Detection of citrus psorosis-ringspot virus using RT-PCR and DAS-ELISA

Psorosis, sometimes also associated with ringspot symptoms, is a widespread and damaging disease of citrus in many parts of the world including South America and the Mediterranean basin. We describe the application of RT-PCR and DAS-ELISA diagnostics to an isolate of citrus ringspot virus (CtRSV-4) and other virus isolates associated with this disease. Fragments of cDNA from bottom-component RNA of CtRSV-4 were cloned and sequenced, and PCR primers were designed, 5'ACAATAAGCAAGACAAC upstream, and 5'CCATGTCACTTCTATTC downstream. RT-PCR experiments using these primers allowed detection of CtRSV-4 in infected citrus leaves down to a tissue dilution of 1/12 800 representing 2 μg of tissue, and less sensitive detection of the related citrus psorosis-associated virus (CPsAV90-1-1) and four other psorosis isolates from Argentina and the USA. In addition, CtRSV-4 particles were partially purified from local lesions in Chenopodium quinoa, and the preparations used to raise a rabbit antiserum. The antiserum was absorbed with extracts of healthy C. quinoa leaves, and a DAS-ELISA kit was prepared and tested for detection of CtRSV-4, CPsAV90–1-1, and other psorosis isolates from Argentina, the USA, Italy and Spain. The ELISA detected CtRSV-4 down to a tissue dilution of 1/1600, and most other psorosis isolates down to dilutions of 1/200–1/800. Three of a total of 20 heterologous isolates were consistently negative. Comparison of the PCR and ELISA results suggests that both methods can be used for detection of a range of psorosis isolates, but that variation of the viruses in the field might cause problems for any one diagnostic test.     

Filamentous flexous particles and serologically related proteins of variable size associated with citrus psorosis and ringspot diseases

Filamentous flexous partic les of unusual morphology, previously associated with several ringspot isolates, were detected also in psorosis A and psorosis B isolates by serologically specific electron microscopy using an antiserum to citrus ringspot. Upon partial purification of six ringspot, six psorosis A, and three psorosis B isolates, a specific protein of 47 kDa was detected in most cases, but two isolates (one psorosis A and one ringspot) had a 46 and a 48 kDa-protein, respectively. These differences in molecular masses were observed when purification was done from different host species or from plants co-inoculated with two isolates differing by their protein size. The three types of protein were serologically related in Western blots. Our results indicate that a common virus with different strains may be involved in psorosis A, psorosis B, and ringspot diseases.     

Partial purification of a virus associated with a Spanish isolate of citrus ringspot

A citrus ringspot isolate from Star Ruby grapefruit (RS-SR) was mechanically transmitted to Chenopodium quinoa. RS-SR was partially purified by differential centrifugation, fractionation in a sucrose gradient, and agarose gel electrophoresis of selected fractions. Infectivity of concentrated extracts on C. quinoa was lost in individual fractions of the gradient, but it was recovered by combining a top and a bottom component. Both components contained a 48-kDa protein not found in similar preparations from healthy plants. After further purification the 48 kDa protein was detected at the top edge of the agarose gel. In the initial experiments a 38-kDa protein was found in the same fractions that later contained the 48-kDa protein. An antiserum obtained to the 38 kDa protein reacted in Western blots with both the 38- and the 48-kDa proteins, whereas another antiserum raised to the Florida isolate CRSV-4 (also containing a 48-kDa protein) did not react with the 38-kDa protein, indicating that the latter was probably a degradation product of the 48-kDa protein. Filamentous flexous particles were observed by serologically specific electron microscopy in crude extracts from RS-SR-infected C. quinoa plants. These results indicate that RS-SR is associated with a two-component virus similar to those associated with several psorosis and ringspot isolates, and serologically related to CRSV-4.     

Effect of temperature on RNA silencing of a negative‐stranded RNA plant virus: Citrus psorosis virus

Citrus psorosis virus (CPsV), genus Ophiovirus, causes a bark scaling disease of citrus. CPsV virions are kinked filaments with three negative‐stranded RNA molecules (vRNA) and a 48 kDa coat protein. The effect of temperature on symptom expression, virus accumulation and RNA silencing was examined in sweet orange seedlings (Citrus sinensis) graft‐inoculated with three different CPsV isolates and grown in a glasshouse at 26/18°C or 32/26°C (day/night). Most plants kept in the cooler glasshouse showed a shock reaction in the first flush with shoot necrosis, and then moderate to intense chlorotic flecking and spotting in young leaves, whereas plants incubated at 32/26°C did not exhibit shoot necrosis, and young leaf symptoms were milder. Virus titre estimated by ELISA and by northern and dot blot hybridization paralleled symptom intensity, with significantly higher virus accumulation in plants incubated at 26/18°C. The amount of CPsV‐derived small RNAs (CPsV‐sRNAs) slightly increased at 32/26°C, with the ratio of CPsV‐sRNA/vRNA being higher at 32/26°C than at 26/18°C. These results suggest that (i) CPsV infection induces RNA silencing in citrus plants, (ii) symptom intensity is associated with virus accumulation, and (iii) temperature increase enhances the RNA silencing response of citrus plants and decreases virus accumulation.     

