Molecular Identification of a Phytoplasma Associated with Witches'-Broom Disease of Black Raspberry in Oregon and Its Classification in Group 16SrIII, New Subgroup Q

Plants of Rubus occidentalis (black raspberry) ‘Munger’ exhibiting symptoms of black raspberry witches'-broom (BRWB) disease were observed in commercial fields in Oregon (1). Symptoms were often severe, leading to death of infected plants, and a phytoplasma (mycoplasmalike bodies) was observed in ultrathin sections of diseased plants (1). In the current work, the association of phytoplasma with BRWB was assessed using the polymerase chain reaction (PCR) for specific amplification of phytoplasmal rDNA. DNA template for use in the PCR was extracted from plants as described elsewhere (2). Phytoplasmal 16S rDNA was amplified from diseased black raspberry plants in PCR primed by primer pair P1/P7 and reamplified in nested PCR primed by primer pair R16F2n/R2 (F2n/R2) by a method described previously (2). These results indicated the presence of a phytoplasma, designated BRWB phytoplasma, in the diseased plants. Identification of BRWB phytoplasma was accomplished by restriction fragment length polymorphism (RFLP) analysis of DNA amplified in PCR primed by F2n/R2. Phytoplasma classification was done according to the system of Lee et al. (3). On the basis of collective RFLP patterns of the amplified 16S rDNA, the BRWB phytoplasma was classified as a member of group 16SrIII (group III, X-disease phytoplasma group). The HhaI RFLP pattern of BRWB 16S rDNA differed from that of its close relative, clover yellow edge (CYE) phytoplasma. The RsaI RFLP pattern of BRWB rDNA differed from that of rDNA from all phytoplasmas previously described in group III. Based on these results, BRWB phytoplasma was classified in a new subgroup, designated subgroup Q (III-Q) within group III. The 1.8 kbp DNA product of PCR primed by primer pair P1/P7 was cloned and its nucleotide sequence determined. The sequence was deposited in GenBank under Accession no. AF302841. Results from putative restriction site analysis of the cloned and sequenced rDNA were in excellent agreement with the results from enzymatic RFLP analysis of uncloned rDNA amplified from BRWB diseased black raspberry. Sequence similarity between the 1.8 kbp rDNA of BRWB phytoplasma and that of CYE phytoplasma was 99.4%. The nucleotide sequence data support the conclusion that the BRWB phytoplasma is related to, but is distinct from, other strains that are classified in group III. These findings contribute knowledge about the diversity of phytoplasmas affiliated with group III and provide information to aid the diagnosis of BRWB disease. References: (1) R. H. Converse et al. Plant Dis. 66:949, 1982. (2) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

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Molecular Identification and Classification of a Phytoplasma Associated with Phyllody of Strawberry Fruit in Maryland

Plant Disease ◽

10.1094/pdis.2001.85.3.335b ◽

2001 ◽

Vol 85 (3) ◽

pp. 335-335 ◽

Cited By ~ 7

Author(s):

R. Jomantiene ◽

J. L. Maas ◽

R. E. Davis ◽

E. L. Dally

Keyword(s):

