Broom In Iran Research Articles

Association of a phytoplasma from ribosomal subgroup 16SrXII‐A with walnut witches’‐broom in Iran

Walnut trees displaying symptoms similar to those caused by phytoplasmas, including foliar chlorosis, yellowing and shortening of internodes, were found in a fruit orchard in the north of Fars province, Iran in 2020 (Fig. 1). Samples were collected and analysed by PCR to detect and identify possible phytoplasma(s) associated with diseased plants. Four diseased and four asymptomatic walnut trees were sampled and analysed for the presence of phytoplasma. Total DNA was extracted from 0.1 g of midribs using the CTAB method. Nested PCR was performed using universal phytoplasma primers P1/P7 (Deng & Hiruki, 1991) in the first round, and R16F2n/R16R2 (Smart et al., 1996) in the second. PCR products of the expected size, c. 1.2 kb, were obtained from all diseased plants but not from the symptomless ones. The nucleotide sequence was determined directly and submitted to GenBank (Accession No. MZ005313). A BLAST search showed that the phytoplasma associated with walnut witches’-broom had the highest sequence identity (99.9%) with Iranian alfalfa witches’-broom phytoplasma (KT763373), a member of the ribosomal subgroup 16SrXII-A (‘Candidatus Phytoplasma solani’). Virtual RFLP patterns obtained with iPhyclassifier (https://plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) supported the sequence analysis results. Walnut witches’-broom phytoplasma was first reported in Iowa, USA and was associated with the group 16SrIII (‘Ca. P. pruni’) (Yun & Harrington 2011). In Iran, the 16SrXII phytoplasma group has been reported in alfalfa, almond, aubergine, grapevine, peach, plum and tomato (Siampour et al., 2019). It has also been identified from Cannabis sativa (Sichani et al., 2011) and Salix babylonica (Zamharir & Taheri, 2017). In Fars province, walnut orchards are adjacent to stone fruit orchards and vineyards, so it is likely that this phytoplasma has been transferred from such crops to walnut trees. Since large areas of walnut are grown in Iran, this phytoplasma may be a serious threat to walnut cultivation. To the best of our knowledge, this is the first report of the association of a 16SrXII-A related phytoplasma with walnut witches’-broom globally. The authors would like to thank Dr. Sadegh Sadeghi and Mr. Mohsen Amiri for great help in the field.

First report of a ‘Candidatus Phytoplasma asteris‘‐related strain (16SrI‐B) associated with Morus alba (white mulberry) witches' broom in Iran

White mulberry (Morus alba), belongs to the family Moraceae and is grown domestically and commercially in Iran for use as fresh fruit or dried nuts. During 2018-19, white mulberry plants showing witches' broom (WMWB) symptoms (Fig. 1) were observed sporadically in the Marvast district of Yazd province, Iran. The maximum incidence of disease was 4%. In affected trees, the witches' broom was restricted to some branches and did not affect the whole canopy. Total DNA was extracted from 0.2g of midrib tissue of fresh leaves from a total of 14 diseased plants from five different orchards, and three asymptomatic plants. DNA samples were tested for the presence of phytoplasmas by nested PCR, using primers P1/P7 (Deng & Hiruki, 1; Schneider et al., 4) in the first round, and primers R16F2n/R16R2 (Gundersen & Lee, 3) in the nested round. PCR amplicons of 1.8 and 1.25 kb (respectively) were obtained from all diseased plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 amplicons using RsaI, KpnI, HpaII, HaeIII, AluI, MseI, HhaI, TaqI, BfaI and ThaI restriction endonucleases showed RFLP patterns similar to the 16SrI phytoplasma group. The PCR products from three randomly selected WMWB plants were sequenced and showed 100% sequence identity with each other and 99.6% identity with ‘Ca. P. asteris’ (M30790). One R16F2n/R16R2 16S rDNA sequence from the Marvast WMWB strain was submitted to GenBank (Accession No. MT075612). BLAST searches showed that the WMWB phytoplasma shared more than 99% sequence identity with those of ‘Ca. P. asteris’ and related strains. Phylogenetic analysis using Mega7 software confirmed that the phytoplasma strain associated with WMWB clustered within the 16SrI phytoplasma clades closest to ‘Ca. P. asteris’ (M30790) (Fig. 2). Many phytoplasma strains related to ‘Candidatus Phytoplasma aurantifolia’ (from carrot and sunflower), ‘Ca. Phytoplasma australasia’ (from alfalfa, almond, beetroot, marigold, sesame, squash and tamarisk), ‘Ca. Phytoplasma trifolii’ (from alfalfa, aubergine, cucumber and tomato), ‘Ca. Phytoplasma solani’ (from bindweed and grapevine) and ‘Ca. Phytoplasma omanensis’ (from bindweed and peach) have been reported previously in Yazd province. A 16SrI-B related phytoplasma has been reported associated with Eruca sativa (rocket) phyllody in Yazd province (Esmailzadeh Hosseini et al., 2). To our knowledge, this is the first report of a ‘Ca. Phytoplasma asteris’ (16SrI-B) strain associated with witches' broom of Morus alba in Iran.

