Characterization of Citrus tristeza virus isolates in northern Iran

The biological and molecular properties of four Citrus tristeza virus (CTV) isolates isolated from infected Satsuma trees imported from Japan, and growing in citrus groves in northern Iran (Mahdasht orchards, Mazandaran Province), were investigated. CTV-infected samples were collected from sweet orange trees and grafted onto Alemow (Citrus macrophylla Wester) seedlings. On indicator plants, these isolates produced various symptoms including vein clearing and stem pitting on Mexican lime, Alemow, and Citrus hystrix, and yellowing and stunting on sour orange and grapefruit seedlings. Citrus samples were also surveyed for CTV using serological tests. The coat protein (CP) gene of these isolates was amplified using specific primers, yielding an amplicon of 672 bp for all isolates. Sequence analysis showed 98%–99% sequence homology of Iranian isolates with the Californian CTV severe stem-pitting isolate SY568 and 97%–98% homology with the Japanese seedling yellows isolate NUagA. The Iranian isolates were compared by restriction fragment length polymorphism (RFLP) analysis of the CP amplicon for further classification.     

Biological characterization of citrus tristeza virus isolates by in vitro tissue cultures

Stem segments from Mexican lime, sweet orange, grapefruit, Citrus excelsa and alemow, infected with five citrus tristeza virus (CTV) isolates, were cultured in vitro . Regeneration of roots and shoots were modified as a result of infection. The effect of CTV on the morphogenesis of stem segments cultured in vitro depended on the CTV isolate and the plant host, and showed a correlation with the in vivo effects observed in biological indexing. Evaluation of the morphogenic response of stem segments of Mexican lime and grapefruit can be used as an additional tool for the biological characterization of CTV isolates. The symptoms on sweet orange plants obtained from regenerated shoots indicated that CTV is unevenly distributed in the host plant cells and that the regeneration process may be utilized as a tool to separate strains from complex field isolates.     

应用改进的多克隆抗体Western blot技术研究柑桔速衰病毒蛋白

 本文利用多克隆抗体发展了一种无背景的Western blot技术并用于研究柑桔速衰病毒(CTV)的蛋白。结果表明,利用CTV兔多克隆抗体1212和1052发展的Western blot技术可以检测到感染CTV的墨西哥酸橙或Citrus excelsa植株内CTV的4种蛋白P1、P2、P3和P4。在健株或病株内杂交反应均无非特异性背景。从不同寄主上分离到的CTV不同分离物的蛋白条带是不同的。利用1212和1052抗体均可以检测到感染6个CTV分离物的墨西哥酸橙幼苗内的P1、P2和P3。利用1052抗体能检测到感染严重型分离物T36、T3和Mm2的墨西哥酸橙幼苗内微弱的P4,但感染轻型分离物T30、T26和T4的幼苗内则检测不到。利用1212抗体检测不到P4。在C. excelsa内,1212和1052抗体均能检测到感染所有分离物的病株内的P1。在感染T3、T26、T4或Mm2的病株内能检测到P2,但在感染T30和36分离物的病株内则检测不到。在感染T36、T3、T26、T4和Mm2的病株内可检测到P3,但在感染T30的病株内则检测不到。在大多数植物内,P1、P2、P3和P4的分子量分别约为25kDa,24kDa,21kDa和18kDa。在感染T36分离物的C. excelsa植株体内,P1和P3的分子量分别约为27kDa和22kDa,比感染其它分离物的C. excelsa和墨西哥酸橙内的P1和P3分子量略大。P1、P2和P3的分子量与利用单克隆抗体检测的CP,CP1和CP2的分子量相等。因此,P1、P2和P3可能是CTV的外壳蛋白CP,CP1和CP2。P4的特性不清楚。研究结果也表明,利用特异性的多克隆抗体进行的Western blot是研究CTV的一种有用的技术。应用该技术,病株内不同的CTV分离物或株系就可以通过特异性蛋白条带区分开来。     

Detection and characterization of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> stem pitting isolates in Jamaica

An island wide survey for Citrus tristeza virus (CTV) in citrus orchards across Jamaica (13 regions) was conducted over 2 years. Trees (1, 885) showing virus-like symptoms as well as asymptomatic trees were randomly sampled for testing by ELISA and 55 samples from the 6 major citrus growing regions were graft inoculated on indicator plants. Most samples (74%) reacted to polyclonal antibodies against CTV in ELISA, while 20% were positive in tests using monoclonal antibodies specific to severe CTV strains. Samples collected from the 6 major citrus growing regions produced vein clearing and stem pitting symptoms on Mexican lime indicator plants (87%). In addition, stem pitting symptoms were induced on Duncan grapefruit, sweet orange, sour orange or sweet orange grafted on sour orange. Nucleotide sequencing of the coat protein gene sequences isolated from these samples indicated high identities (88 to 95.5%) among the Jamaican isolates and previously reported stem pitting strains from Central and North America and Eurasia (88 to 100%). The results suggest a shared ancestry with isolates from other geographical locations, rather than geographical speciation, and presumably separate CTV introductions into Jamaica.     

