Peach witches’-broom, an emerging disease associated with ‘Candidatus Phytoplasma phoenicium’ and ‘Candidatus Phytoplasma aurantifolia’ in Iran

During field surveys carried out from 2013 to 2017 in the eight main peach producing provinces of Iran, symptoms of a phytoplasma-like peach witches'-broom disease (PWIB), inducing severe yellowing, little leaf, internode shortening, crown and stem witches\u2019-broom, decline, and death, were observed. The aim of this work was to identify and characterize the agent(s) associated with PWIB by biological assays and molecular analyses. PWIB agents were successfully transmitted under controlled conditions from scions of in field-affected peach trees, exhibiting severe or mild symptoms, to peach and bitter almond seedlings inducing phytoplasma-like symptoms. A 16S rDNA fragment of 1250 bp was amplified by nested-PCR from all PWIB-affected trees and grafted seedlings. Nucleotide sequence identity, presence of species-specific signature sequences, in silico RFLP, single nucleotide polymorphisms, and phylogenetic analyses of 16S rDNA allowed the assignation of the phytoplasma strains identified in seven Iranian provinces in peach trees with severe PWIB symptoms to four SNP genetic lineages of \u2018Ca. P. phoenicium\u2019 (subgroup 16SrIX-B and its variant). PWIB phytoplasma strains identified in Abarkooh (Yazd province) in peach trees with mild symptoms were attributed to the species \u2018Ca. P. aurantifolia\u2019 (subgroup 16SrII-C). This report of a wide spread of \u2018Ca. P. phoenicium\u2019 in association with PWIB in Iran supported its capability of adaptation to a broad range of fruit tree species, such as peach, nectarine, and apricot. As \u2018Ca. P. phoenicium\u2019 and \u2018Ca. P. aurantifolia\u2019 are the etiological agents of other important plant diseases in Iran and neighbouring countries, further investigations are needed to determine the role played by peach in their epidemiological pathways

Molecular and biological characterization of a 16SrII phytoplasma associated with carrot witches\u2019 broom in Iran

During 2011-14 surveys, a witches\u2019 broom (CarWB) disease was observed in carrot fields of Chahgeer and Salim Abad in Abarkooh and Khatam regions (Yazd province, Iran). The main symptoms of CarWB disease were little leaf, yellowing, proliferation of shoots from taproot, stunting of taproot, virescence, phyllody, leaf reddening and witches\u2019 broom. The disease agent was transmitted by graft and dodder to periwinkle inducing phytoplasmatype symptoms. Orosius albicinctus leafhopper was identified as a natural vector of CarWB disease since it was able to transmit the agent from naturally witches\u2019 broom infected carrot to healthy alfalfa and carrot plants. Direct and nested polymerase chain reaction assays using phytoplasma-specific primer pairs amplified products of expected size from symptomatic carrot in the field and symptomatic grafted, dodder and vector inoculated plants. Restriction fragment length polymorphism and phylogenetic analyses of partial 16S rRNA gene sequence showed that phytoplasmas associated with CarWB disease in Yazd province belong to peanut witches\u2019 broom (16SrII) phytoplasma group, subgroup C. Considering the disease vector is found to be the same of alfalfa witches\u2019 broom infecting fields in the same areas, alfalfa seems to have an important role in the epidemiology of CarWB disease spreading in Yazd province. This is the first report of association of a 16SrII phytoplasma with carrot witches\u2019 broom disease

Characterization of 16SrII group phytoplasmas associated with alfalfa (Medicago sativa) witches\u2019 broom disease in diverse areas of Iran.

