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Laboratory Manual

Biocontrol


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ISLAMIC UNIVERSITY OF GAZA

DEPARTMENT OF BIOTECHNOLOGY

BIOCONTROL

LABORATORY MANUAL

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: +962-2-

7201071. E-mail address: [email protected]


Department of Applied Biological Sciences, Jordan University ofScience and Technology, P.O. Box 3030, Irbid- 22110, Jordan.

Phone: +962-2-7201000-Ext. 23460; Fax: +962-2-7201071. E-mail

address: [email protected]


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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.


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: +962-2-

7201071. E-mail address: [email protected]


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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 chemicalsare 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 SaadounDept. 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: [email protected]; [email protected]


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Dept. of Biotechnology

Biocontrol Lab

Contents Pages

Introduction and Orientation -Review of Microbial Techniques 7-13

Isolation of Soil Streptomycetes From Gaza 14-15

In Vitro Control of Bacterial Phytopathogens (Agrobacterium

tumefaciens, Pseudomonas, Erwinia and Corynebacterium) by Soil

Streptomycetes

16-18

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

Plant Pathogens

19-22

In Vitro Control of Food Associated Fungi by Soil Streptomycetes

Isolated From Gaza

23-25

Toxicity Preparations of Bacillus thuringiensis Towards Drosophila

melanogasterand Culex sp.

26-29

Phytotoxin-Producing Soil Streptomycetes and their Role to Control

Weeds

30-33

In Vitro Control of Fungal Phytopathogens by Chitinase-Producing

Streptomycetes

34-37

Continue

Biological Control of Orobanche by Fungi Isolated from Diseased

Specimens in Gaza

38-41

Appendices 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, biologicalcontrol 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 withagricultural 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 Science46: 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. Phytopathology35: 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.


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Dept. of Biotechnology

Biocontrol Lab

Lab Schedule

The aim of this lab course is to provide an understanding of the application of beneficialorganisms as promising biocontrol agent(s) against several disease causative agents.

Week Exercise Pages

1 Introduction and Orientation -

2 Review of Microbial Techniques 7-13

3 Isolation of Soil Streptomycetes From Gaza 14-15

4 In Vitro Control of Bacterial Phytopathogens (Agrobacterium

tumefaciens, Pseudomonas, Erwinia and Corynebacterium) by Soil

Streptomycetes

16-18

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

Plant Pathogens

19-22

6 In Vitro Control of Food Associated Fungi by Soil Streptomycetes

Isolated From Gaza

23-25

7 Mid Term Exam -

8 Toxicity Preparations of Bacillus thuringiensis Towards Drosophila

melanogasterand Culex sp.

26-29

9 Continue

10 Phytotoxin-Producing Soil Streptomycetes and their Role to Control

Weeds

30-33

11 Continue -

12 In Vitro Control of Fungal Phytopathogens by Chitinase-Producing

Streptomycetes

34-37

13 Continue14 Biological Control of Orobanche by Fungi Isolated from Diseased

Specimens in Gaza

38-41

15 Continue -

16 Final Exam


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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 andtransferring 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 contaminatedwater 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 itcontacts the loop);4. Holding the culture container in the other hand, remove the cover by grasping it betweenthe 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 orderto 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 personusing 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 itemscontaminated with microbes should be autoclaved before washing or disposal.

PLATING

Isolation of individual microbial types may be obtained by dilution methods. The dilution, areduction 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 celldilution 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.


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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 asfollows:

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 patternholding 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. Allow the plate to stand, cool, and solidify;6. 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 beclearly 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 asmall 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.


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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 forthe 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 asemisolid 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 acrystal violet-iodine complex after leaching with an alcohol solvent, these cells generally have


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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,Bacillussubtilis, Staphylococcus aureus

Broth mix of: Staphylococcus aureus andEscherichia 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 eachof 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 outfirst 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 isolatedcolonies 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)


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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 and the plates streaked for isolation as

sources for microorganisms to stain

1. Make gram stains of the E. coli, S. aureus,B. subtilis and the mixed culture according tothe procedure described by your instructor. Observe these stains under the microscopeusing the oil immersion magnification.

2. Make a spore stain of the cultures assigned to you. Observe it under the microscope usingthe 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 10

5)


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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 08

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.


