In silico characterization, docking, and simulations to understand host-pathogen interactions in an effort to enhance crop production in date palms.

Food safety remains a significant challenge despite the growth and development in agricultural research and the advent of modern biotechnological and agricultural tools. Though the agriculturist struggles to aid the growing population's needs, many pathogen-based plant diseases by their direct impact on cell division and tissue development have led to the loss of tons of food crops every year. Though there are many conventional and traditional methods to overcome this issue, the amount and time spend are huge. Scientists have developed systems biology tools to study the root cause of the problem and rectify it. Host-pathogen protein interactions (HPIs) have a promising role in identifying the pathogens' strategy to conquer the host organism. In this paper, the interactions between the host Rhynchophorus ferrugineus (an invasive wood-boring pest that destroys palm) and the pathogens Proteus mirabilis, Serratia marcescens, and Klebsiella pneumoniae are comprehensively studied using protein-protein interactions, molecular docking, and followed by 200 ns molecular dynamic simulations. This study elucidates the structural and functional basis of these proteins leading towards better plant health, production, and reliability.

The value of blue-green algae (Spirulina platensis) as a nutritive supplement and toxicant against almond moth [Cadra cautella (Lepidoptera: Pyralidae)].

Blue-green algae, Spirulina platensis is a well-known algal formulation known for its beneficial effects on the growth and development in several types of organisms. Although it is used as a food supplement, it possesses significant toxic effects on growth and development of organisms. This study assessed the positive/negative impacts of S. platensis on almond moth, Cadra cautella (almond moth) that is a serious pest of date fruits and other grains under laboratory conditions. The S. platensis powder were mixed with diet and newly hatched C. cautella larvae were fed. The larvae were observed on alternate days to record the data. The diet was changed once a week. The S. platensis proved very good nutrition supplement at lower dose. Whereas, moderate and high mortality was noted for 5 and 10% formulations, respectively. Moreover, larval span was significantly altered by different formulations and lower formulation (1%) resulted in shorter larval period compared to the rest of the formulations. Although 33% mortality was recorded under 5% S. platensis formulation, however, the larvae which reached to adult stage, copulated, and females laid more eggs. Furthermore, the highest mortality (90%) was observed under 10% S. platensis formulation and a few larvae reached adult stage; thus, no data on pupal period and reproductive traits was recorded for this formulation. These findings proved that S. platensis can be used as nutritional supplement as well as a toxic substance to manage C. cautella in date storage. However, future studies on this are needed to reach concrete conclusions.

Carbon Dioxide-Enriched Atmosphere to Control Oryzaephilus surinamensis L. On Stored Saqie Date Fruits.

<b>Background and Objective:</b> Control of the stored date insects by an eco-friendly method is essential to maintain the fruit for prolonged periods, especially that dates are eaten fresh. The search for a safe method, as an alternative to commonly used chemical pesticides, is needed. The objective is to investigate CO₂ enriched atmosphere on the Mortality Percentage (MP) of the Saw-toothed grain beetle, <i>O. surinamensis</i>, life stages with special emphasis on reducing damage to stored dates. <b>Materials and Methods:</b> The effect of high levels of CO₂ as an alternative control method against the Saw-toothed grain beetle, <i>Oryzaephilus surinamensis</i> L., at different life stages was studied on infested 'Saqie' dates. Four CO₂ pressures (25, 50, 75 and 90 kPa, balance is nitrogen) were tested for 6, 12, 18, 24, 48, 72 and 96 hrs intervals. The response of different life stages of <i>O. surinamensis</i> to the different treatments varied according to CO₂ level, developmental stage and exposure period. <b>Results:</b> Mortality (%) was higher during the larval stage, followed by adults, pupae and eggs, in descending order. The larvae and adult stages were more sensitive to CO₂ treatment than the pupal and egg. Exposure time was more effective on eggs, larval and adult MP than the CO₂ atmosphere level. Mortality% at 96 hrs exposure time was almost 100% with CO₂ atmospheres of 50, 75 and 90 kPa. Mathematical equations were developed to model the relationship between mortality% and CO₂ treatments using multiple regression analyses for each life stage. <b>Conclusion:</b> The results confirmed that CO₂ could be applied to final food products during packaging to control the residual occurrence of insect pests after storage and before the packaging process to prevent further infestation in the final packages.

