Phytoplasma Etiology Research Articles

Phytoplasma etiology and epidemiology of wheat streak and stunting disease in Central India.

The online version contains supplementary material available at 10.1007/s13205-024-04114-3.

Detecção de 'CA. Phytoplasma phoenicium' em plantas de pervinca (Cathranthus roseus) em Uttar Pradesh, Índia

Cathranthus roseus also known as periwinkle, an ornamental plant contains several medicinal values, was found with the symptoms of little leaf and witches’ broom at Shahjahanpur location with the incidence of up to 8%. The phytoplasma etiology was confirmed through scanning electron microscopy examination in all the four-leaf samples. Molecular analysis through PCR with universal primer pairs P1/P6 followed by nested PCR with R16F2n/R16r2 primers yielded ~1.2kbp amplicons in all the four symptomatic leaf samples. One amplicon was eluted, purified, sequenced, and used in BLASTn searches, which showed maximum identity of periwinkle isolate with several isolates of 16SrIX group of phytoplasma. Further, phylogenic analysis and in silico RFLP confirmed the association of 16SrIX-C subgroup phytoplasma in little leaf and witches broom plants which is the first report from India.

Multilocus sequence analysis of a ‘Candidatus Phytoplasma australasia’-related strain associated with peanut little leaf disease in India

Severe little leaf and excessive shoot proliferation symptoms typical of the peanut little leaf (PnLL) disease were observed in different peanut (Arachis hypogaea) fields, variety K-6, at Kadiriand Gooty regions, of Andhra Pradesh, India, during the monsoon season of 2018–19. Disease incidence was 2–3%. The phytoplasma etiology of the recorded disease was confirmed by amplifying ~1.25 kb DNA products of 16S rRNA gene from symptomatic plants using nested polymerase chain reaction with phytoplasma specific primers, P1/P7 and R16F2n/R16R2, respectively. A further confirmation of the phytoplasma etiology was obtained by amplification of 840 bp, 1094 bp and 465 bp DNA fragments using phytoplasma specific primers targeting secA, tuf and SAP11 genes, respectively. Sequence and phylogenetic analyses of the amplified 16S rRNA, secA, tuf and SAP11 genes revealed that the detected phytoplasma is a member of the peanut witches’-broom phytoplasma group or 16SrII group (‘Candidatus Phytoplasma australasia’). Also, on the basis of computer-simulated RFLP (= in silico RFLP) analysis of amplified 16S rRNA gene, the detected phytoplasma was assigned to subgroup D (16SrII-D). Parthenium hysterophorus plants showing witches’ broom and Cleome viscosa plants with little leaf symptoms, both collected in the mentioned peanut fields were also infected by similar strain of phytoplasma which proved to be identical with each of the molecular marker employed to the peanut-infecting agent in India. This is the first report on the association of ‘Ca. P. australasia’ (16SrII-D subgroup phytoplasma) with PnLL disease in India.

Identification and characterization of Candidatus Phytoplasma trifolii (16SrVI-D) inducing shoot proliferation disease of potato in India

Potato (Solanum tuberosum) is a widely cultivated and commercially important vegetable crop widely grown in India. Profuse shoot proliferation symptoms were observed in potato plants of two varieties, Kufri Khyati and Kufri Surya at the Research plots of All India Co-ordinated Research Project on Potato, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, India during February, 2017. The symptomatic plants were tested for phytoplasma etiology. Nested and semi-nested PCR assays primed by primer pairs P1/P7 and 3Far/3Rev for 16S rRNA gene and secAF1/R3 for secA gene amplified DNA fragments of 1.3 kb (3Far/3Rev) and 840 bp (secAF1/R3), respectively from all the symptomatic potato samples but not from any asymptomatic plants. The 16S rDNA sequences of potato tuber (Acc. no. KY815100, Kufri Khyati; MG566064, Kufri Surya) and shoot phytoplasma strains (Acc. no. KY815099, Kufri Khyati; MG566063, Kufri Surya) shared 100% nucleotide sequence identity with 16S rDNA sequence of identified phytoplasma strains belonging to 16SrVI-D subgroup associated with Lens culinaris witches’ broom (Acc. no. KY439869), brinjal little leaf (Acc. no. KX284698) and Brassica oleracea var. capitata witches’ broom (Acc. no. KX671553) in pair wise sequence comparison. Sequence comparison of 840 bp of secA gene of potato phytoplasma strains from tuber of variety, Kufri Khyati (Acc. no. KY815101) and variety Kufri Surya (Acc. no. MG566065) showed 99% sequence identity to secA gene sequences of brinjal little leaf phytoplasma strains (Acc. no. KX610808, KX894792, KY064175), which belonged to 16SrVI-D subgroup. Phylogenetic analysis of 16S rRNA and secA gene sequences, and virtual RFLP analysis of 16S rDNA sequences of potato shoot proliferation phytoplasma strain confirmed their clustering and grouping with strains of clover proliferation subgroup D. Our results confirmed potato as a new host of 16SrVI-D subgroup of phytoplasmas in the world. The 16SrVI-D strain is wide spread in India and cause significant yield losses and hence poses serious socio-economic threat to commercially grown potato crops.

