Woody Indexing Research Articles

Biological assays for plant viruses and other graft‐transmissible pathogens diagnoses: a review

Biological assays, often referred to as woody indexing and herbaceous indexing, may be used to detect and identify plant viruses and other graft‐transmissible pathogens to ensure that plants for planting are free from regulated pests. However, many factors can influence the results of biological indexing, which affect the analytical specificity, analytical sensitivity and reliability (repeatability and reproducibility) of these tests. This review focuses on the following: the environmental factors, the role of the inoculum source, the choice of the indicator plant, the expression of symptoms, the number of repetitions, the use of positive controls and healthy controls, the means of transmission of pathogens, the differentiation of strains, the influence of isolates or strains of pathogens, mixed infections, and the bulking of samples for testing. Some indicator plants which are highly susceptible to many viruses and virus‐like diseases may be used to detect a wide range of pathogens. However, comparing the results from bioassays with those from serological or molecular tests has shown the limits for detection and identification of many pathogens. It is concluded therefore that biological assays should only be considered for use in conjunction with serological or molecular tests to detect and identify pathogens. Bio‐amplification by indicator plants to amplify a pathogen to levels that can be detected by another test method remains of interest.

First Report of Infection of Cherry Rusty Mottle Associated Virus in Portuguese Laurel (Prunus lusitanica) in Washington State.

During late spring of 2012 in Snohomish County of Washington State, chlorotic yellow leaf blotch symptoms suggestive of a virus infection were observed on Portuguese laurel (Prunus lusitanica) planted in a hedge row. Leaf samples from representative trees were initially tested for the presence of Cherry leaf roll virus (CLRV) and Plum pox virus (PPV) by ELISA with antibodies specific to CLRV and general potyvirus, respectively (Agdia, Inc., Elkhart, IN). The ELISA test yielded negative results for both viruses. Reverse transcription (RT)-PCR was pursued to detect other viruses known to infect Prunus spp., namely American plum line pattern virus (APLPV), Apple chlorotic leafspot virus (ACLSV), Cherry mottle leaf virus (CMLV), Cherry raspleaf virus (CRLV), Cherry virus A (CVA), Prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV), as well as CLRV and PPV. None of these viruses were detected. However, RT-PCR with a generic primer pair Fovea2/AdPr (3) that amplifies the coat protein (CP) coding sequence and 3'-untranslated regions (3'-UTR) of several members of the family Betaflexiviridae yielded a 1.4-kb amplicon that was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. KF356396). The sequences from three clones were 99.8% identical to each other at the nucleotide level. Comparison of the consensus CP coding region with the nucleotide sequence database revealed 86 to 93% identity to Cherry rusty mottle associated virus (CRMaV), and only 73 to 75% identities to Cherry necrotic rusty mottle virus (CNRMV) (1) and 71 to 76% identities to Cherry green ring mottle virus (CGRMV) (4) isolates from sweet cherry (P. avium). This result suggested that the cloned fragment represents a strain of CRMaV. Prunus avium 'Bing' and 'Sam,' and P. serrulata 'Kwanzan' were grafted with bark patches from the symptomatic tree and observed for induction of cherry rusty mottle disease (CRMD) symptoms. Ninety days after grafting, symptoms typical of CRMD consisting of chlorotic yellow mottle appeared on 'Bing,' 'Sam,' and 'Kwanzan' indicators. Small necrotic spots also appeared on the leaves of the latter. Angular necrotic lesions on 'Sam' and epinasty of 'Kwanzan' that are diagnostic symptoms of cherry necrotic rusty mottle disease (CNRMD) and cherry green ring mottle disease (CGRMD), respectively, were absent from graft inoculated indicators. Further RT-PCR tests on the indicators using primers specific to CNRMV, CGRMV, and CRMaV (2) yielded negative results for CNRMV and CGRMV but showed positive amplification for CRMaV. The results of the woody indexing corroborate the presence of CRMaV but the absence of CNRMV and CGRMV in the symptomatic Portuguese laurel. To our knowledge, this is the first report of CRMaV in Portuguese laurel in the United States and the first description of symptoms associated with CRMaV in this host. As a potential reservoir of CRMaV, Portuguese laurel could play an important component in management of CRMD in cherry production areas where this ornamental cherry is also present.

