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

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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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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First Report of Little cherry virus 1 Infecting Sweet Cherry in Ontario, Canada

Plant Disease ◽

10.1094/pdis-04-21-0798-pdn ◽

2021 ◽

Author(s):

Aaron Simkovich ◽

Susanne Kohalmi ◽

Aiming Wang

Keyword(s):

San Diego ◽

Sweet Cherry ◽

The United States ◽

Eastern Region ◽

Rt Pcr ◽

Degenerate Primers ◽

Sequence Alignments ◽

First Report ◽

Blastn Search ◽

Cherry Trees

The Niagara fruit belt is one of the richest fruit-producing areas in Canada, contributing to 90% of Ontario's tender fruits such as peach, plum and sweet cherry. Little cherry virus 1 (LCV1) of the genus Velarivirus is a causal agent of little cherry disease which has devastated cherry crops in many regions (Eastwell and Bernardy 1998, Jelkmann and Eastwell, 2011). From 2013 to 2018, foliar symptoms indicative of viral infection such as leaf deformation, ringspot, mottling, vein clearing, and reddening were found on sweet cherry trees grown in the Niagara region. To determine if these trees were infected by a virus, small RNAs (sRNAs) were isolated from separately pooled asymptomatic and symptomatic leaves using the mirPremier microRNA isolation kit (Sigma Aldrich Canada, Oakville, ON). The sRNAs were used to create two libraries (four leaves per library) with the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA). The sRNA libraries were separately sequenced with the MiSeq Desktop Sequencer (Illumina, San Diego, CA). In total, 5,380,196 reads were obtained and Trimmomatic (Bolger et al. 2014) was used to remove adaptors. The remaining 4,733,804 clean reads were assembled into contigs using Velvet 0.7.31 (Zerbino and Birney, 2008) and Oases 0.2.09 (Schulz et al. 2012) with minimum length of 75 nt (Supplementary Table 1). A BLASTn search (Altschul et al. 1997) of the contigs identified the presence of Cherry virus A (genus: Capillovirus), two members of the Ilarvirus genus (Prunus necrotic ringspot virus and Prune dwarf virus) in both libraries. LCV1 was only found in contigs derived from the symptomatic library. Of the clean reads, 22,016 were assembled into six contigs (with lengths ranging from 86 to 116 nt, Supplementary Table 1) mapping to LCV1, covering 7.07% of the viral genome. To confirm LCV1 infection, primers were designed from the assembled contigs and used for reverse transcription polymerase chain reaction (RT-PCR). Amplicons were sequenced and the terminal sequences were determined using 5’ and 3’ RACE Systems (Invitrogen, Burlington, ON). Degenerate primers were designed from multiple sequence alignments of published LCV1 genomes for amplification and primer walking to obtain the sequence of LCV1 (Table S2). The complete genome sequence of LCV1 has a length of 16,934 nt and was deposited in GenBank (accession no. MN508820). A BLASTn search showed that this isolate is nearly identical (99.6% sequence identity) to an isolate from California (accession no. MN131067). To determine the incidence of infection, a field survey was performed at the same location during spring months of 2014 to 2018 using RT-PCR with primers specific to the viral coat protein gene (Supplementary Tale 2). Among 46 cherry trees sampled, two (4.3%) trees were infected with LCV1 and showed negative results with CVA, PNRSV and PDV. Both trees displayed mild suturing of primary and secondary veins (Supplementary Figure 1). LCV1 has been identified in Western stone fruit producing regions (British Columbia in Canada, and Washington, California, and Oregon in the United States of America). To the best of our knowledge, this is the first report of LCV1 in any eastern region of Canada. The low incidence of LCV1 suggests that this virus is not widespread in this region. Routine monitoring and detection of LCV1 is required to prevent this devastating cherry disease from spreading in this region.