First report of occurrence of Candidatus Phytoplasma trifolii-related strain causing witches’ broom disease of chilli in India

During February 2017, 20% of the chilli plants in fields at the Indian Agricultural Research Institute, New Delhi, India showed witches’ broom symptoms of suspected phytoplasma etiology. Phytoplasma association was confirmed by utilizing a nested PCR assay with universal primer pairs P1/P7 and 3F/3R that target the phytoplasma 16S rRNA gene. The chilli witches’ broom phytoplasma (CWBP) sequence (Acc. No. KY612251) shared 100% nucleotide sequence identity with the 16S rDNA sequences of identified strains of ‘Candidatus Phytoplasma trifolii’ subgroup D (16SrVI-D) phytoplasmas associated with Lens culinaris’ witches’-broom (Acc. No. KY439869), brinjal little leaf (Acc. No. KX284698), Brassica oleracea var. capitata witches’ broom (Acc. No. KX671553) and Apium graveolens white leaf (Acc. No. KX671551) in pair wise sequence comparisons. The CWBP strain also clustered with strains of ‘Candidatus Phytoplasma trifolii’ subgroup D in phylogenetic comparison analysis. This is the first report of association of ‘Ca. P. trifolii’ subgroup D with witches’ broom disease of chilli in India.

Molecular characterization of phytoplasma of 16SrI-B group association with Acalypha indica in India

The typical phytoplasma symptoms of little leaf, yellowing, chlorosis, witches' broom, and stunting growth were observed on Acalypha indica plants during the field survey conducted at Lucknow and surrounding districts in year 2015-2016. To confirm the association and possibility of phytoplasma etiology, PCR assays were performed using universal primer pairs (P1/P6) and nested primer pairs (R16F2n/R2) in a total of five diseased samples along with control. A~1.2Kb amplicon was observed in nested PCR assay in diseased sample; however, no band was observed in control sample. The positive amplicons were sequenced for 16S rDNA and used for the virtual RFLP analysis and phylogenetic studies. BLASTn search showed 99-100% sequence identities with the 'Candidatus phytoplasma asteris' members (16SrI group)and phylogenetic analysis showed closest relationship with member of 16SrI group. The virtual RFLP assigned it as a member of 16SrI-B subgroup. This is the first record of phytoplasma association of 'Ca. P. asteris' subgroup B with A. indica in the world.

Molecular characterization, vector identification and sources of phytoplasmas associated with brinjal little leaf disease in India.

Brinjal little leaf (BLL) is a widespread disease of phytoplasma etiology in India that induces severe economic losses. Surveys were conducted in eight brinjal-growing states of India during July 2014 to September 2015 and eighteen BLL samples showing little leaf, phyllody and witches' broom symptoms were collected for phytoplasma identification. Presence of phytoplasmas was confirmed in all the eighteen BLL samples using polymerase chain reaction with phytoplasma-specific primer pairs (P1/P6, R16F2n/R16R2). Pair wise sequence comparison and phylogenetic relationship of 16S rRNA gene sequences of BLL phytoplasma strains confirmed that sixteen out of eighteen BLL strains belonged to clover proliferation phytoplasma (16SrVI) group and two BLL strains (GKP-A and GKP-B) from Gorakhpur, Uttar Pradesh, were classified under 16SrII group. Further virtual RFLP analysis of 16S rDNA sequences allowed finer classification of BLL strains into 16SrII-D and 16SrVI-D subgroups. BLL phytoplasma strains belonging to 16SrVI-D subgroup were found as the most widespread phytoplasma strains associated with BLL disease in India. 16SrVI-D subgroup phytoplasma association with two symptomatic weed species viz. Cannabis sativa subsp. sativa at Noida, Uttar Pradesh and Portulaca oleracea at IARI fields, New Delhi was also confirmed by nested PCR assays with similar set of phytoplasma-specific primers, pairwise 16S rDNA sequence comparison, phylogeny and virtual RFLP analysis. Out of five identified leafhopper species from BLL-infected fields at Noida, Uttar Pradesh and Delhi, only Hishimonas phycitis was identified as carrier and natural vector of 16SrVI-D subgroup of phytoplasmas by nested PCR assays, sequence comparison, phylogeny, virtual RFLP analysis and transmission assays.