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

AbstractA 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.

‘Candidatus Phytoplasma phoenicium’ associated with apricot yellows and peach witches’ broom in Iran

‘<i>Candidatus</i> Phytoplasma phoenicium’ associated with apricot yellows and peach witches’ broom in Iran

First report of a ‘Candidatus Phytoplasma trifolii‘‐related strain associated with rapeseed witches' broom in Iran

Rapeseed (Brassica napus) is an economically important oilseed crop grown in many countries worldwide. During recent years rapeseed cultivation has developed in regions of Iran. In April 2018, symptoms similar to phytoplasma-associated diseases including witches' broom (Fig. 1), stunting and little leaves (Fig. 2) were observed in rapeseed in several areas of Hormozgan province, Iran. To identify the association of phytoplasma with the diseased plants, total genomic DNA was extracted from midribs of eight symptom-bearing and two symptomless plants using the CTAB method. The samples were analysed for phytoplasma DNA by direct PCR using the phytoplasma universal primer pair P1/P7 and nested PCR using primers P1/P7 (first round) followed by R16F2n/R16R2 (second round) (Gundersen & Lee, 2). Amplicons of the expected sizes (1800 and 1250 bp) were amplified with direct and nested PCR in all symptom-bearing plants but not from symptomless plants or sterile distilled water used as negative controls. A positive nested PCR product was directly sequenced and deposited in GenBank under accession no. MH430092. A BLAST search using the sequenced fragment showed that the phytoplasma associated with rapeseed witches' broom (RsWB) had 100% nucleotide identity with the corresponding sequence of several members (KX773529, MG788318 and AM260486) of the 16SrVI phytoplasma group and 99.8% identity with the type strain, ‘Candidatus Phytoplasma trifolii’ (AY390261). The virtual RFLP revealed that the RsWB phytoplasma belonged to the 16SrVI-A subgroup. There are several reports regarding the association of aster yellows group (16SrI) phytoplasmas with rapeseed (e.g. rapeseed phyllody phytoplasma) in Iran (Salehi et al., 6) and other countries (Maliogka et al., 4; Olivier et al., 5). To our knowledge, this is the first report of natural infection of rapeseed by a 16SrVI phytoplasma in Iran and most probably in the world. Phytoplasmas belonging to the 16SrVI-A have been previously reported on several economically vegetable crops such as tomato and cucumber in Iran (Esmailzadeh Hosseini et al., 1; Jamshidi et al., 3). Therefore, the infected volunteer rapeseed around tomato and cucumber fields may act as an alternative host for this phytoplasma. The authors would like to thank Dr. Majid Siampour for his excellent technical assistance.

Association of a ‘Candidatus Phytoplasma solani’-related strain with bindweed witches’ broom in Iran

Association of a ‘<i>Candidatus</i> Phytoplasma solani’-related strain with bindweed witches’ broom in Iran

First report of a ‘Candidatus Phytoplasma phoenicium‘‐related strain (16Sr IX) associated with Salix witches' broom in Iran

In Iran Salix acmophylla, S. aegyptiaca, S. alba and S. babylonica are trees traditionally grown in urban areas. Over the last few years witches' broom symptoms (Fig. 1) were observed in 12 out of 20 trees of S. alba growing along the Chalus Road in Alborz province, Iran. The symptomatology observed suggested the presence of a phytoplasma; there have been previous reports of an aster yellows group phytoplasma (16SrI-B) in Salix tetradenia (black mountain willow) in China (Muo et al., 6), a clover proliferation phytoplasma (16SrVI) in Salix bebbiana, S. discolor, S. exigua and S. petiolaris in Canada (Khadhair & Hiruki, 5), and a stolbur group phytoplasma (16SrXII) in Salix babylonica in Spain (Alfaro-Fernandez et al., 1). Leaf samples from the twelve trees (S. alba) showing witches' broom symptoms and five asymptomatic trees were collected in different areas in Alborz province. Leaf tissue was subjected to DNA extraction immediately after collection according to the procedure described by Doyle & Doyle (2). The partial 16Sr DNA was amplified with phytoplasma universal primers using primers P1/tint in the first round. The resultant PCR products were diluted with sterile distilled water (1:29) prior to nested PCR using primers R16F2/R2 (Gundersen & Lee, 4). Nested PCR gave positive results from all twelve Salix trees with witches' broom symptoms. No PCR products were obtained from the five asymptomatic Salix trees or negative controls. The PCR product from one randomly selected positive Salix sample (reference BT18) was cloned, sequenced and submitted to GenBank (Accession No. KX500119). It showed a 99% sequence identity to the reference isolate of ‘Candidatus P. phoenicum’ (AF515636), and is therefore a 'Ca. P. phoenicum'-related strain. Phylogenetic analysis with selected reference strains indicated that the phytoplasma clustered together with member strains of 'Ca. P. phoenicium' (16SrIX) (Fig. 2). Other hosts of ‘Ca. P. phoenicium’ include almond in Lebanon, and numerous hosts including almond, grapevine, peach, pistachio, Bidens alba and Chrysanthemum morifolium in Iran (Ghayeb Zamharir et al., 3). To our knowledge this is the first report of a ‘Ca. P. phoenicum‘-related strain associated with Salix worldwide.

