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

Plant Disease ◽  
2000 ◽  
Vol 84 (2) ◽  
pp. 198-198 ◽  
Author(s):  
R. Jomantiene ◽  
R. E. Davis ◽  
L. Antoniuk ◽  
J. Staniulis

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.

Plant Disease ◽  
1999 ◽  
Vol 83 (5) ◽  
pp. 488-488 ◽  
Author(s):  
R. D. Peters ◽  
M. E. Lee ◽  
C. R. Grau ◽  
S. J. Driscoll ◽  
R. M. Winberg ◽  
...  

Samples of alfalfa (Medicago sativa L.) leaves and stems showing symptoms of inter-veinal chlorosis and purpling, commonly associated with insect feeding, were collected from 8 sites in central and southern Wisconsin in autumn of 1998. Samples were frozen within 24 h of collection. Approximately 0.3 g of plant tissue from each sample was used for total DNA extraction according to the protocol of Zhang et al. (4), with minor modifications in grinding procedures and reagent volumes to optimize results. Nested polymerase chain reaction (PCR) was carried out by amplification of 16S rDNA with the universal primer pairs R16mF2/R16mR1 followed by R16F2n/R16R2 as described by Gunder-sen and Lee (1). Undiluted total sample DNA was used for the first amplification; PCR products were diluted (1:30) in sterile water prior to final amplification. Alfalfa DNA and sterile water were used as negative controls; DNA from phytoplasma causing X-disease in peach (CX) served as a positive control. Fragments of 16S rDNA from putative phytoplasmas amplified by PCR with the primer pair R16F2n/R16R2 were characterized by restriction endonuclease digestion (3). The resulting restriction fragment length polymorphism (RFLP) patterns were compared with patterns for known phytoplasmas described by Lee et al. (3). Products of nested PCR were also purified and sequenced with primers R16F2n/R16R2 and an automated DNA sequencer (ABI 377XL; C. Nicolet, Biotechnology Center, University of Wisconsin-Madison). Of 51 samples of alfalfa assessed, one sample from Evansville, WI, yielded a nested PCR product of the appropriate size (1.2 kb), indicating the presence of phytoplasma. Digestion of this product with various restriction enzymes produced RFLP patterns that were identical to those for phytoplasmas in the aster yellows phytoplasma subgroup 16SrI-A (3). Alignment of the DNA sequence of the nested PCR product from the positive sample with sequences found in the GenBank sequence data base (National Center for Biotechnology Information, Bethesda, MD) with the BLAST sequence similarity function confirmed this result. Although other phytoplasma strains (particularly those causing witches'-broom) have been reported to infect alfalfa (2), this is the first report of the presence of the aster yellows phytoplasma in the alfalfa crop. Vectors involved in transmission and the potential agronomic impacts of aster yellows phytoplasma in alfalfa are topics of current investigation. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) A.-H. Khadhair et al. Microbiol. Res. 152:269, 1997. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) Y.-P. Zhang et al. J. Virol. Methods 71:45, 1998.


Plant Disease ◽  
2001 ◽  
Vol 85 (10) ◽  
pp. 1121-1121 ◽  
Author(s):  
R. E. Davis ◽  
E. L. Dally ◽  
R. H. Converse

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.


Plant Disease ◽  
2002 ◽  
Vol 86 (10) ◽  
pp. 1177-1177 ◽  
Author(s):  
A. Alminaite ◽  
R. E. Davis ◽  
D. Valiunas ◽  
R. Jomantiene

