Biocontrol

Laboratory Manual

Prof. Ismail Saadoun

ISLAMIC UNIVERSITY OF GAZA DEPARTMENT OF BIOTECHNOLOGY

BIOCONTROL LABORATORY MANUAL

Prof. Dr. Ismail Saadoun Department of Applied Biological Sciences, Jordan University of Prof. Dr. Ismail Saadoun Science and Technology, P.O. Box 3030, Irbid- 22110, Jordan. Department of Applied Biological Sciences, JordanFax: +962-2-7201071. E-mail Phone: +962-2-7201000-Ext. 23460; University of Science and Technology, P.O. Box 3030, Irbid- 22110, Jordan. Phone: +962-2-7201000-Ext. 23460; Fax: +962-2address: isaadoun@just.edu.jo 7201071. E-mail address: isaadoun@just.edu.jo

Copyright 2008. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission of the author.

Prof. Dr. Ismail Saadoun Department of Applied Biological Sciences, Jordan University of Science and Technology, P.O. Box 3030, Irbid- 22110, Jordan. Phone: +962-2-7201000-Ext. 23460; Fax: +962-27201071. E-mail address: isaadoun@just.edu.jo

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PREFACE This manual has been designed for an undergraduate level laboratory sessions in biocontrol technology. The manual is divided into experiments that belong to a particular category. An experiment will be carried out each week and some times may be continued in the week after. It should be noted that the first exercise in this manual require a repetition of basic techniques, and most results call for observations and tabulations. Prior to each lab session, careful orders and preparations are required which can be found in the procedure or the appendix sections. Each experiment contains the following basic sections: Introduction Background and principles behind the assays performed. Procedure A detailed description of the materials, equipment needed to conduct the experiment and the method to be followed. Detailed listing of laboratory media, cultures, and special chemicals are also included. Results The experimental analysis data are lay out as tables and figures. Reports of the field visits are also included as instructed. References and further readings A listing of useful articles and books is also provided. Appendix Media, buffers and solutions used in each experiment are provided. Their composition and companies which supply them are also included.

Prof. Dr. Ismail Saadoun Dept. of Biotechnology and Genetic Engineering Dept. of Applied Biological Sciences Jordan University of Science and Technology Irbid-22110, JORDAN Tel: (Work) 962-2-7201000, Ext. 23460 Fax: 962-2-7201071 E-mail: isaadoun@just.edu.jo; isaadoun_1962@yahoo.com

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ISLAMIC UNIVERSITY OF GAZA Dept. of Biotechnology Biocontrol Lab

Contents Introduction and Orientation Review of Microbial Techniques Isolation of Soil Streptomycetes From Gaza In Vitro Control of Bacterial Phytopathogens (Agrobacterium tumefaciens, Pseudomonas, Erwinia and Corynebacterium) by Soil Streptomycetes Effect of Parasitic Seed Plant (Orobanche spp.) Extracts on Human and Plant Pathogens In Vitro Control of Food Associated Fungi by Soil Streptomycetes Isolated From Gaza Toxicity Preparations of Bacillus thuringiensis Towards Drosophila melanogaster and Culex sp. Phytotoxin-Producing Soil Streptomycetes and their Role to Control Weeds In Vitro Control of Fungal Phytopathogens by Chitinase-Producing Streptomycetes Continue Biological Control of Orobanche by Fungi Isolated from Diseased Specimens in Gaza Appendices

Pages 7-13 14-15 16-18 19-22 23-25 26-29 30-33 34-37 38-41 42

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INTRODUCTION
It has been long recognized by scientists dealing with living organisms the significance of their indigenous genetic resources. In one aspect of application relevant to that aspect is searching for novel and beneficial organisms of some significance to the sustainability of human life and their environment. Plant pathogens and their management were of great concern to human societies, since there were burst pesticides applications. Hence, biological control of plant disease gained wider attention and raised good hope and anticipation into a safe and environment friendly alternative. In the modern and future management practices of plant diseases, there is an urgent need for novel plant disease management different from the currently used agrochemicals regarding their mode of action and strategy of their application. Problems of deleterious effects on the environment of chemical fertilizers, pesticides, antibiotics in addition to the appearance of organisms with resistance resulting from high application rates of these agrochemicals (Cohen and Coffey, 1986; Fruh et al., 1996 and Knight et al., 1997) are the most significant reasons for creation of such new strategies. Chemical and physical methods were established to solve these problems. However, these problems cannot be solved completely without using biological methods and technologies in coordination with agricultural production (Regunold et al., 1990; Parr and Hornick, 1992a). Biological control became highly desired practice since there was growing concern about the use of chemical pesticides and their long-term adverse environmental effects. Therefore, searching the environment for novel and beneficial organism(s) significant as biocontrol agent(s) is very important to the sustainability of human life and their environment (Higa, 1994 and Knight, et al 1997). It is very legitimate to lunch intensive efforts in searching for promising biological control agent(s) against several disease causative agents. References Cohen, Y. and Coffey, M.D. 1986. Systemic fungicides and the control of oomycetes. Ann. Rev. Phytopath., 24: 311-338. Fruh, T., P. Chemla, J. Ehrler and Farooq, S. 1996. Natural products as pesticides: two examples of strereoselective synthesis. Pesticide Science 46: 37- 47.
Higa, T. 1994. Effective microorganisms: A new dimension for Nature Farming. P. 20-22. In J.F. Parr, S.B. Hornick, and C.E. Whitman (ed.) Proceedings of the First International Conference on Kyusei Nature Farming. USDA, Washington, D.C., USA.

Knight, S.C., V.M. Anthony, A.M. Brady, A.J. Greenland, S.P. Heaney, D.C. Murray, K.A. Powell, M.A. Schulz, C.A. Sinks, P.A. Worthington and D. Youle, 1997. Rationale and perspectives on the development of fungicides. Ann. Rev. Phytopathology 35: 349-372. Parr, J.F., and Hornick, S.B. 1992a. Agricultural use of organic amendments: A historical perspective. Amer. J. Alternative Agric., 7: 181-189.

ISLAMIC UNIVERSITY OF GAZA Dept. of Biotechnology Biocontrol Lab Lab Schedule The aim of this lab course is to provide an understanding of the application of beneficial organisms as promising biocontrol agent(s) against several disease causative agents. Week 1 2 3 4 Exercise Introduction and Orientation Review of Microbial Techniques Isolation of Soil Streptomycetes From Gaza In Vitro Control of Bacterial Phytopathogens (Agrobacterium tumefaciens, Pseudomonas, Erwinia and Corynebacterium) by Soil Streptomycetes Effect of Parasitic Seed Plant (Orobanche spp.) Extracts on Human and Plant Pathogens In Vitro Control of Food Associated Fungi by Soil Streptomycetes Isolated From Gaza Mid Term Exam Toxicity Preparations of Bacillus thuringiensis Towards Drosophila melanogaster and Culex sp. Continue Phytotoxin-Producing Soil Streptomycetes and their Role to Control Weeds Continue In Vitro Control of Fungal Phytopathogens by Chitinase-Producing Streptomycetes Continue Biological Control of Orobanche by Fungi Isolated from Diseased Specimens in Gaza Continue Final Exam Pages 7-13 14-15 16-18 19-22 23-25 26-29 30-33 34-37 38-41 -

5 6 7 8 9 10 11 12 13 14 15 16

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Review of Microbial Techniques

