Pythium and Globisporangium species associated with cucumber rhizosphere causing damping-off and their effects on cucumber seed decay in Oman.

Pythium sensu lato (s.l.) is a pathogenic oomycete. The present study was conducted to isolate and identify Pythium s.l. species associated with the rhizosphere and roots of greenhouse-growing cucumbers showing damping-off symptoms in 10 Omani governorates (provinces). A total of 166 isolates were recovered from 276 rhizosphere soil and root samples and were identified based on the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit I (COX I) gene region. Pythium aphanidermatum, P. myriotylum, Globisporangium spinosum, Globisporangium sp.1 (isolates Kb003/PySyCu-1 and Kb004/PySyCu-2), and Globisporangium sp.2 (isolate Ib002R) were identified. Among these species, P. aphanidermatum was the most abundant species, represented by 143 isolates (86.1%), followed by G. spinosum with 18 isolates (10.8%), Globisporangium sp.1 and P. myriotylum each with 2 isolates (2.4%), and Globisporangium sp.2 with 1 isolate (0.6%). Pathogenicity tests were also conducted for 38 isolates, including P. aphanidermatum (25), P. myriotylum (2), Globisporangium sp.2 (1), G. spinosum (8), and Globisporangium sp.1 (2). Among the tested isolates, only Globisporangium sp.2 isolate was avirulent, and none of the seeds were rotted at the end of the treatment. However, the other species induced the symptoms of seed decay with the incidence ranged from 86.7 to 100%. Phylogenetic analyses were conducted based on 222 ITS and 53 COX I sequences, and confirmed morphological identification. In addition, the genetic diversity of 93 P. aphanidermatum isolates was assessed via the amplified fragment length polymorphism (AFLP) method. The analysis produced 93 genotypes and 449 polymorphic loci. Pythium aphanidermatum populations were found to have moderate levels of genetic diversity (H = 0.2) and a moderate Shannon information index (I = 0.3793). Analysis of molecular variance (FST = 0.1, P = 0.0) revealed a moderate level of genetic differentiation among P. aphanidermatum isolates between Oman governorates. The sensitivity of 15 P. aphanidermatum isolates was evaluated against hymexazol at different concentrations (10, 100, and 1000 ppm). The results revealed that P. aphanidermatum could grow well at concentrations of up to 100 ppm hymexazol. However, hymexazol at 1000 ppm retarded the growth of P. aphanidermatum. This study showed that P. aphanidermatum is the most prevalent species in greenhouses in Oman and exhibited a moderate level of genetic diversity. Most of the isolates exhibited differences in tolerance to hymexazol but showed no resistance.

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.

An Insight into Emerging Begomoviruses and their Satellite Complex causing Papaya Leaf Curl Disease.

Papaya leaf curl disease (PaLCD) was primarily detected in India and causes major economic damage to agriculture crops grown globally, seriously threatening food security. Begomoviruses are communicated by the vector Bemisia tabaci, and their transmission efficiency and persistence in the vector are the highest, exhibiting the widest host range due to adaptation and evolution. Symptoms induced during PaLCD include leaf curl, leaf yellowing, interveinal chlorosis, and reduced fruit quality and yield. Consequently, plants have evolved several multi-layered defense mechanisms to resist Begomovirus infection and distribution. Subsequently, Begomovirus genomes organise circular ssDNA of size ~2.5-2.7 kb of overlapping viral transcripts and carry six-seven ORFs encoding multifunctional proteins, which are precisely evolved by the viruses to maintain the genome-constraint and develop complex but integrated interactions with a variety of host components to expand and facilitate successful infection cycles, i.e., suppression of host defense strategies. Geographical distribution is continuing to increase due to the advent and evolution of new Begomoviruses, and sweep to new regions is a future scenario. This review summarizes the current information on the biological functions of papaya-infecting Begomoviruses and their encoded proteins in transmission through vectors and modulating host-mediated responses, which may improve our understanding of how to challenge these significant plant viruses by revealing new information on the development of antiviral approaches against Begomoviruses associated with PaLCD.

