A Rapid Protocol for Evaluating Prunus Germplasm for Tomato Ringspot Virus Resistance

Rooted cuttings of `Halford' and `Redhaven' peaches [Prunus persica (L.) Batsch] and `Stanley' (Prunus domestica L.) and `Marianna 2624' (P. cerasifera × P. munsoniana) plums were planted in soil containing ≈38 tomato ringspot virus-(TomRSV) infested nematodes (Xiphinema americanum sensu lato Cobb) per 100 cc. Test- and control-plant sap extracts were made from root and leaf tissues after 10, 22, and 34 weeks. Aliquots of these samples were assayed by mechanical inoculation to Chenopodium quinoa Willd. Total nucleic-acid extracts prepared from the remainder of each sample were analyzed by dot blot hybridization using a cRNA probe for TomRSV. The bioassay identified one `Stanley' and two `Redhaven' infected plants. Hybridization results indicated that two of two `Stanley', three of three `Halford', five of five `Redhaven', and zero of six `Marianna 2624' were infected. Our results demonstrate the sensitivity of molecular hybridization for TomRSV detection in Prunus and substantiate the TomRSV resistance of `Marianna 2624'.

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Identification of Genes Regulated by Low Temperature in Pachysandra terminalis Sieb. et Zucc Using cDNA Differential Display

HortScience ◽

10.21273/hortsci.40.7.1995 ◽

2005 ◽

Vol 40 (7) ◽

pp. 1995-1997

Author(s):

Suping Zhou ◽

Roger J. Sauve ◽

Abdulah Abdulah

Keyword(s):

Differential Display ◽

Blot Hybridization ◽

Leaf Tissue ◽

Northern Blot Hybridization ◽

Dot Blot ◽

Novel Genes ◽

Cold Tolerant ◽

Reverse Northern ◽

Stress Related Genes ◽

And Control

Complementary Deoxyribonucleic Acid (cDNA) differential display and reverse Northern dot blot were used to identify genes in Pachysandra terminalis Sieb. & Zucc., a cold-tolerant plant, that are regulated by low temperatures. Rooted cuttings were obtained from stock plants that had been maintained in a greenhouse at 24 °C. These cuttings were subjected to the following cold treatments: 2 weeks at 12 °C, 48 hours at 4 °C, 48 hours at 0 °C, and 4 hours at –1 °C. Following leaf tissue analysis of treated and control plants, some stress-related genes and many novel genes were identified. Northern blot hybridization demonstrated that all novel genes were regulated by the cold treatments.

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A cRNA Probe Detects Prunus Necrotic Ringspot Virus in Three Peach Cultivars after Micrografting and in Peach Shoots following Long-term Culture at 4 °C

HortScience ◽

10.21273/hortsci.34.2.346 ◽

1999 ◽

Vol 34 (2) ◽

pp. 346-347 ◽

Cited By ~ 6

Author(s):

K. Heuss ◽

Q. Liu ◽

F.A. Hammerschlag ◽

R.W. Hammond

Keyword(s):

Prunus Persica ◽

Blot Hybridization ◽

Ringspot Virus ◽

Shoot Cultures ◽

Crna Probe ◽

Reading Frame ◽

Prunus Necrotic Ringspot Virus ◽

Repeated Use ◽

Graft Success

As part of a program to develop transgenic peach (Prunus persica L. Batsch) cultivars with resistance to Prunus necrotic ringspot virus (PNRSV), we are testing a system for measuring virus in peach shoot cultures. Micrografting in vitro is used for inoculation and slot-blot hybridization, with a digoxigenin (DIG)-labeled cRNA probe complementary to the 5′ open reading frame (ORF) of PNRSV RNA 3, for detection. In this study, we investigated whether infected shoots maintain virus infection over long periods of culture at 4 °C and if PNRSV-infected `Suncrest' shoot cultures can serve as graft bases to transmit virus equally well into cultivars Nemaguard, Springcrest, and Suncrest. The results of RNA hybridization analysis showed that virus was present in extracts of leaf samples from 2-year-old PNRSV-infected `Suncrest' shoots that had been subjected to varying lengths of incubation at 4 °C in the dark, suggesting that infected shoots can be maintained for repeated use. Rates of graft success were higher in heterografts between `Suncrest' bases and tips of `Springcrest' or `Nemaguard' than in autografts between `Suncrest' and `Suncrest', and there was equal efficacy of graft inoculation from `Suncrest' into these three cultivars.

