Optimization and production of antifungal hydrolysis enzymes by streptomyces aureofaciens against Colletotrichum gloeosporioides of mango

doi:10.4236/as.2011.22021

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Vol.2, No.2, 146-157 (2011)

doi:10.4236/as.2011.22021

Agricultural Science s

Optimization and production of antifungal hydrolysis

enzymes by Streptomyces aureofaciens against

Colletotrichum gloeosporioides of mango

Wafaa Mohamed Haggag1*, Enas Mostafa Mohamed2, Ahamed Mohamed El Azzazy2

1Department of Plant Pathology National Research Center, Cairo, Egypt; *Corresponding Author: wafaa_haggag@yahoo.com

2Biotechnology and Genetic Engineering Unit, National Research Center, Cairo, Egypt.

Received 9 March 2011; revised 15 March 2011; accepted 28 March 2011.

ABSTRACT

We isolated naturally occurring actinomycetes

with an ability to produce metabolites having

antifungal property against, Colletotrichum glo-

eosporioides, the causal agent of mango anth-

racnose. One promising strain was strong an-

tifungal activity, was selected for further studies.

Based on the physiological and biochemical ch-

aracteristics, the bacteria l strain was identical to

Streptomyces aureofaciens. Culture filtrate col-

lected from the exponential and stationary ph-

ases inhibited the growth of fungus tested, in-

dicating that growth suppression was due to

extracellular antifungal metabolites present in

culture filtrate. Isolate highly produced extrace-

llular chitinase and β-1,3-glucanase during the

exponential and late exponential phases, re-

spectively. In order to standardize the metabo-

lite production some cultural conditions like di-

fferent incubation time in hours, pH, carbon

sources and concentratio ns and nitro gen s o ur c e

were determined. During fermentation, growth,

pH and hydrolysis enzymes production were

monitored.Treatment with bioactive components

exhibited a significantly high protective activity

against development of anthracnose disease on

mango trees and increased fruit yi eld.

Keywords: Antifungal; Colletotrichum

Gloeospori oi des; Mango Anth ra cno se A nd

Streptomyces Aureofaciens

1. INTRODUCTION

Mango suffers from several diseases at all stages of its

life. Anthracnose disease caused by Colletotrichum

gloeosporioides (Glomerella cingulata Spauld & Sch-

renk) is one of the most common and serious diseases of

mango (Mangifera indica L.) in the tropics [1]. The dis-

ease occurs at any stage of fruit growth and the pathogen

causes the disease on a wide range of hosts such as apple,

pear, guava and mango [2]. Flower blight, fruit rot, and

leaf spots are among the symptoms of this disease [3].

Severe infection destroys the entire inflorescence result-

ing in no setting of fruits. Young infected fruits develop

black spots, shrivel and drop off. Fruits infected at ma-

ture stage carry the fungus into storage and cause con-

siderable loss during storage, transit and marketing . The

most visible evidence of disease occurs on postharvest

mango fruit by latent infection which usually results in

commercial losses [4]. Disease control methods include

the prophylactic use of fungicides such as benomyl, pro-

chloraz, mancozeb, carbendazim, iprodione and thia-

bendazol. Chemical fungicides not only may pollute the

atmosphere but also can be environmentally harmful, as

the chemicals spread out in the air and accumulate in the

soil, level and development of pathogen resistance [5].

Therefore, microbe-based biocontrol methods are one

alternative way to control diseases in place of agroche-

micals. Various microbial antagonists have been investi-

gated as potential antifungal biocontrol agents of plant

diseases. Many species of actinomycetes, especially those

belonging to the genus Streptomyces (Gram-positive,

mycelia-forming soil bacteria), are well known as bio-

control agents that inhibit or lyse several soilborne and

airborne plant pathogenic fungi [6,7]. The antifungal

potential of extracellular metabolites of Streptomyces

strains against some fungi was previously reported from

different locations of the world. The antagonistic activity

of Streptomyces to fungal pathogens is usually related to

the production of extracellular hydrolytic enzymes [8].

