Detection Of Phytoplasma Research Articles

Detection, characterization and management of brinjal little leaf disease in Assam

Aim: To record the prevalence, detection, molecular and biological characterization of phytoplasma associated with brinjal little leaf disease in Assam and its management. Methodology: Roving survey was conducted during the 2018-2020 brinjal growing seasons to record the disease incidence and symptoms associated with the disease. Insect and dodder transmission studies were carried out along with tetracycline tests. Further, molecular detection of all the samples collected during the survey and after dodder and insect transmission studies were done through PCR assays by using universal primers. Results: The disease incidence varied from 3.62 to 23.63 per cent in surveyed districts. Transmission studies revealed that Hishimonus phycitis (Dist.) is acting as a vector of brinjal little leaf phytoplasma and found to be transmitted by dodder (Cuscuta campestris). The phylogenetic analysis indicated that the Brinjal little leaf phytoplasma understudy was associated with ‘Candidatus phytoplasma trifolii’ and plants like Datura stramonium play a major role as potential alternate host of the phytoplasma. Further, studies on the effect of the antibiotic tetracycline hydrochloride in managing the disease elucidated that the antibiotic was effective only for temporary remission of symptoms. Interpretation: This study reveals a broad picture about the incidence, phytoplasma associated with the disease, putative leafhopper vector transmitting the phytoplasma in the field, alternate host harbouring the phytoplasma, and effect of antibiotic on the disease suppression, which will be useful to develop effective management strategies.

Insect vectors transmission of phytoplasma to vegetables in Eastern Visayas Philippines

Phytoplasma diseases are found affecting vegetables in Eastern Visayas, Philippines. Identifying the insect vectors that transmit phytoplasma in the field is necessary for the management of this disease. This study was conducted to identify the vectors of phytoplasma affecting bitter gourd, loofah, and string beans. Insects associated with these vegetables that had shown phytoplasma symptoms in the field were collected, mass-reared, and the progenies of mass reared insects were used for transmission to healthy host plants. Phytoplasma detection was done through PCR and nest PCR assays. Phytoplasma was positively transmitted to healthy bitter gourd plants by a cicadellid leafhopper, Hishimonus sp. and brown planthopper, Ricania speculum. The ~ 1.25Kb phytoplasma-specific band was amplified in these insects. The phytoplasmas transmitted by the Hishimonus sp. and R. speculum produced two different types of symptoms to the bitter gourd, and rDNA sequence analysis separated them into two clusters which confirmed that the phytoplasmas belong to two different strains. Although the Aphis gossypii and Aphis craccivora also produced symptoms in bitter gourd and string beans, and the pathogen was detected in the insects, whether they vectored phytoplasma to their respective hosts needs further study, since the symptoms produced were quite different from the ones produced by the cicadellid leafhopper and Ricania speculum.

Detection, Identification, and Molecular Characterization of a 16SrII-V Subgroup Phytoplasma Associated with Nicotiana plumbaginifolia.

Nicotiana plumbaginifolia Viviani, commonly known as curl-leaved tobacco, is an annual herbaceous plant belonging to Solanaceae family. This plant is native to Mexico, South America, and parts of the Caribbean and has been reported to be present in Taiwan since 2006. In March 2021, N. plumbaginifolia Viviani, found in Yunlin County, Taiwan, was observed to have phyllody, virescence, and witches'-broom, which is consistent with the disease symptoms caused by phytoplasma infection. Samples of the healthy and symptomatic plants were collected for analysis of the causal agent associated with the diseased N. plumbaginifolia Viviani. Under transmission electron microscopy, the phytoplasma-like pleomorphic bodies were found in the sieve tubes of the diseased plants. The 16S ribosomal RNA (rRNA)-based phylogenetic analysis and the iPhyClassifier-based virtual restriction fragment length polymorphism study demonstrated that the phytoplasma identified in this study can be classified into the 16SrII-V subgroup, which is similar to the peanut witches'-broom phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain. Further identification of SAP54/PHYL1 and SAP11 homologs in the phytoplasma explain the disease symptoms of phyllody, virescence, and witches'-broom observed in diseased N. plumbaginifolia Viviani. The discovery of new phytoplasma plant hosts has gained scientific importance in light of the attempt to unravel an efficient strategy to fight the rapid spread of this disease, which poses a threat to the agricultural sector and food security in Taiwan.

