scholarly journals A Phytoplasma Closely Related to the Pigeon Pea Witches'-Broom Phytoplasma (16Sr IX) Is Associated with Citrus Huanglongbing Symptoms in the State of São Paulo, Brazil

2008 ◽  
Vol 98 (9) ◽  
pp. 977-984 ◽  
Author(s):  
D. C. Teixeira ◽  
N. A. Wulff ◽  
E. C. Martins ◽  
E. W. Kitajima ◽  
R. Bassanezi ◽  
...  
Keyword(s):  
16S Rdna ◽  
External Source ◽  
Pigeon Pea ◽  
Sao Paulo ◽  
Rdna Sequence ◽  
Universal Primers ◽  
Insect Vector ◽  
São Paulo ◽  
16S Rdna Sequence ◽  
Pcr Products

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of São Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and ‘Candidatus Liberibacter asiaticus’. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.

scholarly journals A Sida sp. Is a New Host for “Candidatus Phytoplasma brasiliense” in Brazil

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 363-363 ◽  
Author(s):  
B. Eckstein ◽  
J. C. Barbosa ◽  
J. A. M. Rezende ◽  
I. P. Bedendo
Keyword(s):  
16S Rdna ◽  
Sequence Similarity ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Sao Paulo ◽  
São Paulo ◽  
New Host ◽  
Nucleotide Sequence Alignment ◽  
Pcr Products

Sida is a genus of flowering herbs in the family Malvaceae, which includes several species that are weeds in Brazil. Plants of a Sida sp. exhibiting symptoms characterized by stunting, chlorosis, small leaves, and witches'-broom, indicative of infection by phytoplasmas, were found in a field previously cultivated with tomato, located in the region of Campinas, State of São Paulo, in December 2008. To demonstrate the presence of phytoplasmas in diseased tissues, DNA was extracted from shoots and leaves from three symptomatic and eight asymptomatic plants. Nested PCR was performed using primers P1/Tint followed by primer pair R16F2n/R16R2 (1). DNA fragments of 1.2 kb, corresponding to 16S rDNA, were amplified only for DNA from two symptomatic samples. Phytoplasma identification was initially carried out by restriction fragment length polymorphism (RFLP) analysis through digesting the PCR products with the restriction enzymes AluI, HhaI, HaeIII, HpaII, MseI, and RsaI. The two phytoplasma isolates found to be infecting a Sida sp. showed identical RFLP patterns, which were indistinguishable from the phytoplasma previously reported in association with hibiscus (Hibiscus rosa-sinensis) witches'-broom in Brazil (2). Nucleotide sequence alignment revealed that 16S rDNA of both phytoplasma isolates found in a Sida sp. (GenBank Accession No. HQ230579) shared 99.9% sequence similarity with 16S rDNA from hibiscus witches'-broom phytoplasma (HibWB) (GenBank Accession No. AF147708). HibWB is the representative of the 16SrXV group and it was proposed as a putative species nominated “Candidatus Phytoplasma brasiliense” (2). The disease is frequently observed in hibiscus plants used as ornamentals in the states of São Paulo (4) and Rio de Janeiro (2). “Ca. Phytoplasma brasiliense” has only been reported in Brazil to be infecting hibiscus (2,4) and periwinkle (Catharanthus roseus) (3). The presence of a phytoplasma belonging to group 16SrXV in a Sida sp. expands its natural host range. The role of this weed as a potential source of inoculum for crops should be investigated. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) H. G. Montano et al. Int. J. Syst. Evol. Microbiol. 51:1109, 2001. (3) H. G. Montano et al. Plant Dis. 85:1209, 2001. (4) E. G. Silva et al. Summa Phytopathol. 35:234, 2009.

scholarly journals First Report of Group 16SrXII Phytoplasma Causing Stolbur Disease in Potato Plants in the Eastern and Southern Anatolia Regions of Turkey

Plant Disease ◽  
2010 ◽  
Vol 94 (11) ◽  
pp. 1374-1374 ◽  
Author(s):  
S. Eroglu ◽  
H. Ozbek ◽  
F. Sahin
Keyword(s):  
16S Rdna ◽  
Rdna Sequence ◽  
Universal Primers ◽  
Effective Control ◽  
Diseased Plant ◽  
Eastern Anatolia ◽  
Universal Primer ◽  
16S Rdna Sequence ◽  
First Report ◽  
Potato Plants

