First Report of Kidney Bean Little Leaf Disease Associated with 16SrII Group Phytoplasma in China

Kidney bean (Phaseolus vulgaris) is an important cash crop in China. It is widely grown in the Yunnan Province in the southwest region. In November of 2008, a new disease was observed on kidney bean plants grown in Yuanmou County. Affected plants displayed symptoms that included numerous twisted lateral shoots with abundant, tiny trifoliate leaves that were approximately one-tenth the size of healthy leaves. Some affected leaves, which were slightly distorted and oblong to oval, were evident on diseased plants and appeared pale green. On plants with little leaf symptoms, flowers were poorly formed, withered or aborted, and no pods were present. Surveys conducted in areas affected by disease revealed the presence of the disease in approximately 10% of the plants. DNA was extracted from 0.1 g of petioles and midveins harvested from both diseased and symptomless plants with the Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Plants were evaluated for phytoplasma infection by a nested PCR assay with phytoplasma specific ribosomal operon primer pair P1/Tint followed by R16F2/R16R2 (2,3). An rDNA product of approximately 1,250 bp was amplified from seven of seven diseased plants, whereas no products were amplified from symptomless plants or a negative control devoid of DNA. Digests of nested PCR products (approximately 200 ng) with endonucleases AluI, ScaI, or EcoRI revealed no differences in restriction fragment length polymorphism (RFLP) among diseased plants. The amplicon was cloned and sequenced (GenBank Accession No. GQ336993). Comparison of in silico RFLP profiles with published profiles showed that kidney bean little leaf phytoplasma is a member of peanut witches' broom group 16SrII. Blast analysis of the kidney bean little leaf phytoplasma 16S rDNA sequence revealed that this strain is most similar (99.0%) to Syringa oblata yellows phytoplasma (Accession No. FJ263629) and to other phytoplasmas classified as group 16SrII members. Previously, phytoplasmas identified as 16SrII strains have been reported as probable cause of cactus witches' broom (1) and crotalaria witches' broom (4) in China. To our knowledge, this is the first report of a 16SrII phytoplasma infecting the kidney bean in China. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) I.-M Lee et al. Phytopathology 83:834, 1993. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996 (4) Z. H. Wang et al. Plant Pathol. 57:364, 2008.

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First Report of a Group 16SrII Phytoplasma Associated with Witches'-Broom of Eggplant in Oman

Plant Disease ◽

10.1094/pdis-10-10-0761 ◽

2011 ◽

Vol 95 (3) ◽

pp. 360-360 ◽

Cited By ~ 9

Author(s):

A. M. Al-Subhi ◽

N. A. Al-Saady ◽

A. J. Khan ◽

M. L. Deadman

Keyword(s):

Nested Pcr ◽

Solanum Melongena ◽

Positive Control ◽

Rrna Gene ◽

Direct Pcr ◽

First Report ◽

Pcr Product ◽

Pcr Products ◽

Broom Disease ◽

Phytoplasma Infection

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman. References: (1) A. J. Khan et al. Phytopathology 92:1038, 2002. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA, 81:8014, 1984.

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First Report of Two Distinct Phytoplasma Species, ‘Candidatus Phytoplasma cynodontis’ and ‘Candidatus Phytoplasma asteris,’ Simultaneously Associated with Yellow Decline of Wodyetia bifurcata (Foxtail Palm) in Malaysia

Plant Disease ◽

10.1094/pdis-04-13-0412-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1504-1504 ◽

Cited By ~ 7

Author(s):

N. Naderali ◽

N. Nejat ◽

Y. H. Tan ◽

G. Vadamalai

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Rflp Analysis ◽

Rrna Gene ◽

Gene Sequences ◽

First Report ◽

White Leaf ◽

General Decline ◽

Pcr Products ◽

Plant Pathol

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV ‘Candidatus Phytoplasma cynodontis’ group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the ‘Candidatus Phytoplasma asteris’ 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia. References: (1) N. Nejat et al. Plant Pathol. 58:1152, 2009. (2) N. Nejat et al. Plant Pathol. J. 9:101, 2010. (3) Y. P. Zhang et al. J. Virol. Meth. 71:45, 1998. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of Witches'-Broom Disease of Sesame (Sesamum indicum) in Oman

