Phylogenetic relationships and DNA barcoding of nine endangered medicinal plant species endemic to Saint Katherine protectorate.

A high degree of endemism has been recorded for several plant groups collectively in Saint Katherine Protectorate (SKP) in the Sinai Peninsula. Nine endangered endemic plant species in SKP were selected to test the variable abilities of three different DNA barcodes; Riboluse-1,5- Biphosphate Carboxylase/Oxygenase Large subunit (rbcL), Internal Transcribed Spacer (ITS), and the two regions of the plastid gene (ycf1) as well as Start Codon Targeted (SCoT) Polymorphism to find the phylogenetic relationships among them. The three barcodes were generally more capable of finding the genetic relationships among the plant species under study, new barcodes were introduced to the National Centre for Biotechnology Information (NCBI) for the first time through our work. The barcode sequences were efficient in finding the genetic relationships between the nine species. However, SCoT polymorphism could only cluster plant species belonging to the same genus together in one group, but it could not cluster plant species belonging to the same families except for some primers solely. RbcL was the most easily amplified and identified barcode in eight out of the nine species at the species level and the ninth barcode to the genus level. ITS identified all the species to the genus level. Finally, ycf1 identified six out of the eight species, but it could not identify two of the eight species to the genus level.

Comparative Analyses of Four Chemicals Used to Control Black Mold Disease in Tomato and Its Effects on Defense Signaling Pathways, Productivity and Quality Traits.

The field application of safe chemical inducers plays a vital role in the stimulation of systematic acquired resistance (SAR) of plants. In this study, the efficacy use of three and six field applications with chitosan, lithovit, and K-thiosulfate at 4 gL-1 and salicylic acid at 1.5 gL-1 in improving tomato productivity, quality, and modifying the defense signaling pathways to the Alternaria alternata infection was investigated. Salicylic acid was the most effective in vitro where it completely inhibited the growth of Alternaria alternata. The highest yield quantity was recorded with six applications with Chitosan followed by Salicylic acid; also, they were the most effective treatments in controlling the Alternaria alternata infection in tomato fruits. The maximum increase in chitinase and catalase activity of tomato fruits was observed at five days after inoculation, following treatment with six sprays of salicylic acid followed by chitosan. The transcript levels of seven defense-related genes: ethylene-responsive transcription factor 3 (RAP), xyloglucan endotransglucosylase 2 (XET-2), catalytic hydrolase -2 (ACS-2), proteinase inhibitor II (PINII), phenylalanine ammonia-lyase 5 (PAL5), lipoxygenase D (LOXD), and pathogenesis-related protein 1 (PR1) were upregulated in response to all treatments. The highest expression levels of the seven studied genes were recorded in response to six foliar applications with chitosan. Chitosan followed by salicylic acid was the most effective among the tested elicitors in controlling the black mold rot in tomato fruits. In conclusion, pre-harvest chitosan and salicylic acid in vivo application with six sprays could be recommended as effective safe alternatives to fungicides against black mold disease in tomato fruits.

Gene-targeted molecular phylogeny, phytochemical profiling, and antioxidant activity of nine species belonging to family Cactaceae.

Cactaceae plant family comprises over 130 genera and 2000 species of succulent flowering plants. The genera Mammillaria and Notocactus (Parodia), which have medicinal and nutritional applications as well as aesthetic appeal, are considered to be among the major genera of the family. Several species of both genera show morphological and chemical similarities and diversities according to environmental conditions and genotypes. Here, we assessed the genetic relationships of nine species belonging to two major genera Mammillaria and Notocactus under the family Cactaceae, using two modern gene-targeting marker techniques, the Start Codon Targeted (SCoT) Polymorphism and the Conserved DNA-Derived Polymorphism (CDDP). Besides, we screened the various phytochemicals and evaluated the antioxidant activities of the nine species of cacti. Five out of the 10 SCoT and eight CDDP primers used to screen genetic variations within the nine species yielded species-specific reproducible bands. The entire 156 loci were detected, of which 107 were polymorphic, 26 were monomorphic, and 23 were unique loci. The nine species were categorized into two groups based on the dendrogram and similarity matrix. Phytochemical profiling revealed that sterols, triterpenes, flavonoids, and tannins were found in all the tested species. Additionally, two Notocactus species (N. shlosserii and N. roseoluteus) and one Mammillaria species (M. spinosissima) revealed a considerable antioxidant activity. Our results demonstrated that gene-targeting marker techniques were highly powerful tools for the classification and characterization of the nine investigated species, despite displaying high similarities at both morphological and phytochemical levels.

