Protein interaction mapping reveals widespread targeting of development-related host transcription factors by phytoplasma effectors.

Phytoplasmas are pathogenic bacteria that reprogram plant host development for their own benefit. Previous studies have characterized a few different phytoplasma effector proteins that destabilize specific plant transcription factors. However, these are only a small fraction of the potential effectors used by phytoplasmas; therefore, the molecular mechanisms through which phytoplasmas modulate their hosts require further investigation. To obtain further insights into the phytoplasma infection mechanisms, we generated a protein-protein interaction network between a broad set of phytoplasma effectors and a large, unbiased collection of Arabidopsis thaliana transcription factors and transcriptional regulators. We found widespread, but specific, interactions between phytoplasma effectors and host transcription factors, especially those related to host developmental processes. In particular, many unrelated effectors target specific sets of TCP transcription factors, which regulate plant development and immunity. Comparison with other host-pathogen protein interaction networks shows that phytoplasma effectors have unusual targets, indicating that phytoplasmas have evolved a unique and unusual infection strategy. This study contributes a rich and solid data source that guides further investigations of the functions of individual effectors, as demonstrated for some herein. Moreover, the dataset provides insights into the underlying molecular mechanisms of phytoplasma infection.

The observation of taxonomic boundaries for the 16SrII and 16SrXXV phytoplasmas using genome-based delimitation.

Within the 16SrII phytoplasma group, subgroups A-X have been classified based on restriction fragment length polymorphism of their 16S rRNA gene, and two species have been described, namely 'Candidatus Phytoplasma aurantifolia' and 'Ca. Phytoplasma australasia'. Strains of 16SrII phytoplasmas are detected across a broad geographic range within Africa, Asia, Australia, Europe and North and South America. Historically, all members of the 16SrII group share ≥97.5 % nucleotide sequence identity of their 16S rRNA gene. In this study, we used whole genome sequences to identify the species boundaries within the 16SrII group. Whole genome analyses were done using 42 phytoplasma strains classified into seven 16SrII subgroups, five 16SrII taxa without official 16Sr subgroup classifications, and one 16SrXXV-A phytoplasma strain used as an outgroup taxon. Based on phylogenomic analyses as well as whole genome average nucleotide and average amino acid identity (ANI and AAI), eight distinct 16SrII taxa equivalent to species were identified, six of which are novel descriptions. Strains within the same species had ANI and AAI values of >97 %, and shared ≥80 % of their genomic segments based on the ANI analysis. Species also had distinct biological and/or ecological features. A 16SrII subgroup often represented a distinct species, e.g., the 16SrII-B subgroup members. Members classified within the 16SrII-A, 16SrII-D, and 16SrII-V subgroups as well as strains classified as sweet potato little leaf phytoplasmas fulfilled criteria to be included as members of a single species, but with subspecies-level relationships with each other. The 16SrXXV-A taxon was also described as a novel phytoplasma species and, based on criteria used for other bacterial families, provided evidence that it could be classified as a distinct genus from the 16SrII phytoplasmas. As more phytoplasma genome sequences become available, the classification system of these bacteria can be further refined at the genus, species, and subspecies taxonomic ranks.

History and Current Status of Phytoplasma Diseases in the Middle East.

Phytoplasmas that are associated with fruit crops, vegetables, cereal and oilseed crops, trees, ornamental, and weeds are increasing at an alarming rate in the Middle East. Up to now, fourteen 16Sr groups of phytoplasma have been identified in association with more than 164 plant species in this region. Peanut witches' broom phytoplasma strains (16SrII) are the prevalent group, especially in the south of Iran and Gulf states, and have been found to be associated with 81 host plant species. In addition, phytoplasmas belonging to the 16SrVI, 16SrIX, and 16SrXII groups have been frequently reported from a wide range of crops. On the other hand, phytoplasmas belonging to 16SrIV, 16SrV, 16SrX, 16SrXI, 16SrXIV, and 16SrXXIX groups have limited geographical distribution and host range. Twenty-two insect vectors have been reported as putative phytoplasma vectors in the Middle East, of which Orosius albicinctus can transmit diverse phytoplasma strains. Almond witches' broom, tomato big bud, lime witches' broom, and alfalfa witches' broom are known as the most destructive diseases. The review summarizes phytoplasma diseases in the Middle East, with specific emphasis on the occurrence, host range, and transmission of the most common phytoplasma groups.

Association of the 16SrII-D Phytoplasma with African Marigold (Tagetes erecta) Phyllody in Oman.

