Younes M Rashad

Sunflower (Helianthus annuus L.) has economic value worldwide mainly for edible oil production and other various industrial purposes. Fusarium root rot, caused by Fusarium solani (Mart.) Sacc., is the most important disease of sunflower plants and causes considerable economic losses. Seed treatment with a turmeric aqueous extract was tested for control of Fusarium root rot. Gas chromatography-mass spectrometry analysis of the turmeric aqueous extract revealed the presence of three major constituents; ar-curcumin, camphor and α-turmerone. Greenhouse experiments showed that disease incidence and severity were significantly reduced after treatment with turmeric extract. Moreover, growth parameters were also improved 2 and 4 weeks after inoculation. In addition, treatment with turmeric extract triggered the plant immune system as indicated by the induction of the phenolic content and activity of antioxidant enzymes (peroxidase and phenylalanine ammonia lyase). Molecularly, differential display-PCR of the sunflower plants showed distinct profiles of gene expression in response to the applied treatments. Of the four bands randomly selected for sequencing and identification, three up-regulated genes that encode defense related proteins (glutathione S-transferase 6, ascorbate peroxidase, and defensin). A time-course real-time quantitative PCR was carried out on mRNA of the defense-related genes defensin and chitinase of the treated sunflower seedlings. After 14 days, treatment with turmeric extract enhanced the expression levels of chitinase and defensin genes by 9.62 and 4.34 folds, respectively. Based on these results, we recommend treatment of seeds with turmeric extract for controlling Fusarium root rot of sunflower.

The methanol extracts of five medicinal plants (Eucalyptus tereticornis Sm., Ammi visnaga (L.) Lam., Azadirachta indica A. Juss., Rheum Palmatum L., and Adansonia digitata L.) were screened in vitro for their antifungal activity against Rhizoctonia solani Kühn, the causal agent of Rhizoctonia root rot of maize. All tested extracts showed antifungal activity with varied extents. Ammi visnaga (khella) extract was superior to other tested extracts comparing with the untreated control. Observations using transmission electron microscopy exhibited dramatic ultrastructural changes in the hyphal cell as a response to the exposure to the antifungal khella extract. Gas chromatography-mass spectrometric analysis of khella extract showed the presence of 69 chemical compounds. The antifungal properties of the extracts were mainly attributed to their content of coumarins and fatty acids. In the greenhouse experiment, the treatment of maize plants with khella extract at 15 % recorded the lowest disease incidence of Rhizoctonia root rot. Results of DD-PCR showed varied up- and down- regulations of some genes as a response to the treatment with khela extract. Identification of the randomly selected genes from DD-PCR revealed that they were defense-related genes such as S-domain class receptor-like kinase 3 and glutathione-S-transferase 1. On the other hand, the real-time PCR showed an induction in the gene expression of the pathogenesis-related proteins chitinases (2.36 fold) and thaumatin-like proteins (8.99 fold) by treatment with khella extract at the rate of 15 % higher than that observed at 10 and 20 %, indicating a triggering effect of the plant immune system against the R. solani infection in concentration-dependent manner. Considering the efficient, low-cost and eco-friendly characteristics of khella extract, we suggest an use of this extract for the control of Rhizoctonia root rot disease of maize

A field survey was conducted to investigate the correlation between arbuscular mycorrhizal (AM) fungi and soil properties in four different districts of Dakahlia governorate, Egypt. Among the 40 plant samples collected from different soils, 32 were colonized with AM fungi in varied extents. While, few plant species belonging to Chenopodiaceae, Zygophyllaceae, Cruciferae, Cyperaceae and Labiatae were found to be non-mycorrhizal. Our investigations indicated that soil salinity appeared to be a limiting factor for the occurrence of AMF spores and mycorrhizal colonization. While, soil pH level had no direct effect on the mycorrhizal colonization or number of spores in the rhizosphere soil. Increases in soil organic matter content was favored mycorrhizal colonization and spores number. Available phosphorus content was positively correlated with either spores number or the percentage of mycorrhizal colonization. Extracted AMF spores were belonged to two genera Glomus and Scutellospora. Sp...