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.     

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.     

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.     

福建省柑橘褐斑病菌的分离、鉴定和系统进化分析

为明确在福建省南平市的橘柚和三明市的温州蜜橘上发现的疑似柑橘褐斑病的病原菌种类,采用组织分离法获得纯化菌株,通过回接法验证菌株的致病性,利用形态学特征对病原菌进行初步鉴定,并采用最大似然法以多聚半乳糖醛酸酶基因endoPG为靶标对本研究以及国内外已报道的链格孢菌株构建系统发育树,分析其遗传多样性。结果表明,从病组织中共分离获得26株纯培养菌株,经形态学鉴定均为链格孢菌Alternaria spp.。利用分生孢子液接种橘柚离体嫩叶发现,有22株菌株能侵染橘柚叶片并产生与田间相似的褐斑病症状,确认该病害为链格孢引起的柑橘褐斑病。系统发育树分析结果显示,分离所得的26株菌株均聚在已报道的4个柑橘链格孢进化分支Clade1～Clade4中,其中21株菌株聚在国内特有的分支Clade4中,有3株菌株和1株菌株分别聚在国内外兼有的分支Clade3和Clade1中,1株菌株聚在国外特有的分支Clade2中,表明在福建省采集的这些柑橘褐斑病菌均为链格孢菌,且遗传多样性较丰富。     

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.     

Genetic Marker Analysis of a Global Collection of Isolates of Citrus tristeza virus: Characterization and Distribution of CTV Genotypes and Association with Symptoms

ABSTRACT Genetic markers amplified from three noncontiguous regions by sequence specific primers designed from the partial or complete genome sequences of Citrus tristeza virus (CTV) isolates T3, T30, T36, and VT were used to assess genetic relatedness of 372 isolates in an international collection. Eighty-five isolates were judged similar to the T3 isolate, 81 to T30, 11 to T36, and 89 to VT. Fifty-one isolates were mixed infections by two or more identifiable viral genotypes, and 55 isolates could not be assigned unequivocally to a group defined by marker patterns. Maximum parsimony analysis of aligned marker sequences supported the grouping of isolates on the basis of marker patterns only. Specific disease symptoms induced in select citrus host plants were shared across molecular groups, although symptoms were least severe among isolates grouped by markers with the T30 isolate and were most severe among isolates grouped by markers with the T3 isolate. Isolates assigned the same genotype showed variable symptoms and symptom severity. A classification strategy for CTV isolates is proposed that combines genetic marker patterns and nucleotide sequence data.     

湖南柑橘衰退病调查分析及弱毒系筛选

 The investigation showed that stem-pitting Citrus tristeza virus (CTV)occurred commonly in citrus production areas in several varieties of Hunan Province. Accurate detection of CTV strains was performed by p23/PCR method, PCR and the results indicated that the most samples were infected with several CTV isolates. Three mild strains were isolated and their pathogenicity was identified by biological identification, it indicated that p23/PCR groups had uniformity with the pathogenicity of CTV isolates. Furthermore, three mild isolates were tested in the cross protection by analysis of biological symptoms and composition of p23 gene. Different protecting effects were observed among these strains and W17 mild isolate was effective.     

First report of <Emphasis Type="Italic">Citrus psorosis virus</Emphasis> in Japan

Citrus psorosis virus (CPsV) was detected from citrus trees for the first time in Japan. The diagnosis was confirmed by molecular, serological, and biological indexing. RT-PCR detected CPsV from two citrus trees among ca. 200 tested. Both trees were variety Shiranui of [Citrus unshiu Marc. × C. sinensis (L.) Osb.] × C. reticulata Blanco, and neither had the bark scaling symptom typical of CPsV. The CPsV isolate could be genetically related to those from Spain, Italy, Florida, and California.     

Symptom expression and variation of rose mosaic

Three isolates ofPrunus necrotic ringspot virus (PNRV) causing line pattern in rose and one causing yellow mosaic were compared on four greenhouse and on five outdoor rose cultivars with an isolate of the virus causing Stecklenberger disease in morello and with an isolate of apple mosaic virus (AMV) from apple. All PNRV isolates were readily transmissible through budding in rose (up to 100%). AMV was hard to transmit from apple to rose, but as easily from rose to rose as the PNRV isolates. In the greenhouse, first symptoms were produced 28 days after budding. Budding of outdoor roses in early September usually gave first symtoms next spring, but sometimes not until late during the second year after budding. In the course of time symptom production was erratic and often on a few leaves only. Nature and severity of symptoms greatly depended on cultivar and to a lesser extent on the isolate used. Symtomatologically all isolates, including AMV, behaved like members of a continuum, with AMV in certain cultivars causing characteristic rose (yellow) mosaic but usually being more virulent on rose.     

