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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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