Amgad A. Saleh

Characterization of Novel Trichoderma asperellum Isolates to Select Effective Biocontrol Agents Against Tomato Fusarium Wilt

The use of novel isolates of Trichoderma with efficient antagonistic capacity against Fusarium oxysporum f. sp. lycopersici (FOL) is a promising alternative strategy to pesticides for tomato wilt management. We evaluated the antagonistic activity of 30 isolates of T. asperellum against 4 different isolates of FOL. The production of extracellular cell wall degrading enzymes of the antagonistic isolates was also measured. The random amplified polymorphic DNA (RAPD) method was applied to assess the genetic variability among the T. asperellum isolates. All of the T. asperellum isolates significantly reduced the mycelial growth of FOL isolates but the amount of growth reduction varied significantly as well. There was a correlation between the antagonistic capacity of T. asperellum isolates towards FOL and their lytic enzyme production. Isolates showing high levels of chitinase and β-1,3-glucanase activities strongly inhibited the growth of FOL isolates. RAPD analysis showed a high level of genetic variation among T. asperellum isolates. The UPGMA dendrogram revealed that T. asperellum isolates could not be grouped by their anta- gonistic behavior or lytic enzymes production. Six isolates of T. asperellum were highly antagonistic towards FOL and potentially could be used in commercial agriculture to control tomato wilt. Our results are consistent with the conclusion that understanding the genetic variation within Trichoderma isolates and their biochemical capabilities are required for the selection of effective indigenous fungal strains for the use as biocontrol agents.

Molecular characterization of early blight disease resistant and susceptible potato cultivars using random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers

Potato early blight disease caused by Alternaria solani is one of the major factors limiting potato production worldwide. Developing highly resistant cultivars is the most effective way to control the disease. In this study, 20 random amplified polymorphic DNA (RAPD) and 6 simple sequence repeats (SSR) primers were applied to assess the genetic diversity and to identify molecular markers associated with resistance to early blight disease in resistant (Cara, Spunta and Valor), moderately resistant (Hermes and Atlantic) and susceptible (Atlas, Desiree and Lady-Rosetta) potato cultivars. The results showed that 296 and 29 clear fragments were amplified by RAPD and SSR, respectively. SSR revealed higher frequency of polymorphic bands (93.1%) than RAPD (57.4%). The clustering pattern obtained from the analyses of two marker systems showed a similar distribution of the tested potato cultivars. Interestingly, the resistant potato cultivars were grouped together in the dendrograms generated from RAPD and SSR clustering analyses. RAPD primers OPY-07, OPG-05, OPA-10 and OPA- 18 generated five unique fragments (OPY-07 880bp , OPG-05 949bp , OPA-10 1000bp , OPA-18 2030bp and OPA- 18 1128bp ) presented only in resistant cultivars. In addition, all resistant cultivars and moderate resistant cultivar (Hermes) shared two markers; one RAPD (OPA-18 1070bp ) and the other SSR (STI57 209bp ). On the other hand, RAPD primer UBC-17 generated specific fragment UBC-17 720bp presented only in susceptible cultivars and moderate resistant cultivar (Atlantic). This study demonstrate that RAPD and SSR markers can be effectively used to assess the genetic variation among potato cultivars in order to develop molecular markers associated with early blight disease to improve potato breeding programs. Key words : Alternaria solani, cluster analysis, resistance, Solanum tuberosum.

