scholarly journals First Report of a Group 16SrVII-C Phytoplasma Associated with Shoot Proliferation of Sunn Hemp (Crotalaria juncea) in Brazil

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1652-1652 ◽  
Author(s):  
D. Flôres ◽  
A. P. O. Amaral Mello ◽  
N. S. Massola Junior ◽  
I. P. Bedendo
Keyword(s):  
16S Rdna ◽  
Consensus Sequence ◽  
Shoot Proliferation ◽  
Rflp Analysis ◽  
Vector System ◽  
Electron Microscopic ◽  
New Host ◽  
Crotalaria Juncea ◽  
Transmission Electron ◽  
Sunn Hemp

Sunn hemp (Crotalaria juncea) is widely grown in tropical and subtropical regions. In Brazil, this species is commonly used for green manure, since this legume is an efficient nitrogen fixer that produces organic residues for soil improvement. In July of 2012, C. juncea exhibiting intense shoot proliferation, leaf malformation, shortened internodes, and generalized yellowing were found in an experimental field located in Piracicaba, State of São Paulo, Brazil. The incidence was about 1 to 2% and the diseased plants were distributed at random. Since these symptoms are indicative of infection by phytoplasmas, the present study aimed to detect and identify the phytoplasma. Four symptomatic and two asymptomatic plants were sampled. Small segments of leaf veins were prepared for microscopy, as previously reported (1), and observations were made using a Jeol (Akishima/Japan) model Jem-1011 transmission electron microscope. Total DNA was extracted from leaves using a commercial kit (DNeasy Plant Mini, Qiagen Inc.), and nested PCR assays were performed with primers, P1/Tint followed by R16F2n/R16R2 (2). The initial assumption that disease symptoms were associated with phytoplasma was confirmed by PCR amplification of 1.2 kb DNA fragments from the 16S rDNA gene. In contrast, no amplicon was generated with PCR using template DNA from asymptomatic plants. The phytoplasma detected from each symptomatic sample was considered to be an isolate. PCR products were purified and cloned in Escherichia coli DH5α, using the pGEM-T Easy Vector System I (Promega). Three isolates were selected and the cloned 16S rDNA sequences from three colonies of each isolate were sequenced. Since no sequence polymorphisms were found, a majority consensus sequence was selected for each isolate. These sequences were identical and one of them, designated CrSP-Br01 (crotalaria shoot proliferation) with 1,249 bp (GenBank Accession KC756947), was used as representative of the sunn hemp phytoplasma. The 16S rDNA nucleotide sequence of this phytoplasma shared 100% sequence identity with the reference phytoplasma for subgroup VII-C (Argentinian Alfalfa witches'-broom phytoplasma, AY147038). According to the in silico RFLP analysis for delineation of subgroups (3), which is based on virtual RFLP patterns and similarity coefficient calculation, the C. juncea phytoplasma was classified as a member of group 16SrVII, subgroup C. Phylogenetic analysis supported that this phytoplasma is closely related to the representative of subgroup 16SrVII-C, since both phytoplasmas emerged from the same branch. Transmission electron microscopic examination revealed the presence of phytoplasmas by visualization of pleomorphic and round bodies 100 to 400 nm in diameter, in the phloem vessels of symptomatic plants. The present study reports the first occurrence of a 16SrVII-C phytoplasma in Brazil. In addition, C. juncea was identified as a new host for phytoplasmas belonging to this subgroup. References: (1) A. B. Maunsbach and B. A. Afzelius. Biomedical Electron Microscopy. Illustrated Methods and Interpretations. Page 381-426, San Diego, Academic Press, 1999. (2) M. C. C. Rappussi et al. Eur. J. Plant Pathol. 133:829, 2012. (3) W. Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007.

scholarly journals First Report of Phytoplasmas Groups 16SrI and 16SrXV in Crotalaria juncea in Brazil

Plant Disease ◽  
2014 ◽  
Vol 98 (7) ◽  
pp. 990-990 ◽  
Author(s):  
L. F. Bianco ◽  
E. C. Martins ◽  
R. S. Toloy ◽  
D. A. B. Coletti ◽  
D. C. Teixeira ◽  
...  
Keyword(s):  
Shoot Proliferation ◽  
Rflp Analysis ◽  
New Host ◽  
Crotalaria Juncea ◽  
First Report ◽  
Tropical Plant ◽  
Leaf Yellowing ◽  
Pcr Products ◽  
Sunn Hemp ◽  
Low Incidence

