Isolation of Soil Streptomycetes From Gaza

1. Isolation of Soil Streptomycetes From Gaza

2. Introduction Streptomyces is the best recognized genus in the family Streptomycetaceae, because of its wide distribution in nature especially in soil. Further, this genus harbours agreat number of the most important producer of antibiotic and other secondary metabolites. Therefore, various attempts were made to isolate these valuable organisms.

3. The aim of this experiment is to isolate streptomycetes common in Gaza soils. Your instructor and TA will keep the recovered streptomycetes under proper conditions for further experiments.

4. PROCEDURE Sampling: - Collect soil samples from cultivated or uncultivated soils from different locations in Gaza. -Take these samples with an auger up to 10 cm depth, after removing approximately 3 cm of the soil surface. - Place them in polyethylene bags, close tightly and store in a refrigerator.

5. Isolation: - Mix the samples thoroughly and sieve them by 2 mm pore size mesh . - Suspend samples of 1g in 100 ml distilled water on a water-bath shaker (140 rpm, 30 min). - Dilute serially up to 10-6 and spread (0.1 ml) over the surface of glycerol nitrate casein agar plates with sterile L-shaped glass rods. - Triplicate plates will be used for total streptomycete count.

6. - Incubate the plates at 27 ºC and determine the number of colonies after 10 days. Dilutions that gave 20-200 colonies will be chosen for the isolation. - Select some colonies then purify them by repeated streaking.

7. Characterisation of the isolates: - Streptomyces colonies are characterised morphologically and physiologically following the directions given for the International Streptomyces project (ISP) - Determine the general morphology on oatmeal agar plates, incubated in the dark at 27 ºC for 21days, by direct light microscopy examination of the surface of cultures.

8. - Preserve these isolates under proper conditions for further experiments.

9. RESULTS

10. In Vitro Control of Bacterial Phytopathogens (Agrobacterium tumefaciens, Pseudomonas,Erwinia and Corynebacterium) by Soil Streptomycetes

11. Introduction Crown gall, caused by Agrobacterium tumefaciens, is a significant worldwide distributed disease in nurseries and fruit orchards . A.tumefaciens isolates from several tumors showed virulence variation .

12. One way to treat this disease is by using biological methods, such as using nonpathogenic strains of Agrobacterium. A common method is by using antimicrobial agents .Streptomyces isolates were also tested for their in vitro inhibitory activity against A. tumefaciens and were the focus of several studies to control crown gall disease

13. PROCEDURE Cultures: - The T.A. will provide you with some Streptomyces cultures that have been isolated and purified previously.

14. Antimicrobial activity: • Perform this is test by plate diffusion method against Agrobacterium tumefaciens. Other bacterial phytopathogens like Pseudomonas, Erwinia, Corynebacterium, and Xanthomonas can also be tested. - These isolates can be provided from other labs which work with phytopathogens.

15. Grow the above test organisms in 250 ml flasks containing 50 ml nutrient broth medium and incubate at 27 ºC with shaking at 100 rpm for overnight. - Inoculate the culture by a sterile cotton swab on the surface of Mueller-Hinton agar plates.

16. - Cut discs (9 mm in diameter) from the oatmeal agar cultures with the Streptomyces isolates grown on for 10 days. - By a sterile cotton swab inoculate the surface of Mueller-Hinton agar with the above test organism.

17. Place the oatmeal agar culture disc on the surface of Mueller-Hinton agar cultures. - Incubate at 27 ºC, and then check the inhibition zones after 24 hrs.

18. RESULTS Table 1. Action of the most active Streptomyces strains on the phytopathogen, A. tumefaciens.

19. Effect of Parasitic Seed Plant (Orobanche spp.) Extracts on Human and Plant Pathogens

21. Introduction Orobanche species are destructive root parasites of many economic crops that cause serious problems to farmers in Europe and Middle East countries . Saadoun and Hameed reported the presence of some bioactive metabolites in the tissue of O. cernua. They showed that the tissue of this parasitic seed plant has a remarkable activity against some pathogenic bacteria. Also, the activity against the crown gall and soft rot phytopathogens; Agrobacterium tumefaciens and Erwinia, respectively, has been explored .

