运用一步法RT-PCR检测柑橘鳞皮病毒及其在橘橙不同部位中的全年分布

2005年5月到2006年4月逐月采样,运用一步法RT-PCR检测Citrus psorosis virus(CPV)在Dweet橘橙苗木叶片和枝皮中的分布。保存在控温温室中的Dweet橘橙病株中老叶、老皮、嫩叶和嫩皮全年都可以检测出CPV;保存在网室中的Dweet橘橙病株中老叶、老皮全年均能检测到CPV,而夏梢的嫩叶、嫩皮不能稳定地检测出CPV,春、秋梢的嫩叶、嫩皮均可检测到CPV,表明一步法RT-PCR检测CPV最佳取样部位为老叶和老皮。     

柑橘衰退病、裂皮病和碎叶病的多重RT-PCR检测方法研究

 本研究以广东省农业科学院果树研究所隔离网室内通过嫁接分别感染CTV、CEVd和CTLV的柑橘树皮为实验材料,建立了以oligo(dT)为反转录引物的RT-PCR检测体系,成功检测到在病毒RNA 3'末端带有ploy_(A)加尾的CTV和CTLV。实验过程中,发现不具有ploy_(A)加尾的环状类病毒CEVd,同样可以用oligo(dT)反转录的RT-PCR检测出来,通过测序分析,其同源性在96%以上,并发现在CEVd序列中有一个富含A的区段。在此基础上,进一步研究了同时检测CTV、CEVd和CTLV的多重RT-PCR检测体系,该方法能为这3种病害的检测简化步骤、节省时间、降低成本。     

柑桔裂皮病类病毒分子生物学研究进展

本文就柑桔裂皮病类病毒(citrus exocortis viroid,CEVd)的分类地位,分子结构及其生物学鉴定、聚丙烯酰胺凝胶电泳分析和分子杂交、分子生物学检测等方法进行了综述。     

柑橘锈皮虫的发生与防治

柑橘锈皮虫又名爆皮虫 ,属鞘翅目、吉丁虫科。以幼虫蛀食主干和主枝。受害部位首先产生流胶现象 ,继而树皮爆裂 ,形成层中断 ,阻碍养分输送 ,引起枝干枯死 ,乃至全株死亡。柑橘锈皮虫 1年发生 1代。以不同虫龄幼虫在树干木质部浅处越冬 ,翌年 3,4月各龄幼虫先后在树干内化蛹 ,5～ 7月成虫羽化出洞 ,先在树冠啃食嫩叶 ,有假死性 ,卵多产在树干的裂缝处 ,6～ 8月为产卵盛期 ,幼虫孵化后 ,危害树皮表面呈芝麻状油渍点 ,随后有泡沫状流胶物质出现。随着幼虫龄期的增加 ,被害部分产生不规则隧道 ,隧道内充满虫粪 ,使树皮和木质部分离枯死而爆裂。…     

衢江区柑橘溜皮虫的发生与防治

2004年9月，在开展柑橘有害生物疫情普查时，发现浙江衢州市云溪乡堰头村部分橘园的橘树大多已开始枯萎，树皮成片脱落，脱落树皮的树干上可以明显看到有虫子吃过的痕迹。一些橘树看上去挺好，但只要把树干的皮一剥开，就可以看到里面有不少虫子。部分村民的房前屋后，堆放着许多枯死的橘树，光溜溜的树干上布满了虫孔。后经省、市昆虫专家的现场调查和鉴定，这些是柑橘溜皮虫造成的。     

柑橘绿藻病药剂防治试验

近年来,柑橘绿藻病在秀山县发生较普遍,个别果园发生较重。为了探索柑橘绿藻病的防治方法,秀山县植保植检站组织开展了柑橘绿藻病药剂防治试验。试验结果表明,石硫合剂、冰醋酸液、生石灰浸泡液对柑橘绿藻病均有较好的防治效果。药后40 d,29%石硫合剂水剂150倍液和100倍液对柑橘绿藻病的防治效果分别为70.8%和74.6%,生产上可以示范推广应用。     

