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

Suspected 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 detection and identification of phytoplasmas associated with Catharanthus roseus,Pinus eldarica,and Petunia hybrida in southeastern Iran's urban green spaces

Given the potential for urban green spaces to provide fresh and healthy environments for humans, exploring the issues that threaten plants in these places is crucial. Phytoplasma-related symptoms were encountered on some plants in urban green spaces in the province of Kerman, southeastern Iran, between 2017 and 2019. Affected periwinkles and petunias exhibited phytoplasma disease symptoms, including virescence, phyllody, and witches'-broom. However, ball or disc-like shoot proliferation symptoms were noticed on the trunks and branches of pine trees. PCR was performed with phytoplasma-detecting universal primers, targetting and amplifying the 16S rRNA gene, and determining whether phytoplasmas are implicated in the symptomatic plants. The infection of the symptomatic plants was confirmed using nested-PCR amplification of expected DNA sizes for phytoplasmas. No product, however, was amplified from sampled symptomless plants. The sequencing of nested-PCR products was performed to obtain sequences encasing the standard F2nR2 fragments. The resulted sequences were submitted to iPhyClassifier, the universal phytoplasma classification platform, for the taxonomic assignment of the found phytoplasmas compared with previously identified ‘Candidatus Phytoplasma’ species, groups, and subgroups. The results revealed that phytoplasma strains related to the species ‘Ca. P. trifolii’ (16SrVI-A subgroup) infect periwinkles and pines. However, strains from the species ‘Ca. P. aurantifolia’ (16SrII-D subgroup) and ‘Ca. P. phoenicium’ (16SrIX-C subgroup) were found in petunias and periwinkles, respectively. To the best of our knowledge, phytoplasmas from the 16SrVI-A and 16SrII-D subgroups are the first reported to infect these plants in Kerman province, while a related strain from the subgroup 16SrIX-C is the first recorded to infect periwinkles in Iran and the second in the world.     

Detection and characterisation of phytoplasma strains associated with field bindweed witches’ broom disease in Iran

Abstract

During 2013–2015 surveys in Fars, Lorestan and Yazd provinces (Iran), a field bindweed witches’ broom (FBWB) disease was observed. The main symptoms were reduction of leaves size, yellowing, internode shortening, witches’ broom and stunting. The agent of FBWB was dodder transmitted to periwinkle plants inducing phytoplasma-type symptoms. Amplifications of nearly 1.8 and 1.2 kbp were, respectively, obtained from 15 symptomatic bindweed plants and 28 symptomatic dodder-inoculated periwinkles. Virtual RFLP analyses showed that the phytoplasma detected belonged to 16SrXII-A subgroup, and it was the same in all the samples examined; phylogenetic analyses confirmed it as a ‘Candidatus Phytoplasma solani’-related strain. This is the first report of 16SrXII-A phytoplasmas presence in bindweed plants showing witches’ broom symptoms in Fars, Lorestan and Yazd provinces. As a perennial widespread weed, it may act as a 16SrXII-A phytoplasma source for alfalfa, grapevine, Sophora alopecuroides, tomato, hemp and Japanese spindle reported diseases in these Iranian provinces.     

Characterization of 16SrII-D subgroup associated phytoplasmas in new host plants in Egypt

Samples of three plant species displaying phytoplasma symptoms were collected from Kafrelsheikh and Al-Gharbia governorates during 2014. Witches’ broom and virescence symptoms were observed in periwinkle (Catharanthus roseus). Onion (Allium cepa) plants showed yellowing, streaks and twisting and Opuntia abjecta with proliferation and cylindrical of cladodes. Total DNA was extracted from symptomatic and asymptomatic plants, and phytoplasma were detected in all 12 symptomatic plants collected through direct and nested PCR assays with primers P1/P7 and R16F2n/R16R2. The results of phylogenetic analysis revealed that the phytoplasma isolates belong to 16SrII group. With a nucleotide identity greater than 98.7% with three members of 16SrII group, Papaya yellow crinkle, Y10097; “Ca. P. aurantifolia”, U15442; and peanut witches’ broom, Al33765, the strains identified in this study are “Ca. P. aurantifolia”-related strains. Virtual RFLP analysis of the 16S rRNA gene sequences with 17 restriction enzymes confirmed that the phytoplasma isolates belong to the “Candidatus Phytoplasma australasia” 16SrII-D subgroup. To the best of our knowledge, periwinkle, onion and Opuntia abjecta are considered new hosts for 16SrII group in Egypt.     