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First Report of Peach latent mosaic viroid in Peach Trees in Argentina

Plant Disease ◽

10.1094/pdis-92-7-1137c ◽

2008 ◽

Vol 92 (7) ◽

pp. 1137-1137 ◽

Cited By ~ 3

Author(s):

A. M. Nieto ◽

L. Di Feo ◽

C. F. Nome

Keyword(s):

Prunus Persica ◽

Blot Hybridization ◽

Summer Season ◽

Partial Purification ◽

Dot Blot ◽

First Report ◽

Preliminary Identification ◽

Pcr Product ◽

Local Sequence ◽

Peach Trees

Peach trees (Prunus persica L.) with yellowing symptoms on leaves were found in December (summer season) of 2005 in commercial fields in Colonia Caroya, Córdoba, Argentina. This symptom resembled Peach latent mosaic viroid (PLMVd). The viroid-affected trees usually showed vein banding and yellowing symptoms on leaves, fruit suture splitting, delays in budding, flowering and fruit ripening, reduced foliage density, and a spindly appearance. PLMVd is mainly transmitted by the propagation of infected budwood and to a lesser extent by pruning tools and aphids (1). Extraction and partial purification of nucleic acids was made from symptomatic leaves (2) collected during the indicated summer season from seven peach trees from commercial plots. Preliminary identification of the viroid was made by dot-blot hybridization with a PLMVd probe (provided by R. Flores). The positive plants were tested also by reverse transcription (RT)-PCR with PLMVd primers RF-43 (5′-CTGGATCACACCCCCCTCGGAACCAACCGCT-3′) and RF-44 (5′-TGTGATCCAGGTACCGCCGTAGAAACT-3′), which amplify the complete genome. All identified positive samples yielded the correct sized PCR product and four were cloned and sequenced. This local sequence (GenBank Accession No. EU429320) had 97% nucleotide sequence identity with a reported PLMVd sequence (GenBank Accession No. M83545), confirming the identity of the local pathogen as Peach latent mosaic viroid. This result suggests that the source of the pathogen may have been infected budwood introduced from Europe. To our knowledge, this is the first report of this viroid in Argentina. References: (1) R. Flores et al. Mol. Plant Pathol. 7:209, 2006. (2) V. Pallás et al. J. Gen. Virol. 68:3201, 1987.

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Detection of Tomato Ringspot Virus in Nectarine Trees Using ELISA and Transcribed RNA Probes

HortScience ◽

10.21273/hortsci.26.10.1290 ◽

1991 ◽

Vol 26 (10) ◽

pp. 1290-1292 ◽

Cited By ~ 2

Author(s):

C.A. Powell ◽

A. Hadidi ◽

J.M. Halbrendt

Keyword(s):

Nucleic Acid ◽

Prunus Persica ◽

Leaf Tissue ◽

Ringspot Virus ◽

Nucleic Acid Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Tomato Ringspot Virus ◽

Dot Hybridization ◽

Bark Tissue ◽

Stem Pitting

The ability of 32P-labeled transcribed cRNA probes to detect tomato ringspot virus (TmRSV) RNA in nucleic acid extracts from roots, bark, and leaves of nectarine (Prunus persica [L.] Batsch) trees with the Prunus stem-pitting disease was assessed and compared with detection of TmRSV antigen by enzyme-linked immunosorbent assay (ELISA) in the same tissues. Neither TmRSV-specific nucleic acid nor antigen was detected in nectarine leaf tissue. ELISA detected TmRSV antigen in root extracts from 71% of the diseased trees, while dot hybridization detected virus-specific nucleic acid in 18% of the same samples. However, ELISA detected TmRSV antigen in only 47% of bark extracts; whereas TmRSV-specific nucleic acid was detected in 100% of the bark extracts from samples collected at or near the soil line. When nucleic acid extracts from bark were prepared from various locations on diseased trees and tested for TmRSV-specific nucleic acid by dot hybridization, there was an almost perfect correlation between the presence of stem-pitting symptoms and the detection of TmRSV nucleic acid. Detection of TmRSV RNA from the bark tissue of rootstock suckers from TmRSV-infected `Delicious'/MM.lO6 apple (Malus × domestica Borkh.) trees was unsuccessful using dot hybridization. The viral RNA, however, was usually detected in either leaf or root tissue of these same trees.

