Field Evaluation of Promising Indigenous Entomopathogenic Fungal Isolates against Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae).

The rate of the sounds (i.e., substrate vibrations) produced by the movement and feeding activity of red palm weevil (RPW) pest infestations in a date palm tree was monitored over time after trees were separately treated with injection of entomopathogenic fungal isolates, Beauveria bassiana and Metarhizium anisopliae, or water treatment as the control. The activity sensing device included an accelerometer, an amplifier, a digital recorder, and a signal transmitter that fed the data to a computer that excluded background noise and compared the rates of bursts of movement and feeding sound impulses among treated trees and controls. Observations were made daily for two months. The rates of bursts were representative of the feeding activity of RPW. The unique spectral pattern of sound pulses was typical of the RPW larval feeding activity in the date palm. The microphone confirmed that the same unique tone was produced in each burst. Two months after fungal injection, the RPW sound signal declined, while the RPW sound signal increased in the control date palms (water injection). The mean rates of bursts produced by RPW decreased to zero after the trees were injected with B. bassiana or M. anisopliae compared with the increased rates over time in the control treatment plants.

Occurrence and Distribution of Tomato Brown Rugose Fruit Virus Infecting Tomato Crop in Saudi Arabia.

During the growing season of 2021-2022, a total of 145 symptomatic tomato leaf and fruit samples were collected from different locations in Riyadh Region, Saudi Arabia, showing a moderate-to-severe mosaic with dark green wrinkling, blistering, narrowing, and deformation with necrosis spot on tomato leaves, while irregular brown necrotic lesions, deformation, and yellowing spots rendering the fruits non-marketable were observed on tomato fruits. These samples were tested serologically against important tomato viruses using enzyme-linked immunosorbent assay (ELISA), and the obtained results showed that 52.4% of symptomatic tomato samples were found positive for Tomato brown rugose fruit virus (ToBRFV), wherein 12 out of 76 samples were singly infected; however, 64 out of 145 had mixed infection. A sample with a single infection of ToBRFV was used for mechanical inoculation into a range of different host plants; symptoms were observed weekly, and the presence of the ToBRFV was confirmed by ELISA and reverse transcription-polymerase chain reaction (RT-PCR). A total RNA was extracted from selected ELISA-positive samples, and RT-PCR was carried out using specific primers F-3666 and R-4718, which amplified a fragment of 1052 bp. RT-PCR products were sequenced in both directions, and partial genome nucleotide sequences were submitted to GenBank under the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities (99-99.5%) among them and 99-100% identity with ToBRFV isolates in different countries. A ToBRFV isolate (MZ130503) was selected for mechanical inoculation and to evaluate symptom severity responses of 13 commonly grown tomato cultivars in Saudi Arabia. All of the tomato cultivars showed a wide range of symptoms. The disease severity index of the tested cultivars ranged between 52% and 96%. The importance ToBRFV disease severity and its expanding host range due to its resistance breaking ability was discussed.

Interaction estimation of pathogenicity determinant protein ßC1 encoded by Cotton leaf curl Multan Betasatellite with Nicotiana benthamiana Nuclear Transport Factor 2.

Background: Begomovirus is one of the most devastating pathogens that can cause more than 90% yield loss in various crop plants. The pathogenicity determinant ßC1, located on the betasatellite associated with monopartite begomoviruses, alters the host signaling mechanism to enhance the viral disease phenotype by undermining the host immunity. The understanding of its interacting proteins in host plants to develop disease symptoms such as curly leaves, enations, vein swelling, and chlorosis is crucial to enhance the disease resistance in crop plants. The current study was designed to reveal the contribution of ßC1 in disease pathogenicity and to unveil potential interacting partners of ßC1 protein in the model plant Nicotiana benthamiana. Methods: The ßC1 gene was cloned in pGKBT7 and used as bait against the cDNA library of N. benthamiana and its pathogenesis was tested against the healthy plant and the plants infiltrated with empty vectors. The yeast two-hybrid-based screening was performed to find the interacting factors. Successful interacting proteins were screened and evaluated in various steps and confirmed by sequence analysis. The three-dimensional structure of the Nuclear Transport Factor 2 (NTF2) protein was predicted, and in-silico protein-protein interaction was evaluated. Furthermore, protein sequence alignment and molecular phylogenetic analysis were carried out to identify its homologues in other related families. In-silico analyses were performed to validate the binding affinity of ßC1 protein with NTF2. The 3D model was predicted by using I-TASSER and then analyzed by SWISS MODEL-Workspace, RAMPAGE, and Verify 3D. The interacting amino acid residues of ßC1 protein with NTF2 were identified by using PyMOL and Chimera. Results: The agroinfiltrated leaf samples developed severe phenotypic symptoms of virus infection. The yeast-two-hybrid study identified the NTF2 as a strong interacting partner of the ßC1. The NTF2 in Solanaceae and Nicotiana was found to be evolved from the Brassica and Gossypium species. The in-silico interaction studies showed a strong binding affinity with releasing energy value of -730.6 KJ/mol, and the involvement of 10 amino acids from the middle portion towards the C-terminus and five amino acid residues from the middle portion of ßC1 to interact with six amino acids of NTF2. The study not only provided an insight into the molecular mechanism of pathogenicity but also put the foundation stone to develop the resistance genotypes for commercial purposes and food security.

