Isolation and Characterization of Streptomyces species

with in vitro and in vivo Activities against some

Phytopathogenic Fungi

A Thesis Submitted to the University of Khartoum in Fulfillment of the

requirements for the Degree of Master of Science in Botany (Microbiology)

By

Rihab Eshag Mohammed Alhasan

B.Sc. (Honours) Botany

Supervisor Dr. Adil Ali El Hussein

Co. Supervisor Dr. Suhair Ahmed Abd Elwahab

Department of Botany

Faculty of Science

University of Khartoum

Jan, 2010

Dedication To my:

Lovely mother,…

Wonderful father,…

Great brothers and sister,…

Dear friends,…

All who make me smile,…

With my love

Table of contents

Page Acknowledgement……………………………………...................... i

Abstract……………………………………………………………... ii

Abstract in Arabic…………………………………………………... iv

List of Tables ………………………………………………………. vi

List of Plates………………………………………………………... vii

1.Introduction……………………………………………………… 1

2.Literature Review……………………………………………… 5

2.1.The Actinomycetes……………………………………………... 5

2.2.Streptomyces…………………………………………................. 6

2.2.1.Classification of Streptomyces………………………………... 7

2.2.2.Life Cycle of Streptomyces…………………………………… 7

2.2.3.Habitat………………………………………………………… 8

2.2.4.Nutrition………………………………………………............. 9

2.2.5.Isolation of Streptomyces……………………………………... 10

2.2.6.Importance of Streptomyces…………………………………... 11

2.3.Antibiotics………………………………………………………. 12

2.3.1.Major groups of antibiotics and their mode of action………… 13

2.3.1.1.Beta –Lactam group………………………………………… 13

2.3.1.2.Aminoglycosides……………………………………………. 14

2.3.1.3.Tetracyclines………………………………………………... 14

2.3.1.4.Macrolides……………………………………………........... 14

2.3.1.5.Sulphonamides and Trimethoprim………………………….. 15

2.3.1.6.Rifamycins…………………………………………….......... 15

2.3.1.7.Polypeptides………………………………………………… 16

2.3.1.8.Azoles…………………………………………………......... 16

2.3.2.Classification of antibiotics…………………………………… 16

2.3.2.1. According to their origin…………………………………… 16

2.3.2.1.1.Antibiotics produced by Streptomyces……………………. 16

2.3.2.1.2.Antibiotics produced by other bacteria…………………… 17

2.3.2.1.3.Antibiotics produced by fungi……………………………. 17

2.3.2.1.4. Antibiotics produced by chemical synthesis……………. 17

2.3.2.1.5. Antibiotics produced by semi-synthesis………………… 17

2.3.2.2. According to their effect…………………………………… 18

2.4.Fermentation techniques used in antibiotics production………... 18

2.5. Some important fungal diseases of Sudanese crops…………… 18

2.5.1. Alternaria alternata Tomato early blight……………………. 18

2.5.2.Alternaria leaf spot of sesame………………………………... 20

2.6.Macrophomina phaseolina sesame charcoal rot………………... 21

2.7.Drechslera leaf spot of Sorghum…………………….................. 22

3.Materials and Methods………………………………………….. 23

3.1.Source and Isolation of actinomycetes isolates………………… 23

3.2.Isolation of some phytopathogenic fungi for in-vitro and in vivo

bioassays……………………………………………………….

24

3.2.1.Blotter Method………………………………………………... 25

3.2.2.Tissue transplanting method………………………………….. 25

3.2.3.Single spore isolation of the suspected pathogenic fungi ……. 26

3.2.3.1. Isolation of Drechslera halodes, Alternaria alternata, and

Alternaria sesami …………………………………………..

26

3.2.3.2.Single spore isolation of Macrophomina phaseolina………. 26

3.3.Characterization and identification of the tested fungi…………. 27

3.4.Testing the virulence of the isolated phytopathogens…………... 27

3.4.1.Pathogenicity of Drechslera halodes on sorghum plants…….. 27

3.4.2.Pathogenicity of Alternaria alternata and A. sesami on

tomato and sesame plants…………………………………….

28

3.4.3.Pathogenicity of Macrophomina phaseolina on sesame

plants………………………………………………….............

28

3.5.Preliminary screening of Streptomyces isolates for antifungal

activity………………………………………………………….

29

3.6.Characterization of the active Streptomyces isolates…………… 29

3.6.1.Cultural characteristics of the active Streptomyces isolates … 30

3.6.2.Microscopical characteristics…………………………………. 30

3.6.2.1.Gram straining……………………………………………… 30

3.6.2.1.Mycelial morphology……………………………………….. 31

3.6.2.3.Motility test……………………………………………......... 31

3.6.3.Biochemical Tests…………………………………………….. 31

3.6.3.1.Oxidase test……………………………………………......... 31

3.6.3.2.Catalase test………………………………………………… 32

3.6.3.3.Utilization of different carbon sources……………………... 32

3.6.3.4.Aerobiosis…………………………………………………... 32

3.6.3.5.Starch hydrolysis test……………………………………….. 33

3.6.3.6.Urease test…………………………………………………... 33

3.6.3.7.Gelatin liquefaction……………………………………......... 34

3.6.3.8.Organic Acid Test…………………………………………... 34

3.6.3.9.Reduction of nitrate to nitrite……………………………….. 34

3.6.3.10.Casein hydrolysis………………………………………….. 35

3.6.3.11.H2S production…………………………………………….. 35

3.7.Antibiotic production by Streptomyces in submerged cultivation

using Bennet broth………………………….. …………...........

35

3.8.In vitro antifungal activities of R92broth extract……………….. 36

3.9.In vivo anti-fungal activity…………………………………….... 37

4.Results and Discussion…………………………………………... 39

4.1.Isolation of Streptomyces……………………………………….. 39

4.2.Isolation and characterization of plant pathogenic fungi……….. 44

4.2.1Characteristics of isolated fungi……………………………….. 44

4.2.1.1.Drechslera halodes……………………………………......... 44

4.2.1.2.Alternaria alternata………………………………………… 44

4.2.1.3.Alternaria sesami…………………………………………… 44

4.2.1.4.Macrophomina phaseolina……………………………......... 49

4.3.The virulence of the isolated phytopathogens………………….. 49

4.3.1.Symptoms of Drechslera halodes leaf spot on sorghum

plants…………………………………………………………

49

4.3.2.Symptoms of Alternaria alternata early blight on tomato

plants…………………………………………………………

51

4.3.3.Symptoms of Alternaria sesami leaf spot on sesame

plants…………………………………………………………

51

4.3.4.Symptoms of Macrophomina phaseolina charcoal rot on

sesame plants………………………………………………....

51

4.4.Screening of Streptomyces for antifungal activities…………… 52

4.5.Characterization of Streptomyces isolates……………………… 58

4.5.1.Cultural characteristics………………………………………... 58

4.5.2.Microscopical characteristics…………………………………. 62

4.5.3.Biochemical characteristics…………………………………... 62

4.6.In vitro antifungal activities of R92broth extract……………….. 65

4.7.In vivo activity of R92 extract against Sorghum Drechslera leaf

spot and Alternaria tomato early blight………………………..

73

4.8.In vivo effect of R92 broth extract on the development of

sorghum leaf spot and tomato early blight symptoms…………

73

4.9. Summary and Recommendations ……………………………... 79

5.References………………………………………………………... 82

i

Acknowledgments Most of all I thank "Almighty Allah", with countless thanks and gratitude, for all his blessings, and for giving me this great opportunity to complete this work. I take pride in acknowledging Dr. Adil Ali El-Hussein, Botany Department, Faculty of Science, University of Khartoum. His valuable guidance and encouragement enabled me to carry out this research work successfully. I would like to express profound gratitude to my co-supervisor Dr. Suhair Ahmed Abd Elwahab for her encouragement and guidance during this study. I would like to express my sincere gratitude to Dr. Marmar Abd El-Rahman El-Siddeg, Department of Botany, Faculty of Science, University of Khartoum, for her continuous help and encouragement. I would like to express profound gratitude to Dr. Ashraf for his continuous help Iam also very much thankful to all of my colleagues and friends in the Botany Department Last, but not least, I wish to express my sincere gratitude to members of my beloved family, who supported me in every stage of my life.

ii

Abstract

This study was designed and conducted to evaluate the in vitro and in vivo

inhibitory activities of indigenous Streptomyces isolates against four virulent

locally isolated phytopathogenic fungi: Alternaria alternata (tomato early

blight), Drechslera halodes (sorghum leaf spot), Alternaria sesami (sesame

leaf spot) and Macrophomina phaseolina (sesame charcoal rot).

A collection of 104 Streptomyces isolates were recovered from soil samples

gathered from different regions in Sudan. Streptomyces isolates were

screened for their abilities to inhibit the growth of the four phytopathogens.

Eighty three of the isolates have shown inhibitory effect against one or more

of the tested fungi, the growth inhibition zones recorded were in the range of

1-34 mm, 15 of them produced inhibition zone diameters of more than 10

mm and were considered as potential isolates for control. The remaining 21

isolates showed no inhibitory effect against the tested pathogens.

The potential isolates were furtherly characterized by cultural, microscopical

and biochemical characteristics. Isolates were found to be filamentous with

aerial hyphae differentiating into conidiospore. Filaments were aerobic,

Gram positive, non- motile and were all acid fast negative. All of the isolates

were able to express catalase, oxidase, urease and nitrate reductase. In

addition, they were also able to hydrolyze starch and to utilize rhaminose,

arabinose, glucose, galactose, fructose, sucrose, maltose, lactose and

mannitol as sole carbon sources. However, a wide range of variation was

iii

shown by the isolates in some other biochemical tests such as: production of

H2S on Kilgler Iron Agar medium, hydrolysis of casein, formation of

organic acids and liquefaction of gelatin.

Isolate R92, isolated from Wad Medani (Gezira State), strongly inhibited the

hyphal growth and sporulation of all tested fungi with the greatest inhibition

zone diameters (34mm) recorded against Alternaria alternata.

The broth culture filtrate of isolate R92 was extracted with isopropanol and

the crude extract was tested for in vitro and in vivo activities against the test

fungi. The inhibition zone diameters recorded for R92 broth extract

measured (17-19mm) compared to those recorded for standard commercial

antifungal agents (4-9mm) indicate the strong potency of the former.

The in vivo tests demonstrated that R92 culture broth extract has strong

control efficacy against sorghum leaf spot and tomato early blight. The

crude extract have prevented the incidence of the diseases and suppressed

their development as well.

However, further advanced studies are necessary to identify isolate R92, to

purify the bioactive compound/s produced and to test its in vivo efficacy

under field conditions.

iv

المستخلص

in خلويالو in vitroالمعملي الُمثبطين االثرين لتقييمصممت هذه الدراسة

vivoمن بكتيريا معزولة لسالالت Streptomyces للنبات الممرضة فطرياتمن ال أربع على

تبقع ( Drechslera halodes، )اللفحة المبكرة في الطماطم( Alternaria alternata: وهي

Macrophomina (و) تبقع األوراق في السمسم( Alternaria sesami ، )في الذرة االوراق

phaseolina ذورتفحم ).في السمسم الج

من مناطق مختلفة ُجمعتمن عينات تربة Streptomyces بكتيرياساللة من 104ُعزلت

. تحت اإلختبار الفطريات وثم ُأجري مسح إلختبار مقدرة هذه السالالت على تثبيط نم. بالسودان

أقطار .ثالث و ثمانون ساللة أظهرت أثرًا مثبطًا ضد واحدة او اآثر من األربع فطريات المختبرة

خمسة عشر ساللة حيث سجلت . ملم 34-1 من نطاقات التثبيط لهذه السالالت إنحصرت في المدى

درة ترميلمل العشرةنطاقات تثبيط تفوق أقطار هامن الالت ذات مق برت س ى و اعت ة عل آامن

ة رى ساللة ذآر ان اإلحدى و عشرينالجدير بال .المكافح أي لنمو مثبطًانشاطًا تظهر أي لم األخ

. برةتالمخ الفطريات من

المجهرية و الكيموحيوية للخمسة عشر ساللة , تم التعرف على الخصائص المزرعية

طة اجكشف إلتت ذات هيفات هوائية, آانت السالالت خيطية النش موجبة , ثيم آونيديهجرا نت

، آما أظهرت جميع السالالت مقدرتها على Acid fastغير متحرآة و سالبة لصبغة , لصبغة جرام

، و اإلنزيم المختزل urease ، اليورييزoxidase، االوآسديز catalaseإنتاج إنزيمات الكتاليز

، وإستخدامstarchنشا باإلضافة إلى قدرتها ايضًا على تحلل ال nitrate reductase.للنترات

، Galactoseوز تالجالآ، Glucose الجلكوز، Arabinoseاالرابينوز ، Rhaminose الرامانوز

ول تو المان Lactoseوز تالالآ، Maltoseوز تالمال، Sucroseالسكروز ، Fructoseالفرآتوز

Mannitol إلختبارات آما أظهرت العزالت تباينًا واسعًا في بقية ا. آمصدر وحيد للكربون

v

قدرتها على تحلل ، production H2Sإنتاج آبريتيد الهيدروجين : الكيموحيوية مثل

إسالة الجالتين و acid formation organic ، تكوين األحماض العضوية caseinالكيزين

gelatin liquefaction.

ة ) ية الجزيرة وال( من مدينة ود مدني ةاخوذو التي ُعزلت من عينة التربة الم R92 العزل

ىأظهرت الفطري وآذلك في تثبيط تكوين أبواغ الفطر و ذلك لجميع وفعالية في تثبيط النم أعل

ملم ضد فطر 34تثبيط وهو النطاق ل قطر الفطريات تحت االختبار، و قد سجلت هذه الساللة اآبر

Alternaria alternata.