柑橘皮乙醇提取物中抑菌成分的分离与鉴定

以茶云纹叶枯病病原菌(Colletotrichum camelliae Massee)为指示菌种,采用活性跟踪法对柑橘皮乙醇提取物进行分离,并利用现代波谱技术(1H-NMR)对已分离的化合物进行结构鉴定.分别采用“生长速率法”和“抑菌圈法”测定柑橘皮乙醇提取物中4种萃取物和5个化合物对茶云纹叶枯病病原菌的抑制作用.结果表明:当石油醚部位浓度为0.8 mg/mL,其对茶云纹叶枯病病原菌的抑制率达80.9％,其E50达0.163 8 mg/mL;当二氯甲烷部位浓度为1.12 mg/mL时,其对茶云纹叶枯病病原菌的抑制率为60.6％,EC50达0.764 3 mg/mL;在供试浓度为1 000μg/mL时,5个化合物中5-羟基6,7,8,3',4 '五甲氧基黄酮和柠檬苦素的抑菌性较强,抑菌直径分别达13.47和16.05 mm.     

上海柑橘黑点病田间流行与降雨关系研究

自2009年起柑橘黑点病逐渐成为上海地区影响柑橘果实商品价值的最主要病害,严重制约上海市柑橘产业的效益。为了解该病的田间流行动态以及病害发生与降雨量的关系,2010年—2018年连续9年进行了病害发生动态调查和分析。结果表明,上海地区6月上旬至8月下旬是果实感病期,其中,6月中旬至7月中旬为发病高峰期;雨水是黑点病发生的必要条件,果实生长期不同阶段的降雨参数,尤其是6月下旬的降雨量与病情指数密切相关。本文以病情发展的3个时间节点(7月15日、8月15日和9月10日)的病情指数为因变量,以这3个时间点之前各旬的降雨量、降雨日为自变量筛选变量建立了多元线性回归方程,根据分析结果,采用6月下旬的降雨量等气象因子建立的多元线性回归方程,可以比较准确地预测7、8月中旬和9月10日即病害发展稳定期的田间病情。     

宜昌市柑橘砂皮病发生现状与防治对策

柑橘砂皮病近年来宜昌市发生较为严重,影响了柑橘产业健康发展。本文调查了宜昌市柑橘砂皮病的发生分布现状,主要症状表现和流行特点,认为橘园管理水平下降,清园不到位导致菌源基数的积累,加之气象条件比较有利于发病,可以从田间管理培壮树势,清洁果园减少病源、科学施药防治到位等措施上减少发病,最后对促进柑橘产业健康发展提出了几点建议。     

应用多重PCR技术同步检测柑橘4种重要嫁接传播病原

 柑橘衰退病毒(Citrus tristeza virus,CTV),柑橘碎叶病毒(Citrus tatter|leaf virus,CTLV),柑橘裂皮病类病毒(Citrus exocortis viroid,CEVd)和柑橘黄龙病(Huanglongbing, HLB)亚洲种病原(Candidatus liberobacter asiaticus)是重要的柑橘嫁接传播病原。本文建立了同时检测HLB病菌、CTV、CEVd 和CTLV 4种柑橘嫁接病原的一步法、双温多重PCR检测技术体系,同时在体系中设置内参基因。应用该体系快速评价了4种嫁接传播病原在田间侵染情况,结果表明28个田间样品CTV、CEVd、CTLV和HLB感染率分别为89.3 %、17.9 %、10.7 %和28.6 %,接近半数样品为混合感染。并且将该方法应用于快速评价茎尖嫁接苗病毒的脱除情况。     

Detection of Citrus psorosis virus in field trees by direct tissue blot immunoassay in comparison with ELISA, symptomatology, biological indexing and cross-protection tests