Identification of phytoplasmas associated with sesame phyllody disease in southeastern Iran

Phyllody disease is a threat to sesame production in Kerman province, southeastern Iran. RFLP analysis of PCR products of phytoplasma-specific 16S rRNA gene (1.8 kb) and phylogenetic analyses of 16S-23S rDNA spacer region (SR) sequence indicated that the predominant agent associated with sesame phyllody in Kerman province is a phytoplasma with 100% similarity with eggplant big bud, and peanut witches’-broom phytoplasmas, members of “Candidatus Phytoplasma aurantifolia” from Iran and China, respectively. Among the samples tested, only one strain (SPhSr1), had a unique RFLP profile and its SR was 100% similar in nucleotide sequence with the phytoplasma carried by Orosius albicinctus and Helianthus annus witches’-broom phytoplasma from Iran, members of “Ca. Phytoplasma trifolii”. Virtual RFLP patterns of SPhJ2 (representative of the predominant PCR-RFLP profiles) SR sequence were identical to those of peanut witches’-broom phytoplasma (16SrII-A, JX871467). However, SPhSr1 SR sequence patterns resemble (99.7%) those of vinca virescence phytoplasma (16SrVI-A, AY500817).     

Detection, Identification and Molecular Characterization of a Phytoplasma Associated with Arabian Jasmine (Jasminum sambac L.) Witches' Broom in Oman

Arabian jasmine (Jasminum sambac L.) plants showing witches’ broom (WB) symptoms were found in two regions in the Sultanate of Oman. Polymerase chain reaction (PCR) amplification of the 16S rRNA gene and the 16S–23S spacer region utilizing phytoplasma‐specific universal and designed primer pairs, and transmission electron microscopy of phytoplasma‐like structures in phloem elements confirmed phytoplasma infection in the symptomatic plants. PCR products primed with the P1/P7 primer pair were 1804 bp for jasmine witches’ broom (JasWB) and 1805 bp for alfalfa (Medicago sativa L.) witches’ broom (AlfWB). Actual and putative restriction fragment length polymorphic analysis indicated that jasmine and AlfWB phytoplasmas were molecularly indistinguishable from each other and closely related to papaya yellow crinkle (PYC), as well as being distinct from lime WB (LWB) and Omani alfalfa WB (OmAlfWB) phytoplasmas. A sequence homology search of JasWB and AlfWB showed 99.8% similarity with PYC from New Zealand and 99.6% similarity with each other (JasWB/AlfWB). The jasmine and AlfWB phytoplasmas were also shown to be related to the peanut WB group (16SrII) of 16S rRNA groups based on a phylogenetic tree generated from phytoplasma strains primed with the P1/P7 primer pair and representing the 15 phytoplasma groups.     

Molecular Characterization of a Phytoplasma Associated with Potato Witches’‐broom Disease in Iran

Potato plants showing symptoms suggestive of potato witches’‐broom disease including witches’‐broom, little leaf, stunting, yellowing and swollen shoots formation in tubers were observed in the central Iran. For phytoplasma detection, Polymerase Chain Reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7, followed by primer pair R16F2n/R16R2. Random fragment length polymorphism analysis of potato phytoplasma isolates collected from different production areas using the CfoI restriction enzyme indicated that potato witches’‐broom phytoplasma isolate (PoWB) is genetically different from phytoplasmas associated with potato purple top disease in Iran. Sequence analysis of the partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma trifolii’ is associated with potato witches’‐broom disease in Iran. This is the first report of potato witches’‐broom disease in Iran.     

Detection and identification of phytoplasma associated with witches’ broom and little leaf disease in Arundo donax: first report from India

During the survey of two successive years 2012–2013, in nearby places of Gorakhpur districts, Uttar Pradesh, India, Arundo donax plants were found to be exhibiting witches’ broom, excessive branching accompanied with little leaf symptoms with considerable disease incidence. Nested PCR carried out with universal primers pair R16F2n/R16R2 employing the PCR (P1/P7) product as a template DNA (1:20) resulted in expected size positive amplification ~1.2 kb in all symptom-bearing plants suggested the association of phytoplasma with witches’ broom disease of Narkat plants. BLASTn analysis of the 16S rRNA gene sequence showed the highest (99%) sequence identity with Candidatus phytoplasma asteris (16SrI group). In phylogenetic analysis, the sequence data showed close relationships with the members of 16SrI phytoplasma and clustered within a single clade of 16SrI group and closed to B subgroup representatives. This is a first report of 16Sr I-B group phytoplasma associated with witches’ broom accompanied with little leaf disease of Narkat in India.     