بواسطة ايزوبروبانول ل على وسط غذائي سائ R92 الساللة يعرزاستخلص ناتج ت

isopropanol نطاق التثبيط قطر ضد الفطريات تحت اإلختبار، آان خلويًاو اختبر معمليًا و

القياسية والتي ملم مقارنًة بالمضادات الفطرية التجارية 19-17 منالمسجل لهذه الساللة في المدى

.ملم مما يدل على قوة فعالية المستخلص 9- 4 سجلت في المدى من

ةأثبتت التجارب الحقلية أن لمستخلص أثرًا قويًا لمنع االصابة بمرض تبقع R92 العزل

االوراق في الذرة ومرض اللفحة المبكرة في الطماطم آما ثبت ايضًا أن لذلك المستخلص أثرًا

.واضحًا في تثبيط تطورهذين المرضين

رورةيتضح من نتائج هذه الدراسة هذه إجراء العديد من التجارب المتقدمة لتشخيص ض

حتى يتسنى هذه الساللة المرآبات الفعالة التي تنتجها /المرآب تنقية الساللة والتعرف عليها آما يلزم

. استخدامها وتطبيقها في التجارب الحقلية بصورة أوسع

vi

List of Tables

Page Table 1: Streptomyces presumptive isolates…………………………. 40

Table 2: Microscopical characteristics of isolated fungi …………..... 46

Table 3: Inhibition zones diameters (mm) shown by different

Streptomyces isolates against plant pathogenic fungi ……

53

Table 4: Cultural characteristics of potential Streptomyces isolates… 59

Table 5: Microscopical characteristics of Streptomyces isolates…….. 63

Table 6: Biochemical characteristics of Streptomyces isolates……… 66

Table 7: Carbohydrate utilization by different Streptomyces isolates.. 69

Table 8: Inhibition zone diameters (mm) shown by R92 broth extract

against phytopathogenic fungi.............................................

71

Table 9: infection % of sorghum and tomato plants treated with

Streptomyces R92 extract before inoculation with the tested

fungal pathogens……………………………………………

74

Table 10: Effect of R92 extract on the development of disease

symptoms on infected sorghum and tomato as reflected by

the diameters of spot area…………………………………

77

vii

List of Plates

Page Plate1: Filamentous mycelium of Streptomyces and their colonies on

GAA…………………………………………………………..

43

Plate 2: Isolation of pathogenic fungi………………………………... 45

Plate 3: Spores and pycnidia of the isolated pathogenic fungi………. 48

Plate 4: Disease symptoms shown by different fungi on different

plant species…………………………………………………..

50

Plate 5: Effect of Streptomyces isolates on different phytopathogenic

fungi………………………………………………………...

56

Plate 6: Colonies of different Streptomyces isolates on GAA medium 60

Plate 7: Microscopical characteristics of Streptomyces isolates……... 64

Plate 8: Biochemical characteristics of Streptomyces isolates……… 67

Plate 9: In vitro antifungal activities of R92broth extract…………… 72

Plate 10: In vivo effect of R92 broth extract on the incidence of

Sorghum Drechslera leaf spot and disease……………

75

Plate 11: In vivo effect of R92 broth extract on the incidence of

tomato Alternaria early blight disease ……………..

76

Plate 12: in vivo effect of R92 broth extract on the development of

sorghum leaf spot and tomato early blight symptoms………………...

78

1

1. Introduction

Streptomyces, Greek adjective streptos (pliant or bent) myces (fungus)

therefore, Streptomyces means pliant or bent fungus (Pridham and Treesner,

1974). Streptomyces species are gram-positive filamentous bacteria that

belong to the Actinommycetales. They are characterized by the ability to

form reproductive mycelium from vegetative mycelium in soil culture

(Kawamoto et al., 2001). The filamentous growth and the branching of

Streptomyces mycelia differentiate these organisms from the true bacteria

(Waksman and Lechevalier, 1953). Commonly, the genus Streptomyces has

slender coenocytic hyphae, the aerial mycelium at maturity forms chains of

three to many spores (Goodfellow and Cross, 1984).

Members of the genus are soil inhabitants. They are common in wet than in

dry areas having a pH of about 6.5-8.0, and with the exception of few

species that cause mycetoma; Streptomyces are saprophytes (Murray et al.,

1995). They may be found on vegetation, food products, manures, peat,

water basins, composts, silage, fresh water and river bottoms, dust and plant

residues (Waksman, 1950; Waksman and Lechevalier, 1953).

For many years, members of Actinomycetales were classified with fungi,

with which they share parallel evolution, but to which they are completely

unrelated (Levy et al., 1973). Various keys for the identification of

Streptomyces have been suggested and the most common of them employs

four criteria: colour of aerial mycelium, spore chain morphology, structure

of spore surface, and melanin formation. Pridham (1976) used utilization of

carbon sources and the nature of secondary metabolites production

(principally antibiotics) for recognition of species. The data of the

2

International Streptomyces Project (ISP) recognized 450 species of

Streptomyces and Streptoverticillium (Shirling and Gottlieb, 1972; Kim et

al., 2004).

Streptomyces is the largest antibiotic-producing genus in the microbial

world discovered so far. Of the nine thousand antibiotics used against

bacteria and fungi, 66% are produced by members of Streptomyces (Kron-

Wendisch and Kutzner, 1992). Fungi produce a large number, contributing

approximately 18% of the antibiotic producers and yielding about 8% of the

total (Champness, 2000). The number of antimicrobial compounds reported

from the genus Streptomyces per year has increased almost exponentially for

about two decades. Reports have shown that this group of microorganisms

will remain an important source of antibiotics (Watve et al., 2001).

Generally, new bioactive products from microbes continue to be discovered

at an amazing pace: 500 per year (Dworkin et al., 2006).

As a result of the increasing prevalence of antibiotic-resistant pathogens and

the pharmacological limitation of antibiotics, there is exigency for new

antimicrobial substances. In fact, many of the known antibiotics produced by

members of the family Bacillaceae are polypeptides, which have proven

generally to be somewhat unstable and difficult to purify. Antibiotics

produced by fungi, with a few notable exceptions, are generally found to be

too toxic for treatment of eukaryotes including plants (Casida, 1968).

However, the antibiotics produced by Streptomyces are comparatively

recognized as generally safe and stable (O'Grady et al., 1997). For this

reason Streptomyces screening for the production of new antibiotics has

been intensively pursued for many years by a number of scientists

(Waksman, 1961; Lacey, 1973; McCarthy and Williams, 1990; Saadoun and

Gharaibeh, 2003 and Wu et al., 2009). This resulted in the characterization

3

and purification of about 6000 distinct antibiotic substances from

Streptomyces species (Champness, 2000).

Among the different types of drugs prevailing in the market, antifungal

antibiotics are very few but significant and have an important role in the

control of mycotic plant and animal diseases (Dhanasekaran et al., 2008).

The search for new, safer, broad- spectrum antifungal agents with greater

potency has been progressing. The reason for this is that when compared to

antibacterial, fungi, like plant cells, are eukaryotes and therefore agents that

inhibit protein, RNA or DNA biosynthesis in fungi have greater potential for

toxicity on plant as well (Georgopapadakou and Tkacz, 1994).

Recent reports have shown that Streptomyces continue to remain an

important source of antifungals examples included: 24-Demethyl-

bafilomycin C1 (Lu and Shen, 2004), Levorin (Kozhuharova et al., 2008),

Phenyl - 1- napthyl- phenyl acetamide and DPTB16 (Dhanasekaran et al.,

2008), and (6S,8aS,9S,11S,12aR)-6-hydroxy-9,10-dimethyldecahydrobenzo

[d] azecine- 2,4,12(3H)- trione (Wu et al., 2009).

About 80% of plant diseases can be traced to fungi (Someya, 2008). In

Sudan; fungi infect some important crops and cause serious diseases that

lead to great losses in the production of these crops. Examples include:

Alternaria early blight on tomato, Alternaria leaf spot on sesame,

Macrophomina charcoal rot on sesame, Drechslera leaf spot on sorghum,

Colletotrichum tissue necrosis in beans, D. maydis leaf blight on corn,

(Kendrick, 2001) and Fusarium wilt caused by Fusarium spp. (Kim, et al.,

2005).

Continuous screening of Streptomyces for secondary metabolites production

can possibly reveal a novel antifungal agent which can be used to treat one

or more of such plant diseases. Hence the main objective of this study has

4

been screen locally isolated Streptomyces for production of potent antifungal

agents that can be used to control some selected important fungal plant

pathogens. The practical steps to a chieve this goal is:

1. Selective isolation of Streptomyces species from different soils.

2. Screening of these isolates for antifungal agents production by the

agar debussing method using different plant pathogenic fungi isolated

from different infected plants.

3. Characterization of the potential isolates using cultural, microscopical,

and biochemical traits.

4. Production of the antifungal agent by Streptomyces in submerged

culture using Bennet broth as a production medium.

5. Extraction of the antifungal agent from the fermentation broth.

6. Studying the in vivo antifungal effect of the potential extracts on

seedlings infected by locally isolated and identified phytopathogenic

fungi.

5

2. Literature Review

2.1 The Actinomycetes:

Microorganisms are found in the soil, air, and water. They represent an

important class of biodiversity. Although some of the microorganisms are

pathogenic, much of them are beneficial to humanity in different aspects

including, degradation of organic materials, nitrogen fixation, production of

vitamins, enzymes and antibiotics.

The first chemotherapeutically effective antibiotic (penicillin) was

discovered by Alexander Fleming in 1929. Since then about nine hundred

antibiotic substances are known, the majority (66%) is produced by

members of the genus Streptomyces which belongs to the eubacterial order

Actinomycetales (Champness, 2000; Roubos et al., 2001).

Actinomycetes are Gram positive bacteria; they are widely distributed in

terrestrial environments, from which they are easily isolated (Meyers et al.,

2003). They produce fine mycelium; its hyphae never exceed 1.5µm

(Labeda et al., 1997). Many Actinomycetes produce spores that are different

from the endospores of Bacilli, not only in the method of formation but also

in being mildly resistant to heat. The combination of aerial growth and

sporulation usually confer a cottony or powdery texture on the surface of the

colony. Colonies which lack aerial mycelium are either glossy or matt

(Karandikar et al., 1997).

Actinomycetes have considerable economic importance and in nature they

contribute to the mineralization of organic residues. Only a few are plant

pathogens but a number cause debilitating or even lethal infections of animal

6

and Man. They produce 85% of the known antibiotics including substances

active against bacteria, fungi, protozoa, rickettsiae, viruses and neoplasmas.

They also generate a great variety of biologically important substances in

their environment such as vitamins, pigments and enzymes (Tetsuo et al.,

2000).

According to Lacey (1973) the order Actinomycetales contains four families

namely: Mycobacteriaceae, Actinomycetaceae, Actinoplanaceae and

Streptomycetaceae. This latter family forms true vegetative mycelium which

produces conidiospores and does not fragment into small segments.

Streptomycetaceae comprises three genera as follows:

1- Micromonospora: conidia formed singly at the terminal end of short

conidiophores, never in chains of spores, no growth at 50 to 60ºC.

2- Thermoactinomyces: Similar to Micromonospora except for growth

at 50-65ºC.

3- Streptomyces: conidia in chains on aerial hyphae.

2.2 Streptomyces:

The genus Streptomyces comprises the most mold-like of Actinomycetes

that it produces conidia on aerial hyphae. Like some mold colonies, the

surface of the Streptomyces colony has a powdery appearance. The branched

cells are aerobic, gram-positive and some species are thermophilic. The cell

wall of Streptomyces contains peptidoglycan and is consequently susceptible

to lysozymes (Levy et al., 1973).

7

2.2.1 Classification of Streptomyces:

The 7th edition of Bergey’s Manual of Systematic Bacteriology classified

Streptomyces as follows:

Division: Protophyta-primitive plants.

Class: Schizomycetes- bacteria.

Order: Actinomycetales (slender, often branching, mold-like cells which

may form spores).

Family: Streptomycetaceae (conidia formed on sporophores as described by

Wyss et al., 1963 and Kmpfer, 2006).

2.2.2 Life Cycle of Streptomyces:

The life cycle of Streptomyces begins with the germination of a single spore,

which produces one or more multi-nucleoid filaments (Hardisson et al.,

1978). This will elongate and branch on the surface and into the substrate or

culture medium to form a vegetative mycelium. Hyphal growth is by quasi-

exponential growth kinetics (Chater, 1993). The complex network of

filaments will continue penetrating the medium, utilizing the available

organic molecules with the use of extracellular hydrolytic enzymes. This

motility of the Streptomyces vegetative filaments gives it a big advantage

compared to other less motile bacteria when it comes to colonizing solid

substrates in the soil. In response to appropriate signals, believed to include

the exhaust of nutrient supplies in the surrounding environment, the

substrate mycelium will break the surface barrier and aerial hyphae are

formed. Aerial growth coincides with the onset of secondary metabolism in

cultures grown on solid media (Chater, 1989). The continuation of the aerial

growth is supported by the utilization of the vegetative mycelium. When the

extension of the aerial hyphae stops; their multigenomic tips undergo

8

synchronous multiple septation to give rise to unigenomic prespore

compartments (Schwedock et al., 1997; Ryding et al., 1998). Mature spores

are held together in chains of about 50 and they develop a characteristic grey

pigment as they mature (McGregor, 1954). Unlike the endospores of other

Gram-positive bacteria, such as Bacillus and Clostridium, Streptomyces

exospores are not resistant to extreme heat or pH and are less dormant;

however, they are fairly resistant to desiccation.