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First Report of Little cherry virus 2 from Sweet Cherry in Poland

Plant Disease ◽

10.1094/pdis-92-9-1366a ◽

2008 ◽

Vol 92 (9) ◽

pp. 1366-1366 ◽

Cited By ~ 3

Author(s):

B. Komorowska ◽

M. Cieślińska

Keyword(s):

Coat Protein ◽

Sweet Cherry ◽

Prunus Avium ◽

Late Summer ◽

Rt Pcr ◽

Cherry Tree ◽

First Report ◽

Nontranslated Region ◽

Methyltransferase Gene ◽

Cherry Trees

Little cherry disease (LChD) is a serious viral disease of sweet (Prunus avium) and sour (P. cerasus) cherry trees. Infection of sensitive cultivars results in small, angular, and pointed fruits with reduced sweetness. In late summer, leaves show a characteristic red coloration or bronzing of the surfaces. One Ampelovirus species, Little cherry virus 2 (LChV-2) (2), and one unassigned species in the Closteroviridae, Little cherry virus 1 (LChV-1) (3), have been associated with LChD. Twenty-seven sour and sweet cherry trees of six varieties from orchards located in several regions of Poland were tested for LChV-1 and LChV-2. Leaf samples were taken either from trees showing fruit symptoms or from asymptomatic trees during the summer of the 2006 growing season. RNA was isolated from the leaves with an RNeasy Kit (Qiagen, Hilden, CA), and reverse transcription (RT)-PCR was performed using primer pairs LCV1U/LCV1L and LCV2UP2/LCV2LO2, which are specific for a 419-bp fragment of the LChV-1 3′ nontranslated region and a 438-bp fragment of the LChV-2 methyltransferase gene, respectively (1). The primer pair L2CPF (5′-GTTCGAAAGTGTTTCTTGAT-3′) and L2CPR (5′-GCAACAGAAAAACATATGACTCA-3′) was designed from existing LChV-2 sequences (GenBank Accession Nos. AF416335 and NC_005065) to amplify the entire LChV-2 coat protein (CP) gene (nucleotides 13,007 to 14,134). The amplified cDNA fragments of LChV-2 genome were ligated to the bacterial vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA), which was used to transform Escherichia coli TOP10 competent cells following the manufacturer's protocol. Both strands of three clones for each amplified LChV-2 genome fragment were sequenced with an automated nucleotide sequencer at the Institute of Biochemistry and Biophysics in Warsaw. RT-PCR results showed that 6 of 27 trees were infected, with LChV-1 detected in five sweet cherry trees and LChV-2 singly infecting one sweet cherry tree cv Elton (isolate C4/14). The nucleotide sequence of the 438-bp methyltransferase gene fragment of isolate C4/14 showed 86, 85, and 84% identity to GenBank Accession Nos. AF333237, AF531505, and AJ430056, respectively, all previously reported LChV-2 sequences from cherry trees. Sequence analysis of the 1,088-bp coat protein gene showed 89 to 91% and 92 to 93% nucleotide and amino acid identity, respectively, with the aforementioned three LChV-2 isolates. The tree infected with LChV-2 was indexed by graft transmission to the woody indicator, Prunus avium cv. Canindex, which showed reddening of the leaves characteristic of LChD 3 months after inoculation. Since cherry production in Poland is 230,000 t per year, the disease may have a significant economic impact because the affected fruits are unsuitable either for consumption or sale. To our knowledge, this is the first report of LChV-2 in Poland. References: (1) M. E. Rott and W. Jelkmann. Phytopathology 91:261, 2001. (2) M. E. Rott and W. Jelkmann. Arch. Virol. 150:107, 2005. (3) M. Vitushkina et al. Eur. J. Plant Pathol. 103:803, 1997.