Direct identification of fungi associated with indoor and outdoor ornamental plants by utilizing PCR assay

Direct identification of fungi associated with indoor and outdoor ornamental plants by utilizing PCR assay

Genomic characterization of Iranian ʻCandidatus Phytoplasma phoeniciumʼ using next‐generation sequencing

AbstractPeach trees showing witches’‐broom disease symptoms in the northwest of Iran were sampled for phytoplasma detection. PCR assays and Sanger sequence analyses indicated that ʻCandidatus Phytoplasma phoeniciumʼ was associated with peach witchesʼ‐broom disease. Virtual RFLP analyses of the 16S rRNA gene indicated that ʻCa. Phytoplasma phoeniciumʼ strain, which was prevalent in the northwest of Iran belonged to 16SrIX‐C subgroup. For the genomic characterization of Iranian ʻCa. Phytoplasma phoeniciumʼ strain, total DNA extracted from the infected peach trees was subjected to Illumina next‐generation sequencing. The NGS sequencing resulted in 41157647 read pairs of raw data. The raw reads length was 150 bp and the insert size was 350 bp. Assembly and genes clustering finally resulted in 750 contigs with a total size of 228345 bp. The GC content of sequence was 33.2%, N50 value was 259 and L50 value was 256. According to NCBI‐ PGAP annotation the genome of Prunus persica phytoplasma PP2 contained 410 genes including 377 CDSs with protein, 10 rRNAs, nine tRNAs and 14 pseudogenes.

Amplificação isotérmica mediada por loop para detecção de patógenos de plantas

Disease control is crucial to minimize potential losses in agriculture and thereby maintain high crop yield. However, for its effectiveness, the pathogen must be detected early and correctly in the production fields. Different methods of diagnosis can be used, from those based on symptoms to molecular tests. Loop-mediated isothermal amplification (LAMP) is a molecular technique that has been widely used in several biological fields, due to the ease with which it can be applied. The reaction can be carried out in a single thermal condition, due to the use of Bst DNA polymerase, isolated from the bacterium Bacillus stearothermophilus, which has high displacement activity. LAMP is a highly exponential amplification method that produces the target DNA in amounts 109 -1010 times between 45 and 60 minutes at 60-65°C. Its advantages are the visualization of results directly with the naked eye and the fact that it does not need sophisticated equipment for its application. In phytopathology, the technique has been gaining prominence in the detection of fungi, viruses, bacteria, nematodes and phytoplasmas, as well as in the monitoring of fungicide-resistant fungi. LAMP can benefit agriculture so that early, accurate and sensitive diagnostics can be carried out in the fields of cultivation and minimize losses caused by diseases. In this review, we present and discuss LAMP tests, developed for plant pathogens detection, which can be useful for researchers who wish to use the technique in their research area

Candidatus phytoplasma malaysianum (16SrXXXII) associated with Elaeocarpus sylvestris decline in South Korea

Phytoplasma has been newly detected in Elaeocarpus sylvestris, a tree species that grows on the island of Jeju, South Korea. Various symptoms of infection of E. sylvestris have appeared since 2013, including loss of leaf coloration, such as yellowing, darkening and branch dieback. We analyzed leaves and seeds of E. sylvestris sequences infected by phytoplasma, and found partial 16S rRNA sequences to be 99.5%, and secA genes 98.2% correlated with Malaysian periwinkle virescence group strain ELY-BN1, MaPV, MOP, MYD, and TtWB. Analyzing these sequences and through the virtual restriction fragment length polymorphism (virtual-RFLP), we report here the first occurrence of Elaeocarpus decline associated with a phytoplasma strain belonging to 16SXXXII groups, as well as the first detection of phytoplasma in the seeds of E. sylvestris in South Korea.

