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。     

柑橘黄龙病研究进展

柑橘黄龙病是1种系统性侵染的毁灭性病害.近年来,柑橘黄龙病在我国部分柑橘产区为害加重,巳成为制约柑橘产业健康发展的关键因素之一.为了进一步了解该病的发生、发展规律,制定科学、有效的防治技术方案,为农业生产服务,对该病的发生与为害状况、症状表现、防治技术等方面的研究成果进行了概述.     

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。     

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.     

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.     

柑橘黄龙病病原研究进展

本文对柑橘黄龙病(HLB)的发现、认识过程及病原菌的分类、检测鉴定技术进行了综述。HLB是一种十分严重的柑橘病害,主要通过木虱和嫁接进行传播,在不同的地区和国家具有不同的名字,已统一命名为HLB。对引起HLB的原因探索经历了一个漫长的过程,从水害、缺素、镰刀菌、病毒到类菌原体,最后确定为细菌。该病原属于α-亚纲,韧皮部杆菌属,分为亚洲种、非洲种和美洲种3个种,可以通过PCR技术进行检测鉴定。同时,对韧皮杆菌的人工培养以及与黄龙病相关植原体的最新研究进展进行了阐述。     

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.     

湖南省永州地区柑橘慢衰病与黄龙病病原鉴定及分布调查

为明确湖南省柑橘黄化症状与柑橘慢衰病和柑橘黄龙病的相关性,应用形态学和分子生物学方法对湖南省永州地区表现黄化症状的柑橘进行了两种病原的鉴定及分布调查。结果表明,造成该地区柑橘黄化现象的主要原因为柑橘半穿刺线虫和黄龙病菌对柑橘的侵染。所检样本中,柑橘慢衰病平均发生率为82.1%,土壤中的半穿刺线虫群体密度最高达到3 077条/100mL。永州地区柑橘黄龙病病菌属于韧皮部杆菌属类细菌亚洲种,平均检出率为64.3%。柑橘半穿刺线虫和黄龙病菌在柑橘园存在混合侵染现象,混合侵染率为53.6%。柑橘半穿刺线虫在永州地区柑橘产区分布广泛,是造成柑橘黄化症状的重要病因,重视并有效防控柑橘半穿刺线虫已迫在眉睫。