Transcriptomic analysis of Dubas bug (Ommatissus lybicus Bergevin) infestation to Date Palm.

Date palm (Phoenix dactylifera L.) and its fruit possess sociocultural, health and economic importance in Middle East. The date palm plantations are prone to Dubas bug (DB; Ommatissus lybicus DeBergevin; Homoptera: Tropiduchidae) attacks that severely damages the tree's growth and reduces fruit production. However, the transcriptome related datasets are not known to understand how DB activates physiological and gene regulatory mechanisms during infestation. Hence, we performed RNA-Seq of leaf infected with or without DB to understand the molecular responses of date palm seedlings. Before doing that, we noticed that DB infestation significantly increase superoxide anion and malondialdehyde production to two-folds as compared to healthy control. Stress-responsive genes such as proline transporter 2, NADP-dependent glyceraldehyde and superoxide dismutase were found significantly upregulated in infected seedlings. The infection repercussions were also revealed by significantly higher contents of endogenous phytohormonal signaling of jasmonic acid (JA) and salicylic acid (SA) compared with control. These findings persuaded to dig out intrinsic mechanisms and gene regulatory networks behind DB infestation to date palm by RNA-Seq analysis. Transcriptome analysis revealed upregulation of 6,919 genes and down-regulation of 2,695 genes in leaf during the infection process. The differentially expressed genes were mostly belongs to cellular functions (calcium and MAPK), phytohormones (auxin, gibberellins, abscisic acid, JA and SA), and secondary metabolites (especially coumarinates and gossypol). The data showed that defense responses were aggravated by gene networks involved in hypersensitive responses (PAR1, RIN4, PBS1 etc.). In conclusion, the results revealed that date palm's leaf up-regulates both cellular and phytohormonal determinants, followed by intrinsic hypersensitive responses to counter infestation process by Dubas bug.

Susceptibility survey of Ommatissus lybicus (de Bergevin) populations against deltamethrin and fenitrothion in Oman.

Despite of extensive application of insecticides, severe infestation of date palms is reported with Ommatissus lybicus every year. The development of insecticide resistance in the field strains receiving heavy pesticide regimes can be a reason for unsuccessful control. Seventeen field strains of O. lybicus from Oman were appraised for resistance against deltamethrin and fenitrothion in years 2017 and 2018. Eleven field strains depicted susceptibility (RR < 3-fold) while six strains expressed minor (RR = 3-5-fold) to low level (RR = 5-10-fold) of resistance against deltamethrin when compared with lab-susceptible strain. In case of fenitrothion, fourteen field strains exhibited minor to low level of resistance and only two showed susceptibility. Intermediate resistance (RR = 10-40-fold) was also reported in one field strain against fenitrothion. A significant but low resistance (RR = 7.86-fold) was observed in a lab strain selected with deltamethrin for resistance development mechanism studies. The deltamethrin-selected strain exhibited minor resistance development (RR = 3.13-fold) against fenitrothion. Significant reduction in resistance was observed with higher toxicity values when the two pesticides were tested along with piperonyl butoxide (PBO) against all the field collected dubas bug strains. Higher susceptibility of field strains to deltamethrin suggest it a suitable alternate candidate to deter resistance development in O. lybicus.

Overexpression of a Metallothionein 2A Gene from Date Palm Confers Abiotic Stress Tolerance to Yeast and Arabidopsis thaliana.

