Molecular Characterization of the 16S rRNA Gene of Phytoplasmas Detected in Two Leafhopper Species Associated with Alfalfa Plants Infected with Witches' Broom in Oman

Two leafhopper species, Austroagallia avicula and Empoasca sp., were consistently found in alfalfa fields infected with witches’ broom phytoplasma (OmanAlfWB) in the Al-Batinah, Dakhliya, North and South Sharqiya, Muscat, and Al-Bureimi regions of the Sultanate of Oman. Phytoplasmas from both leafhoppers were detected by specific polymerase chain reaction (PCR) amplification of the 16S rRNA gene and the spacer region in direct PCR using P1/P7 primer pairs. Comparative RFLP profiles of the amplified rRNA gene and the spacer region from leafhopper phytoplasmas and from 20 phytoplasma controls yielded patterns referable to phytoplasmas belonging to the peanut witches’ broom group (16SrII group). In particular, extensive RFLP analyses with the endonucleases HpaII, Tru9I, Tsp509I, and RsaI indicated that the phytoplasmas in A. avicula and Empoasca sp. were identical but showed some differences from OmanAlfWB; however, RFLP patterns obtained with TaqI showed the OmanAlfWB and the phytoplasmas from the two leafhoppers to be identical. Direct PCR products amplified from phytoplasma leafhopper DNA using the P1/P7 primer pair were cloned and sequenced yielding 1758 bp and 1755 bp products from A. avicula and Empoasca sp. respectively; the homology of these sequences with OmanAlfWB and papaya yellow crinkle phytoplasmas was more than 98%. A phylogenetic tree based on the 16S rRNA gene and spacer region sequences from 44 phytoplasmas revealed that the phytoplasmas from the leafhoppers clustered with OmanAlfWB, papaya yellow crinkle, and gerbera phyllody phytoplasmas, all belonging to 16SrII group, but were distinct from lime witches’ broom phytoplasma, the most commonly found phytoplasma in the Sultanate of Oman

Ceratocystis manginecans associated with a serious wilt disease of two native legume trees in Oman and Pakistan

A serious wilt disease has recently been found on Prosopis cineraria (Ghaf) in Oman and on Dalbergia sissoo (Shisham) in Pakistan. Disease symptoms on both these native, leguminous hosts include vascular discolouration and partial or complete wilt of affected trees. A species of Ceratocystis was consistently isolated from symptomatic material. Morphological comparisons and analyses of DNA sequence data of the ITS, β-tubulin, and EF 1-α gene regions showed that the Ceratocystis isolates obtained from both tree species represent C. manginecans. This is the same pathogen that is causing the devastating mango sudden decline disease in Oman and Pakistan. This is also the same pathogen that has been reported causing a wilting disease on Acacia mangium in Indonesia. Cross inoculation with C. manginecans isolates from P. cineraria, D. sissoo and mango showed that the fungus can cause disease on all three trees.Tree Protection Cooperative Programme (TPCP), University of Pretoria, South Africa, and the Ministry of Agriculture and Fisheries, Omanhttp://link.springer.com/journal/13313hb201

A new phytoplasma associated with witches\u2019 -broom of Cassia italica in Oman.

In several Oman locations plants of Cassia italica exhibit witches\u2019 broom symptoms. Samples were collected from four locations, and examined for phytoplasma presence. PCR amplification using ribosomal phytoplasma primers followed by RFLP analyses indicates that the phytoplasmas present in these samples were undistinguishable from each other, but differed from those reported in the literature. RFLP and phylogenetic analysis of 16S rRNA gene plus spacer sequence confirmed that the closest phytoplasma relatives were members of the pigeon pea witches\u2019 broom phytoplasma ribosomal group (16SrIX), sharing a 93-97% sequence similarity. Nested PCR experiments using primers amplifying the gene coding for ribosomal protein S3 provided amplification from phytoplasmas detected in C. italica, after sequencing 600 bp in this gene the higher homology showed was 78% with phytoplasmas related to \u2018Candidatus Phytoplasma phoenicium\u2019 and 77% with phytoplasmas belonging to ribosomal group 16SrIX

\u2018Candidatus Phytoplasma omanense\u2019, a phytoplasma associated with witches\u2019 broom of Cassia italica (Mill.) Lam. in Oman.

Samples from plants of Cassia italica exhibiting typical witches\u2019-broom symptoms (Cassia witches\u2019-broom; CWB) were examined for the presence of plant pathogenic phytoplasmas by PCR amplification using universal phytoplasma primers. All affected plants yielded positive results. RFLP analyses of rRNA gene products indicated that the phytoplasmas detected were different from those described previously. Phylogenetic analysis of 16S rRNA gene sequences confirmed that CWB represents a distinct lineage and shares a common ancestor with \u2018Candidatus Phytoplasma phoenicium\u2019. Molecular comparison revealed that the 16S rRNA gene sequences of the four CWB strains (IM-1, IM-2, IM-3 and IM-4) identified in symptomatic C. italica samples were nearly identical (99.6\u2013100% similarity). The closest relatives were members of the pigeon pea witches\u2019-broom phytoplasma ribosomal group (16SrIX; 95\u201397% sequence similarity). On the basis of unique 16S rRNA gene sequences and biological properties, the phytoplasma associated with witches\u2019-broom of C. italica in Oman represents a coherent but discrete novel phytoplasma, \u2018Candidatus Phytoplasma omanense\u2019, with GenBank/DDBJ/EMBL accession number EF666051 representing the reference strain

Unisexual reproduction in Huntiella moniliformis

Sexual reproduction in fungi is controlled by genes present at the mating type (MAT) locus, which typically harbors transcription factors that influence the expression of many sex-related genes. The MAT locus exists as two alternative idiomorphs in ascomycetous fungi and sexual reproduction is initiated when genes from both idiomorphs are expressed. Thus, the gene content of this locus determines whether a fungus is heterothallic (self-sterile) or homothallic (self-fertile). Recently, a unique sub-class of homothallism has been described in fungi, where individuals possessing a single MAT idiomorph can reproduce sexually in the absence of a partner. Using various mycological, molecular and bioinformatic techniques, we investigated the sexual strategies and characterized the MAT loci in two tree wound-infecting fungi, Huntiella moniliformis and Huntiella omanensis. H. omanensis was shown to exhibit a typically heterothallic sexual reproductive cycle, with isolates possessing either the MAT1-1 or MAT1-2 idiomorph. This was in contrast to the homothallism via unisexual reproduction that was shown in H. moniliformis, where only the MAT1-2-1 gene was present in sexually reproducing cultures. While the evolutionary benefit and mechanisms underpinning a unisexual mating strategy remain unknown, it could have evolved to minimize the costs, while retaining the benefits, of normal sexual reproduction.University of Pretoria, the Department of Science and Technology (DST)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology and the Genomics Research Institute (University of Pretoria Institutional Research Theme) and the National Research Foundation of South Africa. specific unique reference number (UID) 83924).http://www.elsevier.com/locate/yfgbi2016-07-31hb201