Genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody in Iran

During 2010-14 surveys in the major sesame growing areas of Fars, Yazd and Isfahan provinces (Iran), genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody were studied. Virtual RFLP, phylogenetic, and DNA homology analyses of partial 16S ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas referable to three ribosomal subgroups, 16SrII-D, 16SrVI-A, and 16SrIX-C. The same analyses using 16S rDNA sequences from sesame phyllody-associated phytoplasmas retrieved from GenBank database showed the presence of phytoplasmas clustering with strains in the same subgroups in other Iranian provinces including Bushehr and Khorasan Razavi. Circulifer haematoceps and Orosius albicinctus, known vectors of the disease in Iran, were tested for transmission of the strains identified in this study. C. haematoceps transmitted 16SrII-D, 16SrVI-A, and 16SrIX-C phytoplasmas, while O. albicinctus only transmitted 16SrII-D strains. Based on the results of the present study and considering the reported presence of phytoplasmas belonging to the same ribosomal subgroups in other crops, sesame fields probably play an important role in the epidemiology of other diseases associated with these phytoplasmas in Iran

Molecular identification and phylogenetic analysis of phytoplasmas associated with alfalfa witches’ broom diseases in the western areas of Iran.

During 2014-2015 phytoplasmas associated with alfalfa witches’ broom (AWB) in the western areas of Iran were characterized by molecular analyses studies. Polymerase chain reaction was carried out using direct and nested as says to amplify the 16S ribosomal gene for phytoplasma identification. RFLP analysis of R16F2n/R16R2 amplicons with selected restriction enzymes and phylogenetic studies showed that AWB associated phytoplasma strains from Salafchegan (Qom province), Arak (Markazi province), Tabriz (East Azerbaijan province), Dehgolan and Qhorveh (Kordestan province), Eghlid (Fars province), Mahidasht (Kermanshah province), Dehdasht (Kohgiluyeh and Boyer-Ahmad province), Borujerd (Lorestan province), Chenar-e Mahmudi and Shahrekian (Chaharmahal and Bakhtiari province) belong to 16SrXII-A subgroup (“stolbur”), while AWB associated phytoplasmas from Jowkar (Hamedan province) belong to 16SrII-D subgroup. Disease incidence was variable in the different areas and the highest infection rate (14.7%) was observed in Chenar-e Mahmudi (Chaharmahal and Bakhtiari province). The highly AWB affected areas were generally located in the warmer parts of each region in sparse fields. This is the first report and characterization of 16SrXII-A phytoplasmas associated with alfalfa witches’ broom diseases in Iran and occurrence of 16SrIID in western areas of Iran

First report of a 16SrII‐D phytoplasma associated with Calendula officinalis

Calendula officinalis, commonly known as marigold, belongs to the family Asteraceae. In Iran, this species is cultivated for ornamental purposes and used for many medicinal, culinary and cosmetic uses. From 2006, Calendula officinalis phyllody (CaoP) has been observed in different areas of Yazd province, Iran and association of a phytoplasma with the disease has been reported (Esmailzadeh Hosseini et al., 2011). Disease incidence of up to 12% and symptoms including leaf size reduction, yellowing, phyllody, virescence, proliferation and sterility in the flower, proliferation of axillary buds along the stem, witches' broom and stunting were observed (Fig. 1). Total DNA was extracted from 0.2 g of fresh leaves from both diseased and symptomless plants. DNA samples were tested for phytoplasma presence by direct PCR using P1/P7 primers (Deng & Hiruki, 1991; Schneider et al., 1995) and nested PCR using primers P1/P7 and R16F2n/R16R2 (Gundersen & Lee, 1996). PCR amplicons of ~1.8 and ~1.25 kb respectively were obtained from six diseased C. officinalis plants from Ashkezar and Yazd (two areas in Yazd province) but not from four symptomless marigold plants. The ~1.25 kb amplicons from the six diseased plants were directly sequenced and shown to have 100% identity. A consensus sequence of 1,248 bp (CaoP1) was deposited in GenBank (Accession No. KU297202). Sequence comparison by BLAST analysis showed the highest sequence identity with members of phytoplasma group 16SrII (peanut witches' broom). Phylogenetic analysis using the neighbourjoining method (MEGA software version 6.0) confirmed that the CaoP1 phytoplasma clustered within the group 16SrII phytoplasma clade, and closer to strains affiliated to the 16SrII-D subgroup than to other strains in the same group (Fig. 2). Computer-simulated analysis with 17 restriction endonucleases using iPhyClassifier (Zhao et al., 2009) showed hat the RFLP pattern derived from the CaoP1 16S rRNA gene was identical to the papaya yellow crinkle strain classified into subgroup 6SrII-D (Y10097). This is the first report of a 16SrII-D phytoplasma associated with Calendula officinalis phyllody disease in Iran. Due to the occurrence and spread of alfalfa witches' broom phytoplasma associated with 16rSrII-D in Iran since 1990 (Esmailzadeh Hosseini et al., 2015) adjacent to plantings of C. officinalis, it is possible that alfalfa play a role in the epidemiology of Calendula officinalis phyllody disease or vice versa. Due to natural ransmission of phytoplasma diseases by leafhoppers and psyllids, studies to identify insect vectors are in progress