Molecular Characterization and Potential Insect Vector of a Phytoplasma Associated with Garden Beet Witches' Broom in Yazd, Iran

In 2002, garden beet witches’ broom (GBWB) phytoplasma was detected for the first time in garden beet plants (Beta vulgaris L. ssp. esculenta) in Yazd, Iran. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphic (RFLP) analysis of PCR‐amplified phytoplasma 16S rDNA were employed for the detection and identification of the phytoplasma associated with garden beet. A phytoplasma belonging to subgroup 16SrII‐E, in the peanut witches’ broom group (16SrII), was detected in infected plants. Asymptomatic plant samples and the negative control yielded no amplification. The result of analysis of the nucleotide sequence of a 1428 bp fragment of 16S rDNA gene from GBWB phytoplasma (GenBank accession number DQ302722 ) was basically consistent with the classification based on RFLP analysis, in which GBWB phytoplasma clustered with phytoplasmas of the 16SrII‐E subgroup. A search for a natural phytoplasma vector was conducted in Yazd in 2004, in an area where garden beet crops had been affected since 2002. The associated phytoplasma was detected in one leafhopper species, Orosius albicinctus, commonly present in this region. The leafhopper O. albicinctus was used in transmission tests to determine its vector status for the phytoplasma associated with GBWB. Two of eight plants that had been fed on by O. albicinctus, showed mild symptoms of GBWB including stunting and reddening of midveins. A phytoplasma was detected in the two symptomatic test plants by PCR using universal primers and it was identified by RFLP as the GBWB phytoplasma. This finding suggests O. albicinctus is a vector of the GBWB phytoplasma.     

Molecular Identification of a Candidatus Phytoplasma asteris Associated with Cabbage Witches’‐Broom in China

In 2010, cabbages (Brassica oleracea L.) showing symptoms of proliferated axillary buds, crinkled leaves and plant stunting with shortened internodes typical to phytoplasma infection were found in a breeding facility in Beijing, China. Three symptomatic plants and one symptomless plant were collected, and total DNA was extracted from the midrib tissue and the flowers. With phytoplasma universal primers R16F2n/R16R2, a special fragment of 1247 bp (16S rDNA) was obtained from all three symptomatic cabbage plants, but not from the one symptomless cabbage plant. The 16S rDNA sequence showed 99% similarity with the homologous genes of the aster yellows group phytoplasma (16SrI group), and the phytoplasma was designed as CWBp‐BJ. Phylogenetic and computer‐simulated restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA gene revealed that CWBp‐BJ belongs to subgroup 16SrI‐B. This is the first report of a phytoplasma associated with cabbage witches’‐broom in China.     

Detection and molecular characterization of phytoplasmas associated with vegetable and alfalfa crops in Qassim region

During a survey conducted in Qassim province, Saudi Arabia, in the year in 2015, 120 samples of carrot (Daucus carota subsp. sativus), onion (Allium cepa), faba bean (Vicia faba), green mustard (Brassica juncea) and alfalfa (Medicago sativa) plants displaying symptoms reminiscent of phytoplasma diseases were collected and tested for phytoplasma infection. Phytoplasma-specific PCR products were only amplified from symptomatic plants by nested-PCR. Disease incidence ranged from 3.14% in alfalfa crop fields 1 year after cultivation to 77.48% in 3-year-old fields. In the five carrot fields sampled in this study, the incidence changed from 3.2% to 100% after 7 months of cultivation. Phylogenetic analysis revealed that all Qassim phytoplasma isolates belong to the 16SrII group. Most of them shared 100% identity with papaya yellow crinkle (16SrII-D Y10097). The results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence confirmed that the phytoplasma of Qassim isolates under study is a member of 16SrII-D subgroup. To the best of my knowledge, the onion and green mustard are considered new hosts for the 16SrII group; therefore, this is the first report on the association of phytoplasma with diseases of faba bean, onion, carrot, mustard and alfalfa in Qassim province, Saudi Arabia.     

Molecular Characterization of Phytoplasma Associated with Rose Witches'-Broom in China

In 2005, rose plants (Rosa rugosa cv. ‘Plena’) exhibiting typical phytoplasma disease symptoms of stunting, yellowing, witches’‐broom and dieback were observed in Pingyin, Shandong Province, China. The disease, rose witches’‐broom (RoWB), is progressively destructive and can be graft‐transmitted. Polymerase chain reaction (PCR), sequencing of PCR products and electron microscopy were used to investigate the possible association of phytoplasma with RoWB. All results indicated that presence of phytoplasma in the symptomatic rose plants. Sequence alignment of 16S rRNA gene, tuf gene and rp gene confirmed that the phytoplasma associated with RoWB is the causal agent of Paulownia witches’‐broom disease, which might be transmitted from the paulownia tree that is several meters away. To our knowledge, this is the first report of the molecular characterization of phytoplasma infecting rose in China.     