Presence and seed transmission of phytoplasmas in tomato fields in Italy

Tomatoes are the second most consumed vegetable, quite often severely affected by phytoplasma-associated diseases worldwide. The identification and confirmation that the phytoplasmas associated with the disease in Southern Italy are also transmitted through seeds was achieved. DNA extractions from mother plants collected in 2020 and 2021 and the seedlings from these plants, tested at different times, allowed the detection of phytoplasmas in both mother plants and seedlings grown under an insect-proof greenhouse. Phytoplasmas enclosed in groups 16SrX-B (‘Candidatus Phytoplasma prunorum’) and 16SrV-A (‘Ca. P. ulmi’) were identified in 2020 samples, and the same phytoplasma groups were identified in the seedlings. Phytoplasmas enclosed in groups 16SrI and 16SrXII were identified in mother plants collected in 2021 and were also identified in the seedlings. Moreover, a difference in germination percentage was observed. The 16SrXII-A group phytoplasmas detected in the mother plants and were identified as variants St5 for the stamp gene, tuf-b1 for the tuf gene and Vm44 for the vmp1 gene. This research is a significant confirmation of previous laboratory results on transmission of phytoplasmas through seeds from several diseased plant species. The epidemiological role of phytoplasma seed transmission is an essential knowledge for efficient management strategy planning for a seed production aimed at reducing the impact of phytoplasma-associated diseases in horticultural relevant crops such as tomatoes.

Phytoplasma detection in “huanglongbing”-infected citrus in the Caribbean

Phytoplasma detection in “huanglongbing”-infected citrus in the Caribbean

Molecular detection of aster yellows phytoplasmas in ashwagandha plants in South India

Ashwagandha is a native medicinal plant of Indian sub-continent grown in different parts of the world for its medicinal value. The little leaf symptoms were observed in ashwagandha plants in Tamil Nadu, India. The nested PCR with the primer pair fU5/rU3, amplified 890 bp fragment of the 16S rRNA region of phytoplasma from five symptomatic leaf samples of ashwagandha plants. The nucleotide sequence analysis of 16S rRNA region revealed that the sequences of the five samples were identical and confirmed 99% nucleotide sequence identity with 16S ribosomal RNA gene sequences of ‘Candidatus Phytoplasma asteris’. This result indicate the presence of aster yellows phytoplasmas in this plants from South India.

Genome-informed design of a LAMP assay for specific detection of aster yellows phytoplasmas occurring in grapevines in South Africa

Genome-informed design of a LAMP assay for specific detection of aster yellows phytoplasmas occurring in grapevines in South Africa

Detection and molecular characterization of phytoplasmas in mango seedlings

Detection and molecular characterization of phytoplasmas in mango seedlings

Detection of phytoplasmas of subgroups 16SrII-C and 16SrII-D in Murraya koenigii with leaf yellowing and flat stem symptoms in India

Detection of phytoplasmas of subgroups 16SrII-C and 16SrII-D in Murraya koenigii with leaf yellowing and flat stem symptoms in India

Detection of blueberry stunt phytoplasma in Eastern Canada using cpn60-based molecular diagnostic assays