Chitinase and β-1,3-glucanase are considered to be im-

portant hydrolytic enzymes in the lysis of fungal cell

walls [9-12]. However, data related to the antagonistic

ability of the extracellular metabolites of Streptomyces

strains to suppress the growth of the fungal pathogens C.

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

147

gloeosporioides having a broad host range are limited.

Thus ,this study investigated to study i) antifungal activ-

ity of the cell-free culture filtrate of this antagonist to

determine whether the production of the extracellular

hydrolytic enzymes is involved in its observed effect ii)

the optimization of antifungal metabolites production

and using selected antagonistic bacteria for the control of

pre- and post-harvest anthracnose on mango fruit.

2. MATERIAL AND METHODS

2.1. Organisms and Media

Isolation of the pathogen from mango fruit with

anthracnose symptom was performed by tissue trans-

planting technique using potato dextrose agar (PDA).

Stock cultures of C. gloeosporioides was maintained

on PDA slants and stored at 4˚C. Actinomycetes were

isolated from the root tissues of mango trees by the

surface-sterilization technique [13]. All cultures were

purified by streak plate technique and confirmed by

colony morphology and screened for their antifungal

activity [14].

2.2. In Vitro A ntifungal Activity of

Extracellular Metabolites in

Cell-Free Culture Filtrates

To prepare the cell-free culture filtrate, the antagonist

was cultured into broth medium and incubated on an

incubator shaker (150 rpm) at 28˚C. The fermentation

broth was collected during the exponential and station-

ary phases. Cells were removed by centrifugation at

8,000 rpm for 20 min at 4˚C. Cell-free supernatant was

filtered aseptically through a sterile membrane with 0.45-

μm pore size and stored at 4˚C. The growth inhibitory

effects of the assay as described previously by Prapag-

dee et al. [15] with some modifications. Minimum In-

hibitory Concentration (MIC) of these compounds were

determined.

2.3. Identification of Isolated Antagonist

One promising isolate which showed a unique, stable

and interesting property of inhibiting only dermatoph-

ytes was selected and characterized. Identification of the

isolate to species level was based on morphological,

cultural, physiological and biochemical characteristics as

described by Taechowisan & Lumyong [16].

2.4. Effect of Cultivation Conditions on

Enzyme Production

Chitinase assay. The reaction mixture contained 1 ml

of 0.1% colloidal chitin in sodium acetate buffer (0.05 M,

pH 5.2) and 1 ml culture filtrate was incubated at 37˚C

for 2 h in a water bath with constant shaking. Suitable

substrate and enzyme blanks were included. Chitinase

activity was assayed by the colorimetric method of [16].

The reaction was terminated by adding 0.1 ml of 0.08 M

potassium tetraborate, pH 9.2 to 0.5 ml of reaction mix-

ture and then boiled in a water bath for 3 min. Then 3 ml

of diluted pdimethyl amino benzaldehyde (p-DMAB,

Sigma Chemicals Company, USA) reagent was added

and again incubated at 37˚C for 15 min. The released

product in the reaction mixture was read at 585 nm in a

spectrophotometer (Hitachi, Japan). One unit of chitinase

activity was defined as the amount of enzyme, which

produces 1 μ mole of N-acetylglucosamine in 1 ml of

reaction mixture under standard assay condition.

β-1,3-Glucanase activity was assayed by colourimet-

ric method of Nelson [17]. Reaction mixtures were in-

cubated at 37˚C for 30 min and were stopped by boiling

for 5 min. One unit of B-1,3-glucanase activity was de-

fined as the amount of enzyme that releases 1 μmol of

reducing sugar equivalents (expressed as glucose) per

min.

