Detection Of Phytoplasma Research Articles

Widespread occurrence of \u2018Candidatus Phytoplasma ulmi\u2019 in elm species in Germany

Background‘Candidatus Phytoplasma ulmi’ is the agent associated with elm yellows and has been categorised in the European Union as a quarantine pathogen. For central and northern European countries, information on the occurrence and distribution of the pathogen and its impact on elms is scarce, so a survey of native elm trees has been conducted in Germany.ResultsAbout 6500 samples from Ulmus minor, Ulmus laevis and Ulmus glabra, were collected nationwide. Phytoplasma detection was performed by applying a universal 16Sr DNA-based quantitative PCR (qPCR) assay and a novel ‘Ca. P. ulmi’ specific qPCR assay targeting the 16S–23S spacer region. Both assays revealed that 28% of the samples were infected by ‘Ca. P. ulmi’, but infection rates of the elm species and regional incidences differed. The phytoplasma presence in the trees was not correlated to disease-specific symptoms. The survey identified a regional disparity of infection which was high in east, south and central Germany, whereas only a few infected sites were found in the western and northern parts of the country. Monitoring the seasonal titre of ‘Ca. P. ulmi’ in an infected tree by qPCR revealed a high colonisation in all parts of the tree throughout the year.Conclusions‘Ca. P. ulmi’ is widely present in elms in Germany. The rare occurrence of symptoms indicates either a high degree of tolerance in elm populations or a low virulence of pathogen strains enabling high infection rates in a long-living host.

Simultaneous Detection and Direct Identification of Phytoplasmas in the Aster Yellows (16SrI), Clover Proliferation (16SrVI) and Stolbur (16SrXII) Groups Using a Multiplex Nested PCR Assay in Potato Plants

Phytoplasmas in the aster yellows (16SrI), clover proliferation (16SrVI) and stolbur (16SrXII) groups have a wide host range with common hosts. In the current study, specific primers based on the 16-23S rRNA spacer region were designed and optimised for the detection and identification of phytoplasmas in these groups in a multiplex nested PCR assay. Fifty-two phytoplasma symptomatic potato plants were tested, and clover proliferation (16SrVI) and stolbur (16SrXII) phytoplasmas detected coinfecting 16 potato plants with single infection of these phytoplasmas in 5 and 21 samples, respectively. ‘Candidatus P. asteris’ (16SrI) was not detected. The specificity of the primers was proven in both single and multiplex PCR assays. Detection of multiple infections can be useful in assisting epidemiological studies and in deciding appropriate disease management strategies.

Molecular detection and characterisation of phytoplasma in Trigonella foenum-graecum and identification of potential insect vectors in Punjab, Pakistan

Molecular detection and characterisation of phytoplasma in Trigonella foenum-graecum and identification of potential insect vectors in Punjab, Pakistan

Molecular detection of a 16srix-e phytoplasma in chickpea affected by witches’ broom disease in Iran

Molecular detection of a 16srix-e phytoplasma in chickpea affected by witches’ broom disease in Iran

Report of ‘Candidatus Phytoplasma hispanicum’ (16SrXIII-E) Associated with Cauliflower Stunt in São Paulo State, Brazil, and Balclutha hebe as Its Potential Vector