Piriformospora indica promotes cucumber tolerance against Root-knot nematode by modulating photosynthesis and innate responsive genes.

Root Knot Nematode (RKN, Meloidogyne incognita) is one of the greatest damaging soil pathogens causes severe yield losses in cucumber and many other economic crops. Here, we evaluated the potential antagonistic effect of the root mutualistic fungus Piriformospora indica against RKN and their impact on vegetative growth, yield, photosynthesis, endogenous salicylic acid (SA) and its responsive genes. Our results showed that P. indica dramatically decreased the damage on shoot and root architecture of cucumber plants, which consequently enhanced yield of infested plants. Likewise, P. indica colonization clearly improved the chlorophyll content and delimited the negative impact of RNK on photosynthesis. Moreover, P. indica colonization exhibited a significant reduction of different vital nematological parameters such as soil larva density, amount of eggs/eggmass, eggmasses, females and amount of galls at cucumber roots. Additionally, the results showed that SA level was significantly increased generally in the roots of all treatments especially in plants infested with RKN alone as compared to control. This suggests that P. indica promoting SA levels in host cucumber plant roots to antagonize the RKN and alleviate severity damages occurred in its roots. This higher levels of SA in cucumber roots was consistent with the higher expressional levels of SA pathway genes PR1 and PR3. Furthermore, P. indica colonization reduces PR1, PR3 and increased NPR1 in roots of RKN infested cucumber plants when compared to non-colonized plants. Interestingly, our in vitro results showed that direct application of P. indica suspension against the J2s exhibited a significant increase in mortality ratio. Our results collectively suggest that P. indica promoting morphological, physiological and SA levels that might together play a major important role to alleviate the adverse impact of RKN in cucumber.

Impact Effect of Methyl Tertiary-Butyl Ether "Twelve Months Vapor Inhalation Study in Rats".

We investigated the early risk of developing cancer by inhalation of low doses (60 µL/day) of methyl tertiary butyl ether (MTBE) vapors using protein SDS-PAGE and LC-MS/MS analysis of rat sera. Furthermore, histological alterations were assessed in the trachea and lungs of 60 adult male Wistar rats. SDS-PAGE of blood sera showed three protein bands corresponding to 29, 28, and 21 kDa. Mass spectroscopy was used to identify these three bands. The upper and middle protein bands showed homology to carbonic anhydrase 2 (CA II), whereas the lower protein band showed homology with peroxiredoxin 2. We found that exposure to MTBE resulted in histopathological alterations in the trachea and the lungs. The histological anomalies of trachea and lung showed that the lumen of trachea, bronchi, and air alveoli packed with free and necrotic epithelial cells (epithelialization). The tracheal lamina propria of lung demonstrated aggregation of lymphoid cells, lymphoid hyperplasia, hemorrhage, adenomas, fibroid degeneration, steatosis, foam cells, severe inflammatory cells with monocytic infiltration, edema, hemorrhage. Occluded, congested, and hypertrophied lung arteries in addition, degenerated thyroid follicles, were observed. The hyaline cartilage displayed degeneration, deformation, and abnormal protrusion. In conclusion, our results suggest that inhalation of very low concentrations of the gasoline additive MTBE could induce an increase in protein levels and resulted in histopathological alterations of the trachea and the lungs.

Isolation, characterization, cloning and bioinformatics analysis of a novel receptor from black cut worm (Agrotis ipsilon) of Bacillus thuringiensis vip 3Aa toxins.