2.5. Optimization of Incubation Period, PH

and Carbon and Nitrogen Sources

Concentration for A ntifungal Met abolite

Production

The optimization of composition of incubation period,

and cultural conditions was carried out based on step-

wise modification of the governing parameters for anti-

fungal production. The cultures were transferred to seed

broth (200 mL of Medium) contained in a 500 mL Er-

lenmeyer flask and incubated at 30˚C on a rotary shaker

(175 rpm) for 6 - 8 hours. A 500 mL Erlenmeyer flask

containing 200 mL of the same seed medium was incu-

bated as specified above. The seed culture was trans-

ferred to a 5 liter fermenter containing each one 3.5 liter

of the three liquid media

2.6. Assay in Liquid Cultures

Bioagents growth was estimated directly by spectro-

photometric measurement of the OD600 (Amax) using a

PM 2A spectrophotometer and dry biomass concentra-

tion (bmax). Changing the pH 3 to 10 in the production

medium the effect of pH was observed. The effect of

cultivation temperature on the antifungal production was

examined at different temperatures starting from 25˚C to

60˚C with 5˚C intervals.

2.7. Effect of Incubation Period on

Antifungal (Enzymes) Production

The effects of incubation period were evaluated by 24

h interval by checking the antifungal activity were also

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

148

done. Hydrolysis enzymes was determined as previous

above. Culture optimization was carried out based on

stepwise modification of the governing parameters for

metabolites production and bioassay test at incubation

time in hours (24, 48, 72, 96 and 120); pH (6.0, 7.0, 7.5,

8.0 and 9.0); carbon sources and concentrations (glucose,

Cellulose, fructose, starch and sucrose) and nitrogen

source (KNO3, NaNO3, (NH4)2SO4, NH4NO3, NH4Cl,

urea, casein, yeast extract and peptone) in three repli-

cates. Fermentation studies were carried out in three

stages. To prepare inocula (b), a loopful spore from a

well-sporulated plate added each 40.0 ml respective seed

medium in 250 ml Erlenmeyer flasks and incubated at

28.0˚C for 48 h on a rotary shaker (150 rpm). After op-

timization of the fermentation parameters, 2.0 ml of the

seed culture (5.0%, v/v) was transferred to 250 ml Er-

lenmeyer flask containing 40.0 ml of the production me-

dium. The yield of the antifungal metabolite was moni-

tored in terms of arbitrary units (AU). Antifungal me-

tabolite production was carried out in 100 ml starch ca-

sein medium (starch 1%, casein 0.1%, KH2PO4 0.05%,

0.05%, MgSO4.7H2O, pH-7) in 500 ml Erlenmeyer

flasks.

2.8. In Vivo Evaluation of Antifungal

Activity

To determine the efficacy of the antifungal metabolite

against anthracnose pathogen, a field experiment was

conducted under natural infested conditions, using the

susceptible cultivars i.e. Sadekia (8˚ yr-old) at Noubaria

region, El Behera and Ismailia Sedekia (15˚ yr-old)

Governorates. Two sprayers were applied at 30 d inter-

vals starting from 1 st March (about one month before

normal flowering) on mango trees in one year. The bio-

active components at active concentration mixed with

0.1% carboxymethyl cellulase (CMC) as sticker was

sprayed using a knapsack sprayer. Trees were sprayed

till run-off with approximately 2˚ L of spray solution per

tree. Treatments were assigned in a randomized com-

plete block design. Trees sprayed with water and fungi-

cide served as a check treatment. Plots consisting of

three mango trees were replicated three times. Irrigation,

fertilization and other cultural practices were carried out

as recommended. The disease incidence was determined

as percentage of infected flowers at 30 days interval

during the growing season (March to July). Fruit yield

(kg/tree) were determined at harvesting date for treat-

ments.

2.8.1. Statistica l Analysis

The collected data were evaluated statistically using

the software SPSS for Windows (release 7.5.1, 20 De-

cember 1996; SPSS Inc., Chicago, IL). Data were sub-

jected to analyses of variance and treatment mean values

were compared by a modified Duncan’s multiple test (P

> 0.05).