枸头橙种子中柑桔衰退-茎陷点病毒的鉴定

 采用双抗体夹心-酶联免疫吸附测定试验(DAS-ELISA)对枸头橙种子中柑桔衰退-茎陷点病毒的带毒情况进行了鉴定。结果表明,从严重感染茎陷点病的枸头橙罹病树采集的种子可检测到柑桔衰退病毒(CTV)。种子带毒量以内种皮为较高,剥皮种子次之,外种皮呈阴性。种子在4℃冰箱内贮藏1~2年后仍带毒,但带毒量有所下降。对带毒种子繁殖的实生苗及嫁接在实生苗上的墨西哥来檬的检测结果,有部分呈阳性,种植在防虫隔离网室内的100余株2年生枸头橙实生苗,用无毒珠心系的墨西哥来檬芽嫁接进行生物鉴定,经2年观察未发现症状     

Separation and interference of strains from a citrus tristeza virus isolate evidenced by biological activity and double-stranded RNA (dsRNA) analysis

Separation of strains of citrus tristeza virus (CTV), differentiated by their double-stranded RNA (dsRNA) profiles, was obtained by graft-inoculating citron plants from a Mexican lime that had been recently aphid- or graft-inoculated with a mild CTV isolate (T-385). Up to 24 sub-isolates with differing dsRNA profiles were obtained from the aphid-inoculated lime. Some of these sub-isolates induced stronger symptoms in several citrus species than the original T-385 isolate. One sub-isolate, T-385-33, was mild in Mexican lime, but induced stem pitting on sweet orange. Inoculation of this isolate on Mexican lime, sour orange and Eureka lemon induced mild or no symptoms when inoculum was taken from citron, but very severe symptoms when the inoculum was from sweet orange. Mexican lime and sweet orange plants co-inoculated with T-385-33 from sweet orange in combination with the other 23 sub-isolates showed mild symptoms. The results obtained suggest that there is natural cross-protection among sub-isolates in the original T-385 isolate.     

Investigation of biological properties, double-stranded RNA patterns and antigen concentration in citrus species infected with citrus tristeza virus

Biological properties and dsRNA patterns of one Cyprus and three Turkish isolates of citrus tristeza virus (CTV) were investigated. In addition, CTV antigen concentration and effect of tissue sampling time from naturally infected Shamouti sweet orange trees grown in the field of Icel Province, Turkey, were also determined. The Cyprus isolate showed vein clearing symptoms on grapefruit, ‘Madam Vinous’ and Mexican lime and stem pitting symptoms on Mexican lime. The three Turkish isolates showed only vein clearing symptoms on Mexican lime. All four isolates showed a full-length major double-stranded RNA (dsRNA) band of 13.3 × 10⁶ Da mol. wt in extracts from infected Madam Vinous sweet orange trees, and major or minor dsRNA bands with 2.0. 0.8 and 0.5 × 10⁶ mol.wt. All seven different citrus varieties inoculated with the Igdir (D) strain contained full-length dsRNA. The additional two dsRNA of 0.8 and 0.5 × 10⁶ mol.wt were also detected as clearly as full-length dsRNA in these hosts, but were weaker inCitrus exelsa and ‘Interdonat’ lemon. Madam Vinous, rough lemon and Mexican lime were the best hosts for dsRNA analysis. ELISA values were highest in April (OD_405nm =0.476), decreased steadily until August, and then increased gradually through December. ELISA values were lowest in July and August (OD_405nm =0.157 and 0.141, respectively). dsRNA recovery from a field tree infected with isolate Igdir D was good in March, April and May and poor in January and February. No dsRNA band was detected in August or September. http://www.phytoparasitica.org posting July 9, 2002.     