CULTURAL TRANSFER The procedure for transferring a microbial sample from a broth or solid medium or from a food sample is basically the same. The sample is collected with a sterile utensil and transferred aseptically to a sterile vessel. Two implements commonly used for collecting and transferring inoculum are the cotton swab and the platinum needle or loop. The swab is used in instances where its soft nature and its fibrous qualities are desired such as in taking a throat mucus sample or in sampling the skin of an apple. A platinum needle or loop is used in those instances where a more concentrated microbial sample is available, such as in a contaminated water sample. A typical culture transfer proceeds as follows: 1. In one hand hold the wire loop as you would a pencil; 2. Heat the wire loop until red; 3. Allow it to cool for a moment (this prevents burning or boiling of the medium when it contacts the loop); 4. Holding the culture container in the other hand, remove the cover by grasping it between the small finger and the palm of the loop holding hand; 5. Flame the container by passing the vessel top through the flame slowly (2 to 3 sec) in order to sterilize the rim; 6. Insert the wire loop and take the sample; 7. Reflame the container top and replace the lid; 8. Open and flame the top of the receiving vessel as you did with the sample vessel; 9. Inoculate the sample into the vessel; 10.Reflame and cap the receiving vessel-; 11.Flame the loop to resterilize it. All vessels used need to be clearly labeled for identification. The date and name of the person using the vessel should be included along with the other pertinent information, (e.g, medium type, control, concentration, etc.). All swabs, medium tubes, culture plates, and other items contaminated with microbes should be autoclaved before washing or disposal. PLATING Isolation of individual microbial types may be obtained by dilution methods. The dilution, a reduction of microbial cell concentration, may be achieved by spreading a small amount of culture across a wide medium surface. This technique is called streaking. Bacterial cell dilution may also be carried out using a series dilution scheme, a small amount of initially concentrated culture is introduced into a volume of medium or physiological saline and then homogeneously dispersed into that volume. Physiological saline (0.85% NaCl) is used to protect cells from sudden osmotic shock thus preventing cell rupture, a sample of the new volume may be redispersed in yet another dilution volume to achieve further cell number reduction, by transferring known volumes of sample culture to known volumes of dilution media, one can calculate the reduction in cell concentration achieved, for example, if one introduced 1 ml of a sample into 9 ml of medium, one would have reduced the initial concentration by a factor of ten. Please refer to a dilution scheme for practice in making dilutions. In dilution schemes one must maintain aseptic technique. All transferring items must be microbe free. All new media or dilution media must be sterile. 1

A pipet is used to transfer volumes of liquid. The pipet should be clean and sterile. It should be equipped with a pipet bulb or propipet so that oral contact and the potential danger of inhaling the microbial sample is avoided. Always place pipets in germicidal washing solution immediately after use. Dilution of cultures made by volume dilution may be plated out in Petri dishes and then incubated to allow the microbes time to grow. A typical plating procedure would be as follows: 1. Pipette 1 or 0.1 ml of a known dilution of a sample into the bottom section (smaller plate) of a sterile Petri dish; 2. Within 20 min add 12-15 ml of warm (46-48c) fluid medium to this Petri dish;

3. Cover the dish; 4. Swirl it gently to disperse the sample throughout the medium, (a figure eight pattern holding the dish flat on the table is the recommended swirl pattern: care should be taken to prevent splashing of the medium onto the lid of the dish); 5. 6. Allow the plate to stand, cool, and solidify; Invert the Petri dish (medium surface pointing down) and incubate in this position.

Petri dishes are incubated upside down to prevent water from condensation from standing on the medium surface during incubation. Pools of surface water would result in the loss of individual surface colonies since bacterial cells forming in the colonies could use the water pools as vehicles to reach the medium. After a period of incubation microbial growth may be observed. If sufficient dilution has been achieved, individual colonies of microbes may be clearly seen. It is assumed that colonies arises from single microbial cells, thus an individual colony represents only one microbial type. This assumes that the microbes in the original culture were not clustered and that a true homogenous dispersion was achieved. (Shaking the solution with glass beads helps to break up cells clusters.) by picking out individual colonies and transferring them to a new sterile medium, microbial isolation can be achieved. Isolation is also achieved using the streaking technique. This involves the aseptic transfer of a small quantity of culture to a sterile Petri dish containing medium. The most common implement for streaking is the wire loop. Streaks should be performed by initially introducing an inoculum of the culture onto a small area of the medium plate surface. This is called the well. After inoculating the well, the transfer loop is reflamed, allowed to cool, and then touched on a remote corner of the plate to remove any heat remaining. Beginning with the sterile loop in the well a streak is made across a corner of the medium surface. (This spreads a bit of the culture out over the mediumdispersing or diluting the culture.) the loop is reflamed, cooled, and the streaking continued until all the available medium surface is utilized. On a typical plate 3-5 streaks can be made. 2

Remember: the streaking loop must be reflamed after each streak. Both processes, streaking and volume dilution, reduce and disperse the cell concentration onto the medium. Upon incubation both dilution procedures should produce isolated colonies of a single strain. The dilution technique has added use, in that upon sufficient dilution, all the colonies from the dilution can be seen as separate individual spots when plated. By counting these spots and multiplying that number by the dilution factor for the plate, one can arrive at an estimate of the number of organisms in the original culture solution. As a rule of thumb only those incubated plates which have between 30 and 300 colonies are used to determine organism concentration in the original culture. Thirty is taken as the lower limit since statistically this many individual colonies are required for accuracy in calculation. Three hundred is taken to be the upper limit] because difficulty is encountered in counting more than this number of colonies accurately. Motility Testing Many microbes are motile. Motility can be checked by inoculating a culture sample into a semisolid medium. This is done with an inoculating needle which is stabbed straight down and pulled straight out of the tube. Upon incubation, a non-motile colony will produce a single line of growth along the needle jab line, while a motile colony will give a wider band of growth. The hanging drop mount is used to check motility. It is prepared by placing a ring of lubricating grease around the rim of the recession in the hanging drop slide. A drop of culture medium or a water suspension of a culture is then placed on a coverslip. The coverslip is inverted so that the drop is clinging to the lower side, and the coverslip is laid to rest on the slidebeing supported by the ring of grease. This mount has the advantages that motility of live, motile microbes can be observed.

Staining A method of biochemical differentiation is staining. Staining operates on the principal that different types of microbes have different chemical constituents making up their cellular components. For example, the Gram stain operates on the principle that some cells retain a crystal violet-iodine complex after leaching with an alcohol solvent, these cells generally have 3

complex membranes which result in retention of the blue complex and are thus called gram positive. Other microbials with less complex membranes are not affected by the mordant, iodine. The dye in these cells is washed out and replaced by a safranin counter-stain (red). These cells are said to be gram negative. There are many other types of cellular dyes. There are basic dyes specific for nuclear material, other cellular elements, and spores. Objectives: This exercise will review the technical skills required to successfully function in an analytical microbiology laboratory. This exercise will enable you to: 1. Transfer cultures, streak plates and inoculate slants; 2. Carry out dilution schemes to obtain microbial counts; 3. Determine microbial motility by two methods; 4. Carry out gram and spore stains; Materials: Broth and slant of: Escherichia coli, Bacillus subtilis, Staphylococcus aureus Broth mix of: Staphylococcus aureus and Escherichia coli Tryptone glucose extract agar (TGEA) 1 ml pipettes Petri plates 99 ml dilution blanks Gram and spore stains Semi-solid agar tubes Procedure: A. Microbial Isolation 1. Flask of agar medium are kept in a 48c oven to maintain their fluidity, label _____ plates of TGEA and pour 115 ml of the medium into these plates and allow them to cool and solidify for streaking and spread plating. 2. The instructors have prepared 4 different types of broth cultures. You will dilute out each of these 4 different cultures, 2 by spread plating techniques and 2 by pour plating methods. Your instructor will explain these procedures, as well as designate which of the cultures are to be spread or pour plated and to what dilution. Dilution schemes should be worked out first on paper to avoid confusion. (Note examples of dilution schemes are given at the end of this exercise) 3. If the TGEA plates prepared in step 1 have solidified, proceed to streaking so that isolated colonies may be observed. Streak out samples from all 4 broth cultures. Which of the cultures are to be spread or pour plated and to what dilution? Dilution schemes should be worked out first on paper to avoid confusion. (Note examples of dilution schemes are given at the end of this exercise) 4

4. When all plates have cooled and solidified, invert and incubate at 37 c for 48 hr. Count the plates from the dilution(s) yielding between 25 and 250 colonies. Calculate the bacterial cell concentration in the original culture. Observe the streak plates. Exchange class data. B. Microbial Motility 1. Obtain 3 tubes of semi-solid agar and inoculate each tube with one of the 3 culture types using an inoculating needle. Omit the mixed culture sure to label each tube, incubate tubes at 37c for 48 hr. C. Staining Use the broth cultures provided sources for microorganisms to stain and the plates streaked for isolation as

1. Make gram stains of the E. coli, S. aureus, B. subtilis and the mixed culture according to the procedure described by your instructor. Observe these stains under the microscope using the oil immersion magnification. 2. Make a spore stain of the cultures assigned to you. Observe it under the microscope using the oil immersion objective. Can you observe distinct spore bodies? If so, are they terminal, subterminal, or central? Are cells swollen at the spore location?