3. RESULTS

3.1. Antifungal Activity

Preliminary screening for antifungal production was

done by streak on agar medium .Subsequent screening of

10 isolates that showed inhibitory activity against test

organism (Table 1). In particular, S. aureofaciens fol-

lowed by S. griseofuscus S2 showed significant activities

against pathogen. The antifungal activity of the purified

active substance against pathogen was determined. The

minimum inhibitory concentration (MIC) was deter-

mined by the diffusion method. The nutrient agar plates,

seeded with test organisms were used for the MIC de-

termination. The response was observed as a clear zone

(mm) around the paper discs (diameter 0.5 mm) loaded

with different concentration of active compound (20 μl)

of each concentration were spotted on paper discs. In

most cases, purified active substance of S. aureofaciens

showed antifungal activity against C. gloeosporioides

growth expressed as zone inhibition. The highest reduc-

tion was recorded at 1:3 concentration. The results in

Tabl e 1 showed the isolate S. aureofaciens was the best

isolate produces antifungal substances than the other

Streptomyces isolates. So, S. aureofaciens was chosen in

this study.

3.2. Enzymes Assays

The general ability of tested S. aureofaciens to pro-

duce secondary metabolites include hydrolysis enzymes

was determined (Figure 1). Exochitinase and β-1,3-glu-

canase appeared to be conman metabolites produced by

the tested BCAs. Maximum production of chitinase

and β-1,3-glucanase by the tested S. aureofaciens in

shaken broth culture occurred after 150 hrs (1.98 Unit/

ml) and (2.9 Unit/ml), respectively.

3.3. Optimization Condition for Antifungal

Metabolite Production

The time course for pH shows slight increase until 72

hrs then a negligible decrease through the remaining

time. The dry weight increases with time through all the

incubation period. The substrate decreases radically from

the beginning of incubation time till 120 hr then almost

stabilize. In the inhibition zone, enzymes curves, Colle-

totrichum follow the same pattern whereas the inhibition

zone, chitinase and β-1,3-glucanase increases with incu-

bation period till it reaches its maximum at 69 hr. The

time course for optical density shows that the maximum

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

149

Table 1. Antifungal activity of Streptomyces spp. against Colletotrichum gloeosporioides.

Spore suspension Purified active substance

Isolate Inhibition zone (cm)Inhibition zone (cm) MIC

Streptomyces aureofaciens 2.6a 2.4a 1:32

S. albidoflavu 1.8c 1.6c 1:2

S. albidoflavus 1.9bc 1.7b 1:2

S. cyanocolor 1.8c 1.7b 1:2

S. griseofuscus S1 1.8c 1.3d 1:1

S. griseofuscus S2 2.1b 1.8b 1:16

S. nodosus, 1.8c 1.5bc 1:2

S. alanosinicus 2.0b 1.7b 1:2

S. rochei 1.8c 1.3d 1:1

S. mutabilis, 1.8c 1.3d 1:1

Values represent the mean percentage of six replicates. Values in each column followed by the same letter are not significantly

different (P < 0.05)

Figure 1. Hydrolysis enzymes activities in different days.

value is obtained at 36 hr incubation time, then a decline

occurs. This is explained in the residual substrate curve

as we notice that a gradual consumption of glucose

occurs till 36 hrs then the glucose concentration re-

mains constant for the rest of incubation time (Figure

2). From the experimental results it was noticed that by

using different types of carbon source, the production

of chitinase and β, 1-3 glucanse (unit/ml) were maxi-

mum with starch as carbon sources. Also, the zone in-

hibition of Colletotrichum was maximum after 48 h

incubation with starch as carbon sources. So, the starch

was chosen as an optimum carbon sources (Figure 3).

By using different concentration of starch for the

growth of Streptomyces, the dry weight concentration

increased after 24 h with increasing the glucose and

fructose concentrations till 5 g/l then decreased (Figure

3). Also, the production of β, 1-3 glucanse (unit/ml)

and Chitinase increased after 48 h by increasing the

stach concentration till 5 g/l then decreased again

(Figure 3). The zone inhibition of Colletotrichum in-

creased gradually by increasing the glucose concentra-

tion till 5 g/l then remained almost constant. From the

previous observations, 5 g/l was chosen as the optimum

glucose concentration (Figure 4). By using different

types of nitrogen source for the growth of Streptomyces

it was noticed that the maximum biomass growth was

obtained using malt extract and soyabean as nitogen

source while the maximum zone inhibition for Colleto-

tricum were obtained with malt extract and CSL. The

maximum production of Chitinase and β, 1-3 glucanse

Enzymes were obtained with peptone and peptone in

equal ratio in the media (Figure 5).