The African marigold (Tagetes erecta L.) is an ornamental, herbaceous plant commonly found in Oman. In 2019, African marigold plants showing phyllody and virescence symptoms, which are typical symptoms of phytoplasmas disease, were found in at Sultan Qaboos University in Oman. Transmission electron microscopy of marigold leaf midrib from phyllody disease plants showed the presence of numerous phytoplasma bodies in the sieve tube of all of the symptomatic samples. DNA was extracted from asymptomatic and symptomatic marigold plant samples, followed by PCR of the 16S ribosomal RNA (rRNA) and imp genes. The PCR assays showed that the symptomatic plants are positive for phytoplasma. The DNA sequence analysis and phylogenetic trees showed that the 16S rDNA and imp gene sequences from all marigold phyllody strains shared 100% sequence identity to 16SrII-D subgroup sequences in the GenBank. This is the first report of a phytoplasma of the 16SrII-D subgroup associated with the African marigold (T. erecta) worldwide.

First report of a subgroup 16SrII-D phytoplasma associated with Opuntia cylindrica fasciated disease in Oman.

Cacti are evergreen perennial succulent plants that are used as ornamental and hedge plants. The fruits and leaves are also used as forage in some areas (Dewir, 2016). Cactus species are susceptible to several pathogens, including phytoplasma. In March 2020, three cactus plants (Opuntia cylindrica) out of ten (30% incidence) exhibited phytoplasma symptoms, including stunted growth, fasciation in stems and cladodes, color changes of the tips of cladodes to purple, and having clusters of highly proliferating cladodes. The plants were located in the Botanic Garden at Sultan Qaboos University, Muscat, Oman (N:23º59'14"; E:58º16'34"). PCR assays were carried out on the DNA samples extracted from young cladodes of three each of symptomatic and asymptomatic plants using phytoplasma-universal 16S rRNA primers, P1/P7 in direct PCR followed by R16F2n/R16R2, P4/P7 in the nested PCR. Distilled water (DW) and Alfalfa witches' broom phytoplasma (AlfWB) were used as negative and positive controls in each assay, respectively. In addition, amplification of the partial translocase protein A (secA) gene in the symptomatic cactus samples was done using SecA-II-F1/SecA-II-R1 (targeting 2140 bp) followed by SecA-II-F1/SecAR4 (targeting 1510 bp) (Al-Subhi et al., 2018). All the symptomatic plants and the positive control were positive for both genes (16S and secA), but no amplification was observed from the asymptomatic samples and DW. Sequence analysis and similarity searches against BLASTn revealed that the phytoplasma 16S rRNA (MT327813) shared 100% sequence identity with that of 'Candidatus Phytoplasma aurantifolia' isolate CB04 (MT555412) from India. The secA gene sequence (MT331815) analysis showed 100% identity with Cicer arietinum phyllody (KX358585). The associated phytoplasma was designated as cactus fasciated phytoplasma (CFP). Phylogenetic trees based on CFP 16Sr rRNA,secA genes, and a combined phylogenetic tree showed clustering of the CFP with the 16SrII-D subgroup phytoplasmas. The association of the aster yellows and peanut witches'-broom phytoplasma groups with other cactus species has already been reported from Lebanon, Mexico, China, Italy and Egypt (Dewir, 2016). The 16SrII phytoplasma in association with O. cylindrica showing fasciated stem has been reported from Egypt (Omar et al., 2014). A series of diverse plant species in association with 16SrII-D phytoplasma has been reported from Oman (Al-Subhi et al., 2018). However, this is the first report of a cactus phytoplasma disease in Oman belonging to the 16SrII-D subgroup phytoplasmas. Some fasciated cactus species are attractive and therefore cultivated as new ornamental plants and transported around the world, which may pose a new threat to other economically important crops.

Characterization of chickpea genotypes of Pakistani origin for genetic diversity and zinc grain biofortification.

BACKGROUND: Intake of food low in essential minerals, like zinc (Zn), is one of the major reasons of malnutrition. Development of genotypes with grains enriched in essential minerals may help to solve the issue of malnutrition. In this study, 16 chickpea genotypes (eight each of desi and kabuli types) of Pakistani origin were evaluated for genetic diversity and grain Zn biofortification potential with and without Zn fertilization. RESULTS: A wide variation was noted for agronomic, physiological, agro-physiological, utilization, and apparent recovery efficiencies of Zn in the chickpea genotypes tested. Genotypes also differed for grain Zn concentration (37.5-48.6 mg kg-1 ), bioavailable Zn (3.72-4.42 mg day-1 ), and grain yield. The highest grain Zn concentration and bioavailable Zn were noted in genotypes NIAB-CH-2016 (47.1 mg kg-1 and 4.30 mg day-1 respectively) and Noor-2013 (48.6 mg kg-1 and 4.38 mg day-1 respectively) among the desi and kabuli types respectively. The same genotypes were the highest yielders. Cluster analysis showed that all (eight) kabuli genotypes grouped together, whereas most (six) of the desi genotypes clustered in a separate group. There was low to moderate genetic diversity (0.149 for desi and 0.104 for kabuli types) and a low level of genetic differentiation between the two chickpea types (0.098). CONCLUSION: Two populations of chickpea had low to moderate genetic diversity, with consistent gene flow. This genetic diversity in both chickpea types allows the breeding gains for improving the grain yield and grain Zn biofortification potential of chickpea genotypes. © 2020 Society of Chemical Industry.