2.2.3 Habitat:

Most Actinomycetes are saprophytic occurring both at the soil surface and at

deeper levels, and forming an important part of the soil microflora. The

characteristics odour of wet soil is supposed to be due to them. They are

particularly numerous in dry alkaline soils, in heavily limed arable soils and

in soils rich in organic matter. They are also abundant in composts, in lake

water and in mud at lake bottoms (Alexander, 1967). Some species, in

manures and composts, are thermophilic and thrive temperatures of 50-65ºC

(Lacey, 1973). Streptomyces are also common on plant remains and as

epiphytes on plants. They have been found in milk and other food stuffs

(Hawker et al., 1960). Several species of Streptomyces are involved in a

symbiotic relationship with species of ants in the genus Attini.

The Streptomyces optimum pH for growth is 6.5-8.0 and the optimum

temperature is about 25-35 ºC although some species can grow at

temperatures within the thermophilic range. The conidiospores, however, are

slightly more temperature resistant than vegetative cells of most bacteria;

heating at 65ºC will kill them in 10 to30 minutes (Locci, 1990).

9

Although antibiotic activity may significantly affect interaction among soil

microbes, information on the ecology of antibiotic-producing microbial

populations in soil is limited. Indeed, the factors that predict the presence of

strong antibiotic inhibitory or resistance activities within the soil microbial

community are poorly understood (Davelos et al., 2004). Actually the

potential effects of antibiotic-producing bacteria on plant health in both

agricultural and non-agricultural soils have been studied (Bent et al., 2003;

Vogel et al., 2003 and Franklin and Mills, 2003).

2.2.4 Nutrition:

The wide range of habitats occupied by Actinomycetes indicates that they

are able to utilize a variety of substances as nutrients. The importance of the

antibiotics produced by this group has led to detailed investigations on the

nutritional requirements of certain species in an attempt to simplify the

medium used in antibiotics production.

As corresponds to their habitat, these bacteria are nutritionally quite versatile

and produce extra cellular hydrolytic enzymes including proteolytic

enzymes that permit the utilization of high molecular weight biopolymers

such as proteins, polysaccharide, fats and other substrates (Nikolova et al.,

2005).

Carbohydrates, such as glucose, maltose, starch and sucrose, certain organic

acids, glycerol, alcohols, amino acids, aromatic compounds and simple

proteins may be used by most species as a source of carbon. Some are able

to decompose and use agar as a source of carbon and energy.

Many species of Streptomyces utilize, with a consequent release of ammonia

which may rapidly make a culture medium too alkaline for further growth,

10

organic nitrogenous substances, including complex proteins. Usually

ammonium salts are less suitable sources of nitrogen. Phosphorus,

potassium, magnesium and other minerals, including traces of zinc and other

heavy metals, are required by Actinomycetes as by other microorganisms

(Balagurunathan, and Radhakrishnan, 2007). This metabolic diversity is due

to their extremely large genome which has hundreds of transcription factors

that control genes expression (Sumby and Smith, 2002; Microbewiki, 2009).

2.2.5 Isolation of Streptomyces:

Streptomyces can be isolated from a wide variety of habitats, but most

isolation procedures involve extraction from soil and cultivation on solid

media. Isolation usually requires an enrichment step followed by plating of a

serial dilution on selective media under specific isolation conditions (Manfio

et al., 1995). The Glycerol Arginine Agar (GAA) medium was found to be

superior to other media, resulting in higher number and proportion of

streptomycete colonies (El-Nakeeb and Lechevalier, 1963).

It has been found that heat treatment of soil (40-50ºC, 2-16 h) leads to a

significant reduction of most bacteria without affecting the colony counts of

Streptomyces (Williams et al., 1972). The addition of CaCO3 (10:1 w/w) to

air-dried soil samples and the subsequent incubation at 26ºC for 7-9 days in

a water-saturated atmosphere can lead to a 100-fold increase of

Streptomycete colonies on isolation plates (Kieser et al., 2000).

The use of media supplemented with an antifungal agent has also been

widely used to suppress fungal growth. The mostly used antibiotics are

Cycloheximide (actidione, 50-100 µg/ml), Pimaricin and Nystatain (each 10-

50 µg/ml) (Williams and Davies, 1965). The use of compounds with

antibacterial activity is restricted because actinomycetes are also sensitive to

11

them. Williams and Davies (1965) found that bacterial flora including

Streptomyces may be suppressed by Polymyxin B (5 µg/ml) and Penicillin (1

µg/ml).

A medium containing Cycloheximide and Nystatin (each 50 µg/ml) to

control fungal growth and Oxytetracycline (25µg/ml) to suppress

Streptomyces and other Actinomycetes genera was suggested by Hanka et al.

(1985), for the selective isolation of streptoverticil-producing

Actinomycetes.

2.2.6 Importance of Streptomyces:

The most interesting property of Streptomyces is its ability to produce

secondary metabolites including antibiotics and other bioactive compounds.

These metabolites are of great value in human and veterinary medicine,

agriculture and as unique biochemical tools (Demain, 1999). Structural

diversity is generally observed in these metabolites that encompass not only

antibacterial, antifungal, antiviral, antimalarial and antitumor compounds

but also metabolites with immunosuppressant, antihypertensive,

antihypercholesterlemic properties (Prescott et al., 1993; Hayakawa et al.,

1996, and Isaka et al., 2002).

A major factor in its prominence as producer of a variety of antibiotics is its

possession of several metabolic pathways for biosynthesis (Arisawa et al.,

1996). Omura et al. (2001) reported the existence of at least 8.7 million base

pairs in the chromosome of Streptomyces avermitilis, this is the largest

bacterial genome discovered so far. The report provides insights into the

intrinsic capabilities of Streptomyces for production of diverse metabolites.

12

Another area in which the production of secondary metabolites by

Streptomyces has proved important is their incorporation into livestock feed

and their use as food preservative (Prescott et al., 1993).

In addition to over six hundred antibiotics, different Streptomyces species

liberate extracellular enzymes such as: proteases (Yang and Wang, 1999); β-

glucosidases (Ozaki and Yamada, 1991); α-amylase (Vukelic et al., 1992);

chitinases (Gupta et al., 1995) and cellulases (Nishio et al., 1981).

Although most Streptomyces are non-pathogenic saprophytes, a few are

associated with plant and human diseases. Streptomyces scabies causes scab

disease in potato and beets (Walker, 1952). In 1954, Brian, listed ninety-six

distinct antibiotics produced by fungi alone and of these, over half were well

characterized. Some, such as griseofulvin and cycloheximide, have been

commercially produced and used as fungicides. Wallen (1955) reported a

promising control of stem rust of wheat by cycloheximide however

phytotoxicity and considerable mammalian toxicity limit the usefulness of

cycloheximide in crop protection.

Streptomyces somaliensis is the only Streptomycetes known to be

pathogenic for humans. It is associated with actinomycetoma, an infection of

subcutaneous tissues that produces tensions and leads to swelling, abscesses

and even bone destruction if untreated (Prescott et al., 1993).

2.3 Antibiotics:

Microbial secondary metabolites are substances that are mainly produced by

microbial genera inhabiting soil and undergoing morphological

differentiation such as actinobacteria, bacilli and fungi, they are not needed

for growth or for other essential process in the cell (Vining, 1990).

Secondary metabolites are reported to have different biological roles giving

13

their producers competitive advantages in microbial community (Gregory

and David, 2003). There are over 23,000 known microbial secondary

metabolites, 42% of which are produced by actinobacteria, 42% by fungi

and 16% by other bacteria (Lazzarini et al., 2001).

Antibiotics are among the most important secondary metabolites produced

by microorganisms. They have low molecular weight and often have unusual

chemical structure that interferes with specific activities in certain types of

organisms. There are many theories trying to explain the reason for

antibiotic production by certain organisms. The most widely accepted one

suggests that antibiotics help the organism to compete with other organisms

in relatively nutrient-depleted environment of the soil. Other hypothesis

suggests the role of the antibiotics synthesis in reducing the redundant level

of intermediates accumulated in the cell after the growth stops (Maplestone

et al., 1992).

2.3.1 Major groups of antibiotics and their mode of action:

2.3.1.1 Beta –Lactam group:

This group of antibiotics includes: Penicillins and Cephalosporins (Hugo and

Russell, 1998). Penicillin is produced by fermentation of molds such as

Penicillium notatum and Penicillium chrysogenum. The most important

Penicillins are the Benzl Penicillin. The Cephalosporins now available have

similar antibacterial activities but are stable at acid pH.

The beta-lactam antibiotics inhibit the last steps in peptidoglcan synthesis,

and hence impede cell wall formation (Stanier et al., 1977; Stephen et al.,

2004).

14

2.3.1.2 Aminoglycosides:

Members of this group contain cyclohexane rings and amino sugars. They

include: Streptomycin, Dihydrostrepomycin, Neomycin, Tobramycin,

Framycetin, Kanamycin and Paromomycin (all produced by Streptomyces)

and Gentamicin which is produced by Micromonospora purpurea. (Hugo

and Russell, 1998).

This group of antibiotics inhibits the bacterial protein synthesis by binding to

the 30S subunit of the bacterial ribosome causing misreading of the genetic

message carried by mRNA, preventing the transfer of the activated amino

acids to the ribosome and consequently blocking the elongation of the

peptide chain.

2.3.1.3 Tetracyclines:

The Tetracyclines consists of eight members, and may be considered as a

group of antibiotics obtained as by-products of the metabolism of various

species of Streptomyces. The Tetracyclines are broad- spectrum antibiotics;

they have a wide range of activity against Gram-positive and Gram-negative

bacteria.

Tetracyclines, like Aminoglycosides, target the bacterial ribosomes and bind

to the 30 S subunit, inhibit binding of aminoacyl- t RNA to ribosomal A site

(Greenwood, 1997).

2.3.1.4 Macrolides:

The Macrolide antibiotics are characterized by possessing molecular

structures that contain large lactone rings linked through glycosidic bonds

with amino sugar. The most important members of this group are

Erythromycin, which is produced by Streptomyces erythraeus (Prescott et

15

al., 1993). Macrolides inhibit bacterial protein synthesis by binding to the

23S rRNA of the 50S ribosomal subunit and so inhibit peptide chain

elongation during protein synthesis (Atlas, 1989).

2.3.1.5 Sulphonamides and Trimethoprim

These are synthetic chemicals belonging to a group of chemically related

compounds, which have the capacity to inhibit the metabolic activities of

certain bacteria. They are structurally related to Sulfanilamide, an analogue

of P-amino-benzoic acid. The latter substance is used in the synthesis of the

cofactor folic acid. Sulfonamides antibiotics compete with P-aminobenzoic

acid for the active site of the enzyme involved in folic acid synthesis. The

failure to synthesize folic acid is detrimental to bacterial cell because folic

acid is essential for the synthesis of purines and pyrimidines used in the

construction of DNA, RNA and other important cell constituents (Prescott et

al., 1993).

2.3.1.6 Rifamycins:

The Rifamycins which are produced by Streptomyces comprise a

comparatively new antibiotic group, and consist of Rifamycin A, B, C, D,

and E. They are active against Gram-positive bacteria including

Mycobacterium tuberculosis and Gram-negative bacteria.

Rifamycins are extremely efficient inhibitor of the bacterial enzyme DNA

dependant RNA polymerase; thus blocking RNA synthesis (Greenwood,

1997; Stephen et al., 2004).

16

2.3.1.7 Polypeptides:

The polypeptide antibiotics comprise a rather diverse group. They include

Bacitracin and Polymyxin which are produced by Bacillus species. These

antibiotics bind to the cell membrane and disrupt its structure and

permeability properties (Stanier et al., 1977).

2.3.1.8 Azoles:

This group which contains an azole ring, include Metronidazole. They act by

the production of short-lived intermediate compounds which are toxic to

DNA (Arisawa et al., 1996). These groups also act by disruption of fungal

cell membrane, causings leakage of cytoplasmic content (Ghannoum and

Rice, 1999).

2.3.2 Classification of antibiotics:

There are several methods used to classify antibiotics, the most common

methods are as follows:

2.3.2.1 According to their origin:

2.3.2.1.1 Antibiotics produced by Streptomyces:

The majority of antibiotics used today are produced by different species of

Streptomyces, examples include: Streptomycin (S. griseus),

Chloramphenicol (S. venezuelae), Tetracycline (S. rimosus), Amphoterricin

B (S. nodusus), Avermectin (S. avermitilis), Erythromycin (S. erythraea),

Viderabine (S.antibioticus), Doxorubicin (S. peucetius), Kanamycin (S.

kanamyceticus), Nystatin (S.noursei) and Carbomycin which is produced by

S.halstedii (Atlas, 1989; Prescott et al., 1993; Newman et al., 2003; Berdy,

2005).

17

Among the Streptomyces, both the quantity and types of antibiotics produced

vary widely among individuals of the same species (Vining, 1990; Hotta and

Okami, 1996). Many Streptomyces produce more than one antibiotic, for

example, Streptomyces coelicolor produces four antibiotics (Actinorhodin,

Undecylprodigiosin, Methylenomycin, and a calcium-dependent antibiotic

(Hopwood, 1988).

2.3.2.1.2 Antibiotics produced by other bacteria:

These include, Gentamicin (Bacillus brevis), Bacitracin (Bacillus

linheniformis), Polymyxin-B (Bacillus polymyxa), Bacteriocins (Escherichia

coli) and Gentamycin which is produced by Micromonospora purpurea

(Prescott et al., 1993).