A cauliflower stunt disease associated with phytoplasmas occurs in crops of cauliflower (Brassica oleracea var. botrytis) in Sao Paulo state (SP), southeastern region of Brazil. The symptoms are characterized by stunting, extensive malformations of floral parts, leaf reddening, and vessel necrosis. 16SrIII-J and 16SrXV-A phytoplasmas have already been reported in association with this disease in this region (Canale and Bedendo 2013; Rappussi et al. 2012). Herein we report the involvement of 16SrXIII-E phytoplasma with cauliflower stunt disease and the presence of this bacteria in Balclutha hebe leafhopper. DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from leaf veins of seven symptomatic and seven asymptomatic cauliflower samples collected in May 2008 from a farm located in Morungaba (SP, –22.882603, –46.755783). In addition, in an attempt to identify a possible vector for the phytoplasma, insects were captured using a sweep net from a cauliflower farm in Sorocaba (SP, –23.391314, –47.507474), where the disease occurred in high incidence levels (Rappussi et al. 2012). The most abundant leafhopper species collected with the net was identified as B. hebe Kirkaldy, 1900 (Hemiptera: Cicadellidae). DNA was also obtained from whole insects using the same extraction kit as we used for plants. Phytoplasma detection was performed with a nested-PCR assay using P1/Tint and R16F2n/R16R2 universal primers specific to the phytoplasma 16S rRNA gene (Lee et al. 1998; Smart et al. 1996). PCR products of the expected size (∼1.2 kb) were obtained from all of the symptomatic cauliflower but not from the collected symptomless samples. We amplified phytoplasma DNA from four out of 15 B. hebe specimens. Two plant amplicons and two leafhopper amplicons were sequenced (Macrogen, Seoul, Korea). Nucleotide sequence alignment revealed that 16S rDNA of the phytoplasma from cauliflower and insect samples shared ˃99% sequence similarity with Papaya apical curl necrosis phytoplasma, a member of the 16SrXIII-E subgroup and previously described by our group (Melo et al. 2013). Restriction fragment length polymorphism analysis using AluI, RsaI, KpnI, HpaII, MseI, HhaI, MboI, and BstUI restriction enzymes and in silico analysis using the iPhyClassifier online tool (Zhao et al. 2009) also allowed confirmation of the identification of the phytoplasma affecting cauliflower plant and leafhopper samples as member of the 16SrXIII-E subgroup (F = 1.00). A phylogenetic tree was generated with sequences of 16S rDNA of distinct subgroups affiliated with the 16SrXIII group, using the neighbor-joining method and 1,000 bootstrap replicates (MEGA software, Tamura et al. 2011), using Acholeplasma laidlawii as an outgroup. The phylogenetic analysis supported the identification of cauliflower and B. hebe phytoplasmas reported in this study as members of 16SrXIII-E subgroup. Sequences of the 16SrXIII-E phytoplasmas were deposited in GenBank as the accession JN818844 (Cauliflower stunt phytoplasma) and JN818843 (B. hebe phytoplasma). The 16SrXIII phytoplasma group (‘Candidatus Phytoplasma hispanicum’) is diverse and apparently restricted to the American continent, affecting a wide range of plant hosts (Davis et al. 2016). B. hebe is a polyphagous generalist species omnipresent in southern and south Brazil. It is reported as a putative vector for Xylella fastidiosa in plum orchards in south Brazil (Hickel et al. 2001). Also, this species was found in grasses within citrus rows in orchards in Sao Paulo, and one specimen was infected with 16SrIX phytoplasma (Marques et al. 2012). B. hebe was abundant within the cauliflower crops in both locations of this study, and its abundance along with our research results indicate that this leafhopper may play a role as a vector for 16SrXIII-E to cauliflower. However, more refined transmission assays employing phytoplasma and insects maintained in a laboratory are needed to prove B. hebe capacity as a phytoplasma vector. Here, we report ‘Ca. P. hispanicum’ strain as one of the associated phytoplasmas with cauliflower stunt disease in Brazil and B. hebe leafhopper as a putative vector.

Detection and Identification of Banana-associated Phytoplasma Using Nested-PCR Method

Phytoplasma is known to be associated with plant diseases in about 300 plant species from various families. Information on the presence of phytoplasma in bananas as one of the pathogens that can cause disease in bananas in Indonesia has never been reported. This research was conducted with the aim to detect the presence of banana phytoplasma by the nested-PCR method and to identify phytoplasma obtained based on the sequence analysis of the 16S rRNA gene. Standard PCR was carried out using P1/P7 primary pairs, followed by nested-PCR using a pair of R16F2n/R16R2m23SR primers separately that could amplify the target 16S rRNA genes in a row at 1600 bp. BLAST analysis shows that the results of phylogenetic analysis of banana phytoplasmic nucleotide cv. manggala from Tasikmalaya and cv. Raja nangka from Banjar has a genetic relationship that is closer to lethal wilt oil palm Phytoplasma (Candidatus Phytoplasma asteris). This phytoplasma belongs to the 16SrI-B group (aster yellows).

