Detection and Elimination of Viruses in Callus, Somatic Embryos and Regenerated Plantlets of Grapevine

The distribution of some grapevine viruses in flower explants, embryogenic and non-embryogenic calli, single somatic embryos and plants regenerated from embryogenic cultures was investigated by RT-PCR and ELISA. Immature anthers and ovaries of the cultivars Grignolino infected by GRSPaV, GLRaV-1 and GVA, Müller-Thurgau infected by GRSPaV and GLRaV-3 and Bosco infected by GRSPaV were cultivated on media inducing indirect somatic embryogenesis. Viruses were detected both in anthers and ovaries. Four months after culture initiation 65.6% of tested calli were infected by at least one virus; high percentages of virus infection were found in calli originating from ovaries. No virus was detected in calli tested 8 months after culture initiation, as well as in single somatic embryos or in embryo-derived plantlets. Somatic embryogenesis confirmed its effectiveness in eliminating phloem-limited grapevine viruses. Regeneration of RT-PCR negative plantlets occurred even when at least a sector of the callus was still infected: the mechanism whereby somatic embryos are freed of some viruses could be related to the rapid proliferation of embryogenic cells within the callus or to the origin of the embryogenic callus from virus-free cells within the original explant.     

Somatic embryogenesis efficiently eliminates viroid infections from grapevines

Indirect somatic embryogenesis is effective at eliminating the most important viruses affecting grapevines. Accordingly, this technique was tested as a method for eradicating two widespread viroids, Grapevine yellow speckle viroid 1 (GYSVd-1) and Hop stunt viroid (HSVd), from four grapevine cultivars. Both viroids were detected by RT-PCR in grapevine floral explants used for initiating embryogenic cultures, as well as in undifferentiated cells of embryogenic and non-embryogenic calli from anthers and ovaries. In contrast, somatic embryos differentiated from these infected calli were viroid-free, and viroids were not detected in embryo-derived plantlets even 3 years after their transfer to greenhouse conditions. A wider spatial distribution of HSVd than GYSVd-1 within proliferating calli was revealed by in situ hybridization, whereas no hybridization signal was detected in the somatic embryos. In addition, GYSVd-1 and HSVd were localised in the nucleus of infected cells, conclusively showing the nuclear accumulation of representative members of Apscaviroid and Hostuviroid genera, which has been only an assumption so far. Somatic embryogenesis was compared to in vitro thermotherapy, a technique routinely used for virus eradication. After thermotherapy, HSVd and GYSVd-1 were detected in all in vitro plantlets of the cultivar Roussan, and in all lines analysed after 3 years of culture in greenhouse. The high efficiency with which somatic embryogenesis may eliminate viroids and viruses from several infected grapevine cultivars, should allow the availability of virus- and viroid-free material, which would be useful not only for sanitary selection but also for basic research on plant-virus and plant-viroid interactions in grapevine.     

<Emphasis Type="Italic">In situ</Emphasis> localization of <Emphasis Type="Italic">Grapevine fanleaf virus</Emphasis> and phloem-restricted viruses in embryogenic callus of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

In grapevine, somatic embryogenesis is particularly effective in eliminating several important virus diseases. However, the mechanism whereby regenerated somatic embryos are freed of the viruses is not clear. The distribution of Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus-3 (GLRaV-3) and Grapevine virus A (GVA) in embryogenic callus of grapevine was investigated by in situ hybridization using digoxygenin-labelled oligonucleotide probes. Four months after culture initiation, in callus originated by GFLV-infected explants we observed a mosaic of infected and uninfected cells, with high concentrations of viruses in some cell groups in peripheral zones of the callus. In addition some abnormal somatic embryos showed a high hybridization signal. In callus originated by GVA- and GLRaV-3-infected explants the viruses were concentrated in few cells surrounded by areas of virus-free cells. The two viruses were generally localized in different clusters of cells inside the callus and the levels of infection were lower than those observed in GFLV-infected callus. No virus was detected in callus nor in somatic embryos after 6 months of culture. The results highlight the difficulties of some viruses at stably invading callus tissues and the differential ability of GFLV to spread in the callus cells compared to the phloem-limited viruses.     

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.     

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.     

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.     

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.     

Uncontrolled Citrus psorosis virus infection in Citrus sinensis transgenic plants expressing a viral 24K-derived hairpin that does not trigger RNA silencing

Citrus psorosis virus (CPsV) is the causal agent of psorosis disease of citrus. Pineapple sweet orange plants were transformed with a hairpin construct derived from the viral 24k gene (lines ihp24K). Contrary to expectations, these lines did not trigger efficient RNA silencing, and when infected with CPsV they showed a phenotype of exacerbated symptoms with a persistent and homogeneous infection without the recovery observed in non-transgenic plants. Ihp24K lines did not behave similarly when challenged with Citrus tristeza virus. All these results indicate that hypersusceptibility is likely related to the specific action of 24K-derived hairpin over CPsV multiplication.     

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.     