2.3.2.1.3 Antibiotics produced by fungi:

Antibiotics produced by Fungi include, Penicillin (Pencillium chrysogenum

and P. notatum), Griseofulvin (Pencillium griseofulvium), Cephalosporins

(Cephalosporium spp.) and Gliotoxins which is produced by Aspergillus

fumigatus (Prescott et al., 1993; Parekh et al., 2000).

2.3.2.1.4 Antibiotics produced by chemical synthesis:

Certain antibiotics are produced wholly through chemical synthetic

processes under specific laboratory conditions, for example

Chloroamphenicol (Stephen et al., 2004).

2.3.2.1.5 Antibiotics produced by semi-synthesis:

In this case, part of the molecule is produced by microbial activity, such as

fermentation process; and the product is then modified by a chemical

process under laboratory conditions (Hugo and Russell, 1998).

18

2.3.2.2 According to their effect:

Antimicrobial agents can affect cells in a variety of ways. Agents that kill

cells are usually given names ending with –cide (for example, fungicide,

bactericide, and so on), whereas those that inhibit growth without directly

killing the cells are given names ending with –stat (for example, fungistatic,

bacteriostatic, and so on) and agents that actually cause cell lysis are called

lytic agents like bacteriolytic (Brock, 1970).

2.4 Fermentation techniques used in antibiotics production:

There are several techniques which are employed for the production of

antibiotics from microorganisms: the first method is the surface culture in

which the producing microorganism is grown on the surface of a liquid

culture medium contained in small vessels (Atlas, 1989). The second method

is the deep submerged culture, in which the producer organism is grown in

deep aerated fermentation tanks and conditions are adjusted to enable mass

growth and production deep within small or large volume media (Prescott et

al., 1993). Alternatively, solid-state fermentation technique is preferably

employed for antibiotics production using cheap solid substrates. In this

respect, Yang and Ling (1989) reported the production of Tetracycline by

solid- state fermentation using sweet potato, whereas Yang and Swei (1996)

used corncob as a solid substrate for the production of oxytetracycline.

2.5 Some important fungal diseases of Sudanese crops:

2.5.1 Alternaria alternata Tomato early blight:

Tomato (Solanum lycopersicum L. “ syn Lycopersicon esculentum Mill.”) is

one of the most important fruit vegetables for humans, and are cultivated

19

across all countries in fields or in protected shelters. It is a member of the

family Solanaceae (Rotem, 1994). Presently, tomato is becoming

increasingly important in Sudan for local consumption and for export. It is

cultivated throughout the year under irrigation in an area that exceeds 36540

hectares with an average yield of 17.57 tons per hectare (AOAD, 2007). The

most important grown cultivars are the canning types such as Strain B,

Strain C, Peto86, Peto111 and CastleRock in addition to few local varieties.

Tomato is a rich source of antioxidants which counteract the adverse effects

of oxidative stress and lead to improved immune function and reduce risk of

infectious diseases (Fawzi et al., 2000).

Tomato is susceptible to many diseases especially fungal diseases, the most

important of which in Sudan is the early blight. The disease is caused by

Alternaria solani (Ellis and Martin 1882) in the humid areas of the world

and by Alternaria tenuis in the drier parts (Kapoor and Hingorani, 1958;

Tandon and Chalurvedi, 1965). In Sudan, Ahmed (2007) identified

Alternaria alternata as the causal agent of tomato early blight in Al saggay,

North Khartoum.

The disease can occur at all stages of the plant growth causing the damping

of seedlings, leaf spots, later collar rot, stem lesions and fruit rot. Infection

of the plant can result in complete loss of the crop as this disease can lead to

complete defoliation of tomato (Peralta et al., 2005).

The early symptoms of the early blight disease appear on the lower

senescent leaves as dark necrotic lesions, then the disease progress upward

as the plant becomes older. The lesions become larger and commonly

showing concentric rings surrounded by a yellow zone (Sherf and MacNab,

1986). Brown to black spots, ¼ to ½ inch in diameter with dark edges,

appear on lower leaves. Spots frequently merge, forming irregular blotches.

20

Leaves turn yellow and dry up when only a few spots are present. The

fungus occasionally attacks fruit at the stem end, causing large, sunken areas

with concentric rings and a black, velvety appearance (Jones, 1991). The

heavy infection on fruits leads to their dropping before maturity. On the

susceptible genotypes the calyx and blossom may also be infected (Pandey

et al., 2003). The causal organism is classified according to Ellis (1974) as

follows:

Sub-division: Deuteromycotina

Class: Hyphomycetes

Order: Moniliales

Family: Dematiaceae

Genus: Alternaria

Species: A. alternata (Fr). Keissler

A. tenuis

A. solani

The Ridomil gold MZ68WG; was used to control early blight in Sudan.

2.5.2 Alternaria leaf spot of sesame: Sesame (Sesamum indicum) L: syn.S.orientale L. is an important oil crop

which originated in East Africa and India (Bedigian, 1985). In 2000, over 15

million acres (6.2 million hectares) were allotted for sesame world wide.

Africa grows 15% of the world’s sesame, in Sudan, Uganda and Nigeria. In

Sudan sesame is cultivated in an area of about 1,450,000 hectares with an

average production of 220,000 metric tons (USDA, 2000).

Diseases of sesame include several fungal leaf spot including that of

Alternaria sesami. The symptoms appear mainly on the leaf blade as small,

brown, round to irregular spots and are responsible for losses in grain yield

21

of the crop (Smith, 1999). Later on the spots enlarge and turn dark with

concentric rings, the appearance of the disease at the seedling stage can

cause post emergence damping off (Saharan et al., 2005). The causal

organism is classified according to Ellis (1971) as follows:

Sub-division: Deuteromycotina

Class: Hyphomycetes

Order: Moniliales

Family: Dematiaceae

Genus: Alternaria

Species: A. sesame

2.6 Macrophomina phaseolina sesame charcoal rot:

Charcoal rot is caused by Macrophomina phaseolina (Tassi) Goid. The

disease is one of the most widespread diseases throughout sesame growing

areas. The fungus can infect the root and lower stem of over 500 plant

species and is widely distributed in the United States (Wyllie, 1988).

Charcoal rot is an important disease during hot, dry weather or when

unfavorable environmental conditions stress the plant. Infected seedlings

show a reddish brown root discoloration which extends up to the stem and

turns dark brown to black. Foliage of infected seedlings can appear off-

color or begin to dry out and turn brown. A twin-stemmed plant may

develop if the fungus kills the growing point. Under cool and wet conditions,

young plants that are infected may survive but carry a latent infection that

will express symptoms later in the season with hot, dry weather. The

microsclerotia are released into the soil as infected tissue decays (Bowers

and Russin, 1999). The complete classification of Macrophomina

phaseolina according to Ainsworth (1973) as follows:

22

Sub-division: Deuteromycotina

Class: Coelomycetes

Order: Sphaeropsidales

Family: Sphaeropsidaceae

Genus: Machrophomina.

Species: phaseolina

2.7 Drechslera leaf spot of Sorghum:

Sorghum (Sorghum bicolor (L) Moench) is recognized as an important crop

throughout the arid tropical and sub-tropical regions of Africa, Asia, and

Central America. In Sudan, sorghum is admittedly considered the most

important cereal crop for nutrition, especially in regions where low rain fall

prevails. The most important production areas of the crop include Eastern

and Western parts of the country (rain fed areas), and under irrigation in

Gezira Scheme and Northern and Western parts of the Sudan (Badi and

Monawar, 1987).

Drechslera leaf spot symptoms appear as a well defined and elongated spots,

which vary in size and color according to sorghum genotype (Borges, 1983).

Leaf spot of Sorghum bicolor (L.) Moench is caused by Drechslera halodes.

(Riccelli, 1980), which is classified as follows:

Sub-division: Deuteromycotina

Class: Hyphomycetes

Order: Moniliales

Family: Dematiaceae

Genus: Drechslera

Species: D. halodes

23

3. Materials and Methods

3.1. Source and isolation of actinomycetes isolates:

Streptomyces used throughout this study were isolated from different soil

samples collected from different locations in 11 States in Sudan. These

locations included: Gureir, Marawi, Dongola (Northern State), Shendi, Al

hosh, Berber, Atbara, El Moswarat (River Nile State), Shambat, beaches of

Blue and White Niles, AL saggay, El kadaro, Jabal Auliaa, Um Dowm

(Khartoum State), El Gedarif, El Fao (Gedarif State), Kassala (Kassala

State), Um Siyala, Sawdiri, El Mazroob, Bara (Northern Kordofan State),

Kenana, Asalaya (White Nile State), Waw, Gabal Khair, El Goor (Western

Bahr Al Ghazal State), El Hilaliya, El Halawen, Madani (Gezira State), , El

Damazeen (Blue Nile State), and areas in Northern Baher El Ghazal State

(Table1). The soil samples were taken from a depth of 15-20 cm after

removing approximately 3 cm of the earth surface, and were then air-dried at

room temperature for two days.

Isolation of Streptomyces was performed by the soil dilution plate technique

(You and Park, 2004). In this technique, one gram of each soil sample was

suspended in nine ml of sterilized distilled water in a pre-sterilized test tube.

Serial aqueous dilutions (10-1-10-7) were prepared by transferring one ml of

the soil suspension into nine ml sterilized distilled water in sterilized test

tubes. 0.1ml from dilutions 10-4, 10-5 and 10-6 were taken and applied

separately into sterilized Petri-dishes and 20ml of warm melted (about 50ºC)

Glycerol Arginine Agar (GAA) medium was then added. Plates were gently

rotated and were then incubated at 27 ºC for 7-14 days.

24

GAA medium was prepared by dissolving in a two litre Erlenmeyer flask

(g/L) 20.0 agar, 12.5 glycerol, 1.0 arginine, 1.0 dipotassium hydrogen ortho-

phosphate, 1.0 sodium chloride, 0.5 hydrated magnesium sulphate, 0.01

hydrated ferric sulphate, 0.001 hydrated copper sulphate, 0.001 hydrated

manganese sulphate and 0.001 hydrated zinc sulphate. Flasks were then

plugged with cotton and autoclaved for 15 minutes at 121ºC (151b/inch2).

The medium was then supplemented with 100ml filter sterilized Nystatin

(50µg/ml) and 100 ml Chloramphenicol (1µg/ml) to inhibit the growth of

fungi and some other non-filamentous bacteria, respectively.

Colonies characteristic of Streptomycetaceae (rough, chalky and with earth

odour) that appeared on the incubated plates were selected, repeatedly

subcultured for purification and preserved in the maintenance medium at

4ºC. Maintenance medium (Glycerol Asparagine Agar) was prepared by

dissolving 20.0g agar, 10.0g glycerol, 1.0g L-aspargine, 1.0g dipotassium

hydrogen ortho-phosphate in one litre of distilled water. One m1 of filter-

sterilized trace elements solution was then added to it. The trace elements

solution was prepared by dissolving 0.1g hydrated ferrous sulphate, 0.1g

hydrated manganese chloride and 0.1g hydrated zinc sulphate in 100ml

sterilized distilled water. Slants of this medium were prepared by dispensing

six ml of molten Glycerol Asparagine Agar into test-tubes. The tubes were

autoclaved at 121ºC (151b/inch2) for 15 minutes and were left to cool in

slanted way and were inoculated each with one of the isolates.

3.2 Isolation of some phytopathogenic fungi for in-vitro and in vivo

bioassays:

Seeds and plant parts were collected in polythene bags from infected crop

plants including Sorghum (Sorghum bicolor (L) Moench, sesame (Sesamum

25

indicum L: (Syn.S.orientale L.), and tomato (Lycopersicon esculentum L.).

The Standard Blotter method was used for isolation of seed-borne fungi

while the Tissue Transplanting method was used for the purpose of isolating

fungi from infected plant parts.

3.2.1 Blotter method:

Seeds were disinfected by dipping in 4% sodium hypochlorite solution for 3

min and were then washed in five changes of sterilized distilled water. Seeds

were sown at fixed distances (according to the size of the seeds) onto

sterilized moistened filter paper (Blotter) placed in glass Petri dishes (9 cm

diameter). The Petri-dishes were incubated for 4-7 days at 28ºC in cooled

incubator. The blotters were kept moistened throughout by aseptically

adding small amount of sterilized distilled water whenever necessary. The

plates were examined daily for the presence of fungal growth (ISTA 1966).

3.2.2 Tissue Transplanting method:

Sections (2-4mm2) of the infected plants parts were disinfected by dipping in

4% sodium hypochlorite solution for 3 min. Sections were then washed in

three changes of sterile distilled water. The sections were placed at

reasonable distances on layers of sterilized moistened filter paper (9cm

diameter) in Petri dishes. The Petri dishes were then incubated in an

incubator for 4-7 days at 28ºC and examined daily for the presence of fungal

growth (Agrios, 2004).

26

3.2.3 Single spore isolation of the suspected pathogenic fungi:

3.2.3.1 Isolation of Drechslera halodes, Alternaria alternata, and

Alternaria sesami:

The Petri-dishes containing the Blotters were placed under a stereoscopic

binocular microscope placed in a laminair flow cabinet. A heap of fruiting

mycelia were transferred to a large square drawn in the back of the Petri-dish

containing filtered Corn Meal Agar (CMA) medium. Individual spores were

transferred from the large square to smaller ones drawn at the back of the

Petri-dish just beneath the larger square. Petri-dishes were then incubated at

28ºC for 24 h. When the individual spores germinated a square was cut in

the agar around each germinating spore using a sterile cork borer. This

square was transferred to a Petri-dish containing a thin layer of filtered

CMA. CMA was prepared by adding 17 g of the medium into a litre of

distilled water. The medium was boiled until completely dissolved,

autoclaved at 121ºC (151b/inch2) for 15 min.