In recent years, a stolbur-like disease has had devastating effects on the yield and marketable quality of potato production in Erzurum (Eastern Anatolia) and Akcakale-Sanliurfa (Southern Anatolia) regions of Turkey. Potato plants exhibited several different symptoms including stunting, upward rolling of the top leaves along with reddish or purplish coloration, chlorosis, shortened internodes, swollen nodes, proliferated axillary buds, aerial tubers, and early plant decline. An extensive survey from 2003 to 2010 was performed and diseased plant samples were collected. Total genomic DNAs were isolated from the leaf mid-veins of the six different symptomatic and two symptomless plants selected. Nested-PCRs, carried out by using phytoplasma-universal primer pair P1/P7 followed by R16F2n/R16R2 (2), amplified 16S rDNA fragments (F2nR2) from only templates derived from symptomatic plants. F2nR2 PCR products from two independent symptomatic plants were cloned and sequenced from both directions with M13 universal primers. The obtained 16S rDNA sequence (GenBank Accession No HM485579) was subjected to virtual restriction fragment length polymorphism (RFLP) analysis using iphyclassifier software (3). Results indicated that the phytoplasma, here identified in association with potato plants, shared best sequence identity (99%) with members of subgroup 16SrXII-A (e.g., GenBank Accession No. EU010006). Moreover, collective RFLP pattern of potato-associated phytoplasma differed from digestion profiles of previously described 16SrXII subgroups, sharing best similarity coefficient (0.94) with the reference phytoplasma strain of subgroup 16SrXII-A (GenBank Accession No. AJ964960). Thus, it was confirmed that potato-associated phytoplasma represents a new 16SrXII subgroup (16SrXII-N). Furthermore, a new primer set (PatsecF/PatsecR) was designed for priming specific PCR-amplification of potato-associated phytoplasma 16S rDNA sequence. PCR reaction was successfully used for specifically detecting stolbur phytoplasma in infected potato plants. The use of this method may help to determine possible alternative hosts and vectors of potato phytoplasma, which is important for development of an integrated management strategy for effective control of this disease in the future. Presence of potato stolbur diseases in the Eastern Anatolia Region of Turkey has previously been reported (1). To our knowledge, this is the first report of occurrence of a 16SrXII group phytoplasma causing potato stolbur diseases caused in the Eastern and Southern Anatolia regions of Turkey. References: (1) A. Citir. J. Turk. Phytopathol. 14:53, 1985. (2) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3)Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

scholarly journals First Report of Herbaspirillum rubrisubalbicans Causing Mottled Stripe Disease on Sugarcane in China

Plant Disease ◽  
2010 ◽  
Vol 94 (3) ◽  
pp. 379-379 ◽  
Author(s):  
ZQ. Tan ◽  
R. Men ◽  
RY. Zhang ◽  
Z. Huang
Keyword(s):  
16S Rdna ◽  
Uv Light ◽  
Rdna Sequence ◽  
Universal Primers ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Potato Dextrose Agar Medium ◽  
16S Rdna Sequence ◽  
First Report ◽  
Herbaspirillum Rubrisubalbicans