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.     

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.     

Aphid Transmission Alters the Genomic and Defective RNA Populations of Citrus tristeza virus Isolates

ABSTRACT A total of 14 Spanish isolates of Citrus tristeza virus (CTV) and 1 isolate from Japan were transmitted by Aphis gossypii, and the subisolates obtained were compared with the source isolates for symptom expression and double-stranded RNA (dsRNA) pattern. Of the 14 Spanish isolates, 9 showed altered dsRNA patterns after aphid transmission but only minor variations in the intensity of symptoms induced on Mexican lime. Northern blot hybridization with complementary DNA (cDNA) probes corresponding to both the 5' and the 3' termini of the CTV genomic RNA (gRNA) showed that the dsRNA bands that could be used to discriminate between the dsRNA pattern of the source and the aphid-transmitted isolates were the replicative forms of defective RNAs (D-RNAs). Conversely, the Japanese isolate and two subisolates obtained from it by aphid transmission had the same dsRNA pattern, but one of the subisolates induced milder symptoms in several hosts. Dot-blot hybridization with cDNA probes representing several regions of the gRNA showed that most of the aphid-transmitted isolates differed from the corresponding source isolate by their hybridization pattern. Our results indicate that aphid transmission often sorts the populations of gRNA variants and D-RNAs present in CTV isolates.     

Reclassification of an isolate of Guignardia citricarpa from New Zealand as Guignardia mangiferae by sequence analysis

Citrus black spot was recorded as present in New Zealand in international databases on the basis of one isolate (ICMP 8336) identified by morphological features as Guignardia citricarpa . This isolate was from a soft rot, not a typical symptom of citrus black spot, on a Seminole tangerine fruit grown in the northern region of New Zealand. Sequence analysis of the internal transcribed spacer (ITS) region (ITS1, 5·8S and ITS2) showed that this isolate was 99% identical to the ITS region of G. mangiferae , a closely related saprotroph. Despite climatic conditions being suitable for this disease, citrus black spot symptoms have never been seen on citrus fruits grown in New Zealand. Thus the absence of symptoms on citrus in New Zealand is probably because G. citricarpa is not present. On the basis of these results, the record of citrus black spot occurring in New Zealand should be re-examined.     

Biological diversity of citrus tristeza virus (CTV) isolates in Spain

A survey of citrus tristeza virus (CTV) isolates was carried out in most citrus-growing areas in Spain. Twenty-two isolates were selected by geographical origin, cultivar of source tree, and symptoms observed on the host or in preliminary tests, and were biologically characterized.
A wide range of variation in transmissibility by aphids and symptom intensity on nine different indicator species or scion-rootstock combinations was observed among CTV isolates. Mexican lime. Citrus macrophylla , and to a lesser extent citron were the most useful hosts for characterizing these isolates, and leaf symptoms and stem pitting were the most discriminating traits. Positive correlation was observed between symptoms induced on Mexican lime and C. macrophylla , but not between the symptoms induced on these indicators under greenhouse conditions and the homologous symptoms on plants grown in the screenhouse. Some of the traits studied enabled us to establish relatively well-defined groups of isolates, but in most cases a continuous range of variation was obtained and no clear group could be defined.     

Occurrence of the Andean strain of potato virus S in imported potato material and its effects on potato cultivars

Infection with potato virus S Andean (PVS^A) and ordinary (PVS^o) strains was found in potato breeder's selection No. 8163-511 imported from West Germany; the two PVS strains were differentiated by their reactions on Chenopodium quinoa Tests on potato leaf samples using enzyme-linked immunosorbent assay followed by inoculation to C quinoa were subsequently used to detect PVS^A and PVS^o in a large-scale survey of imported and domestic potato material. PVS^A was detected in breeders' selections and cultivars imported from the Netherlands and West Germany, but not in domestic certified seed potato stocks or farmers' once-grown stocks. PVS^o was found in both imported and domestic certified stocks, but infection was commoner in the imported ones. When plants of C. quinoa, C. amaranticolor, C. murale and Nicotiana debneyi were inoculated with four isolates of PVS^A, one induced mild symptoms while the reactions of the others ranged from moderate to severe. When plants of different potato cultivars were inoculated with three isolates, the plants were mostly infected without symptoms. However, when tubers from some were grown on, the progeny plants of most of the different combinations of cultivar and isolate of PVS^A developed one or more of the following symptoms: vein deepening, rugosity, interveinal chlorosis, premature senescence and early loss of lower leaves.