Serological detection of Citrus psorosis virus (CPsV) by direct tissue blot immunoassay (DTBIA) and by double (DAS) and triple (TAS) antibody sandwich ELISA, was compared in samples from various citrus varieties growing in the glasshouse and in the field. In young shoots and leaves, CPsV was readily detected by the three procedures, whereas DTBIA detection in old leaves was less consistent. DTBIA detection and ELISA readings in nine different citrus varieties were similar, suggesting that CPsV accumulates to equivalent levels in all of them. In infected field trees from Spain or Italy, CPsV was consistently detected by TAS ELISA, even in samples of old leaves in winter, whereas DTBIA detection in the same trees was reliable only when using young shoots. Detection of CPsV by DTBIA and by DAS and TAS ELISA in previously untested field trees correlated perfectly with psorosis diagnostics based on biological indexing, specifically with the capacity of those sources to cross-protect against challenge inoculation with psorosis B. Some trees without bark scaling were shown to be psorosis-infected by biological indexing and to contain CPsV by serological tests; other trees showing psorosis-like bark or leaf symptoms in the field were shown to be psorosis-free by biological indexing and also CPsV-free by serology. This is the first time that the presence of CPsV has been correlated with psorosis infection as diagnosed by biological indexing.     

Detection of Citrus Psorosis Virus by ELISA, Molecular Hybridization, RT-PCR and Immunosorbent Electron Microscopy and its Association with Citrus Psorosis Disease

Psorosis is a citrus disease of undemonstrated etiology that can be diagnosed by biological indexing on sweet orange seedlings followed by a cross protection test. Its presumed causal agent is Citrus psorosis virus(CPsV), type species of the genus Ophiovirus. We compared detection of CPsV by ELISA, RT-PCR, molecular hybridization and immunosorbent electron microscopy, and examined its association with psorosis disease in 11 biologically characterized isolates and in 47 uncharacterized field sources by observation of field symptoms and by biological indexing including the cross protection test. Detection of CPsV by any of the four procedures always coincided with diagnosis of psorosis by cross protection, but it did not always correlate with observation of symptoms thought to be specific, in field trees or in graft-inoculated indicator plants. Trials to detect CPsV by ELISA, molecular hybridization and RT-PCR in citrus sources from different geographical origins, presumed to be psorosis-infected on the basis of field symptoms or reaction of indicator plants, were sometimes unsuccessful, indicating that psorosis symptoms may be induced by causes other than CPsV.     

Improved detection of citrus psorosis virus using polyclonal and monoclonal antibodies

Citrus psorosis is a serious and widespread disease associated with citrus psorosis virus (CPsV), a novel filamentous negative-stranded virus in the genus Ophiovirus . Laborious and costly indexing on test plants has been the only routine diagnostic method available, but recently an antiserum usable in double antibody sandwich (DAS) ELISA has been prepared. Here, major improvements to the DAS-ELISA protocol, a new purification method, and production of two monoclonal antibodies (mabs) to CPsV, an IgG and an IgM are reported. A highly sensitive triple antibody sandwich (TAS) ELISA making use of the mabs is described. In glasshouse citrus the homologous virus was still detectable at a tissue dilution of 1/6250 in DAS and at 1/31250 in TAS-ELISA. Both the DAS and IgG mab-TAS formats detected all CPsV isolates so far tested (from Argentina, Italy, Lebanon, Spain and the USA). A few isolates were not detected by the IgM mab.     

Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA 2

Citrus psorosis virus (CPsV), the type species of genus Ophiovirus, is the presumed causal agent of a bark scaling disease in citrus plants. CPsV virions are kinked filaments composed of three negative‐strand RNA molecules and a ～48‐kDa coat protein. The virus induces two different syndromes: psorosis A (PsA), characterized by limited bark scaling lesions in the trunk and main limbs, and a more aggressive form of the disease called psorosis B (PsB) with rampant bark lesions affecting even thin branches and chlorotic blotches in old leaves. In the greenhouse, the PsA and PsB syndromes can be induced by graft inoculating healthy citrus seedlings with non‐lesion or with lesion bark inoculum from PsA‐affected field trees. PsA‐ and PsB‐inducing CPsV sub‐isolates obtained by this procedure from the same tree showed identical single‐strand conformation polymorphism (SSCP) profiles in homologous segments of the RNAs 1 and 3, whereas segments of the RNA 2 enabled discrimination between PsA‐ and PsB‐associated sequence variants. SSCP analysis of the RNA 2 population present in different tissues of psorosis‐infected plants showed that: (i) PsA‐inducing isolates contain PsB‐associated sequence variants at low frequency, (ii) the PsB‐associated sequence variant is predominant in blistered twigs and gummy pustules affecting old leaves, characteristic of PsB isolates, and (iii) the PsB‐associated sequence variant accumulates preferentially in bark lesions of the trunk and limbs. SSCP analysis of the RNA 2 population also enabled monitoring of interference between PsA‐ and PsB‐associated variants in plants co‐inoculated with both psorosis types.     