First report of dieback on fig (Ficus carica L.) caused by Lasiodiplodia theobromae in North Al-Batinah governorate of Oman.

Fig (Ficus carica L.) is widely grown in Oman mainly for its nutritional value and health benefits. In May 2020, survey was conducted for fig trees which were showing symptoms of decline in three farms where more than 1500 fig trees were grown. The incidence of the disease was 3 to 4 % of fig trees showing symptoms of dieback of the twigs, death of entire branch and discoloration of vascular tissues were observed in Sohar, North Al-Batinah governorate of Oman. The disease severity was range between 25 to 40 % in most of the infected tree. The death of severely affected trees was observed in about 3 % of the orchards. Symptomatic samples were collected and isolation of the causal agent was performed. Infected tissues were cut into small pieces and surface-sterilized with 1 % NaOCl for 3 min. The tissues were rinsed with sterile distilled water 3-4 times, blotted dry on sterile filter paper and placed on potato dextrose agar (PDA) medium under aseptic conditions. After 2-3 days of incubation of plates at 27°C, pure culture of the fungus was obtained by hyphal tip isolation technique. Two identical isolates were grown in the PDA medium. The fungal colony was dark grey to black colored on PDA medium and the fungus produced dense aerial mycelium and numerous dark colored pycnidia in 25 days at ambient temperature range between 23-25 ℃ and nature photoperiod. Pycnidia were raptured and 20 emerged conidia were measured. The mature conidia were 1-septate, brown, ellipsoid, with thick cell wall and having longitudinal striation. The average measurement of the length of 20 conidia was 22.5-25 (24.9 µm) however the width measured was 12.5-15 (15 µm). To identify the fungus, DNA was extracted from the mycelium using phenol-chloroform-isoamyl alcohol method, following Al-Mahmooli et al. (2015). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and translation elongation factor 1-α (TEF) gene were amplified using ITS-4 and ITS-5 primers for ITS (White et al. 1990), EF1-983F and EF1-2218R and EF1-728F and EF1-986R primers for TEF (Alves et al. 2008), respectively and the amplified products were sequenced. BLASTn analysis of the ITS sequence of the fungal isolate revealed 100% identity with reference sequences of Lasiodiplodia theobromae (Pat.) Griffon & Maubl (MT075438.1). The sequence of TEF gene was 99% identical to the sequences of L. theobromae (XM_035519539.1 and MN461169.1). The nucleotide sequences were submitted to GenBank and accessioned with (MW590660) for ITS and (MZ159970, MZ159971, OM654917) for TEF gene. For phylogenetic analysis, we constructed a combined ITS-TEF dataset, following (Alves et. 2008). Using Maximum Likelihood phylogenetic analysis of the combined ITS-TEF dataset, our specimen falls in a clade formed by the members of L. theobromae (Supplementary materials). Pathogenicity of the fungus of three replicates (three fig seedlings) was established by artificial inoculation of 2- to 3-month-old potted fig seedlings with the fungus by making pin prick injuries on the stem at 5 cm above the soil level with a sterile inoculation needle, placing mycelial discs containing spores and pycnidia (15 × 15 mm) from 20-days old colonies grown on PDA on the injured surface and wrapping with parafilm which removed 48 hrs after establishing of infection. The three inoculated plants showed withering and drying of leaves 14 days after inoculation (DAI) begins near to the inoculation site expend to the younger leaves. Eventually, seedlings were showing dieback and death of entire seedling occurs in 25-28 DAI. The mock-inoculated plants remained symptomless. The fungus re-isolated from the diseased plants was confirmed by sequencing of ITS regions. The prevalence of dieback could be a potential threat to cultivation of fig in Oman. To the best of our knowledge, this is the first report of dieback disease caused by L. theobromae on fig in Oman.

Effects of the Rhizosphere Fungus Cunninghamella bertholletiae on the Solanum lycopersicum Response to Diverse Abiotic Stresses.