Detection and Quantification of the 16SrIV-D Phytoplasma in Leaf Tissue of Common Ornamental Palm Species in Florida using qPCR and dPCR.

Lethal bronzing disease (LBD) is a lethal decline of various palm species caused by the 16SrIV-D phytoplasma. The disease was described in Texas in 2002 but found in Florida in 2006. Since its introduction, the phytoplasma has spread throughout much of the state. Typically, sampling of infected palms involves taking trunk tissue; however, in some instances this is not possible so alternative protocols are needed. In this study, phytoplasma titers were measured in different leaf tissues of infected palm in order to provide stakeholders with more sampling options. In addition, understanding the phytoplasma distribution in the palm canopy can shed light on the pathogen's biology and aid in vector studies. Three species of palm, Phoenix roebelinii, Sabal palmetto, and Syagrus romanzoffiana, were identified and confirmed positive for infection by qPCR analysis. Leaf tissue from these species that represented different stages of decay were sampled and tested by qPCR and dPCR. For each species, phytoplasma was only detectable in the spear leaf tissue that was connected directly to the apical meristem by both qPCR and dPCR. These data are useful by demonstrating that the 16SrIV-D phytoplasma appears to be restricted to the spear leaf so stakeholders who wish to sample palms but cannot sample trunk tissue due to palm size or lack of permission to drill into the trunk have an alternative tissue type to reliably sample. In addition, this information will help improve vector research by knowing where to collect insects that have a higher probably of possessing the phytoplasma.

First Report of Phytoplasma Infecting Pterocarpus indicus in China

Pterocarpus indicus Willd., an adaptable and fast-growing landscape tree, is commonly cultivated in landscaping in southern China. In April 2018, a P. indicus tree with symptoms of flat branches and typical fasciation, which had the same symptoms of phytoplasma infection, was observed in Haikou, Hainan province, China. To determine the presence of phytoplasma, stem samples of symptomatic and asymptomatic P. indicus were subjected to total DNA extraction using the plant Genomic DNA Kit (Tiangen, Beijing, China), followed by nested polymerase chain reaction (PCR) detection of phytoplasma (Jomantiene et al. 1998; Lee et al. 1998). Briefly, the universal primer pair R16mF2/R16mR1 was employed for the initial PCR amplification of 16SrDNA fragment of phytoplasma and primer set R16F2n/R16R2 (Lee et al. 1998) for nested PCR amplification. As expected, the PCR products were generated from the samples of the symptomatic plant but not from the symptomless control samples. The anticipated amplicon was approximately 1.2 kb in size. The PCR products were gel purified using TIANgel Midi Purification Kit (Tiangen, Beijing, China) and cloned into pMD-18T vector (Takara Bio, Dalian, China). Three independent positive clones were subjected to Sanger sequencing. The obtained sequence was deposited into NCBI GenBank database with GenBank accession no. MH727702. A BLASTn search against the nonredundant nucleotide database revealed high sequence identities with the corresponding regions of phytoplasmas of aster yellows phytoplasma group (16SrI). A maximum of 99.7% nucleotide sequence identity with Malvastrum coromandelianum phyllody phytoplasma (GenBank accession no. MF490802) was observed. Moreover, an iPhyClassifier (Zhao et al. 2013) operation was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile for the sequence derived from the symptomatic P. indicus sample, which showed the most similarity with that of the reference strain (GenBank accession no. AP006628) belonging to 16SrI group. However, this strain may represent a new subgroup because the similarity coefficient was only 0.91, which is within the values set for a new subgroup (Biswas et al. 2013). To our best knowledge, this is the first report of phytoplasma infecting P. indicus in China.