Incidence and molecular characterization of a 16SrI-B phytoplasma strain associated with Eruca sativa phyllody in Iran

During 2012-2014 surveys for phytoplasma diseases, a phyllody symptomatology was observed in Eruca sativa in Abarkooh (Yazd province, Iran). The highest disease incidence was 32% in Ali Abad-e Shams (Esfandabad), and infected plants showed crown proliferation, witches\u2019 broom, little leaf, flower virescence, phyllody, sterility, and plant stunting. Total DNA extracted from symptomatic and asymptomatic plants was tested for phytoplasma presence verification by direct PCR using P1/P7 primers and nested PCR using P1/P7 and R16F2n/R16R2 primers pairs. PCR amplicons of about 1.8 and 1.25 kb respectively, were obtained from all symptomatic E. sativa plants, but not from the symptomless ones. Restriction fragment length polymorphism analysis of R16F2n/R2 amplicons using AluI, HhaI, HinfI, HpaII, MseI, RsaI and TaqI restriction enzymes showed profile identical to each other and also to those of 16SrI phytoplasmas indicating that the phytoplasmas, associated with E. sativa phyllody are members of 16SrI group. The consensus sequence of Ali Abad-e Shams E. sativa phyllody strain showed 100% identity with those of the \u2018Candidatus Phytoplasma asteris\u2019-related strains. Phylogenetic analysis confirmed that this phytoplasma clustered within the 16SrI phytoplasma clade closer to the onion yellows mild strain (OY-M) a 16SrI-B phytoplasma. Restriction analysis using iPhy Classifier confirmed that virtual RFLP patterns of the phytoplasma were identical (similarity coefficient 1.00) to OY-M strain. A 16SrI-B related phytoplasma has been reported associated with rapeseed (Brassica napus) phyllody in Iran and Eruca sativa may be a secondary host for the 16SrI-B phytoplasma associated with rapeseed phyllody

Biologic, serologic and molecular characteristics of two 16SrII-C-related phytoplasma strains associated with alfalfa witches\u2019 broom disease in Yazd and Fars provinces, Iran

Alfalfa witches\u2019 broom (AWB) is a limiting factor for alfalfa growth and production in Iran, especially in the tropical regions of the country. AWB phytoplasma strains from two severely affected areas, Chahgeer (Abarkooh, Yazd province) and Juyom (Larestan, Fars province), were compared for main biologic, serologic and molecular characteristics. Based on disease symptoms in alfalfa farms, Chahgeer and Juyom AWB (CAWB and JAWB, respectively) strains were not differentiable. In dodder inoculated periwinkle and tomato plants JAWB phytoplasma induced stronger little leaf compared to the one induced by CAWB phytoplasma. In these experiments the two JAWB and CAWB phytoplasma strains are confirmed as vectored by different leafhopper species, Circulifer haematoceps and Orosius albicinctus respectively. Based on 16S rRNA gene and 16S-23S intergenic spacer region sequences, CAWB and JAWB were not differentiable, however no serologic relationship was observed between the two phytoplasmas in ELISA and DIBA tests using polyclonal antibodies prepared against each of them. Due to the lack of serological relationship, different insect vectors and induction of different symptoms in common host plants, CAWB and JAWB phytoplasmas should be considered as two AWB strains both belonging to 16SrII-C subgroup

Molecular characterization of a new phytoplasma associated with Helianthus annuus phyllody in Iran

Sunflower is an important oil crop in Iran. During two years surveys, sunflower phyllody was observed in fields of Yazd, Fars and Esfahan provinces. Affected plants showed proliferation of abnormal shoots and heads along the stem, virescence and phyllody of the main flowers. Total DNA extracted from fresh sunflower tissues allow to obtain amplification of phytoplasma 16S ribosomal DNA from eight symptomatic plants but not from samples collected from the symptomless ones. Eight P1/P7 DNA fragments amplified from phyllody-affected sunflower plants were separately cloned and sequenced. The obtained 16S rDNA sequences shared 100% identity with each other and an Abarkooh sunflower phyllody (ASP) strain was deposited in the GenBank. Sequence comparison by BLAST analysis showed the highest sequence identity with phytoplasmas in group 16SrII (\u2018Candidatus Phytoplasma aurantifolia\u2019). Phylogenetic analysis confirmed that the ASP phytoplasma clusters with phytoplasmas enclosed in ribosomal group 16SrII and is therefore a \u2018Ca. P. aurantifolia\u2019-related strain. Analysis carried out using the iPhyClassifier showed that the RFLP pattern of ASP phytoplasma was different from all described 16SrII subgroups, having the major similarity coefficient (0.63) with subgroup 16SrII-D. Therefore, considering the RFLP divergences this phytoplasma could be assigned to a new subgroup designed 16SrII-Z