Dilution Calculations Dilution factor = initial dilution x subsequent dilutions x amount plated Count per ml (or g) = reciprocal of dilution factor x colonies counted Example: A sample was diluted initially 1:100 (1 ml of in 99 ml sterile diluent). A subsequent 1:10 dilution (1 ml of the initial dilution into 9 ml sterile water) was prepared. Finally, 0.2 ml of the final dilution plated and 64 colonies were counted on the plate. Initial dilution x subsequent dilutions x amount plated = dilution factor

1/100 x 1/10x0.2 =0.0002 or 10- 2 x 1 0 - 1 x 2 x l 0 - 1 = 2 x 1 0 - 4 reciprocal of dilution factor x colonies counted = count per ml 5000 (or 5 x 103) x 64 = 320,000 (or 3.2 x 105)

A. Plate count results should be reported to two significant figures only. Example: If the dilution factor used was 106 and 212 colonies were counted, the count/ml would be calculated thus, Reciprocal of dilution factor x colonies counted count/ml = count/ml 106 x 212 = 2 . 1 2 x 1 0 8 Then, the answer should be rounded off to 2.1 x 108 colony forming units (CFU) per ml. B. Only those plates with between 25 and 250 colonies should be used to calculate plate counts. Counting Colonies on Plates and Recording Results Refer to the prepared handout for details. References: American Public Health Association. 1985. Standard methods for the examination of dairy products. 15th edition (APHA: N.Y.). Chapter 5, standard plate count method.

Isolation of Soil Streptomycetes From Gaza

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 a great number of the most important producer of antibiotic and other secondary metabolites. Therefore, various attempts were made to isolate these valuable organisms. 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. . 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. Isolation: -Mix the samples thoroughly and sieve them by 2 mm pore size mesh (Retsch, Germany). -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 (Kuster & Williams, 1964) plates with sterile L-shaped glass rods. Triplicate plates will be used for total streptomycete count. -Incubate the plates at 27 C 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. Characterisation of the isolates: Streptomyces colonies are characterised morphologically and physiologically following the directions given for the International Streptomyces project (ISP) (Shirling & Gottlieb, 1966). -Determine the general morphology on oatmeal agar plates, incubated in the dark at 27 C for 21days, by direct light microscopy examination of the surface of crosshateched cultures. -Preserve these isolates under proper conditions for further experiments.

RESULTS Table 1. Distribution of the Streptomyces isolates in different sites. Colour Series Site White Grey Yellow Red Blue Green

Violet

Total

Total

Fig. 1. Diversity of Streptomyces colonies on Starch Casein Nitrate Agar (SCNA) medium. REFERENCES Kuster, E. & S.T. Williams (1964). Selection of media for isolation of streptomycetes, Nature, 202: 928-929. Shirling, E.B. & D. Gottlieb (1966). Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol., 16: 313-340. Further Readings
1-Saadoun, I. and R. Gharaibeh. 2002. The Streptomyces flora of Jordan and its potential as a source of antibiotics active against antibiotic-resistant Gram-negative bacteria. World J. Microbiol. Biotech. 18: 465-470. 2-Saadoun, I., L. Wahiby, Q. Ababneh, Z. Jaradat, M. Massadeh and F. Al-Momani. 2008. Recovery of soil streptomycetes from arid habitats in Jordan and their potential to inhibit multi-drug resistant Pseudomonas aeruginosa pathogens. World J. Microbiol. Biotech. 24: 157-162

In Vitro Control of Bacterial Phytopathogens (Agrobacterium tumefaciens, Pseudomonas, Erwinia and Corynebacterium) by Soil Streptomycetes Introduction Crown gall, caused by Agrobacterium tumefaciens, is a significant worldwide distributed disease in nurseries, vineyards and fruit orchards (2, 10, 12, 15, 17, 19). Interestingly, A. tumefaciens isolates from several tumors showed virulence variation (4). One way to treat this disease is by using biological methods, such as using nonpathogenic strains of Agrobacterium (8, 9, 13). A common method is by using antimicrobial agents (5, 6, 7, 11, 13, 14, 17, 18, 20). 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 (1, 3, 16). PROCEDURE Cultures: -The T.A. will provide you with some Streptomyces cultures that have been isolated and purified previously. 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. -Grow the above test organisms in 250 ml flasks containing 50 ml nutrient broth medium and incubate at 27 C with shaking at 100 rpm for overnight. -Inoculate the culture by a sterile cotton swab on the surface of Mueller-Hinton agar plates. -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. -Place the oatmeal agar culture disc on the surface of Mueller-Hinton agar cultures. -Incubate at 27 C, and then check the inhibition zones after 24 hrs.

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RESULTS Table 1. Action of the most active Streptomyces strains on the phytopathogen, A. tumefaciens.
Isolate No.

Antibiosis A. tumefaciens Erwinia Pseudomonas Corynebacterium

1 2 3 4 5
Record the results in Table 2 as + or -. You can measure also the diameter of the inhibition zone and compare between the isolates.

References [1] Abussaud, M. J., Saadoun, I. M., 1988. Isolation, characterization and taxonomy of Streptomyces sp. isolated from Jordanian soils and antagonistic to Agrobacterium tumefaciens. Egypt. J. Microbiol. 23, 597-609. [2] Al-Momani, F. S., 1986. Isolation, idiotyping and characterization of the genus Agrobacterium from grape nurseries of Jordan Valley and from other infected plants from different location in Jordan. M.Sc. Thesis, Yarmouk University, Jordan. [3] Al-Momani, F., Saadoun, I., Makawi, H. I., 1999. Streptomyces species from Jordan soils with in vitro inhibitory activity against Agrobacterium tumefaciens AB 136 pti 854. Afr. Plant Prot. 5, 129-130. [4] Al-Momani, F., AL-Bashir, S., Saadoun, I., 2006. Distribution of Agrobacterium tumefaciens biovars in Jordan and variation of virulence. Plant Pathol. J. 22, 318-322. [5] Cooksey, D.A., Moore, L.W., 1980. Biological control of crown gall with fungal and bacterial antagonists. Phytopathology 70, 506-509. [6] Herlache, T.C., Triplett, E.W. 2002. Expression of a crown gall biological control phenotype in an avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance genes. BMC Biotechnol. 2, 2-9. [7] Htay, K., Ker, A., 1974. Biological control of crown gall: seed and root inoculation. J. Appl. Bacteriol. 37, 525-530. [8] Kawaguchi, A., Inoue K., Nasu, H. 2005. Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine. J. Gen. Plant Pathol. 71, 422-430. [9] Kawaguchi, A., Inoue K., Nasu, H. 2007. Biological control of grapevine crown gall by non-pathogenic Agrobacterium vitis strain VAR03-1. J. Gen. Plant Pathol. 73, 133-138. [10] Keane, P. J., Kerr, A., New, P. B., 1970. Crown gall of stone fruit. II Identification and nomenclature of Agrobacterium isolates. Aust. J. Biol. Sci. 23, 588-598. [11] Khmel, I.A., Sorokina, T.A., Lemanova, N.B., Lipasova, V.A., Metlitski, O.Z., Burdeinaya, T.V., Chernin, L.S. 1998. Biological control of crown gall in grapevine and raspberry by two Pseudomonas spp. With a wide spectrum of antagonistic activity. Biocont. Sci. Technol. 8, 45-57.

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[12] Ma, D., Yanofsky, M. F., Gordon, M. P., Nester, E.W., 1987. Characterization of Agrobacterium tumefaciens strain isolated from grape vine tumor in China. Appl. Environ. Microbiol. 53, 13338-1343. [13] Moore, L. W., Warren, G., 1979. Agrobacterium radiobacter strain 84 and biological control of crown gall. Ann. Rev. Phytopathol. 17, 163-179. [14] New, P. B. and Kerr, A., 1972. Biological control of crown gall field measurements and glasshouse experiments. J. Appl. Bacteriol. 35, 279-287. [15] Panagopoulas, C. G. and Psallidas P. G., 1973. Characteristic of Greek isolates of Agrobacterium tunefacieus. Appl. Bacteriol. 36, 233-240. [16] Saadoun, I. and AL-Momani, F., 1997. Steptomycetes from Jordan soils active against Agrobacterium tumefaciens. Actinomycetes 8, 2-36. [17] Schroth, M. N. and Moller, W. J., 1976. Crown gall controlled in glass house with a non pathogenic bacterium. Plant Dis. Rep. 60, 275-278. [18] Schroth, M. N., Weinhold, A. R., McCain, A.H., Hilderbrand, D. C. and Ross, N. 1971. Biology and control of Agrobacterium tumefaciens. Hilgradia 40, 537-552. [19] Sule, S., 1978. Biotypes of Agrobacterium tumefaciens in Hungary. J. Appl. Bacteriol. 44, 207-213. [20] Webster, J., Dos Santos, M. and Thomson, J.A., 1986. Agrocin producing Agrobacterium tumefaciens strain active against grapevine isolates. Appl. Environ. Microbiol. 52, 217219.