This study examined the efficiency of fungal strain (Cunninghamella bertholletiae) isolated from the rhizosphere of Solanum lycopersicum to reduce symptoms of salinity, drought and heavy metal stresses in tomato plants. In vitro evaluation of C. bertholletiae demonstrated its ability to produce indole-3-Acetic Acid (IAA), ammonia and tolerate varied abiotic stresses on solid media. Tomato plants at 33 days' old, inoculated with or without C. bertholletiae, were treated with 1.5% sodium chloride, 25% polyethylene glycol, 3 mM cadmium and 3 mM lead for 10 days, and the impact of C. bertholletiae on plant performance was investigated. Inoculation with C. bertholletiae enhanced plant biomass and growth attributes in stressed plants. In addition, C. bertholletiae modulated the physiochemical apparatus of stressed plants by raising chlorophyll, carotenoid, glucose, fructose, and sucrose contents, and reducing hydrogen peroxide, protein, lipid metabolism, amino acid, antioxidant activities, and abscisic acid. Gene expression analysis showed enhanced expression of SlCDF3 and SlICS genes and reduced expression of SlACCase, SlAOS, SlGRAS6, SlRBOHD, SlRING1, SlTAF1, and SlZH13 genes following C. bertholletiae application. In conclusion, our study supports the potential of C. bertholletiae as a biofertilizer to reduce plant damage, improve crop endurance and remediation under stress conditions.

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.

Ampelopsin Confers Endurance and Rehabilitation Mechanisms in Glycine max cv. Sowonkong under Multiple Abiotic Stresses.

The present investigation aims to perceive the effect of exogenous ampelopsin treatment on salinity and heavy metal damaged soybean seedlings (Glycine max L.) in terms of physiochemical and molecular responses. Screening of numerous ampelopsin concentrations (0, 0.1, 1, 5, 10 and 25 µM) on soybean seedling growth indicated that the 1 µM concentration displayed an increase in agronomic traits. The study also determined how ampelopsin application could recover salinity and heavy metal damaged plants. Soybean seedlings were irrigated with water, 1.5% NaCl or 3 mM chosen heavy metals for 12 days. Our results showed that the application of ampelopsin raised survival of the 45-day old salinity and heavy metal stressed soybean plants. The ampelopsin treated plants sustained high chlorophyll, protein, amino acid, fatty acid, salicylic acid, sugar, antioxidant activities and proline contents, and displayed low hydrogen peroxide, lipid metabolism, and abscisic acid contents under unfavorable status. A gene expression survey revealed that ampelopsin application led to the improved expression of GmNAC109, GmFDL19, GmFAD3, GmAPX, GmWRKY12, GmWRKY142, and GmSAP16 genes, and reduced the expression of the GmERF75 gene. This study suggests irrigation with ampelopsin can alleviate plant damage and improve plant yield under stress conditions, especially those including salinity and heavy metals.

Elicitins as molecular weapons against pathogens: consolidated biotechnological strategy for enhancing plant growth.

To fight against pathogens, defense systems in plants mainly depend upon preformed as well as induced responses. Pathogen detection activates induced responses and signals are transmitted for coordinated cellular events in order to restrict infection and spread. In spite of significant developments in manipulating genes, transcription factors and proteins for their involvement in immunity, absolute tolerance/resistance to pathogens has not been seen in plants/crops. Defense responses, among diverse plant types, to different pathogens involve modifications at the physio-biochemical and molecular levels. Secreted by oomycetes, elicitins are small, highly conserved and sterol-binding extracellular proteins with PAMP (pathogen associated molecular patterns) functions and are capable of eliciting plant defense reactions. Belonging to multigene families in oomycetes, elicitins are different from other plant proteins and show a different affinity for binding sterols and other lipids. These function for sterols binding to catalyze their inter-membrane and intra- as well as inter-micelle transport. Importantly, elicitins protect plants by inducing HR (hypersensitive response) and systemic acquired resistance. Despite immense metabolic significance and the involvement in defense activities, elicitins have not yet been fully studied and many questions regarding their functional activities remain to be explained. In order to address multiple questions associated with the role of elicitins, we have reviewed the understanding and topical advancements in plant defense mechanisms with a particular interest in elicitin-based defense actions and metabolic activities. This article offers potential attributes of elicitins as the biological control of plant diseases and can be considered as a baseline toward a more profound understanding of elicitins.