Although the date palm tree is an extremophile with tolerance to drought and certain levels of salinity, the damage caused by extreme salt concentrations in the soil, has created a need to explore stress-responsive traits and decode their mechanisms. Metallothioneins (MTs) are low-molecular-weight cysteine-rich proteins that are known to play a role in decreasing oxidative damage during abiotic stress conditions. Our previous study identified date palm metallothionein 2A (PdMT2A) as a salt-responsive gene, which has been functionally characterized in yeast and Arabidopsis in this study. The recombinant PdMT2A protein produced in Escherichia coli showed high reactivity against the substrate 5'-dithiobis-2-nitrobenzoic acid (DTNB), implying that the protein has the property of scavenging reactive oxygen species (ROS). Heterologous overexpression of PdMT2A in yeast (Saccharomyces cerevisiae) conferred tolerance to drought, salinity and oxidative stresses. The PdMT2A gene was also overexpressed in Arabidopsis, to assess its stress protective function in planta. Compared to the wild-type control, the transgenic plants accumulated less Na+ and maintained a high K+/Na+ ratio, which could be attributed to the regulatory role of the transgene on transporters such as HKT, as demonstrated by qPCR assay. In addition, transgenic lines exhibited higher chlorophyll content, higher superoxide dismutase (SOD) activity and improved scavenging ability for reactive oxygen species (ROS), coupled with a better survival rate during salt stress conditions. Similarly, the transgenic plants also displayed better drought and oxidative stress tolerance. Collectively, both in vitro and in planta studies revealed a role for PdMT2A in salt, drought, and oxidative stress tolerance.

Resistance to commonly used insecticides and phosphine fumigant in red palm weevil, Rhynchophorus ferrugineus (Olivier) in Pakistan.

The red palm weevil Rhynchophorus ferrugineus (Olivier) is an important pest of date palms in many regions of the world. This paper reports the first survey of insecticide resistance in field populations of R. ferrugineus in Pakistan which were collected from seven date palm growing areas across Punjab and Khyber Pakhtunkhwa (KPK) provinces, Pakistan. The resistance was assessed by the diet incorporation method against the formulated commonly used chemical insecticides profenophos, imidacloprid, chlorpyrifos, cypermethrin, deltamethrin, spinosad, lambda-cyhalothrin and a fumigant phosphine. Elevated levels of resistance were recorded for cypermethrin, deltamethrin and phosphine after a long history of insecticide use in Pakistan. Resistance Ratios (RRs) were 63- to 79-fold for phosphine, 16- to 74-fold for cypermethrin, 13- to 58-fold for deltamethrin, 2.6- to 44-fold for profenophos, 3- to 24-fold for chlorpyrifos, 2- to 12-fold for lambda-cyhalothrin and 1- to 10-fold for spinosad compared to a susceptible control line. Resistant R. ferrugineus populations were mainly found in southern Punjab and to some extent in KPK. The populations from Bahawalpur, Vehari, Layyah and Dera Ghazi Khan were most resistant to chemical insecticides, while all populations exhibited high levels of resistance to phosphine. Of the eight agents tested, lower LC50 and LC90 values were recorded for spinosad and lambda-cyhalothrin. These results suggest that spinosad and lambda-cyhalothrin exhibit unique modes of action and given their better environmental profile, these two insecticides could be used in insecticide rotation or assist in phasing out the use of older insecticides. A changed pattern of both insecticides can be used sensibly be recommended without evidence of dose rates and frequencies used.

Synthetic Co-Attractants of the Aggregation Pheromone of the Date Palm Root Borer Oryctes agamemnon.

Laboratory and field investigations to identify and evaluate plant co-attractants of the aggregation pheromone of the date palm pest Oryctes agamemnon are reported. Volatiles emitted by freshly cut palm core and palm core with feeding males, were collected, analyzed by gas chromatography coupled to mass spectrometry and evaluated in olfactometers alone or combined with synthetic pheromone. A collection of palm odor without male effluvia was attractive alone and enhanced attraction to synthetic pheromone in an olfactometer similar to that to a collection of palm odor emitted with feeding males and containing natural pheromone. Behavioral responses to collections of palm volatiles were correlated to the amount of volatiles material in them. Enhancement of the attractiveness of the pheromone was not correlated to chemicals specific to beetle feeding. The chemicals common to the active collections extracts were benzoate esters, mostly ethyl benzoate, anisole derivatives and sesquiterpenes. Blends of the most abundant components of the extracts were evaluated for enhancement of the attractiveness of pheromone (1 µg) in olfactometers at 1 or 10 µg doses. The mixtures were further evaluated by field trapping in Tunisia at 3-10 mg/day using reference (6 mg/day) or experimental pheromone formulations. A mixture of ethyl benzoate, 4-methylanisole and farnesol (1:1:1 w/w at 6.5 mg/day) enhanced captures in pheromone baited traps in 2014 and 2015 and this mixture was as active as the natural palm bait. The practical prospect of the result for the management for O. agamemnon, and other palm beetles is discussed.