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Effect of Parasitic Seed Plant (Orobanche spp.) Extracts on Human and Plant Pathogens Introduction Orobanche species are destructive root parasites of many economic crops that cause serious problems to farmers in Europe and Middle East countries (1, 2). Saadoun and Hameed (3) 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 (4). In this exercise the antibacterial activity of the plant extract (see different species of Orobanche in the plate below) will be investigated.

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Procedure Plant materials -The instructor and T.A. will provide you with the parastic seed plant (Orobanche spp.) (Fig. 1) 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. 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. -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 m pore size millipore filters (Millipore corp., Bedford, MA). 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. 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 (5). 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 l of the water-soluble ethanolic extract. -Use a plate as control with holes containing sterile D. H2O. Dilution method Minimum inhibitory concentration (MIC) will be determined by the dilution method (7, 8). 14

-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 27C for 12 hr. -Record the results by measuring the zones of growth inhibition surrounding the holes. Results Table 1. Activity of Orobanche spp. extract by hole diffusion method. Extract Concentration (g/ml) Pathogen 100000 50000 25000 12500

The above results can be compared to the action of standard test antibiotics Table 2. Inhibitory action of 100 mg/ml concentration of the different Orobanche species extract on human and plant pathogens Pathogen Diameter of Inhibition Zone (mm)b Sp. 1 Sp. 2 Sp. 3

The above results can be compared to the action of standard test antibiotics Table 3. Minimum concentration of the different Orobanche species extract inhibiting human and plant pathogens. Pathogen Diameter of Inhibition Zone(mm)b MIC of the Extracts (mg/ml) Sp. 2 Sp. 3

Sp. 1

Sp. 4

The above results can be compared to the action of standard test antibiotics

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References 1. Abu-Irmaileh B. The Orobanche problem and management in Jordan. 1993. In Biology and management of Orobanche. Proceedings of the third international workshop on Orobanche and related striga research. Amesterdam. The Netherlands, p. 659-62, 2. Pieterse A. 1979. The broomrapes (Orobanchaceae)- a review. Abstracts on Tropical Agriculture 5: 9-35. 3. Saadoun I, Hameed K. 1999. Antibacterial activity of Orobanche cernua extract. J Basic Microb 39: 377-80. 4. Saadoun, I., K. Hameed, F. Al-Momani and Q. Ababneh. 2008. Effect of three-Orobanche spp. extracts on some local phytopathogens, Agrobacterium and Erwinia. Turkish Journal of Biology. 32: 113-117. 5. Hugo W, Russel A. Pharmaceutical Microbiology. Blackwell Scientific Publications, Oxford, London, Edinburg and Melbourne, p. 165, 1977. 6. Bauer A, Kirby W, Sherris J et al. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clinic. Pathol. 45: 493-96, 1966. 7. Baker F, Breach M. Medical Microbiological Techniques. Butterworths, London, Boston, Sydney, Wellington, Durban and Toronto, p. 496, 1980.

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

Introduction Beginning with the discovery of actinomycin (Lechevalier, 1982) 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 (Takizawa et al., 1993). Of these antibiotics, the majority were isolated from the genus Streptomyces (Betina, 1983; Goodfellow and ODonnell, 1989). The traditional approach to isolation has been the use of terrestrial soils, which provide a rich source of these microorganisms (Lechevalier, 1982) with screening programs were and still being initiated in various countries for isolation such antibiotic-producing Streptomyces strains mainly from soil samples. 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 (Wood and Tviet, 1955; Turhan, 1981; Lechevalier, 1982; Grossmann et al. 1986; Tulemisova and Nikitina, 1989). Tulemisova and Nikitina (1989) 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). 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. PROCEDURE Sampling and Isolation: Collection of soil samples and isolation of streptomycetes will be done as described previously. Antifungal Activity: -This will be tested by the Bauer-Kirby method (Bauer et al . 1966) 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. -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.

17

Results Table 1. Antifungal activity of the Streptomyces isolates.

Color Series
Gray No. of isolates No. of active isolates Torula spp. Aspergillus niger Aspergillus niger Trichoderma viride Trichoderma harasmii Aspergillus flavus Penicillium spp.1 White Yellow Green Red Blue Variable Total

Table 2. Action of the most active Streptomyces strains on the food associated fungi.
Isolate No.

Antibiosis Fungus 1 Fungus 2 Fungus 3 Fungus 4

1 2 3 4 5
Record the results in Table 2 as + or -. You can measure also the diameter of the inhibition zone and compare between the isolates.

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References Bauer, A.W., Kirby, W.M., Sherris, J.C., and Turk, M. (1966) Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clinic. Path. 45. 493. Betina, V. (1983) The chemistry and biology of antibiotics. Elsevier Scientific Publication. Colited., New York (USA). Goodfellow, M. and . Odonnell, A.G. (1989). Search and discovery of industrially significant actinomycetes. In Microbial Products: New approaches. Ed., S. Baumberg, I. Hunter and M. Rhods, Cambridge University Press, pp. 343-383. Grossmann, F. and Gulay, T. (1986) Investigation of a great number of actinomycetes isolated on their antagonistic effect against soil-born fungal plant pathogens by an improved method. Phytopathology 116. 238. Lechevalier, H. (1982). The development of applied microbiology at Rutgers. Rutgers. The State University of New Jersey, pp. 1-82. Saadoun, I., and Al-Momani, F. (1997) Steptomycetes from Jordan soils active against Agrobacterium tumefaciens. Actinomycetes 8. 29. Takizawa, M., R.R. Colwell, and Hill, R.T. (1993). Isolation and diversity of actinomycetes in the Chesapeake Bay. Appl. Environ. Microbiol. 59. 997. Tulemisova, E., and Nikitina, T. (1989) Search for actinomycetes antagonists of fungi causing sugar beet root rot. Acta Biotechnology 9. 389. Turhan, G. (1981) A new race of Streptomyces ochraceiscleraticus in the biological control of soil-born plant pathogens: 2. In vivo studies on the possibilities of using cl2-9 isolate against some important diseases. Zeitschlift fur Pflanzenkrankheiten und Pflanzenschutz 88. 422. Wood, R.K.S., and Tviet, M. (1955) Control of plant disease by use of antagonistic organisms. Botanical Review 21. 441. Further Readings
1-Saadoun, I. and F. Al- Momani. 1997a. Streptomycetes from Jordan soils active against Agrobacterium tumefaciens. Actinomycetes 8:29-36. 2-Saadoun, I., F. Al-Momani, H. Malkawi and M.J. Mohammad. 1999 Isolation, identification and analysis of antibacterial activity of soil streptomycetes isolates from North Jordan. Microbios 100: 41-46. 3-Saadoun, I., K. Hameed, F. Al-Momani, H. Malkawi, M. Meqdam and M.J. Mohammad. 2000. Characterization and analysis of antifungal activity of soil streptomycetes isolated from North Jordan. Egyptian Journal of Microbiology 35: 463-471. 4-Aghighi, S., G.H. Shahidi Bonjar and I. Saadoun. 2004. First report of antifungal properties of a new strain of Streptomyces plicatus (strain 101) against four Iranian phytopathogenic Verticillium dahliae, a new horizon in biocontrol agents. Biotechnology. 3(1): 90-97. 5-Aghighi, S., G.H. Shahidi Bonjar, R. Rawashdeh, S. Bataineh and I. Saadoun. 2004. First report of antifungal spectra of activity of Iranian actinomycetes strains against Alternaria solani, Alternaria alternate, Fusarium solani, Phytophthora megasperma, Verticillium dahliae and Saccharomyces cervisiae. Asian J. Plant Sci. 3(4): 463-471. 6- Shahrokhi, S., G.H. Shahidi Bonjar and I. Saadoun. 2005. Biological control of potato isolate of Rhizoctonia solani by Streptomyces olivaceus strain 115. Biotechnology 4 (2): 132-138, 2005. 7-Shahidi Bonjar, G.H., S. Zamanian, S. Aghighi, P. Rashid Farrokhi, M.J. Mahdavi and I. Saadoun. 2006. Antibacterial activity of Iranian Streptomyces coralus strain 63 against Ralstonia solanacearum. J. Biological Sci. 6(1): 127-129. 8-Tahtamouni, ME. W., K.M. Hameed and I. Saadoun. 2006. Biological control of Sclerotinia sclerotiorum using indigenous chitinolytic actinomyctes in Jordan. Plant Pathology Journal 22(2): 107-114. 9-Saadoun, I. and F. Al-Momani. 2008. Susceptibility of local Agrobacterium tumefaciens strains to streptomycetes isolates from Jordan soils. Journal of Basic Microbiology 48 (3): 213-216.