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.

Antagonistic Activity of Endophytic and Rhizosphere Fungi Isolated From Sea Purslane (Sesuvium portulacastrum) Against Pythium Damping off of Cucumber.

This study was conducted to investigate the antagonistic activity of endophytic and rhizosphere fungi isolated from a medicinal plant, Sesuvium portulacastrum, against Pythium aphanidermatum, the cause of damping off of cucumber. A total of 40 endophytic and 19 rhizosphere fungi were isolated from S. portulacastrum. Three endophytic isolates and two rhizosphere isolates gave >50% suppression of P. aphanidermatum in the in vitro dual-culture tests. Scanning electron microscopic studies at the inhibition zone showed hyphae wall damage and abnormal mycelial growth of the genus Pythium. Molecular analysis identified the antagonistic endophytes as Aspergillus insulicola (isolate A435), A. insulicola (A419), and Aspergillus melleus (A412) and the rhizosphere antagonists as Aspergillus terreus (A213) and Aspergillus luchuensis (A116). Except for A116, the culture filtrates of the other antagonists significantly increased the electrolyte leakage from Pythium mycelia, whereas ethyl acetate extracts of A435, A412, and A213 showed significant growth suppression. All five antagonists were able to produce varying amounts of cellulase and ß-glucanase enzymes. However, A435, A412, and A213 showed significantly higher cellulase activity, whereas A435 and A116 showed the highest ß-glucanase activity. Controlled glasshouse growth experiments showed that isolates A435 and A116 resulted in up to 70% control of damping off, whereas isolates A412 and A213 showed 30 to 40% damping-off control. The antagonists A435, A116, and A213 also contributed to increased cucumber shoot length as well as shoot and root dry mass. The synergetic effects of metabolites and hydrolytic enzymes could be the reason for the variation between isolates in the antagonistic activity and cucumber growth promotion. This study reports for the first time A. insulicola, A. melleus, and A. luchuensis as potential biocontrol agents against P. aphanidermatum-induced damping off of cucumber.

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.

Evaluation of indigenous Omani alfalfa landraces for morphology and forage yield under different levels of salt stress.

Alfalfa is the major fodder crop of Sultanate Oman, but salinity is a major problem in its cultivation. Therefore, thirty-four alfalfa (Medicago sativa L.) landraces of Oman were evaluated for morphology and forage yield response to different salinity levels viz. 1 (control), 3, 6, 9, and 12 dS m-1 under greenhouse conditions. The experiment was conducted under a completely randomized design. Different alfalfa landraces responded differently to the five salinity levels for plant height, number of branches, number of leaves, leaflet width, leaflet length, forage fresh weight, and forage dry matter yield. Salt stress caused a reduction in growth and dry matter yield of alfalfa landraces with exception of some, which responded positively to the salinity levels of 3 and 6 dS m-1 compared to control for the number of leaves per plant. Moreover, some landraces had better forage fresh weight and dry matter yield at 6 dS m-1 than 3 dS m-1. Alfalfa landraces OMA 257, OMA, 245, OMA 270, OMA 315, OMA 211, OMA 117, OMA 56, OMA 239, OMA 148, OMA 131, OMA 95, OMA 263, OMA 262, OMA 289 and OMA 220 were designated as salt tolerant based on their overall performance across salinity levels of 6, 9 and 12 dS m-1. However, the landraces OMA 305, OMA 100, OMA 211, OMA 148, OMA 60, OMA 248, OMA 9, OMA 88, and OMA 302 collected were sensitive to 6, 9 and 12 dS m-1 salinity stress. The study showed the variation of alfalfa landraces potential for salinity tolerance, and their potential for cultivation in saline areas and/or use in breeding programs aimed to develop salt tolerant alfalfa genotypes.

Molecular and biological characterization of Chilli leaf curl virus and associated Tomato leaf curl betasatellite infecting tobacco in Oman.

BACKGROUND: In Oman tobacco (Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection. METHODS: Circular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation. RESULTS: The full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites. CONCLUSIONS: The leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.