Black cutworm (BCW) is an economically important lepidopteran insect. The control of this insect by a Bt toxin and the understanding of the interaction between the Bt toxin and its receptor molecule were the objectives of this research work. A gene coding for a Vip3A receptor molecule was identified, characterized, and cloned, from the brush border membrane vesicles (BBMV) of the BCW. The nucleotide sequence analysis of the cloned putative Vip3A-receptor gene revealed that the gene was 1.3-kb long and exhibited no homology with any gene in the gene bank. We succeeded in identifying and characterizing most of the Vip3A-receptor gene sequence; and the nucleotide sequence analysis of the cloned putative Vip3A-receptor gene (accession no. KX858809) revealed about 92% of the expected sequence was recovered, which exhibited no homology with any gene in the GenBank.

Multifunctional activities of ERF109 as affected by salt stress in Arabidopsis.

Transcriptomic analysis was conducted in leaves of Arabidopsis T-DNA insertion ERF109-knocked out (KO) mutant or plants overexpressing (OE) the gene to detect its role in driving expression of programmed cell death- (PCD-) or growth-related genes under high salt (200 mM NaCl) stress. The analysis yielded ~22-24 million reads, of which 90% mapped to the Arabidopsis reference nuclear genome. Hierarchical cluster analysis of gene expression and principal component analysis (PCA) successfully separated transcriptomes of the two stress time points. Analysis indicated the occurrence of 65 clusters of gene expression with transcripts of four clusters differed at the genotype (e.g., WT (wild type), KO ERF109 or OE ERF109 ) level. Regulated transcripts involved DIAP1-like gene encoding a death-associated inhibitor of reactive oxygen species (ROS). Other ERF109-regulated transcripts belong to gene families encoding ROS scavenging enzymes and a large number of genes participating in three consecutive pathways, e.g., phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism and plant hormone signal transduction. We investigated the possibility that ERF109 acts as a "master switch" mediator of a cascade of consecutive events across these three pathways initially by driving expression of ASA1 and YUC2 genes and possibly driving GST, IGPS and LAX2 genes. Action of downstream auxin-regulator, auxin-responsive as well as auxin carrier genes promotes plant cell growth under adverse conditions.

First report of detection of the putative receptor of Bacillus thuringiensis toxin Vip3Aa from black cutworm (Agrotis ipsilon).

Black cutworm (BCW) Agrotis ipsilon, an economically important lepidopteran insect, has attracted a great attention. Bacillus thuringiensis (Bt) is spore forming soil bacteria and is an excellent environment-friendly approach for the control of phytophagous and disease-transmitting insects. In fact, bio-pesticide formulations and insect resistant transgenic plants based on the bacterium Bt delta-endotoxin have attracted worldwide attention as a safer alternative to harmful chemical pesticides. The major objective of the current study was to understand the mechanism of interaction of Bt toxin with its receptor molecule(s). The investigation involved the isolation, identification, and characterization of a putative receptor - vip3Aa. In addition, the kinetics of vip toxin binding to its receptor molecule was also studied. The present data suggest that Vip3Aa toxin bound specifically with high affinity to a 48-kDa protein present at the brush border membrane vesicles (BBMV) prepared from the midgut epithelial cells of BCW larvae.

Developing transgenic wheat to encounter rusts and powdery mildew by overexpressing barley chi26 gene for fungal resistance.

BACKGROUND: The main aim of this study was to improve fungal resistance in bread wheat via transgenesis. Transgenic wheat plants harboring barley chitinase (chi26) gene, driven by maize ubi promoter, were obtained using biolistic bombardment, whereas the herbicide resistance gene, bar, driven by the CaMV 35S promoter was used as a selectable marker. RESULTS: Molecular analysis confirmed the integration, copy number, and the level of expression of the chi26 gene in four independent transgenic events. Chitinase enzyme activity was detected using a standard enzymatic assay. The expression levels of chi26 gene in the different transgenic lines, compared to their respective controls, were determined using qRT-PCR. The transgene was silenced in some transgenic families across generations. Gene silencing in the present study seemed to be random and irreversible. The homozygous transgenic plants of T4, T5, T6, T8, and T9 generations were tested in the field for five growing seasons to evaluate their resistance against rusts and powdery mildew. The results indicated high chitinase activity at T0 and high transgene expression levels in few transgenic families. This resulted in high resistance against wheat rusts and powdery mildew under field conditions. It was indicated by proximate and chemical analyses that one of the transgenic families and the non-transgenic line were substantially equivalent. CONCLUSION: Transgenic wheat with barley chi26 was found to be resistant even after five generations under artificial fungal infection conditions. One transgenic line was proved to be substantially equivalent as compared to the non-transgenic control.