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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 ofStreptomyces 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 soilstreptomycetes from arid habitats in Jordan and their potential to inhibit multi-drug resistant Pseudomonas

aeruginosa pathogens. World J. Microbiol. Biotech. 24: 157-162


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In Vitro Control of Bacterial Phytopathogens (Agrobacterium tumefaciens, Pseudomonas,

Erwinia and Corynebacterium) by Soil Streptomycetes

Introduction

Crown gall, caused byAgrobacterium 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

theirin vitro inhibitory activity againstA. 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 andpurified 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 Corynebacterium1

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 againstAgrobacterium 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 ofAgrobacterium 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

Agrobacteriumradiobacterbiovar 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-pathogenicAgrobacteriumvitis 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 ofAgrobacterium 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 radiobacterstrain 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 ofAgrobacterium tumefaciens. Hilgradia 40, 537-552.

[19] Sule, S., 1978. Biotypes ofAgrobacterium tumefaciens in Hungary. J. Appl. Bacteriol.

44, 207-213.

[20] Webster, J., Dos Santos, M. and Thomson, J.A., 1986. Agrocin producingAgrobacterium

tumefaciens strain active against grapevine isolates. Appl. Environ. Microbiol. 52, 217-219.


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Effect of Parasitic Seed Plant (Orobanchespp.) 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 ofO. cernua. They showed

that the tissue of this parasitic seed plant has a remarkable activity against some pathogenicbacteria. Also, the activity against the crown gall and soft rot phytopathogens;Agrobacterium

tumefaciens andErwinia, 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).


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-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 ofOrobanche 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)

Sp. 1 Sp. 2 Sp. 3


Table 3. Minimum concentration of the different Orobanche species extract inhibiting human

and plant pathogens.

PathogenDiameter of Inhibition Zone(mm)

b

MIC of the Extracts (mg/ml)

Sp. 1 Sp. 2 Sp. 3 Sp. 4



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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 TropicalAgriculture 5: 9-35.

3. Saadoun I, Hameed K. 1999. Antibacterial activity ofOrobanche 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 andErwinia. 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 orderActinomycetales 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 andBotrytis cinerea. Another study on the activity

of soil actinomycetes in Turkey against different fungi revealed the inhibition ofSclerotinia

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 bedone 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.


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Results

Table 1. Antifungal activity of the Streptomyces isolates.Color Series

Gray White Yellow Green Red Blue Variable Total

No. of isolatesNo. of active isolates

Torula spp.Aspergillus niger

Aspergillus niger

Trichoderma virideTrichoderma harasmiiAspergillus flavus

Penicillium spp.1

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 causingsugar beet root rot.Acta Biotechnology 9. 389.

Turhan, G. (1981) A new race ofStreptomyces 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 Agrobacteriumtumefaciens.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

Streptomycesplicatus (strain 101) against four Iranian phytopathogenic Verticilliumdahliae, 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, Verticilliumdahliae and Saccharomycescervisiae.Asian J. Plant Sci. 3(4):463-471.

6- Shahrokhi, S., G.H. Shahidi Bonjar and I. Saadoun. 2005. Biological control of potato isolate ofRhizoctonia

solaniby 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 Streptomycescoralus strain 63 against Ralstoniasolanacearum. J. Biological

Sci. 6(1): 127-129.

8-Tahtamouni, ME. W., K.M. Hameed and I. Saadoun. 2006. Biological control of Sclerotinia sclerotiorumusing 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 tostreptomycetes isolates from Jordan soils. Journal of Basic Microbiology 48 (3): 213-216.


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Toxicity Preparations ofBacillus thuringiensis TowardsDrosophila

melanogaster and Culex sp.

INTRODUCTION

The preference of using biological pesticides over chemical ones has been widely accepted indifferent 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; Vaecket al. 1988). Identified strains ofB.

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 ; Schnepfet 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 melanogasterinsects 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.


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21

-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 ofB. 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 ofCulex 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 toxin-

spore suspension.


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RESULTS

-Determine toxicity of the reference strains ( B. t. israelensis, B. t. kurstaki HD1 and B. t.

aizawai) against the third instar larvae ofD. 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 ofBacillus 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) TheBacillus thuringiensis family tree. InAdvanced engineered pesticidesed. 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 Microbiology57, 2277-2282.