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Toxicity Preparations of Bacillus thuringiensis Towards Drosophila melanogaster and Culex sp.
INTRODUCTION The preference of using biological pesticides over chemical ones has been widely accepted in different parts of the world in crop and forest protection and in insect vector control. The greatest successes in microbial pesticides have come from use of commercial preparations of Bacillus thuringiensis (Bt) that has been shown to be the most successful biological pest control product worldwide with 90-95% of the sales of microbial pesticides are of this bacterial agent (Spear 1987; Carlton, 1988). The popularity of such products are due to high insect toxicity, environmental safety and lack of toxicity to vertebrates. Upon sporulation, this bacterium produces insecticidal proteinaceous crystals that exhibite a wide range of toxicity against different insect orders (Lu et al. 1994; Vaeck et al. 1988). Identified strains of B. thuringiensis from soil samples, plant surfaces, dead insects, and stored grains showed a wide range of specificity against different insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Phthiraptera or Mallophaga, and Acari) and other invertebrates (Nemathelminthes, Platyhelminthes, and Sarcomastigorphora) (Feitelson 1993 ; Schnepf et al. 1998). In this experiment the toxic potential of different B. thuringiensis strains on insects as the fruit fly will be assessed. PROCEDURE Growth of isolates for bioassay: -The instructor will provide you with different reference strains of B. thuringiensis subspecies. -These bacteria can be supplied by. Dr. Marguerite-M. Lecadet (Pasteur institute, Paris, France.). The bacteria are: B. thuringiensis; aizawai, darmstadiensis, israelensis, kenyae, kurstaki, kurstaki HD1, morrisoni; and tolowrthi. -Transfer those producing parasporal bodies to 10 m1 of T3 medium (per liter: 3 g tryptone [Himedia, India], 2 g tryptose, 1.5 g yeast extract, 0.05 M sodium phosphate [pH 6.8], and 0.005 g of MnCl2) in 250-ml Erlenmeyer flasks and incubate for 7 days at 30C with shaking at 250 rpm (Meadows et al. 1992; Travers et al. 1987). -After growth, transfer 10 ml to 10 ml tubes and centrifuge at 5000 rpm for 15 min, then pour off supernatant broth and wash pellets (spores and crystals) three times with sterile distilled water (5000 rpm, 5 min each). -Finally suspende in 3 ml of sterile distilled water. Insects Drosophila melanogaster insects can be supplied by Department of Biological Sciences at the IUG. The flies can be fed a standard medium (Fruit fly medium; formula 4-24, blue, protected with anti-oxidant and mold inhibitor, [Carolina Biological Supply Company], USA). -Flies can be raised in vials containing 4 g of the standard food dissolved in 20 ml of sterile distilled water. 20

-Place 5 males and 5 juvenile females in the vial and keep at 25C. -For Culex sp., the third instar larvae can be collected from a pond at IUG campus and directly transfer to the lab, then classified (Service, 1980).

Bioassy Drosophila melanogaster: -Homogenize 1 g of artificial fruit flies diet in 10 ml of sterile distilled water in an electrical blinder. -Dilute the toxin-spore suspension (one fold serial dilution; 10-1) in sterile distilled water (Karamanlidou et al. 1991). -Place 10 third instar larvae into each well of 24-well plates [Corning Laboratory Science Company, USA]; 0.3 ml of the diet homogenate and 0.7 ml of B. thuringiensis suspension (spore and crystals) are added to each well. -Assay the pathogenicity of each isolate in duplicate for either the original toxin-spore suspension or the diluted ones. -Incubate the plates at 25C for 24 h then mortality is scored in comparison with parallel control that has 0.7 ml sterile distilled water instead of toxin. -Observe the mortality by viewing brown mid-gut of died larvae under dissecting microscope at 10X. Culex sp.: -For mosquitocidal assays, 10 third instar larvae of Culex sp. are added to 4 ml of sterile water in 20 ml vials, then 1ml of the bacterial suspension is added ( Ishii and Ohba 1993; Martin and Travers 1989; Yu et al. 1991). -Perform the assay in duplicate for either the original toxin-spore suspension or the diluted ones. -Score the mortality after incubation at 25C for 24 h in comparison with parallel negative control that has no bacterial suspension and a positive control with reference strains toxinspore suspension.

21

RESULTS -Determine toxicity of the reference strains (B. t. israelensis, B. t. kurstaki HD1 and B. t. aizawai) against the third instar larvae of D. melanogaster. Count the living 3rd instar larvae of Fruit fly and determine the % of a- Living larvae from the total b- Dead larvae from the total -Observe any changes in color of the mid-gut of the larvae under dissecting microscope. -Compare that change with the control. REFERENCES Carlton B (1988) Development of genetically improved strains of Bacillus thuringiensis. In: Biotechnology for crop protection eds. Hedin P, Menn J, Hollingworth R. American Chemical Society, Washington, D. C, pp. 260-279 Feitelson, J. (1993) The Bacillus thuringiensis family tree. In Advanced engineered pesticides ed. Kim, L. pp. 63-72. Marcel Dekker, Inc., New York, N.Y. Ishii, T. and Ohba, M. (1993). Diversity of Bacillus thuringiensis environmental isolates showing larvicidal activity specific for mosquitoes. Journal of General Microbiology 139, 2849-2854. Karamanlidou, G., Lambropoulos, A., Koliais, S., Manousis, T., Ellar, D. and Kastritsis, C. (1991) Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae). Applied and Environmental Microbiology 57, 2277-2282. Lu, h., Rajamohan, F. and Dean, D. (1994) Identification of amino acid residues of Bacillus thuringiensis -endotoxin CryIAa associated with membrane binding and toxicity to Bombyx mori. Journal of Bacteriology 176, 5554-5559. Martin, P. and Travers, R. (1989) Worldwide abundance and distribution of Bacillus thuringiensis isolates. Applied and Environmental Microbiology 55, 2437-2442. Meadows, M., Ellis, D., Butt, J., Jarrett, P. and Burges, H. (1992) Distribution, frequency, and diversity of Bacillus thuringiensis in an animal feed mill. Applied and Environmental Microbiology 58, 1344-1350. Meqdam, M., Youssef, M., Nimri, L., Shurman, A., Rawashdeh, M. and Al-Khdour, M. (1997) Viral gastrointeritis among young children in Northern Jordan. Journal of Tropical Pediatrics 43, 349-352. Schnepf, E., Crickmore, N., Rie, J., Lereculus, D., Baum, J., Feitelson, J., Zeigler, D. and Dean, D. (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews 62, 775-806. Service, M. 1980. A guide to medical entomology. Macmilan Press Ltd, London, pp. 22-26. Spear, B. (1987) Genetic engineering of bacterial insecticides, P. 204-214. In Biotechnology in agricultural chemistry ed. LeBaron, H., Mumma, R., Honeycutt, R., Duesing, J., Phillips, J. and Haas, M. pp. 204-214. American Chemical Society, Washington, D. C. Travers, R., Martin, P. and Reichelderfer, C. (1987) Selective process for efficient isolation of soils Bacillus spp.. Applied and Environmental Microbiology 53, 1263-1266. Vaeck, M., Reynaerts, A., Hofte, H. and Mellaert, H. (1988) Transgenic crop variants resistant to insects, P. 280-283. In Biotechnology for crop protection ed. Hedin, P., Menn, J. and Hollingworth, R. pp. 280-283. American Chemical Society, Washinbgton, D. C. 22

Yu, Y., Ohba, M. and Gill. (1991) Characterization of mosquitocidal activity of Bacillus thuringiensis subsp. fukuokaensis crystal proteins. Applied and Environmental Microbiology 57, 1075-1081. Further Readings
1-Obeidat, M., F. Al-Momani and I. Saadoun. 2000. Diversity of Bacillus thuringiensis in different habitats of Northern Jordan. J. Basic Microbiology. 40 (5-6): 385-388. 2-Saadoun, I., F. Al-Momani, M. Obeidat, M. Meqdam and A. Elbetieha. 2001. Assessment of toxic potential of local Jordanian Bacillus thuringiensis strains on Drosophila melanogaster and Culex sp. (Diptera). J. Applied Microbiology 90 (6): 866-872.