Plant Disease ◽

10.1094/pd-89-0530c ◽

2005 ◽

Vol 89 (5) ◽

pp. 530-530 ◽

Cited By ~ 20

Author(s):

M. A. Al-Sakeiti ◽

A. M. Al-Subhi ◽

N. A. Al-Saady ◽

M. L. Deadman

Keyword(s):

Nested Pcr ◽

Restriction Enzymes ◽

Negative Control ◽

Direct Pcr ◽

First Report ◽

Pcr Products ◽

Broom Disease ◽

Short Shoots ◽

Template Dna ◽

Positive Controls

Sesame is the major oil seed crop in Oman. During 2004, disease symptoms were observed at Nizwa, 175 km south of Muscat. Symptoms included phyllody and excessive development of short shoots and internodes resulting in little leaves. Total genomic DNA was extracted from healthy and symptomatic plants with a modified cetyltrimethylammoniumbromide (CTAB) buffer method (2). DNA samples were assayed by polymerase chain reaction (PCR), with the 16S rDNA amplified using primers P1 and P7. Direct PCR products were used as template DNA for nested PCR with primers R16F2n and R16R2. Direct PCR products were analyzed by restriction fragment length polymorphism (RFLP) with four restriction enzymes, Tru9I, HaeIII, HhaI, and RsaI. DNAs from alfalfa and lime plants infected by witches'-broom phytoplasmas were used as positive controls and DNA from healthy plants and water were negative controls. The results showed the presence of a 1.8-kb product amplified with the direct PCR and a 1.2-kb product of the nested PCR from infected sesame and the positive controls. No PCR product was observed in the negative control. The PCR assay confirmed the presence of phytoplasma causing witches'-broom disease in sesame. The RFLP results showed the sesame phytoplasma to be most similar to the alfalfa phytoplasma, a member of 16SrII group (1). To our knowledge, this is the first report of a phytoplasma of the 16Sr II group causing witches'-broom disease on sesame in the Sultanate of Oman. References: (1) A. J. Khan et al. Phytopathology 92:1038, 2002. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA, 81:8014, 1984.

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First Report of Alfalfa Witches Broom Disease in Oman Caused by a Phytoplasma of the 16Sr II Group

Plant Disease ◽

10.1094/pdis.2001.85.12.1287a ◽

2001 ◽

Vol 85 (12) ◽

pp. 1287-1287 ◽

Cited By ~ 8

Author(s):

A. J. Khan ◽

K. M. Azam ◽

M. L. Deadman ◽

A. M. Al-Subhi ◽

P. Jones

Keyword(s):