Impact of environmental variables on Dubas bug infestation rate: A case study from the Sultanate of Oman.

Date palm cultivation is economically important in the Sultanate of Oman, with significant financial investment coming from both the government and from private individuals. However, a global infestation of Dubas bug (Ommatissus lybicus Bergevin) has impacted the Middle East region, and infestations of date palms have been widespread. In this study, spatial analysis and geostatistical techniques were used to model the spatial distribution of Dubas bug infestations to (a) identify correlations between Dubas bug densities and different environmental variables, and (b) predict the locations of future Dubas bug infestations in Oman. Firstly, we considered individual environmental variables and their correlations with infestation locations. Then, we applied more complex predictive models and regression analysis techniques to investigate the combinations of environmental factors most conducive to the survival and spread of the Dubas bug. Environmental variables including elevation, geology, and distance to drainage pathways were found to significantly affect Dubas bug infestations. In contrast, aspect and hillshade did not significantly impact on Dubas bug infestations. Understanding their distribution and therefore applying targeted controls on their spread is important for effective mapping, control and management (e.g., resource allocation) of Dubas bug infestations.

Impacts of human-related practices on Ommatissus lybicus infestations of date palm in Oman.

Date palm cultivation is economically important in the Sultanate of Oman, with significant financial investments coming from both the government and private individuals. However, a widespread Dubas bug (DB) (Ommatissus lybicus Bergevin) infestation has impacted regions including the Middle East, North Africa, Southeast Russia, and Spain, resulting in widespread damages to date palms. In this study, techniques in spatial statistics including ordinary least squares (OLS), geographically weighted regression (GRW), and exploratory regression (ER) were applied to (a) model the correlation between DB infestations and human-related practices that include irrigation methods, row spacing, palm tree density, and management of undercover and intercropped vegetation, and (b) predict the locations of future DB infestations in northern Oman. Firstly, we extracted row spacing and palm tree density information from remote sensed satellite images. Secondly, we collected data on irrigation practices and management by using a simple questionnaire, augmented with spatial data. Thirdly, we conducted our statistical analyses using all possible combinations of values over a given set of candidate variables using the chosen predictive modelling and regression techniques. Lastly, we identified the combination of human-related practices that are most conducive to the survival and spread of DB. Our results show that there was a strong correlation between DB infestations and several human-related practices parameters (R2 = 0.70). Variables including palm tree density, spacing between trees (less than 5 x 5 m), insecticide application, date palm and farm service (pruning, dethroning, remove weeds, and thinning), irrigation systems, offshoots removal, fertilisation and labour (non-educated) issues, were all found to significantly influence the degree of DB infestations. This study is expected to help reduce the extent and cost of aerial and ground sprayings, while facilitating the allocation of date palm plantations. An integrated pest management (IPM) system monitoring DB infestations, driven by GIS and remote sensed data collections and spatial statistical models, will allow for an effective DB management program in Oman. This will in turn ensure the competitiveness of Oman in the global date fruits market and help preserve national yields.

Transcriptome sequencing and analysis of the coconut leaf beetle, Brontispa longissima.