Sunn hemp (Crotalaria juncea L., Fabaceae) is widely used as a cover crop in sugar cane and citrus plantations in Brazil. C. juncea has been reported in São Paulo State (SPS) by Wulff et al. (3) as a host of the phytoplasma associated with symptoms of huanglongbing (HLB) in citrus, a member of group 16SrIX, that induces witches'-broom in sunn hemp (3). In studying the distribution of group 16SrIX phytoplasma in C. juncea in SPS, we identified this species as a new host of two phytoplasmas. Sunn hemp fields were inspected for symptoms usually associated with phytoplasma infections, such as leaf yellowing, shoot proliferation, witches'-brooms, and virescence. Ninety-nine plant samples were collected and DNA was extracted with the CTAB protocol from stems. Nested PCR was carried out with primers P1/P7, followed by amplification with primers fU3/rU5 (2), both sets being universal for phytoplasma. Asymptomatic sunn hemp samples were used as negative controls and were negative in PCR reactions. PCR products were directly sequenced with primers P1/P7 and fU3/rU5 and phytoplasma identification was conducted with BLASTn and in silico RFLP analysis for delineation of subgroups (4). Plants showing leaf yellowing (three plants; Catanduva County), shoot proliferation (one plant; Ibirá County), or witches'-brooms (one plant; Promissão County) symptoms were found to be infected with the 16SrI phytoplasma group, subgroup S. The 16S rDNA sequence (GenBank Accession No. KF878383) showed 99% identity (E value 0.0) with Candidatus Phytoplasma asteris, Onion yellows phytoplasma OY-M (AP006628), Mulberry yellow dwarf phytoplasma (GQ249410), and Ash witches'-broom phytoplasma (AY566302), among other phytoplasmas from the same group. Sunn hemp plants with shoot proliferation (three plants) carried the 16SrXV phytoplasma group, subgroup A, found in Ibirá (two plants) and Catanduva (one plant) counties, SPS. This sequence (GenBank Accession No. KF878382) displayed 99% identity (E value 0.0) with Ca. P. brasiliense, Hibiscus witches'-broom phytoplasma (AF147708), Guazuma ulmifolia witches'-broom phytoplasma (HQ258882, HQ258883), and Cauliflower stunt phytoplasma (JN818845). Both phytoplasma groups described in this report, 16SrI and 16SrXV, were collected in May 2010 and both have limited geographic distribution and occurred at low incidence. Phytoplasma of group 16SrI (Ca. P. asteris) was identified in C. spectabilis in India (1). To our knowledge, this is the first report of phytoplasmas groups 16SrI and 16SrXV in sunn hemp. References: (1) S. Kumar et al. Plant Dis. 94:1265, 2010. (2) E. Seemüller et al. Int. J. Syst. Bacteriol. 44:440, 1994. (3) N. A. Wulff et al. Tropical Plant Pathol. 34:S7, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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 a 16SrI-C Group Phytoplasma Associated With a Yellows-Type Disease Affecting Willow Plants in China

Plant Disease ◽  
2009 ◽  
Vol 93 (2) ◽  
pp. 197-197 ◽  
Author(s):  
T. Wei ◽  
Y. F. Wu ◽  
K. K. Wu ◽  
W. Hou ◽  
Y. R. Li
Keyword(s):  
16S Rdna ◽  
Rflp Analysis ◽  
Northern China ◽  
Shaanxi Province ◽  
Nucleotide Sequence Analysis ◽  
Molecular Evidence ◽  
Rrna Gene ◽  
Transmission Electron ◽  
Ctab Method ◽  
Salix Babylonica