The reliability of woody indexing for detection of grapevine virus-associated diseases in three different climatic conditions in Australia

Background and Aims In Australia, grapevine varieties are tested for viruses by woody indexing (WI) in combination with other procedures during post-entry quarantine and prior to their introduction into the nucleus collections of high health certification programs. The aim of this study was to determine the reliability and accuracy of WI for virus detection in different climatic conditions. Methods and Results Replicated experiments were conducted in a hot climate, a cool climate and a screenhouse. Indicator plants were inoculated with Grapevine virus A, Grapevine virus B, Grapevine fleck virus, Grapevine leafroll-associated virus (GLRaV) 1, GLRaV-2, GLRaV-3, GLRaV-9 and Rupestris stem pitting-associated virus. Indicators were observed during three years for characteristic symptom development and tested for viruses by reverse-transcription polymerase chain reaction. Virus transmission was not always successful. Symptoms were not always observed in each year at each trial site even if viruses could be detected by reverse-transcription polymerase chain reaction. Conclusions WI reliability was affected by the success of bud-take, transmission of the virus from the candidate bud to the indicator and different climatic conditions. It is recommended that WI be carried out for a minimum of three years in the field. Significance of the Study Understanding the effect of different climatic conditions on WI will improve the reliability of grapevine virus detection in Australia.

Magyarországon előforduló szőlővírusok és a hazai szőlő vírusbetegségek áttekintése

The past fifty years important advances have been made in the field of grapevine virus research, including characterization of pathogens and control measurements. Still the occurrence of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tomato black ring virus (TBRV), Grapevine chrome mosaic virus (GCMV), Alfalfa mosaic virus (AMV), Grapevine Bulgarian latent virus (GBLV), Grapevine fleck virus (GFkV), Grapevine leafroll- associated viruses (GLRaV1-4), Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine rupestris stem pitting- associated virus (GRSPaV) have been reported in Hungary and characterized by conventional methods as woody indexing, herbaceous indexing and serological methods. Among grapevine viruses the Grapevine line pattern virus (GLPV) seems to be uncial; because it was reported only in Hungary. Causal agents of several grapevine diseases, like enation, vein necrosis and vein mosaic remained undiscovered. These virus-like diseases occurred only sporadically, without economic importance.

Molecular divergence of Grapevine virus A (GVA) variants associated with Shiraz disease in South Africa

Shiraz disease (SD) is a highly destructive, insect-transmitted disease of noble grapevine cultivars, such as Shiraz and Merlot, in South Africa. Earlier studies revealed that, of the three molecular groups of GVA that were detected in local vineyards, variants of group II are closely associated with expression of this disease, and variants of group III are commonly present in GVA-infected SD-negative plants [Goszczynski, D.E., 2007. Single-strand conformation polymorphism (SSCP), cloning and sequencing of Grapevine virus A (GVA) reveal a close association between related molecular variants of the virus and Shiraz disease in South Africa. Plant Pathol. 56, 755–762]. Among many GVA variants transmitted to Nicotiana benthamiana from various grapevines with different SD status, a variant of group II, referred to here as GTR1-2, was also isolated from a consistently SD-negative Shiraz plant. The genome of this variant, along with 7 other GVA variants of the 3 molecular groups, including 4 variants of molecular group II associated with strong symptoms of SD, were sequenced. Results of comparative analysis of these genomes, with emphasis on differences between GTR1-2 and other variants of molecular group II, are presented. Among the many differences that were found, a 119 nt ORF2-related fragment was discovered within the native ORF2 of a GVA variant, P163-M5. This variant was isolated from a grapevine used by local industry as a reliable positive control for SD in woody indexing. The variant induced symptoms in N. benthamina that were significantly more severe than those of other variants of molecular group II.

Certification scheme forSambucus

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Viruses of pome fruit trees in Syria