Genetic analysis of 'Candidatus Phytoplasma aurantifolia' associated with witches' broom on acid lime trees.

"Candidatus Phytoplasma aurantifolia" is associated with witches' broom disease of lime in Oman and the UAE. A previous study showed that an infection by phytoplasma may not necessarily result in the physical appearance of witches' broom symptoms in some locations in Oman and the UAE. This study investigated whether phytoplasma strains belonging to "Ca. P. aurantifolia" (based on the 16S rRNA gene analysis) in locations where disease symptoms are expressed are different from phytoplasma in locations where disease symptoms are not expressed. About 21 phytoplasma strains (15 from areas and trees with disease symptoms and six from areas and trees without disease symptoms) were included in the analysis. The study utilized sequences of the imp and SAP11 genes to characterize the 21 strains. Phylogenetic analysis of both genes showed that the 21 strains are similar to each other and to reference strains in GenBank. The study shows that there is a low level of diversity among all phytoplasma strains. In addition, it shows that phytoplasma in places where witches' broom symptoms are not expressed are similar to phytoplasma in places where disease symptoms are expressed. This may suggest that disease expression is not linked to the presence of different phytoplasma strains, but may be due to other factors such as weather conditions.

Detection, Identification, and Molecular Characterization of the 16SrII-D Phytoplasmas Infecting Vegetable and Field Crops in Oman.

Typical symptoms of phytoplasma infection were observed on 11 important crops in Oman that included alfalfa, sesame, chickpea, eggplant, tomato, spinach, rocket, carrot, squash, field pea, and faba bean. To identify the phytoplasmas in these crops, samples from infected and asymptomatic plants were collected, followed by amplifying and sequencing of the 16S ribosomal RNA, secA, tuf, imp, and SAP11 genes. We found that these sequences share >99% similarity with the peanut witches' broom subgroup (16SrII-D). Whereas some sequence variation was found in the five genes among 11 phytoplasma isolates of different crops, all sequences grouped into one clade along with those of other phytoplasmas belonging to the 16SrII-D group. Thus, 16SrII-D phytoplasmas infect a diverse range of crops in Oman. Phytoplasmas in this group have not been reported to occur in carrot, spinach, rocket, and field pea previously. Within Oman, this is the first report of the presence of 16SrII-D phytoplasmas in tomato, spinach, rocket, carrot, squash, field pea, and faba bean. Sequences of the five genes enabled for better distinction of the 16SrII-D phytoplasmas that occur in Oman.

Classification of a new phytoplasmas subgroup 16SrII-W associated with Crotalaria witches' broom diseases in Oman based on multigene sequence analysis.

BACKGROUND: Crotalaria aegyptiaca, a low shrub is commonly observed in the sandy soils of wadis desert and is found throughout all regions in Oman. A survey for phytoplasma diseases was conducted. During a survey in a wild area in the northern regions of Oman in 2015, typical symptoms of phytoplasma infection were observed on C. aegyptiaca plants. The infected plants showed an excessive proliferation of their shoots and small leaves. RESULTS: The presence of phytoplasma in the phloem tissue of symptomatic C. aegyptiaca leaf samples was confirmed by using Transmission Electron Microscopy (TEM). In addition the extracted DNA from symptomatic C. aegyptiaca leaf samples and Orosius sp. leafhoppers were tested by PCR using phytoplasma specific primers for the 16S rDNA, secA, tuf and imp, and SAP11 genes. The PCR amplifications from all samples yielded the expected products, but not from asymptomatic plant samples. Sequence similarity and phylogenetic tree analyses of four genes (16S rDNA, secA, tuf and imp) showed that Crotalaria witches' broom phytoplasmas from Oman is placed with the clade of Peanut WB (16SrII) close to Fava bean phyllody (16SrII-C), Cotton phyllody and phytoplasmas (16SrII-F), and Candidatus Phytoplasma aurantifolia' (16SrII-B). However, the Crotalaria's phytoplasma was in a separate sub-clade from all the other phytoplasmas belonging to Peanut WB group. The combination of specific primers for the SAP11 gene of 16SrII-A, -B, and -D subgroup pytoplasmas were tested against Crotalaria witches' broom phytoplasmas and no PCR product was amplified, which suggests that the SAP11 of Crotalaria phytoplasma is different from the SAP11 of the other phytoplasmas. CONCLUSION: We propose to assign the Crotalaria witches' broom from Oman in a new lineage 16SrII-W subgroup depending on the sequences analysis of 16S rRNA, secA, imp, tuf, and SAP11 genes. To our knowledge, this is the first report of phytoplasmas of the 16SrII group infecting C. aegyptiaca worldwide.