Detection of phytoplasma causing tomato big bud disease in Syria

Detection of phytoplasma causing tomato big bud disease in Syria

Spread and colonization pattern of ‘Candidatus Phytoplasma aurantifolia’ in lime plants [Citrus aurantifolia (Christm.) Swingle] as revealed by real-time PCR assay

Lime witches’-broom (WBDL) is a devastating disease of Mexican lime trees in southern Iran. ‘Candidatus Phytoplasma aurantifolia’ is associated with the disease. Understanding the pattern of colonization and spread of ‘Ca. P. aurantifolia’ within lime trees is critical for its accurate detection during the latent period of the disease. In the present study, the distribution pattern of the WBDL phytoplasma in graft inoculated lime seedlings was investigated under greenhouse conditions and its seasonal variation of concentration in symptomless lime trees was evaluated in an infected orchard using qPCR assay. Repeated sampling from various tissues of graft-inoculated lime seedlings was carried out at various times post-inoculation. In the field, leaf samples were taken from symptomless trees in an infected orchard in March, August and December. Our results showed that after entrance into the lime seedlings, ‘Ca. P. aurantifolia’ moved downward into the roots as well as upward into the apical leaves. The concentration of phytoplasma in apical leaves showed an initial high followed by a decrease before establishment of infection and subsequent symptom appearance. The spread of the phytoplasma from root to the aerial parts was rather slow. Severe witches’ broom symptoms appeared on the grafted branches 10 months after grafting. At this time, the highest phytoplasma DNA concentration was found in descending order in stems and leaves of witches’ brooms, crown, various parts of the root, and stem bark and apical leaves of the symptomless branches. Results of the field experiment revealed that concentration of ‘Ca. P. aurantifolia’ cells in infected trees was highest in August compared to that in December and March. Our result would be useful for rapid detection of WBDL phytoplasma during the latent period of the disease.

Simultaneous detection of coconut lethal yellowing phytoplasmas (group 16SrIV) by real-time PCR assays using 16Sr- and GroEL-based TaqMan probes

Lethal yellowing (LY) represents a serious threat to coconut in Mexico, Central America and the Caribbean islands. It is caused by a phytoplasma of group 16SrIV, and particularly subgroups -A and -D have been the major phytoplasmas affecting coconut palms grown in the southeast of Mexico. Therefore, is important to reliably detect the 16SrIV group of phytoplasmas not only for diagnosis purposes but also to improve the understanding of pathogen-plant-vector pathosystems. The 16S ribosomal operon genes have been particularly used as the targets for probes and primers for universal phytoplasma detection. However, its conservative nature has imposed the search of non-ribosomal genes to support a finer characterization and differentiation of phytoplasmas, particularly those closely related and one alternative has been the GroEL gene for the specific detection of subgroups 16SrIV-A and 16SrIV-D. Sensitive, rapid and reliable singleplex and duplex assays were developed based on real-time PCR (TaqMan) targeting both the GroEL gene and 16S rRNA. The assay performed with higher sensitivity compared to conventional nested-PCR when analysing DNA extracts obtained from LY symptom bearing palms. The LY 16S TaqMan probe assays amplified DNA from both subgroups, while the LY GroEL TaqMan probe assay only amplified DNA from subgroup 16SrIV-A. The duplex TaqMan probe assay is a novel option for the simultaneous detection and quantification of LY phytoplasmas of 16Sr subgroups -A and -D, which are the phytoplasmas that predominantly affect palms in Mexico.

Molecular Characterization and Detection of 16SrIII Group Phytoplasma Associated with Huanglongbing Symptoms.