3.4. Field Experiments

More attention in the last decade has focused on re-

placing synthetic fungicides with new strategies, such as

the use of biological control agents. The efficacies of the

spraying mango trees cv. Sadekia with culture filtrate of

Streptomyces aureofaciens on controlling of anthracnose

disease were determined in season of 2010 in Boheria and

Ismailia Governorates under nature infection conditions.

In control treatment, the diseases incidence was higher on

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

150

leaves and flowers blossom clusters in both regions. Sig-

nificant suppression of diseases incidence were achieved

by applying of culture filtrate of S. aureofaciens compared

to the fungicide and untreated control (Table 2). Applica-

tion of bioactive components of S. aureofaciens gave

completely reduced in all diseases, increased in flowering

and yield of both regions (Table 2).

5. DISCUSSION

In recent years, anthracnose has become a major chal-

lenge to both the pathologists working with the pathogen

and to mango researchers in general. The routine control

measure involving chemical pesticide application leads

to toxicity, residual effect and resistance development by

pathogens. The current situation is mainly focused on

biological control. Since all the commercial mango va-

rieties are susceptible to the disease, biological control

provides an effective, persistent and durable protection

Streptomyces aureofaciens had the ability to exhibit high

[18].

Figure 2. Effect of different nitrogen sources on the growth, inhibition activity and production

of hydrolysis enzymes by S. aureofaciens.

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

151

Figure 2. Cont.

B,1-3 glucanase

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

152

Figure 3. Effect of different carbon sources on the growth, inhibition activity and production

of hydrolysis enzymes by S. aureofaciens.

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

153

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

Glucose. FructoseSucrose CelluloseStarch

B 1-3 glucanse Enzymes (uni

/ ml)

0.2

0.4

0.6

0.8

1.2

Ch itinase Enzym es (unit/ m

1.4

1.6

Glucose. FructoseSucroseCelluloseStarch

Figure 3. Cont.

R2 = 0.9975

0.0000

5.0000

10.0000

15.0000

20.0000

25.0000

01234567

Starch conc. g/l

D ry weig h t g/l

= 1

5.8

5.9

6.1

6.2

6.3

6.4

6.5

6.6

6.7

6.8

01234567

Starch conc. g/l

Figure 4. Effect of different carbon concentration on the growth, inhibition activity and production of hydrolysis enzymes by S.

aureofaciens.

= 0.9949

2.85

2.9

2.95

3.05

3.1

3.15

3.2

3.25

3.3

3.35

01234567

Starch conc. g/l

Inhibition zone (mm) 72h

= 0.9616

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

01234567

Starch co nc . g/ l

Chitinase Enzymes (unit/ ml)

= 1

0.5

1.5

02468

Starch conc. g/l

B 1-3 glucanse Enzymes

(un it / ml )

Figure 4. Cont.

B,1-3 glucanse Enzymes (unit/ml)

Chitinase Enzymes (unit/ml)

Inhibition zone (mm) 72 h

Chitinase Enzymes (unit/ml)

B,1-3 glucan se Enzymes (unit/ml)

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

154

antifungal activity in vitro against Colletotrichum gloeo-

sporioides. The culture filtrate of this strain had also the

ability to in vivo suppress infection of Colletotrichum

gloeosporioides on mango trees. Many species of actin-

omycetes, especially those belonging to the genus Strep-

tomyces , are well known as biocontrol agents that inhibit

or lyse several soilborne and airborne plant pathogenic

fungi [7]. It is well known that Streptomyces sp. can

produce industrially useful compounds, notably wide

spectrum of antibiotics, as secondary metabolites, and

continues to be screened for new bioactive compounds

[12]. Optimization of fermentation media ingredients and

environmental factors for enzyme production is a more

convenient and effective strategy, compared to other re-

cent approaches like molecular techniques, to manifest

the physiological characteristics to synthesis enzymes.

