Characterization of a 16SrII subgroup D phytoplasma strain associated with Calendula officinalis phyllody in Iran

Abstract

plants with phyllody symptoms (CaoP) were observed in Yazd and Ashkezar (Yazd province, Iran) during 2013-2016. Twenty-one symptomatic and four asymptomatic plants were transferred individually to the greenhouse and potted for the biological and molecular characterization of associated phytoplasma. The dodder transmission from symptomatic potted marigold plants, induced virescence, phyllody and witches' broom symptoms in periwinkle. Total DNAs extracted from symptomatic and symptomless plants and dodder-inoculated periwinkles were subjected to nested PCR assay using primer pairs amplifying phytoplasma ribosomal DNA. Expected PCR amplification was detected in all CaoP plant and dodder-inoculated periwinkle samples. RFLP analysis of the amplicons obtained in direct PCR with P1/P7 primers using I,I, I,fI and III restriction enzymes showed profiles identical to each other and related to phytoplasmas in all the 21 positive samples. R16mF2/R16mR2 amplicons from six CaoP plant samples were sequenced where consensus sequences had 100% of identity among each other. R16F2n/R16R2-trimmed sequences (1248 bp) of representative samples from Yazd and Ashkezar were deposited in GenBank under accession numbers KU297202 and MH065715, respectively. BLAST search and phylogenetic analysis showed that the CaoP phytoplasma had 99% homology and clusters with phytoplasmas in group 16SrII. Computer-simulated analysis usingPhyClassifier suggests that the CaoP RFLP 16S rRNA gene pattern was identical to 16SrII-D phytoplasmas subgroup. Phytoplasma strains (16SrII-D) were reported as alfalfa witches' broom disease agent previously in the same geographic areas, and it is possible that alfalfa plays a role in the epidemiology of CaoP disease or vice-versa.

Detection and Molecular Characterization of Phytoplasma Associated with Phyllody Disease on Dimorphotheca pluvialis in Egypt

“…Phytoplasma-induced symptoms were successfully transmitted from naturally infected African daisy plants to healthy periwinkle ones in the greenhouse of the Virus and Phytoplasma Research Department, Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt using dodder transmissions as described by El-Banna et al (2015), with 90% as the efficiency percentage of transmission, that was within the range reported by Ranebennur et al (2022), who noticed the dodder transmission of sesame phyllody disease was reached to be 93.33% (sesame to sesame) and 92.85% (sesame to periwinkle). The exhibited phyllody symptoms were very similar to the previous studies on the transmission phytoplasma using the parasitic dodder plant, such as gazania phyllody disease in gazania plants (Gad et al, 2019), Calendula officinalis phyllody disease (Esmailzadeh Hosseini et al, 2018), rose phyllody disease (Mikhail et al, 2012), sesame phyllody disease (Ranebennur et al, 2022) and safflower phyllody phytoplasma (Salehi et al, 2009). Phytoplasma-infected African daisy plants, exhibited many ultrastructural changes in the vascular bundles with clearly thickness of the cell walls and many necrotic areas between phloem cells in the petioles midribs using light microscopy as mentioned by El-Banna et al (2015).…”

“…The full length of 16S rRNA gene DNA fragments of Dimorphotheca pluvialis phyllody phytoplasma (Dimo-Cairo) from all three Egyptian isolates were 1250 bp in size. Such a value is within the range recorded by other authors for phytoplasma in the ornamental plants, such as rose (Mikhail et al, 2012), pot marigold (Calendula officinalis) (Esmailzadeh Hosseini et al, 2018;Gharouni-Kardani et al, 2020), gazania (Gad et al, 2019), Coreopsis grandiflora (Gharouni-Kardani et al, 2020), Dodonaea viscosa (Mokbel, 2020), lily (Lilium spp.) (Abdel-Salam et al, 2022) and narrowleaf firethorn (Pyracantha angustifolia) plants (Kilic et al, 2022).…”

“…Phytoplasma is considered as a devastating phytopathogenic mollicute in several ornamental plants grown in fields, gardens and greenhouses, causing economic yield losses, especially in the flower productions worldwide (Kilic et al, 2022;Viczián et al, 2023;Bernardini et al, 2022;Salehi et al, 2009;Salehi et al, 2022;Gharouni-Kardani et al, 2020;Esmailzadeh Hosseini et al, 2018;Fernández et al, 2018;Montano et al, 2014;Lee et al, 2012;Bertaccini et al, 2005;Alma et al, 2000;Gautam et al, 2020). In Egypt, phytoplasma was found as Candidatus phytoplasma asteris infecting lilies (Lilium spp.)…”

“…P. astris) associated with severe fasciation symptoms in lily (Lilium spp.) plants (Bertaccini et al, 2005), Candidatus phytoplasma asteris association with leaf yellowing of Wrightia antidysenterica (also known as 'Arctic Snow') (Family Pocynaceae) (Shreenath et al, 2021), Candidatus phytoplasma malaysianum (16SrXXXII) associated with a decline disease affecting the leaves and seeds of E. sylvestris (Family Elaeocarpaceae) (Lee et al, 2022), aster yellows phytoplasma associated with a little leaf disease in papaya (Carica papaya L.) plant (Mitra et al, 2020), Candidatus Phytoplasma australasia associated with little leaf and yellowing symptoms in Pyracantha angustifolia (Family: Rosaceae) (Kilic et al, 2022), Candidatus phytoplasma fraxini' associated with witches' broom disease in Erigeron bonariensis (Montano et al, 2014), Chrysanthemum yellows phytoplasma infecting Arabidopsis thaliana wild type and Atcals7ko mutant plants (Bernardini et al, 2022), Coreopsis grandiflora phyllody phytoplasma infecting pot marigold (Calendula officinalis L., family Asteraceae) and tickseed (Coreopsis grandiflora) (Gharouni-Kardani et al, 2020), Candidatus Phytoplasma asteris associated with yellows disease in gerbera (Gerbera jamesonii) (commonly known as African daisy) (family Asteraceae) (Gautam et al, 2020), Candidatus Phytoplasma asteris' strain associated with phyllody disease in pot marigold (Family Asteraceae) (Salehi et al, 2022;Gharouni-Kardani et al, 2020;Esmailzadeh Hosseini et al, 2018) and jujube witches'broom (JWB) phytoplasma in periwinkle (Cantharanthus roseus) plant (Lee et al, 2012). In Egypt, phytoplasma infecting lilies (Lilium spp.)…”

Pest categorisation of the non‐EU phytoplasmas of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L.

“…Calendula officinalis : leaf size reduction, yellowing, phyllody, virescence, proliferation and sterility in the flower, proliferation of axillary buds along the stem, witches’ broom and stunting (Esmailzadeh‐Hosseini et al., );…”