRNA基因的DNA序列构建的分子系统进化树结果一致.     

广东省马尾松毛虫发生规律的研究

马尾松毛虫在广东省北部地区每年发生3代,南部每年发生4代。根据历年不同地区马尾松毛虫的发生情况进行分析,中部地区发现面积小,年间波动小,其发生呈下降趋势,西南部则是发生面积大,年间波动很小,发生呈增长,趋势一种中间形式的类型区是一个狭长的带。分布于西部边缘地区和中部与东部、北部的过渡区。     

In vitro egg hatching of Diaphorina citri,the vector of Huanglongbing

Citrus greening (Huanglongbing, HLB) is a destructive disease associated with the uncultivable, phloem-limited, gram-negative bacteria Candidatus Liberibacter spp., which affects citrus crops and other Rutaceae species. HLB is rapidly transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). We developed an artificial medium on which D. citri eggs can hatch and first-instar nymphs can feed. The medium could be used to study insect physiology and screen molecules that may interfere with egg hatching and nymph development.     

GC-MS analysis of headspace and liquid extracts for metabolomic differentiation of citrus Huanglongbing and zinc deficiency in leaves of 'Valencia' sweet orange from commercial groves

Introduction – Citrus Huanglongbing (HLB) is considered the most destructive citrus disease worldwide. Symptoms‐based detection of HLB is difficult due to similarities with zinc deficiency. Objective – To find metabolic differences between leaves from HLB‐infected, zinc‐deficient, and healthy ‘Valencia’ orange trees by using GC‐MS based metabolomics. Methodology – Analysis based on GC‐MS methods for untargeted metabolite analysis of citrus leaves was developed and optimized. Sample extracts from healthy, zinc deficient, or HLB‐infected sweet orange leaves were submitted to headspace solid phase micro‐extraction (SPME) and derivatization treatments prior to GC‐MS analysis. Results – Principal components analysis achieved correct classification of all the derivatized liquid extracts. Analysis of variance revealed 6 possible biomarkers for HLB, of which 5 were identified as proline, β‐elemene, (‐)trans‐ caryophyllene, and α‐humulene. Significant (P < 0.05) differences in oxo‐butanedioic acid, arabitol, and neo‐inositol were exclusively detected in samples from plants with zinc deficiency. Levels of isocaryophyllen, α‐selinene, β‐selinene, and fructose were significantly (P < 0.05) different in healthy leaves only. Conclusion – Results suggest the potential of using identified HLB biomarkers for rapid differentiation of HLB from zinc deficiency. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolomic analysis elucidates how shade conditions ameliorate the deleterious effects of greening (Huanglongbing) disease in citrus

Under tropical and subtropical environments, citrus leaves are exposed to excess sunlight, inducing photoinhibition. Huanglongbing (HLB, citrus greening), a devastating phloem-limited disease putatively caused by Candidatus Liberibacter asiaticus, exacerbates this challenge with additional photosynthetic loss and excessive starch accumulation. A combined metabolomics and physiological approach was used to elucidate whether shade alleviates the deleterious effects of HLB in field-grown citrus trees, and to understand the underlying metabolic mechanisms related to shade-induced morpho-physiological changes in citrus. Using metabolite profiling and multinomial logistic regression, we identified pivotal metabolites altered in response to shade. A core metabolic network associated with shade conditions was identified through pathway enrichment analysis and metabolite mapping. We measured physio-biochemical responses and growth and yield characteristics. With these, the relationships between metabolic network and the variables measured above were investigated. We found that moderate-shade alleviates sink limitation by preventing excessive starch accumulation and increasing foliar sucrose levels. Increased growth and fruit yield in shaded compared with non-shaded trees were associated with increased photosystem II efficiency and leaf carbon fixation pathway metabolites. Our study also shows that, in HLB-affected trees under shade, the signaling of plant hormones (auxins and cytokinins) and nitrogen supply were downregulated with reducing new shoot production likely due to diminished needs of cell damage repair and tissue regeneration under shade. Overall, our findings provide the first glimpse of the complex dynamics between cellular metabolites and leaf physiological functions in citrus HLB pathosystem under shade, and reveal the mechanistic basis of how shade ameliorates HLB disease.     

佛冈县不同类型橘园柑橘木虱的种群动态

[目的] 明确佛冈县不同类型橘园柑橘木虱的种群动态,为当地柑橘木虱及柑橘黄龙病的防治工作提供科学依据。[方法] 通过2015-2016年的系统调查,对广东省佛冈县砂糖橘园的柑橘木虱种群消长动态进行研究,对比分析了不同处理措施对柑橘木虱种群数量的影响。[结果] 1-3月柑橘木虱发生危害较轻,随着嫩梢增多,其种群数量开始上升,6-9月为种群发生高峰期,期间出现多个发生高峰,10月之后种群数量逐渐进入消退期,种群数量维持在较低水平;2015、2016年佛冈县柑橘木虱的季节性消长动态基本一致,但种群发生量存在明显差别;3种不同类型橘园内柑橘木虱种群发生数量也存在显著差异,常年失管橘园柑橘木虱种群发生数量最高,常规水肥管理橘园次之,二者均显著高于常规水肥管理及化学防治橘园。[结论] 化学防治可在一定程度上控制柑橘木虱的种群数量,及时清理失管橘园对控制柑橘木虱和柑橘黄龙病传播至关重要。     