The coconut leaf beetle, Brontispa longissima, is a destructive pest of palm plants. Although its ecological and biological characteristics are well understood, its genetic information remains largely unknown. To advance our understanding of its molecular ba-sis of biology and ecology, we sequenced and analyzed its whole transcriptome by using high-throughput Illumina paired-end sequencing technology. Approximately 8.08 Gb of clean reads were generated in a single run, which were assembled by using Trinity into 41,652 unigenes with an average length of 932 bp. By sequence similarity searches for known proteins, 23,077 (55.4%) unigenes were annotated by BLASTx searches against the NCBI non-redundant protein database. Of the unigenes assembled, 18,153 and 13,733 were assigned to Gene Ontology and Clusters of Orthologous Groups of proteins, respectively. In addition, 10,415 unigenes were mapped onto 247 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. These transcriptomic resources will facilitate gene identification and elucidate the molecular mechanisms of biological and ecological aspects under-lying this palm pest, in order to design a new control strategy.

Comparative efficacy of CO2 and ozone gases against Ephestia cautella (Lepidoptera: Pyralidae) larvae under different temperature regimes.

Comparative efficacy of three different modified atmospheres: 100% CO2, 75% CO2 + 25% N2, and 22 ppm ozone were examined against larval mortality of the almond moth, Ephestia cautella (Walker) (Lepidoptera: Pyralidae) at temperature regimes of 25°C and 35 ± 2°C and 60 ± 5% relative humidity, and 9:15 dark and light. Wandering young larval instars, which are fast growing, large enough in size and considered as more tolerant to modified atmosphere, were collected directly from the rearing culture, placed inside pitted date fruits of vars.: "Khudri," "Ruziz," and "Saqie," were treated with aforementioned gases for 24, 48, and 72 h. The immediate and delayed larval mortality was recorded after each exposure timing. Ozone possessed the strongest fumigant toxicity causing 100% mortality with all varieties, at 25 and 35°C after 24 h exposure and was more effective than 75% CO2 that caused 83 and 100% immediate mortality with variety ruziz at 25 and 35°C, respectively. Extending the treatments exposure time to 72 h, 100% mortality was recorded by exposing larvae to any of the studied gases at 25 and 35°C. These results suggest that gases and temperature used in this study can be effectively used to control E. cautella in dates and stored grains.

Identification of Proteins Modulated in the Date Palm Stem Infested with Red Palm Weevil (Rhynchophorus ferrugineus Oliv.) Using Two Dimensional Differential Gel Electrophoresis and Mass Spectrometry.

A state of the art proteomic methodology using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI TOF) has been employed to characterize peptides modulated in the date palm stem subsequent to infestation with red palm weevil (RPW). Our analyses revealed 32 differentially expressed peptides associated with RPW infestation in date palm stem. To identify RPW infestation associated peptides (I), artificially wounded plants (W) were used as additional control beside uninfested plants, a conventional control (C). A constant unique pattern of differential expression in infested (I), wounded (W) stem samples compared to control (C) was observed. The upregulated proteins showed relative fold intensity in order of I > W and downregulated spots trend as W > I, a quite interesting pattern. This study also reveals that artificially wounding of date palm stem affects almost the same proteins as infestation; however, relative intensity is quite lower than in infested samples both in up and downregulated spots. All 32 differentially expressed spots were subjected to MALDI-TOF analysis for their identification and we were able to match 21 proteins in the already existing databases. Relatively significant modulated expression pattern of a number of peptides in infested plants predicts the possibility of developing a quick and reliable molecular methodology for detecting plants infested with date palm.

Diversity and phylogenetic analysis of endosymbiotic bacteria of the date palm root borer Oryctes agamemnon (Coleoptera: Scarabaeidae).

