Assunta Bertaccini

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.

Detection and differentiation of the coconut lethal yellowing phytoplasma in coconut-growing villages of Grand-Lahou, Côte d’Ivoire

Surveys for the Cote dIvoire lethal yellowing (CILY) phytoplasma were conducted in eight severely CILY-affected villages of Grand-Lahou in 2015. Leaves, inflorescences and trunk borings were collected from coconut palms showing CILY symptoms and from symptomless trees. Total DNA was extracted from these samples and tested by nested polymerase chain reaction/RFLP and sequence analysis of the 16S rRNA, ribosomal protein (rp) and the translocation protein (secA) genes. The CILY phytoplasma was detected in 82.9% of the symptom-bearing palms collected from all the surveyed villages and from all the plant parts. Trunk borings were recommended as the most suitable plant tissue type for sampling. Results indicate that the CILY phytoplasma may have a westward spread to other coconut-growing areas of Grand-Lahou. CILY phytoplasma strains infecting coconut palms in the western region of Grand-Lahou exhibited unique single nucleotide polymorphisms on the rp sequence compared to the strains from the eastern region. Moreover, single nucleotide polymorphisms on the SecA sequence distinguished the CILY phytoplasma from the Cape St. Paul Wilt Disease phytoplasma in Ghana, and the Lethal Yellowing phytoplasma in Mozambique.

General phytoplasma detection by a q-PCR method using mycoplasma primers

Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field-infected plant samples showed a single peak at 82.5xa0°C (±0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: Candidatus Liberibacter solanacearum, Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except Ca. L. solanacearum that was amplified with a melting temperature at 85xa0°C. Absolute quantification of phytoplasma titer was calculated using standard curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 106 to 103 according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10-6 for conventional PCR and 10-7 for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material.

Molecular and biologic characterization of a phytoplasma associated with Brassica campestris phyllody disease in Punjab province, Pakistan

A phytoplasma-associated disease was identified in Brassica campestris (sarson) plants during a survey conducted in Punjab province of Pakistan in 2014–2016. The symptomatic plants showed characteristic symptoms of phyllody and witches’ broom. Phytoplasma presence was detected by polymerase chain reaction on 16S ribosomal and tuf DNAs, followed by RFLP analysis and sequence comparison of the 16S rRNA and tuf genes. The phytoplasma detected was classified in a new ribosomal subgroup designed 16SrIX-H. The phytoplasma presence in phloem tissues of symptomatic sarson samples was also confirmed through light microscopy and transmission studies to healthy plants through dodder and the leafhopper Orosius albicinctus. This is the first report of identification of 16SrIX-H subgroup phytoplasma associated with sarson and its natural vector in Pakistan.

Association of Eriophyes dimocarpi (Acari: Eriophyidae) with longan witches’ broom disease in Vietnam

Abstract Longan witches’ broom (LgWB) syndrome was observed in all longan-growing provinces of Vietnam. Key symptoms include: small, stunted shoots with curved, rolled up margins, deformed leaves with blisters and hairy patches on the underside; abnormal development of flower structures, flower abortion, failure to produce fruits or developing small fruits. During 2012–2015, field surveys and study of the relationship between LgWB symptoms and Eriophyes dimocarpi mites presence were carried out. Moreover, molecular analysis, electron microscopy observations, E. dimocarpi inoculations, pruning of affected spikelets and miticide experiments were performed. An average of 36.58% of longan seedlings inoculated with E. dimocarpi mites showed typical symptoms of LgWB at about 27.5 days after inoculation. The use of several pesticides in different combinations showed high efficacy on E. dimocarpi mites elimination and of symptomatology incidence reduction. These results suggest a significant association of E. dimocarpi with LgWB syndrome in Vietnam.