22. In this exercise the antibacterial activity of the plant extract (see different species of Orobanche in the plate below) will be investigated.

23. Procedure Plant materials -The instructor and T.A. will provide you with the parastic seed plant (Orobanche spp after they collect it from several fields in Gaza. - Dry the shoots of mature plant at room temperature then blend them with an electrical blender.

24. Preparation of extracts - Place approximately 100 g of a blended plant parts (shoots) in a 2L beaker and then impregnate with 1 liter of 96% ethanol. - Cover the beaker with a glass dish and leave at room temperature for 72 hrs. - Remove the glass cover and subject the ethanol-impregnated plant to heat radiation from tungsten lamp to aid in ethanol evaporation.

25. Preparation of extracts - Dissolve the final extracted material in the appropriate volume of sterile distilled water to obtain a concentration of 100 mg/ml. - Filter sterilized through 0.45 μm pore size millipore filters .

26. Test organisms -Different human (E. coli, S. aureus) and plant pathogen cultures (A. tumefaciense, Erwinia) - grown in nutrient broth for overnight at 27 °C will be provided to you by the T.A.

27. Antimicrobial testing Antimicrobial activity of the ethanolic extract of the whole plant will be determined by the (hole-plate diffusion method) and the (dilution method)

28. Hole-plate diffusion method - Get tubes containing 20 ml of nutrient agar then pour into Petri dishes. - Inoculate the plates with the appropriate test organism using a sterile swab. - Remove cores of 6 mm diameter from the agar by a sterile glass test tube. - Fill up the holes with 50 μl of the water-soluble ethanolic extract. - Use a plate as control with holes containing sterile D. H2O.

29. Dilution method - Dilute the stock plant extract (100 mg/ml) under aseptic conditions in sterile distilled water to obtain different concentrations. - Incubate the agar plates with the bacterial pathogens for overnight at 27°C for 12 hr. - Record the results by measuring the zones of growth inhibition surrounding the holes.

30. In Vitro Control of Food Associated Fungi by Soil Streptomycetes Isolated From Gaza

31. Introduction Beginning with the discovery of actinomycin in 1940, the order Actinomycetales has produced many commercially important bioactive compounds. It has been estimated that approximately two-thirds of naturally occurring antibiotics have been isolated from actinomycetes .Of these antibiotics, the majority were isolated from the genus Streptomyces .

32. The traditional approach to isolation has been the use of terrestrial soils, which provide a rich source of these microorganisms with screening programs were and still being initiated in various countries for isolation such antibiotic-producing Streptomyces strains mainly from soil samples.

33. The antagonistic activity of these organisms and other microorganisms against some phytopathogens has been suggested that they could represent useful agents for the control of plant diseases . identified several Streptomyces isolates antagonistic to phytopathogenic fungi. They showed that the majority of them were active against Fusarium solani and Botrytis cinerea. Another study on the activity of soil actinomycetes in Turkey against different fungi revealed the inhibition of Sclerotinia sclerotiorum, Rhizoctonia solani and Alternaria alternata by 90%, 17% and 14% of the isolates, respectively (Grossmann and Gulay, 1986).

34. In this experiment, the antifungal activity of local isolates of actinomycetes that have been recovered previously against several food associated fungi and molds will be conducted.

35. PROCEDURE Sampling and Isolation: Collection of soil samples and isolation of streptomycetes will be done as described previously.

36. Antifungal Activity: -This will be tested by the Bauer-Kirby method against several food associated fungi and molds such as: Torula spp., Aspergillus niger, Trichoderma viride, Trichoderma harasmii, Aspergillus flavus and Penicillium spp. - Grow isolates of actinomycetes on oatmeal agar for 10 days.

37. - Add 0.1 ml of semisolid nutrient agar (0.2%) containing cells of the test fungi and immediately spread over the surface agar by a sterile L-shape glass rod. - Transfer 3 discs (5 mm in diameter) of oatmeal agar cultures on nutrient agar. - Incubate the plates at 28 °C, then visually detect the inhibition zones after 48 hrs.