A survey was conducted to evaluate the sanitary status of pome fruit trees in Syria during spring 2003 and 2004 in 6 governorates: Damascus, Al‐Qunaitara and Al‐Sweida (Southern region), Homs and Hama (Central region) and Latakia (Costal Western region), as the main production areas of pome fruits. Leaf samples from 1077 apples, 54 pears and 14 quince were collected and tested for the presence of Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV) in 70 commercial orchards and 3 varietal collections by ELISA. Results showed that the virus infection rates were 34 and 2% in apple and pear, respectively. Quince trees were found to be virus tested free. ACLSV was prevailing on apple with 34%, whereas ASGV and ApMV were found in 2 and 0.2% of tested trees, respectively. Pear trees were infected only with ACLSV (2%). 21 apples and 15 pears representative budwood samples were indexed by grafting on the following indicators: (i) Malus pumila cvs. Virginia Crab and Radiant for apple and (ii) M. pumila cv. V. Crab and Pyrus communis cv. Nouveau Poiteau for pear. The virus infection rates by woody indexing were much higher than ELISA, Apple stem pitting virus (ASPV) and ASGV were found in 86 and 82% of apple tested samples, whereas they were 80 and 60% of pear tested samples, respectively. Additional RT‐PCR testing carried out for a limited number of samples confirmed the high incidence of ACLSV ASPV, ASGV and the presence of ApMV. This is the first report on pome fruit viruses in Syria, indicating an unsatisfactory sanitary status of the industry. As a consequence, a certification program is recommended for producing locally healthy propagating material.

New Variant of Little cherry virus Associated with Little Cherry Disease of Sweet Cherry in British Columbia, Canada.

Little cherry disease (LChD) occurs in most cherry growing areas in the world. Infection of sensitive cultivars results in small fruit with poor color, angular shape, and insipid flavor. Three viruses associated with LCD have been described: (i) Little cherry virus-1 (LChV-1) first found and described in Germany (4); (ii) LChV-2 an isolate obtained from the United States (2); and (iii) LChV-3 first found and described in British Columbia (1). Despite similarities in symptom development in orchard trees and woody indexing, the three viruses have distinct molecular sequences (3). LChV-2 and -3 share greater homology with each other than either does with LChV-1. For many years, the British Columbia Ministry of Agriculture, Fisheries and Food (BCMAFF) in conjunction with Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre (AAFC-PARC) has conducted a survey to monitor the incidence and spread of LChD in the Okanagan and Kootenay valleys of British Columbia, Canada. Until recently, testing for LChD used woody indexing on indicator trees, Prunus avium cv. Lambert (fruit symptoms) and cvs. Canindex1 and Sam (foliar symptoms). Recently, incidence of LChD has been evaluated using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) developed at AAFC-PARC (3), and reverse transcription-polymerase chain reaction (RT-PCR) tests based on sequence data from LChV-3 (1). During the 1999 survey, orchard trees displaying symptoms typical of LChD tested negative for LChV-3 using ELISA and RT-PCR. Also, trees that formerly tested positive for LChD by woody indexing also tested negative for LChV-3 using RT-PCR and ELISA. Two hundred ninety-three trees were subsequently tested for LChV-1 by RT-PCR using the primer set LCV3EC/LCV16659 (4). A 276-bp fragment corresponding to the extreme 3' untranslated region (3' UTR) of the LChV-1 genome was amplified by RT-PCR from 140 of the trees. The RT-PCR amplicon from one sample (#99-68B from Peachland) was sequenced and using a BLASTn search, LChV-1 was identified as the most probable match (E value 4e-59). Sequence alignment using ClustalX identified two regions of high sequence homology; bases 2 to 70 (92%) and bases 99 to 239 (95%). The intervening region displayed much lower homology (61%). The overall homology of the amplicon was 88% compared to the corresponding region in LChV-1. Divergence between the published sequence of LChV-1 and the sequence of the new LChV isolate (tentatively named LChV-4) was investigated. Seven sets of primers constructed on the basis of sequence data from various regions of the LChV-1 genome (two sets from each of the RNA-dependent RNA polymerase, heat shock 70 protein homologue, and coat protein) failed to yield RT-PCR products when tested with the LChV-4 isolate. LChV-4 is clearly related to LChV-1 within the 3'-UTR but complete sequencing is required to determine the overall relationship with other viruses causing LChD. The discovery of a new isolate of LChV in British Columbia may require a reevaluation of the epidemiology of LChD and disease management strategy.