Detection and identification of phytoplasmas associated with declining Liquidambar styraciflua trees in Colombia

Liquidambar styraciflua was introduced in Bogotá (D. C.) Colombia, in the 1990s as an urban tree. Yellowing and deformation of the tree crowns began to appear in 2007, leading to the hypothesis of a possible phytoplasma infection. To detect and identify phytoplasmas, samples were collected from 21 liquidambar trees from Bogota in 2009, 2010 and 2011 and subjected to nested PCR, RFLP and sequencing analyses of 16S ribosomal DNA. Nested PCR assays with group-specific primers and RFLP analyses identified phytoplasmas belonging to the 16SrI-B (aster yellows), 16SrV-B (jujube witches’ broom), 16SrVII-A (ash yellows), 16SrIX (pigeon pea witches’ broom) and 16SrXII-A (“stolbur”) groups/subgroups, in single or mixed infections. Sequence analysis and phylogenetic evaluation of samples with single phytoplasma infections confirmed the presence of phytoplasmas related to ‘Candidatus Phytoplasma asteris’, ‘Ca. P. fraxini’, and ‘Ca. P. solani’. In 2010 and 2013 two further surveys involving 100 trees each time were carried out to estimate the prevalence of the disease: the average prevalence with severe symptoms was 28%, with mild symptoms, 56%, and with minor symptoms 16% of the trees. No symptomless trees were observed. Phytoplasma presence in liquidambar trees from Bogota carries economic, environmental and epidemiological consequences, and urgent measures should be implemented to avoid the spreading of the disease to other tree species and to the agricultural areas surrounding the city.

Detection and characterization of phytoplasmas infecting five plant species in Egypt

Samples of orange, hibiscus, peach, olive and pepper plants showing symptoms of dwarf branches with shortened internodes, leaf deformation and chlorosis, proliferation of axillary buds, yellowing, twisting and streaks together with samples from asymptomatic plants of the same age were collected from El-Behira and Alexandria Governorates, Egypt during 2015–2016. The samples were tested to verify phytoplasma presence by nested PCR assays using the ribosomal gene amplifying primers R16F2n/R2 on the P1/P7 1: 30 diluted amplicons as template. Amplification bands were obtained only from samples collected from symptomatic plants, and the RFLP analyses with informative restriction enzymes indicated that detected phytoplasmas belonged to 16SrII-D subgroup. These phytoplasmas are reported for the first time to infect olive trees, and for the first time in Egypt in peach, orange and hibiscus plants.

New phytoplasma subgroup identified from Arecaceae palm species in Grand-Lahou, Côte d’Ivoire

Abstract Coconut palms in the south coastal littoral of Grand-Lahou have long been affected by the Côte d’Ivoire lethal yellowing (CILY), associated with subgroup 16SrXXII-B ‘Candidatus Phytoplasma palmicola’-related strains. Recently, foliar discolouration and yellowing symptoms were observed in oil palms (Elaeis guineensis), Rônier palms (Borasus aethiopium), and raffia palms (Raphia vinifera) located in the vicinity of coconut palms (Cocos nucifera) showing CILY-like symptoms in coconut-growing villages of Grand-Lahou. Immature leaf samples were randomly collected from symptom-bearing palms, including coconut palms. Total DNA was extracted and used as a template for a nested PCR assay with universal primers that target the phytoplasma 16S rRNA gene, and the translocation protein (secA) genes. The R16F2n/R2 and SecA amplicons from representative samples of each palm species were purified, cloned, and sequenced. R16F2n/R2 sequences were over 99% identical to phytoplasmas enclosed in group 16SrXXII. Based on iPhyclassifier analyses with 17 restriction enzymes, the phytoplasmas found in C. nucifera, E. guineensis, B. aethiopium and R. vinifera were identical to each other. However, a unique Sau3AI RFLP pattern was observed, and similarity coefficients were lower than 0.97, and so a new 16SrXXII subgroup, 16SrXXII-C, was assigned. Phylogeny based on both R16F2n/R2 and SecA sequences supported this RFLP classification. The presence of the subgroup 16SrXXII-C in the same area where coconut palms are severely infected by the subgroup 16rSXXII-B could lead to different epidemiological constraints that may affect the severity of the coconut disease. Thus, the 16SrXXII-C phytoplasma poses a new threat for other Arecaceae species, which may impact CILY management and control in Grand-Lahou.