3.2.3.2 Single spore isolation of Macrophomina phaseolina:

Suspected pycnidia of M. phaseolina were removed with sterile forceps

from diseased seedlings, and then crushed in sterile water in a Petri-dish to

release pycnidiospores. A loop full of dilute pycnidiospores suspension was

spread on the surface of water agar plates. After 24 hours incubation, the

dishes were examined under a stereoscopic binocular microscope (16-40X)

for the presence of sprouting pycnidiospores. Using a sterile cork- borer,

discs of agar with a single germinating pycnidiospores were cut, transferred

to Potato Dextrose Agar (PDA) and incubated at 28-30ºC. PDA was

prepared by adding 15g of the medium to one litre distilled water and

autoclaving at 121ºC (151b/inch2) for 15 min.

27

3.3 Characterization and identification of the tested fungi:

Morphological characteristics such as colour, shape and spores chain

morphology for each isolated fungus were examined under a stereoscopic

binocular microscope to a magnification of up to 50 X.

Slides for microscopic observation were either prepared in lactophenol or in

water in the case of dark-coloured fungal spores. Small cylindrical pieces

(6mm diameter) of uninoculated CMA medium were removed by a cork

borer and inserted on the surface of a thin layer of CMA in another Petri-

dish. The top of the cylindrical pieces were inoculated with the fungal

growth and covered with sterilized cover slips. After few days, the fungal

growth on the cover slip was gently stained with cotton blue and mounted in

lactophenol for examination.

3.4 Testing the virulence of the isolated phytopathogens:

Pathogenicity of Drechslera halodes, Alternaria alternata, Alternaria.

sesami and Macrophomina phaseolina was tested on their respective hosts

as follows:

3.4.1 Pathogenicity of D. halodes on sorghum plants:

Four weeks old plants were sprayed with 150ml spores suspension (4×104

spores per ml) of D. halodes. The inoculated plants were placed in a moist

plastic chamber at 100% relative humidity and 28±5ºC. After 24h, the plants

were removed from the chamber, transferred to the green house and

examined daily for the presence of disease symptoms (Borges, 1983).

28

3.4.2 Pathogenicity of A. alternata and A. sesami on tomato and sesame

plants:

The abilities of A. alternata and A. sesami to infect and induce disease

symptoms, each on its respective host, were studied according to the

procedure reported by Foolad et al. (2000). In this procedure, tomato and

sesame leaves from five-week old seedlings, were injured by rubbing the

leaves between thumb and fingers (Poysa and Tu, 1996). The seedlings were

then sprayed with spore suspension having a concentration of 4.1×103 and

4.5×103 spores/ml in the case of A. alternata and A. sesame, respectively.

Plants were then incubated for 24h at 100% relative humidity (RH).

Seedlings were transferred to the green house and incubated at 70-80 RH

and 25±5ºC and examined daily for appearance of disease symptoms.

Spore count was done according to the procedure of Dimas et al. (1998), in

which ten microlitre of spore suspension were counted using the Neubauer

chamber (hemocytometer). The cover slip and chamber were cleaned with a

detergent, washed thoroughly with distilled water, swapped with 70%

ethanol and dried. The chamber was charged with spore suspension under

test. After spores had settled, the chamber was placed under a compound

microscope (40X), spores in the 4 large corner’s squares (each containing 16

small squares) were counted.

The following formula was used for calculating the number of spores per ml.

No of spores per ml = spores count ×dilution factor× 2.5 ×105.

3.4.3 Pathogenicity of Macrophomina phaseolina on sesame plants:

Soil was inoculated with M. phaseolina inoculum which prepared by cutting

16 discs from the edge of a colony on CMA with a sterile cork borer (6 mm

diameter). The discs were then put in a flask containing corn meal- sand

29

mixture, incubated for 10 days at 30ºC and was shaken every 2 days. Pots,

which were filled with sterile soil, were inoculated each with 40g of the

fungal culture and were then sown by the sterile seeds of sesame (Mihail,

1992).

3.5 Preliminary screening of Streptomyces isolates for antifungal

activity:

Antifungal activities of the Streptomyces isolates were tested in vitro against

the four pathogenic plant fungi which were previously isolated from

different infected parts of plant species.

Antifungal activities of pure Streptomyces isolates were performed

according to the Agar Debussing method as described by Taechowisan et al.

(2005). In this method, a plug of mycelium of the tested fungus was placed

onto the center of CMA medium in a Petri-dish. Inoculated plates were then

seeded with the Streptomyces isolates by spotting Streptomyces mycelium on

the four edges of the agar surface (see plate5 page 46). The plates were then

incubated at 28ºC and the inhibition zones diameters were measured and

recorded in mm after 7-9 days.

3.6 Characterization of the active Streptomyces isolates:

The active Streptomyces isolates (having inhibition zone diameters of more

than 10 mm) were selected and characterized following the methods given

by the International Streptomyces Project ISP (Shirling and Gottlib, 1966;

Locci, 1990).

30

3.6.1 Cultural characteristics of the active isolates:

Active isolates were cultured on Nutrient Agar plates (prepared by

dissolving 28g of Nutrient Agar medium in one litre of distilled water,

autoclaved for 15 minutes at 121ºC (151b/inch2) and incubated at 28ºC for 7-

14 days). Colony characteristics such as colour, shape, elevation, margin,

consistency and transparency were determined visually or by placing the

Petri-dish containing the pure individual colonies under a stereomicroscope.

The descriptive terminologies followed were those suggested by ISP

(Shirling and Gottlib, 1966; Locci, 1990).

3.6.2 Microscopical characteristics:

The following microscopical tests were performed on the active

Streptomyces isolates.

3.6.2.1 Gram straining:

Gram staining of 24h old nutrient agar cultures was performed as described

by Collins et al. (1995). A loopful of the bacterium was transferred and

mixed with a drop of sterilized distilled water at the center of a clean glass

slide. Bacterial suspension obtained was spread onto a glass slide with a

sterilized loop to obtain a thin film (smear). The slide was left to air-dry and

then fixed by passing over a flame (3-5 times).

The smear was flooded with crystal violet – ammonium oxalate complex dye

for one minute. The dye was prepared by dissolving 2g crystal violet in 20ml

of 95% ethanol and mixed with 0.8g of ammonium oxalate dissolved in

80ml of distilled water. The solution was then left to stand for 24 h. The

smear was covered with Gram's idoine solution (prepared by dissolving 2g

of potassium iodide and 1g of iodine in 300ml of distilled water) for one

31

minute, washed under running tap water, and ethanol was added until no

more stain comes away, the slide was washed with tap water and counter

stained with safranin dye (prepared by dissolving 0.25g of safranin to 10ml

of 95% ethanol, then made up to 100ml with distilled water for 30 seconds.

Excess safranin was washed off with tap water, the smear was blot-dried and

examined under a compound microscope using oil immersion lens. The

result was recorded as Gram positive or Gram negative.

3.6.2.2. Mycelial morphology:

The same preparations used for determination of Gram reaction were

employed for the determination of the presence or absence of aerial mycelial

and conidia.

3.6.2.3. Motility test:

A semi-solid transparent Motility Agar medium consisting of 3g agar

dissolved in 150 ml of Nutrient Broth medium was dispensed in test tubes

and autoclaved. The tubes were left to set in a vertical position. The medium

was inoculated with straight wire, making a single stab down the center of

the tube to about half the depth of the medium. After incubating overnight,

motile bacteria typically give diffuse, hazy growths that spread throughout

the medium rendering it slightly opaque while non- motile bacteria generally

give growths that are confined to the stab-line (Collee et al., 1996).

3.6.3 Biochemical Tests:

3.6.3.1 Oxidase test:

Commercial discs (Hi-Media, India) were used to determine the expression

of oxidase enzyme. One colony from 24 hours Nutrient Agar culture was

scraped with a wooden stick and rubbed on the oxidase disc. The

32

development of purple colour within 20 seconds was recorded as a positive

reaction for the expression of cytochrome oxidase (Collins et al., 1993).

3.6.3.2 Catalase test:

Colonies grown on Nutrient Agar were examined for catalase expression by

adding a drop of 6% hydrogen peroxide to the colony. Appearance of gas

bubbles within 10 seconds was considered as a positive result (Prescott et

al., 1993).

3.6.3.3 Utilization of different carbon sources:

The abilities to utilize different carbohydrates as sole carbon sources was

tested according to the procedure of Collee et al. (1996). In this procedure,

3g of Peptone Water powder were dissolved in 200ml of distilled water and

2.5ml of bromothymol blue were added. The medium was then distributed

into tubes (5ml each) and autoclaved. Each carbohydrate tested (10%) was

sterilized at 110ºC (151b/inch2) for 10 minutes. A volume of 0.3ml of the

tested carbohydrate was then added aseptically to the Peptone Water

medium. Each tube was then inoculated with one of the isolates and

incubated for 24h at 37ºC. The change of the medium colour to yellow

indicates acid production and consequently CHO utilization. The

carbohydrates tested were: glucose, galactose, fructose, rhaminose, lactose,

maltose, sucrose, arabinose and mannitol.

3.6.3.4 Aerobiosis:

The test for aerobiosis was carried out by using 10 ml aliquots of sterilized

Thioglycolate Broth medium (29.8g in 1000 ml distilled water) in test tubes.

33

The broth medium was inoculated with a loopfull of the bacterium and the

tubes were left undisturbed for 2 days.

Aerobiosis was indicated as aerobic, microaerophilic, anaerobic and

facultative anaerobic by observing the growth and specifying whether it

occurred at the surface, sub-surface, bottom, or throughout the broth

respectively (Collins et al., 1995).

3.6.3.5 Starch hydrolysis test:

Starch Agar plates, which were prepared by adding 20ml of 10% aqueous

solution of soluble starch to 100ml of melted Nutrient Agar, were inoculated

with one of each of the tested active Streptomyces isolates. The inoculated

plates were incubated for 5 days and were then flooded with iodine solution.

Starch hydrolysis is indicated by clear zones around the bacterial growth

(Collins et al., 1995).

3.6.3.6 Urease test:

Urea medium (Atlas,1997) was prepared by dissolving 0.9g of a mixture

containing (g) agar, 12.0; sodium chloride, 5.0; disodium hydrogen

phosphate (Hydrated), 1.98; glucose, 1.0; casein, 1.0; Magnesium phosphate

(Hydrated), 0.5; phenol red, 0.012 in 95ml of distilled water in 250 ml

conical flask. The mixture was sterilized by autoclaving, cooled to 55ºC,

then 5ml of 40% filter-sterilized aqueous solution of urea were added. The

medium was distributed aseptically into 15ml aliquots in sterilized glass

vials. Each vial was then heavily inoculated with one of each of

Streptomyces isolates and incubated at 35ºC. The gradual change of the

medium colour from pale orange to pink was considered as a positive test

(Collins et al., 1995).

34

3.6.3.7 Gelatin liquefaction:

Nutrient Gelatin (38.4g) was dissolved in 300ml of distilled water,

distributed in test tubes (4ml each) and autoclaved. The medium was

inoculated with Streptomyces and incubated at 30ºC for 7 days. The tubes

were then transferred to the refrigerator, left overnight and gelatin

liquefaction was then noted (Collins et al., 1995).

3.6.3.8 Organic Acid Test:

Organic Acid medium was made of three solutions, solution A (4g agar, 20g

glucose, 1.2g yeast extract, 0.25g MgSO4. 7H2O, 0.012g bromo-cresol

purple in 400ml of distilled water), solution B (0.534g Na2HPO4. 2H2O,

0.272 g KH2 PO4 in 500ml of distilled water) and solution C (1g CaCO3 in

100 ml of distilled water). Both solutions A and B were autoclaved each in a

separate flask and then mixed, solution C was distributed into test tubes each

containing 0.2ml and autoclaved. Then, 1.80 ml of solutions A and B

mixture were added aseptically to solution C in the test tubes. The tubes

were then inoculated each with one of the isolates. Isolates that produce

organic acids change the colour of the medium to purple within 5-15 days.

3.6.3.9 Reduction of nitrate to nitrite: Nitrate medium was prepared by dissolving (g/L) glycerol, 5; agar, 4; NaCl,

2; KNO3, 1; Na2HPO4.7H2O, 0.534; MgSO4. 7H2O, 0.5; KH2PO4, 0.272; and

1.0m1 of trace elements solution (prepared by dissolving 0.1g FeSO4.H2O,

0.1 MgCl2.H2O and ZnSO4.H2O in 100ml distilled water) in one litre of

distilled water. One ml of the medium was distributed into test tubes and

autoclaved. After 24 h, each tube was inoculated with one of the isolates

under test and incubated. Seven days later, two drops of Griess –Hosvay

35

reagent was added to each test tube, the development of a red colour

indicates a positive result (Atlas, 1997).

3.6.3.10 Casein hydrolysis:

Casein Hydrolysis medium (Williams and Cross,1971) was prepared from

two solutions: Solution A (prepared by dissolving 10g skimmed milk

powder in 100ml distilled water) and solution B (prepared by dissolving 2g

agar in 100ml distilled water). Solutions A and B were autoclaved

separately, allowed to cool to about 50ºC and combined before they were

poured into sterile plates. Plates were then inoculated each with one of the

isolates and incubated at 28ºC for 3 days. Appearance of a clear zone around

each colony indicates a positive result.

3.6.3.11 H2S production:

Production of H2S was tested using Kligler Iron Agar (KIA) medium which

was prepared by dissolving 8.62g of KIA medium in 150ml distilled water

and boiled to dissolve completely. The medium was then divided into test

tubes (3ml each) and autoclaved. The tubes were allowed to solidify in a

slanting way and were then heavily inoculated each with one of the isolates.