Molecular characterization of phytoplasmas of 'Clover proliferation' group associated with three ornamental plant species in India.

Suspected phytoplasma symptoms of little leaf, yellowing, chlorosis, phyllody, witches’ broom, and stunting were observed on ten different ornamental plant species at New Delhi, Andhra Pradesh, Haryana, Bengaluru, and Pune, India, during March to July 2016. To investigate the possibility of phytoplasma etiology, PCR assays were performed using universal primer pairs (P1/P7 followed by 3Far/3Rev) specific to the phytoplasma 16Sr RNA gene. First round PCR amplification with primer pair P1/P7 did not yield expected 1.8 kb product of 16S rRNA region from any of the 17 symptomatic samples. However, 1.3 Kb amplicons were observed in nested PCR assays with 3Far/3Rev primer pair in symptomatic leaf samples of Hibiscus rosa-sinensis L. (Pune isolate), Saponaria officinalis L. (Pune isolate), and Allamanda cathartica L. (Delhi isolate). No amplifications were observed in any of the other tested symptomatic and non-symptomatic plant samples either in first round or second round of nested PCR assays with phytoplasma specific primer pairs. Pairwise sequence comparison of 16S rDNA sequences of the five positive phytoplasma strains of A. catharica, H. rosa-sinensis, and S. officinalis in the present study revealed 99–100% sequence identities with strains of ‘clover proliferation’ (16SrVI) group. Phylogenetic and virtual RFLP analysis of 16S rDNA sequences of the five identified phytoplasma strains belonging to three ornamental species further confirmed their clustering and grouping with member strains of ‘clover proliferation’ subgroup D. This is the first record of the phytoplasma association of ‘clover proliferation’ subgroup D with H. rosa-sinensis, S. officinalis, and A. cathartica in the world.

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

AbstractSuspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.

Molecular Characterization of Aster Yellows Subgroup 16SrI‐B Phytoplasma in Verbena × hybrida

AbstractSymptoms resembling phytoplasma disease were observed on Verbena × hybrida in Alanya, Turkey, during October 2013. Infected plants were growing as perennials in a flower border and showed symptoms of discoloured flowers, poor flower clusters, inflorescences with a small number of developed flowers and thickened fruit stalks. Electron microscopy examination of the ultra‐thin sections revealed polymorphic bodies in the phloem tissue of leaf midribs. The phytoplasma aetiology of this disease was confirmed by polymerase chain reaction of the 16S rRNA gene, the 16–23S rRNA intergenic spacer region and the start of the 23S rRNA gene using universal phytoplasma‐specific primer pair P1A/P7A, two ribosomal protein (rp) genes (rpl22 and rps3) (the group‐specific primer pair rp(I)F1A/rp(I)R1A) and the Tuf gene (group‐specific fTufAy/rTufAy primers) generating amplicons of 1.8 kbp, 1.2 kbp and 940 bp, respectively. Comparison of the amplified sequences with those available in GenBank allowed classification of the phytoplasma into aster yellows subgroups 16SrI‐B, rpI‐B and tufI‐B. This is the first report about molecular detection and identification of natural infection of the genus Verbena by phytoplasma and occurrence of the aster yellows group phytoplasma on an ornamental plant in Turkey.

The first Stolbur Phytoplasma occurrence on two St. John's Worth species (Hypericum perforatum L. and Hypericum barbatum L.) in Serbia

The symptoms indicating phytoplasma like leaf yellowing, reddening and early drying were observed on two St. John’s worth species (Hypericum perforatum L. and Hypericum barbatum L.) on infected fields (Pancevo, Indjija and Stara Pazova) in Serbia in 2008. Electron microscopy examination of the ultra-thin sections revealed the presence of numerous polymorphic phytoplasma-like bodies in the phloem tissue of leaf midribs and petioles. The phytoplasma etiology was confirmed by polymerase chain reaction (PCR) using 3 sets of primers (P1/P7, P1/16S-Sr and R16F2n/R16R2). Restriction fragment length polymorphism (RFLP) analysis of amplification products of 1.2 kb (obtained with R16F2n/R2 primer pair) in 51 from 60 symptomatic plants, indicated the presence of 16SrXII-A phytoplasma subgroup from all three affected localities. Sequence of R16F2n/R2 amplicon for representative phytoplasma H. perforatum L. isolate Hp22 was deposited in the GeneBank with accession number JQ033928. This is the first report of the natural occurrence of Stolbur phytoplasma in two cultivated St. John’s worth species in Serbia. Key words: Hypericum perforatum, Hypericum barbatum, phytoplasma diseases, 16SrXII-A subgroup.