Detection, characterisation and transmission by Macrosteles leafhoppers of watercress yellows phytoplasma in Hawaii

A new yellows disease of watercress (Nasturtium officinale) in Hawaii has symptoms of reduced leaf size, leaf yellowing and crinkling, and occasionally witches’ brooms. This disease is found on all watercress farms on Oahu but has not yet been found on other Hawaiian islands. Watercress plants were tested for phytoplasma infection by polymerase chain reaction assays using phytoplasma‐specific primers. Amplicons of the expected sizes were produced from all symptomatic plants but not from healthy plants raised from seed. Phylogenetic analysis of the 16S rRNA gene indicated that watercress yellows was caused by a phytoplasma in the aster yellows group, with sequence similarity to onion yellows from Japan. Six weed species collected from the vicinity of affected watercress farms, Amaranth sp., Eclipta prostrata, Emilia sonchifolia, Plantago major, Myriophyllum aquaticum and Sonchus oleraceus, were also determined to be hosts of this phytoplasma. Leafhoppers, identified as Macrosteles sp. (Hemiptera, Cicadellidae), collected from symptomatic watercress transmitted this phytoplasma to watercress, plantain and lettuce (Lactuca sativa) in greenhouse experiments.     

Occurrence and Characterization of a 16SrII‐D Subgroup Phytoplasma Associated with Parsley Witches’ Broom Disease in Iran

During 2010–2013 surveys for the presence of phytoplasma diseases in Yazd province (Iran), a parsley witches’ broom (PrWB) disease was observed. Characteristic symptoms were excessive development of short spindly shoots from crown buds, little leaf, yellowing, witches’ broom, stunting, flower virescence and phyllody. The disease causative agent was dodder transmitted from symptomatic parsley to periwinkle and from periwinkle to periwinkle by grafting inducing phytoplasma‐type symptoms. Expected length DNA fragments of nearly 1800 and 1250 bp were, respectively, amplified from naturally infected parsley and experimentally inoculated periwinkle plants in direct polymerase chain reaction (PCR) using phytoplasma primer pair P1/P7 or nested PCR using the same primer pair followed by R16F2n/R16R2 primers. Restriction fragment length polymorphism and phylogenetic analyses of 16S rRNA gene sequences showed that the phytoplasma associated with PrWB disease in Yazd province belong to 16SrII‐D phytoplasma subgroup. This is the first report of association of a 16SrII‐related phytoplasma with PrWB disease in Iran.     

Detection and Identification of Elm Yellows Group Phytoplasma (16SrV) Associated with Alfalfa Witches’ Broom Disease

In July, 2011, alfalfa plants were observed in Yangling, Shaanxi Province, China with typical witches’ broom symptoms. The presence of phytoplasma was confirmed by transmission electron microscopy and a nested PCR, which amplified a 1.2‐kb fragment using universal primer pairs P1/P6 followed by R16F2n/R2. Sequence, phylogeny and RFLP analyses showed that the alfalfa witches’ broom disease was associated with a phytoplasma of group 16SrV, subgroup V‐B. This is the first record of the 16SrV phytoplasma group infecting alfalfa plants.     

Molecular Identification of a 16SrII‐A Group‐Related Phytoplasma Associated with Cinnamon Yellow Leaf Disease in China

Chinese cinnamon (Cinnamomum cassia Presl), an evergreen tree native to China, is a multifaceted medicinal plant. The stem bark of cinnamon is used worldwide in traditional and modern medicines and is one of the most popular cooking spices. In recent years, cinnamon with pronounced yellow leaf symptoms has been observed in their natural habitat in Hainan, China. Phytoplasmas were detected from symptomatic cinnamon trees via polymerase chain reaction using phytoplasma universal primers P1/P7 followed by R16F2n/R16R2. No amplification products were obtained from templates of asymptomatic cinnamon trees. These results indicated a direct association between phytoplasma infection and the cinnamon yellow leaf (CYL) disease. Sequence analysis of the CYL phytoplasma 16S rRNA gene determined that CYL phytoplasma is a ‘Candidatus Phytoplasma australasiae’‐related strain. Furthermore, virtual restriction fragment length polymorphism pattern analysis and phylogenetic studies showed that CYL phytoplasma belongs to the peanut witches’‐broom (16SrII) group, subgroup A. This is the first report of a 16SrII group phytoplasma infecting cinnamon under natural conditions.     