Symptoms of phyllody of flowers and general plant yellowing indicating possible phytoplasma infection were observed in diseased plants of hairy willow-weed (Epilobium hirsutum L., family Onagraceae) growing in a meadow at Harku Village near Tallin, Estonia. DNA was extracted from diseased E. hirsutum using a Genomic DNA Purification Kit (Fermentas AB, Vilnius, Lithuania) and used as a template in nested polymerase chain reaction (PCR). Ribosomal (r) DNA was initially amplified in PCR primed by phytoplasma universal primer pair P1/P7 (4) and reamplified in PCR primed by nested primer pair 16SF2n/16SR2 (F2n/R2) (1) as previously described (2). Products of 1.8 kbp and 1.2 kbp were obtained in PCR primed P1/P7 and F2n/R2, respectively, from all four symptomatic plants examined. These data indicated that the diseased E. hirsutum plants were infected by a phytoplasma, termed epilobium phyllody (EpPh) phytoplasma. The 16S rDNA amplified in PCR primed by nested primer pair F2n/R2 was subjected to restriction fragment length polymorphism (RFLP) analysis using restriction endonucleases AluI, MseI, HpaI, HpaII, HhaI, RsaI, HinfI, and HaeIII (Fermentas AB). On the basis of the collective RFLP profiles, EpPh phytoplasma was classified in group 16SrI (aster yellows phytoplasma group), subgroup B (aster yellows phytoplasma subgroup), according to the phytoplasma classification scheme of Lee et al. (3). The 1.8-kbp rDNA product of P1/P7-primed PCR, which included 16S rDNA, 16S-23S intergenic spacer region, and the 5′ -end of 23S rDNA, was cloned in Escherichia coli using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, Ca) according to manufacturer's instructions and sequenced. The sequence was deposited in the GenBank database as Accession No. AY101386. This nucleotide sequence shared 99.8% sequence similarity with a comparable rDNA sequence (GenBank Accession No. AF322644) of aster yellows phytoplasma AY1, a known subgroup 16SrI-B strain. The EpPh phytoplasma sequence was highly similar (99.9%) to operons rrnA (GenBank Accession No. AY102274) and rrnB (GenBank Accession No. AY102273) from Valeriana yellows (ValY) phytoplasma infecting Valeriana officinalis plants in Lithuania. ValY phytoplasma was found to exhibit rRNA interoperon sequence heterogeneity (D. Valiunas, unpublished data). To our knowledge, this is the first report to reveal E. hirsutum as a host of phytoplasma and to demonstrate the occurrence of a plant pathogenic mollicute in the northern Baltic region. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) R. Jomantiene et al. HortScience 33:1069, 1998. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) B. Schneider et al. Phlogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. Page 369 in: Molecular and Diagnostic Procedures in Mycoplasmology, Vol 1, R. Razin, and J. G. Tully eds. Academic Press, San Diego, 1995.


Plant Disease ◽  
2001 ◽  
Vol 85 (7) ◽  
pp. 804-804 ◽  
Author(s):  
D. Valiunas ◽  
A. Alminaite ◽  
J. Staniulis ◽  
R. Jomantiene ◽  
R. E. Davis

Phytoplasma strains that belong to group 16SrI (aster yellows phytoplasma group), subgroup A (I-A, North American tomato big bud phytoplasma subgroup) were discovered in diverse plant species in Lithuania. Plants in which the strains were found exhibited symptoms characteristic of infections by phytoplasma. Carrot (Daucus sativus) with carrot proliferation disease exhibited symptoms of proliferation of the crown, chlorosis of young leaves, and reddening of mature leaves. Diseased phlox (Phlox paniculata) exhibited symptoms of virescence and leaf chlorosis. Diseased sea-lavender (Limonium sinuatum) exhibited abnormal proliferation of shoots, chlorosis of young leaves, reddening of mature leaves, and degeneration of flowers. Diseased hyacinth (Hyacinthus orientalis) exhibited chlorosis of leaves and undeveloped flowers. Diseased Aconitum sp. exhibited proliferation of shoots. Phytoplasma-characteristic ribosomal (r) DNA was detected in the plants by use of the polymerase chain reaction (PCR). The rDNA was amplified in PCR primed by primer pair P1/P7 and reamplified in nested PCR primed by primer pair R16F2n/R16R2 (F2n/R2), as previously described (1). The phytoplasmas were classified through restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, amplified in the nested PCR primed by F2n/R2, using single endonuclease enzyme digestion with AluI, MseI, KpnI, HhaI, HaeIII, HpaI, HpaII, RsaI, HinfI, TaqI, and Sau3AI. Collective RFLP patterns indicated that all detected phytoplasma strains were affiliated with subgroup I-A. The 16S rDNA amplified from the phytoplasma (CarrP phytoplasma) in diseased carrot was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank data library (GenBank accession no. AF291682). The 16S rDNAs of CarrP and tomato big bud (GenBank acc. no. AF222064) phytoplasmas shared 99.8% nucleotide sequence similarity. Phytoplasmas belonging to group 16SrIII (3), group 16SrV (D. Valiunas, unpublished data), and subgroup I-C in group 16SrI (2,3) occur in Lithuania. This report records the first finding of a subgroup I-A phytoplasma in the Baltic region and expands the known plant host range of this phytoplasma subgroup. References: (1) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (2) Jomantiene et al. Phytopathology 90:S39, 2000. (3) Staniulis et al. Plant Dis. 84:1061, 2000.