The first detection of ‘Candidatus Phytoplasma trifolii’ in Rhododendron hybridum

Four Rhododendron hybridum plants (from cvs Moravanka and Don Juan), all exhibited symptoms of shortened axillary shoots, reduced leaves with vein clearing and yellowing, undeveloped flowers, and general stunting in a rhododendron nursery garden in southern Bohemia in 2007. Electron microscopy examination of ultra-thin sections revealed the presence of numerous polymorphic phytoplasma-like bodies in the phloem tissue of leaf midribs and petioles. The phytoplasma etiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal phytoplasma primers. Restriction fragment length polymorphism (RFLP) analysis of amplification products obtained with a R16F2/R16R2 primer pair from all symptomatic plants indicated the presence of phytoplasma from the 16SrVI-A subgroup. A detailed comparison of the amplified sequences and phylogenetic analysis confirmed that the phytoplasma belonged to the subgroup 16SrVI-A (clover proliferation phytoplasma group). This is the first report of the natural occurrence of ‘Candidatus Phytoplasma trifolii’ in plants of Rhododendron hybridum.

CORN REDDENING: THE DISEASE AND BREEDING FOR RESISTANCE

Corn reddening (CR), observed for the first time in Serbia in 1957, has occurred sporadically there ever since, especially in the Banat area. However, in 2002 and 2003 severe outbreaks took place in late July-early August. Initial symptoms of CR consist in the apperance of a red-violet color on the leaves, leaf sheaths, husks and the bare portion of the internodes. Discolourations typically appear at the milk maturity stage, being strongest on top leaves, around the mid-rib and along the edges of the leaf blade, from base to tip. The ears are underdeveloped and kernels are shrivelled. Soon after symptom development, affected plants wilt, the foliage dessicates rapidly, most of the red pigmentation disappears, and affected plants eventually die. Controversy on the biotic or abiotic nature of CR seems to have been settled by the recent discovery of a possible phytoplasma aetiology. Whatever the cause, an interesting source of apparent resistance to CR was identified in a local maize population which may be used in breeding programmes.

‘Candidatus Phytoplasma lycopersici’, a phytoplasma associated with ‘hoja de perejil’ disease in Bolivia

New diseases known locally as 'hoja de perejil' of tomato (Lycopersicon esculentum Mill) and 'brotes grandes' of potato (Solanum tuberosum L.) were first recognized in surveys of production fields in Bolivia during 2000-2003. Alfalfa (Medicago sativa) witches' broom and little leaf diseases of native weeds Morrenia variegata and mora-mora (Serjania perulacea) were also identified near to production fields. Phytoplasma aetiology was attributed to each of these diseases following detection and initial identification of aster yellows group (16SrI) phytoplasmas in all five diseased plant species. While potato, alfalfa and mora-mora plants contained indistinguishable 16SrI-B strains, 'hoja de perejil' (THP) and morrenia little leaf (MVLL)-associated phytoplasma strains shared 97.5 % 16S rRNA gene sequence similarity with 'Candidatus Phytoplasma asteris' and related strains and <95 % similarity with all other 'Candidatus Phytoplasma' species. Phylogenetic analysis of 16S rRNA gene sequences indicated that the THP and MVLL phytoplasmas represent a novel lineage within the aster yellows (16SrI) group and, on the basis of unique 16S rRNA gene sequences, we propose that THP and MVLL phytoplasmas represent 'Candidatus Phytoplasma lycopersici', with THP as the reference strain.

Aetiology of Opuntia ficus‐indica malformations and stunting disease

AbstractThe phytoplasma aetiology of epidemic stunting of cladodes and stunted growth observed in a cactus pear plantation in Carlentini (Sicily, Italy) was investigated with graft inoculation trials and PCR/restriction fragment length polymorphism analyses of 16S ribosomal RNA (rRNA) gene and S3 ribosomal protein gene. After sequencing of the 1525‐bp from 16S rRNA gene of both naturally infected and graft‐inoculated symptomatic cactus pear samples, phytoplasma TS belonging to ribosomal subgroup 16SrII‐C were identified as aetiological agents of this worldwide spread disease.

