First Report of Plum bark necrosis stem pitting-associated virus in Stone Fruit Trees in China

Plum bark necrosis and stem pitting disease was first observed on a ‘Black Beaut’ plum (Prunus salicina Lindl.) in the United States in 1986 and later is several other countries. Plum bark necrosis stem pitting-associated virus (PBNSPaV; genus Ampelovirus, family Closteroviridae), the putative causal agent of the disease, infects many stone fruit species and causes decline, gummosis, flattening of scaffold branches, and stem necrotic pits in some diseased trees (1,3). An investigation of the incidence of PBNSPaV on stone fruit trees in China was conducted during 2009 and 2010. Leaf samples were collected from 47 trees, including peach (P. persica L. Batsch), nectarine (P. persica L. var. nucipersica Schneider), plum (P. domestica L.), ornamental plum (P. cerasifera Ehrb), sweet cherry (P. avium L.), and flowering cherry (P. serrulata L.), grown in Hubei, Henan, and Shandong provinces in central and northern China. Most of sampled trees showed trunk gummosis or stem pitting. The presence of PBNSPaV was tested by reverse transcription (RT)-PCR using primer set PBN195F/PBN195R (5′-CTGGTCTTCCTGCTACTCCTT-3′/5′-AAGCCCACAATCTCAGAGCG-3′) designed for the detection of the coat protein (CP) gene of the virus. Total RNA was extracted from leaves using a CTAB protocol reported by Li et al. (2). Products of the expected size of 190 bp were amplified from 20 samples, including seven cultivated peach, four ornamental peach, one nectarine, two plum, one ornamental plum, three sweet cherry, and two flowering cherry samples. All trees positive for PBNSPaV showed stem pitting symptoms on the base of the trunk. To further confirm these results, a 590-base region of the heat shock protein 70 homolog (HSP70h) gene was amplified by RT-PCR using primers HSP-P1/HSP-P2 (5′-GGAATTGACTTCGGTACAAC-3′/5′-TCGAAAGTACCACCACCGAA-3′). Amplicons of the expected size were cloned into the vector pMD18-T (TaKaRa, Dalian, China) and sequenced by Genscript Corp. (Nanjing, China). Sequences of 18 PBNSPaV isolates were deposited in GenBank with Accession Nos. JF810177–JF810194. Sequence comparisons showed that the partial HSP70h gene from the Chinese PBNSPaV isolates shared 82.2 to 100% nucleotide (nt) and 94.0 to 100% amino acid (aa) similarities between them and 83.6 to 99.1% nt and 94 to 100% aa similarities with the corresponding region of the other PBNSPaV isolates deposited in GenBank. In July 2010, peach GF305 seedlings were inoculated by side grafting with budwoods from two PBNSPaV-positive ornamental peach plants. In June 2011, grooving symptom was observed on the stems of the seedlings and the virus was detected by RT-PCR. The results further confirmed PBNSPaV infection in China. These results show that PBNSPaV and the associated disease occur in main cultivated and ornamental Prunus species in China. Given the importance and the devastating symptoms of the disease, it is important to prevent virus spread by using virus-tested propagation materials. References: (1) M. Al Rwahnih et al. Arch. Virol. 152:2197, 2007. (2) R. Li et al. J. Virol. Methods 154:48, 2008. (3) D. B. Marini et al. Plant Dis. 86:415, 2002.