Plant Disease ◽  
2001 ◽  
Vol 85 (4) ◽  
pp. 447-447 ◽  
Author(s):  
I.-M. Lee ◽  
R. A. Dane ◽  
M. C. Black ◽  
Noel Troxclair

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.


2001 ◽  
Vol 2 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Wolfgang Kraatz ◽  
Ulf Thunberg ◽  
Bertil Pettersson ◽  
Claes Fellström

AbstractDNA was extracted from colonic biopsies of 33 patients with and three without evidence of intestinal spirochetosis (IS) in the large bowel. The biopsies were subjected to PCR. A pair of primers, generating a 207 bp fragment, were designed to detect specifically the 16S rDNA gene ofBrachyspira. PCR products of the expected size were obtained from 33 samples with histologic evidence of IS. The PCR amplicons were used for sequencing. The sequences obtained were aligned to the corresponding 16S rRNA sequences of five type strains ofBrachyspira. The sequences of 23 PCR products were 99–100% identical with the correspond-ingB.aalborgitype strain sequence. Two cases showed 99–100% sequence similarity with the type strain ofB.pilosicoliP43/6/78. Six cases could not be referred to any of the known species ofBrachyspira. Two PCR products gave incomplete sequences.


2011 ◽  
Vol 61 (6) ◽  
pp. 1360-1369 ◽  
Author(s):  
Arvind Kumar Gupta ◽  
Mahesh Shantappa Dharne ◽  
Ashraf Yusuf Rangrez ◽  
Pankaj Verma ◽  
Hemant V. Ghate ◽  
...  

Two Gram-negative-staining, aerobic, non-motile, rod-shaped bacteria, designated strains FFA1T and FFA3T, and belonging to the class Gammaproteobacteria were isolated from the gastrointestinal tract of adult flesh flies (Diptera: Sarcophagidae). Phylogenetic analysis of 16S rRNA gene sequence data placed these two strains within the genus Ignatzschineria with similarities of 98.6 % (FFA1T) and 99.35 % (FFA3T) to Ignatzschineria larvae L1/68T. The level of gene sequence similarity between strains FFA1T and FFA3T was 99 %, 97.15 % and 78.1 % based on the 16S rRNA, 23S rRNA and gyrB gene sequences, respectively. Strains FFA1T and FFA3T shared 24 % DNA–DNA relatedness. DNA–DNA hybridization revealed a very low level of relatedness between the novel strains (22 % for strain FFA1T and 44 % for strain FFA3T) and I. larvae L1/68T genomic DNA. The respiratory quinone was Q-8 in both novel strains. The DNA G+C contents were 41.1 mol% and 40.1 mol% for strains FFA1T and FFA3T, respectively. The cell membrane of both strains consisted of phosphatidylglycerol, phosphatidylethanolamine, phospholipids and aminophospholipid. The major fatty acids for both strains were C16 : 0, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), CyC19 : 0ω8c and C14 : 0. The results of DNA–DNA hybridization between the two new strains and I. larvae L1/68T, in combination with phylogenetic, chemotaxonomic, biochemical and electron microscopic data, demonstrated that strains FFA1T and FFA3T represented two novel species of the genus Ignatzschineria for which the names Ignatzschineria indica sp. nov. (type strain FFA1T = DSM 22309T = KCTC 22643T = NCIM 5325T) and Ignatzschineria ureiclastica sp. nov. (type strain FFA3T = DSM 22310T = KCTC 22644T = NCIM 5326T) are proposed.