Blueberry stunt phytoplasma (BBSP; ‘Candidatus Phytoplasma asteris’) is an insect-vectored plant pathogen that causes severe yield losses in blueberry (Vaccinium corymbosum), which is the most valuable fruit crop in Canada. Rapid, field-based diagnostic assays are desirable tools for the control of BBSP, as part of an integrated, proactive approach to production management termed biovigilance. We designed and validated a chaperonin-60 (cpn60)-targeted LAMP assay for detection of BBSP, providing a rapid, low cost, field-deployable diagnostic option. Our validation demonstrates that the assay is reproducible, with high analytical specificity and improved sensitivity when compared with 16S rRNA nested PCR. We applied the validated LAMP assay to nearly 2000 blueberry samples from Québec and Nova Scotia over three growing seasons (2016–2018). Our surveys revealed that BBSP is present in most sites across both provinces, though detection of the pathogen in individual plants varied in different tissues across sampling dates and across years, and evidence of spread between plants was limited. To quantify pathogen load in select plants, we designed additional qPCR and ddPCR assays, also based on cpn60. We found that pathogen load fluctuates in individual plants, both within and between growing seasons. Finally, we designed an interactive map to visualize the results of our surveys. These results provide a validated diagnostic assay that can be used as part of a biovigilance strategy for detecting and controlling infections caused by BBSP.

First detection and molecular characterization of grapevine phytoplasmas in Kosovo

AbstractA search for phytoplasma‐associated diseases was conducted for the first time in the main grapevine‐growing localities of the Dukagjini plain in Kosovo. A total of 144 samples were collected from grapevine cultivars displaying leaf yellowing, reddening, discolouration and irregular wood ripening, and analysed using nested and quantitative PCR assays. These assays showed that 35.4% of samples belonging to eight cultivars were positive to the presence of phytoplasmas in the 16SrXII group. The 16S rDNA phytoplasma sequences obtained from 15 samples shared identity greater than 99.5% with ‘Candidatus Phytoplasma solani’. Sequence analysis of the tuf gene showed that the strains found in Kosovar grapevines are in the tuf‐type b1 group, sharing 99.6% to 99.8% identity with ‘Ca. P. solani'‐related strains associated with the “bois noir” grapevine disease in many European countries; the secY gene sequences, on the other hand, shared 100% identity with ‘Ca. P. solani' strains from Bosnia and Herzegovina, Serbia, Croatia and Turkey. This study constitutes the first report on the presence and molecular characterization of phytoplasmas in Kosovar vineyards. Based on these results, it is recommended that testing for phytoplasma be included in the certification program for grapevine in Kosovo.

A new host record for Candidatus Phytoplasma cynodontis (16Sr XIV-A) associated with phyllody and fasciation of linseed (Linum usitatissimum) from India.

Linseed commonly called as flaxseed (Linum usitatissimum Linn.) is an important oilseed crop cultivated widely in Northern parts of Karnataka. During, 2019 (January-February), a characteristic disease was noticed with symptoms that resembled phytoplasma or like disease symptoms. The incidence was ranged from 6·5 to 16·5% in the experimental station of Raichur Agricultural University. The typical symptoms observed were virescence of floral parts, fasciation of the inflorescence axis, phyllody, stunted and flattened stem with reduced leaves. Symptomatic and healthy samples were collected and processed for molecular detection of phytoplasma. Total DNA was isolated from four infected plants and two healthy plants. The 16S rDNA region was amplified using P1/P7 followed by R16F2n/R16R2 primer pair which showed the amplification of expected amplicon size from all four infected samples. Furthermore, the SecA gene was amplified using SecA1/SecA3 primers. The PCR amplified products were subjected for direct sequencing from both directions and the consensus sequences were obtained and nBLAST search analysis revealed that the 16Sr RNA and SecA sequences were sharing maximum similarity (100%) with the reference sequence of Ca. P. cynodontis. The sequences were analysed phylogenetically by constructing a Phylogram independently by NJ method along with reference sequence of 16S rRNA region and SecA region retrieved from GenBank database showed that the phytoplasma sequence from linseed phyllody of the present study placed in a distinct clade along with reference sequence of "Ca. P. cynodontis" thus confirming the identity phylogenetically. Furthermore, iPhyClassifier and virtual RFLP proved that the phytoplasma belonged to 16SrXIV (subgroup A) phytoplasma. Previously linseed is known to be associated with 16SrII-D phytoplasma but the association of the 16SrXIV-A group of phytoplasma is not reported so far. Therefore, this is the new host record for Ca. P. cynodontis (16SrXIV-A) phytoplasma associated with linseed stem fasciation, phyllody from India.