Identification of RAPDs associated with resistance to lethal yellowing of the coconut ( Cocos nucifera L.) palm

Lethal yellowing (LY) is a disease of phytoplasma etiology that has devastated coconut ( Cocos nucifera L.) cultivation for over a century. The only effective means for controlling LY is replanting with resistant germplasm. Breeding coconuts for any desirable traits is hindered by the long generation time, low multiplication rate, and ineffective clonal propagation of this crop. Additionally, the lack of genotypes adequate for identifying markers linked with LY resistance demands alternative approaches. We identified three coconut populations which could be used for this purpose, and comprised the susceptible West African Tall (WAT), the resistant Malayan Yellow Dwarf (MYD), and a resistant population of Atlantic Tall (AT) plants. This latter material was closely related to WAT, and both of them were distantly related to MYD. The objective of this work was to use those populations for identifying RAPDs associated with LY resistance. RAPDs were considered as associated with that trait if their frequencies were high in MYD and AT, and low in WAT. A total of 82 RAPDs could differentiate the DNA pools from MYD and WAT, and 12 of them appeared at frequencies ≥0.85 in MYD, and ≤0.15 in WAT. Five of such markers were in AT at frequencies of 0.80 (−B4 570) or 1 (−A11 990, −B11 1140, −AL3 1160 and −AL7 350). The results are discussed in terms of the prospects for marker-assisted selection of LY-resistant plants.

First report of Spartium witches’ broom disease in Spain

Symptoms of witches’ broom and decline were observed on Spanish broom ( Spartium junceum ) in Catalonia, Spain. The appearance of witches’ brooms, which developed from axillary buds on this woody perennial shrub, was followed by a drying of foliage. Affected plants eventually withered and died within a few years. A similar disease of S. junceum described in Italy was attributed to a phytoplasma aetiology (Marcone et al ., 1996). Samples from both affected and symptomless plants were analysed for phytoplasma infection by a nested polymerase chain reaction (PCR) assay employing rRNA gene primer pairs P1/P7 (Smart et al ., 1996) followed by U5/U3 (Lorenz et al ., 1995) or R16F2/R2 (Lee et al ., 1995). A PCR product of expected size (1·2 kb) was amplified by nested R16F2/R2-primed PCR from all nine affected plant sample DNAs, whereas no product was amplified from four symptomless plant samples. No products were obtained from either plants with or without symptoms by nested U5/U3-primed PCR. R16F2/R2 products from two S. junceum plants (T-500 and T-701) were analysed further by reamplification of products by PCR using nested 16S rRNA gene primer pair R16(V)F1/R1 or R16(X)F1R1 (Lee et al ., 1995). A 1·1-kb product was obtained from both plants only with the apple proliferation phytoplasma (16SrX) group-specific primer pair. Identification and classification of S. junceum -infecting phytoplasmas as strains belonging to 16SrX group, subgroup D, were determined by restriction fragment length polymorphism analysis of R16F2/R2-primed rDNA products using six endonuclease enzymes. Phylogenetic analysis of 16S rDNA gene sequences also delineated phytoplasma strain T-701 (EMBL accession no. AJ430067) as a 16SrX group strain most closely related to the phytoplasma associated with Spartium witches’ broom in Italy. This is the first report of a phytoplasma disease of S. junceum in Spain.

Association of aster yellows subgroup 16SrI‐B phytoplasmas with a disease of Rehmannia glutinosa var. purpurea

A new phytoplasma disease of Rehmannia glutinosa var. purpurea was observed in the Czech Republic in 1998. Infected plants showing severely proliferating shoots, leaves reduced in size with vein clearing and chlorosis, shortened internodes and virescent petals died in advanced stages of the disease. Electron microscopy examination of the ultra‐thin sections revealed the presence of numerous polymorphic bodies in phloem tissue of leaf midribs and petioles. The disease was successfully transmitted from infected plant via a dodder bridge into periwinkle (Catharanthus roseus). The phytoplasma aetiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal primers R16F2/R16R2. Restriction fragment length polymorphism (RFLP) analysis of amplification products indicated the presence of aster yellows related phytoplasmas (16SrI‐B) in naturally infected samples of R. glutinosa var. purpurea and in symptomatic periwinkle after dodder transmission of the agent. A comparison of the amplified sequence with 17 sequences available in the GenBank confirmed the classification of the phytoplasma in the subgroup 16SrI‐B. This is the first report of natural occurrence of phytoplasma‐associated disease in R. glutinosa var. purpurea.