Biological control of plant pathogens could reduce of

this concern. More recently an increased number of re-

searches focused on the potential of some types of bacte-

ria, yeast, and actinomycetes as a biocontrol agent

[9,11,6]. Regardless of the fermentation process that is

used to grow cells, it is necessary to monitor and control

parameters starting from the selection of optimum carbon

and nitrogen sources and including inoculum volume,

moisture content, pH, temperature, incubation period etc.

Changes in one of these parameters can have a dramatic

effect on the yield of cells and the stability of protein

product. The meaning of optimization in this context

needs careful consideration of the environmental and

nutritional parameters for growth and production. Me-

dium formulation is the foremost step for designing suc-

cessful laboratory experiments for yield enhancement.

alt extract(10 g

)+Yeast extract (4 g

)

alt extract(10 g

)+ Peptone (2.61 g

)

alt extract(10 g

)+ Soya beans (2.8 g

)

alt extract(10 g

)+ C

(11.88 m

Yeast extract (4 g

) + Peptone (0.2 g

)

Yeast extract (4 g

) + C

(0.91 m

Yeast extract (4 g

) + Soya B

eans

( 0.214 g

)

Peptone ( 1.41 g

) + Soya beans (1.51 g

)

Peptone ( 1.41 g

) + C

(6.394 m

Soya beans (1.51 g

) + C

(6.394 m

Yeast extract (4.31 g

)

alt extract (140.67 g

)

Peptone (2.81 g

)

(12.79 g

)

Soya beans

(3.014 g

)

Malt extract(10 g)+Yeast extract (4 g)

Malt extract(10 g)+ Peptone (2.61 g)

Malt extract(10 g)+ Soya beans (2.8 g)

Malt extract(10 g)+ CSL (11.88 ml)

Yeast extract (4 g) + Peptone (0.2 g)

Yeast extract (4 g) + CSL (0.91 ml)

Yeast extract (4 g) + Soya Beans ( 0.214 g)

Peptone ( 1.41 g) + Soya beans (1.51 g)

Peptone ( 1.41 g) + CSL (6.394 ml)

Soya beans (1.51 g) + CSL (6.394 ml)

Yeast extract (4.31 g)

Malt extract (140.67 g)

Peptone (2.81 g)

CSL (12.79 g)

Soya beans (3.014 g)

Dry wei g ht g / l

Figure 5. Effect of different nitrogen sources on the growth, inhibition activity and produc-

tion of hydrolysis enzymes by S. aureofaciens.

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

155

0. 5

1. 5

2. 5

Malt extract (10 g)+Yeast extract (4 g)

Malt extract(10 g)+ Peptone (2.61 g)

Malt extract(10 g)+ Soya beans (2.8 g)

Malt extract (10 g)+ CSL (11.88 ml)

Yeast extract (4 g) + Peptone (0.2 g)

Yeast extract (4 g) + CSL (0.91 ml)

Yeast extract (4 g) + Soya Beans ( 0.214 g)

Peptone ( 1.41 g) + Soya beans (1.51 g)

Peptone ( 1.41 g) + CSL (6.394 ml)

Soya beans (1.51 g) + CSL (6.394 ml)

Yeast extract (4.31 g)

Malt extract (140.67 g)

Peptone (2. 81 g)

CSL (12.7 9 g)

Soya beans (3. 014 g)

C olletotricum Zone inhibition (c m

)

0. 1

0. 2

0. 3

0. 4

0. 5

0. 6

0. 7

0. 8

0. 9

Malt ext ract(10

g)+Yeast extract (4 g)

Malt extract(10

g)+ Peptone (2.61 g)

Malt extract(10

g)+ Soya beans (2.8 g)

Malt ext ract(10

g)+ CSL

(11.88

ml)

Yeast extract (4 g) +

Peptone (0.2 g)

Yeast extract (4 g) + CSL

(0.91

ml)