16S Rdna ◽

Sequence Similarity ◽

Rflp Analysis ◽

Close Relative ◽

Rflp Pattern ◽

Strawberry Fruit ◽

Group Iii ◽

Site Analysis ◽

Leaf Chlorosis ◽

Clover Phyllody

Several phytoplasmas have been reported to be associated with phyllody of strawberry fruit, including clover yellow edge, clover proliferation, clover phyllody, eastern and western aster yellows, STRAWB2, strawberry multicipita, and Mexican periwinkle virescence phytoplasmas. Plant symptoms in addition to phyllody may include chlorosis, virescence, stunting, or crown proliferation. In this report we describe a new phytoplasma in association with strawberry leafy fruit (SLF) disease in Maryland. Diseased plants exhibited fruit phyllody, floral virescence, leaf chlorosis, and plant stunting. Phytoplasmal 16S rDNA was amplified from SLF diseased plants by using the polymerase chain reaction (PCR) primed by primer pair P1/P7 and was reamplified in nested PCR primed by primer pair R16F2n/R2 (F2n/R2) as previously described (1). These results indicated the presence of a phytoplasma, designated SLF phytoplasma. Identification of SLF phytoplasma was accomplished by restriction fragment length polymorphism (RFLP) analysis of DNA amplified in PCR primed by F2n/R2, using endonuclease enzyme digestion with AluI, HhaI, KpnI, HaeIII, MseI, HpaII, RsaI, and Sau3AI. Phytoplasma classification was done according to the system of Lee et al. (2). RFLP analyses of rDNA amplified in three separate PCRs gave identical patterns. On the basis of collective RFLP patterns of the amplified 16S rDNA, the SLF phytoplasma was classified as a member of group 16SrIII (group III, X-disease phytoplasma group). The HhaI RFLP pattern of SLF 16S rDNA differed from that of the apparently close relative, clover yellow edge (CYE) phytoplasma, and all other phytoplasmas previously described in group III. Based on these results, SLF phytoplasma was classified in a new subgroup, designated subgroup K (III-K), within group III. The 1.2 kbp DNA product of PCR primed by primer pair F2n/R2 was sequenced, and the sequence deposited in GenBank under Accession No. AF 274876. Results from putative restriction site analysis of the sequence were in agreement with the results from actual enzymatic RFLP analysis of rDNA amplified from phylloid strawberry fruit. Although the sequence similarity between the 1.2-kbp fragment from the 16S rDNA of SLF phytoplasma and that of CYE phytoplasma was 99.9%, the Hha1 RFLP pattern of SLF rDNA supports the conclusion that the SLF phytoplasma may be closely related to, but is distinct from, CYE and other strains that are classified in group III. These findings contribute knowledge about the diversity of phytoplasmas affiliated with group III and the diversity of phytoplasmas associated with diseases in strawberry. References: (1) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

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First Report of Phytoplasmas in Soybean, Alfalfa, and Lupinus sp. in Lithuania

Plant Disease ◽

10.1094/pdis.2000.84.2.198c ◽

2000 ◽

Vol 84 (2) ◽

pp. 198-198 ◽

Cited By ~ 11

Author(s):

R. Jomantiene ◽

R. E. Davis ◽

L. Antoniuk ◽

J. Staniulis

Keyword(s):

16S Rdna ◽

Type Strain ◽

Sequence Data ◽

Sequence Similarity ◽

Dna Amplification ◽

Plant Host ◽

Nucleotide Sequence Data ◽

Aster Yellows ◽

Veinal Necrosis ◽

Aster Yellows Phytoplasma

Plants of cultivated soybean (Glycine max) and alfalfa (Medicago sativa) in Dotnuva and of wild Lupinus sp. in Ledakalnis, Lithuania, exhibited symptoms that suggested phytoplasmal infections. Soybean plants were of normal growth habit but exhibited veinal necrosis. Alfalfa and Lupinus plants exhibited stunting, abnormally small leaves, and witches'-broom symptoms. Diseases in the plants were termed soybean veinal necrosis (SVN), alfalfa stunt (AlfS), and Lupinus stunt (LupS), respectively. The presence of phytoplasmas in diseased plants was assessed using polymerase chain reaction (PCR) for amplification of phytoplasma-specific 16S rDNA. A phytoplasma-characteristic 1.2-kbp DNA fragment was amplified from all diseased plants but not from known healthy plants in nested PCRs in which the first DNA amplification was primed by primer pair P1/P7 and reamplification of DNA was primed by primer pair F2n/R2 (2,4). Products from the nested PCR primed by F2n/R2 were subjected to restriction fragment length polymorphism (RFLP) analysis, and the RFLP patterns obtained were compared with patterns previously published (1–4). On the basis of AluI, HaeIII, HhaI, HpaI, KpnI, MseI, and RsaI RFLP patterns, the SVN and LupS phytoplasmas were classified in group 16SrIII (peach X-disease phytoplasma group), subgroup B (III-B, type strain clover yellow edge phytoplasma), and the AlfS phytoplasma was classified in group 16SrI (aster yellows phytoplasma group), subgroup B (I-B, type strain aster yellows phytoplasma). Nucleotide sequences were determined for 16S rDNA fragments amplified from SVN and AlfS phytoplasmas in nested PCRs primed by F2n/R2. The sequences were deposited in GenBank under Accession nos. AF177383 for SVN and AF177384 for AlfS. Sequence similarity between the 16S rDNAs of SVN and Canadian clover yellow edge (strain CYE-C, GenBank Accession no. AF175304) phytoplasmas was 99.8%; sequence similarity between 16S rDNAs of AlfS and aster yellows (strain SAY, GenBank Accession no. M86340) phytoplasmas was 99.6%. The SVN phytoplasma 16S rDNA shared 100% sequence similarity with a 16S rDNA from the Lithuanian clover yellow edge (CYE-L, GenBank Accession no. AF173558) phytoplasma. The nucleotide sequence data supported the conclusion that the SVN and AlfS phytoplasmas were closely related to strains classified in subgroups III-B and I-B, respectively. Our findings extend the known geographic ranges of phytoplasma subgroups I-B and III-B to northern Europe, including Lithuania, and expand the known plant host ranges of these pathogens. References: (1) R. E. Davis et al. Int. J. Syst. Bacteriol. 47:262, 1997. (2) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (3) R. Jomantiene et al. HortScience 33:1069, 1998. (4) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