Development of Resistance to Hymexazol Among Pythium Species in Cucumber Greenhouses in Oman.

A study was conducted to characterize the common Pythium spp. in greenhouses in Oman and their level of resistance to hymexazol, a widely used fungicide in the country. Pythium isolates were obtained from soil samples, cocopeat bags, and cucumber roots collected from seven regions in the country. Identification of 80 Pythium isolates to the species level using sequences of the internal transcribed spacer region of the ribosomal RNA showed that they belong to four species: Pythium aphanidermatum (77 isolates), P. spinosum (1 isolate), P. myriotylum (1 isolate), and P. catenulatum (1 isolate). Investigating the aggressiveness of three Pythium spp. on cucumber showed that P. aphanidermatum, P. myriotylum, and P. spinosum are pathogenic. Phylogenetic analysis of P. aphanidermatum isolates showed that most of the isolates obtained from cocopeat clustered separately from isolates obtained from soil and roots. This may indicate a difference in the origin of the cocopeat isolates. Evaluating the resistance of 27 P. aphanidermatum isolates to hymexazol showed that most isolates were sensitive (0.9 to 31.2 mg liter-1) whereas one isolate was resistant (142.9 mg liter-1). This study is the first to report P. myriotylum and P. catenulatum in Oman. It is also the first to report the development of resistance to hymexazol among P. aphanidermatum populations from greenhouses. Growers should use integrated disease management strategies to avoid further development of resistance to hymexazol.

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.

Characterizing bread wheat genotypes of Pakistani origin for grain zinc biofortification potential.

BACKGROUND: Zinc (Zn) is essential for all life forms and its deficiency is a major issue of malnutrition in humans. This study was carried out to characterize 28 wheat genotypes of Pakistani origin for grain zinc biofortification potential, genetic diversity and relatedness. RESULTS: There was low genetic differentiation among the tested genotypes. However, they differed greatly in yield-related traits, grain mineral (Zn, calcium (Ca) and protein) concentrations and Zn bioavailability. Zinc application increased the concentration of Zn in wheat grain (32.1%), embryo (19.8%), aleurone (47%) and endosperm (23.7%), with an increase in bioavailable Zn (22.2%) and a reduction in phytate concentration (6.8%). Application of Zn also enhanced grain protein and Ca concentrations. Among wheat genotypes, Blue Silver had the highest concentration of Zn in grain, embryo, aleurone and endosperm, with high bioavailable Zn, while Kohinoor-83 had low phytate concentration. CONCLUSION: Wheat genotypes of Pakistan are genetically less diverse owing to continuous focus on the development of high-yielding varieties only. Therefore genetically diverse wheat genotypes with high endospermic Zn concentration and better grain yield should be used in breeding programs approaches, aiming at improving Zn bioavailability. © 2018 Society of Chemical Industry.

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.

Illumina MiSeq sequencing analysis of fungal diversity in stored dates.

BACKGROUND: Date palm has been a major fruit tree in the Middle East over thousands of years, especially in the Arabian Peninsula. Dates are consumed fresh (Rutab) or after partial drying and storage (Tamar) during off-season. The aim of the study was to provide in-depth analysis of fungal communities associated with the skin (outer part) and mesocarp (inner fleshy part) of stored dates (Tamar) of two cultivars (Khenizi and Burny) through the use of Illumina MiSeq sequencing. RESULTS: The study revealed the dominance of Ascomycota (94%) in both cultivars, followed by Chytridiomycota (4%) and Zygomycota (2%). Among the classes recovered, Eurotiomycetes, Dothideomycetes, Saccharomycetes and Sordariomycetes were the most dominant. A total of 54 fungal species were detected, with species belonging to Penicillium, Alternaria, Cladosporium and Aspergillus comprising more than 60% of the fungal reads. Some potentially mycotoxin-producing fungi were detected in stored dates, including Aspergillus flavus, A. versicolor and Penicillium citrinum, but their relative abundance was very limited (<0.5%). PerMANOVA analysis revealed the presence of insignificant differences in fungal communities between date parts or date cultivars, indicating that fungal species associated with the skin may also be detected in the mesocarp. It also indicates the possible contamination of dates from different cultivars with similar fungal species, even though if they are obtained from different areas. CONCLUSION: The analysis shows the presence of different fungal species in dates. This appears to be the first study to report 25 new fungal species in Oman and 28 new fungal species from date fruits. The study discusses the sources of fungi on dates and the presence of potentially mycotoxin producing fungi on date skin and mesocarp.