Rice is among the most important cereals in the world, it is life for thousands of millions of people and it is deeply embedded in the cultural heritage of their societies. Brown spot - caused by Bipolaris oryzae - is one of the important rice diseases in the world. Widespread, reported in all rice growing countries of Asia, America and Africa. It can be a serious disease causing a considerable yield loss. Chemical control may be available to reduce the effects of brown spot disease on young plants effectively and extensively, but field application of these chemicals may not always be desirable. Excessive and improper use of these pesticides presents a menace to the health of humans, animals and environment. So that, the present work aimed to replacement the undesirable and unsafe chemical control by another effective, inexpensive and safe options for rice brown spot disease control. Two ways were suggested here to overcome this problem, the biological control and the induction of s...

Effectiveness of arbuscular mycorrhizal fungi in the protection of common bean plant (Phaseolus vulgaris L.) against Fusarium root rot disease was investigated in the present study under natural conditions in pot experiment. A mixture of arbuscular mycorrhizal fungi consists of propagated units of Glomus mosseae, Glomus intraradices, Glomus clarum, Gigaspora gigantea and Gigaspora margarita in suspension form (106 unit.L-1 in concentration) was used at dilution of 5 ml.L-1 water. The obtained results demonstrated that, arbuscular mycorrhizal colonization significantly reduced the percentage of disease severity and incidence in infected bean plants. On the other hand, mycorrhizal colonization significantly increased the tested growth parameters and mineral nutrient concentrations. While, infection with Fusarium root rot disease negatively affected on the mycorrhizal colonization level in bean roots. Finally, mycorrhizal colonization led to a significant increase in the phenolic content and the activities of the investigated defense related enzymes (Phenylalanine ammonia-lyase, Polyphenol oxidase and Peroxidase enzyme). From the obtained results, it can be concluded that the application of arbuscular mycorrhizal fungi as a biocontrol agent played an important role in plant resistance and exhibit greater potential to protect bean plants against the infection with F. solani.

Antifungal activity of ethanol-water extracts of four medicinal plants, cinnamon (Cinnamomum verum Presl.), anise (Pimpi-nella anisum L.), black seed (Nigella sativa L.) and clove (Syzygium aromaticum L. Merr. & Perry.) was investigated against pea (Pisum sativum L.) root-rot fungus Rhizoctonia solani. In vitro antifungal activity test shown a high growth inhibition at concentration (4%) of each plant extract. The highest antifungal activity was recorded for clove extract which causes complete growth inhibition at concentration of 1%. Efficacy of clove extract on disease incidence of Rhizoctonia root-rot of pea was investigated in the greenhouse pot experiment. Clove extract at concentration 4% as well as the chemical fungicide recorded highly significant increase in the percentage of survived plants (40 and 48%, respectively) and highly significant decrease in disease incidence.

Interaction between arbuscular mycorrhizal fungi as a bio-agent and Rhizocto-nia root rot disease of common bean plant was investigated in this study under natural conditions in pot experiment. A mixture of Egyptian formulated AM (Multi-VAM) in suspension form (1 × 10 6 unit L −1 in concentration) was used at dilution of 5 ml L −1 water. The results demonstrated that colonization of bean plants with AM fungi significantly increased growth parameters, yield parameters and mineral nutrient concentrations and reduced the negative effects on these parameters as well as both disease severity and disease incidence. Different physical and biochemical mechanisms have been shown to play a role in enhancement of plant resistance against Rhizoctonia solani, namely, improved plant nutrition, improved plant growth, increase in cell wall thickening, cytoplasmic granulation, and accumulation of some antimicrobial substances (phenolic compounds and defense related enzymes).

One hundred and twenty-eight strains of actinomycetes were isolated from different soils in Riyadh region, Saudi Arabia. All isolates were screened for their antifungal activities against Rhizoctonia solani, Fusarium solani, Fusarium verticillioides, Alternaria alternata and Botrytis cinerea. A potent antagonist against all tested phytopathogenic fungi, designated RDS28, was selected and identified as Streptomyces spororaveus RDS28 according to analysis of 16S rRNA gene sequence. Factors affecting the production of the antifungal compounds were investigated. The results showed that incubation of S. spororaveus RDS28 for 72 h at 31°C and initial pH 7.5 on a medium containing glucose as a carbon source and proline as a nitrogen source gave the best antifungal antibiotic production. The culture filtrate of S. spororaveus RDS28 was extracted and purified and rechecked for their in vitro antifungal activities against the tested fungi. It can be concluded that, the antifungal antibiotic produced by S. spororaveus RDS28, demonstrated an obvious inhibitory effect on the tested pathogenic fungi.