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Analysis of Double-Stranded RNAs from Cherry Trees with Stem Pitting in California

Plant Disease ◽

10.1094/pdis.1998.82.8.871 ◽

1998 ◽

Vol 82 (8) ◽

pp. 871-874 ◽

Cited By ~ 6

Author(s):

Yun-Ping Zhang ◽

J. K. Uyemoto ◽

B. C. Kirkpatrick

Keyword(s):

Sweet Cherry ◽

Prunus Avium ◽

Sour Cherry ◽

Rt Pcr ◽

Cloning And Sequencing ◽

Green Ring ◽

Flowering Cherry ◽

Polymerase Chain ◽

Stem Pitting ◽

Dsrna Species

Five distinct dsRNA species were recovered from Bing sweet cherry (Prunus avium (L.) L.) trees with stem pitting symptoms. A 4.7-kilobase pair (kbp) dsRNA was isolated from mahaleb rootstock (P. mahaleb L.); an unrelated 4.7-kbp dsRNA, always co-purified with a 1.3-kbp dsRNA, and a 9-kbp dsRNA were from Bing cherry. In addition, an 8.5-kbp dsRNA found in diseased Shirofugen flowering cherry and in Bing cherry was identified as sour cherry green ring mottle virus (CGRMV). The larger, 8.5- and 9.0-kbp dsRNA species were graft-transmissible, while the smaller ones were non-transmissible and appeared cryptic in nature. Reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for each dsRNA species by cloning and sequencing cDNA synthesized from the dsRNA templates. When several diseased collections were assayed by RT-PCR, approximately 14% reacted positively with primers for the 9.0-kbp dsRNA or CGRMV. Although CGRMV and the 9.0-kbp dsRNA caused wood-marking symptoms in graft-inoculated Mazzard (P. avium) seedling trees, no xylem or canopy symptoms developed in grafted Bing cherry. The causal agent or agents of cherry stem pitting have not been identified.

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Serological and molecular detection of Prune dwarf virus infecting stone fruits of Charmahal-va-Bakhtiari province, a central region of Iran

International Journal of Plant Biology ◽

10.4081/pb.2013.e4 ◽

2013 ◽

Vol 4 (1) ◽

pp. 4

Author(s):

Nourolah Soltani ◽

Jamshid Hayati ◽

Ghobad Babaei ◽

Maryam Ebrahim Qomi

Keyword(s):

Sweet Cherry ◽

Fruit Trees ◽

Coat Protein Sequence ◽

Elisa Test ◽

Stone Fruit ◽

Stone Fruits ◽

Rt Pcr ◽

Leaf Chlorosis ◽

Fruit Orchards ◽

Das Elisa

<em>Prune dwarf</em> virus (PDV) is one of the major positive RNA viruses which cause economical damages in stone fruit trees. The symptoms of PDV vary between different stone fruits namely sour and sweet cherry, almond, peach, apricot and plum including leaf narrowing, leaf chlorosis, vein clearing, mosaic, leaf whitening, leathery leaf, bushy branches and stunt trees. During the years 2011 and 2012, 251 leaf samples were collected for detection of PDV in stone fruit orchards of Charmahal-va-Bakhtiari province. DAS-ELISA test proved PDV presence serologically. Then, total RNA were extracted and tested by two-step RT-PCR which replicated partial and full coat protein sequence of PDV. One hundred and eighty one out of total samples (251 samples) showed PDV infection using serological and two-step RT-PCR assays, hence, incidence of PDV in Charmahal-va-Bakhtiari province was confirmed. This is the first report of PDV in stone fruit orchards of Charmahal-va-Bakhtiari province and in Iran.

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First Report of Apricot latent virus and Plum bark necrosis stem pitting-associated virus in Apricot from Spain

Plant Disease ◽

10.1094/pdis-94-2-0275a ◽

2010 ◽

Vol 94 (2) ◽

pp. 275-275 ◽

Cited By ~ 8

Author(s):

A. García-Ibarra ◽

P. Martínez-Gómez ◽

M. Rubio ◽

F. Dicenta ◽

A. Soler ◽

...