Allelic variation of melanocortin-1 receptor locus in Saudi indigenous sheep exhibiting different color coats

Objective This study was designed to characterize the DNA polymorphisms of the melanocortin-1 receptor (MC1R) gene in indigenous Saudi Arabian sheep breeds exhibiting different color coats, along with individuals of the Sawaknee breed, an exotic sheep imported from Sudan. Methods The complete coding region of MC1R gene including parts of 3′ and 5′ untranslated regions was amplified and sequenced from three the indigenous Saudi sheep; Najdi (generally black, n = 41), Naeimi (generally white with brown faces, n = 36) and Herri (generally white, n = 18), in addition to 13 Sawaknee sheep. Results Five single nucleotide polymorphisms (SNPs) were detected in the MC1R gene: two led to nonsynonymous mutations (c.218 T>A, p.73 Met>Lys and c.361 G>A, p.121 Asp>Asn) and three led to synonymous mutations (c.429 C>T, p.143 Tyr>Tyr; c.600 T>G, p.200 Leu>Leu, and c.735 C>T, p.245 Ile>Ile). Based on these five SNPs, eight haplotypes representing MC1R Ed and E+ alleles were identified among the studied sheep breeds. The most common haplotype (H3) of the dominant Ed allele was associated with either black or brown coat color in Najdi and Sawaknee sheep, respectively. Two other haplotypes (H6 and H7) of Ed allele, with only the nonsynonymous mutation A218T, were detected for the first time in Saudi indigenous sheep. Conclusion In addition to investigating the MC1R allelic variation in Saudi indigenous sheep populations, the present study supports the assumption that the two independent nonsynonymous Met73Lys and Asp121Asn mutations in MC1R gene are associated with black or red coat colors in sheep breeds.

Variation in a molecular marker for resistance of Saudi date palm germplasm to Fusarium oxysporum f. sp. albedinis the causal agent of Bayoud disease

Fusarium oxysporum f. sp. albedinis (FOA) is a seed- and soil-borne vascular wilt pathogen that causes a very serious and destructive disease of date palm (Bayoud disease). FOA is not currently known to occur in Saudi Arabia, but the disease has been moving eastward from its origin in Morocco and Algeria. The main objective of this study was to evaluate Saudi Arabian date palm germplasm at the molecular level to assess the presence of R or S plasmid. We evaluated 711 trees representing 42 date palm varieties in Saudi Arabia with a mitochondrial molecular marker and sequenced 64 of the diagnostic PCR amplicons. Most of the trees (628/711) carried the R-plasmid which is associated with Bayoud resistance against FOA fungus. The other 83 trees carried the S-plasmid and are predicted to be susceptible. All amplifications from putatively resistant trees had the same DNA sequence. Moreover, the sequence of the Saudi resistance amplicon was identical to the sequence of Moroccan resistance DNA publically available in NCBI-GenBank database. All the sensitive trees from Saudi Arabia shared a common DNA sequence. This sequence differed from the Moroccan susceptible one at seven unique mutations including five single nucleotide polymorphisms (SNPs) and two insertion/deletion (indels) regions. The uniformity of the sequences means that the introduction of new germplasm to Saudi Arabia will be easily detected. The relatively high frequency of putative resistance means that if/when this disease enters the Kingdom that most trees should be resistant.

Management of Asiatic Citrus Canker Under Field Conditions in Saudi Arabia Using Bacteriophages and Acibenzolar-S-Methyl

Applications of formulated bacteriophages with skim milk and sucrose or nonformulated bacteriophages combined with acibenzolar-S-methyl (ASM) were compared with copper bactericides applications for suppressing Asiatic citrus canker (ACC) caused by Xanthomonas citri subsp. citri (Xcc) on leaves under greenhouse and field conditions in Saudi Arabia. Bacteriophages were applied one day prior to inoculation of Mexican lime (Citrus aurantifolia) plants with Xcc, then twice a week until the end of the trials. Copper hydroxide was applied once prior to inoculation and then every seven days afterward, whereas ASM was applied one week prior to inoculation and then every 21 days afterward. Under greenhouse conditions, the incidence of ACC on leaves was reduced significantly from 75.2 to 12.8% or 18.3% for plants treated with copper hydroxide or bacteriophages in combination with ASM, respectively. Applications of formulated phages in combination with ASM as soil drench under field conditions significantly decreased disease incidence by 14.8% (Trial 1) and 16.8% (Trial 2) compared with untreated control plants. Overall, the Xcc-inoculated plants treated with bacteriophages + ASM combination showed significant ACC reduction under greenhouse and field conditions. The bacteriophages + ASM combination tested in these trials can be an effective tool in the integrated management programs of Asiatic citrus canker disease.