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First Report of Cherry necrotic rusty mottle virus Infecting Sweet Cherry Trees in Korea

Plant Disease ◽

10.1094/pdis-07-13-0723-pdn ◽

2014 ◽

Vol 98 (1) ◽

pp. 164-164 ◽

Cited By ~ 3

Author(s):

I. S. Cho ◽

G. S. Choi ◽

S. K. Choi ◽

E. Y. Seo ◽

H. S. Lim

Keyword(s):

Sweet Cherry ◽

Mottle Virus ◽

Post Inoculation ◽

Rt Pcr ◽

First Report ◽

Coat Protein Region ◽

Pcr Products ◽

Leafroll Virus ◽

Leaf Virus ◽

Cherry Trees

Cherry necrotic rusty mottle virus (CNRMV), an unassigned member in the family Betaflexiviridae, has been reported in sweet cherry in North America, Europe, New Zealand, Japan, China, and Chile. The virus causes brown, angular necrotic spots, shot holes on the leaves, gum blisters, and necrosis of the bark in several cultivars (1). During the 2012 growing season, 154 sweet cherry trees were tested for the presence of CNRMV by RT-PCR. Samples were randomly collected from 11 orchards located in Gyeonggi and Gyeongsang provinces in Korea. RNA was extracted from leaves using the NucliSENS easyMAG system (bioMérieux, Boxtel, The Netherlands). The primer pair CGRMV1/2 (2) was used to amplify the coat protein region of CNRMV. Although none of the collected samples showed any notable symptoms, CNRMV PCR products of the expected size (949 bp) were obtained from three sweet cherry samples from one orchard in Gyeonggi province. The PCR products were cloned into a pGEM-T easy vector (Promega, Madison, WI) and sequenced. BLAST analyses of the three Korean sequences obtained (GenBank Accession Nos. AB822635, AB822636, and AB822637) showed 97% nucleotide sequence identity with a flowering cherry isolate from Japan (EU188439), and shared 98.8 to 99.6% nucleotide and 99.6 to 100% amino acid similarities to each other. The CNRMV positive samples were also tested for Apple chlorotic leaf spot virus (ACLSV), Cherry mottle leaf virus (CMLV), Cherry rasp leaf virus (CRLV), Cherry leafroll virus (CLRV), Cherry virus A (CVA), Little cherry virus 1 (LChV-1), Prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV) by RT-PCR. One of the three CNRMV-positive samples was also infected with CVA. To confirm CNRMV infection by wood indexing, Prunus serrulata cv. Kwanzan plants were graft-inoculated with chip buds from the CNRMV-positive sweet cherry trees. At 3 to 4 weeks post-inoculation, the Kwanzan plants showed quick decline with leaves wilting and dying; CNRMV infection of the indicators was confirmed by RT-PCR. To our knowledge, this is the first report of CNRMV infection of sweet cherry trees in Korea. Screening for CNRMV in propagation nurseries should minimize spread of this virus within Korea. References: (1) R. Li and R. Mock. Arch. Virol. 153:973, 2008. (2) R. Li and R. Mock. J. Virol. Methods 129:162, 2005.

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First Report of Cherry mottle leaf virus Infecting Cherry in China

Plant Disease ◽

10.1094/pdis-01-14-0058-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1161-1161 ◽

Cited By ~ 1

Author(s):

Y. X. Ma ◽

J. J. Li ◽

G. F. Li ◽

S. F. Zhu

Keyword(s):