Keyword(s):

The United States ◽

Full Length ◽

Latent Virus ◽

Stone Fruit ◽

Pcr Analysis ◽

Rt Pcr ◽

First Report ◽

Cp Gene ◽

Fruit Samples ◽

Stem Pitting

Representing 2% of world production, 20,000 ha of apricot (Prunus armeniaca L.), are cultivated in Spain, primarily in the southeast. A survey was conducted during the spring of 2008 in orchards in the region of Murcia to assess the incidence of several stone fruit viruses. Leaf and fruit samples from 160 trees from 40 orchards were collected randomly for reverse transcription (RT)-PCR analysis. Total RNA extracted (3) from leaves and fruits was tested by a multiplex one-step RT-PCR protocol with a mix of primers that detect eight distinct viruses (4). Amplicons of 250 bp expected for Plum bark necrosis stem pitting-associated virus (PBNSPaV), corresponding to part of the heat shock 70 protein gene, were obtained from four trees and amplicons of 700 bp expected for Apricot latent virus (ApLV), corresponding to part of the coat protein (CP) gene, were obtained from two trees. In all cases, amplicons were obtained using RNA extracted from leaf and fruit tissues. RT-PCR results were confirmed by uniplex RT-PCR with primers specific for each virus and dot-blot hybridization with virus-specific digoxygenin-labeled RNA probes (2). To further characterize the new viruses, we designed primers to amplify specifically the CP gene of ApLV (5′-CCCGACCATGGCTACAAGC-3′ and 5′-TTGCCGTCCCGATTAGGTTG-3′) and the minor CP gene of PBNSPaV (5′-GAACAAACTACAGCAGCACC-3′ and 5′-CAAGGGTAGGACGGGTAACGC-3′). Amplicons of 1,500 and 950 bp corresponding to the ApLV and PBNSPaV CP genes, respectively, were purified from agarose gels and cloned in the pTZ57R plasmid (Fermentas, Burlington, Ontario, Canada). Blastp analysis of the full-length ApLV CP sequence from one infected tree (GenBank Accession No. GQ919051) revealed 86% amino acid (aa) similarity to the single full-length ApLV CP sequence available (No. AAC16234) and 79 and 66.9% similarity to Peach sooty ringspot virus (No. AAG48314) and Apple stem pitting virus (No. NP604468), respectively. Identity/similarity analysis of the full-length PBNSPaV minor CP genes using the Matrix Global Alignment Tool software, version 2.02 (1), revealed 98.8 to 99.6% aa similarity between the Spanish PBNSPaV isolates (Nos. GQ919047, GQ919048, GQ919049, and GQ919050) and 97.1 to 97.4% with the PBNSPaV isolate from the United States (No. EF546442). None of the six infected trees were associated with any particular field symptoms. Five infected trees were cv. Búlida and one was native cv. Murciana, which was infected with ApLV. All infected trees were located in geographically separated orchards. The incidence of ApLV and PBNSPaV was 1.25 and 2.5%, respectively. The low incidence of both viruses together with the scattered geographic distribution could be due to the recent introduction of virus-contaminated plants, although we cannot exclude that it is a consequence of a low dissemination rate. Even though no symptoms were observed, we cannot discard that the infection could affect fruit production or flowering or even cause a synergistic effect in mixed infection with other stone fruit viruses, a risk especially relevant considering the total area of cultivated apricot. To our knowledge, this is the first report of ApLV and PBNSPaV in Spain. References: (1) J. J. Campanella et al. BMC Bioinformatics 4:29, 2003. (2) M. C. Herranz et al. J. Virol. Methods 124:49, 2005. (3) D. J. Mackenzie et al. Plant Dis. 81:222, 1997. (4) J. A. Sánchez-Navarro et al. Eur. J. Plant Pathol. 111:77, 2005.

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First Report of Cherry necrotic rusty mottle virus on Stone Fruit Trees in China

Plant Disease ◽

10.1094/pdis-09-12-0836-pdn ◽

2013 ◽

Vol 97 (2) ◽

pp. 290-290 ◽

Cited By ~ 6

Author(s):

J. F. Zhou ◽

G. P. Wang ◽

L. N. Qu ◽

C. L. Deng ◽

Y. Wang ◽

...