Lu, h., Rajamohan, F. and Dean, D. (1994) Identification of amino acid residues ofBacillus

thuringiensis-endotoxin CryIAa associated with membrane binding and toxicity to Bombyx

mori.Journal of Bacteriology176, 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

Microbiology58, 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 Reviews62, 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 Biotechnologyin 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

soilsBacillus spp..Applied and Environmental Microbiology53, 1263-1266.

Vaeck, M., Reynaerts, A., Hofte, H. and Mellaert, H. (1988) Transgenic crop variants

resistant to insects, P. 280-283. InBiotechnology for crop protection ed. Hedin, P., Menn, J.

and Hollingworth, R. pp. 280-283. American Chemical Society, Washinbgton, D. C.


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Yu, Y., Ohba, M. and Gill. (1991) Characterization of mosquitocidal activity ofBacillus

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 ofBacillus 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 melanogasterand Culexsp. (Diptera). J. Applied

Microbiology 90 (6): 866-872.


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24

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 andTakematsu, 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 post-

emergence. 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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25

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) thenaseptically 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-shapeglass 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. 33:439-

446.

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.)Microbesand 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 AgriculturalDevelopment. 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.

YamadaO., 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.

Isolate

Cucumber Ryegrass

Gera%

% Gb R.Lc

Mm% R.L S.L

dMm

% S.L Ger.%

% G.L R.LMm

% R.L S.LMm

% S.L

Cont.

1

2

3

4

5

6a

Ger: Germinationb%: Percent decrease over the controlcR.L: Radicle length

dS.L: Shoot length


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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 streptomycetesbeing 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 orN-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 re-

suspend the chitin, and the suspension is blended at high speed for about 10 min. Autoclavedfilter 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.


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-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 ofchitinase activity in these isolates, andthe 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.

Chitinolytic activity ( Xa)

Group1

(5 10 mm)

Group2

(2.1 4 mm)

Group3

(1 2 mm)

Isolate

X

Isolate

X

Isolate X

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


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A B


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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, andcharacterization 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

Leeuwenhoek79: 285289Tahtamouni 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 Leeuwenhoek78: 315321


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32

Biological Control of Orobanche by Fungi Isolated from Diseased Specimens in Gaza

Introduction

Species of the genusOrobanche areparasitic 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 (Al-

Khazraji 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 semi-

arid 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 ahaustorium, or tubercule (Hameed and Foy 1991), and shoot(s). This process takes about 5-7

weeks (Saghiret. 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 (Saghiret. 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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33

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 solutionprior 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

IsolatedFungi

Fungi isolated from diseased Orobanche Stems

O. ramosa O. cernua O. egyptiaca O. crenata


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Table 2. Pathogenicity of the isolated fungi on healthy stem segments ofOrobanche

Isolated

Fungi

Disease ratings of fungal infection on Orobanche

After 2 and 5 days incubation

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),24-255- 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 ofOrobanche 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 Orobancheproblem. In Proceedings of the 3rd

International Workshop on Orobanche and Related Striga Research ( A.H. Peterse, J. A. C.


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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 ofOrobanche 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 oftomato 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 ofOrobanche 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 Company Per Liter

Casein Difco, USA 0.3g

Starch Riedel-de Haen, Germany 10.0g

KNO3 GCC, UK 2.0g

MgSO4.7H2O Chemlab, England 0.05g

FeSO4.7H2O Chemlab, England 0.01g

CaCO3 Chemlab, England 0.02g

NaCl Merck, Germany 2.0g

K2HPO4 Laboratory Rasayan, India 2.0g

Agar Himedia, India 18.0g

2. Oatmeal Agar (pH = 7.2)

Oatmeal Quaker, UK 20.0g

Agar Himedia, India 18.0g

Trace salt solution: 1.0 ml

FeSO4.7H2O Chemlab, England 0.1g/100ml

MnCl2.4H2OBDH, England

0.1g/100ml

ZnSO4.7H2O BDH, England 0.1g/100ml

Distilled Water 100ml

3. Mueller-Hinton Agar

Muller Hinton Agar Himedia, India 30.0 g

D. H2O 1 L

4- T3 medium

(Per liter)

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.

bio control lab gaza

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