When huanglongbing (HLB) was found in Brazil in 2004, 'Candidatus Liberibacter americanus' was infecting most of the trees while 'Ca. L. asiaticus' was present in a minor proportion. Currently, 'Ca. L. asiaticus' is the predominant bacterium associated with HLB in citrus trees in São Paulo (SP) and Minas Gerais (MG) States, the major citrus-growing regions in Brazil. A phytoplasma from the 16SrIX group was associated with HLB symptoms in Brazil in 2007, in plants free of Liberibacter spp. In this report, HLB samples testing negative for 'Ca. L. asiaticus', 'Ca. L. americanus', and 16SrIX phytoplasma were infected with 16SrIII phytoplasmas. Coinfection with 'Ca. L. asiaticus' and 16SrIII was also found. The 16S ribosomal RNA (rRNA) gene sequences from 22 samples were obtained and sequenced, confirming that the 16SrIII group phytoplasma is associated with HLB symptoms in SP and MG States. Ten single-nucleotide polymorphisms (SNPs) were found in the 1,427-bp 16S rRNA gene sequences from 16SrIII phytoplasmas from citrus, whereas none was detected in 16S rRNA gene sequences among 16SrIX phytoplasma from citrus. Ribosomal protein (rp) rpsSrplVrpsC gene sequences were amplified with 16SrIII group-specific primers, sequenced from a subset of nine samples, and assembled into three groups based on eight SNPs. SNPs in 16S rRNA gene and rp gene sequences are common in 16SrIII phytoplasmas from other hosts and this phytoplasma group is widespread in South America. 16SrIII phytoplasmas highly related are commonly found in Melia azedarach, a widespread tree in Brazil and Argentina. The finding of a new phytoplasma associated with HLB symptoms belonging to the 16SrIII group reinforces the need to develop diagnostic tools to assess HLB-associated microbiomes.

Molecular detection and identification of phytoplasmas in a novel 16SrI subgroup in sunflowers and cocklebur weeds

Sunflowers (Helianthus annuus L.) exhibiting symptoms of stem fasciation and cluster of multiple inflorescences were observed in Baicheng, Jilin province, China. Coincidently, cockleburs (Xanthium strumarium L.), a weed in the sunflower fields, showing similar symptoms were observed as well. Based on successful amplification of 16S rRNA and secY genes of phytoplasmas, the sunflower fasciation disease (SFD) and cocklebur fasciation disease (CFD) were determined to be associated with phytoplasmas. Based on virtual and actual RFLP analysis of the F2nR2 region of the 16S rRNA genes, the SFD and CFD associated phytoplasmas were classified into a novel 16SrI subgroup, designated 16SrI-AI. This was the first report of SFD associated phytoplasma in China, and the first evidence of phytoplasma infecting cocklebur worldwide.

Nested and TaqMan® probe based quantitative PCR for the diagnosis of Ca. Phytoplasma in coconut palms

The root (wilt) disease caused by phytoplasma (Ca. Phytoplasma) is one of the major and destructive occurs in coconut gardens of Southern India. As this organism could not be cultured in vitro, the early detection in the palm is very much challenging. Hence, proper early diagnosis and inoculum assessment relay mostly on the molecular techniques namely nested and quantitative PCR (qPCR). So, the present study qPCR assay conjugated with TaqMan® probe was developed which is a rapid, sensitive method to detect the phytoplasma. For the study, samples from different parts of infected coconut palms viz., spindle leaflets, roots and the insect vector-leaf hopper (Proutista moesta) were collected and assessed by targeting 16S rRNA gene. Further, nested PCR has been carried out using p1/p7 and fU5/rU3 primers and resulted in the amplification product size of 890bp. From this amplified product, specifically a target of 69bp from the 16S rRNA gene region has been detected through primers conjugated with Taqman probe in a step one instrument. The results indicated that the concentration of phytoplasma was more in spindle leaflets (8.9 × 105g of tissue) followed by roots (7.4 × 105g of tissue). Thus, a qPCR approach for detection and quantification of coconut phytoplasma was more advantageous than other PCR methods in terms of sensitivity and also reduced risk of cross contamination in the samples. Early diagnosis and quantification will pave way for the healthy coconut saplings selection and management under field conditions.