Certification programme for production of virus-free propagating material of grapevine and its results in Hungary

In Hungary, detection of virus and virus-like diseases of grapevine began in 1960's at the Research Institute for Viticulture and Enology by János Lehoczky and his colleagues. At present, sixteen virus and virus-like diseases of Vitis vinifera are known to occur in Hungary.&#x0D; Regular virological screening of grapevine varieties started in 1972. The present system of screening (visual selection, indexing, ELISA) has been established using methods with continuous improvement according to recommendations of international organizations.&#x0D; In the first year symptomless grapevine plants are selected and marked during surveys carried out twice in the vegetation period: at about flowering and in the second half of September. At the first selection time plants are sampled for ELISA.&#x0D; In the spring of the second year, overwintered canes are checked by woody indexing on 8 indicator species in the field.&#x0D; In the third and fourth years the nursery is evaluated twice again. At the end, the marked grapevine plants, giving negative results on all indicators in every case, are considered virus-free.&#x0D; In autumn of the fourth year, the virus-free material is planted out under screenhouse and also in a special mother block (nuclear stock) for maintenance and propagation.&#x0D; Mother blocks of virus-free scion varieties have been established on 2 ha and those of rootstock varieties on 0.5 ha planted with the following number of varieties included in the national list: 71 European scion — and 12 rootstock varieties or variety candidates/clones. It is necessary to increase the area of Pre-base, Base and Certified stocks exclusively with tested virus-free (clean) material.

Detection of Prunus necrotic ringspot virus in almond: effect of sampling time on the efficiency of serological and biological indexing methodologies

The reliability of enzyme-linked immunosorbent assay (ELISA) and indexing methodologies for the detection of Prunus necrotic ringspot virus (PNRSV) in almond was assessed under local conditions. Thirteen field-grown almond trees were sampled fortnightly throughout the growing season. Petal and leaf homogenates were used for ELISA and to inoculate herbaceous indicator species, while buds collected from October onwards were budded to woody indicators. ELISA reliably detected PNRSV in petals and young leaves from bud-break until the cessation of stem elongation. While herbaceous indexing was not as reliable as ELISA, woody indicators could differentiate positive from negative samples reliably throughout the testing period. However, for mass screening of foundation plantings, nursery stock and elite germplasm, woody indexing is too costly and slow to give results. The use of ELISA can overcome these limitations but the timing of sample collection is critical. Petal or leaf tissue collected early in the season will yield the most reliable results.

Rapid and Sensitive Detection of Apple stem pitting virus in Apple Trees Through RNA Amplification and Probing with Fluorescent Molecular Beacons

ABSTRACT Currently, detection of Apple stem pitting virus (ASPV; genus Foveavirus) in apple trees for certification purposes occurs by woody indexing. This method requires a minimum of 12 to 24 weeks in greenhouse testing to up to 2 years in field testing. In this paper, the development of a single tube AmpliDet RNA system for the rapid gel-free detection of ASPV in apple tree tissues is described. The system relies on the specific amplification of the viral RNA by nucleic acid sequence-based amplification and the simultaneous fluorescent detection of the amplification product through molecular beacons. A sensitivity of a minimum of 100 molecules of transcript RNA was obtained by the ASPV-specific AmpliDet RNA. All biologically characterized ASPV isolates from a field trial and 12 of 14 isolates from a plant virus collection were readily detected with this AmpliDet RNA system. In addition, the efficiency of this method for detecting ASPV in 'Golden Delicious' and 'Gravenstein' apple trees was compared throughout the year with mechanical inoculation onto Nicotiana occidentalis 37B, a candidate indicator for ASPV. This revealed that only AmpliDet RNA consistently detected the virus in bark tissue, irrespective of the season. Season-specific tissues such as buds, petals, and fruits, but not leaves, also were reliable sources for detection of ASPV by the AmpliDet RNA system.

Detection and identification of phytoplasmas in peach based on woody indexing and molecular methods

Symptoms resembling phytoplasma disease have been observed on peach trees in a seed-source plantation of stone fruits in south Hungary quite recently. In this publication we report on the results of woody indexing of symptomatic peach trees on GF 305 indicator in the field and under greenhouse conditions as well as on molecular studies. Phytoplasma infection detected on GF 305 indicators in greenhouse and field indexing was confirmed by PCR. Nested PCR was conducted using universal primer pairs followed by group and subgroup specific primers for the second amplification. RFLP analysis of nested PCR products was performed using Rsal restriction enzyme. Based on the results of molecular studies it can be concluded that phytoplasmas, belonging to the European stone fruit yellows subgroup (16SrX-B) were identified in peach trees. Further studies on symptomatic peach trees originating from different parts of Hungary are in progress.

First Report of Grapevine Kober Stem Grooving in the United States.