Organisms that produce H2S blacken the medium (Collee et al., 1996).

3.7 Antibiotic production by Streptomyces in submerged cultivation

using Bennet broth:

R92 Streptomyces isolate which showed the highest activity against all

tested fungi in the screening experiment was used for antibiotic production

in submerged culture using Bennet Broth medium. Bennet Broth was

prepared by dissolving 2.1g of the medium into 150ml distilled water in

250ml Erlenmeyer flask, and autoclaving at 121ºC (151b/inch2) for 15

36

minutes. A pure colony from 14-days old R92 culture was transferred to

Bennet Agar medium, pH 7.2, and incubated at 28 ºC. After ten days, a pure

colony was used to inoculate 150 ml of Bennet Broth in 250 ml Erlenmeyer

flasks. Flasks were incubated at room temperature (28-30ºC) for 30 days

onto an orbital shaker operating at 180 rpm (Hoon et al., 2002).

At the end of the incubation period, the fermentation broth culture was

mixed with isopropanol (1:1 v/v) and centrifuged. Supernatant was

concentrated under reduced pressure in a freeze- drier, and 250µg of the

extract were dissolved in 5ml of dimethyl sulfoxide to give a final

concentration of 50µg/ml (Hoon et al., 2002). To this, 500ml of water:

methanol (19:1 v/v) and 125 ml of Tween 80 were added. This extract

preparation was later used for testing in vitro and in vivo antifungal

activities.

3.8 In vitro antifungal activities of R92broth extract:

A piece of a well-grown CMA cultures of Drechslera halodes, Alternaria

alternata, Alternaria sesami and Macrophomina phaseolina were cut with a

cork borer, placed each in the midst of a separate freshly prepared agar

plates and incubated for 2 days. Filter paper discs (6 mm diameter) were

loaded, each with ten µl of each of three selected commercial antibiotics and

R92 broth culture extract. The discs were left to dry and placed onto the agar

plates. The plates were incubated for 3 days at 24ºC and the diameters of

growth inhibition zones were measured, recorded and compared with those

of the commercial antibiotics (Jain and Jain, 2005).

37

3.9 In vivo anti-fungal activity:

Seedlings of sorghum and tomato were selected for testing the in vivo

antifungal activity of R92broth culture extract preparation. Seeds of these

crops were surface disinfected in 4% sodium hypochlorite for 3 minutes,

washed five times with sterilized distilled water and were then sown in

sterile soils in vinyl pots. The soil was sterilized in an oven for 3 h at 180ºC

for two successive days. Seedlings were irrigated regularly and left in the

green house at 25±5ºC for 5 weeks (Hoon et al., 2002).

Raised seedlings were sprayed with R92 broth culture extract and left to

grow for 24 h. Sorghum and tomato seedlings were then inoculated with D.

halodes and A. alternata spores suspension (103-107 spores/ml) respectively.

Seedlings were then incubated in the dark for one day at 25 ±2ºC and 100%

relative humidity, transferred to the green house and left to grow under the

conditions of 70-80% RH, 25 ±2ºC with 12h of light per day. The

experiment was arranged in a randomized complete block design with three

replicates. A control set, in which seedlings were sprayed with Tween 80

and water-methanol alone instead of Streptomyces extract preparation, was

included. Inoculated seedlings were examined daily for the appearance of

disease symptoms. The percentage of the leaf area covered by the expected

lesions (necrotic area) on the inoculated seedlings was estimated and

compared to that estimated for the control.

In another in vivo experiment, a set of seedlings of each plant were

inoculated each with its respective spores suspension (103-107 spores/ml) of

the test fungi and incubated in the dark for one day at 25 ±2ºC and 100%

relative humidity. The seedlings were then transferred to the green house,

left to grow under the conditions of 70-80% RH, 25 ±2 ºC with 12h of light

38

per day for 72 h. The diameters of necrotic spots were measured and

recorded. Seedlings of each test plants (sorghum and tomato) were then

divided into two lots, the first lot was sprayed with R92 extract preparation,

left for 24 hours and the spot diameter was re-measured and recorded.

However, the second lot was not sprayed with R92 broth culture extract but

left to grow for 24h and its spot diameter was also re-measured and

recorded. The experiment was arranged in a randomized complete block

design with three replicates (ten plants in each replicate). A control set in

which seedlings were sprayed with fungal spore suspension alone instead of

Streptomyces extract preparation was included. The diameter of the spots

area on the inoculated seedlings was estimated and compared to that

estimated for control seedlings. The % increment in spot diameter was

calculated and compared in both lots.

39

4. Results and Discussion

4.1. Isolation of Streptomyces:

In this study one hundred and four isolates of Streptomyces were recovered

from soil samples collected from different localities in Sudan. Each isolate

was given a number prefixed with R (Table1). Twenty one of the isolates

were recovered from soil samples collected from different locations in

Khartoum State, 16 from River Nile State, 14 (Northern Kordofan State), 10

(Gezira State), 9 (each of the Northern and Western Bahr Al Ghazal States),

7 (Gadarif State), 6 (White Nile State), 5 (Bahr El Ghazal State), 4 (Blue

Nile State) and 3 from Kassala State. All of the isolates were considered as

Streptomyces depending on their mycelia growth nature (plate1a) and on

their abilities to grow on Glycerol Arginine Agar (GAA) supplemented with

Nystatin (50 µg/ml) and with 1 µg/ml of penicillin (Plate1b). This medium

seems to be specific and sensitive for Streptomyces since it contains glycerol

that most actinomycetes use as a sole carbon source. Nystatin reduces fungal

growth (Porter et al., 1960) whereas penicillin reduces the development of

non-filamentous bacteria and actinomycetes other than Streptomyces

(O’Grady et al., 1997).

Isolation of Streptomyces from all soil samples tested indicates the

dominance of Streptomyces in these soils. These isolates were preserved on

Glycerol Asparagine Agar slant, and screened later for their abilities to

inhibit the growth and multiplication of some plant pathogenic fungi viz

Alternaria alternata, Alternaria sesami, Drechslera halodes, and

Macrophomina phaseolina.

40

Table 1: Streptomyces presumptive isolates

Isolate

Source of soil sample

R1 Gureir

Northern State

R2 R3 R4 Marawi R5 R6 R7 Dongola R8 R9

R10 Shendi

River Nile State

R11 R12 R13 Al hawsh R14 R15 R16 Berber R17 R18 R19 Atbara R20 R21 R22 R23 El Moswarat R24 R25 R26 Shambat

Khartoum state

R27 R28 R29 R30 Beach of Blue and

White Nile R31 R32 R33

41

Table 1: Continued Isolate Source of soil sample R34 As saggay

Khartoum State

R35 R36 R37 El kadaro R38 R39 R40 Jabal Awliaa R41 R42 R43 R44 Um Dawm R45 R46 R47 El Gadarif

Gedarif State

R48 R49 R50 Al Fao R51 R52 R53 R54 Kassala

Kassala State R55 R56 R57 Um Siyala

Northern Kordofan State

R58 R59 R60 Sawdiri R61 R62 R63 El Mazroob R64 R65 R66 R67 Barah R68 R69 R70

42

Table 1: Continued Isolate Source of soil sample R71 Kenana

White Nile State

R72 R73 R74 Asalaya R75 R76 R77 Waw

Western Baher Al Ghazal State

R78 R79 R80 Jabal Kair R81 R82 R83 El Goor R84 R85 R86 El Hilaliya

Gezira State

R87 R88 R89 El Halawen R90 R91 R92 Wad Medani R93 R94 R95 R96 Al Damazin

Blue Nile State R97 R98 R99

R100 areas in Baher El Gazal

Northern Baher El Ghazal State

R101 R102 R103 R104

43

a: Branched mycelia of Streptomyces.

b: Streptomyces on GAA medium

Plate1: Filamentous mycelium of Streptomyces and their colonies on GAA

Streptomyces

Streptomyces

Streptomyces

44

4.2 Isolation and characterization of plant pathogenic fungi:

Isolates of four species of plant pathogenic fungi were recovered from some

seeds and other infected plant parts obtained from different localities in

Sudan using the standard Blotter and Tissue Transplanting methods (Plate 2a

and b). These isolates were characterized as Alternaria alternata, Alternaria

sesami, Drechslera halodes, and Macrophomina phaseolina following the

descriptions by Ellis (1971) and Cloud and Rupe (1991). Results are shown

in Table 2.

4.2.1 Characteristics of the isolated fungi:

4.2.1.1 Drechslera halodes:

Drechslera colonies are black, conidiophores are solitary, brown and with a

thickness of 5.9µ (5-8µ). Conidia are straight and have pseudosepta, with

hyaline end cells that are cut off by thick dark septa. Intermediate cells are

golden brown and the hilum is distinctly protuberant (Table 2, Plate 3a).

4.2.1.2 Alternaria alternata:

Colonies are black, conidiophores are single or in groups, simple or

branched, straight, golden brown and smooth, 3.6µ thick (3-6µ). Conidia

formed in long, branched chains with short conical peak which was pale to

mid golden brown, 3.1µ long (less than ⅓ the length of the conidia),

Smooth, with 6-11 transverse septa (up to 8), and several longitudinal septa,

27.3µ long (20-63µ), 9.8µ thick (9-18µ) (Table 2, Plate 3b).

4.2.1.3 Alternaria sesami:

Colonies are black, conidiophores are solitary or in small groups, straight,

septate, rather pale brown, smooth and have 6.4µ thick (5-9µ). Conidia are

45

a: Blotter method (Sesame seeds)

Sorghum leaves Tomato leaves

b: Tissue transplanting method

c: Single spore isolation

Plate 2: Isolation of pathogenic fungi

46

Table 2: Microscopical characteristics of isolated fungi

Drechslera

halodes

Alternaria

alternata

Alternaria

sesami

Macrophomina

phaseolina

Colonies Colonies

colour

Black Black Black Grey to black

Conidia Solitary or

in chains

Solitary Solitary or

short chains

Chains Solitary

Shape Straight Straight,

obclavate,

ellipsoidal

Obclavate,

ellipsoidal,

obpyriform,

ovoid

Oval

Colour Golden

brown

Pale to mid

golden

brown

Pale to mid

golden

brown

Hyaline

Margin Smooth Smooth or

verruculose

Smooth Smooth

Length

diameter

* 27.3µ 99.5µ 17 µ

Thick 14.2µ 9.8µ 16.6µ 9 µ

type of

septa

Pseudo septa True septa True septa -

No. of

longitudinal

or oblique

septa

- Several or

many

Several -

47

Table 2: Continued

Drechslera

halodes

Alternaria

alternata

Alternaria

sesami

Macrophomi

na phaseolina No. of

transverse

septa

- 8 6-11 -

Peak - 3.1µ 51.8µ -

Protuberant

hillum

+ - - -

Conidio-

phores

Color Brown Brown to

yellowish

brown

Mid golden

brown

Pale brown to

hyaline or

brown

Shape Straight to

geniculate

Straight Straight Rod like

Length * 18.5µ 25.1µ 10 µ

Thick 5.9µ 3.6µ 6.4µ -

Pycnidia Color - - - Black

Shape - - - Globose

Length - - - 117 µ

Thickness - - - -

Ostiole - - - Present

Microscler

otia

Colour - - - Black

Shape - - - Irregular

Margin - - - Smooth

* ≡ present but not tested; - ≡ absent

48

a: Drechslera halodes b: Alternaria alternata

c: Alternaria sesami d: Macrophomina phaseolina

( Pycnidia)

Plate 3: Spores and pycnidia of the isolated pathogenic fungi.

49

solitary but are some times found in chains, straight. The conidium body is

ellipsoidal, peak 110.8µ in length (up to twice as long as the body). Conidia

pale to mid golden brown, smooth, with 6-11 transverse septa (6-11), and

several longitudinal septa. 99.5µ long (90-260µ), 16.6µ thick (14-16µ) in

the broadest part (Table 2, Plate 3c).

4.2.1.4 Macrophomina phaseolina:

Colonies are grey to black, pycnidia are initially immersed in host tissue,

they are 100-200 µ in diameter; dark to greyish, becoming black with age;

globose or flattened globose; with an inconspicuous or definite truncate

ostiole. The pycnidia are bear simple, rod-shaped, conidiophores 10 µ (10-

15 µ) long. Conidia117 µ long (14-33 x 6-12 µ), single celled, hyaline, and

elliptic or oval. Microsclerotia of M. phaseolina are jet black in colour and

are smooth and round to oblong or irregular (Plate 3d).

4.3 The virulence of the isolated phytopathogens:

Pathogenicity of Drechslera halodes, Alternaria alternata, Alternaria

sesami and Macrophomina phaseolina was tested on their respective hosts

as follows:

4.3.1 Symptoms of D. halodes leaf spot on sorghum plants:

Sorghum plants sprayed with 150ml spores suspension (4×104 spores per ml)

of D. halodes have shown disease symptoms after 24 hours. Symptoms

appeared as small lesions, spherical in shape but later became surrounded by

a dark brown-reddish purple border (Plate 4). Such symptoms were also

described for D. halodes leaf spot by Khan et al. (2001).

50

a: Drechslera leaf spot on sorghum leaves b: Alternaria early blight on tomato leaves

c: Alternaria leaf spot on sesame capsule

Plate 4: Disease symptoms shown by different fungi on different plant

species.