Phytoplasma detection in date palm plant tissues by colony isolationfollowed by molecular analyses

In the last years, some reports of phytoplasmas presence in date palms have appeared indicating that these pathogens have a growing importance in this crop species. The present work was conducted to verify the possibility to isolate phytoplasmas infecting date palms in Saudi Arabia as alternative and/or complementary method to the amplification of 16Sr ribosomal gene. The comparison of phytoplasma detection from field infected materials and by isolation and colonies in culture indicates that the complexity of phytoplasma associated diseases is greater than believed and it is very likely that the symptomatology observed is the result of complex of microorganisms (mainly phytoplasmas) interaction. Further studies following this methodology would help in the better understanding of this complexity of phytoplasmas toward designing appropriate tools for the containment and management of these epidemic diseases to reduce their economic impact.

Phytoplasma detection and quantification: Make it easy

When high numbers of analyses are required a rapid sample preparation method coupled to RT-qPCR allows to reduce the time and the costs for the phytoplasma detection in plant samples. A comparison between DNA and RNA contribution to qPCR detection revealed the higher input of the latter ensuring the maintenance of the sensitivity and specificity of the assay. The protocol has been validated for the detection and quantification of ‘Candidatus Phytoplasma mali’, ‘Ca. P. pyri’, ‘Ca. P. prunorum’, ‘Ca. P. solani’ and “flavescence doree” phytoplasma by qPCR, RT-qPCR, ddPCR and ddRT-PCR techniques based on TaqMan chemistry.

Genome-informed design of LAMP assays for specific detection of aster yellows phytoplasmas

Genome sequence data is becoming more available and represents a source of potential target sequences for specific molecular tests to verify the feasibility and efficiency of genome-inform test design the attempt to identify novel specific sequences in the publicly available genomes of aster yellows phytoplasmas and use them to design primers for a spcific LAMP assays. Coupling RUCS automated analysis with PrimerExplorer V5 primer designer and applying criteria for selection of assays at each stage of the process resulted in 13 promising LAMP assays targeting four newly identified regions specific to aster yellows phytoplasmas.

Molecular study of a phytoplasma associated with safflower fasciation in Iran

During the growing seasons of 2017 and 2018, a disease occurred in safflower fields in the south Khorasan province of Iran. Affected plants exhibited extensive fasciation, formation of bushy growth, small leaves, phyllody and shortened internodes. For detection of the putative phytoplasma(s), polymerase chain reaction (PCR) assays were performed using universal phytoplasma primers pairs. Nested PCR analyses revealed that all symptomatic safflowers plants showing fasciation symptoms tested positive for phytoplasma presence, while asymptomatic plants were negative. Nucleotide sequence analysis identified a phytoplasma belonging to the peanut witches’ broom group (16SrII). Phylogenetic analysis confirmed the clustering of the safflower fasciation phytoplasma (SaP) with ‘Candidatus Phytoplasma aurantifolia’-related strains.

Molecular detection of phytoplasmas infecting date palm in Saudi Arabia and Jordan

Date palm is one of the most important cash crops in Saudi Arabia and Jordan. Symptoms of mild leaf stunting, yellow streaking and a reduction in fruit and stalk size had been observed in date palm orchards in both countries. In Jordan a total of 262 healthy and symptomatic samples were collected from different date palm cultivars, weeds growing under date palm trees and other different plant species possible phytoplasma hosts growing in the orchards in addition to leafhoppers, while 567 leaf samples were collected from Saudi Arabia. Nested PCR was performed using phytoplasma P1/P7 and R16F2n/R2 primer pairs. BLAST analysis of the obtained sequences of samples from Jordan revealed over 98% sequence identity with different reference phytoplasma strains deposited in the GenBank and 98–100% sequence identity with ‘Candidatus Phytoplasma ulmi’-related strains. A 16SrII phytoplasma, ‘Candidatus Phytoplasma aurantifolia’-related strain was identified in the samples from Saudi Arabia.

Molecular identification of phytoplasmas in Russian vineyards

Phytosanitary surveys of vineyards in the Krasnodar region were carried out for phytoplasma detection. Symptomatic samples were tested by nested PCR. According to the restriction fragment length polymorphism analysis the identified phytoplasma belongs to the “stolbur” subgroup 16SrXII-A.