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A Novel Geminivirus of Ipomoea indica (Convolvulacae) from Southern Spain

Plant Disease ◽

10.1094/pdis.1999.83.5.486b ◽

1999 ◽

Vol 83 (5) ◽

pp. 486-486 ◽

Cited By ~ 36

Author(s):

G. K. Banks ◽

I. D. Bedford ◽

F. J. Beitia ◽

E. Rodriguez-Cerezo ◽

P. G. Markham

Keyword(s):

Nucleotide Sequence ◽

Mosaic Virus ◽

Vegetative Propagation ◽

Sequence Data ◽

Sequence Similarity ◽

Yellow Vein ◽

Southern Spain ◽

Universal Primers ◽

Nucleotide Sequence Data ◽

Q Biotype

A cutting of Ipomoea indica displaying yellow vein symptoms was collected from Nerja in southern Spain in 1995, rooted, and maintained by vegetative propagation under glasshouse conditions at the John Innes Centre, Norwich. Although this member of the Convolvulaceae is native to the New World, it has escaped from cultivation as an ornamental and has now been naturalized in many tropical and warm temperate regions of the world, such as southern Spain. The same plant was found to host a population of whiteflies that were also brought back to containment facilities, and maintained in colony. Total plant DNA was extracted from the I. indica plant and universal primers for begomovirus A component (1) were used to amplify an approximately 2.8-kb fragment that was cloned and sequenced. The sequence is available in the DDJB, EMBL, and GenBank nucleotide sequence data bases under accession number AJ132548. A GENEMBL search with the complete sequence of the clone showed 70.8% identity to the AC1 gene of Ageratum yellow vein virus (AYVV). A search with the coat protein gene sequence showed highest homology to tomato leaf curl virus from southern India, another monopartite virus. Typical geminivirus vein yellowing symptoms, nucleotide sequence similarity, and EM detection of geminate virus particles strongly suggest that a geminivirus is present in this plant. The low level of homology to other sequenced geminiviruses suggests that it is an uncharacterized Begomovirus sp. With degenerate DNA-B primers (2), no B component has so far been detected. This virus is provisionally named Ipomoea yellow vein virus (IYVV). With techniques already established for identifying Bemisia spp. (3), the whiteflies collected with this Ipomoea plant were confirmed as Bemisia tabaci. Transmission studies to healthy I. indica showed that this whitefly population (named biotype S), the Q biotype from Spain, and the B biotype from Israel were all unable to transmit IYVV to healthy I. indica, tobacco, tomato, or nightshade. This may be due to many years of vegetative propagation of the host plant as an ornamental, resulting in loss of virus transmissibility by insects, which has occurred with Abutilon mosaic virus (AbMV) and honeysuckle yellow vein mosaic virus (HYVMV). This is the first report of a novel geminivirus on I. indica. It highlights the importance of weeds as hosts and potential reservoirs of both viruses and pests. We acknowledge support from the British Council, The Royal Society, BBSRC, and MAFF. References: (1) R.W. Briddon and P. G. Markham. Mol. Biotechnol. 1:202, 1994. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) R. C. Rosell et al. Ann. Entomol. Soc. Am. 90:575, 1997.

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