51

4.3.2 Symptoms of A. alternata early blight on tomato plants:

Spots which appear on the lower leaves of tomato plants as a result of

spraying with 4.1×103 spores suspension of A. alternata were brown to

black, ¼ to ½ inch in diameter, with dark edges, frequently merge forming

irregular blotches. Dark concentric rings often appear in leaf spots. Leaves

turn yellow and often dry up when only a few spots are present (Plate 4b).

The symptoms, described here were also mentioned for by Koike et al.

(2007) tomato A. alternata early blight.

4.3.3 Symptoms of A. sesami leaf spot on sesame plants:

Symptoms as a result of inoculating sesame plants with 150 ml (4.5×103

spores/ml) spores suspension of Alternaria sesami appeared mainly on the

leaf blade as brown, small, and round to irregular spots (Plate 4c). These

results are comparable to the results reported for the same disease by

Ojiambo et al. (2000).

4.3.4 Symptoms of M. phaseolina charcoal rot on sesame plants:

Inoculation of soil with M. phaseolina prior to sowing sesame seeds has

resulted in pre emergence mortality of the embryo or the small seedling.

Some strains of M. phaseolina were reported to kill the embryo of the seeds

(Khan el al., 2000).

52

4.4 Screening of Streptomyces for antifungal activities:

Streptomyces isolates were screened for their abilities to inhibit the growth

of the isolated fungi. Screening was performed by agar debussing method

and the diameters of growth inhibition zones were measured in millimeters

for each of the Streptomyces isolates; the results are shown in Table 3 and

Plate 5. Approximately 80℅ (83 isolates) of the tested isolates have shown

potent in vitro antifungal activities against all tested pathogens. The highest

activities were shown by isolate R92 against Alternaria alternata (34 mm

diameters), Drechslera halodes (33 mm), Macrophomina phaseolina

(33mm) and 32mm against Alternaria sesami. It is also evident in Table 1

that isolates R39 and R43 have shown strong activities against all tested

phytopathogens with inhibition zone diameters ranging between 21.5 and

22.0mm. Twelve of the isolates (R1, R2, R6, R8, R9, R10, R13, R15, R19,

R28, R29 and R37) have shown moderate inhibitory effect against the tested

fungi with inhibition zones diameters in the range of 11-19 mm. Forty six of

the isolates have shown weak inhibitory effect against one or more of the

tested fungi with inhibition zone diameters ranging between 5and10 mm.

Twenty two of the Streptomyces isolates have shown very weak inhibitory

effect against one or more of the tested fungi with inhibition zone diameters

between 0.1 and 4.9 mm.

Although, 20.2 % of the isolates have completely failed to inhibit the

growth of any of the tested fungi, it is possible that they produce other useful

compounds that were not identified in this study. Gullo et al. (2006)

reported that 10-20 gene clusters that code for secondary metabolite

production were present in actinomycetes especially Streptomyces spp.,

however, their expression is clearly determined by the culture conditions

53

Table 3: Inhibition zones diameters (mm) shown by different

Streptomyces isolates against plant pathogenic fungi

Isolate code Drechslera halodes

Alternaria alternata

Alternaria sesami

Macrophomina phaseolina

RI 14 15 14 16 R2 15 14 15 15 R3 9.0 10 8.0 7.0 R4 7.0 6.0 5.0 6.0 R5 6.0 7.0 6.0 8.0 R6 17 17 16 15 R7 3.0 4.0 3.0 3.0 R8 17 19 19 16 R9 17 18 17 19 R10 15 16 16 14 R11 2.0 1.0 1.0 1.0 R12 2.0 02 02 03 R13 16 16 15 14 R14 0.0 0.0 0.0 0.0 R15 16 13 13 14 R16 0.0 0.0 0.0 0.0 R17 1.0 2.0 1.0 1.0 R18 0.0 0.0 0.0 0.0 R19 17 16 18 19 R20 9.0 10.0 7.0 7.0 R21 0.0 0.0 0.0 0.0 R22 2.0 1.0 0.0 1.0 R23 0.5 0.0 0.0 0.0 R24 0.0 0.0 0.0 0.0 R25 7.0 6.0 6.0 9.0 R26 3.0 3.0 4.0 3.0 R27 2.0 1.0 1.0 1.0 R28 18 16 18 17 R29 19 19 17 19 R30 6.0 7.0 7.0 7.0 R31 0.0 0.0 0.0 0.0 R32 0.0 0.0 0.0 0.0 R33 10.0 10.0 9.0 10.0 R34 7.0 6.0 8.0 6.0

54

Table 3: Continued R35 7.0 7.0 6.0 5.0 R36 8.0 6.0 7.0 6.0 R37 15 15 16 15 R38 0.0 0.0 0.0 0.0 R39 21 20 22 23 R40 4.0 6.0 5.0 6.0 R41 6.0 5.0 6.0 4.0 R42 6.0 6.0 6.0 6.0 R43 20 23 22 23 R44 8.0 6.0 7.0 6.0 R45 8.0 8.0 8.0 8.0 R46 6.0 5.0 5.0 6.0 R47 0.0 0.0 0.0 0.0 R48 1.0 1.0 1.0 1.0 R49 0.0 0.0 0.0 0.0 R50 0.0 0.0 0.0 0.0 R51 0.0 0.0 0.0 0.0 R52 0.0 0.0 0.0 0.0 R53 7.0 6.0 7.0 6.0 R54 2.0 3.0 2.0 3.0 R55 7.0 7.0 7.0 6.0 R56 9.0 9.0 9.0 8.0 R57 9.0 8.0 9.0 7.0 R58 0.0 0.0 0.0 0.0 R59 8.0 8.0 8.0 9.0 R60 5.0 5.0 5.0 5.0 R61 1.0 1.0 1.0 1.0 R62 2.0 3.0 3.0 3.0 R63 0.0 0.0 0.0 0.0 R64 4.0 4.0 4.0 4.0 R65 10.0 9.0 10.0 10.0 R66 9.0 10.0 10.0 9.0 R67 5.0 4.0 5.0 4.0 R68 10.0 9.0 10.0 9.0 R69 5.0 5.0 5.0 5.0 R70 3.0 5.0 4.0 5.0 R71 2.0 3.0 2.0 3.0 R72 9.0 9.0 8.0 9.0

55

Table 3: Continued R73 0.0 0.0 0.0 0.0 R74 5.0 5.0 4.0 5.0 R75 9.0 10.0 10.0 10.0 R76 4.0 4.0 4.0 4.0 R77 2.0 2.0 2.0 2.0 R78 0.0 0.0 0.0 0.0 R79 10.0 10.0 10.0 9.0 R80 6.0 5.0 6.0 6.0 R81 6.0 7.0 6.0 6.0 R82 7.0 8.0 7.0 6.0 R83 7.0 8.0 7.0 6.0 R84 7.0 6.0 7.0 7.0 R85 5.0 5.0 5.0 5.0 R86 8.0 8.0 8.0 8.0 R87 5.0 5.0 5.0 5.0 R88 6.0 4.0 5.0 5.0 R89 4.0 5.0 5.0 5.0 R90 7.0 8.0 7.0 7.0 R91 0.0 0.0 0.0 0.0 R92 33.0 34.0 32.0 33.0 R93 2.0 3.0 2.0 2.0 R94 8.0 8.0 9.0 8.0 R95 0.0 0.0 0.0 0.0 R96 9.0 9.0 8.0 9.0 R97 5.0 5.0 4.0 5.0 R98 0.1 0.0 0.0 0.0 R99 9.0 9.0 9.0 9.0 R100 9.0 9.0 9.0 8.0 R101 2.0 2.0 1.0 2.0 R102 0.0 0.0 0.0 0.0 R103 0.0 0.0 0.0 0.0 R104 8.0 7.0 7.0 8.0

56

R94 on D. halodes R13 on M. phaseolina

R89 on D. halodes R7 on A. alternata

R57 on D. halodes D. halodes control

Plate 5: Effect of Streptomyces isolates on different phytopathogenic fungi.

57

R1 on A. sesame R2 on D. halodes

R92 on A. alternata R16 on D. halodes

R5on D. halodes R39 on M. phaseolina

Plate 5: Effect of Streptomyces isolates on different phytopathogenic fungi

(continued).

58

adopted for these organisms. It is likely that other antimicrobial metabolites

might be found (Porter, 1971).

4.5 Characterization of Streptomyces isolates:

The 15 Streptomyces isolates that showed great potentialities for antifungal

production were selected and characterized.

Characterization was done according to the methods of the International

Streptomyces Project (Shirling and Gottlib, 1968) and Bergey’s Manual of

Systematic Bacteriology (Bergy and Holt, 1994).

4.5.1 Cultural characteristics:

The results of cultural characterization after 7days of incubation on GAA are

shown in Table 4 and Plate 6. The colonies of the tested isolates were all

dry, opaque and the majority (ten isolates) was round in shape. During the

first 10-14 days, colonies were small and with smooth surfaces, later the

aerial mycelium developed and appeared granular and powdery. Almost, all

of the isolates have whitish colonies but later they produced a variety of

pigments that coloured the vegetative and aerial mycelia. Four of the isolates

produced white- coloured mycelia, nine, grey mycelia; one blue mycelium

and another produced a brown mycelium. Colonies elevations were either

umbonate (R2, R8, R19, R37 and R92), flat (R1, R6, R9, R10 and R28),

convex (R13, R15 and R43), raised (R29) or drop like (R39). Colony edges

were either smooth (12 isolates), filamentous (two isolates) or wrinkled (one

isolate). The colonies morphology displayed by the isolates on GAA

medium are typical to those described by Lacey (1973); Williams et al.

(1989); and Anderson and Wellington (2001) for actinomycetes.

59

Table 4: Cultural characteristics of potential Streptomyces isolates.

Isolate Code

Shape or configuration

Chromo-genesis Edge Opacity Elevation Surface Consistency

R1 Round White with gray margin Smooth Opaque Flat Powdery Dry

R2 Filamentous Grey Filamentous Opaque Umbonate Powdery Dry

R6 Concentric White with Yellow margin Filamentous Opaque Flat Powdery Dry

R8 Round Grey Smooth Opaque Umbonate Powdery Dry

R9 Round Grey Smooth Opaque Flat Powdery Dry

R10 Filamentous Grey Smooth Opaque Flat Powdery Dry

R13 Round Grey Smooth Opaque Convex Powdery Dry

R15 Filamentous Brown Smooth Opaque Convex Powdery Dry

R19 Round Grey Smooth Opaque Umbonate Powdery Dry

R28 Round White Smooth Opaque Flat Powdery Dry

R29 Round Blue with white margin Smooth Opaque Raised Powdery Dry

R37 Round Gray with white margin Wrinkled Opaque Umbonate Powdery Dry

R39 Round Grey Smooth Opaque Drop-like Powdery Dry

R43 Round White Smooth Opaque Convex Powdery Dry

R92 Concentric Grey Smooth Opaque Umbonate Powdery Dry

60

R6 R30

R11 R29

R4 R101

Plate 6: Colonies of different Streptomyces isolates on GAA medium.

61

R77 R20

R50 R82

R23 R62

Plate 6: Colonies of different Streptomyces isolates on GAA medium

(continued).

62

4.5.2 Microscopical characteristics:

The results of microscopical characterization are shown in Table 5. It is clear

that all of the tested isolates were filamentous, non motile, Gram positive,

and acid fast negative (plate 7). Diameters of the vegetative hyphae were in

the range of 0.5-2.0µm. Hyphae were branched but fragmented and verticils

were not detected. At maturity, the aerial hyphae of all isolates differentiated

into long spiral chains of spherical to cylindrical spores. Such characters are

typical of the actinomycetes group (Goodfellow and Williams, 1983; Cross,

1989 and Anderson and Wellington, 2001).

4.5.3 Biochemical characteristics:

Streptomyces is one of the best recognized genera of the whole order of

Actinomycetales because of its wide distribution in nature, especially in

soils. Many bacterial genera including Streptomyces are not only

morphologically and microscopically identical but yield colonies which are

not clearly distinguishable. The biochemical activities of such pure cultures

frequently allow genera and species characterization and identification

(Lacey, 1973; Gillies and Dods, 1984). Moreover, the selection of a range of

biochemical tests to be used depends upon the diversity and nature of the

group of bacteria understudy.

In this study biochemical characterization of the isolates involved 10

diagnostic characters that are recommended by the International

Streptomycete Project (ISP), and were successfully utilized by various

63

Table 5: Microscopical characteristics of Streptomyces isolates.

Test

Isolate

Gram stain Acid-fast

stain

Motility Aerial

mycelium

Conidia

R1 +ve -ve -ve Present present

R2 +ve -ve -ve ″ ″

R6 +ve -ve -ve ″ ″

R8 +ve -ve -ve ″ ″

R9 +ve -ve -ve ″ ″

R10 +ve -ve -ve ″ ″

R13 +ve -ve -ve ″ ″

R15 +ve -ve -ve ″ ″

R19 +ve -ve -ve ″ ″

R28 +ve -ve -ve ″ ″

R29 +ve -ve -ve ″ ″

R37 +ve -ve -ve ″ ″

R39 +ve -ve -ve ″ ″

R43 +ve -ve -ve ″ ″

R92 +ve -ve -ve ″ ″

64

a: Gram staining: Gram positive

b: Streptomyces conidia and conidiophore

c: Acid fast negative

Plate 7: Microscopical characteristics of Streptomyces isolates.

65

investigators in the field (Anderson and Wellington, 2001; Oskay et al.,

2004). Results of biochemical investigations are shown in Table 6 and Plate

8, in which it is clear that all tested isolates were aerobic. They were capable

of hydrolyzing starch and expressing catalase, oxidase, nitrate reductase and

urease enzymes. These results are in line with results reported by Anderson

and Wellington (2001) for their Streptomyces isolates. The isolates showed a

great deal of variability in their abilities to produce H2S on TSI medium, to

hydrolyze casein, produce organic acids from carbohydrate fermentation and

to liquefy gelatin.