Variability among Spanish citrus tristeza virus isolates revealed by double-stranded RNA analysis

A survey for citrus tristeza virus (CTV) strains, based on double-stranded RNA (dsRNA) analysis, was carried out in five locations on the eastern citrus-growing area of Spain. CTV was recovered from 137 trees of different ages, citrus species and varieties, sampled in 53 orchards. The best months for dsRNA recovery were April, May, September, October, and November, and the highest dsRNA yield was obtained from sweet orange cultivars. Sixteen dsRNA profiles differing by the number and/or position of subgenomic bands were detected. One of these profiles was detected in more than half the trees analysed. Maximum diversity of dsRNA patterns was found in the location with the oldest citrus orchards and the highest CTV incidence (Alzira-Carcaixent). In many instances, several dsRNA profiles were detected in neighbouring trees of the same orchard, notably in Alzira-Carcaixent, where 70% of the plots sampled contained more than one profile. The possible causes for the diversity of CTV isolates found in this specific area are discussed.     

广西柑橘衰退病和黄脉病调查及其病原病毒的遗传多样性分析

为明确柑橘衰退病毒(citrus tristeza virus, CTV)和柑橘黄脉病毒(citrus yellow vein clearing virus, CYVCV)在广西柑橘上的发生?分布及其遗传变异情况, 于2020年至2021年对百色?北海?崇左?贵港?桂林?河池?贺州?来宾?柳州?南宁?梧州和玉林等12个柑橘产区进行了病毒病调查?采用RT-PCR对采集样品进行了病毒检测, 并基于病毒分离物外壳蛋白(coat protein, CP)基因的核苷酸序列进行比对分析, 构建系统发育树?结果表明:采集的737份柑橘样品中, CTV的检出率为20.62%, CYVCV检出率为18.32%, CTV的检出率略高于CYVCV?病毒复合侵染的现象在采集的柑橘样品中普遍存在, CTV和CYVCV复合侵染率高达34.50%?对RT-PCR产物测序共获得12个CTV分离物和6个CYVCV分离物的CP基因序列?遗传多样性分析发现, CTV和CYVCV的CP基因序列都较保守, CTV分离物的遗传进化与地理来源?寄主来源均没有明显相关性, 但CYVCV分离物的遗传进化与地理位置具有相关性, 而与寄主来源无明显相关性?上述研究结果可为深入了解CTV和CYVCV在广西的流行情况以及柑橘病毒病的检疫和防控提供参考?     

摩洛哥脐血橙植株上一种机械传播性病毒的鉴定

 本文采用叶片接种法和枝条接种法对25个柑桔种类体细胞杂种或品种进行了抗脚腐病鉴定,结果表明:体细胞杂种中枳+酸橙、枳+甜橙为高抗类型;酸橙+兰普来檬、枳+印度酸桔;蚝壳刺+哈姆甜橙、酸橙+澳洲指桔为抗性;酸橙+印度酸桔为中抗。建立了叶片接种法的抗病性分级标准。初步分析了柑桔体细胞杂种的抗性规律。     

6种柑橘类植物对柑橘衰退病毒分离株TR-L514变异的影响

 柑橘衰退病毒(Citrus tristeza virus,CTV)存在复杂的株系分化现象,运用弱毒株交叉保护防治柑橘衰退病时需了解不同类型柑橘对CTV构成的影响。作者将CTV分离株TR-L514嫁接接种到6类柑橘植物上获得了18个亚分离株,并对这18个亚分离株和TR-L514进行了指示植物鉴定,p25基因的限制性片段长度多态性(RFLP)和单链构象多态性(SSCP)分析,p23基因的序列比较。结果表明,CTV株系对不同柑橘类植株的适应性存在差异。TR-L514嫁接到不同柑橘类植株其构成会发生变化,在甜橙上可以同时检测出p25//HinfⅠ RFLP第1和6组群,并具有比在其它4类柑橘上更为复杂的SSCP谱型构成,因此甜橙可能更适宜于CTV的增殖。TR-L514及18个亚分离株的p23基因序列差异可能与不同类柑橘植株的适应性有关。     

Molecular characterization of Citrus yellow mosaic badnavirus (CMBV) isolates revealed the presence of two distinct strains infecting citrus in India

Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the etiologic agent of yellow mosaic disease of citrus in India. The disease was initially reported from the southern parts of India and has now spread to other parts of the country. It is a serious disease of sweet orange (Citrus sinensis) in southern India, where it causes significant yield losses. During a recent survey of citrus groves in the Nagpur region, central India, characteristic mosaic symptoms were observed in mandarin orange (Citrus reticulata) and sweet orange. Virus transmission studies, electron microscopy, PCR amplification and sequencing of cloned PCR products from samples showing mosaic symptoms confirmed the presence of a badnavirus. The CMBV–Nagpur isolate could be transmitted to the Rangpur lime (C. limonia) and acid lime (Citrus aurantifolia) by graft inoculation. Sequence analysis of a segment of ORF-III region and intergenic region (IR) of the viral genome revealed that CMBV–Nagpur isolate formed a distinct clade along with some previously reported isolates that are known to infect acid lime and Rangpur lime. CMBV isolates that infect citrus species other than the acid lime and Rangpur lime formed a second clade. Based on the transmission studies and phylogenetic analyses, it was concluded that at least two strains of CMBV exist in India currently.     