Sweet Potato ◽

Infected Plant ◽

Rflp Analysis ◽

Negative Control ◽

Sultanate Of Oman ◽

Nested Polymerase Chain Reaction ◽

Aster Yellows ◽

First Report ◽

Pcr Products ◽

Plant Pathol

Alfalfa (Medicago sativa L.) is a primary forage crop in the Sultanate of Oman. A new disease of alfalfa in Oman is characterized by proliferation of shoots and yellowing of leaves in 1- to 2-year-old plants and tillering of stems in 4- to 5-year-old plants. Annual losses due to this disease are estimated at more than US$ 23 million. Samples of healthy and infected alfalfa plants were collected from different regions. Total DNA was extracted according to Khadhair et al. (1), with minor modifications. Amplification of 16S rDNA was done using a nested polymerase chain reaction (PCR) approach with primers P1/P7 and R16F2n/R16R2. DNA from healthy leaves and sterile water was used as a negative control, while DNA from periwinkle infected with faba bean phyllody (16SrII-C), aster yellows (16SrI), tomato big bud (16SrII-D), sweet potato little leaf (16SrII-D), catharanthus phyllody (16SrVI), and sesame phyllody (16SrII-A) were used as positive controls and for restriction fragment length polymorphism (RFLP) comparisons. Nested 1.25-kb PCR products from infected plant samples were subjected to RFLP analysis with restriction endonucleases RsaI, AluI, HaeIII, HhaI, EcoRI, TaqI, Tru9I, and Sau3AI. The analysis showed that the alfalfa witches' broom phytoplasma (AWBP) belonged to the 16SrII group (peanut witches' broom) and that the AWBP was most similar to sweet potato little leaf (16SrII-D) but distinct from “Candidatus Phytoplasma aurantifolia,” the cause of lime witches' broom in Oman. Other phytoplasmas infecting alfalfa have been reported from Europe and North America (1,3), but they belong to the 16SrVI (clover phyllody) and 16SrI (aster yellows) groups. An alfalfa witches' broom reported from Italy (2) forms a separate grouping (4). To our knowledge, this is the first report of a phytoplasma from the peanut witches' broom group infecting alfalfa in the Sultanate of Oman. References: (1) A. H. Khadhair et al. Microbiol. Res. 152:259, 1997. (2) C. Marcone et al. J. Plant Pathol. 79:211, 1997. (3) R. D. Peters et al. Plant Dis. 83:488, 1999. (4) E. Seemuller et al. J. Plant Pathol. 80:3, 1998.

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First Report of a “Candidatus Phytoplasma australiense”-Related Strain in Lucerne (Medicago sativa) in Australia

Plant Disease ◽

10.1094/pd-91-0111a ◽

2007 ◽

Vol 91 (1) ◽

pp. 111-111 ◽

Cited By ~ 7

Author(s):

M. A. Getachew ◽

A. Mitchell ◽

G. M. Gurr ◽

M. J. Fletcher ◽

L. J. Pilkington ◽

...

Keyword(s):

Medicago Sativa ◽

Nested Pcr ◽

The United States ◽

Plant Sample ◽

Related Strain ◽

Universal Primer ◽

Separate Species ◽

First Report ◽

Pcr Products ◽

Plant Pathol

Australian lucerne yellows (ALuY), a phytoplasma-associated disease, is a major problem in Australia that causes the pasture seed industry millions of dollars in losses annually (3). Samples were collected from lucerne (Medicago sativa L.) plants exhibiting symptoms indicative of ALuY (4) in a seed lucerne paddock (cv CW 5558) at Griffith, southwestern New South Wales (NSW), Australia, in November 2005 and again in January 2006. Samples were kept at 4°C and processed within 36 h of collection. Total DNA was extracted from approximately 0.3 g of leaf midribs and petioles of each plant sample and used as template in a nested PCR assay with phytoplasma universal primer pairs P1/P7 and fU5/m23sr. PCR products resulting from the first amplification were diluted (1:30) with sterile distilled water (SDW) before reamplification with fU5/m23sr. DNA of Australian tomato big bud (TBB) phytoplasma and SDW were used as positive and negative assay controls, respectively. Ten of fifteen plant samples collected in November tested positive for phytoplasma DNA. Restriction digestion profiles of nested PCR amplicons with HpaII endonuclease were the same for all symptomatic plants but differed from the control. Phytoplasma identity was determined by sequencing two nested PCR products that yielded identical sequences. One was deposited in the GenBank database (Accession No. DQ786394). BLAST analysis of the latter sequence revealed a >99.6% similarity with “Candidatus Phytoplasma australiense” (L76865) and related strains papaya dieback (Y10095), phormium yellow leaf (U43570), strawberry green petal (AJ243044), and strawberry lethal yellows (AJ243045). Direct PCR with primers FP 5′-GCATGTCGCGGTGAATAC-3′ and RY 5′-TGAGCTATAGGCCCTTAATC-3′ designed to specifically amplify DNA of “Ca. P. australiense” detected the phytoplasma in 8 of 40 samples collected in January. Whether this phytoplasma is the etiological agent solely responsible for ALuY is currently under investigation. “Ca. P. asteris” and stolbur group (16SrXII) phytoplasmas have been reported in lucerne in the United States (2) and Italy (1), respectively. Within the stolbur group 16SrXII, “Ca. P. australiense” and stolbur phytoplasma are regarded as separate species and both are distinct from “Ca. P. asteris”, a group 16SrI strain. To our knowledge, this is the first report of a “Ca. P. australiense” related strain in lucerne. References: (1) C. Marzachi et al. J. Plant Pathol. 82:201, 2000. (2) R. D. Peters et al. Plant Dis. 83:488, 1999. (3) L. J. Pilkington et al. Australas. Plant Pathol. 28:253, 1999. (4) L. J. Pilkington et al. First report of a phytoplasma associated with ‘Australian lucerne yellows’ disease. New Disease Report. Online publication at http://www.bspp.org.uk/ndr/jan2002/2001-46.asp .