2007 ◽  
Vol 21 (6) ◽  
pp. 569 ◽  
Author(s):  
Mark B. Schultz ◽  
Sarah A. Smith ◽  
Alastair M. M. Richardson ◽  
Pierre Horwitz ◽  
Keith A. Crandall ◽  
...  

Nucleotide sequence data from the mitochondrial 16S rDNA region were utilised to investigate phylogenetic relationships and species boundaries among Australian freshwater crayfish belonging to the genera Engaeus Erichson, 1846, Geocharax Clark, 1936 and Gramastacus Riek, 1972. Geocharax and Gramastacus were found to be monophyletic genera but one species currently assigned to Engaeus may belong to another genus. Relationships between the three existing genera were not resolved. Analysis of species boundaries within Geocharax suggests that there are an additional two species in this genus, and our analysis of Gramastacus indicates that undescribed populations from central New South Wales may comprise a second species. The data provide at least one instance of a taxon crossing the Great Dividing Range and provide confirmation of previously proposed hypotheses seeking to explain trans-Bass Strait distributions of species.


2004 ◽  
Vol 54 (3) ◽  
pp. 819-825 ◽  
Author(s):  
Ying Li ◽  
Yoshiaki Kawamura ◽  
Nagatoshi Fujiwara ◽  
Takashi Naka ◽  
Hongsheng Liu ◽  
...  

On the basis of phenotypic and genotypic characteristics and 16S rRNA gene sequence analysis, novel species belonging to the genera Sphingomonas and Brevundimonas were identified from samples taken from the Russian space laboratory Mir. Strain A1-18T was isolated from the air. 16S rDNA sequence analysis showed that strain A1-18T formed a coherent cluster with Sphingomonas sanguinis, Sphingomonas parapaucimobilis, Sphingomonas paucimobilis and Sphingomonas roseiflava with sequence similarity of 97·5–98·6 %. Similar to other Sphingomonas species, the G+C content was 66·1 mol%, but DNA–DNA hybridization rates at optimal temperatures among these related species were only 24·7–51·7 %. Strain A1-18T can be differentiated biochemically from related species. Strain W1-2BT was isolated from condensation water. It forms a distinct lineage within the genus Brevundimonas, forming a coherent cluster with Brevundimonas vesicularis, Brevundimonas aurantiaca and Brevundimonas intermedia. 16S rDNA sequence similarities were 98·6–99·5 % and the G+C content was 66·5 mol%, similar to other Brevundimonas species, but DNA–DNA relatedness was only 50·2–54·8 %. Strain W1-2BT also showed some differential biochemical properties from its related species. A series of polyphasic taxonomic studies led to the proposal of two novel species, Sphingomonas yabuuchiae sp. nov. (type strain A1-18T=GTC 868T=JCM 11416T=DSM 14562T) and Brevundimonas nasdae sp. nov. (type strain W1-2BT=GTC 1043T=JCM 11415T=DSM 14572T).


2004 ◽  
Vol 54 (4) ◽  
pp. 1025-1029 ◽  
Author(s):  
C. Marcone ◽  
K. S. Gibb ◽  
C. Streten ◽  
B. Schneider

Spartium witches'-broom (SpaWB), buckthorn witches'-broom (BWB) and allocasuarina yellows (AlloY) are witches'-broom and yellows diseases of Spartium junceum (Spanish broom), Rhamnus catharticus (buckthorn) and Allocasuarina muelleriana (Slaty she-oak), respectively. These diseases are associated with distinct phytoplasmas. The SpaWB, BWB and AlloY phytoplasmas share <97·5 % 16S rDNA sequence similarity with each other and with other known phytoplasmas, including the closely related phytoplasmas of the apple proliferation group. Also, the SpaWB, BWB and AlloY phytoplasmas each have a different natural plant host. Based on their unique properties, it is proposed to designate the mentioned phytoplasmas as novel ‘Candidatus’ species under the names ‘Candidatus Phytoplasma spartii’, ‘Candidatus Phytoplasma rhamni’ and ‘Candidatus Phytoplasma allocasuarinae’, respectively.


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