Capsid Protein ◽

Sweet Cherry ◽

Restriction Site ◽

Protein Gene ◽

Capsid Protein Gene ◽

First Report ◽

Pcr Product ◽

Sweet Cherry Cultivars ◽

Leaf Virus ◽

Cherry Trees

Cherry mottle leaf virus (CMLV) is a member of the genus Trichovirus (family Betaflexiviridae). CMLV infects several species of the genus Prunus including cherry (Prunus avium) and peach (P. persica) (2,3). It is spread via budding and grafting with infected wood and can be transmitted from infected bitter cherry (P. emarginata), or infected but symptomless peach trees to healthy sweet cherry trees by the bud mite (Eriophyes inaequalis) (1). On susceptible sweet cherry cultivars, CMLV causes symptoms such as chlorotic mottle-leaf pattern, distortion, puckering of younger leaves, and small fruits that ripen late (1), which may lead to severe economic losses in some cultivars. Cherry is one of the most important fruit tree species in North China, and Shandong Province is one of the major cherry production areas. In June 2013, a survey of possible CMLV presence was conducted in a cherry orchard planted in 1996 in Zoucheng city, Shandong. The sweet cherry cultivars in this orchard included Black Tartarian, Bing, Hongdeng (a hybrid between cvs. Napoleon and Huangyu), and others; the rootstock cultivar utilized to graft these cultivars was mountain cherry (P. tomentosa). During the survey, characteristic symptoms on leaves such as leaf mottling, distortion, and puckering similar to those caused by CMLV were observed on some trees of the cv. Hongdeng, and the symptomatic trees accounted for ~10% of the total trees of this cultivar. Five symptomatic cherry leaf samples and three healthy-looking cherry leaf samples of cv. Hongdeng were collected. Total RNA extracted from the leaf samples using RNeasy plant mini kit (Qiagen Inc., Valencia, CA) was subjected to first strand cDNA synthesis with the reverse primer CMLV-3R (5′-CTCGAGAACACAGAGATTTGTCGAGAC-3′, sequence in italics indicates restriction site XhoI) and M-MLV reverse transcriptase (Promega, Madison, WI) according to the manufacturer's instruction. The cDNA was then used as template in the PCR assay using primers CMLV-5F (5′-GGATCCATGTCGGCGCGATTGAATC-3′, sequence in italics indicates restriction site BamHI) and CMLV-3R, which amplify the genome fragment including the capsid protein gene of CMLV. The expected PCR product ~590 bp was amplified from all five symptomatic samples, while no such PCR product was amplified from the symptomless samples. The PCR products were cloned into pMD18-T vector (TaKaRa, Dalian, China). Three positive clones for each of the five amplicons were sequenced in both directions. Sequence alignment and nucleotide BLAST analysis of the sequences revealed that they were 99% to 100% identical to the corresponding capsid protein gene sequence of a cherry isolate of CMLV (GenBank Accession No. AF170028) and 85% identical with that of the peach wart strain of CMLV (KC207480). Our results confirm the infection of cherry trees by CMLV in Shandong. To our knowledge, this is the first report of CMLV on cherry in China. As the spread of CMLV by mite vector in the field is rare (1), and no bud mite outbreak had occurred in this orchard in the past years, so it is possible that virus-infected propagation materials are largely responsible for the spread of this virus. Considering the importance of cherry cultivation in China, this report prompts the need to survey the occurrence of this virus in Shandong and other provinces, and the need to develop more effective management strategies such as the use of certified virus-free nursery stocks to reduce the impact of CMLV. References: (1) J. E. Adaskaveg et al. Diseases. Page 61 in: UC IPM Pest Management Guidelines: Cherry. University of California ANR Publication 3444, 2014. (2) D. James et al. Arch. Virol. 145:995, 2000. (3) T. A. Mekuria et al. Arch. Virol. 158:2201, 2013.

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First Report of Stem and Bulb Nematode Ditylenchus dipsaci on Garlic in New Mexico

Plant Health Progress ◽

10.1094/php-12-16-0069-br ◽

2017 ◽

Vol 18 (2) ◽

pp. 91-92

Author(s):

Jason M. French ◽

Jacki Beacham ◽

Amanda Garcia ◽

Natalie P. Goldberg ◽

Stephen H. Thomas ◽

...

Keyword(s):

International Trade ◽

New Mexico ◽

Sequence Data ◽

The State ◽

Economic Losses ◽

First Report ◽

Important Pest ◽

Ditylenchus Dipsaci ◽

Production Areas ◽

Stem And Bulb Nematode

Taken together, symptoms present, microscopic characterization, and ITS-1 sequence data indicate New Mexico garlic samples infested with Ditylenchus dipsaci, making this the first known report of this pest in the state. This discovery is significant because D. dipsaci can be a persistent pest and has the potential to cause significant economic losses on agronomically important hosts including onion, garlic, and alfalfa. Its longevity in the soil and international trade issues will be concerns for producers. Monitoring of production areas in the region will be performed to determine if this was an isolated and contained introduction or if this important pest has become established in New Mexico.