Identification and molecular characterization of cotton leaf curl Gezira betasatellite and two distinct begomoviruses infecting papaya trees in the Kingdom of Saudi Arabia.

Begomovirus is the largest genus in the family Geminiviridae and constitutes more than 445 virus species. Begomoviruses are characterized by single-stranded circular genomes with monopartite or bipartite components and transmitted by whitefly (Bemisia tabaci). Begomoviruses cause severe diseases in many economically important crops throughout the world. Typical symptoms of a begomovirus infection including severe leaf curling, vein thickening, vein darkening and reduced leaf size were observed in papaya plants in the Dammam district of the Eastern Province of Saudi Arabia during the growing season in 2022. A total of 10 samples were collected, and total genomic DNA was isolated from naturally infected papaya tree samples and subjected to PCR amplification using universal diagnostic primers for begomoviruses and associated satellites. Three PCR-amplified genomic components of begomoviruses and betasatellite namely P61Begomo (645 bp), P62Begomo (341 bp) and P62Beta (563 bp) were sent for Sanger DNA sequencing to Macrogen Inc. These partial viral genome sequences were submitted to Genbank database and accession numbers ON206051, ON206052 and ON206050 were assigned to P61Begomo, P62Begomo and P62Beta respectively. Phylogenetic analysis and pairwise nucleotide sequence identity studies identified P61Begomo was identified as Tomato yellow leaf curl virus, P62Begomo as DNA A component of a bipartite begomovirus Watermelon chlorotic stunt virus and P62Beta as begomovirus associated betasatellite; Cotton leaf curl Gezira betasatellite. To the best of our knowledge, this is the first report of a begomovirus complex infecting papaya (Carica papaya) in the Kingdom of Saudi Arabia.

Identification of Tomato black ring virus from tomato plants grown in greenhouses in Saudi Arabia.

A survey was conducted in Al-Kharj governorate, Riyadh region to identify viruses causing variety of virus-like symptoms on tomato plants. A total of 135 samples were collected from symptomatic tomato plants. Symptoms included mottling, deformation, necrosis of leaves and fruits. Eighteen viruses were tested by DAS-ELISA. Tomato black ring virus (TBRV) was the virus of concern as it was not detected in Saudi Arabia before and was detected in 52.6% of the collected samples in this study. RT-PCR was used to confirm detection of TBRV and to sequence the amplified products to determine molecular characteristics of this virus. In the host range test study that was performed using a purified isolate of TBRV, sixteen out of the twenty two tested plants showed symptoms. Brassica oleracea was not infected by this virus. Gel electrophoreses (2% agarose) yielded fragments of 978 bp of coat protein gene of TBRV. Nucleotide sequences of purified RT-PCR products for three TBRV Saudi isolates were deposited in the GenBank with the following accession numbers MT274656, MT274657, and MT274658. These isolates of TBRV indicated a close Phylogenetic relationship of (99-100%) among themselves and with five isolates from Poland (95-98%) but a distant relationship of 85% with isolates from England and Lithuania deposited in the GenBank. This is the first report for detection and molecular characterization of TBRV infecting tomato plants in Saudi Arabia.

First Report of Tomato black ring virus (TBRV) on Tomato (Solanum lycopersicum) in Saudi Arabia.

Tomato is a popular vegetable crop that is cultivated worldwide. It is also one of the most important crops in Saudi Arabia. In 2017, the area in which tomato was grown in Saudi Arabia was estimated to be 13317 ha and produced 306389 tons. Al Kharj Governorate in Riyadh region contributes the highest production of greenhouse tomatoes in Saudi Arabia (Ministry of Env. WTR & AGRI., 2017). In fall 2015, striking virus-like symptoms (mottling, leaf rolling, yellowing, and deformation, black strip on the stem, cracking on fruits, deformation, mottling, and mummification with severe yield losses) were observed on greenhouse tomato plants in several farms in Al Kharj Governorate. Samples were collected within the period of fall 2015 and the summer of 2017. The collected samples were tested serologically using enzyme linked immunosorbent assay (ELISA) for identification of the causal agent(s) using kits and protocols from AC Diagnostics Inc (Fayetteville, Arkansas, UAS). Out of 18 common tomato viruses tested, 14 viruses were detected in tomato plants in the region. The greatest concern was the presence of Tomato black ring virus (TBRV) as this was the first detection in Saudi Arabia and displayed the highest frequency of detection among all other detected viruses. Seventy-one out of the 135 tested samples were positive for TBRV. To confirm the presence of TBRV in the infected tomato samples, total RNA was extracted from positive samples and tested by RT-PCR with the newly designed primer pair F-TBRV (5'-GCAAACCAACGCTCTATGTTGT-3')/R-TBRV (5'-AGAGCCAAACTGGAATGGTAGG-3') that is specific to the CP gene of TBRV. RT-PCR products of 978 bp in length were successfully obtained from the naturally infected tomato plants. One of the detected isolates was used to inoculate Chenopodium amaranticolor with the aim of obtaining a pure isolate from single local lesions that could be later used for propagation and maintenance in Nicotiana tabacum. A host range experiment was conducted using mechanical inoculation with the single-lesion isolate of TBRV on four replicates of 14 different plant species in parallel with healthy controls (Brunt et. al. 1996). Three weeks post-inoculation, varying reactions and symptoms ranging from local lesion to plant death, depending on host species, were observed on the tested plants (Supplementary Table 1). Host range results were largely similar to those reported in previous studies (Sneideris et al. 2012, and Rymelska et al. 2013). The presence of TBRV was confirmed both by ELISA and RT-PCR. Nucleotide sequences obtained from PCR products of selected samples were submitted to the GenBank and assigned the following accession numbers: MT274656, MT274657, and MT274658. Saudi isolates of TBRV were found to share 99-100% of their nucleotide sequences. They had the highest similarity of 98% with the Polish isolates (MG458221 and KX977561) and the lowest similarity of 85% with isolates from Lithuania (KF678369, and KF678370). To the best of our knowledge, this is the first report of occurrence of TBRV in Saudi Arabia. Since this virus is transmitted by seeds, it may have entered through imported seeds and spread in greenhouses through mechanical means. A survey of the different agricultural regions is encouraged to determine the incidence, distribution, and damage induced by this virus in Saudi Arabia.