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First Report of “Candidatus Phytoplasma Asteris”-Related Strains Infecting Chinaberry Trees with Leaf Yellowing Symptoms in Vietnam

Plant Disease ◽

10.1094/pd-90-0527a ◽

2006 ◽

Vol 90 (4) ◽

pp. 527-527 ◽

Cited By ~ 3

Author(s):

N. A. Harrison ◽

M. L. Carpio ◽

E. Boa

Keyword(s):

Restriction Fragment ◽

Nested Pcr ◽

First Record ◽

Positive Control ◽

Negative Control ◽

Melia Azedarach ◽

Leaf Sample ◽

Crown Shape ◽

Pcr Products ◽

Phytoplasma Infection

Although no loss of crown shape or unusual growth were evident on two mature Chinaberry trees (Melia azedarach L.) located near the citadel in central Hué city, Vietnam, leaves on both trees displayed distinctive interveinal yellowing during September 2003. This symptom was reminiscent in appearance to foliar discoloration previously observed on mature Chinaberry trees in El Torno, Santa Cruz, Bolivia that was subsequently attributed to phytoplasma infection of these trees (2). Eight samples of yellowed leaves were collected from affected trees and preserved by pressing and drying for later analysis. Total nucleic acids were extracted from 0.5 g of each leaf sample and assayed for phytoplasma DNA using a polymerase chain reaction (PCR) assay with phytoplasma universal rRNA primer pair P1 and P7 (4). No visible product was generated from any Chinaberry sample while a product of expected size (1.8 kb) was obtained from DNA of a periwinkle plant (Catharanthus roseus (L.) G. Don) infected with “Candidatus Phytoplasma asteris”-related strain eastern aster yellows (EAY) and included as a known positive control in the assay. After P1/P7-primed products were reamplified by PCR with nested phytoplasma universal 16S rRNA primer pair R16mF2/R16mR1 (1), a 1.4-kb product of predicted size was obtained from the eight samples and EAY positive control, whereas no product was obtained from DNA of seed-grown healthy periwinkle included as a negative control. Digests of nested PCR products (1.4 kb) with HaeIII or MseI endonuclease, and electrophoresis of digests through 8% polyacrylamide gels, revealed no apparent differences in restriction fragment patterns among products from Chinaberry samples. However, HaeIII and MseI patterns differed from those obtained by digestion of nested PCR products from EAY, a known 16SrI-A subgroup phytoplasma (3), with these enzymes. Chinaberry phytoplasmas were definitively identified as group 16SrI strains after reevaluation of samples by a PCR incorporating ribosomal protein (rp) gene primer pair rpF1/rpR1 and reamplification of resulting products with nested 16SrI group-specific primer pair rp(I)F1A/rp(I)R1A (3). A 1.2-kb product of expected size was obtained from all Chinaberry samples and EAY positive control only. Restriction fragment length polymorphism patterns produced by DraI or SspI endonuclease digests of nested PCR products (1.2 kb) revealed no differences among Chinaberry samples, although patterns associated with each enzyme differed from those observed for the EAY positive control. Sequence comparison and phylogenetic analysis of Chinaberry yellows phytoplasma (CbY-V) 16Sr DNA (GenBank Accession No. AY863003) determined this strain to be most closely related (99.65%) to Epilobium phyllody phytoplasma, a 16SrI-B subgroup strain (3). However, based on analysis of rp gene sequences (GenBank Accession No. DQ321823), strain CbY-V was judged most similar (99.59%) to cabbage proliferation, a well characterized 16SrI-B subgroup, rpI-B subgroup phytoplasma (3). To our knowledge, this is the first record of phytoplasma infection of Chinaberry, a common urban shade tree in Vietnam. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) N. A. Harrison et al. Plant Pathol. 52:147, 2003. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004 (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