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First Report of Transmission of Little cherry virus 2 to Sweet Cherry by Pseudococcus maritimus (Ehrhorn) (Hemiptera: Pseudococcidae)

Plant Disease ◽

10.1094/pdis-12-12-1115-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 851-851 ◽

Cited By ~ 15

Author(s):

T. A. Mekuria ◽

T. J. Smith ◽

E. Beers ◽

G. W. Watson ◽

K. C. Eastwell

Keyword(s):

Sweet Cherry ◽

Prunus Avium ◽

Washington State ◽

Replicase Gene ◽

Insect Vector ◽

Significant Finding ◽

Rt Pcr ◽

First Report ◽

Plant Pathol ◽

Pseudococcus Maritimus

Little cherry virus 2 (LChV2; genus Ampelovirus, family Closteroviridae) is associated with Little Cherry Disease (LCD), one of the most economically destructive diseases of sweet cherry (Prunus avium (L.)) in North America (1). Since 2010, incidence of LCD associated with LChV2 confirmed by reverse transcription (RT)-PCR assays has increased in orchards of Washington State. LChV2 was known to be transmitted by the apple mealybug (Phenacoccus aceris (Signoret)) (3). However, the introduction of Allotropus utilis, a parasitoid platygastrid wasp (2) for biological control, contributed to keeping insect populations below the economic threshhold. In recent years, the population of grape mealybug (Pseudococcus maritimus (Ehrhorn)) increased in cherry orchards of Washington State (Beers, personal observation). Since grape mealybug is reported to transmit Grapevine leafroll associated virus 3 (Ampelovirus) in grapevine (4), this study investigated whether this insect would also transmit LChV2. A colony of grape mealybugs on Myrobalan plum (Prunus cerasifera Ehrh.) trees was identified visually and morphologically from slide mounts. In a growth chamber, first and second instar crawlers were fed on fresh cut shoots of sweet cherry infected with a North American strain (LC5) of LChV2. After an acquisition period of 7 days, 50 crawlers were transferred to each young potted sweet cherry trees, cv. Bing, confirmed free from LChV2 by RT-PCR. This process was repeated in two trials to yield a total of 21 potted trees exposed to grape mealybug. One additional tree was left uninfested as a negative control. After 1 week, the trees were treated with pesticide to eliminate the mealybugs. Two to four months after the inoculation period, leaves were collected from each of the recipient trees and tested by RT-PCR for the presence of LChV2. To reduce the possibility of virus contamination from residual mealybug debris on leaf surfaces, the trees were allowed to defoliate naturally. After a 3-month dormant period, the new foliage that emerged was then tested. Two sets of primers: LC26L (GCAGTACGTTCGATAAGAG) and LC26R (AACCACTTGATAGTGTCCT) (1); and LC2.13007F (GTTCGAAAGTGTTTCTTGA) and LC2.14545R (CATTATYTTACTAATGGTATGAC) (this study) were used to amplify a partial segment of the replicase gene (409 bp) and the complete (1,080 bp) coat protein gene of LChV2, respectively. Of 21 trees tested, 18 yielded positive results for LChV2. The reaction products from six randomly selected trees were cloned and the virus identity was verified by sequencing. The sequences of RT-PCR amplicons from both primer pairs showed ≥99% identity to LChV2, strain LC5 (GenBank Accession No. AF416335). The result confirmed that P. maritimus transmits LChV2, a significant finding for this cherry production region. Grape mealybug is of increasing concern in the tree fruit industry because it is difficult to control in established orchards. The presence of infested orchards that serve as reservoirs of both LCD and this insect vector present a challenge for management. To the best of our knowledge this is the first report to show transmission of LChV2 by grape mealybug. References: (1) K. C. Eastwell and M. G. Bernardy. Phytopathology 91:268, 2001. (2) C. F. W. Muesbeck. Can Entomol. 71:158, 1939. (3) J. R. D. Raine et al. Can. J. Plant Pathol. 8:6, 1986. (4) R. Sforza et al. Eur. J. Plant Pathol. 109:975, 2003.