Occurrence and molecular characterization of a \u2018Candidatus Phytoplasma omanense\u2019-related strain associated with Prunus persica yellowing and decline in Iran

In 2014, peach (Prunus persica) yellowing and decline symptoms were observed in some fruit tree in Mehriz (Yazd province, Iran). The disease was named Mehriz peach yellowing and decline (MPYD) and characteristic symptoms were yellowing, leaf curl, witches\u2019 broom and decline. Disease incidence was variable and up to 64% of presence was observed. Direct polymerase chain reaction was carried out on DNAs extracted from symptomatic and asymptomatic samples using using P1/P7 followed in nested PCR by R16mF2/R16mR2 and R16F2n/R16R2 to amplify the 16S ribosomal gene for phytoplasma identification. RFLP analysis of 13 amplicons with AluI, HaeIII, HhaI, HpaI, HpaII, KpnI, RsaI, MseI and Taq1 restriction enzymes showed profiles identical to those of phytoplasmas belonging to 16SrXXIX-A subgroup that is enclosing \u2018Candidatus Phytoplasma omanense\u2019. Five samples from different orchards were directly sequenced and after alignment and assembling, the resulting sequences were trimmed and compared. Since they were 100% identical to each others only one was submitted to GenBank as MPYD phytoplasma under accession no. \u2026\u2026\u2026\u2026\u2026... Sequence comparison by BLAST analysis (www.ncbi.nlm.nih.gov) showed the highest sequence identity with \u2018Ca. P. omanense\u2019 classified in 16SrXXIX-A subgroup and previously associated with cassia witches\u2019 broom in Oman. Phylogenetic analysis using neighbour-joining method (MEGA software version 7.0) confirmed that the MPYD phytoplasma is a \u2018Ca. P. omanense\u2019-related strain. Computer-simulated analysis with 17 restriction endonucleases using iPhyClassifier further confirmed that the RFLP pattern of MPYD phytoplasma 16S rRNA gene was identical (similarity coefficient 1.00) to the reference pattern of 16SrXXIX-A subgroup (GenBank accession number: EF666051) and that is therefore a member of 16SrXXIX-A subgroup. This is first report this phytoplasma in a fruit tree species in Iran and worldwide. Formerly 16SrXXIX-A and \u2013B subgroups were detected in Cassia italica witches\u2019 broom in Oman and Convolvulus arvensis witches\u2019 broom in Iran respectively. This finding is enclosing the \u2018Ca. P. omanense\u2019 among phytoplasma strains infecting also fruit trees

Occurrence of a \u2018Candidatus Phytoplasma omanense\u2019-related strain in bindweed showing a witches\u2019 broom disease in Iran

In 2014, bindweed witches\u2019 broom and dwarfing symptoms were observed in some bindweed plants grown in alfalfa fields in Bafg (Yazd province, Iran). Direct polymerase chain reaction using P1/P7 and nested PCR using P1/P7 followed by R16mF2/ R16mR2 and R16F2n/R16R2 amplify the 16S ribosomal gene and RFLP analysis of several R16F2n/R16R2 amplicons with AluI, HaeIII, HhaI, HpaI, HpaII, KpnI, RsaI, MseI and Taq1 restriction enzymes showed profiles in which phytoplasma strains belonging to 16SrXXIX group were identified together with 16SrXII-A phytoplasmas by comparison of profiles and confirmed after nested PCR/RFLP analyses with R16(I)F1/R1 primers. Three samples from different fields were directly sequenced using nested PCR products obtained with P1/P7 and R16mF2/R16mR2. Sequence comparison by BLAST analysis showed the highest sequence identity with phytoplasmas in group 16SrXXIX (\u2018Candidatus Phytoplasma omanense\u2019) and phylogenetic analysis confirmed that the phytoplasma could be considered a \u2018Ca. P. omanense\u2019-related strain. While the computer-simulated analysis with the 17 restriction endonucleases in the iPhyClassifier indicated identity of this strain with \u2018Ca. P. omanense\u2019, a virtual digestion using BcgI and AatII enzymes allow to enclose it in a new subgroup designed 16SrXXIX-B