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First Report of a Group 16SrII Phytoplasma Infecting Chickpea in Oman

Plant Disease ◽

10.1094/pd-90-0973c ◽

2006 ◽

Vol 90 (7) ◽

pp. 973-973 ◽

Cited By ~ 8

Author(s):

N. A. Al-Saady ◽

A. M. Al-Subhi ◽

A. Al-Nabhani ◽

A. J. Khan

Keyword(s):

Nested Pcr ◽

Animal Feed ◽

Restriction Enzymes ◽

Universal Primers ◽

Polymorphism Analysis ◽

Disease Symptoms ◽

First Report ◽

Pcr Products ◽

Polymerase Chain ◽

Group 2

Chickpea (Cicer arietinum), locally known as “Dungo”, is grown for legume and animal feed mainly in the interior region of Oman. During February 2006, survey samples of chickpea leaves from plants showing yellows disease symptoms that included phyllody and little leaf were collected from the Nizwa Region (175 km south of Muscat). Total nucleic acid was extracted from asymptomatic and symptomatic chickpea leaves using a cetyltrimethylammoniumbromide method with modifications (3). All leaf samples from eight symptomatic plants consistently tested positive using a polymerase chain reaction assay (PCR) with phytoplasma universal primers (P1/P7) that amplify a 1.8-kb phytoplasma rDNA product and followed by nested PCR with R16F2n/R16R2 primers yielding a product of 1.2 kb (2). No PCR products were evident when DNA extracted from healthy plants was used as template. Restriction fragment length polymorphism analysis of nested PCR products by separate digestion with Tru9I, HaeIII, HpaII, AluI, TaqI, HhaI, and RsaI restriction enzymes revealed that a phytoplasma belonging to group 16SrII peanut witches'-broom group (2) was associated with chickpea phyllody and little leaf disease in Oman. Restriction profiles of chickpea phytoplasma were identical with those of alfalfa witches'-broom phytoplasma, a known subgroup 16SrII-B strain (3). To our knowledge, this is the first report of phytoplasma infecting chickpea crops in Oman. References: (1) A. J. Khan et al. Phytopathology, 92:1038, 2002. (2). I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998 (3) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA. 81:8014, 1984.

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First Report of an Aster Yellows Phytoplasma Associated with Cabbage in Southern Texas

Plant Disease ◽

10.1094/pdis.2001.85.4.447b ◽

2001 ◽

Vol 85 (4) ◽

pp. 447-447 ◽

Cited By ~ 6

Author(s):