Evaluating LAMP assays for detection of phytoplasmas classified in different ribosomal groups

Evaluating LAMP assays for detection of phytoplasmas classified in different ribosomal groups

Detection and molecular characterization of phytoplasmas based on 16s rDNA gene region by phylogenetic and in silico RFLP analysis of local grapevine cultivars in Şanlıurfa and Adıyaman

During the autumn seasons of 2016-2019, several surveys were carried out in grapevine (Vitis vinifera L.) cultivation areas in Şanlıurfa and Adıyaman provinces of South-eastern part of Turkey, to identify grapevine plants exhibiting phytoplasma-like symptoms including yellowing, small leaf formation, chlorosis, short internodes, severe redness and inward curling and to detect and characterize the causal agent responsible for these symptoms. Purified DNA obtained from collected samples was examined for the highly conserved phytoplasma 16S rDNA gene via nested-PCR with the universal phytoplasma-specific primer sets. DNA amplification via nested-PCR/RFLP analyses with some restriction enzymes confirmed the suspected correlations between the disease symptoms observed and phytoplasma presence in the samples. F2n/R2 amplicons of the phytoplasma strains obtained from the samples showed 99.99% homology with each other and 99% homology with phytoplasma DNA partial sequences belonging to some groups deposited in the GeneBank database. Sequencing and phylogenetic analysis revealed that collected isolates have 99% sequence identity with ‘Candidatus phytoplasma solani’ (16SrXII-A) and ‘Ca.P. asteris’ (16SrI-B). According to our knowledge, the results of this study is the first report of the phylogenetic relationship of phytoplasmas infecting different grapevine cultivars based on 16S rDNA gene. Genetic diversity of genes other than 16S rDNA of the pathogen causing these infections is under study.

Phytoplasma diseases of plants: molecular diagnostics and way forward.

Phytoplasmas are obligate phytopathogenic bacteria associated with devastating diseases in hundreds of crops across the world. They have been responsible for huge economic losses in many crop plants for decades now. Isolation and establishment of axenic culture of phytoplasma in complex media is a recent progress in phytoplasma research. Earlier methods for phytoplasma disease detection included symptom profiling, microscopy, serology and dodder transmission studies. With advancement in the field of molecular biology, phytoplasma diagnostics and characterisation witnessed radical improvement. Starting from PCR amplification which often necessities a nested PCR on account of low titre of phytoplasmas, to the closed tube quantitative PCR assays and then the ddPCR, an array of diagnostics have been developed for phytoplasma. The isothermal diagnostic platforms are the latest addition to this and the Loop Mediated Isothermal Amplification (LAMP) assay has been applied for the detection of phytoplasma from several hosts. The futuristic approach in phytoplasma detection will be very likely provided by an integration of nanotechnology and molecular diagnostics. Phytoplasma disease management majorly relies on early detection, vector control, use of disease free planting materials and cultivation of resistant varieties. Hence understanding the molecular mechanism of phytoplasma-host interaction is as important as timely and accurate detection, in the management of phytoplasma diseases. Further, the changing climatic scenario and global warming may lead to an upsurge in the phytoplasma diseases spread and severity across the world, making disease management even more challenging.

Association of 16SrII-C subgroup phytoplasma related strains with witches’-broom symptoms in Sweet orange and Orlando tangelo in Iran

Sweet orange (Citrus sinensis) and Orlando tangelo (C. paradisi × C. reticulata) trees showing witches’-broom symptoms were observed during 2018 in southern Kerman province, Iran. Detection of phytoplasmas in the symptomatic trees was confirmed by nested PCR assays using the phytoplasma specific primers. Sequence analysis indicated that the phytoplasmas associated with the witches’-broom affected trees belong to the 16SrII-C subgroup phytoplasma. This is the first report of a 16SrII-C phytoplasma associated with witches’-broom symptoms of Sweet orange and Orlando tangelo trees in the world.