Molecular Diagnostic Tools for Detection and Differentiation of Phytoplasmas Based on Chaperonin-60 Reveal Differences in Host Plant Infection Patterns
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Phytoplasmas ('Candidatus Phytoplasma' spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S–23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60) gene sequence from a diverse array of 'Ca.Phytoplasma' spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from 'Ca.Phytoplasma' spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns.Keywords:
Phytoplasma
Multilocus sequence typing
Phytoplasma
Cutting
Aster yellows
Cane
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Lethal bronzing (LB) is a fatal palm disease caused by the phytoplasma 'Candidatus Phytoplasma aculeata'. This disease causes significant economic losses in palm industries and landscapes. The American palm cixiid, Haplaxius crudus, recently was identified as the vector of the phytoplasma. However, knowledge about LB phytoplasma transmission is limited due to the lack of a method to generate phytoplasma-infected insects in the laboratory. In this study, the acquisition and transmission of the LB phytoplasma by H. crudus were investigated. Successful acquisitions of the phytoplasma by H. crudus were observed at 2 days acquisition access period on LB-infected palm spear leaves. Analyses revealed increased phytoplasma infection rates of H. crudus with longer acquisition access periods and latent periods. A significantly higher phytoplasma infection rate was shown after various acquisition access periods and latent periods than the infection rate of the field-collected H. crudus population. Transmission of the phytoplasma from LB-infected spear leaves to sucrose media by H. crudus also was observed using digital PCR assays. These results further support the vector status of H. crudus and offer valuable information to understand LB phytoplasma transmission. Additionally, these results generate a critical baseline for future LB phytoplasma-vector research by providing a way to generate vectors with high phytoplasma infection rates in the laboratory setting.
Phytoplasma
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Molecular Detection of Mulberry Phytoplasma in Guangzhou and Primary Study of its 16S rDNA Diversity
Based on the 16S-23S rDNA sequence of phytoplasma,a set of universal primers P1/P7 and a set of nested primers Rm16F2/Rm16R1 were employed to perform nested-PCR detection for pathogenic phytoplasma in mulberry leaf tissues sampled from two variety-collecting bases in Guangzhou,China.It was confirmed that some mulberry varieties in these bases were phytoplasma-infected.The products of nested-PCR,16S rDNA fragments,were then analyzed by restricted fragment length polymorphism with KpnⅠ and MspⅠ and by 6% polyacylamide gel electrophoresis.The result showed that there were three types of RFLP bands which indicated the diversity of mulberry phytoplasma.The obtained 16S rDNA fragments were sequenced and then compared with the reported phytoplasma 16S rDNA.The nucleotide identities were from 83.3% to 99.9% between the detected phytoplasma and others.According to 16S rDNA sequence the phylogenetic tree was constructed.Based on the above results,we deduced that the phytoplasma causes mulberry dwarf disease in Guangzhou belongs to group 16S rⅠ.
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In India phytoplasma associated with plants including crops, fruits, trees, ornamental, sugarcane, grasses and weeds was increasing at alarming rate. Recent evidences showed that phytoplasma associated with pulse crops in India. Phytoplasma cause diseases in pulse crops are chances to causing severe losses. In earlier days very few phytoplasma diseases were identified in India based on bright field, fluorescence, electron microscope observations, tetracycline treatment and to a lesser extent by serological assays. Among these, microscopic methods do not attain pathogen identification, and all of them are not always sufficiently sensitive to detect phytoplasma infections in low titer hosts. Today detection of phytoplasma based on molecular methods including PCR assays are efficiently carried out in India and based on this technique phytoplasma infection on pulse crops was reported in India. The “Candidatus Phytoplasma asteris”, “Candidatus Phytoplasma aurantifolia” belong to 16SrI and 16SrII group of phytoplasmas are the major groups associated with pulse crops reported to be infected with phytoplasma throughout India. In this paper, we have discussed on phytoplasma disease on pulse crops in India.
Phytoplasma
Candidatus
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Since 2005,large area of commercial eucalypt plantations have exhibited so called slow-growing symptoms in Guangdong and Guangxi.The study was carried out to confirm whether this disease is caused by phytoplasma.Both direct-PCR and nest-PCR were employed to detect the phytoplasma by cloning 16 SrDNA from samples with typical symptoms.The results showed that there was no phytoplasma detected from the samples indicated phytoplasma is not the causal agent of the disease.