Fifteen seed samples of local alfalfa (Medicago sativa L.) cultivar (Hegazy) collected from fields at different governorates of the Riyadh region in Saudi Arabia were screened for their seed-borne mycoflora. Standard moist blotter and deep-freezing blotter methods recommended by the International Seed Testing Association, in addition to an innovative alkaline seed-bed method were applied. A total of 24 genera and 35 species of fungi were isolated using the above-mentioned techniques. Alternaria alternata, Cladosporium sp., Aspergillus sp., Stemphylium sp. and Penicillium sp. were the genera most commonly isolated. Among the various techniques adopted for detection, the alkaline NaOH method was found to be effective and yielded the maximum number of pathogenic fungi. Syndromes of seed discoloration were also investigated. The alkaline seed-bed method using NaOH was used to detect saprophytic and pathogenic seed-borne fungi associated with discolored seeds. A total of 15 genera and 26 species of fungi were isolated. Cladosporium sp., Alternaria alternata and Aspergillus species were the saprophytic species detected most, while Stemphylium botryosum and Fusarium incarnatum were common path-ogenic fungi found on discolored seeds. The average of incidence and occurrence percentages of most detected fungi were higher in discolored seeds than in normal seeds. Seed germination was also affected significantly by discoloration. The data also indicate that seed discoloration decreases seed germination significantly (by 26.3–60%).

Verticillium dahliae is a fungal plant pathogen that attacks a wide range of plants including anise causing a wilt disease. The pathogen grows slowly on anise seeds using the standard moist blotter (SMB) or deep-freezing blotter (DFB) methods. In these methods saprophytes and fast growing fungi impair the detection of such slow growing fungi. An attempt was carried out to overcome this problem on anise seeds by applying alkaline seed-bed (ASB) technique for detecting slow growing seed-borne fungi. Data showed significant increase in the incidence levels of V. dahliae (3.4 and 4.73%) when alkaline KOH or NaOH technique was applied as compared to 0.2 and 0.7%, in SBM and DFB methods, respectively. Moreover, the ASB technique proved to be an effective option for early detecting the pathogen on anise seeds after 4 days of incubation in compared to other methods. The in vitro study indicated that the alkaline pH is the favored condition by the fungus. However, V. dahliae was able to grow in a wide range of pH (3.5 to 12.5) with a pH optimum of 8 at which the highest growth rate was recorded (0.96 g.L-1 /day). The maximum glucose coefficient was achieved at pH 8, while the highest glucose utilization was recorded at pH 12.5. These results suggest that ASB technique is recommended for the detection of the alkalophilic V. dahliae.

Nine isolates of Rhizoctonia solani were obtained from different plant types (common bean, broad bean, bell pepper, tomato and cucumber). The obtained isolates were assigned to AG according to hyphal anastomosis. Of these, six isolates belonged to AG 2-2 IIIB, while the other three isolates belonged to AG 4 HG-I. Molecular identification using 18S-rRNA gene showed that all the isolates were R. solani with sequence identity 99% which revealed that these isolates comes from one ancestor, but the analysis based on rRNA-18S sequences failed to group them into different distinct groups on the bases of AGs. Pathogenicity test on common bean under greenhouse conditions showed that all isolates have the potency to cause seed rot, pre-emergence, post-emergence damping-off and root rot diseases, where they caused mortality ranged from 13.33 to 100%. On the other hand, four bean cultivars were tested (Giza 3, Giza 6, Contendor and Rajma). All tested bean cultivars manifested the disease symptoms but Giza 3 was the most susceptible one. It showed the highest value of total mortality (94.07%).