Yeast extract (4 g) + Soya Beans ( 0.214

Peptone ( 1.41

g) +

Soya beans (1.51 g)

Peptone ( 1.41 g) + CSL

(6.394

ml)

Soya beans (1.51

g) +

CSL

(6.394

ml)

Yeast extract (4.31

Malt extract (140.67

Peptone (2.81

CSL

(12.79

Soya beans (3.014

Chitinase Enzymes (unit/ ml

)

0. 2

0. 4

0. 6

0. 8

1. 2

1. 4

1. 6

Malt extract(10

g)+ Yeast extract (4 g)

Malt extract(10

g)+ Peptone (2.61 g)

Malt extract(10

g)+ Soya beans (2.8 g)

Malt extract(10

g)+ CSL

(11.88

ml)

Ye ast extract (4 g) +

Peptone (0.2 g)

Yeast extract (4 g) +

CSL

(0.91

ml)

Yeast extract (4 g) + So ya Beans ( 0.214

Peptone ( 1.41

g) +

Soya beans (1.51

Peptone ( 1.41 g) + CSL

(6.394

ml)

Soya beans (1.51

g) +

CSL

(6.394

ml)

Ye ast extract (4.31

Malt extract (140.67

Peptone (2.81

CSL

(12.79

Soya beans (3.014

B 1-3 glucanse Enzymes (u nit/ ml

)

Figure 5. Cont.

B,1-3 glucanse Enzymes (unit/ml)

W. M. Haggag et al. / Agricultural Sciences 2 (2011) 146-157

156

Table 2. Efficacy of foliar sprays with bioactive substance of Streptomyces aureofaciens on the incidence of anthracnose disease and

yield of Sadika and Ewais mango cultivars.

Percentage of disease reduction % Fruit yield

Noubaria Ismailia Noubaria Ismailia

Leaves Fruits Leaves Fruits

Treatment

Ewais Sadikka Ewais Sadikka EwaisSadikkaEwaisSadikka Ewais Sadikka EwaisSadikka

Control -- -- -- -- -- -- -- --

8.5d 12.9d 11.5d21.3c

Culture filtrate of

Streptomyces aureofa-

ciens 98.8a 99.7a100a 100a 97.9a98.8a 99.8a100a 29.6a 33.6a 36.8a42.7a

Fungicide (Benomyl) 76.9b 74.6b 75.7b 74,6b 66.8b73.9b 77.6b75.7b 18.5b 21.5b 20.7b26.4b

LSD 8.87c 6.46c 8.54c 6.46c 8.38c6.54c 7.76c7.43c 4.65c 6.54e 5.41e6.49d

Values represent the mean percentage of six replicates. Values in each column followed by the same letter are not significantly different (P < 0.05)

The medium components play a vital role in the produc-

tion of antibiotic s and enzymes. In general, the produc-

tivity of microbial metabolites is closely related to the

fermentation process used. In addition to physiological

and genetic characteristics of strain, the medium compo-

sition plays an important role in the improvement of

productivity. The changes of nutrients and their concen-

trations have different effects on the accumulation of

different metabolites, which are controlled by intracellu-

lar effectors. Where, the carbon and nitrogen source can

dramatically influence antibiotic formation [19].

Openly accessible at

Spray application of bacterial filtrate on mango

treesprovided greater efficacy for controlling anthrac-

nose disease suggested that the bacterial produce some

antifungal enzymes for protecting the fruit against the

pathogen. This strain is promising for industrial applica-

tion since they grow quickly in broth condition in simple

and of a low cost process to enhance production yield,

and the excreted enzymes are frequently required for

industrial applications. Therefore, it is thought to be

considered as potential industrial candidate for effective

saccharification process.

6. ACKNOWLEDGEMENTS

This project was supported financially by the Science and Technol-

ogy Development Fund (STDF), Egypt, Grant No 216 under title:

Development of Bioproducts as Bio-fungicides for Controlling of

Major Foliar Diseases of Some Economic Horticultural Crops, from

2009-2012; PI. Wafaa M. Haggag.

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