In May of 2008, a phytoplasma-like disease was observed on willows (Salix babylonica Linn) grown in the Shaanxi Province. Affected plants showed yellowed leaves with green veins and dieback. Incidence of the disease was less than 10%. Samples were collected from 10 symptomatic and five asymptomatic willow plants from five different areas in Shaanxi Province. Total DNA was extracted from 0.5 g of leaf midrib and stem phloem tissue with a modified cetyltrimethylammoniumbromide (CTAB) method (3). Resulting DNA extracts were analyzed by a nested PCR assay using phytoplasma 16S rRNA gene primer pairs R16mF2/R16mR1 followed by R16F2n/R16R2 (1), which amplified a 1,452- and a 1,246-bp product, respectively. Sequences of amplicons were almost the same. Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with AluI, MseI, HhaI, HpaI, RsaI, HinfI, and TaqI endonucleases (2) indicated that all symptomatic plants were infected by a phytoplasma belonging to aster yellows group (16SrI) subgroup C (16SrI-C) ‘Candidatus Phytoplasma asteris’. None of the symptomless plants tested positive. Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession No. FJ179166) confirmed the results on the basis of RFLP analyses. Subsequently, the presence of the phytoplasmas in symptomatic plants was also confirmed by transmission electron microscopy. To our knowledge, this is the first molecular evidence of the presence of a phytoplasma associated with a yellows-type disease of willows in northern China and its association with aster yellow group 16SrI, subgroup 16SrI-C. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Inst. J. Syst. Bacteriol. 48:1153, 1998. (3) Y. Qi et al. Biotechnol. Bull. 4:44, 2004.

scholarly journals Corn with Symptoms of Reddening: New Host of Stolbur Phytoplasma

Plant Disease ◽  
2006 ◽  
Vol 90 (10) ◽  
pp. 1313-1319 ◽  
Author(s):  
B. Duduk ◽  
A. Bertaccini
Keyword(s):  
16S Rdna ◽  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Gene Sequences ◽  
New Host ◽  
Intergenic Spacer Region ◽  
New Finding ◽  
Polymerase Chain ◽  
Stolbur Phytoplasma ◽  
Uncertain Etiology

Recurrent epiphytotic outbreaks of a disease of uncertain etiology known as reddening of corn (Zea mays) have occurred in some areas of Serbia during the last 50 years. Affected plants show early and abnormal ripening, dry precociously, and have poor, shriveled grains. Using molecular tools, phytoplasmas were detected in diseased plants and their identity was subsequently deduced as a subgroup 16SrXII-A strain by a variety of supporting assays involving restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S rDNA and tuf gene sequences, selective amplification of phytoplasma DNA using primer pair G35p/m, similarity of 16-23S intergenic spacer region (SR) sequences, and similarity and phylogenetic analysis of 16S rDNA gene sequences. Presence of stolbur phytoplasmas in corn with reddening symptoms is a new finding not only for Serbia: it is the first report of stolbur phytoplasma in this species worldwide.

A Reproducible Selective Stain for Cardiac Sarcoplasmic Reticulum

1974 ◽  
Vol 32 ◽  
pp. 214-215
Author(s):  
R. A. Waugh ◽  
J. R. Sommer
Keyword(s):  
Complex System ◽  
Electron Microscope ◽  
Sarcoplasmic Reticulum ◽  
Transmission Electron Microscope ◽  
Electron Microscopic ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Transmission Electron

Cardiac sarcoplasmic reticulum (SR) is a complex system of intracellular tubules that, due to their small size and juxtaposition to such electron-dense structures as mitochondria and myofibrils, are often inconspicuous in conventionally prepared electron microscopic material. This study reports a method with which the SR is selectively “stained” which facilitates visualizationwith the transmission electron microscope.

Transmission Electron Microscopy of Protein Macromolecules

1971 ◽  
Vol 29 ◽  
pp. 424-425
Author(s):  
Henry S. Slayter
Keyword(s):  
Biological Systems ◽  
Metal Vapor ◽  
Resolving Power ◽  
Electron Microscopic ◽  
Chemical Methods ◽  
Molecular Weights ◽  
The Past ◽  
Physical Chemical ◽  
Transmission Electron

Electron microscopic methods have been applied increasingly during the past fifteen years, to problems in structural molecular biology. Used in conjunction with physical chemical methods and/or Fourier methods of analysis, they constitute powerful tools for determining sizes, shapes and modes of aggregation of biopolymers with molecular weights greater than 50, 000. However, the application of the e.m. to the determination of very fine structure approaching the limit of instrumental resolving power in biological systems has not been productive, due to various difficulties such as the destructive effects of dehydration, damage to the specimen by the electron beam, and lack of adequate and specific contrast. One of the most satisfactory methods for contrasting individual macromolecules involves the deposition of heavy metal vapor upon the specimen. We have investigated this process, and present here what we believe to be the more important considerations for optimizing it. Results of the application of these methods to several biological systems including muscle proteins, fibrinogen, ribosomes and chromatin will be discussed.