In this work, a wide survey and collection of agricultural soils from different habitats of the Al-Kharj region, Saudi Arabia, were conducted. 110 isolates of actinomycetes were isolated from these samples. Among them, 25 actinomycetes isolates were found to be antagonistic to Fusarium oxysporum, Macrophomina phaseolina and Sclerotium rolfsii. Only one isolate, denoted RDS16 had the strongest antagonistic activity against all of the tested fungi. This isolate was identified as Streptomyces tendae RDS16 based on morphological, cultural, physiological and molecular analyses. In addition, the optimal culture conditions for production of antifungal metabolites were investigated. The obtained results showed that incubation of S. tendae RDS16 for 4 days at 30°C and an initial pH of 7.5 on a fish-meal extract medium containing galactose as carbon source and casein as nitrogen source exhibited the highest production of antifungal compounds.

A time course study was conducted to investigate disease development and molecular defense response in common bean (Phaseolus vulgaris L.) plants colonized by a mixture of five arbuscular mycorrhizal (AM) fungi, namely, Glomus mosseae, G. intraradices, G. clarum, Gigaspora gigantea, and Gigaspora margarita, and post-infected with the soil-borne pathogen Rhizoctonia solani. Results showed that pre-colonization of bean plants by AM fungi significantly reduced disease severity and disease incidence. DNA fingerprinting using the differential display technique revealed a genetic polymorphism (86.8 %) in bean plants that resulted from the colonization by AM fungi. Two genetic mechanisms were recorded: (1) switching on of new genes and (2) induction of other active genes, including the defense genes chitinase and β-1,3-glucanase, to a highly expressed state.

Management and transmission of four seed-borne pathogenic fungi namely, Colletotrichum trifolii, Rhizoctonia solani, Fusarium equiseti and F. incarnatum were investigated on alfalfa plants. The pathogenicity test on alfalf plant showed that R. solani and C. trifolii caused high percentages of rotted seeds and seedlings mortality (26.45, 26.1% and 31.6, 21.1% , respectively). However, no significant differences were observed between the treatments with F. equiseti and F. incarnatum when compared with the control treatment. Transmission of the pathogenic fungi from seed to mature plant of alfalfa was investigated in this study. Results indicate that the recovery percentages of the tested pathogens gradually decreased from root apex up to the first internodes below the shoot tip, but did not reach to the shoot apex. In order to control these fungi, different concentrations of sodium metabisulphite (SM), sodium salicylate (SS) and hydroquinone (HQ) were tested in vitro. Treatment with SM at 10 mM completely inhibited the growth of all isolated fungi. Under greenhouse conditions, soaking alfalfa seeds in a water solution of hydroquinone at 12mM showed to be the most effective treatment in reducing seed rot and seedling mortality percentages and increasing seedling survival percentages. Application of the tested treatments presented significant increases in growth parameters, photosynthetic pigments (chlorophyll a and b and carotenoids) in leaves and total phenol in alfalfa plants. This study therefore recomended the use of HQ and SM as potential and promising antifungal agents in the protection of alfalfa plants against the tested seed-borne fungi.

In vitro antimicrobial activity of methanolic extracts of six medicinal plants
(black seed, camphor, cloocynth, clove, ginger and white cedar) were investigated against pathogenic bacteria and fungi at 3, 6 and 9% concentrations. Results obtained in this study showed that the six plant extracts exhibited varied extents of antimicrobial activity against the tested organisms even at low concentration. Among the tested plant extracts, ginger recorded the highest antifungal activity against Alternaria radicina, Fusarium oxysporum, F. solani, Macrophomina phaseolina, Nigrospora oryzae, Phoma destructiva, Rhizoctonia solani and Sclerotium rolfsii at 9% concentration. Whilst, the maximum antibacterial activity was achieved by black seed extract at 9% concentration against the Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and Grampositive bacteria (Staphylococcus aureus and Streptococcus pneumonia). Chemical compositions of ginger and black seed methanolic extracts were studied using GC-MS analysis and resulted in the identification of 19 and 17 compounds, respectively. Further studies are needed to investigate the antimicrobial potentiality of the active constituents singly or in mixtures with other antibiotics.