First report of a phytoplasma associated with Periploca aphylla witches’ broom in Iran

Periploca aphylla (Asclepiadaceae), is a large erect branched shrub, grown on different altitudes, in desert or in dry, insolated grounds, on rocky and sandy slopes throughout Southwest Asia. Periploca aphylla is commonly used for feeding camels and goats. Its latex is applied as a poultice in tumours and swellings, while the decoction of the bark is used as a purgative or against fever (Kazimierz, 1966). In September 2008, symptoms of witches’ broom, proliferation and shoot dieback were observed on P. aphylla in Hormozgan province, Iran. The affected plants developed dense clusters of highly proliferating branches with shortened internodes. Total DNA was extracted separately from 0·5 g of shoots of ten symptom-bearing and five symptomless plants using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 1990). The samples were analysed for phytoplasma DNA by both direct and nested PCR assays using phytoplasma universal 16S rDNA gene primer pairs P1/P7 and R16F2n/R16R2 that amplify fragments of 1800 bp and 1250 bp, respectively. A 1250 bp PCR product was amplified with nested PCR from all symptom-bearing, but not from symptomless, plants. Two amplicons were cloned using cloning commercial kit #K1214 (Fermentas), and sequenced. The partial 16S rDNA sequence (GenBank Accession No. GQ249159) of the phytoplasma associated with P. aphylla witches’ broom was most closely related (98% sequence identity) to salt cedar witches’ broom phytoplasma, ‘Candidatus Phytoplasma tamaricis’ (FJ432664) (Zhao et al., 2009). Moreover, P. aphylla witches’ broom phytoplasma showed a 96% sequence identity with strains of group 16SrX, apple proliferation, including a ‘Ca. Phytoplasma pyri’-related strain from subgroup 16SrX-C, previously associated with pear decline in Iran (Salehi et al., 2008), which has been reported in almost all pear growing areas of Fars, Isfahan, Tehran and Khorasan provinces. This is the first report of a phytoplasma associated with P. aphylla witches’ broom.

Molecular identification of a phytoplasma associated with Russian olive witches’ broom in Iran

Russian olive trees (Elaeagnus angustifolia) showing witches’ broom symptoms typical of phytoplasma infection were observed in the Urmia region of Iran. A phytoplasma named Russian olive witches’ broom phytoplasma (ROWBp-U) was detected from all symptomatic samples by amplification of the 16S rRNA gene and 16S/23S rDNA spacer region using the polymerase chain reaction (PCR) which gave a product of expected length. DNA from symptomless plants used as a negative control yielded no product. The sequence of the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U showed 99% similarity with the homologous genes of members of the aster yellows group. We also detected a phytoplasma in neighboring alfalfa plants (AlWBp-U) showing severe witches’ broom symptoms. An 1107 bp PCR product from the 16S rRNA gene showed 99% homology with the corresponding product in ROWBp-U, suggesting the presence of the same phytoplasma actively vectored in the area. Further observations showed that Russian olive trees with typical ROWB symptoms were present in an orchard near Tehran which is located over 530 km south-east of the original Urmia site. The corresponding sequence of this phytoplasma (ROWBp-T) showed 99% homology to that of the ROWBp-U. A sequence homology study based on the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U and other phytoplasmas showed that ROWBp-U is most closely related to the 16SrI group. To our knowledge, this is the first report of a phytoplasma infection in a member of the Elaeagnaceae.

'Candidatus phytoplasma phoenicium' sp. nov., a novel phytoplasma associated with an emerging lethal disease of almond trees in Lebanon and Iran.

Almonds (Prunus amygdalus) represent an important crop in most Mediterranean countries. A new and devastating disease of almond trees in Lebanon was recently reported, characterized by the development of severe witches'-brooms on which no flowers or fruits developed, and leading to tree death within a few years. A phytoplasma was detected in diseased trees by PCR amplification of rRNA operon sequences, and RFLP patterns of amplified DNA indicated that the phytoplasma belonged to the pigeon pea witches'-broom (PPWB) group. In the present work, the presence of a phytoplasma in symptomatic plants was confirmed by electron microscopy; this phytoplasma was graft-transmissible to almond, plum and peach seedlings. The phytoplasma was characterized by sequence analysis of rRNA genes and was shown to be different from the phytoplasmas previously described in the PPWB group. A 16S rDNA phylogenetic tree identified the almond tree phytoplasma as a member of a distinct subclade of the class Mollicutes. Oligonucleotides have been defined for specific detection of the new phytoplasma. The almond phytoplasma from Lebanon was shown to be identical to a phytoplasma that induces a disease called 'almond brooming' in Iran, but different from another PPWB-group phytoplasma that infects herbaceous annual plants in Lebanon. Based on its unique properties, the name 'Candidatus Phytoplasma phoenicium' is proposed for the phytoplasma associated with almond witches'-broom in Lebanon and Iran.