Isolation of Pseudomonas syringae pv. Tomato strains causing bacterial speck disease of tomato and marker-based monitoring for their virulence.

BACKGROUND: The bacterial speck disease of tomato caused by a bacterial pathogen Pseudomonas syringae pv. tomato is a most important disease causing severe crop losses. METHODS AND RESULTS: Present study was conducted to investigate and characterize the population diversity of P. syringae pv. tomato pathogen isolated from infected tomato plants from various regions of Egypt. Significant variation among the isolates was observed which demonstrated considerable virulence. All isolates were pathogenic and the CFU population recovered from inoculate tomato leaves by isolate Pst-2 was higher than other isolates. Genetic disparity among the isolates was investigated by PCR analysis by amplifying hrpZ gene using random amplified polymorphic DNA (RAPD), sequence-related amplified polymorphism (SRAP), and inter-simple sequence repeats (ISSR) markers. The amplified products for ITS1 were found to have 810 bp length whereas 536 bp length was observed for hrpZ gene using primer pairs (1406-f/23S-r) and (MM5-F, MM5-R) respectively. The restriction analysis of amplified regions "ITS" and hrpZ by using 5 and 4 endonucleases respectively demonstrated slight variation among the bacterial isolates. The results of RAPD, ISSR and SRAP showed higher polymorphism (60.52%) within the isolates which may assist for successful characterization by unique and specific markers based on geographical distribution, origin and virulence intensity. CONCLUSION: The results of present study suggested that the use of molecular approach may provide successful and valuable information to differentiate and classify P. syringae pv. tomato strains in future for the detection and confirmation of pathogenicity.

SERS study of classical and newly ß-lactams-metal complexation based on in situ laser-induced coral reefs-like silver photomicroclusters: In vitro study of antibacterial activity.

The influence of metal complexation of two polar ß-lactam antibiotics was investigated using surface enhanced Raman spectroscopy (SERS) technique. SERS method was applied to track the structural changes and the degradation behaviour of the studied compounds upon Zinc (II) ions-complexation. In situ laser-induced coral reefs-like photomicroclusters have been utilized as a SERS platform. The produced coral reefs-like photomicroclusters were characterized using scanning electron microscopy (SEM) and transmission electron microscope (TEM). The antibacterial efficiency of the antibiotics was investigated and compared before and after metal complexation using two techniques; agar well diffusion and growth curve. To provide a detailed elucidation of the complexation reaction, mass fragmentation of metal- antibiotics complexes was investigated using liquid chromatography/mass spectrometric (LC/MS) technique. It was found that metal complexation of classical ß-lactam antibiotic (Ticarcillin) promoted the rate of its degradation, leading to a decrease of the antibacterial efficiency. On the other side, the antibacterial activity of the newly developed ß-lactam (Faropenem) has been greatly enhanced via metal-complexation reaction.

Capturing Wheat Phenotypes at the Genome Level.

Recent technological advances in next-generation sequencing (NGS) technologies have dramatically reduced the cost of DNA sequencing, allowing species with large and complex genomes to be sequenced. Although bread wheat (Triticum aestivum L.) is one of the world's most important food crops, efficient exploitation of molecular marker-assisted breeding approaches has lagged behind that achieved in other crop species, due to its large polyploid genome. However, an international public-private effort spanning 9 years reported over 65% draft genome of bread wheat in 2014, and finally, after more than a decade culminated in the release of a gold-standard, fully annotated reference wheat-genome assembly in 2018. Shortly thereafter, in 2020, the genome of assemblies of additional 15 global wheat accessions was released. As a result, wheat has now entered into the pan-genomic era, where basic resources can be efficiently exploited. Wheat genotyping with a few hundred markers has been replaced by genotyping arrays, capable of characterizing hundreds of wheat lines, using thousands of markers, providing fast, relatively inexpensive, and reliable data for exploitation in wheat breeding. These advances have opened up new opportunities for marker-assisted selection (MAS) and genomic selection (GS) in wheat. Herein, we review the advances and perspectives in wheat genetics and genomics, with a focus on key traits, including grain yield, yield-related traits, end-use quality, and resistance to biotic and abiotic stresses. We also focus on reported candidate genes cloned and linked to traits of interest. Furthermore, we report on the improvement in the aforementioned quantitative traits, through the use of (i) clustered regularly interspaced short-palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene-editing and (ii) positional cloning methods, and of genomic selection. Finally, we examine the utilization of genomics for the next-generation wheat breeding, providing a practical example of using in silico bioinformatics tools that are based on the wheat reference-genome sequence.

Genetic Diversity Studies and Identification of Molecular and Biochemical Markers Associated with Fusarium Wilt Resistance in Cultivated Faba Bean (Vicia faba).