Genome-wide In Silico Analysis, Characterization and Identification of Microsatellites in Spodoptera littoralis Multiple nucleopolyhedrovirus (SpliMNPV).

In this study, we undertook a survey to analyze the distribution and frequency of microsatellites or Simple Sequence Repeats (SSRs) in Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV) genome (isolate AN-1956). Out of the 55 microsatellite motifs, identified in the SpliMNPV-AN1956 genome using in silico analysis (inclusive of mono-, di-, tri- and hexa-nucleotide repeats), 39 were found to be distributed within coding regions (cSSRs), whereas 16 were observed to lie within intergenic or noncoding regions. Among the 39 motifs located in coding regions, 21 were located in annotated functional genes whilst 18 were identified in unknown functional genes (hypothetical proteins). Among the identified motifs, trinucleotide (80%) repeats were found to be the most abundant followed by dinucleotide (13%), mononucleotide (5%) and hexanucleotide (2%) repeats. The 39 motifs located within coding regions were further validated in vitro by using PCR analysis, while the 21 motifs located within known functional genes (15 genes) were characterized using nucleotide sequencing. A comparison of the sequence analysis data of the 21 sequenced cSSRs with the published sequences is presented. Finally, the developed SSR markers of the 39 motifs were further mapped/localized onto the SpliMNPV-AN1956 genome. In conclusion, the SSR markers specific to SpliMNPV, developed in this study, could be a useful tool for the identification of isolates and analysis of genetic diversity and viral evolutionary status.

Development of insect resistant maize plants expressing a chitinase gene from the cotton leaf worm, Spodoptera littoralis.

Due to the importance of chitinolytic enzymes for insect, nematode and fungal growth, they are receiving attention concerning their development as biopesticides or chemical defense proteins in transgenic plants and as microbial biocontrol agents. Targeting chitin associated with the extracellular matrices or cell wall by insect chitinases may be an effective approach for controlling pest insects and pathogenic fungi. The ability of chitinases to attack and digest chitin in the peritrophic matrix or exoskeleton raises the possibility to use them as insect control method. In this study, an insect chitinase cDNA from cotton leaf worm (Spodoptera littoralis) has been synthesized. Transgenic maize plant system was used to improve its tolerance against insects. Insect chitinase transcripts and proteins were expressed in transgenic maize plants. The functional integrity and expression of chitinase in progenies of the transgenic plants were confirmed by insect bioassays. The bioassays using transgenic corn plants against corn borer (Sesamia cretica) revealed that ~50% of the insects reared on transgenic corn plants died, suggesting that transgenic maize plants have enhanced resistance against S. cretica.

Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects.

BACKGROUND: Wheat is considered the most important cereal crop all over the world. The wheat weevil Sitophilus granarius is a serious insect pests in much of the wheat growing area worldwide and is responsible for significant loss of yield. Avidin proteins has been proposed to function as plant defense agents against insect pests. RESULTS: A synthetic avidin gene was introduced into spring wheat (Triticum aestivum L.) cv. Giza 168 using a biolistic bombardment protocol. The presence and expression of the transgene in six selected T0 transgenic wheat lines were confirmed at the molecular level. Accumulation of avidin protein was detected in transgenic plants compared to non-transgenic plants. Avidin transgene was stably integrated, transcribed and translated as indicated by Southern blot, ELISA, and dot blot analyses, with a high level of expression in transgenic wheat seeds. However, no expression was detected in untransformed wheat seeds. Functional integrity of avidin was confirmed by insect bioassay. The results of bioassay using transgenic wheat plants challenged with wheat weevil revealed 100 % mortality of the insects reared on transgenic plants after 21 days. CONCLUSION: Transgenic wheat plants had improved resistance to Sitophilus granarius.