Lucerne transient streak virus; a Recently Detected Virus Infecting Alfafa (Medicago sativa) in Central Saudi Arabia.

A survey was conducted to determine the status of Lucerne transient streak virus (LTSV) in three high-yielding alfalfa regions in central Saudi Arabia (Riyadh, Qassim, and Hail) during 2014. Three hundred and eight symptomatic alfalfa, and seven Sonchus oleraceus samples were collected. DAS-ELISA indicated that 59 of these samples were positive to LTSV. Two isolates of LTSV from each region were selected for molecular studies. RT-PCR confirmed the presence of LTSV in the selected samples using a specific primer pair. Percentage identity and homology tree comparisons revealed that all Saudi isolates were more closely related to each other but also closely related to the Canadian isolate-JQ782213 (97.1-97.6%) and the New Zealand isolate-U31286 (95.8-97.1%). Comparing Saudi isolates of LTSV with ten other sobemoviruses based on the coat protein gene sequences confirmed the distant relationship between them. Eleven out of fourteen plant species used in host range study were positive to LTSV. This is the first time to document that Trifolium alexandrinum, Nicotiana occidentalis, Chenopodium glaucum, and Lathyrus sativus are new host plant species for LTSV and that N. occidentalis being a good propagative host for it.

Molecular characterization of a naturally occurring intraspecific recombinant begomovirus with close relatives widespread in southern Arabia.

BACKGROUND: Tomato leaf curl Sudan virus (ToLCSDV) is a single-stranded DNA begomovirus of tomato that causes downward leaf curl, yellowing, and stunting. Leaf curl disease results in significant yield reduction in tomato crops in the Nile Basin. ToLCSDV symptoms resemble those caused by Tomato yellow leaf curl virus, a distinct and widespread begomovirus originating in the Middle East. In this study, tomato samples exhibiting leaf curl symptoms were collected from Gezira, Sudan. The associated viral genome was molecularly characterized, analyzed phylogenetically, and an infectious clone for one isolate was constructed. FINDINGS: The complete genomes for five newly discovered variants of ToLCSDV, ranging in size from 2765 to 2767-bp, were cloned and sequenced, and subjected to pairwise and phylogenetic analyses. Pairwise analysis indicated that the five Gezira isolates shared 97-100% nucleotide identity with each other. Further, these variants of ToLCSDV shared their highest nucleotide identity at 96-98%, 91-95%, 91-92%, and 91-92% with the Shambat, Gezira, Oman and Yemen strains of ToLCSDV, respectively. Based on the high maximum nucleotide identities shared between these ToLCSDV variants from Gezira and other previously recognized members of this taxonomic group, they are considered isolates of the Shambat strain of ToLCSDV. Analysis of the complete genome sequence for these new variants revealed that they were naturally occurring recombinants between two previously reported strains of ToLCSDV. Finally, a dimeric clone constructed from one representative ToLCSV genome from Gezira was shown to be infectious following inoculation to tomato and N. benthamiana plants. CONCLUSION: Five new, naturally occurring recombinant begomovirus variants (>96% shared nt identity) were identified in tomato plants from Gezira in Sudan, and shown to be isolates of the Shambat strain of ToLCSDV. The cloned viral genome was infectious in N. benthamiana and tomato plants, and symptoms in tomato closely resembled those observed in field infected tomato plants, indicating the virus is the causal agent of the leaf curl disease. The symptoms that developed in tomato seedlings closely resembled those observed in field infected tomato plants, indicating that ToLCSDV is the causal agent of the leaf curl disease in Gezira.

Viral metagenomics: analysis of begomoviruses by illumina high-throughput sequencing.

Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.