23

Phytotoxin-Producing Soil Streptomycetes and their Role to Control Weeds

Introduction Many isolation and screening attempts have been done on streptomysetes to find microbial metabolites with bioherbicidal potentials (Arai et al., 1976; Defrank and Putnam, 1985; Li et al., 2003; Mallik, 1997; Mishra et al., 1987; Murao and Hayashi, 1983; Sekizawa and Takematsu, 1983, Takahashi et al., 1995). Anisomycin, which is produced by Streptomyces toyocaensis was the first commercially used phytotoxin (Yamada et al., 1972). Bialaphos, which is produced by Streptomyces hygroscopicus (Mallik, 2001) and Streptomyces viridochromogenes (Charudattan et al., 1996), represents the first patented microbial bioherbicide. Arai et al. (1976) reported the production of two bioherbicides, herbicidans A and B, by Streptomyces saganonesis that are selective against many dicotyledonous plants. Gougerotin is another plant growth inhibitor produced by Streptomyces sp No.179 (Murao and Hayashi, 1983). Babaczinski et al. (1991) reported vulgamycin as phytotoxin against dicotyledonous weeds and grasses if applied postemergence. Phosphenothrixin that is produced by Saccharothrix sp.ST-888 inhibits the germination of gramineous and broadleaved weeds (Takahashi et al., 1995). Herbimycin represents a potent herbicidal activity when used pre-emergence, against flat-sedge (Cyperus microiria Steud.) (Sekizawa and Takematsu, 1983). All these discovered phytotoxins from streptomycetes represent a wide range of plant inhibitory compounds that are naturally degraded in the environment, which may restrict the regular undesirable consequences of using agrochemicals such as, accumulation, biomagnifications, and excessive persistence (Heisey and Putnam, 1990). The intensive and the arbitrary usage of herbicides lead to the development of resistant weed species to some of those herbicides (Mallik, 2001). Moreover, biotechnological techniques were utilized for testing the possibility of transferring the genes of phytotoxin production to a plant pathogen in an attempt to become sufficiently acceptable for control a weed target (Charudattan et al., 1996). There are about 300 common weed species that cause crop losses world wide (Hoagland, 1990). Weed control depends mainly on conventional hand weeding and tillage (Abu Irmaileh, 2000; Salim and Mokhtar, 2000) and the strategies at this stage are more logical in taking early precautions and avoiding future negative consequences on the environment. Therefore, biological control of weeds represents a logical alternative to the agrochemicals. Procedure Use Streptomyces isolates that have been and purified before. The T.A. will provide you with the cultures growing of SCNA plates. Phytotoxic activity assay: Bioassays of the isolated streptomycetes for their phytotoxicity will be performed using two indicator plant seeds namely: cucumber (Cucumis sativus L.) (UPC, 0-21496-28630-3, Lowes, Texas) and ryegrass seeds (Lolium perenne L.) (Local cultivar Beit Alpha) (Mallik, 2001).

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Surface sterilization of the indicator plant seeds. -Surface sterilize the cucumber and ryegrass seeds by immersing them for 5 min inside 2% and 25% solutions of sodium hypochlorite (Clorox commercial containing 6.5% of NaClO), respectively. -In both cases, add 20 l of Tween 20 to break the water surface tension. -Apply vacuum using vacuum pump to facilitate thorough surface sterilization. -Wash the seeds 3 times with sterilized distilled water for 1.5 min at each wash in order to get rid of the residual hypochlorite and Tween 20. -Blott the surface sterilized seeds between double layers of sterilized cheesecloth and transfer to sterilized glass Petri dishes to be used in the bioassay experiments. Screening of streptomycete isolates for their bioherbicidal activity. -Scrap the growth of each streptomycete isolate from SCNA plates (28 C for 10 days) then aseptically transfer into 5 ml vials containing 2 ml of sterilized distilled water and mix with vortex. -Place aliquot of 0.4 ml from each isolate cell suspension in the center of SCNA plates (duplicate) and spread using L-shape glass rod over a 2 cm wide strip along the diameter of those plates. -Non-inoculated SCNA plates will served as controls. -Incubate for 3 weeks at 28 C for 3 weeks. -Place 6 sterilized each of the cucumber or ryegrass seeds on one side of the culture strip. -Incubate the plates in dark at 28 C for 4 days. -Observe seed germination and calculate germination percentages. -Measure average length of radicles and shoots using Vernier caliper.

References AbuIrmaileh B. E. 2000. Weed management in the Near East - general view. pp. 19-25. In: P.G. Americanos, B.E. Abu- Irmaileh and A.R. Saghir (eds), Improved Weed Management in the Near East. Arab Organization of Agricultural Development. Khartoum-Sudan. Arai M., T. Haneishi, N. Kitahara, R. Enokita, K. Kawakubo and Y. Kondo. 1976. Herbicidans A and B, two new antibiotics with herbicidal activity, I. Producing organism and biological activities. J. Antibiotics 29: 863-869.

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Babaczinski P., M. Dorgerloh, A. Lbberding, H.J. Santel, R.R. Schmidt and C. Wnsche. 1991. Herbicidal activity and mode of action of vulgamycin. Pestic. Sci. 33: 439446. Charudattan R., V.J. Prange and J.T. DeValerio. 1996. Exploration of the use of the Bialaphos Genes for improving bioherbicidal efficacy. Weed Technol. 2: 625-636. Defrank J. and A.R. Putnam. 1985. Screening procedures to identify soil borne actinomycetes that can produce herbicidal compounds. Weed Sci. 33: 271-274. Hoagland R. E. 1990. Microbes and microbial products as herbicides, an overview, pp. 2-52. In: R.E. Hoagland (ed.) Microbes and Microbial Product as Herbicides. ACS Symposium No 439. American Chemical Society, Washington, DC. Li Y., Z. Sun, X. Zhuang, L. Xu, S. Chen and M. Li. 2003. Research progress on microbial herbicides. Crop Protec. 22: 247-252. Mallik M. A. B. 1997. Isolates of soil actinomycetes with potential for phytotoxin production. J. Chem. Ecol. 23: 2683-2693. Mallik M. A. B. 2001. Selective isolation and screening of soil microorganisms for metabolites with herbicidal potential. J. Crop Allelo. Agroeco. 4: 219-236. Salim A. A. and M. Mokhtar. 2000. A comparison study of weed control methods in plum orchards, pp. 115-119. In: P.G. Americanos, B.E. Abu- Irmaileh and A.R. Saghir (eds), Improved Weed Management in the Near East. Arab Organization of Agricultural Development. Khartoum-Sudan. Sekizawa Y. and T. Takematsu. 1983. How to discover new antibiotics for herbicidal use, pp. 261-268. In: N. Takahashi, H. Yoshioka, T. Misato and S. Matsunaka (eds.), Pesticide Chemistry: Human Welfare and the Environment, Vol. 2. Natural Producer, Pergamon Press. Oxford, U.K. Takahashi E., T. Kimura, K. Nakamura, M. Arahira and M. Iida. 1995. Phosphonothrixin, a novel herbicidal antibiotic produced by Saccharathrix sp. ST 888, I. Taxonomy, fermentation isolation and biological properties. J. Antibio. 48: 1124-1129. Yamada O., Y. Kaise, F. Futatsuya, S. Ishida, K. Ito, H. Yamamoto and K. Munakata. 1972. Studies on plant growth regulating activities of anisomycin and toycamycin. Agric. Biol. Chem. 36: 2013-2015.