Keyword(s):

Sweet Cherry ◽

Prunus Persica ◽

Sour Cherry ◽

Fruit Trees ◽

Stone Fruit ◽

Mottle Virus ◽

Rt Pcr ◽

Cherry Tree ◽

First Report ◽

Pcr Products

During the growing seasons of 2010 through 2012, leaf tissues from 206 stone fruit trees, including one flowering cherry, three sour cherry, six nectarine (Prunus persica L. var. nucipersica Schneider), 14 apricot, 24 plum (P. domestica L.), 41 sweet cherry, and 117 peach [P. persica (L.) Batsch] trees, grown in six provinces of China, were randomly collected and tested for the CNRMV infection by RT-PCR. Out of those sampled trees, 37 showed shot holes and vein yellowing symptoms. Total RNA was extracted from leaves using the CTAB protocol reported by Li et al. (2). The primer pair CGRMV1/CGRMV2 (1) was used to amplify a fragment of 949 bp from CNRMV genome, which includes the CP gene (804 bp). PCR products with the expected size were detected in one sweet cherry, one apricot, one peach, one plum, and two sour cherry plants. However, no correlation between PCR data and symptom expression could be found. PCR products were cloned into the vector pMD18-T (TaKaRa, Dalian, China). Three independent clones from each isolate were sequenced by Genscript Corp., Nanjing, China, and sequences were deposited in the GenBank under accession nos. JX491635, JX491636, JX491637, JX648205, and JX648206. Results of sequence analysis showed that sequences of the five CNRMV isolates shared the highest nt (99.0 to 99.6%) and aa (98.9 to 100%) similarities with a cherry isolate from Germany (GenBank Accession No. AF237816). The sequence of one isolate from a peach tree (JX648205) was divergent and shared only 84.7 to 86.1% nt and 94.4 to 95.1% aa similarities with those cp sequences. Clones intra each isolate shared more than 99% nt similarities. To confirm CNRMV infection, seedlings of peach GF 305 were graft-inoculated with bud-woods from a peach and a sweet cherry tree, which was positive to CNRMV and also two other viruses: Cherry green ring mottle virus (CGRMV) and Plum bark necrosis stem pitting-associated virus (PBNSPaV), as tested by RT-PCR. Grafted seedlings were kept in an insectproof greenhouse and observed for symptom development. In May of the following year, some newly developed leaves of inoculated seedlings showed vein yellowing, ringspot, and shot hole symptoms. Results of Protein A sandwich (PAS)-ELISA using an antiserum raised against the recombinant CP of a CNRMV isolate (unpublished) and RT-PCR confirmed CNRMV infection in inoculated trees. In addition to CNRMV, tested seedlings were also found to be infected with CGRMV and PBNSPaV by RT-PCR. To our knowledge, this is the first report on the occurrence of CNRMV on stone fruit trees in China, and also the first record of the CNRMV infection in peach and plum plants. Given the economic importance of its hosts and the visible symptoms of the viral disease, it is important to prevent the virus spread by using virus-tested propagation materials. References: (1) R. Li and R. Mock. J. Virol. Methods 129:162, 2005. (2) R. Li et al. J. Virol. Methods 154:48, 2008.

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First Report of Cherry virus A in Sweet Cherry Trees in China

Plant Disease ◽

10.1094/pdis-93-4-0425b ◽

2009 ◽

Vol 93 (4) ◽

pp. 425-425 ◽

Cited By ~ 4

Author(s):

W.-L. Rao ◽

Z.-K. Zhang ◽

R. Li

Keyword(s):