江西柑橘主产区柑橘衰退病毒分离株组群分析

为检测江西柑橘主产区柑橘衰退病毒分离株组群的构成情况,运用限制性片段长度多态性(RFLP)对收集自江西柑橘14个主产区果园的CTV分离株进行分析。发现209份样品的CP/HinfⅠ酶切结果中182份样品表现出单一CP/HinfⅠRFLP谱型,占鉴定样品总数的87.1%,其中以CP/HinfⅠRFLP第3和第1组群的分离株构成为主,分别占样品总数的55.5%和26.8%;混合CP/HinfⅠRFLP组群样品占12.9%。本次检测中发现有1个分离株为第4组群,5个分离株为第5组群,可能为潜在的弱毒分离株。本次试验中检测的江西柑橘样品以CTV单一组群感染为主。     

East Adriatic—a reservoir region of severe <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> strains

Citrus tristeza virus (CTV) represents one of the major threats to citrus production worldwide. In the East Adriatic region, CTV symptoms are mostly absent due to traditional citrus grafting on trifoliate orange (Poncirus trifoliata), a CTV-tolerant rootstock. Therefore, the virus has been continuously spreading by the propagation of infected material. The genetic variability of CTV was studied on nineteen citrus samples, collected from orchards in the coastal region of Croatia, Montenegro and Albania, that previously tested positive by ELISA and immunocapture RT-PCR. Single-strand conformation polymorphism of the amplified coat protein gene demonstrated the presence of different CTV variants in each amplicon, while sequence analysis of cloned CP gene variants confirmed their clustering into six out of the seven phylogenetic groups so far delineated. Four of these groups include sequences of severe quick decline, seedling yellows and stem-pitting (SP) isolates, thought to be found only rarely in the Mediterranean region. Regardless of the lack of symptoms in the field, CTV isolates from the East Adriatic displayed high genetic variability and pathogenic potential, additionally confirmed by biological characterisation. The high percentage of mixed infections suggest the potential for further diversification and a greater risk of severe variants spreading into new areas.     

Use of phages for identifying the citrus canker bacterium Xanthomonas campestris pv. citri in Taiwan

Phages CP115 and CP122, which were isolated from canker lesions on grapefruit and Liucheng sweet orange, respectively, showed a high degree of specificity with respect to lysis of test bacterial strains. When used jointly, they lysed 135 (97·8%) out of 138 Xanthomonas campestris pv. citri strains isolated from the canker lesions on leaves, twigs, and fruits of various citrus species, cultivars, and hybrids grown throughout Taiwan, but they did not lyse other X. campestris pathovars and other phytopathogenic bacteria, nor other bacteria isolated from soil, clinical or environmental samples. Of 252 CP115/CP122-sensitive and 78 CP115/CP122-resistant bacterial strains with colony characteristics typical of or similar to those of X. campestris pv. citri , isolated from canker lesions of various citrus plants in diverse growing regions in Taiwan, 250 (99·2%) and 76 (97·4%) strains were pathogenic and non-pathogenic, respectively, when inoculated into Liucheng sweet orange or Mexican lime. Thus, phages CP115 and CP122, when used jointly, appear to be applicable for identifying X. campestris pv. citri in Taiwan.     

First detection of a virulent strain of Citrus tristeza virus (Closteroviridae) in a citrus orchard of Chlef Valley (Algeria)

A large‐scale survey of Citrus tristeza virus (CTV) was carried out from 2016 to 2018 in the Chlef Valley, one of the main citrus growing areas in Algeria. In this study a total of 1680 citrus trees from 93 commercial orchards were sampled. The collected samples were tested by direct tissue blot immunoassay analysis and by the double antibody sandwich enzyme‐linked immunosorbent assay technique, and 54 trees were identified as being infected with CTV. This result confirmed that 54 trees were infected by the virus, corresponding to an infection rate of 3.21% throughout the studied area. Five of these local CTV sources were chosen for further molecular investigations to determine the genotype associated with the CTV isolates now spreading in the Chlef area. Characterization with multiple molecular markers showed the presence of the T30 and VT genotypes. This result allowed confirmation of the presence of a virulent strain belonging to the VT genotype. The other CTV isolates were similar to those from the Mitidja region, which showed 99% nucleotide identity with the Spanish mild CTV isolate. This early finding of a strain belonging to the VT genotype is an issue for Algerian citrus producers and needs rapid actions to be taken by the National phytosanitary services, extending the surveillance to other citrus production regions and uprooting the infected trees.     