Alkaline protease enzyme was produced under solid state fermentation by Fusarium solani. Various agro-wastes were evaluated to check their potential utilization in the solid state fermentation for protease production. In this connection, the results showed that rice straw was the best agro-waste. The best enzyme activity was achieved at 7th day of incubation, pH 8, temperature 45 °C, inoculum’s size 200 μl/100 ml, agitation speed at 140 rpm, and KNO3 as nitrogen source. The molecular weight of the obtained purified enzyme is 30 KDa. A protease inhibitor protein was isolated and purified from Rumex vesicarius L. The molecular weight of the inhibitor protein is 46 KDa and it composes of 17 amino acids. The minimum inhibitory concentration was found at 20 μl of the inhibitor per 40 μl of the enzyme. The obtained results showed that the inhibitor can withstand the heat at 70°C for 30 min, and pH (5-9) for 72 h at 4°C without loss of activity but it cannot be activated at low acidity less than pH 5 and very high temperature
more than 70°C. This study will serve to increase the understanding of factors that
control the production and inhibition of protease enzyme which may help in many
industrial aspects.

In this study, a wide survey was conducted along Saudi Arabia. One hundred soil samples were collected from different 18 governorates representing different climatic conditions. Five hundred and seventy strains of actinomycetes were isolated from the collected soil samples. Among them, 225 were found to be antagonistic to the pathogenic fungus (Fusarium oxysporum f. sp. lycopersici) with varying degrees. Only one isolate designated E44G, had the strongest antagonistic activity against the tested fungus. The taxonomic status of this isolate was established using Phenotypic and molecular methods. The optimum nutritional and environmental conditions were studied to produce the maximum yield of antifungal activity. The highest antifungal activity was obtained at 2 nd day of incubation, 7.5 pH, and 30 °C. Glucose was the best carbon source and yeast extract was the best nitrogen source. In the present study, we report on a potential Streptomyces strain which has antifungal and antibacterial activities and holds the potential for use in studies of bioactive compounds as well as for possible use in biological control of fungal and bacterial diseases of important crops.

Five pathogenic fungi (Alternaria alternata, Bipolaris sorokiniana, B. spicifera, Fusarium avenaceum and F. graminearum) were evaluated for seed discoloration, shriveling, germination and seedling vigor of wheat. Three wheat cultivars (Yecora Rojo, Logame and Sammah), two inoculation times (at anthesis and 12 days later) and two environmental conditions (wet and dry) were used in this investigation. Considerable differences were recorded between the tested fungi for seed discoloration, shriveling, germination and seedling vigor at each treatment. Fungal mycelia were also assessed in wheat grains via estimation of chitin and ergosterol contents. For each fungal species, significant differences between samples were detected. It is evident that visual characteristics of seed provide an imperfect guide to the extent that seed is infected by fungi. Chemical analysis for either chitin or ergosterol, if developed for routine use, could well be helpful in identifying potentially samples on which additional analyses for specific mycotoxins could be carried out.

The antifungal activity of Streptomyces griseorubens E44G against Rhizoctonia solani, the causal agent of root rot disease of corn, was investigated. The mycelial growth of R. solani was inhibited by S. griseorubens E44G, indicating that it has an antifungal potential. The antagonist, S. griseorubens E44G, was detected to have proteolytic activity, using the method of casein hydrolysis. Moreover, the protease production was optimized under submerged conditions. The purification and precipitation of protease were achieved by ammonium sulphate and Sephadex G-100 gel filtration chromatography. Protease activity was detected spectrophotometrically based on the production of tyrosine. The molecular weight of the enzyme (35 kDa) was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis . The optimum activity of the enzyme was detected at pH 8.5 and 60
C. The results indicated that the enzyme was thermostable and retained full
activity even after 1 hour of incubation at 60
C. The purified enzyme substantially inhibited the growth of R. solani, indicating that this enzyme may be actually involved in the antagonistic process.

The in vitro and in vivo antifungal potential of extracts of three wild medicinal plants, (Acacia nilotica (L.) Delile, Achillea fragrantissima (Forssk.) Sch.Bip. and Calotropis procera (Aiton) W. T. Aiton) was examined against Al-ternaria solani, the causal agent of the early blight of tomato. Aqueous or ethanol extracts of all tested plants reduced the mycelial growth and conidium germination of A. solani in vitro. Ethanol extracts were more effective against the pathogen than the aqueous extracts. Extract of C. procera exhibited more antifungal potential against the pathogen than other plant extracts. Observations by scanning and transmission electron microscopy showed dramatic alterations in the morphology and ultrastructure of A. solani when treated with the ethanol extract of C. procera at a concentration of 20%. Phytochemical screening confirmed the presence of many bioactive constituents in the extracts which were in greater amounts in C. procera than the other two plants. In a plot experiment, both types of extracts from C. procera reduced disease severity. Tomato fruit yield was increased after the treatment with the plant extracts.