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Table I Effect of Streptomyces isolates on seed germination, radicle and shoot growth of cucumber and ryegrass assessed by the agar plate screening method. Cucumber Isolate
Cont. 1 2 3 4 5 6
a Gera % % Gb R.Lc Mm % R.L S.Ld Mm % S.L Ger. % % G.L R.L Mm

Ryegrass
% R.L S.L Mm % S.L

Ger: Germination %: Percent decrease over the control c R.L: Radicle length d S.L: Shoot length
b

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In Vitro Control of Fungal Phytopathogens by Chitinase-Producing Streptomycetes

Introduction A wide variety of bacteria are known as hydrolytic enzyme producers, with streptomycetes being the best known enzyme producers (Vinogradova and Kushnir, 2003). Streptomycetes produce stable mycelia which are capable of secreting an array of different extracellular enzymes including cellulases, chitinases and xylanases. They produce such enzymes to degrade naturally occurring macromolecules, and thus to help in their growth and survival (Christodoulou et al., 2001; Williamson et al., 2000). As chitinolytic microorganisms, streptomycetes are those organisms that are capable of degrading chitin solely by hydrolysis of glycosidic bonds (Goody, 1990). In fact, Streptomyces strains are regarded as the major producers of chitinases in soil (Tanabe et al. 2000). Most streptomycetes secrete a number of chitinases hydrolyzing chitin to its oligomers; chitooligosaccharides, chitobiose or N-acetylglucosamine. Such oligomers can be utilized as carbon or nitrogen sources (Schrempf, 2001). A number of biological control methods to control soilborne plant pathogens were postulated using microbial antagonists (Adams and Ayers, 1982; El-Tarabily et al, 2000; and Tahtamouni et al, 2006). Such approach was targeted to cut down on the application of chemical fungicides. Therefore, chitinase producing organisms received increasing attention during the last decades. Their potential in biocontroling the fungal phytopathogens was sought to be promising, since chitin is the major constituent of the cell walls of many plant pathogenic fungi including S. sclerotiorum (; Gupta et al, 1995; El-Tarabily et al, 2000 and Tahtamouni et al, 2006).

Procedure Preparation of Colloidal chitin Colloidal chitin is prepared from partially purified chitin from crab shells (Sigma) by blending of 40 g in a blender then dissolving in 400 ml of concentrated HCl by stirring for 30 to 50 min. The chitin is precipitated as colloidal suspension by adding it slowly to 2 liters of water at 5 to 10 C. The suspension is collected by filtration with suction on a coarse filter paper and then washed by suspending it in about 5 liters of tap water and re-filtering. Washing is repeated at least three times or until the pH of the suspension is about 3.5. Water content of the chitin is determined by drying a sample at 100 C. For use sufficient water is added to resuspend the chitin, and the suspension is blended at high speed for about 10 min. Autoclaved filter cake or aqueous suspension could be stored indefinitely at room temperature. Plates are incubated at 28 C for 8 days.

Screening for chitinase-producing streptomycetes. -All streptomycetes pure isolates that were recovered previously here in this laboratory or other labs will be screened for their efficiency of chitinase production. 28

-Suspend each isolate in sterile vial containing 3 ml distilled water, to give a spore suspension of 107 spores/ml. -Place a drop of 0.1 ml volume of the spore suspension in the center of a colloidal chitin agar (CCA) media, which is specified for screening of chitinolytic actinomycetes, according to Hsu and Lockwood (1975). -The appearance of chitin clearing zone around those colonies is indicative of the presence of chitinase activity in these isolates, and the difference between clearing zone diameter and the colony diameter is a measure for the enzyme activity. In Vitro bioassay of the chitinase producing streptomycetes against S. sclerotiorum This test can be done on two kinds of media (CCA and SCNA). -Inoculate by streaking the active chitinase-producing Streptomyces isolates on one side of the CCA or SCNA plates. -Incubate at 28 C for 7 days. -Cut a disc of 6 mm diameter from an active growing S. sclerotiorum culture then transfer onto the opposite side of the Streptomyces culture plates approximately 25 mm away from the bacterial growth. -Use control plates containing discs of the fungal pathogen growth only. -Evaluate the magnitude of the antagonistic activity for each isolate in terms of the distance the fungus grew (X1) toward the colony of the Streptomyces in contrast to the distance of the fungal growth when it is present alone (X2), according to the equation of X = X2- X1. Results Table 1. Distribution of the most active chitin-degrading Streptomyces isolates. Group1 (5 10 mm) Isolate X
a

Chitinolytic activity ( Xa) Group2 (2.1 4 mm) Isolate X

Group3 (1 2 mm) Isolate

X (chitinase activity) = the clearing zone diameter -colony diameter.

29

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References Adams OB, Ayers WA. 1982. Biological control of Sclerotinia lettuce drop in field by Sporidesmium sclerotivorum. Phytopathology. 72: 485-8. El-Tarabily, K., Soliman, M., Nassar, A., Al-Hassani, H., Sivasithamparam, K., McKenna, F. and Hardy, GE StJ. 2000. Biological control of Sclerotinia minor using a chitinolytic bacterium and actinomycetes. Plant Pathology. 49: 573-583. Christodoulou E., F. Duffner and C.E. Vorgias. 2001. Overexpression, purification, and characterization of a thermostable chitinase (Chi40) from Streptomyces thermoviolaceus OPC-520. Prot Exp Purif. 23: 97105 Goody G.W. 1990. Physiology of microbial degradation of chitin and chitosan. Biodeg. 1: 177-190 Gupta, R., Saxena, R., Chaturvedi, P.and Virdi, J. 1995. Chitinase production by Streptomyces viridificans: its potential in fungal cell wall lysis. Journal of Applied Bacteriology. 78: 378-383. Hsu S.K., and J.I. Lockwood. 1975. Powder chitin agar as a selective medium for enumeration of actinomycetes in water and soil. Appl Microbiol. 29: 422-426 Schrempf H. 2001. Recognition and degradation of chitin by streptomycetes. Antonie van Leeuwenhoek 79: 285289 Tahtamouni M.E.W., K.M. Hameed and I.M. Saadoun. 2006. Biological control of Sclerotinia sclerotiorum using indigenous chitinolytic actinomycetes in Jordan. Plant Pathol J. 22: 107-114 Tanabe T., T. Kawase, T. Watanabe, Y. Uchida and M. Mitsutomi. 2000. Purification and Characterisation of a 49-kDa Chitinase from Streptomyces griseus HUT 6037. J Bios Bioeng. 1: 27-32 Vinogradova S.P. and S.N. Kushnir. 2003. Biosynthesis of hydrolytic enzymes durig cocultivation of macro- and micromycetes. Appl Biochem Microbiol. 39: 573-575 Williamson N., P. Brian and E.M.H. Wellington. 2000 Molecular detection of bacterial and streptomycetes chitinases in the environment. Antonie van Leeuwenhoek 78: 315321

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Biological Control of Orobanche by Fungi Isolated from Diseased Specimens in Gaza Introduction Species of the genus Orobanche are parasitic flowering plants, holoparasites that attach to the root of their host, green plants. They develop haustoria on the root of their hosts. The parasite sends its shoots above ground in order to expose its flowers and facilitate seed dissemination. Those parasites represent an increasing threat to several vegetable and fruit crops (AlKhazraji et al., 1987 and 1989) in a wide range of cultivated lands leading to yield losses up to 100% in the host plant in several places worldwide (Sauerborn 1991). They are also widely distributed in other parts of the Arab countries as well as other countries within arid and semiarid regions (Parker, 1994). Combating and controlling these parasites has been a difficult task due to the narrow margin of selectivity of the available herbicides, between the host and the parasite in case of the chemical control (Garcia Torres et.al., 1994). Also, due to the nature of this parasite, as it produces a vast number of seeds ( Saghir, et. al., 1973a). They are capable of staying dormant in soil for many years waiting for the germination stimulant exuded from a plant root (Saghir et al., 1973b). The germinated seed ought to be in contact with the root to develop a haustorium, or tubercule (Hameed and Foy 1991), and shoot(s). This process takes about 5-7 weeks (Saghir et. al., 1973b), which means that the crop plant, the host has been suffering for the duration of the period prior to the emergence of the parasite. The negative impact of the parasite upon the host plant was closely related to the developmental stage of both parasite and its host (Manschadi et al., 1996). Therefore, considerable efforts were needed in control measures geared against non-germinated and/or germinated Orobanche seeds, in order to prevent initiation of infection such as deep plowing (Petzoldt et al., 1994). Further, the use of trap crop or decoy plants (Saghir et. al., 1973b), and utilization of inherited resistance genetic resources against the infection by these parasites are needed for control. Biological control of Orobanche and other weeds (Fayadh et. Al., 1990) has been advocated for integrated management of such problems and it includes measures such as the use of insect predators on Orobanche (Kruschel and Klien 1995) and pathogenic fungi (Thomas et al., 1998). Looking for biological control agents against Orobanche (Bedi 1991) and Striga spp. (Kroschel et al., 1996) was articulated upon field observation of disease on parasitic seed plants and isolation of the causal pathogen(s). The potential of that agent(s) was further investigated for their feasibility in future biological control programs (Thomas et al., 1998). Many commercial biological control agents based on the use of pathogenic fungi have been developed for weed control such as controlling weed in rice and soybean, for example (Bowers 1986). Procedure Collection of diseased Orobanche specimens. -Stems, of different species of Orobanche (O. ramosa, O. crenata, O. cernua, and O. egyptiaca) will be collected during several field trips in Gaza. These stems will be sampled for diseased Orobanche plants showing disease symptoms such as wilting, dry or soft rot at the base of the stem, complete blight of the stems with black floral parts and ovules. These samples can be collected from tomato, eggplant, and faba-bean vegetable crops infected with Orobanche.