应用农杆菌介导法获得转大麦黄矮病毒GPV株系复制酶基因ORF2小麦植株

以生产用5种基因型小麦为材料,利用农杆菌介导法将大麦黄矮病毒GPV株系复制酶基因ORF2转化愈龄40d的幼胚愈伤组织,经过G418筛选后,共再生出9株抗性植株,拓宽了小麦农杆菌转化的受体基因型。PCR及Southern杂交分析证实大麦黄矮病毒复制酶基因ORF2已经整合到小麦基因组中。初步抗病性检测结果显示转基因植株对GPV株系具有抗性。研究结果还表明,受体基因型、外植体的生理状态对农杆菌侵染起着至关重要的作用。     

Production of desert locust feeding deterrents from in vitro cultured neem (Azadirachta indica)

Callus was initiated from cotyledon, hypocotyl and shoot tip explants of neem seedlings (Azadirachta indica A. Juss.) that were cultured on Murashige and Skoog (MS) agar medium supplemented with MS vitamins, casein hydrolysate, indole-3-acetic acid and benzylamino purine. Shoots regenerated from hypocotyl-derived callus only. Mature 10-12-week-old callus regenerated both shoots and somatic embryos (SEm). The antifeedant activity of different types of callus, leaves and SEm was determined with larvae of the desert locust Schistocerca gregaria in a no-choice feeding bioassay. In vitro tissue-cultured neem had antifeedant properties against the desert locust. To the best of our knowledge the antifeedant activity of neem somatic embryos extracts is reported here for the first time.     

New genes for disease resistances through somatic hybridization

Somatic hybridization, a process of combining protoplasts from different plants, can provide new sources of disease resistances for plants. In the case of wild and cultivatedSolanum species, the hybrids express resistances from each partner in the fusion and can often be crossed with cultivars to improve agronomic characteristics of the tubers. Restriction fragment length polymorphism (RFLP) analyses can provide a means for determining that the plants being investigated are actually hybrids as well as a means for following the introgression of DNA into progeny lines. These points are addressed in this paper with specific reference to somatic hybrids betweenSolanum brevidens and potato.     

Identification of the Chinese Box Orange (Severinia Buxifolia) as an Alternative Host of the Bacterium Causing Citrus Huanglongbing

The Chinese box orange (Severinia buxifolia) was shown by graft-inoculation and psyllid-transmission tests to be an alternative host of the bacterium causing citrus Huanglongbing (HLB). A PCR-based assay for detection of the HLB bacterium (HLBB) was used to monitor HLBB. In graft-inoculation tests, the Chinese box orange (CBO) grafted with HLBB-infected scions of Luchen sweet orange (LSO) were positive for HLBB, 2–3 months after grafting. The back-grafting test demonstrated that HLBB-infected CBO scions could transmit HLBB back to LSO hosts via grafting. In psyllid-transmission tests, psyllids (insect vectors) transmitted HLBB to CBO plants, in which HLBB could be detected 3–4 months after inoculation. Acquisition-access tests of psyllids revealed that HLBB-free psyllids can acquire HLBB from diseased CBO hosts and can transmit HLBB back to the LSO plants. A field survey verified the presence of HLBB-infected CBO plants in the vicinity of citrus orchards. In this paper, CBO is shown to be a susceptible host plant in which HLBB can exist and replicate. It is also a donor plant from which HLBB can be transmitted to citrus hosts by grafting or by psyllid vectors.     

Properties of a citrus isolate of olive latent virus 1, a new necrovirus

A virus was recovered by sap transmission from plants of several citrus species exhibiting or not symptoms of chlorotic dwarf (CCD), a disease recently reported from Eastern Mediterranean Turkey. The virus was identified as an isolate of olive latent virus 1 (OLV-1), originally described as a possible sobemovirus. The citrus isolate of OLV-1 (OLV-1/Tk) possesses biological, morphological, physico-chemical, and ultrastructural properties similar, if not identical to those of the OLV-1 type strain and is also serologically indistinguishable from it. In addition, OLV-1/Tk has many properties, especially physico-chemical, in common with serotypes A and D of tobacco necrosis necrovirus (TNV-A and TNV-D). However, OLV-1/Tk is only very distantly related serologically to both TNV-A and D, suggesting that it can be regarded as a distinct species in the genusNecrovirus. OLV-1/Tk could not be detected in citrus tissues by ELIS A or dot-blot molecular hybridization, probably because of the extremely low virus concentration. By contrast, limited virus recovery was obtained by sap inoculation and fair detection rates were afforded by PCR. OLV-1/Tk was identified in 54 of 92 (59%) citrus plants affected by CCD and in 14 of 49 (28%) symptomless plants. These results do not support the notion that there is a cause-effect relationship between OLV-1/Tk and CCD, even though the more frequent association of this virus with diseased plants remains intriguing.     

Somatic embryogenesis from anthers of the autochthonous <Emphasis Type="Italic">Vitis vinifera</Emphasis> cv. Domina leads to <Emphasis Type="Italic">Arabis mosaic virus</Emphasis>-free plants

Attempts to conserve and utilise autochthonous grapevine germplasm in modern breeding programmes, are sometimes faced with the challenge that virus-free plants of old grapevine varieties and clones are hard to find. From 50 year-old vineyards in Frankonia the Vitis vinifera cv. Domina was selected showing particularly interesting loose-bunch architecture with fewer berries. However this valuable germplasm was carrying an Arabis mosaic virus (ArMV) infection requiring a reliable and effective method to produce healthy mother plants for clonal selection. Somatic embryogenesis was established from anthers as the most promising technical approach. The absence of ArMV in 46 regenerated plant lines was confirmed by ELISA and IC-RT PCR, repeated after different time intervals in vitro and in vivo after acclimatisation, and after one dormancy period under glasshouse conditions. Morphologically, all grapevines appeared true-to-type, and a screening of 20 plants by flow cytometry to determine the ploidy level and to exclude the risk of undesired genetic variability confirmed that all tested plants were diploid. Field evaluations of the initially selected bunch traits are currently underway.