Factors affecting incidence and severity of leaf spot disease on lettuce caused by Septoria birgitae

In the 1990s during wet seasons a new disease causing brown leaf spots on lettuce (Lactuca sativa) was found for the first time in many lettuce‐growing areas of Austria and Germany. The causal agent, a new pathogenic species called Septoria birgitae, may be responsible for total crop loss. To study how temperature, inoculum density and leaf wetness period influence disease incidence and severity of leaf spot on lettuce caused by S. birgitae, we carried out in vivo experiments in growth chambers and in the field. Additionally, we evaluated the relevance of infected plant debris acting as a primary inoculum source in soil for subsequent crops. S. birgitae produces spores over a wide temperature range between 5°C and 30°C, and can infect plants at temperatures between 10°C and 30°C, with an optimum between 20°C and 30°C. Spores of S. birgitae at a density of at least 10³ conidia mL^–1 are essential for disease outbreak on lettuce. Because leaf wetness is crucial for releasing conidia from pycnidia, we studied the impact of leaf wetness duration on disease development under various temperature conditions. For relevant leaf spot disease development on lettuce in vivo, a leaf wetness duration of at least 24 h and temperatures higher than 10°C were necessary. Leaf spot disease development in the field required several leaf wetness periods longer than 20 h at approximately 15°C at the beginning of crop cultivation. Incorporating S. birgitae infected plant debris in soil as a primary inoculum was not relevant for leaf spot disease outbreak in the next year. However, in cases of continuous cropping of lettuce on the same field and in the same season, Septoria‐infected lettuce debris may become more relevant.     

Symptom-based diagnosis of Huanglongbing (citrus greening) disease by PCR in sweet orange (Citrus sinensis Osbeck) and acid lime (Citrus aurantifolia Swingle)

Huanglongbing (HLB), previously known as citrus greening disease, is one of the most destructive diseases of citrus and responsible for the decline of citrus orchards in Andhra Pradesh (AP) and other citrus growing areas in the country. Polymerase chain reaction (PCR) amplification of 1160 bp fragment of 16S rDNA of HLB was observed in mottling symptoms, yellow vein symptoms, symptoms mixed with both citrus yellow mosaic virus and citrus greening and zinc deficiency-like symptoms; however, no amplification was observed in iron deficiency like symptoms. As the disease shows a variety of symptoms in infected trees, PCR detection could be useful for resolving confusion in identification of HLB between actual deficiency and deficiency like symptoms caused by HLB. In acid lime the midrib and veins of the leaves with both mottling and yellow vein symptoms were suitable for isolation of DNA and used as a template in the PCR test.     

Chemical and behavioral analysis of the cuticular hydrocarbons from Asian citrus psyllid,Diaphorina citri

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the vector of the phloem‐inhabiting bacterium, Candidatus Liberibacter asiaticus, which is presumed to cause HLB in Florida citrus. Laboratory and field studies were conducted to examine the behavioral responses of male and female D. citri to their cuticular extracts. In olfactometer assays, more male D. citri were attracted to one, five, or 10 female cuticular extract equivalent units than blank controls. The results were confirmed in field studies in which clear or yellow traps baited with 10 female cuticular extract equivalent units attracted proportionately more males than clear traps baited with male cuticular extract or unbaited traps. Analyses of cuticular constituents of male and female D. citri revealed differences between the sexes in chemical composition of their cuticular extracts. Laboratory bioassays with synthetic chemicals identified from cuticular extracts indicated that dodecanoic acid attracted more males than clean air. Traps baited with dodecanoic acid did not increase total catch of D. citri as compared with blank traps at the dosages tested; however, the sex ratio of psyllid catch was male biased on traps baited with the highest lure loading dosage tested (10.0 mg).     

深圳兽类物种资源调查及其影响因素分析

2014年4月至2015年10月对深圳市不同区域的兽类进行实地调查,共调查到8目15科32属49种,其中翼手目4科12属24种占比达49.0%,啮齿目3科8属13种占比26.5%,食肉目3科5属5种占比10.2%,鼩形目1科3属3种占比6.1%,其他4个目各1种,分别占比2.0%;发现1种广东省兽类新纪录,8种深圳市兽类新纪录。按景观生态以及生境情况,将深圳市划分为11个区域,通过相关性分析发现,不同区域兽类物种丰富度与其人口密度呈负相关,与植被条件以及距市区距离呈显著正相关,但是与生境斑块面积没有显著相关性;多元回归分析表明,植被条件为最主要的影响因素,且植被条件、距市区距离、人口密度三者间具有相关性。与此同时,对所调查到兽类的栖息生活类型、区系特征、濒危现状等进行了分析。     

广东内伶仃岛薇甘菊危害与生态因子的关联度分析

用灰色关联度分析对广东内伶仃岛草本群落、草灌(或灌草)丛群落和灌木林受薇甘菊危害与环境因子的关联性进行分析。结果表明薇甘菊对草本群落的危害与环境因子的关联度中主要受土壤水分和电导率的影响较大,对草灌丛群落的危害与各环境因子的关联度差异不大,但其中受土壤含水量、pH值和群落透光度的影响稍大些,对灌丛群落的危害与各环境因子的关联度差异也不大,其中受土壤有机质、群落透光度和土壤含水量的影响较大。各环境因子对不同群落受薇甘菊危害的影响由强到弱的顺序为土壤含水量>土壤pH值、有机质含量>群落透光度、土壤养分。