White mold, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is the most globally significant disease of Jerusalem Artichoke (JA) causing considerable losses during storage periods. The aim of this study was to evaluate the use of a natural essential oil for control of white mold disease, sprouting suppression and quality maintaining of JA tubers under storage periods. In vitro fungitoxicity was investigated using two natural essential oils; clove and sweet wormwood at concentrations of (2, 3, 4 and 5%). Clove oil at 2% inhibited completely the fungal growth of S. sclerotiorum. Chemical composition of clove essential oil was studied using GLC-MS analysis and resulted in identification of 12 compounds. The major components were eugenol and eugenol acetate (81.6 and 9%, respectively). Along 120 day of storage, treatment of JA tubers with clove oil led to a significant decrease in the disease severity, sprouting percentage and weight loss. On the other hand, this treatment enhanced the dry matter and contents of carbohydrates, protein, inulin and total phenols as well as the activity of peroxidase and polyphenol oxidase enzymes of JA tuber compared with the untreated-infected tubers. Based on the obtained results, use of clove oil and peat moss when storing JA tubers at room temperature can be recommended due to its eco-safety and saving of the cooling energy.

The efficiency of two isolates of Trichoderma harzianum (WKY1 and
WKY5) as bio-control agents against anthracnose disease in
sorghum was investigated. In vitro, T. harzianum WKY1 isolate
showed superiority in terms of inhibition of both mycelial growth
and spore germination of Colletotrichum sublineolum, the causative
agent of sorghum anthracnose, as well as induction of the sorghum
seed germination over T. harzianum WKY5 isolate. The culture
filtrate of the selected isolate (T. harzianum WKY1) was analysed
using GC-MS system to determine their chemical constituents.
Twenty-nine components with varied existence percentages were
identified. Although T. harzianum WKY1 produced the
phytohormone indole-3-acetic acid (IAA) on tryptophan free
medium, a marked dependency on tryptophan for the production
of IAA was noticed. Nutritional components were optimized for
maximizing IAA production using the central composite design. The
optimum levels were 1.06, 29.86 and 2.93 g L−1 from tryptophan,
sucrose and NaNO3, respectively, with a maximum IAA biosynthesis
(138.9 μg mL−1) after five days of incubation. Production of IAA in
the culture filtrate of T. harzianum WKY1 was qualitatively and
quantitatively analysed by LC-MS system using a reference
standard of IAA. Under greenhouse conditions, application of T.
harzianum WKY1 and/or its filtrate reduced greatly the disease
severity as well as improved the plant growth of sorghum. From
the present data, we can recommend the application of T.
harzianum WKY1 as a dual purpose bio-agent for biological control
of anthracnose disease and plant growth promotion.

In vitro antimicrobial activity of methanolic extracts of six medicinal plants (black seed, camphor, cloocynth, clove, ginger and white cedar) were investigated against pathogenic bacteria and fungi at 3, 6 and 9% concentrations. Results obtained in this study showed that the six plant extracts exhibited varied extents of antimicrobial activity against the tested organisms even at low concentration. Among the tested plant extracts, ginger recorded the highest antifungal activity against Alternaria radicina, Fusarium oxysporum, F. solani, Macrophomina phaseolina, Nigrospora oryzae, Phoma destructiva, Rhizoctonia solani and Sclerotium rolfsii at 9% concentration. Whilst, the maximum antibacterial activity was achieved by black seed extract at 9% concentration against the Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus and Streptococcus pneumonia). Chemical compositions of ginger and black seed methanolic extracts were studied using GC-MS analysis and resulted in the identification of 19 and 17 compounds, respectively. Further studies are needed to investigate the antimicrobial potentiality of the active constituents singly or in mixtures with other antibiotics.