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Isolation of the disease inciting fungi. -Place segments, 1-2 cm long of diseased stem and seeds inside 10% sodium hypochlorate solution for 2-3 minutes to be surface-sterilized. -Rinse them with sterilized distilled water. -Plate the surface sterilized materials on acidified potato dextrose agar (PDA), malt agar (MA) and cornmeal agar (CMA) mycological media. -Isolate the fungal growth associated with diseased Orobanche into pure culture. -Characterized and identify the isolated fungi to genus and species. Pathogenecity test of the isolated fungi. -Inoculate the isolated fungi on healthy stem segments in order to test the pathogenecity of the isolated fungi upon Orobanche materials. -Healthy stem segments of 2-3 cm long are first surface sterilized with hypochlorate solution prior to inoculation. -Inoculate each stem segment by transferring a tiny portion of the fungal growth with the aid of an inoculating needle to the surface of the agar and making 3-5 pricks on that spot. -Place the inoculated stems in a moist chamber and incubate inside an incubator at 25 C. -Observe the segments for disease development within the next 2 days and again 5 days later on. Disease assessments. -Disease incidence and severity can be rated on the basis of the symptom development and an arbitrary scale ranging from zero (0), no disease observed to (100) where the whole segment being affected by the fungus, respectively. -Record the range of disease severity according to the scale on each segment of the Orobanche species and for each fungus. Results Table 1. The incidence of fungi associated with Orobanche stems from open field in Gaza showing disease symptoms Fungi isolated from diseased Orobanche Stems Isolated O. ramosa O. cernua O. egyptiaca O. crenata Fungi

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Table 2. Pathogenicity of the isolated fungi on healthy stem segments of Orobanche Disease ratings of fungal infection on Orobanche Isolated After 2 and 5 days incubation Fungi F. G. % T. Macer. T. discolor D. I. % D. S. 0-100 2 5 2 5 2 5 2 5 2 5

Control F. G. = fungal growth, T. Macer. = Tissue maceration, or tissue deterioration at the inoculation point detected with the aid of a needle, T. Discolor. = Color change of tissue to of normal wax color or black, D. I. = Disease incidence, D. S. = disease severity 0 = no disease to 100 = complete colonization, N. G. = no fungal growth. References 1. Al-Khazraji, T. O, Hameed, K. M. and Saghir, A. R. 1987. Effects of inoculum density of Orobanche seeds in soil on tomato and tobacco. Zanco 5:73-84. 2- Al-Khazraji, T. O., Y. I. Ielya, and K. M. Hameed (1989).Orobanche (Orobanche ramosa L.) as a parasite on Apricot trees (Prunus arneniaca L.) in Iraq. Arab Journal of Plant Protection 7: 3- Bedi, J.S. (1994). Further studies on control of sunflower broomrape with Fusarium oxysporum f. sp. orthoceras- a potential mycoherbicide. In Proceedings of the 3rd International Workshop on Orobanche and Related Striga Research ( A.H. Peterse, J. A. C. Verkleig, and S. J. ter Borg, Eds.), pp 539-544. Royal Tropical Institute, Amsterdam, The Netherlands. 4- Bowers, R. C.(1996). Commercialization of collego-An industrialists view. Weed Sci. 34(suppl.1),24-25 5- Fayadh, A. H., K. M. Hameed, and H. A. Al-ani (1990). Dodder (Cuscuta campestris Yunck) blight caused by Alternaria alternata ( Fr.) Keissler and Geotricum candidum Link ex. Pres. Arab Journal of Plant Protection 8: 55-59 6- Garcia Torres, L., M. Castejon-Munoz, and F. Lopez-Granados (1994a) The problem of Orobanche and its management in Spain. In Proceedings of the 3rd International Workshop on Orobanche and Related Striga Research ( A.H. Peterse, J. A. C. Verkleig, and S. J. ter Borg, Eds.), pp 623-627. Royal Tropical Institute, Amsterdam, The Netherlands. 7- Garcia Torres, L., F.Lopez-Granados, and M. Castejon-Munoz (1994b). Pre-emergence herbicides for the control of broomrape (Orobanche cernua Loefl.) in sunflower ( Helianthus annus L). Weed Res. 34: 395-402. 8- Hameed, K. M., and Foy, C. L. 1991. Observations on the primary haustorium formation by germinated Orbanche ramosa seeds in relation to suscept and non- suspect plant. In: Ransom, J. K., Musselman, L. T. ,Worsham, A. D., and Parker, C. (eds,), Proceeding of the Fifth Internation Symposium on Parasitic Weeds, Nairobi, Kenya. Pages: 36-42. 9- Kroschel, J., and O. Klien (1995). Biological control of Orobanche spp. with Phytomyza orobanchia Kalt., a review. In: J. Kroschel, J., M. Abderabihi, H. Betz (eds). Advances in Parasitic Weed Control at Onfarm Level. Vol. II. Joint Action to Control Orobanche in the WANA Region. Margrat Verlag, Weikersheim, Germany, 135-159. 10- Parker, C. (1994). The present state of the Orobanche problem. In Proceedings of the 3rd International Workshop on Orobanche and Related Striga Research ( A.H. Peterse, J. A. C. 34

Verkleig, and S. J. ter Borg, Eds.), pp 17-26. Royal Tropical Institute, Amsterdam, The Netherlands. 11- Petzoldt, K., Y. Nemli, and J. Snyde (1994)Integrated control of Orobanche cumana In Proceedings of the 3rd International Workshop on Orobanche and Related Striga Research ( A.H. Peterse, J. A. C. Verkleig, and S. J. ter Borg, Eds.), pp 422-449. Royal Tropical Institute, Amsterdam, The Netherlands. 12- Saghir, A. R., C. L. Foy, and K. M. Hameed (1973a). Herbicie effects on parasitism of tomato by Hemp Broomrape. Weed Science 21: 253-257. 13- Saghir, A. R., C. L. Foy, K. M. Hameed, C. R. Drake and S. A. Tolin (1973b). Studies on the biology and control of Orobanche ramosa L. Proc. Eur. Weed Res. Coun. Symp. Parasitic Weeds, Malta. Paper No 197: 106-116. 14- Thomas, H., J. Saurborn, D. Muller-Stover, A. Zeigler, J. S. Badi and J. Kroschel (1998). The potential of Fusarium oxysporum f.sp.orthoceras as abiological control agent for Orobanche cumana in sunflower. Biological control 13: 41-48.

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Appendices Appendix 1: Media Composition:

1. Starch Casein Nitrate Agar (pH = 7.2)

Components Casein
Starch KNO3 MgSO4.7H2O FeSO4.7H2O CaCO3 NaCl K2HPO4 Agar 2. Oatmeal Agar

Company
Difco, USA Riedel-de Haen, Germany GCC, UK Chemlab, England Chemlab, England Chemlab, England Merck, Germany Laboratory Rasayan, India Himedia, India

Per Liter 0.3g 10.0g 2.0g 0.05g 0.01g 0.02g 2.0g 2.0g 18.0g

(pH = 7.2)

Oatmeal
Agar Trace salt solution: FeSO4.7H2O MnCl2.4H2O ZnSO4.7H2O Distilled Water 3. Mueller-Hinton Agar Muller Hinton Agar D. H2O 4- T3 medium (Per liter)

Quaker, UK
Himedia, India Chemlab, England BDH, England BDH, England

20.0g 18.0g 1.0 ml 0.1g/100ml 0.1g/100ml 0.1g/100ml 100ml

Himedia, India

30.0 g 1L

3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 M sodium phosphate [pH 6.8], and 0.005 g of MnCl2.

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