Faba bean (Vicia faba L.) is one of the most important legume crops in Egypt. However, production of faba bean is affected by several diseases including fungal diseases. Fusarium wilt incited by Fusarium oxysporum Schlecht. was shown to be the most common wilt disease of faba bean in Assiut Governorate. Evaluation of 16 faba bean genotypes for the resistance to Fusarium wilt was carried out under greenhouse conditions. Three molecular marker systems (inter-simple sequence repeat [ISSR], sequence related amplified polymorphism [SRAP], and simple sequence repeat [SSR]) and a biochemical marker (protein profiles) were used to study the genetic diversity and detect molecular and biochemical markers associated with Fusarium wilt resistance in the tested genotypes. The results showed that certain genotypes of faba bean were resistant to Fusarium wilt, while most of the genotypes were highly susceptible. The percentage of disease severity ranged from 32.83% in Assiut-215 to 64.17% in Misr-3. The genotypes Assiut-215, Roomy-3, Marut-2, and Giza-2 were the most resistant, and the genotypes Misr-3, Misr-1, Assiut-143, Giza-40, and Roomy-80 performed as highly susceptible. The genotypes Assiut-215 and Roomy-3 were considered as promising sources of the resistance to Fusarium wilt. SRAP markers showed higher polymorphism (82.53%) compared with SSR (76.85%), ISSR markers (62.24%), and protein profile (31.82%). Specific molecular and biochemical markers associated with Fusarium wilt resistance were identified. The dendrogram based on combined data of molecular and biochemical markers grouped the 16 faba bean genotypes into three clusters. Cluster I included resistant genotypes, cluster II comprised all moderate genotypes and cluster III contained highly susceptible genotypes.

Identification of charcoal rot resistance QTLs in sorghum using association and in silico analyses.

Charcoal rot disease, a root and stem disease caused by the soil-borne fungus Macrophomina phaseolina (Tassi) Goid., is a major biotic stress that limits sorghum productivity worldwide. Charcoal rot resistance-related parameters, e.g., pre-emergence damping-off%, post-emergence damping-off%, charcoal rot disease severity, and plant survival rates, were measured in a structured sorghum population consisting of 107 landraces. Analysis of variance of charcoal rot resistance-related parameters revealed significant variations in the response to M. phaseolina infection within evaluated accessions. Continuous phenotypic variations for resistance-related parameters were observed indicating a quantitative inheritance of resistance. The population was genotyped using 181 simple sequence repeat (SSR) markers. Association analysis identified 13 markers significantly associated with quantitative trait genes (QTLs) conferring resistance to charcoal rot disease with an R2 value ranging between 9.47 to 18.87%, nine of which are environment-specific loci. Several QTL-linked markers are significantly associated with more than one resistance-related parameter, suggesting that those QTLs might contain genes involved in the plant defense response. In silico analysis of four novel major QTLs identified 11 putative gene homologs that could be considered as candidate genes for resistance against charcoal rot disease. Cluster analysis using the genotypic data of 181 SSR markers from 107 sorghum accessions identified 12 main clusters. The results provide a basis for further functional characterization of charcoal rot disease resistance or defense genes in sorghum and for further dissection of their molecular mechanisms.

Genetic Variation and Biological Control of Fusarium graminearum Isolated from Wheat in Assiut-Egypt.

Fusarium graminearum Schwabe causes Fusarium head blight (FHB), a devastating disease that leads to extensive yield and quality loss of wheat and other cereal crops. Twelve isolates of F. graminearum were collected from naturally infected spikes of wheat from Assiut Egypt. These isolates were compared using SRAP. The results indicated distinct genetic groups exist within F. graminearum, and demonstrated that these groups have different biological properties, especially with respect to their pathogenicity on wheat. There were biologically significant differences between the groups; with group (B) isolates being more aggressive towards wheat than groups (A) and (C). Furthermore, Trichoderma harzianum (Rifai) and Bacillus subtilis (Ehrenberg) which isolated from wheat kernels were screened for antagonistic activity against F. graminearum. They significantly reduced the growth of F. graminearum colonies in culture. In order to gain insight into biological control effect in situ, highly antagonistic isolates of T. harzianum and B. subtilis were selected, based on their in vitro effectiveness, for greenhouse test. It was revealed that T. harzianum and B. subtilis significantly reduced FHB severity. The obtained results indicated that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in FHB and should be harnessed for further biocontrol applications. The accurate analysis of genetic variation and studies of population structures have significant implications for understanding the genetic traits and disease control programs in wheat. This is the first known report of the distribution and genetic variation of F. graminearum on wheat spikes in Assiut Egypt.

Resistance Potential of Bread Wheat Genotypes Against Yellow Rust Disease Under Egyptian Climate.

Yellow rust (stripe rust), caused by Puccinia striiformis f. sp. tritici, is one of the most destructive foliar diseases of wheat in Egypt and worldwide. In order to identify wheat genotypes resistant to yellow rust and develop molecular markers associated with the resistance, fifty F8 recombinant inbred lines (RILs) derived from a cross between resistant and susceptible bread wheat landraces were obtained. Artificial infection of Puccinia striiformis was performed under greenhouse conditions during two growing seasons and relative resistance index (RRI) was calculated. Two Egyptian bread wheat cultivars i.e. Giza-168 (resistant) and Sakha-69 (susceptible) were also evaluated. RRI values of two-year trial showed that 10 RILs responded with RRI value >6 <9 with an average of 7.29, which exceeded the Egyptian bread wheat cultivar Giza-168 (5.58). Thirty three RILs were included among the acceptable range having RRI value >2 <6. However, only 7 RILs showed RRI value <2. Five RILs expressed hypersensitive type of resistance (R) against the pathogen and showed the lowest Average Coefficient of Infection (ACI). Bulked segregant analysis (BSA) with eight simple sequence repeat (SSR), eight sequence-related amplified polymorphism (SRAP) and sixteen random amplified polymorphic DNA (RAPD) markers revealed that three SSR, three SRAP and six RAPD markers were found to be associated with the resistance to yellow rust. However, further molecular analyses would be performed to confirm markers associated with the resistance and suitable for marker-assisted selection. Resistant RILs identified in the study could be efficiently used to improve the resistance to yellow rust in wheat.