An electron microscopic study of the intra-myocardial lesions in cases of acute myocardial infarction

1978 ◽  
Vol 36 (2) ◽  
pp. 484-485
Author(s):  
Masahiro Ono ◽  
Kaoru Aihara ◽  
Gompachi Yajima
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Coronary Vessels ◽  
Vascular Wall ◽  
Vascular Changes ◽  
Electron Microscopic ◽  
Main Trunk ◽  
Mechanical Destruction ◽  
Transmission Electron ◽  
Myocardial Lesions

The pathogenesis of the arteriosclerosis in the acute myocardial infarction is the matter of the extensive survey with the transmission electron microscopy in experimental and clinical materials. In the previous communication,the authors have clarified that the two types of the coronary vascular changes could exist. The first category is the case in which we had failed to observe no occlusive changes of the coronary vessels which eventually form the myocardial infarction. The next category is the case in which occlusive -thrombotic changes are observed in which the myocardial infarction will be taken placed as the final event. The authors incline to designate the former category as the non-occlusive-non thrombotic lesions. The most important findings in both cases are the “mechanical destruction of the vascular wall and imbibition of the serous component” which are most frequently observed at the proximal portion of the coronary main trunk.

A Comparative Study of Fractographic Replicas

1974 ◽  
Vol 32 ◽  
pp. 566-567
Author(s):  
Loren Anderson ◽  
Pat Pizzo ◽  
Glen Haydon
Keyword(s):  
Electron Microscopy ◽  
Electron Microscopic Examination ◽  
Direct Examination ◽  
Electron Microscopic ◽  
Reliable Technique ◽  
Service Failures ◽  
Transmission Electron ◽  
Mode Of Failure ◽  
Scanning Electron Microscopic ◽  
Scanning Electron

Transmission electron microscopy of replicas has long been used to study the fracture surfaces of components which fail in service. Recently, the scanning electron microscope (SEM) has gained popularity because it allows direct examination of the fracture surface. However, the somewhat lower resolution of the SEM coupled with a restriction on the sample size has served to limit the use of this instrument in investigating in-service failures. It is the intent of this paper to show that scanning electron microscopic examination of conventional negative replicas can be a convenient and reliable technique for determining mode of failure.

Studies of Circular DNA Using a New Electron Microscopic Technique

1973 ◽  
Vol 31 ◽  
pp. 596-597
Author(s):  
C. N. Gordon
Keyword(s):  
Electron Microscopy ◽  
Aqueous Salt Solution ◽  
Contour Length ◽  
Cleavage Surface ◽  
Mica Substrate ◽  
Electron Microscopic ◽  
Replica Technique ◽  
Circular Dna ◽  
Transmission Electron ◽  
Electron Microscopic Technique

Gordon and Kleinschmidt have described a new preparative technique for visualizing DNA by electron microscopy. This procedure, which is a modification of Hall's “mica substrate technique”, consists of the following steps: (a) K+ ions on the cleavage surface of native mica are exchanged for Al3+ ions by ion exchange. (b) The mica, with Al3+ in the exchange sites on the surface, is placed in a dilute aqueous salt solution of DNA for several minutes; during this period DNA becomes adsorbed on the surface. (c) The mica with adsorbed DNA is removed from the DNA solution, rinsed, dried and visualized for transmission electron microscopy by Hall's platinum pre-shadow replica technique.In previous studies of circular DNA by this technique, most of the molecules seen were either broken to linears or extensively tangled; in general, it was not possible to obtain suitably large samples of open extended molecules for contour length measurements.

Sign in / Sign up

Export Citation Format

Share Document