Alfalfa witches\u2019 broom (AWB) is one of the most important alfalfa diseases in Iran. To characterize 16SrII group phytoplasmas associated with this disease, symptomatic and asymptomatic plants were collected during 2013-2015 and subjected to direct and nested polymerase chain reaction (PCR) using P1/P7, R16mF2/R16mR2 and R16F2n/R16R2. PCR amplicons of ~1.8, ~1.4 and ~1.25 kb respectively, were obtained only from all symptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons showed that the phytoplasma associated with AWB disease were members of 16SrII group subgroups 16SrII-D and -C. Blast analysis of these amplicon sequences and sequence homology of collected strains and strain sequences retrived from GenBank (AWB strains Chahgeer, Juyom and Bushehr) confirmed that AWB phytoplasmas collected from Bafg, Ardakan, Bahabad and Herat (Yazd province), Nikshahr (Sistan-Baluchestan), Bam, Zarand, Jiroft (Kerman province), Bushehr (Bushehr province), Tabas (South Khorasan province), Jowkar (Hamedan province) and Zardenjan (Esfahan province) cluster with phytoplasma strains enclosed in the 16SrII-D subgroup, while AWB strains from Chahgeer (Yazd province) and Juyom (Fars province) cluster with phytoplasma strains in the 16SrII-C subgroup. Based on these results the predominant strains of 16SrII phytoplasmas associated with AWB disease in Iran were classified in the 16SrII-D subgroup. In Ashkezar and Abarkouh in Yazd province entire alfalfa farm was infected with witches\u2019 broom disease. In 3 year alfalfa stands in Ashkezar alfalfa farms were plowed due to high incidence of the diseas

Detection and characterisation of phytoplasma strains associated with field bindweed witches\u2019 broom disease in Iran

During 2013\u20132015 surveys in Fars, Lorestan and Yazd provinces (Iran), a field bindweed witches\u2019 broom (FBWB) disease was observed. The main symptoms were reduction of leaves size, yellowing, internode shortening, witches\u2019 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 \u2018Candidatus Phytoplasma solani\u2019-related strain. This is the first report of 16SrXII-A phytoplasmas presence in bindweed plants showing witches\u2019 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 provinc

Molecular differentiation of 16SrIX-I phytoplasmas detected in Onobrychis viciifolia leaf yellowing in Iran from phytoplasmas in 16SrIX-J subgroup

From 2014 in Chaharmahal and Bakhtiari province, Iran, Onobrychis viciifolia plants were observed with symptoms of yellowing, little leaf and reddening of leaflet margins and dwarfing and up to 32% of disease incidence was observed. Total DNA extracted from symptomatic and asymptomatic plants was tested by direct and nested PCR using P1/P7 followed by R16mF2/ R16mR2 and R16F2n/R2 primer pairs. Amplicons of about 1.8, 1.4 and 1.25 kbp were obtained from all symptomatic plants, but not from the asymptomatic ones. Restriction fragment length polymorphism analysis on R16F2n/R16R2 amplicons showed identical restriction profiles in all samples, referable to those of phytoplasma strains in the 16SrIX group. One sample from Kiar (KY) was directly sequenced and the fragment corresponding to the R16F2n/R16R2 amplicon was submitted to GenBank (Accession Number KX461906). Sequence comparison by BLAST analysis showed the highest sequence identity with pigeon pea witches\u2019 broom phytoplasmas. Phylogenetic analysis confirmed that KY phytoplasma clustered with \u2018Candidatus Phytoplasma phoenicium\u2019 with 99.4% sequence identity to the reference strain for this phytoplasma (GeneBank Accession number AF515636), and is therefore, a \u2018Ca. P. phoenicium\u2019-related strain. Computer-simulated analysis using iPhyClassifier showed that the RFLP pattern of KY phytoplasma was different from those of all phytoplasma subgroups in 16SrIX group and the highest similarity was 0.95 with 16SrIX-E (GeneBank Accession number GQ925918) with differences in AluI and TaqI restriction enzymes patterns; the strain was enclosed in the 16SrIX-I subgroup. However a chicory bushy stunt phytoplasma showing identical profile with KY phytoplasma was proposed later as a new subgroup as 16SrIX-J (GeneBank Accession number KY986922). Molecular comparison of the two phytoplasma sequences allow to verify that the two subgroups are differentiated by four single nucleotide polymorphisms at the 3\u2019 of the 16S rDNA sequence not enclosing restriction sites. Since the phytoplasma in O. viciifolia from Iran is well distinguished by RFLP analyses from all reported subgroup but the 16SrIXJ, it could be confused with it in epidemiological studies if the sequences are not compared. Phytoplasmas in the 16SrIX-I subgroup are quite represented in Iran where they were reported in Lactuca serriola phyllody and Bushehr eggplant big bud, however for their differentiation from the 16SrIX-J phytoplasma reported in Saudi Arabia in chicory it will be necessary the sequence comparison or the development of new specific primers on other phytoplasma genes

Occurrence and characterization of a 16SrII-D subgroup phytoplasma associated with parsley witches\u2019 broom disease in Iran.