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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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First Report of Colletotrichum higginsianum Causing Anthracnose of Arugula (Eruca sativa) in Florida

Plant Disease ◽

10.1094/pdis-09-13-0926-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1269-1269 ◽

Cited By ~ 2

Author(s):

J. S. Patel ◽

M. I. Costa de Novaes ◽

S. Zhang

Keyword(s):

Sequence Data ◽

Morphological Characteristics ◽

Its Region ◽

The United States ◽

Economic Losses ◽

Infected Leaf ◽

Eruca Sativa ◽

First Report ◽

Small Water ◽

Colletotrichum Higginsianum

Arugula (Eruca sativa) is grown in Florida and is an important component in packaged salad products. During spring 2013, leaf lesions on arugula caused significant economic losses in Miami-Dade County, Florida. Symptoms initially appeared as small water-soaked lesions that later became circular, sunken, and white in the center with a dark brown to black halo, up to 4 mm in diameter. Acervuli were found under a dissecting microscope on infected leaf lesions with black spines or setae. Occasionally, small, circular, often longitudinal dark brown spots appeared on leaf branches. Leaf tissues (5 × 5 mm) from lesion margins were surface sterilized in 0.9% sodium hypochlorite for 10 min, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA). PDA plates were incubated at 21°C under 24-h fluorescent lights for 4 to 6 days. The fungus initially produced gray mycelium followed by orange conidial mass. Hyphae of the fungus were septate and hyaline. After 5 to 7 days, the fungus produced acervuli with dark brown to black setae (75 to 130 μm long) (n = 20). Conidia were found in the colonies, which were single celled, oblong, hyaline, and 12 to 25 × 4 to 6 μm (n = 20). The cultural and morphological characteristics of the conidia were similar to those for Colletotrichum higginsianum Sacc (1). To further confirm the species of the isolates, the sequence of the ITS region of rDNA, chitin synthase 1 (CHS1), and actin (ACT) was amplified from isolates 05131 and 05132 using primer pairs ITS 1 and ITS 4 (4), CHS-79F and CHS-354R, and ACT-512F and ACT-783R (3), respectively. The sequenced data of each locus were deposited in GenBank with accessions KF550281.1, KF550282.1, KJ159904, KJ159905, KJ159906, and KJ159907. The resulting sequence of ITS showed 100% identity with sequences of C. higginsianum in JQ005760.1, and sequence of ACT gene showed 100% identity with C. higginsianum in JQ005823.1. The sequence of ACT gene and ITS region had ≤99% identity with other closely related Colletotrichum spp. CHS1 gene had 100% identity with JQ005781.1 belonging to C. higginsianum, and one accession JQ005783.1 belonging to C. fuscum. However, ACT gene and ITS region does not share 100% identity with C. fuscum and therefore, sequence data from three loci proves that isolated pathogen is C. higginsianum. All the above mentioned accessions that shared 100% identity with sequences of isolates used in our study have been previously used to represent the species in the C. destructivum clade in a systematics study (2). To confirm its pathogenicity, a suspension of isolate 05132 at 5 × 105 conidia/ml was sprayed on leaves of five arugula plants until runoff. The other five arugula plants sprayed with water served as non-inoculated controls. Both inoculated and non-inoculated plants were separately covered with a plastic bag to maintain high humidity for 24 h at 27 ± 5°C under natural day/night conditions in the greenhouse. Symptoms first appeared 3 to 4 days after inoculation as small water-soaked lesions, which became sunken with dark brown to black margins. Small circular and longitudinal dark brown spots were also seen on leaf branches as seen initially on naturally infected arugula. No symptoms developed on non-inoculated control plants. C. higginsianum was re-isolated from the lesions with the same morphological characteristics as described above, fulfilling Koch's postulates. To our knowledge, this is the first report of C. higginsianum causing anthracnose of arugula in Florida. This pathogen may potentially affect the salad industry in the United States. References: (1) A. J. Caesar et al. Plant Dis. 94:1166, 2010. (2) P. F. Cannon et al. Stud. Mycol. 73:181, 2012. (3) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report of Potato mop top virus Causing Potato Tuber Necrosis in Colorado and New Mexico

Plant Disease ◽

10.1094/pdis-08-14-0819-pdn ◽

2015 ◽

Vol 99 (1) ◽

pp. 164-164 ◽

Cited By ~ 6

Author(s):