Identification,occurrence, incidence and transmission of phytoplasma associated with Petunia violacea witches’ broom in Iran

A petunia witches’ broom (PvWB) disease, characterized by phyllody, virescence, witches’ broom, little leaf and yellowing, was observed in municipal lands and parks in Bandar Abbas, Hormozgan province, Iran. The disease was present with an average incidence of 20%. PCR and sequencing analysis carried out on selected samples from symptomatic plants showed the presence of a phytoplasma associated with the disease. The molecular comparison of the 16S ribosomal gene indicated 99% sequence identity with the one of “Candidatus Phytoplasma australasia”. This phytoplasma was transmitted to healthy petunia plants under experimental conditions by the leafhopper Orosius albicinctus that was then demonstrated to be a vector of this phytoplasma.     

Molecular detection of Candidatus Phytoplasma spp. causing witches’ broom disease of acid lime (Citrus aurantifolia) in India

Phytoplasma infected acid lime plants in India develop characteristic symptoms like small chlorotic leaves, multiple sprouting and shortened internodes. Leaves drop prematurely and infected branches have distorted twigs resembling witches’ broom appearance which eventually show die-back symptoms. During its first report in 1999, witches’ broom disease identification was made on the basis of symptomatology and electron microscopy. However, molecular techniques have proved to be more accurate and reliable for phytoplasma detection than the conventional methods. During survey in the year 2010 six samples were collected from infected acid lime plants showing typical field symptoms from Vidarbha region of Maharastra. Initially, phytoplasma bodies were observed in phloem tissues of all six symptomatic samples under JEM 100S transmission electron microscope and all these six samples were subsequently screened using different set of phytoplasma specific universal primers by nested PCR, a widely recommended molecular technique for phytoplasma detection. In the present study P1/P7 “universal” phytoplasma-primer set was used for first round of PCR and amplified products were processed separately for nested PCR with three different nested primer pairs viz. R16F2n/R16R2, R16mF2/R16mR1 and fU5/rU3. The presence of phytoplasma was confirmed in all six suspected samples and one representative ~1.2 kb size amplicon was sequenced and deposited in GenBank as Candidatus Phytoplasma species AL-M (JQ808143). This is the first report of PCR based molecular detection of phytoplasma-induced witches’ broom disease of acid lime (WBDL) in India. Further molecular evaluation to determine the identity to the species level is in progress.     

Aster Yellows Phytoplasma Identified in Scentless Chamomile by Microscopical Examinations and Molecular Characterization

Scentless chamomile (Matricaria perforata Mérat) plants were commonly found infected with a yellows-type disease caused by phytoplasma in several fields in Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf, stem, root and flower petiole tissues examined by electron microscopy. Application of 4′6-diamidino-2-phenylindole- 2HCl (DAPI) staining techniques confirmed the presence of the phytoplasma in these tissues. These observations were supported by polymerase chain reaction (PCR) assays, using two primer pairs, P1/P6 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows and potato witches′-broom (PWB) DNA phytoplasma samples served as positive controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair P1/P6 gave the expected PCR products of 1.5 kb. Based on a nested-PCR assay using the latter PCR products, as templates, and a specific primer pair R16(1)F1/R1 designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected chamomile and AY samples but not from PWB phytoplasma and healthy chamomile controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected scentless chamomile sample.     

Detection and identification of ‘Candidatus Phytoplasma asteris’ in Brassica rapa subsp. pekinensis in Poland

Severe growth abnormalities, including leaf yellowing, sprout proliferation and flower virescence and phyllody, were found on Brassica rapa subsp. pekinensis plants in Poland. The presence of phytoplasma in naturally infected plants was demonstrated by polymerase chain reaction assay employing phytoplasma universal P1/P7 followed by R16F2n/R16R2 primer pairs. The detected phytoplasma was identified using restriction fragment length polymorphism analysis (RFLP) of the 16S rRNA gene fragment with AluI, HhaI, MseI and RsaI endonucleases. After enzymatic digestion, all tested samples showed restriction pattern similar to that of ‘Candidatus phytoplasma asteris’. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Sequences of the 16S rDNA gene fragment of analysed phytoplasma isolates were nearly identical. They revealed high nucleotide sequence identity (>98%) with corresponding sequences of other phytoplasma isolates from subgroup 16SrI‐B, and they were classified as members of ‘Candidatus phytoplasma asteris’. This is the first report of the natural occurrence of phytoplasma‐associated disease in plants of Chinese cabbage.