Regarding the utilization of different carbohydrates as sole carbon sources,

all of the isolates were able to utilize all of the carbohydrates under test

(Table 7). This should not be surprising, because it is generally reported that

Streptomyces spp. possess several metabolic pathways that are supported by

large-sized genomes (Huang et al., 1998). For example, S. avermitilis

possesses the largest bacterial genome consisting of 8.7 million base pairs

(Ōmura et al., 2001). This provides insights into the intrinsic abilities of

Streptomyces to utilize different carbohydrate sources.

4.6 In vitro antifungal activities of R92broth extract:

Isolate R92 which showed strong in vitro antifungal activities against all of

the tested fungi was selected for antibiotics production in submerged culture

using Bennet broth medium. Fermentation broth culture was extracted in

isopropanol. The extract was freeze-dried and re-dissolved in methanol

before loaded in filter paper discs for antifungal bioassay. The exhibited

activity against fungi by R92 extract was measured and compared to those

66

Table 6: Biochemical characteristics of Streptomyces isolates. Test

Isolate

Catalase Oxidase H2 S Production

Nitrate reductase

Casein hydrolysis

Organic Acid formation

Gelatin liquefaction

Urease Starch hydrolysis

Aerobiosis

R1 + + + + + + + + + + + + + + + + + + + + + + + + + + +

R2 + + + + + + + + + + + + + + + + + + + + + + + + + +

R6 + + + + + + - + + + + + + + + + + + + + + + + + + +

R8 + + + + + + + + + + - + + + + + + + + + + + + +

R9 + + + + + + - + + + + + + + - + + + + + + +

R10 + + + + + + - + + + + + + + + + + + + + + + + + + + +

R13 + + + + + + + + + + + + + + + + + + + + + + + + + +

R15 + + + + + + - + + - - + + + + + + + + + +

R19 + + + + + + + + + + + + + - + + + + + + + + + + +

R28 + + + + + + - + + + + + + + + + + + + + + + + + + +

R29 + + + + + + + + + + + + + + + + + + + + + + + + + + +

R37 + + + + + + - + + + + + + + + + + + + + + + + +

R39 + + + + + + - + + + + + + + + + + + + + + + + +

R43 + + + + + + - + + + + + + + - + + + + + + + +

R92 + + + + + + - + + - + + + + + + + + + + + +

+ mid + + moderate + + + vigorous - negative

67

Nitrate reduction: Urease expression: Right: control Right: positive result (pink colour) Left: positive result (red colour) Left: control

Gelatin liquefaction: Lower: positive result Upper: negative result

Plate 8: Biochemical characteristics of Streptomyces isolates

68

H2S production: Organic acid formation: Right: positive result Right: positive result Left: control Left: control

Starch hydrolysis: clear zone

indicates Positive result.

Plate 8: biochemical tests (continued).

69

Table 7: Carbohydrate utilization by different Streptomyces isolates.

+ = positive result

Pentoses Hexoses Disaccharides Polydric alcohol

Isolate

Rhaminose Arabinose Glucose Galactose Fructose Sucrose Maltose Lactose Mannitol

R1 + + + + + + + + + R2 + + + + + + + + + R6 + + + + + + + + + R8 + + + + + + + + + R9 + + + + + + + + +

R10 + + + + + + + + + R13 + + + + + + + + + R15 + + + + + + + + + R19 + + + + + + + + + R28 + + + + + + + + + R29 + + + + + + + + + R37 + + + + + + + + + R39 + + + + + + + + + R43 + + + + + + + + + R92 + + + + + + + + +

70

recorded for some commercially available antifungal agents. It is evident

that R92 extract has a strong inhibitory effect which is reflected by the

inhibition zones diameters recorded (Table 8 and Plate 9). The inhibition

zone diameters (mm) recorded were: 19 against D. halodes, 18 against A.

alternata, 18 against A. sesami, and 17 against M. phaseolina. In

comparison, the commercial antifungal agents tested have shown inhibition

zone diameters in the range of 4 to 9 mm against the tested fungi.

According to Prescott et al. (1993) an inhibition zone range of 7to 14mm

does not categorize the extract as effective. The results presented for the R92

extract are clearly better than the results recorded for the selected

commercial antibiotics tested. Results are also comparable to the results

recorded by Ouhdouch et al. (2001) in Morocco, Hoon et al. (2002) in

Korea, Elnaggar et al. (2001) in Egypt, and Khamna et al. (2009) in Japan

for their Actinomycetes isolates against some phytopathogenic fungi.

71

Table 8: Inhibition zone diameters (mm) shown by R92 broth extract against phytopathogenic

fungi

Antibiotics Drechslera

halodes

Alternaria

alternata

Alternaria sesami Macrophomina

phaseolina

Nystatin 7.0 6.0 6.0 9.0

Mycosat 4.0 5.0 4.0 6.0

Itracon 5.0 3.0 4.0 5.0

R92 extract 19.0 18.0 18.0 17.0

Methanol 0.0 0.1 0.0 0.0

Isopropanol 0.0 0.0 0.0 0.0

DMSO 0.0 0.0 0.0 0.0

72

R92 extract

Nystatin

ItraconMycosat

Methanol

DMSO

a: R92broth extract

b: Control (A. alternata)

Plate 9: In vitro antifungal activities of R92broth extract

73

4.7 In vivo activity of R92 extract on the incidence of sorghum leaf spot

and tomato early blight:

Sorghum and tomato seedlings sprayed with R92 broth extract were left to

grow for 24 hours before they were sprayed with spore suspension of D.

halodes and A. alternata, respectively. The seedlings were then examined

daily for incidence of disease symptoms and the results are shown in Table 9

and Plates 10 and 11. Like the negative control seedlings (seedlings

uninoculated with spore suspension), all seedlings treated with R92 broth

culture extract are free of infection i.e recording zero infection percentage.

The positive control seedlings (not treated with R92 extract but inoculated

with spore suspension) on the other hand, have recorded 100% infection.

This clearly demonstrated the ability of R92 extract to prevent disease

incidence on sorghum and tomato seedlings due to infection by D. halodes

and A. alternata respectively.

4.8 In vivo activity of R92 broth extract on the development of sorghum

leaf spot and tomato early blight symptoms:

Table 10 and Plate 12 show the development of disease symptoms,

expressed as % increment in the diameter of leaf spot in sorghum and tomato

seedlings sprayed with R92 extract 72 hours after to their infection by D.

halodes and A. alternata, respectively. Sorghum and tomato seedlings

sprayed with R92 broth extract have shown less % increment in diameters of

leaf spot. The increment is 37.9 % In the case of sorghum sprayed with R92

broth extract compared to 251.7% for the non-sprayed seedlings. Similarly

sprayed and non-sprayed tomato seedlings have recorded 110.0 % and 230.0

% increments respectively. This indicates the strong curing value of the

74

Table 9: infection % of sorghum and tomato plants treated with Streptomyces R92 extract before inoculation with the tested fungal pathogens: Replicates %

infectionSorghum seedlings

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

-ve Control

- - - - - - - - - - - - - - - - Zero

+ve Control

+ + + + + + + + + + + + + + + + 100

R92 extract

- - - - - - - - - - - - - - - - Zero

Tomato seedlings

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

-ve Control

- - - - - - - - - - - - - - - - Zero

+ve Control

+ + + + + + + + + + + + + + + + 100

R92 extract

- - - - - - - - - - - - - - - - Zero

-ve Control ≡ Seedlings not treated with R92 extract and uninoculated with fungal spore suspension +ve Control ≡ Seedlings not treated with R92 extract but inoculated with the fungal spore suspension R92 extract ≡ Seedlings treated with R92 extract and inoculated with fungal spore suspension

75

(a) (b)

(c)

sorghum seedlings

a: seedlings treated with R92 extract before inoculated with D. halodes suspension b: seedlings untreated with R92 extract but inoculated with spore suspension. c: seedlings untreated and uninoculated. *note the presence of leaf spot symptoms in (b) and its absence in (a) and (c). Plate 10: In vivo effect of R92 broth extract on the incidence of Sorghum

Drechslera leaf spot disease.

76

(a) (b)

(c)

tomato seedlings

a: seedlings treated with R92 extract before inoculated with A. alternata suspension b: seedlings untreated with R92 extract but inoculated with spore suspension. c: seedlings untreated and uninoculated. *note the presence of leaf spot symptoms in (b) and its absence in (a) and (c). Plate 11: In vivo effect of R92 broth extract on the incidence of tomato

Alternaria early blight disease.

77

Table 10: Effect of R92 extract on the development of disease symptoms on infected sorghum and tomato as reflected by the diameters of spot area

Treatments

Diameters of spot area in mm % Increment in spot diameters

After 72 hours After 96 hours

Mean and SD

Mean and SD

Sorghum seedlings

sprayed withR92

extract

2.9* ±0.76

4.3 ±1,40

37.9

Sorghum control

seedlings

2.9 ±0.76

10.2 ±1.38

251.7

Tomato seedlings

sprayed withR92

extract

1.0 ±0.21

2.1 ±0.29

110.0

Tomato control

seedlings

1.0 ±0.21

3.3 ±0.41

230.0

*mean and standard deviation of 30 spots

78

a: Development of symptoms on sorghum seedlings

Left: inoculated seedlings treated with R92 extract.

Right: inoculated seedlings untreated with R92 extract.

b: : Development of symptoms on tomato seedlings

Left: inoculated seedlings treated with R92 extract.

Right: inoculated seedlings untreated with R92 extract.

Plate 12: in vivo effect of R92 broth extract on the development of sorghum leaf spot and

tomato early blight symptoms.

79

tested extract against sorghum leaf spot diseases, the results are also in line

with the results of the in vitro experiment.

4.9 Summary and Recommendations: Streptomyces is the largest antibiotic producing genus in the microbial world

discovered so far. Approximately 60% of the antibiotics developed for

agriculture were isolated from species of this genus (Tanaka and Omera,

1993). The number of antimicrobial compounds reported from Streptomyces

and usually increased almost exponentially for about two decades to reach

its maximum in 1970 and with a substantial decline in the late 1980's and

1990's. Recently, Watve et al. (2001) presented a mathematical model

which estimated the total number of antimicrobial compounds that this

genus is capable of producing to be in the order of a 100.000, a tiny fraction

of this has been unearthed so far. Bioactive compounds continue to be

discovered from microbes at an amazing pace: 500 per year (Dworkin et al.,

2006), this means that if the screening efforts are maintained, novel

antibiotics are expected to be discovered regularly. It should be emphasized

that, the search for a metabolite of pharmaceutical interest requires a large

number of isolates (Sahin and Ugur, 2003).

About 80% of plant diseases are traced to fungi (Someya, 2008) which in

Sudan (as in other countries) cause very serious crop diseases. Streptomyces

species can there fore contribute significantly to agricultural fungicides.

In this study, soils were specifically collected from different locations in

Sudan. Most of the soil samples collected were from different agricultural

locations. This was based on the assumption that actinomycetes diversity

may be influenced by the diversity of cultivated plant species as these

80

bacteria grow profusely in the humus and leaf litter layers (Oskay et al.,

2004). Although our screening efforts have been limited to few sampling

sites, yet they revealed many actinomycetes. One hundred and four

presumptive isolates when screened for antifungal activity against four

different plant pathogenic fungi, 83 of them produced antifungal substances

with growth inhibition zones in the range of 1-34mm. Any isolate which

produced an inhibition zone diameter of more than 10mm against any of the

tested fungi have been considered as a potential antifungal producer and was

thus characterized by cultural, morphological and physiological traits.

Based on the results of characterization, all of these isolates were classified

in the order, Actinomycetales: family Streptomycetaceae and genus

Streptomyces.

Isolate R92 which showed strong antifungal activities against all of the

tested pathogens was selected for antibiotic production in submerged culture

using Bennet broth medium. The crude extract of R92 broth was

comparatively more effective than the tested commercial antifungal agents

viz Nystatin, Mycosat and Itracon.

The ability of R92 extract to prevent diseases incidence on sorghum and

tomato seedlings due to infection by D. halodes and A. alternata was studied

in vivo. The in vivo control efficacies of these diseases were substantial; the

disease incidence in both cases was zero%. In addition, the progress and

development of D. halodes leaf spot (on sorghum) and A. alternata early

blight (on tomato) were greatly suppressed and restricted due to the

application of the crude extract.

In conclusion, R92 was found to be very effective not only in in vitro

inhibition of spore germination and mycelial growth of D. halodes, A.

alternata, A. sesami and M. phaseolina but also in preventing both incidence

81

and diseases development. The results are comparable with similar world

wide investigation. For example, Hoon et al. (2002) reported 98% control of

Magnaporthe grisea by one (BG2-53) of his actinomycetes isolates. Results

of this study have also demonstrated that the isolation of Streptomyces

species from diverse geographical locations in Sudan may present significant

capacity for antifungal agents production. Therefore recommend a long term

screening programme in order to discover a novel antibiotic. This should be

accompanied by a systematic approach to evaluate and optimize production

under different culture conditions using locally available substrates.

Parameters such as temperature, pH, sugar and nitrogen concentration in the

culture media should be carefully studied and adjusted under our conditions.

According to Desai et al. (2002) the pH value affects the production of

antibiotics at shake flasks fed culture. Also the production of antibiotics is

improved by the addition of phosphate, at a sub-optimal level, to the culture

media (Raytadpadar and Paul; 2001).

82

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