Expression of phytoplasma‐induced witches’ broom disease symptoms in acid lime (Citrus aurantifolia) trees is affected by climatic conditions

Witches’ broom disease (WBD), caused by ‘Candidatus Phytoplasma aurantifolia’, is a serious disease of acid lime (Citrus aurantifolia) in Oman and the UAE. However, little is known about the distribution of phytoplasma and the expression of WBD symptoms in different geographical locations. A survey was carried out in 18 districts in Oman and the UAE covering 143 orchards and 5823 acid lime trees. ‘Candidatus Phytoplasma aurantifolia’ was detected in acid lime in all the 18 surveyed districts. However, the development of typical symptoms of WBD was only observed in 12 districts. Districts in which the phytoplasma was present but symptoms were not expressed were located either in desert areas or in areas characterized by semitropical conditions. Phylogenetic analysis of 16 phytoplasma isolates from trees developing WBD symptoms and six phytoplasma isolates from trees with no WBD symptoms showed that all isolates share an identical 16S rRNA sequence, belonging to subgroup II‐B. Quantitative PCR analysis showed that the concentration of phytoplasma is significantly higher (8800–801 000 copies) in leaves developing WBD symptoms compared to 2–268 copies in symptomless leaves from the same trees and 8–874 copies in acid lime trees from areas where disease symptoms were not expressed. The lack of expression of WBD symptoms under certain environmental conditions may suggest that symptom development and phytoplasma are affected by certain unfavourable environmental conditions. These findings could provide a basis for managing WBD through encouraging lime cultivation under climatic conditions less conducive to WBD symptom expression.     

Postbloom fruit drop of citrus and key lime anthracnose are caused by distinct phylogenetic lineages of Colletotrichum acutatum

Colletotrichum acutatum causes two diseases of citrus, postbloom fruit drop (PFD) and Key lime anthracnose (KLA). PFD is a disease restricted to flowers of sweet orange and most other citrus, and symptoms include petal necrosis, abscission of developing fruit, and the formation of persistent calyces. KLA is a disease of foliage, flowers, and fruits of Key lime only, and symptoms include necrotic lesions on leaves, fruits, twigs, flowers, and blight of entire shoots. The internal transcribed spacers 1 and 2 and the gene encoding the 5.8S ribosomal RNA subunit within the nuclear ribosomal cluster (ITS) and intron 2 of the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) were sequenced for isolates from PFD-affected sweet orange and KLA-affected Key limes collected in the United States (Florida), Brazil (S?o Paulo), Mexico, Belize, Costa Rica, and the Dominican Republic to determine if there are consistent genetic differences between PFD and KLA isolates over the geographic area where these diseases occur. Based on the sequence data, isolates clustered into two well-supported clades with little or no sequence variation among isolates within clades. One clade (PFD clade) contained PFD isolates from all countries sampled plus a few isolates from flowers of Key lime in Brazil. The other clade (KLA clade) contained KLA isolates from Key lime foliage from all countries sampled and one isolate from flowers of sweet orange in Mexico. In greenhouse inoculations with PFD and KLA isolates from Florida, isolates from both clades produced PFD symptoms on Orlando tangelo flowers, but KLA-clade isolates produced significantly less severe symptoms. PFD-clade isolates were not pathogenic to Key lime foliage, confirming previous studies. The differentiation of PFD and KLA isolates into two well-supported clades and the pathogenicity data indicate that PFD and KLA are caused by distinct phylogenetic lineages of C. acutatum that are also biologically distinct. PFD is a recently described disease (first reported in 1979) relative to KLA (first reported in 1912) and it had been proposed that strains causing PFD evolved from strains causing KLA eventually losing pathogenicity to Key lime foliage. We reject the hypothesis that PFD strains have diverged from KLA strains recently based on estimated divergence times of haplotypes and it appears that PFD and KLA strains have been dispersed throughout the Americas independently in association with each host.