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Four out of six known potato diseases attributed to phytoplasma infection were previously reported to occur in Russia based on a combination of biological properties such as symptomatology and/or vector relationships and electron microscopy of infected phloem tissue. In 2007, the first molecular identification of potato diseases causing symptoms including purple top, round leaves, stunting, bud proliferation and formation of aerial tubers was carried out using PCR methods. A nested PCR using primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasma in infected potato samples. PCR products were digested singly with several restriction enzymes. Comparison of RFLP profiles with published profiles was used for identification of the putative phytoplasma detected. The majority of 49 PCR positive potato samples showed RFLP profiles of 16S rDNA sequences very similar or identical to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A, and only two showed RFLP profiles similar to those of aster yellow phytoplasma strains ('Candidatus Phytoplasma asteris') belonging to aster yellows phytoplasma group (16SrI), subgroup 16SrI-A and 16SrI-B. The results demonstrated that stolbur phytoplasma is prevalent in several potato growing regions of Russia.
Phytoplasma
Aster yellows
TaqMan
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The presence of phytoplasmas in apple trees with proliferation symptoms, rubbery wood symptoms and no symptoms was determined by using polymerase chain reaction assays with primers amplifying phytoplasma 16S rRNA gene. Phytoplasmas were detected in all trees with proliferation symptoms. Positive tests for phytoplasma in the group of trees with rubbery wood symptoms and of those without symptoms revealed a relatively high incidence of latent phytoplasma infection. Using restriction fragment length polymorphism analysis, phytoplasma of the same identity - apple proliferation phytoplasma (subgroup 16SrX-A) - was recorded in all positively tested trees.
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In order to diagnose and differentiate jujube witches' broom (JWB) phytoplasma rapidly, oligonucleotide primer pair, 16Sr(V) F/R, for polymerase chain reactions (PCRs) was designed on the basis of 16S rRNA sequences of JWB phytoplasma. The PCR employing phytoplasma universal primer pair P1/P7 consistently amplified DNA in all tested phytoplasma isolates. But no phytoplasma DNA was detected from healthy jujube seedlings. The nested PCR, the primer pair 16S(V) F/R, about 460 bp fragment, amplified DNA in all tested JWB and related phytoplasmas including ligustrum witches' broom phytoplasma of the 16S rRNA group V, but no DNA amplification was detected from other phytoplasma strains such as groups 16SrI (Aster yellows) and 16SrXII (Stolbur group) in which mulberry dwarf phytoplasma and chrysanthemum witches' broom phytoplasma belong to, respectively. The same results were obtained from both Korean and Chinese isolates of JWB phytoplasma. Nested-PCR using phytoplasma universal primer pair P1/P7 and 16SrV group-specific primer pair 16S(V) F/R could detect group V phytoplasmas rapidly and easily, in particular JWB phytoplasma.
Phytoplasma
Broom
Aster yellows
Primer (cosmetics)
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Abstract Phytoplasmas, prokaryotic wall-less microorganisms, are important pathogens of several plant species in most parts of the world. Phytoplasmas have been reported associated with various symptoms on hundreds of plant species. Witches’ broom disease (WBD) is one of the most common disease symptoms, which is caused by phytoplasma strains belonging to different phytoplasma groups. Symptoms of the disease differ from one host to the other as well as from one phytoplasma strain to the other. However, WBD symptoms are usually characterized by the production of a large number of small leaves, accompanied in some host plants by the production of several branches/shoots. Phytoplasma strains belonging to more than 13 groups and 39 subgroups have been reported associated with WBD in more than 116 plant species. Most of the phytoplasma strains causing WBD symptoms in plant species belong to the 16SrII and 16SrI groups, mainly 16SrII-D and 16SrI-B subgroups. The current review provides information on the different types of phytoplasma strains associated with WBD symptoms in ornamental plants, medicinal plants, forest trees, weeds, vegetable crops, field crops, and fruit trees. Emphasis is on WBD on acid limes, almonds, peanuts, jujube, and cassava that have resulted in significant economic losses in different countries. Description of the symptoms, phytoplasma groups, and management options is also provided for some of the diseases.
Phytoplasma
Broom
Ornamental plant
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Frogskin is the most limiting disease of cassava crops in Colombia, causing losses in production up to 90%. Since this disease was associatated with 16SrIII phytoplasma presence, a study was carried out to isolate this phytoplasma using liquid and solid culture media. Root, petiol, stem, leaf and cutting tissues of cassava affected by frogsking were employed as source materials. Molecular and microscopy techniques were applied to verify the phytoplasma growth and to discard other microorganism s presence. The results showed that the media consistently allow phytoplasma growth, and colonies in solid medium were obtained. PCR, qPCR and sequencing tests confi rmed the presence of 16SrIII group phytoplasmas in both liquid and solid culture media. Additionally, the isolation of a pigeon pea witches’ broom phytoplasma strain (group 16SrIX) was obtained from stems, petioles and fl owers of symptomatic Catharanthus roseus confi rming the effectiveness of the medium in the phytoplasma isolation and culture. This is the fi rst isolation of fi eld-collected phytoplasma strains in groups 16SrIII and 16SrIX in America that confi rm and corroborate the previous results in phytoplasma cultivation achieved on micropropagated and fi eld-collected phytoplasma infected samples.
Phytoplasma
Isolation
Limiting
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