I.-M. Lee ◽

R. A. Dane ◽

M. C. Black ◽

Noel Troxclair

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Restriction Enzymes ◽

Diagnostic Procedures ◽

Acid Extraction ◽

Insect Vector ◽

Aster Yellows ◽

First Report ◽

Pcr Products ◽

Aster Yellows Phytoplasma

In early spring 2000 carrot crops in southwestern Texas were severely infected by an outbreak of phyllody associated with aster yellows phytoplasma. Cabbage crops that had been planted adjacent to these carrot fields began to display previously unobserved symptoms characteristic of phytoplasma infection. Symptoms included purple discoloration in leaf veins and at the outer edges of leaves on cabbage heads. Proliferation of sprouts also occurred at the base of the stem and between leaf layers of some plants, and sprouts sometimes continued to proliferate on extended stems. About 5% of cabbage plants in the field exhibited these symptoms. Two symptomless and four symptomatic cabbage heads were collected in early April from one cabbage field. Veinal tissues were stripped from each sample and used for total nucleic acid extraction. To obtain specific and sufficient amount of PCR products for analysis, nested PCR was performed by using primer pairs (first with P1/P7 followed by R16F2n/R16R2) (1,2) universal for phytoplasma detection. A specific 16S rDNA fragment (about 1.2 kb) was strongly amplified from the four symptomatic but not from the two asymptomatic samples. The nested PCR products obtained from the four symptomatic samples were then analyzed by restriction fragment length polymorphism (RFLP) using the restriction enzymes MseI, HhaI, and HpaII, and the RFLP patterns were compared to the published patterns of known phytoplasmas (1). The resulting RFLP patterns were identical to those of a phytoplasma belonging to subgroup B of the aster yellows phytoplasma group (16SrI). These RFLP patterns were also evident in putative restriction sites observed in a 1.5 kbp nucleotide sequence of the 16S rDNA. This is the first report of aster yellows phytoplasma associated disease symptoms in cabbage in Texas. The occurrence of cabbage proliferation coincided with the presence of high populations of the insect vector, aster leafhopper. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) B. Schneider et al. 1995. Molecular and Diagnostic Procedures in Mycoplasmology, Vol. I. Academic Press, San Diego, CA.

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First Report of Grapevine Pinot gris virus (GPGV) in grapevine in France

Plant Disease ◽

10.1094/pdis-10-14-1008-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 293-293 ◽

Cited By ~ 25

Author(s):

M. Beuve ◽

T. Candresse ◽

M. Tannières ◽

O. Lemaire

Keyword(s):

Large Scale ◽

De Novo ◽

Pinot Noir ◽

Rt Pcr ◽

Wine Quality ◽

First Report ◽

Italian Isolate ◽

Pcr Products ◽

Grapevine Pinot Gris Virus ◽

Plant Pathol

Grapevine Pinot gris virus (GPGV), belonging to the genus Trichovirus of the family Betaflexiviridae, was first identified by siRNA sequencing in northern Italy in 2012, in the grapevine varieties Pinot gris, Traminer, and Pinot Noir, which exhibited mottling and leaf deformation (1), and in asymptomatic vines, with a lower frequency. Since 2012, this virus has also been reported in South Korea, Slovenia, Greece (3), Czech Republic (2), Slovakia (2), and southern Italy (4). In 2014, GPGV was identified by Illumina sequencing of total RNAs extracted from leaves of the Merlot variety (Vitis vinifera) grafted onto Gravesac rootstock originated from a vineyard in the Bordeaux region of France. This Merlot plant exhibited fanleaf-like degeneration symptoms associated with Tomato black ring virus (TBRV) infection. Cuttings were collected in 2010 and maintained thereafter in a greenhouse. The full-length genome was assembled either de novo or by mapping of the Illumina reads on a reference GPGV genome (GenBank FR877530) using the CLC Genomics workbench software (CLC Bio, Qiagen, USA). The French GPGV isolate “Mer” (7,223 nucleotides, GenBank KM491305) is closely related to other European GPGV sequences; it exhibits 95.4% nucleotide identity with the reference Italian isolate (NC_015782) and 98 to 98.3% identity with Slovak isolates (KF134123 to KF134125). The higher divergence between French and Italian GPGV isolates was mainly due to differences in the 5′ extremity of the genome, as already shown with the Slovak GPGV isolates. RNA extracted from phloem scrapings of 19 cv. Merlot vines from the same plot collected in 2014 were analyzed by RT-PCR using the specific primer pair Pg-Mer-F1 (5′-GGAGTTGCCTTCGTTTACGA-3′) and Pg-Mer-R1 (5′-GTACTTGATTCGCCTC GCTCA-3′), designed on the basis of alignments of all available GPGV sequences from GenBank. The resulting amplicon of 770 bp corresponded to a fragment of the putative movement protein (MP) gene. Seven (35%) of the tested plants gave a strong positive amplification. Three RT-PCR products were directly sequenced and showed 99.3 to 99.5% identity within the MP gene of the GPGV-Mer isolate. Given the mixed viral infection status of the vines found infected by GPGV, it was not possible to associate a specific symptomatology with the presence of GPGV. Furthermore, similar RT-PCR tests were also performed on RNA extracts prepared from two plants of cv. Carignan that originated from a French grapevine collection, exhibiting fanleaf-like symptoms without any nepovirus detection. These samples similarly gave a strong positive amplification. The sequences obtained from the two Carignan vines showed 98.4 and 97.8% identity with the GPGV-Mer isolate. To our knowledge, this is the first report of GPGV in France. GPGV has been discovered in white and red berry cultivars, suggesting that its prevalence could be important in European vineyards (2). Further large-scale studies will be essential to determine the world prevalence of GPGV and to evaluate its potential effects on yield and on wine quality, as well as to shed light on GPGV epidemiology. Of particular concern is whether, like the other grapevine-infecting Trichovirus, Grapevine berry inner necrosis virus (GPGV) can be transmitted by the eryophid mite Colomerus vitis. References: (1) A. Giampetruzzi et al. Virus Res. 163: 262, 2012. (2) M. Glasa et al. Arch. Virol. 159: 2103, 2014. (3) G. P. Martelli, J. Plant Pathol. 96: S105, 2014. (4) M. Morelli et al. J. Plant Pathol. 96:431, 2014.