Genetic analysis reveals diversity and genetic relationship among Trichoderma isolates from potting media, cultivated soil and uncultivated soil.

BACKGROUND: Trichoderma is one of the most common fungi in soil. However, little information is available concerning the diversity of Trichoderma in soil with no previous history of cultivation. This study was conducted to investigate the most common species and the level of genetic relatedness of Trichoderma species from uncultivated soil in relation to cultivated soil and potting media. RESULTS: A total of 24, 15 and 13 Trichoderma isolates were recovered from 84 potting media samples, 45 cultivated soil samples and 65 uncultivated soil samples, respectively. Analysis based on the internal transcribed spacer region of the ribosomal RNA (rRNA) and the translation elongation factor gene (EF1) indicated the presence of 9 Trichoderma species: T. harzianum (16 isolates), T. asperellum (13), T. citrinoviride (9), T. orientalis (3), T. ghanense (3), T. hamatum (3), T. longibrachiatum (2), T. atroviride (2), and T. viride (1). All species were found to occur in potting media samples, while five Trichoderma species were recovered from the cultivated soils and four from the uncultivated soils. AFLP analysis of the 52 Trichoderma isolates produced 52 genotypes and 993 polymorphic loci. Low to moderate levels of genetic diversity were found within populations of Trichoderma species (H = 0.0780 to 0.2208). Analysis of Molecular Variance indicated the presence of very low levels of genetic differentiation (Fst = 0.0002 to 0.0139) among populations of the same Trichoderma species obtained from the potting media, cultivated soil and uncultivated soil. CONCLUSION: The study provides evidence for occurrence of Trichoderma isolates in soil with no previous history of cultivation. The lack of genetic differentiation among Trichoderma populations from potting media, cultivated soil and uncultivated soil suggests that some factors could have been responsible for moving Trichoderma propagules among the three substrates. The study reports for the first time the presence of 4 Trichoderma species in Oman: T. asperellum, T. ghanense, T. longibrachiatum and T. orientalis.

RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its associated betasatellite.

BACKGROUND: Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus (family Geminiviridae) is responsible for heavy yield losses for tomato production around the globe. In Oman at least five distinct begomoviruses cause disease in tomato, including TYLCV. Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite). RNA interference (RNAi) can be used to develop resistance against begomoviruses at either the transcriptional or post-transcriptional levels. RESULTS: A hairpin RNAi (hpRNAi) construct to express double-stranded RNA homologous to sequences of the intergenic region, coat protein gene, V2 gene and replication-associated gene of Tomato yellow leaf curl virus-Oman (TYLCV-OM) was produced. Initially, transient expression of the hpRNAi construct at the site of virus inoculation was shown to reduce the number of plants developing symptoms when inoculated with either TYLCV-OM or TYLCV-OM with ToLCB-OM to Nicotiana benthamiana or tomato. Solanum lycopersicum L. cv. Pusa Ruby was transformed with the hpRNAi construct and nine confirmed transgenic lines were obtained and challenged with TYLCV-OM and ToLCB-OM by Agrobacterium-mediated inoculation. For all but one line, for which all plants remained symptomless, inoculation with TYLCV-OM led to a proportion (≤25%) of tomato plants developing symptoms of infection. For inoculation with TYLCV-OM and ToLCB-OM all lines showed a proportion of plants (≤45%) symptomatic. However, for all infected transgenic plants the symptoms were milder and virus titre in plants was lower than in infected non-transgenic tomato plants. CONCLUSIONS: These results show that RNAi can be used to develop resistance against geminiviruses in tomato. The resistance in this case is not immunity but does reduce the severity of infections and virus titer. Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.