'Candidatus Phytoplasma omanense', associated with witches'-broom of Cassia italica (Mill.) Spreng. in Oman.

Samples from plants of Cassia italica exhibiting typical witches'-broom symptoms (Cassia witches'-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 'Candidatus Phytoplasma phoenicium'. 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 (99.6-100 % similarity). The closest relatives were members of the pigeon pea witches'-broom phytoplasma ribosomal group (16SrIX; 95-97 % sequence similarity). On the basis of unique 16S rRNA gene sequences and biological properties, the phytoplasma associated with witches'-broom of C. italica in Oman represents a coherent but discrete novel phytoplasma, 'Candidatus Phytoplasma omanense', with GenBank/DDBJ/EMBL accession number EF666051 representing the reference strain.

A divergent isolate of tomato yellow leaf curl virus from Oman with an associated DNA beta satellite: an evolutionary link between Asian and the Middle Eastern virus-satellite complexes.

Tomato is cultivated in the coastal region of Al-Batinah, in the Sultanate of Oman, during the winter season, to meet the high demand for fresh produce in the domestic market. In order to identify the causal agent of a widespread disease associated with infestations of the whitefly Bemisia tabaci (Genn.) leaves were collected from tomato plants showing symptoms characteristic of the disease in Al-Batinah during 2004 and 2005. Total nucleic acids were isolated from the tomato leaves and used as the template for Phi29 DNA polymerase amplification of begomoviral circular DNA. Putative full unit length begomoviral DNA multimers were digested with Nco I and cloned into the plasmid vector pGEM7Zf+. The complete nucleotide (nt) sequence was determined as 2,765 bases, indicative of a monopartite begomoviral genome. A comparison of the genome sequence for the seven field isolates examined, indicated that they shared 99% nt identity. The virus from Oman was most closely related to TYLCV-IR at 91% nt identity, a monopartite begomoviral species described previously from Iran. Based on the guidelines of the ICTV the Oman isolate has been designated TYLCV-Om and is considered an isolate of TYLCV-IR. A satellite DNA (satDNA beta), was amplified by polymerase chain reaction using degenerate primers and cloned, and the DNA sequence was determined. Analysis of the complete nt sequence of 1,371 bases indicated that the satDNA shared 88.5% similarity with its closest relatives, which are DNA beta molecules from tomato in Pakistan. This is the first report of a satDNA beta associated with the TYLCV species. The TYLCV-Om and associated satDNA, thus represent a begomovirus-complex at the Asian-Middle East crossroads that quiet uniquely share geographical and genetic hallmarks of both.

Ceratocystis omanensis, a new species from diseased mango trees in Oman.

Mango (Mangifera indica) sudden decline is an important disease in Oman, which is closely associated with infections by Ceratocystis fimbriata and Lasiodiplodia theobromae. Another Ceratocystis species has also been found associated with symptoms on diseased trees. In this study, we identify that Ceratocystis based on morphology and DNA sequences. Morphological comparisons showed that the fungus from dying mango trees in Oman is similar to C. moniliformis. Both fungi have distinct hat-shaped ascospores, disc-shaped plates at the bases of the ascomatal necks and spines on the ascomatal bases. However, comparison of DNA sequences for ITS1-2, the 5.8S RNA gene, the beta-tubulin gene, and Transcription Elongation Factor (EF1-alpha) gene, confirmed that the fungus from Oman is distinct from C. moniliformis and other related species. Phylogenetically, this fungus formed one of four strongly supported sub-clades. The other sub-clades included isolates of C. bhutanensis, C. moniliformis and C. moniliformopsis, respectively. Based on morphological characteristics and differences in DNA sequences for three gene regions, we conclude that the Ceratocystis sp. from wounds on mango in Oman is a new species, for which we provide the name Ceratocystis omanensis sp. nov.