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Monitoring Prunus Necrotic Ringspot Virus Infection by Hybridization with a CRNA Probe following in Vitro Grafting

HortScience ◽

10.21273/hortsci.30.4.876b ◽

1995 ◽

Vol 30 (4) ◽

pp. 876B-876

Author(s):

K. Heuss-La Rosa ◽

R. Hammond ◽

J.M. Crosslin ◽

C. Hazel ◽

F. Hammerschlag

Keyword(s):

Prunus Persica ◽

Blot Hybridization ◽

Ringspot Virus ◽

Crna Probe ◽

Slot Blot Hybridization ◽

Elisa Testing ◽

Prunus Necrotic Ringspot Virus ◽

Coat Protein Mediated Resistance ◽

In Vitro Micrografting

In vitro micrografting was tested as a technique for inoculating peach [Prunus persica (L.) Batsch] with prunus necrotic ringspot virus (PNRSV). Cultured `Suncrest' shoots derived from a naturally infected tree (as indicated by ELISA testing) maintained virus in vitro, with virus concentrations in growing tips and folded leaves being several times those of fully expanded leaves. The infected shoots served as graft bases and the source of virus. Grafted tips were derived from `Suncrest' trees that had tested negative for the virus. Leaf samples were collected from the tips following grafting and analyzed for the presence of virus by slot-blot hybridization with a digoxigenin-labeled cRNA probed derived from PNRSV RNA 3. Rates of successful grafting were 55% and 73% in three trials and PNRSV was found in all tips analyzed. Virus concentrations approximated those found in the source shoots, suggesting that this method should be useful for screening transformed peach shoots for coat protein-mediated resistance to PNRSV.

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Penggunaan Pelacak DNA untuk Deteksi Papaya ringspot virus dengan Metode Hibridisasi Asam Nukleat

Jurnal Fitopatologi Indonesia ◽

10.14692/jfi.14.3.89 ◽

2018 ◽

Vol 14 (3) ◽

pp. 89

Author(s):

Irsan Nuhantoro ◽

Sri Hendrastuti Hidayat ◽

Kikin Hamzah Mutaqin

Keyword(s):

Nucleic Acid ◽

Detection Method ◽

Blot Hybridization ◽

High Specificity ◽

Dna Probe ◽

Ringspot Virus ◽

Nucleic Acid Hybridization ◽

Disease Spread ◽

Papaya Ringspot Virus ◽

Dot Blot

Use of DNA Probe for Detection of Papaya ringspot virus Using Nucleic Acid Hybridization MethodPapaya ringspot caused by Papaya ringspot virus (PRSV) is one of the most destructive diseases of papaya. The disease had not been found in Indonesia, until disease outbreak in Nangroe Aceh Darussalam was reported in 2012. Since then, the disease spread rapidly in most papaya growing areas in Sumatera, Java and Bali. Papaya ringspot virus (PRSV) is generally detected using serological or polymerase chain reaction methods. Improvement in detection method is necessary to facilitate a more reliable tool for controlling the spread of PRSV. The aim of the research was to construct DNA probe for development of detection method based on nucleic acid hybridization. Molecular characterization based on HCPro gene sequence indicated high homology (97.88 to 99.05%) among PRSV isolates from Boyolali (Central Java), Medan (North Sumatera), Sleman (Yogyakarta) and Tabanan (Bali). Two DNA clones of HCPro gene were selected for probe construction and the probes were then labeled using PCR DIG-dioxigenin. Optimization of nucleic acid dot blot hybridization method to achieve strongest positive reaction was developed, i.e. using stringency washes at 1×SSC, 0.1% SDS, incubation at 60 oC for 15’. The DNA probe for PRSV has a high specificity and sensitifity; it could detect PRSV at the lowest concentration of nucleic acid (0.062 µg µL-1).