Sweet Cherry ◽

Community Gardens ◽

Mottle Virus ◽

Replicase Gene ◽

Rt Pcr ◽

First Report ◽

Type Isolate ◽

The Family ◽

Flowering Cherry ◽

Cherry Trees

Plants in the genus Prunus of the family Rosaceae are important fruit and ornamental trees in China. In June of 2007, sweet cherry (Prunus avium) trees with mottling and mosaic symptoms were observed in a private garden near Kunming, Yunnan Province. Twenty-four samples, six each from sweet cherry, sour cherry (P. cerasus), flowering cherry (P. serrulata), and peach (P. persica) were collected from trees in private and community gardens in the area. The peach and sour and flowering cherry trees did not show any symptoms. Total nucleic acids were extracted using a cetyltrimethylammoniumbromide (CTAB) extraction method, and the extracts were tested for the following eight viruses by reverse transcription (RT)-PCR: American plum line pattern virus, Apple chlorotic leaf spot virus, Cherry green ring mottle virus, Cherry necrotic rusty mottle virus, Cherry virus A (CVA), Little cherry virus 1, Prune dwarf virus, and Prunus necrotic ringspot virus. Only CVA was detected in two symptomatic sweet cherry trees by RT-PCR with forward (5′-GTGGCATTCAACTAGCACCTAT-3′) and reverse (5′-TCAGCTGCCTCAGCTTGGC-3′) primers specific to an 873-bp fragment of the CVA replicase gene (2). The CVA infection of the two trees was confirmed by RT-PCR using primers CVA-7097U and CVA-7383L that amplified a 287-bp fragment from the 3′-untranslated region (UTR) of the virus (1). Amplicons from both amplifications were cloned and sequenced. Analysis of the predicted amino acid sequences of the 873-bp fragments (GenBank Accession Nos. EU862278 and EU862279) showed that they were 98% identical with each other and 97 to 98% with the type isolate of CVA from Germany (GenBank Accession No. NC_003689). The 286-bp sequences of the 3′-UTR (GenBank Accession Nos. FJ608982 and FJ608983) were 93% identical with each other and 93 to 98% with the type isolate. The sequence indicated that the three isolates were very similar and should be considered to be the same strain. CVA is a member of the genus Capillovirus in the family Flexiviridae and has been previously reported in Europe, North America, and Japan. The contribution of CVA to the symptoms observed and its distribution in China remain to be evaluated. To our knowledge, this is the first report of CVA in sweet cherry in China. References: (1) M. Isogai et al. J. Gen. Plant Pathol. 70:288. (2) W. Jelkmann. J. Gen. Virol. 76:2015, 1995.

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Bacteriosis of Stone Fruit Trees in Britain VI. Field Observations on Bacteriosis of Sweet Cherry Trees

Journal of Pomology and Horticultural Science ◽

10.1080/03683621.1938.11513489 ◽

1938 ◽

Vol 15 (1) ◽

pp. 33-48

Author(s):

H. Wormald

Keyword(s):

Sweet Cherry ◽

Fruit Trees ◽

Stone Fruit ◽

Field Observations ◽

Cherry Trees

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Genetic diversity of Prunus necrotic ringspot virus infecting stone fruit trees grown at seven regions in China and differentiation of three phylogroups by multiplex RT-PCR

Crop Protection ◽

10.1016/j.cropro.2015.04.001 ◽

2015 ◽

Vol 74 ◽

pp. 30-36 ◽

Cited By ~ 11

Author(s):

H.G. Cui ◽

H.Z. Liu ◽

J. Chen ◽

J.F. Zhou ◽

L.N. Qu ◽

...