Narrow, red stripes were observed on leaves and sheaths of sugarcane in 2007 in DanZhou County of Hainan Province and XuWen County of GuangDong Province, China. Stripes were parallel to the leaf veins. Some stripes were short (2 to 10 cm) and some were >1 m long, extending from the base of leaves. Width of the stripes was 2 to 4 mm. Symptoms varied with the cultivar. Cv. Taiwang 25, which was the most affected, exhibited red stripes and stalk death from the apex. Cvs. Taiwang 26 and Guang Dong 00236 were slightly affected with only red stripes. Symptoms on cv. Taiwang 22 were mottled stripes. Severe losses were observed in the infected fields that were planted with cv. Taiwang 25, but there were no obvious losses in fields planted with the other three cultivars. Isolations were made from 10 individual plants from different cultivars and provinces that had red stripes, two of which also had apex death. Five independent bacterial isolates were obtained from tissue showing the red stripe symptoms on potato dextrose agar medium. The percentage of positive samples was 50%. No bacteria were obtained from necrotic apex tissue. Bacterial cells were 0.92 to 1.55 × 0.20 to 0.22 μm slightly curved rods that were motile with one to two polar flagella. Colonies on nutrient agar were 2 to 3 mm in diameter, circular, smooth, entire, and milky white. Colonies on King's medium B were nonfluorescent under 365-nm UV light. Five bacterial strains were inoculated by injecting bacterial suspensions (1 × 108 CFU/ml) into the base of the leaves of 6-month-old cv. Taiwang 25 plants (1). Red stripes appeared 7 to 10 days after inoculation and bacteria were reisolated. The reisolated bacteria were identical to the original strains in colony morphology and 16S rDNA sequence. A hypersensitive response appeared within 24 h when 1 × 108 CFU/ml bacteria suspensions were infiltrated into tobacco leaves. Approximately 1,000-bp DNA fragments were amplified with universal primers UP1 (5′-TACGTGCCAGCAGCCGCGGTAATA-3′) and UP2 (5′-AGTAAGGAGGGTATCCAACCGCA-3′) (3). Primers UP1 and UP2 are complementary to nucleotide sequence 509 to 523 and 1541 to 1522, respectively, of the Escherichia coli 16S rDNA gene. The fragment amplified by these primers was approximately 1,032 bp. The 16S rDNA sequences of the five strains were deposited in GenBank as Accession Nos. GQ476791–5. They all shared 99% nucleotide identity with the type strain of Herbaspirillum rubrisubalbicans (GenBank No. AJ238356.1). All five strains were identified as H. rubrisubalbicans on the basis of 16S rDNA sequence and pathogenicity to sugarcane, and the disease was identified as mottled stripe disease (2). Since we were not able to isolate bacteria from necrotic apex tissue, this symptom on cv. Taiwang 25 may not be related to the H. rubrisubalbicans infection. To our knowledge, this is the first report of mottled stripe disease in China. References: (1) H. M. A. EI-Komy et al. Folia Microbiol. 48:787, 2003. (2) A. S. Saumtally et al. A Guide to Sugarcane Diseases. P. Rott et al., eds. CIRAD and ISSCT, Montpellier, France, 2000. (3) Yan Zhi Yong et al. Chin. J. Epidemiol. 24:296, 2003.

scholarly journals First Report of Syringa oblata and S. reticulata Leafroll Disease in China

Plant Disease ◽  
2009 ◽  
Vol 93 (3) ◽  
pp. 322-322
Author(s):  
Z. N. Li ◽  
H. Min ◽  
Y. Yan ◽  
Z. Zhao ◽  
W. J. Wu ◽  
...  
Keyword(s):  
16S Rdna ◽  
Rflp Analysis ◽  
Universal Primers ◽  
University Campus ◽  
Sequence Comparisons ◽  
16S Rdna Sequence ◽  
First Report ◽  
Pcr Products ◽  
Leafroll Disease ◽  
Total Dna

Syringa oblata is an important ornamental tree widely grown in China. In September of 2008, S. oblata plants exhibiting symptoms of leafroll and yellowing were found in a garden on the Northwest A&F University campus. Samples were collected from this site. Total DNA was extracted from 0.5 g of phloem tissue from leaf midribs and stems of each sample. DNA samples were analyzed with a nested PCR assay using phytoplasma 16S rDNA universal primers R16mF2/R16mR1 followed by specific primers R16F2n/R16R2 (1), which amplified a 1,452- and 1,246-bp product, respectively. We tested all 30 lilac samples, 20 of which had symptoms of leafroll and yellowing. These produced the expected 1,452- and 1,246-bp PCR products In contrast, the remaining 10 samples from symptomless trees yielded no PCR products. We also surveyed another lilac variety (Syringa reticulata), which is widely grown on the campus, and tested 50 samples with the above method. Again, 1.4- and 1.2-kb PCR products were amplified from all 30 trees displaying leafroll and yellowing symptoms, but not from the other 20 samples from symptomless trees. A comparative analysis of sequences derived from the two hosts showed that the phytoplasmas infecting them were most similar (>99%) to paulownia witches'-broom (PaWB) phytoplasma (GenBank Accession No. EF199937). Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with endonucleases AluI and MseI indicated that all symptomatic plants were infected by the phytoplasmas belonging to aster yellow group (16SrI) subgroup D (16SrI-D) PaWB phytoplasma (2). 16S rDNA sequence comparisons and RFLP analysis of the cloned 16S rDNA from S. oblata (GenBank Accession No. FJ445224) and S. reticulate (GenBank Accession No. FJ445225) indicated that the phytoplasmas infecting them were nearly identical (99.8% identity). To our knowledge, this is the first report of the presence of the phytoplasma associated with a leafroll disease of S. oblata and S. reticulata in China. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

scholarly journals First Report of a Huanglongbing-Like Disease of Citrus in Sao Paulo State, Brazil and Association of a New Liberibacter Species, “Candidatus Liberibacter americanus”, with the Disease