During 2010\u20132013 surveys for the presence of phytoplasma diseases in Yazd province (Iran), a parsley witches\u2019 broom (PrWB) disease was observed. Characteristic symptoms were excessive development of short spindly shoots from crown buds, little leaf, yellowing, witches\u2019 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

First report of 16SrVI-A and 16SrXII-A phytoplasmas associated with alfalfa witches\u2019 broom disease in Iran

In 2014-15 surveys, alfalfa witches\u2019 broom (AWB) disease was observed in a number of Iranian provinces. The main symptoms were crown proliferation, witches' broom, little leaf, flower virescence, phyllody, sterility and stunting. To investigate the phytoplasma presence, total DNAs were extracted from 132 symptomatic and 19 asymptomatic alfalfa plants and were tested by direct PCR using P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) and nested PCR using R16F2n/R16R2 (Gundersen and Lee, 1996) primers. Amplicons were obtained only from all the symptomatic alfalfa plants and RFLP analyses with KpnI, AluI, HaeIII, HhaI, HpaII, MseI, RsaI, TaqI and HpaI enzymes showed the presence of two profiles. Mixed infections were never detected. From AWB samples collected in East Azarbayijan and Zanjan provinces, six amplicons were directly sequenced and showed 100% identity to each other (GenBank accession No. KU240021). The second profile was present in samples from Qom, Markazi, East Azarbayijan, Kordestan, Fars, Kermanshah, Kohgiluyeh and Boyer-Ahmad, Lorestan and Chaharamahal and Bakhtiari provinces; the 15 amplicons from Tabriz, Dehgolan, Arak, Qhorveh, and Shahrekian showed 100% identity in each area and formed 5 groups having 99% identity (GenBank accession Nos. KT763372, KT763371, KT781662, KT763373 and KT750060, respectively). The KU240021 strain sequence and its virtual RFLP indicated that it could be enclosed in the 16SrVI-A subgroup. The five-sequence group showed the highest homology with 16SrXII-A phytoplasma subgroup that was confirmed by virtual RFLP and phylogenetic analysis (MEGA 6.0). This is the first report of 16SrVI-A and 16SrXII-A phytoplasmas associated with AWB in Iran

An up to date status of alfalfa witches\u2019 broom disease in Iran.

Among the cultivated forage crops, alfalfa (Medicago sativa) is very important in Iran. Nearly 28 years ago, symptoms of alfalfa witches\u2019 broom (AWB) was reported from Jiroft, southern of Iran. Since then, the disease was reported from other parts of Iran and the epidemic of AWB was recorded in Chahgeer, Yazd in 1997. Now AWB disease is widespread all over the country especially in tropical and subtropical areas. The majority of phytoplasma strains reported from alfalfa in Iran were classified in 16SrI, 16SrII, 16SrVI and 16SrXII groups. The major symptoms reported were little leaf, internode shortening, flower virescence, phyllody, proliferation and sterility, witches\u2019 broom, leaf yellowing, leaf curling and death. Orosius albicinctus was identified as natural vector of the disease in Fars and Yazd provinces. Prosopis farcta and Cardaria draba were reported as natural host plants of the AWB phytoplasma. AWB disease reduced plant heights, leaf area, shoot dry and fresh weight and seed yields of alfalfa crop. So far no effective control strategies are in practice; however use of dense planting and field resistant alfalfa varieties are recommended for preventing AWB disease

Identification of a phytoplasma associated with pomegranate little leaf disease in Iran

During 2012e2014 surveys for the presence of phytoplasma diseases in Fars province (Iran), pomegranate little leaf symptoms were observed in several orchards in Khafr and Neyriz areas. Samples collected from symptomatic plants positively reacted in nested PCR assays using P1/P7 followed by R16F2n/R16R2 primer pairs producing the expected 1,250 bp DNA fragments. Real and virtual RFLP analysis showed that the sequences of phytoplasma strains from Khafr and Neyriz (KPLL and NPLL strains, respectively) were identical to each other and belong to 16SrII phytoplasma group, subgroup D. Phylogenetic analysis of the R16F2n\u2044R16R2 DNA region confirmed that KPLL and NPLL phytoplasmas were enclosed in the same clade as other 16SrII-D subgroup phytoplasmas. This is the first reported occurrence of a 16SrII phytoplasma infecting pomegranate trees