The last decade has brought extensive new plantings of grapevines (Vitis vinifera L.) to California vineyards. In some vineyards, severe viruslike problems have been observed (2). To ascertain possible causal agent(s), extensive testing for grapevine viruses was initiated. Testing included a 2-year woody index on indicators V. rupestris cv. Saint George, LN 33, V. vinifera cv. Cabernet Franc, and Kober 5BB. To our knowledge, this is the first field survey for grape virus diseases in the United States that includes woody indexing on Kober 5BB, a relatively new indicator for the grapevine rugose wood diseases (1). A Sonoma County field selection of Sauvignon Blanc grafted to the rootstock Freedom was included in these tests; the 2-year-old vines exhibited stunting, leaf rolling, leaf yellowing, wood necrosis, and pitting symptoms at the graft union. When indexed on the four indicators, the following disease symptoms were observed: stem pitting on V. rupestris; corky bark on LN 33; leafroll on Cabernet Franc; and stem grooving on Kober 5BB. An enzyme-linked immunosorbent assay confirmed the presence of grapevine leafroll-associated viruses 2, and 3, as well as Grapevine Virus A, which is associated with Kober stem grooving. The Sauvignon Blanc selection appears to be infected with several grapevine viruses, as is often the case when virus symptoms in vineyards are severe. Among these disease agents, only Kober stem grooving is reported to cause the severe pitting and grooving symptoms observed on the indicator Kober 5BB (1). This is the first report of Kober stem grooving in the United States.

Comparison between woody indexing and a rapid hybridisation assay for the diagnosis of little cherry disease in cherry trees*

SummarySweet cherry (Prunus avium) and sour cherry (Prunus cerasus) trees from orchards in the Kootenay and Okanagan Valleys of British Columbia, Canada were assayed for the presence of little cherry disease by three different methods: Northern blot analysis of double‐stranded RNA, woody indexing for fruit symptoms on sweet cherry cv. Lambert, and woody indexing for foliar symptoms on cv. Canindex 1. Results of the three methods were in agreement for 85% of the samples. Of the 78 orchard trees tested, double‐stranded RNA isolated from 48 trees hybridised with a radiolabeled cloned probe specific for little cherry disease. When the 48 trees were tested by woody indexing, buds from 41 trees induced fruit symptoms on cv. Lambert, but only 32 yielded foliar symptoms on cv. Canindex 1 under the conditions of the experiment. Of the 30 orchard trees that did not yield a positive response to the Northern blot analysis, 26 samples were negative on cv. Lambert and 26 were negative on cv. Canindex 1. Northern blot analysis of the 78 cv. Lambert indicator trees revealed that there was an absolute correlation between the presence of little cherry disease‐associated double‐stranded RNA and the development of typical little cherry disease symptoms on the indicator trees. Reliability of woody indexing of orchard samples was impaired by poor transmission of the disease from the inoculating bud to the indicator tree. Woody indexing with cv. Canindex 1 was particularly prone to a large number of apparently erroneous negative results. Of the three protocols used, diagnosis of little cherry disease by Northern blot analysis was found to be the most reliable and offered a greatly accelerated means of diagnosis.

Comparative Effectiveness of Symptomatology and ELISA For Detecting Two Isolates of Grapevine Leafroll on Graft-inoculated Cabernet franc

An enzyme-linked immunosorbent assay (ELISA) was compared to the standard woody indexing procedure for effectiveness in detecting grapevine leafroll associated virus (GLRaV) 3 and 4 in infected grapevines of cvs. Pinot noir (PN) and Thompson Seedless (TS). Four plants per plot of individual Cabernet franc (CF) indicator plants were each graft-inoculated with buds from 24 vines of PN and TS infected with the GLRaV 3 and 4, respectively. Inoculated indicators were established in the field, observed periodically for symptoms, and assayed by ELISA. All plots showed GLRaV symptoms and positive ELISA reactions by the end of the first full growing season for GLRaV 3 and by the end of the second full growing season for GLRaV 4. Results on an individual plant basis were similar, whereby 92% to 95% of CF inoculated with GLRaV 3 or GLRaV 4 were positive for symptoms and ELISA. ELISA reactions for GLRaV 3 were consistently higher than those for GLRaV 4. Virus transmission was not dependent on growth of inoculated buds. We conclude that ELISA is useful for rapid detection of GLRaV 3 and 4 in dormant wood of source grapevines and in inoculated CF indicators.