BACKGROUND: The date palm root borer Oryctes agamemnon (Coleoptera: Scarabaeidae) is one of the major pests of palms. In Saudi Arabia, both larvae and adults of Oryctes are particularly troublesome, especially during the establishment of young date palm orchards. Endosymbiotic bacteria are known to have a key role in food digestion and insecticide resistance mechanisms, and therefore are essential to their host insect. Identification of these bacteria in their insect host can lead to development of new insect pest control strategies. RESULTS: Metagenomic DNA from larval midgut of the date palm root borer, O. agamemnon, was analyzed for endosymbiotic bacterial communities using denatured gradient gel electrophoresis (DGGE) utilizing 16S rRNA genes. The DGGE fingerprints with metagenomic DNA showed predominance of eleven major operational taxonomic units (OTUs) identified as members of Photobacterium, Vibrio, Allomonas, Shewanella, Cellulomonas, and Citrobacter, as well as uncultured bacteria, including some uncultured Vibrio members. DGGE profiles also showed shifts in the dominant bacterial populations of the original soil compared with those that existed in the larval midguts. The endosymbiotic bacterial community was dominated by members of the family Vibrionaceae (54.5%), followed by uncultured bacteria (18.2%), Enterobacteriaceae (9.1%), Shewanellaceae (9.1%), and Cellulomonadaceae (9.1%). Phylogenetic studies confirmed the affiliation of the dominant OTUs into specified families revealed by clustering of each phylotype to its corresponding clade. Relative frequency of each phylotype in larval midguts revealed predominance of Vibrio furnisii and Vibrio navarrensis, followed by uncultured bacterial spp., then Cellulomonas hominis, Shewanella algae, and Citrobacter freundii. CONCLUSION: Analysis of metagenomic DNA for endosymbiotic bacterial communities from the midgut of Oryctes larvae showed strong selection of specific bacterial populations that may have a key role in digestion, as well as other benefits to the larvae of O. agamemnon. Determination of the distinct endosymbiotic community structure and its possible biological functions within the insect could provide us with basic information for future pest control research.

Modeling the effects of climate on date palm scale (Parlatoria blanchardi) population dynamics during different phenological stages of life history under hot arid conditions.

The date palm scale (DPS) Parlatoria blanchardi is a serious pest due to the damage it inflicts on its host tree (Phoenix dactylifera). To develop an effective control against DPS in arid regions, it is essential to know its bio-ecology including population dynamics and climatic factors influencing the duration and timing of life history and also the densities of different phenological stages (crawlers, first and second instars nymphs, adult males, and adult females). Monitoring of biological cycle and population dynamics of the pest were achieved through weekly counts of DPS densities on leaflets sampled at different position of date palm trees in an oasis of Ouargla region (Algerian Sahara Desert). Within this hyper-arid region, DPS established four generations per year, the most important was the spring generation. Two overlapping generations occurred in spring-early summer and two in autumn-early winter; these two pairs of generations were interspersed by two phases of high-mortality rates, the first corresponds to winter cold and the second refers to the extreme heat of summer. Statistical analysis of the effects of the studied climatic conditions (minimum, maximum and mean temperatures, precipitation, humidity, wind, rain days, and climatic indices) on the DPS densities at different phenological stages showed great variability from one stage to another. Among these, adult females were the most affected by climate factors. For the total DPS population, high values of minimum temperatures negatively affected population density, while high maximum temperatures, hygrometry, and De Martonne aridity index showed a positive influence.

Identification of pheromone synergists for Rhynchophorus ferrugineus trapping systems from Phoenix canariensis palm volatiles.

Trapping systems for the red palm weevil, Rhynchophorus ferrugineus Olivier, rely on the use of natural plant odor sources to boost the attractiveness of the aggregation pheromone. The identification of the key odorants involved in attraction is essential in the development of a synthetic pheromone synergist to replace the nonstandardized use of plant material in traps. Canary Islands date palms (Phoenix canariensis) have become preferred hosts for R. ferrugineus in Europe; thus, the volatile profile of different P. canariensis plant materials, including healthy and infested tissues, is investigated in the present work by means of solid phase microextraction (SPME-GC-MS), aimed to identify pheromone synergists. The electroantennography (EAG) response of the compounds identified was recorded, as well as the preliminary field response of several EAG-active compounds. The so-called "palm esters" (ethyl acetate, ethyl propionate, ethyl butyrate, and propyl butyrate) elicit the strongest EAG responses but performed poorly in the field. Mixtures of esters and alcohols give evidence of better performance, but release rates need further optimization.