Occurrence of Citrus psorosis virus in Campania, southern Italy

Citrus psorosis virus (CPsV), genus Ophiovirus, is associated with a severe disease of citrus worldwide. Double antibody sandwich (DAS) ELISA using a polyclonal antiserum, and triple antibody sandwich (TAS) ELISAs, employing the IgG monoclonal antibody (mab) 13C5, and the IgM mab 2A3, were used to detect CPsV in orchards of different citrus varieties in Campania, southern Italy. TAS ELISA with 13C5 detected all the infections detected by DAS ELISA. Overall, 14% of trees younger than 15 years were positive, but only 1% of older trees, suggesting that infected propagating material has been increasingly used in recent years, in the absence of certification. Highest infection rates were in younger trees of sweet orange (22.8%) and clementine (18.6%). CPsV could easily be detected at all seasons of the year tested (June–January); these and earlier results indicate that TAS ELISA using 13C5 is a sensitive, broad-spectrum and reliable diagnostic method useful for routine tests and certification programmes. Of 44 field isolates responding strongly to DAS ELISA and 13C5-TAS ELISA, mab 2A3 gave similar results with 29 isolates, but gave low values with the others, thus providing a degree of differentiation among isolates. To confirm that the ELISA tests were indeed detecting CPsV, samples of 42 ELISA-positive plants were analysed by ISEM in a blind test, and in 38 of these, characteristic virus particles were clearly seen. Although CPsV was frequently and consistently detected in the area sampled, bark scaling symptoms were not seen: possible reasons for this are discussed.     

Elimination of Citrus psorosis virus by somatic embryogenesis from stigma and style cultures

Somatic embryogenesis was used to eliminate Citrus psorosis virus (CPsV) from three citrus species (common mandarin, sweet orange and Dweet tangor), all of which regenerated somatic embryos with different embryogenic potential from stigma and style explants. CPsV was detected by double antibody sandwich‐indirect‐enzyme‐linked immunosorbent assay (DASI-ELISA) in explants and embryogenic callus, but was not detected in any of the plants obtained from somatic embryos, even 24 months after regeneration. Loss of juvenile characters (disappearance of thorns) was observed in the first year of growth and was retained in plants propagated by grafting from thornless stems. Somatic embryogenesis appears to be a very promising technique for the production of healthy citrus stocks.     

柑橘花期蓟马的种类与鉴定

为明确柑橘花期蓟马种类,以柑橘园为实验基地,系统调查橘园中蓟马的种类。经形态学鉴定共发现3种蓟马,且3种蓟马的形态分别与棕榈蓟马（Thrips palmi Karny）、淡色蓟马（Thrips flavus Schrank）和华简管蓟马（Haplothris chinesis Pfiesner）极其相似。对3种蓟马分别进行单头虫体的基因组DNA提取,对其ITS2基因序列进行PCR扩增并测序,测序结果登入NCBI数据进行比对,结果表明3种蓟马分别与棕榈蓟马（Thrips palmi Kamy）、淡色蓟马（Thrips flavus Schrank）和华简管蓟马（Haplothris chinesis Priesner）的相似度均达99％。     

柑桔“以螨治螨”生物防治技术研究

柑桔病虫害发生种类多，为害重，生产上喷药次数多，果品存在农药残留高，为了使我市柑桔达到无公害生产水平，减少化学农药使用量，提高柑桔品质，笔者通过释放捕食螨Amblyseius spp．建立捕食螨种群，同时结合使用生物农药和电子灭虫灯，以及白花草Ageratum conyzoides L．等栽培措施，达到了长期持续控制柑桔病虫发生的目的，而且减少我县柑桔生产的农药残留问题，降低生产成本，减少环境污染。     

利用小RNA深度测序技术鉴定上海地区‘红美人’柑橘病毒种类

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。