Mitochondrial Molecular Markers for Resistance to Bayoud Disease in Date Palm

Bayoud disease, caused by Fusarium oxysporum f. sp. albedinis, is a very serious and destructive disease to date palm. Screening of date palm germplasm for resistance to bayoud disease is a crucial step to avoid or alleviate the disease consequences. Fortunately, it was discovered that there are two mitochondrial plasmid-like DNA molecules associated with susceptibility or resistance to bayoud disease. In this chapter, we present a fast, simple, and reliable technique to screen date palm germplasm for the presence of these mitochondrial molecular markers associated with susceptibility or resistance to bayoud.

Microbiome engineers: Grazers, browsers, and the phytobiome stampede

We define microbiome engineers as species that modify the microbiome associated with other host species via changes in the physical environment, potentially including the creation of dispersal networks for microbiome consortia across multiple hosts. Grazers such as bison are indirect plant microbiome engineers through alteration of the structure of plant communities in grasslands and forests. They also can directly engineer plant microbiomes if they distribute microbial consortia. Direct engineering may include simpler examples such as the role of truffle-eating animals in structuring forest mycorrhizal communities, as well as more complex roles in structuring the evolution of bacterial, fungal, and viral microbiome networks. Grazers and browsers may have important historic and current roles in engineering microbiomes by (a) stabilizing and homogenizing microbiomes in their preferred plant species, and (b) selecting for microbiomes in their preferred plant species that are differentiated from microbiomes in plant species they rarely consume.

Trichoderma asperellum strains confer tomato protection and induce its defense-related genes against the Fusarium wilt pathogen

Fusarium wilt of tomato (FW) caused by Fusarium oxysporum f. sp. lycopersici (FOL) is a major challenge for tomato production worldwide. For sustainable management of FW, the potential of five strains of Trichoderma asperellum was evaluated under greenhouse conditions. The results indicated that FOL infected plants treated with T. asperellum strains significantly reduced disease incidence and severity compared with FOL-only infected plants. The reduction of wilt disease on plants treated with T. asperellum strains was accompanied by a significant reduction in FOL populations in tomato stems and rhizosphere. Moreover, the application of T. asperellum promoted tomato plant growth irrespective of the presence or absence of FOL. Two strains of T. asperellum (TS-12 and TS-39) that showed the best performance in minimizing disease development and increases in plant growth parameters were selected for elucidating their ability in triggering tomato defense mechanisms. The expression levels of defense-related genes, chitinase (SlChi3), β-1,3-glucanase (SlGluA) and PR-1 (SlPR-1a) were significantly increased in the stems and roots of Trichoderma treated, FOL infected plants, compared with FOL-only infected ones. These results indicate that the application of T. asperellum strains TS-12 and TS-39 can be used as an alternative strategy to manage FW through their antagonistic activities and abilities to induce systemic resistance.

Integration of Pseudomonas fluorescens and salicylic acid improves citrus canker disease management caused by Xanthomonas citri subsp citri-A*

The individual and combined effects of Pseudomonas fluorescens (Pf) and salicylic acid (SA) were investigated for control of citrus bacterial canker (CBC). Both treated plants with copper hydroxide and untreated ones were used as controls. Mexican lime (Citrus aurantifolia) seedlings were treated with SA at 10 mM, Pf and distilled water. Plants were initially inoculated with Xanthomonas citri subsp citri 72 h post treatments. Results indicated that the Pf and SA treatment controlled CBC more effectively compared to separately applying Pf or SA. The application of Pf in combination with SA significantly reduced lesion number per leaf (72%) and disease severity (84%). Significant changes in the activities of peroxidase and catalase were found. In conclusion, the integration of Pf with SA complements each other and can be applied to manage citrus canker disease in conjunction with other control programmes.