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First Report of Aster Yellows (16SrI-B) Associated with Potatoes in Alaska

Plant Disease ◽

10.1094/pdis-12-10-0913 ◽

2011 ◽

Vol 95 (6) ◽

pp. 767-767

Author(s):

J. H. McBeath ◽

P. J. Laski ◽

M. Cheng

Keyword(s):

Nested Pcr ◽

Disease Transmission ◽

Sequence Data ◽

Restriction Enzymes ◽

Reference Pattern ◽

Aster Yellows ◽

First Report ◽

Group I ◽

Pcr Product ◽

Pcr Products

During a disease survey conducted in 2009 in Alaska, one potato plant (Solanum tuberosum) with symptoms characteristic of aster yellows, such as apical leaves rolling inward, leaves turning yellow or purple, and presence of aerial tubers, was found in a commercial field. Total DNA was extracted from leaves, stems, and roots of the symptomatic and symptomless plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. A nested PCR was carried out with the first round primer pair P1/P7 followed by second round primer pair R16F2n/R16R2 (1,3). An approximate 1.2-kb PCR product was amplified from the symptomatic plant, but not symptomless plants. The PCR products from R16F2n/R16R2 were digested using restriction enzymes AluI, BfaI, BstUI, HhaI, HpaI, KpnI, MseI, and RsaI. The restriction fragment length polymorphism (RFLP) patterns were compared with those from known phytoplasma strains (1) and they matched the patterns for aster yellows subgroup B (16SrI-B). After P1/P7 amplification, the nested PCR product of primer pair P1A/16S-SR (2) was purified with a MiniElute Gel Extraction kit (Qiagen), sequenced by GENEWIZ (South Plainfield, NJ), and the sequence data analyzed by iPhyClassifier software (4). The results indicated that the sequence (GenBank Accession No. HQ599231) had 99.65% similarity to ‘Candidatus Phytoplasma asteris’ reference strain (GenBank Accession No. M30790). The RFLP similarity was identical (coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank Accession No. NC 005303). To our knowledge, this is the first report on the molecular identification of aster yellows phytoplasma associated with potatoes in Alaska. The source of the phytoplasma and pathway of disease transmission is currently under investigation. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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