I. Mallik ◽

N. C. Gudmestad

Keyword(s):

New Mexico ◽

North Dakota ◽

Consensus Sequence ◽

The United States ◽

Economic Losses ◽

Acid Extraction ◽

Rt Pcr ◽

First Report ◽

Tuber Necrosis ◽

Potato Mop Top Virus

Potato mop top virus (PMTV) is considered the type member of the genus Pomovirus. PMTV is an important pathogen of potato vectored by the plasmodiophorid Spongospora subterranea f. sp. subterranea (Sss), which causes powdery scab of potato (1). Sss and PMTV are usually associated with cool and humid environments. PMTV-infected potato tubers generally exhibit internal hollow necrotic spots or concentric rings, and the virus is known to cause significant economic losses in Northern Europe, North and South America, and Asia (4). PMTV in the United States was first reported in Maine (2). Potato (Solanum tuberosum L.) tubers cv. FL2048 and cv. Atlantic were sent to our laboratory from fields in Saguache County in Colorado and in San Juan County in New Mexico, respectively, during the spring of 2013. The tubers from both locations had multiple, internal, concentric, necrotic arcs and circles. Internal tissue with necrotic lesion from six symptomatic tubers from each location were crushed in liquid nitrogen followed by ribonucleic acid extraction using a Total RNA Isolation kit (Promega Corp., Madison, WI). These extracts were tested by reverse transcription (RT)-PCR using three different sets of previously published primers for molecular detection of PMTV. The primer set H360/C819 targeting the coat protein (CP) on RNA 3 of PMTV yielded an amplicon (H360-CO and H360-NM) of 460 bp (4). The second set of primers, pmtF4/pmtR4 (5), amplified a 417-bp product (PMTF-CO and PMTF-NM) in RNA 2, and the third set, PMTV-P9/PMTV-M9 (3), designed to amplify the region encoding an 8-KD cysteine-rich protein in RNA 3 of PMTV, yielded a 507-bp amplicon (PMTV9-CO and PMTV9-NM). The amplicons generated from RT-PCR using all three sets were cloned (PGEMT-easy) and sequenced. Since the sequences from symptomatic tuber extracts from each location were identical to their respective primer sets, a consensus sequence from each primer set was submitted to National Center for Biotechnology Information (NCBI) GenBank. Sequences obtained from the H360/C819 primer set (GenBank Accession Nos. KM207013 and KM207014 for H360-CO and H360-NM, respectively) were 100% identical to the corresponding CP regions of PMTV isolates from North Dakota (HM776172). Sequences from the pmtF4/pmtR4 primer set (KM207015 and KM207016 for PMTF-CO and PMTF-NM, respectively) were 100% identical to the corresponding protein in RNA2 of PMTV isolates from North Dakota (GenBank HM776171), and sequences from the PMTV-P9/PMTV-M9 primer set (KM207017 and KM207018 for PMTV9-CO and PMTV9-NM respectively) were 99% identical to the corresponding protein in RNA3 of PMTV isolates (AY187010). The 100-99% homology of the sequences from this study to the corresponding PMTV sequences published in NCBI confirmed the occurrence of symptoms in the tubers from both Colorado and New Mexico due to PMTV. None of the symptomatic tubers tested positive for Tobacco rattle virus, Tomato spotted wilt virus, Alfalfa mosaic virus, Potato leafroll virus, or the necrotic strains of Potato virus Y by RT-PCR. To our knowledge, this is the first report of PMTV in potato in states of Colorado and New Mexico. References: (1) R. A. C. Jones and B. D. Harrsion. Ann. Appl. Biol. 63:1, 1969. (2) D. H. Lambert et al. Plant Dis. 87:872, 2003. (3) T. Nakayama et al. Am. J. Pot. Res. 87:218, 2010. (4) J. Santala et al. Ann. Appl. Biol. Online publication. DOI: 10.1111/j.1744-7348.2010.00423.x (5) H. Xu et al. Plant Dis. 88:363, 2004.

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