Plant Disease ◽  
2005 ◽  
Vol 89 (1) ◽  
pp. 107-107 ◽  
Author(s):  
D. C. Texeira ◽  
J. Ayres ◽  
E. W. Kitajima ◽  
L. Danet ◽  
S. Jagoueix-Eveillard ◽  
...  
Keyword(s):  
New Species ◽  
16S Rdna ◽  
Sweet Orange ◽  
Sieve Tube ◽  
Sao Paulo ◽  
Rdna Sequence ◽  
16S Rdna Sequence ◽  
Paulo State ◽  
Candidatus Liberibacter ◽  
Symptomatic Leaf

Huanglongbing (HLB) (ex-greening) is one of the most serious diseases of citrus. The causal agent is a noncultured, sieve tube-restricted α-proteobacterium, “Candidatus Liberibacter africanus” in Africa and “Candidatus Liberibacter asiaticus” in Asia (2). The disease has never been reported from the American continent. However, Diaphorina citri, the Asian psyllid vector of HLB, is found in South, Central, and North America (Florida and Texas). Early in 2004, leaf and fruit symptoms resembling those of HLB were observed in several sweet orange orchards near the city of Araraquara, Sao Paulo State. Leaf mottling on small and large leaves was the major symptom. Shoots with affected leaves were yellowish. Fruits were small and lopsided, contained many aborted seeds, and appeared more severely affected than were plants infected with classic HLB. Forty-three symptomatic samples and twenty-five samples of symptomless sweet orange leaves from five farms were analyzed for the presence of the HLB-liberibacters using polymerase chain reaction (PCR) with two sets of HLB-specific primers for amplification of 16S rDNA (2,3) and ribosomal protein genes (1). None of the 43 symptomatic leaf samples gave a positive PCR amplification, while HLB-affected leaves from the Bordeaux HLB collection produced the characteristic amplicons with both sets of primers. The 43 symptomatic and the 25 symptomless leaf samples were then analyzed using PCR with universal primers for amplification of bacterial 16S rDNA (4). All symptomatic leaf samples, but none of the symptomless leaf samples, yielded the same 16S rDNA amplification product, indicating the presence of a bacterium in the symptomatic leaves. This was confirmed using the observation of a sieve tube restricted bacterium by electron microscopy. The 16S rDNA product was cloned, sequenced, and compared with those of “Ca. L. africanus” and “Ca. L. asiaticus”. While the 16S rDNAs of these two liberibacter species have 97.5% sequence identity, the 16S rDNA sequence of the new bacterium shared only 93.7% identity with that of “Ca. L. asiaticus” and 93.9% with that of “Ca. L. africanus”. The 16S rDNA sequence of the new bacterium had a secondary loop structure characteristic of the α subdivision of the proteobacteria and possessed all the oligonucleotide signatures characteristic of the liberibacters. For these reasons, the new bacterium is a liberibacter and is sufficiently different phylogenetically from known liberibacters to warrant a new species, “Candidatus Liberibacter americanus”. Specific PCR primers for amplification of the 16S rDNA of the new species have been developed. They were able to detect “Ca. L. americanus” in 214 symptomatic leaf samples from 47 citrus farms in 35 municipalities, while the “old” species, “Ca. L. asiaticus”, has been found only four times within the 47 farms. References: (1) A. Hocquellet et al. Mol. Cell. Probes, 13:373, 1999. (2) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

16S rDNA Sequence based Phylogenetic Analysis of Some Bacterial Phytoplasma

2012 ◽  
Vol 3 (3) ◽  
pp. 302-304
Author(s):  
G. D.Sharma G. D.Sharma ◽  
◽  
* Dhritiman Chanda ◽  
D.K. Jha D.K. Jha
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rdna ◽  
Rdna Sequence ◽  
16S Rdna Sequence

scholarly journals Erratum to: Deodorization of pig slurry and characterization of bacterial diversity using 16S rDNA sequence analysis

2014 ◽  
Vol 52 (12) ◽  
pp. 1056-1056
Author(s):  
Ok-Hwa Hwang ◽  
Sebastian Raveendar ◽  
Young-Ju Kim ◽  
Ji-Hun Kim ◽  
Tae-Hun Kim ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Bacterial Diversity ◽  
16S Rdna ◽  
Rdna Sequence ◽  
Pig Slurry ◽  
16S Rdna Sequence ◽  
16S Rdna Sequence Analysis

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

Plant Disease ◽  
2001 ◽  
Vol 85 (4) ◽  
pp. 447-447 ◽  
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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