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Specific Detection of Cherry Mottle Leaf Virus Using Digoxigenin-Labeled cDNA Probes and RT-PCR

Plant Disease ◽

10.1094/pdis.1999.83.3.235 ◽

1999 ◽

Vol 83 (3) ◽

pp. 235-239 ◽

Cited By ~ 16

Author(s):

D. James ◽

W. Jelkmann ◽

C. Upton

Keyword(s):

Reverse Transcription ◽

Sequence Data ◽

Blot Hybridization ◽

Chenopodium Quinoa ◽

Virus Genome ◽

Dot Blot ◽

Genome Search ◽

Specific Amplification ◽

Cloned Cdna ◽

Leaf Virus

Cherry mottle leaf virus (CMLV)-associated double-stranded RNA (dsRNA) was isolated from the propagation host Chenopodium quinoa. The dsRNA band, with a molecular weight estimated at 7.0 × 106 Da, was used to produce cDNA. Two recombinant plasmids from the cloned cDNA library were identified that specifically bound with CMLV-associated RNA in dot blot hybridization studies. The cDNA inserts were sequenced, and oligonucleotide primers were designed that specifically amplify an 848-bp fragment of the CMLV genome by reverse-transcription polymerase chain reaction. Also, a poly(T) primer was reliably used for reverse transcription, with specific amplification using the CMLV primers, suggesting polyadenylation of the virus genome. Search of the database revealed some sequence homology of the partially characterized genome of CMLV with that of apple chlorotic leafspot virus. Additional sequence data are required, however, to establish the taxonomic position of the filamentous CMLV.

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Monitoring Prunus Necrotic Ringspot Virus Infection by Hybridization with a cRNA Probe after in vitro Micrografting

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.120.6.928 ◽

1995 ◽

Vol 120 (6) ◽

pp. 928-931 ◽

Cited By ~ 6

Author(s):

Kathleen Heuss-LaRosa ◽

Rosemarie Hammond ◽

James M. Crosslin ◽

Christine Hazel' ◽

Freddi A. Hammerschlag

Keyword(s):

Prunus Persica ◽

Blot Hybridization ◽

Ringspot Virus ◽

Shoot Cultures ◽

Crna Probe ◽

Elisa Testing ◽

Prunus Necrotic Ringspot Virus ◽

Coat Protein Mediated Resistance ◽

In Vitro Micrografting

In vitro micrografting was tested as a technique for inoculating peach [Prunus persica (L.) Batsch] shoot cultures with Prunus necrotic ringspot virus (PNRSV). Cultured `Suncrest' shoots derived from a naturally infected tree (as indicated by ELISA testing) maintained virus in vitro, with virus concentrations in growing tips and folded leaves being several times those of fully expanded leaves. Infected shoots served as graft bases and source of the virus. Grafted tips were derived from `Suncrest' trees that had tested negative for the virus. Leaf samples were collected from the tips following grafting and analyzed for the presence of virus by slot-blot hybridization with a (DIG)-labeled cRNA probe derived from PNRSV RNA 3. Rates of successful grafting ranged from 55% to 73% in three trials and PNRSV was found in all tips analyzed. Virus concentrations approximated those found in source shoots, suggesting that in vitro micrografting should be useful for screening transformed peach shoots for coat protein-mediated resistance to PNRSV. Chemical name used: digoxigenin (DIG).

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Detection of Hepatitis a Virus and other Enteroviruses in Wastewater and Surface Water Samples by Gene Probe Assay

Water Science & Technology ◽

10.2166/wst.1991.0071 ◽

1991 ◽

Vol 24 (2) ◽

pp. 267-272 ◽

Cited By ~ 12

Author(s):

S. Dubrou ◽

H. Kopecka ◽

J. M. Lopez Pila ◽

J. Maréchal ◽

J. Prévot

Keyword(s):

Surface Water ◽

Cell Cultures ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Limit Of Detection ◽

Blot Hybridization ◽

Noncoding Region ◽

Gene Probe ◽

Dot Blot ◽

Lower Efficiency

Enteroviruses were specifically detected by dot blot hybridization when using poliovirus type 1 (PV1) derived subgenomic radiolabeled cRNA probes (riboprobes) in environmental water specimens and in the cell cultures in which the viruses were amplificated. The riboprobe corresponding to the 5' noncoding sequence detected the majority of enteroviruses. Hepatitis A virus (HAV) was specifically detected by an HAV cRNA probe corresponding to the 5' noncoding region of its genome. By this test, the limit of detection of coxsackievirus B5 and echovirus 7 seeded in mineral water was 103 to 104 PFU/spot. In cell cultures, positive signals were observed in the lysates of cells infected by one PFU. Higher positive signals were obtained with a short PV1 probe (nt 221-670) corresponding to the 5' noncoding region, which is a well preserved sequence among the enteroviruses, than with PV1 genomic probe. Hybridization allowed a good detection of enteroviral RNAs in wastewater specimens, but with a lower efficiency in surface water. In this case, amplification of viruses in the cell cultures gave significant hybridization results.

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