Keyword(s):

Genetic Diversity ◽

Fruit Trees ◽

Ringspot Virus ◽

Stone Fruit ◽

Rt Pcr ◽

Prunus Necrotic Ringspot Virus

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First Report of Little cherry virus 2 in Flowering and Sweet Cherry Trees in China

Plant Disease ◽

10.1094/pdis-10-10-0766 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1484-1484 ◽

Cited By ~ 4

Author(s):

W.-L. Rao ◽

F. Li ◽

R.-J. Zuo ◽

R. Li

Keyword(s):

Sweet Cherry ◽

Sequence Data ◽

Community Gardens ◽

Economic Losses ◽

Helicase Domain ◽

Rt Pcr ◽

Viral Origin ◽

First Report ◽

Flowering Cherry ◽

Cherry Trees

Many viruses infect Prunus spp. and cause diseases on them. During a survey of stone fruit trees in 2008 and 2009, flowering cherry (Prunus serrulata) and sweet cherry (P. avium) trees with foliar chlorosis and reddening, stem deformity, and tree stunting were observed in private orchards in Anning and Fumin counties of Yunnan Province. Some sweet cherry trees with severe symptoms yielded small and few fruits and had to be removed. Leaf samples were collected from 68 flowering cherry and 30 sweet cherry trees, either symptomatic or asymptomatic, from private orchards and community gardens in Kunming and counties Anning, Chenggong, Fumin, Jinning, Ludian and Yiliang. Total nucleic acids were extracted with a CTAB extraction method and tested by reverse transcription (RT)-PCR assay using virus-specific primers. Little cherry virus 2 (LChV-2), Cherry virus A (CVA), Prunus necrotic ringspot virus (PNRSV), and Prune dwarf virus (PDV) were detected and infection rates were 68.4, 16.3, 9.2, and 7.1%, respectively. Infection of LChV-2 was common in all counties except Ludian where the orchards were healthy. Of 68 infected trees, 29 were found to be infected with LChV-2 and CVA, PDV or PNRSV. LChV-2 was detected in this study by RT-PCR using a pair of novel primers, LCV2-1 (5′-TTCAATATGAGCAGTGTTCCTAAC-3′) and LCV2-4 (5′-ACTCGTCTTGTGACATACCAGTC-3′), in 59 flowering cherry (87%) and 8 sweet cherry (27%) trees, respectively. The primer pair was designed according to alignment of three available LChV-2 sequences (GenBank Nos. NC_005065, AF416335, and AF333237) to amplify the partial RNA-dependent RNA polymerase gene (ORF1b) of 781 bp. The amplicons of selected samples (Anning26 and Yiliang60) were sequenced directly and sequences of 651 bp (GenBank No. HQ412772) were obtained from both samples. Pairwise comparisons and phylogenetic analysis of the sequences show that the two isolates are identical to one another and share 92 to 96% at the amino acid (aa) sequence level to those of other isolates available in the GenBank database. The sequence data confirm that these isolates are a strain of LChV-2 and genetic variation among different strains is relatively high (2). Biological and serological assays are not available for the LChV-2 detection; therefore, the LChV-2 infections of these trees were further confirmed by RT-PCR using primer pair LCV2-5 (5′-TGTTTGTGTCATGTTGTCGGAGAAG-3′) and LCV2-6 (5′-TGAATACCCGAGAACAAGGACTC-3′), which amplified the helicase domain (ORF1a) of ~451 bp. The amplicons from samples Anning26 and Yiliang60 were cloned and sequenced. The 408-bp sequences (excluding primer sequences) were 92 to 98% identical at the aa sequence level to those of other isolates, confirming again their viral origin. LChV-2 (genus Ampelovirus, family Closteroviridae) (4) has been associated with little cherry disease (LChD), a widespread viral disease of sweet and sour cherries (1,3). The virus is transferred between geographic areas mainly by propagated materials. Ornamental and sweet cherries are important crops in China and LChD has the potential to cause significant economic losses. Thus, certified clean stock should be used to establish new orchards. To our knowledge, this is the first report of LChV-2 in cherries in China. References: (1) N. B. Bajet et al. Plant Dis. 92:234, 2008. (2) W. Jelkmann et al. Acta Hortic. 781:321, 2008. (3) B. Komorowska and M. Cieslińska, Plant Dis. 92:1366, 2008. (4) M. E. Rott and W. Jelkmann. Arch. Virol. 150:107, 2005.

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