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1 Effect of Some Resistance Inducing Agents and Antagonistic Fungi on the Linear Growth of Macrophomina Phaseolina and Fusarium oxyosporum the Causal Agents of Charcoal Rot and Wilt of Sesame In vitro A.M.M.Mahdy*,Nawal A.Eisa*, A.A.El-Wakil**, Faten M. Abdel-Latif,* and D.A.El-Wakil ** *Agric.Botany Dept.,Fac.Agric.,Moshtohor,Benha Univ.,Egypt. **Plant Pathol.Res.Inst.,Agric.,Res.Center,Giza,Egypt. ABSRACT Macrophomina phaseolina and Fusarium oxyosporum were isolated from sesame seeds by using Agar plate and blotter methods. Pathogenicity test using sesame seeds cv Giza 32 illustrated that M. phaseolina and F. oxyosporum were the causal agents of charcoal rot and wilt diseases. Laboratory experiments were carried out to study the effect of 7 chemical inducers, 5 plant extracts and 5 Trichoderma spp. isolates on the linear growth (LG) of M. phaseolina and F. oxysporum.Studying the effect of different concentrations of some inducing resistance agents on the linear growth (mm) of the 4 isolates for both fungi showed that the linear growth of the tested isolates was significantly suppressed by all chemical inducers i.e. Potassium Chloride, Hydrogen Peroxide, Acetic Acid, Butyric Acid, Tanic Acid, Salicylic Acid and Bion, respectively. Different concentrations of both filtered and/or autoclaved plant extracts significantly reduced(LG) of the 4 isolates of M. phaseolina and the 4 isolates of F.oxysporum in vitro. Etracts of cumin and thyme caused a high inhibition of mycelial growth at conc.of 55%.The antagonistic effect of T. harzianum ,T. viride and T.hamatum against the two fungi showed a clear suppresion in (LG) by T.harzianum isolates(T1,T2,T3andT4) for the isolates of M.phaseolina (M1,M5,M8 and M10), respectively. Key words: Sesame, inducing resistance compounds, natural plant extracts, Trichoderma spp. M.phaseolina, F. oxysporum INTRODUCTION Inducing resistance agents of different concentrations were investigated for their effect on the fungal growth of different plant pathogens (Datar., 1999; Orober et al., 1998). Moreover, effect of autoclaved and filtered plant extracts on fungal growth were also successfully used by earlier researchers (Bankole and Adebanjo,1995 and Hilal et. al., 2002). Antagonistic fungi; i.e.,Trichoderma harzianum , Trichoderma viridi and Trichoderma

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Page 1: Materials and methods...2 hamatum were evaluated for their antagonistic effect against fungal growth (Deshmukh and Raut , 1992;Dinakaran and Marimuthu ,1997). This study was conducted

1

Effect of Some Resistance Inducing Agents and Antagonistic Fungi on the

Linear Growth of Macrophomina Phaseolina and Fusarium oxyosporum the

Causal Agents of Charcoal Rot and Wilt of Sesame In vitro

A.M.M.Mahdy*,Nawal A.Eisa*, A.A.El-Wakil**, Faten M. Abdel-Latif,*

and D.A.El-Wakil **

*Agric.Botany Dept.,Fac.Agric.,Moshtohor,Benha Univ.,Egypt.

**Plant Pathol.Res.Inst.,Agric.,Res.Center,Giza,Egypt.

ABSRACT

Macrophomina phaseolina and Fusarium oxyosporum were isolated from sesame

seeds by using Agar plate and blotter methods. Pathogenicity test using sesame seeds cv Giza

32 illustrated that M. phaseolina and F. oxyosporum were the causal agents of charcoal rot

and wilt diseases. Laboratory experiments were carried out to study the effect of 7 chemical

inducers, 5 plant extracts and 5 Trichoderma spp. isolates on the linear growth (LG) of M.

phaseolina and F. oxysporum.Studying the effect of different concentrations of some inducing

resistance agents on the linear growth (mm) of the 4 isolates for both fungi showed that the

linear growth of the tested isolates was significantly suppressed by all chemical inducers i.e.

Potassium Chloride, Hydrogen Peroxide, Acetic Acid, Butyric Acid, Tanic Acid, Salicylic

Acid and Bion, respectively. Different concentrations of both filtered and/or autoclaved plant

extracts significantly reduced(LG) of the 4 isolates of M. phaseolina and the 4 isolates of

F.oxysporum in vitro. Etracts of cumin and thyme caused a high inhibition of mycelial growth

at conc.of 55%.The antagonistic effect of T. harzianum ,T. viride and T.hamatum against the

two fungi showed a clear suppresion in (LG) by T.harzianum isolates(T1,T2,T3andT4) for the

isolates of M.phaseolina (M1,M5,M8 and M10), respectively.

Key words: Sesame, inducing resistance compounds, natural plant extracts,

Trichoderma spp. M.phaseolina, F. oxysporum

INTRODUCTION

Inducing resistance agents of different concentrations were investigated for their effect

on the fungal growth of different plant pathogens (Datar., 1999; Orober et al., 1998).

Moreover, effect of autoclaved and filtered plant extracts on fungal growth were also

successfully used by earlier researchers (Bankole and Adebanjo,1995 and Hilal et. al.,

2002). Antagonistic fungi; i.e.,Trichoderma harzianum , Trichoderma viridi and Trichoderma

Page 2: Materials and methods...2 hamatum were evaluated for their antagonistic effect against fungal growth (Deshmukh and Raut , 1992;Dinakaran and Marimuthu ,1997). This study was conducted

2

hamatum were evaluated for their antagonistic effect against fungal growth (Deshmukh and

Raut , 1992;Dinakaran and Marimuthu ,1997).

This study was conducted to evaluate the asses of some different chemical inducers,

plant extracts and some bioagents on the linear growth of M. phaseslina and F. oxysporum

isolates under laboratory conditions.

MATERIALS AND METHODS

Isolation and identification of sesame seed-borne fungi:

Isolation of the causal pathogenic fungi was carried out on PDA medium by agar plate

and blotter methods in Seed Pathology Dept., Plant Pathology Research Institue, ARC, Giza,

Egypt. Identification of the isolated fungi was verified according to their morphological

features using the descriptions of Barnett and Hunter (1998).

The developed fungi were isolated, purified using the hyphal tip and/or the single

spore techniques (Hildebrand, 1938). The pure cultures were also maintained on PDA slants

kept at 5 oC for further studies.

Pathogenicity tests:-

Pathogenicity of isolates of M. phaseolina and F. oxysporum, isolated from sesame

seeds was tested under greenhouse conditions using seeds of sesame cv. Giza-32 planted in

soil infested with the desired fungal isolate.

Plastic pots (25 cm diameter) were filled with formalin disinfested soil. Inocula were

prepared by growing the desired isolate at 25oC. for two weeks on autoclaved sand sorghum

medium. The prepared inoculum was added to the potted soil at the rate of 3.0% weight,

mixed thoroughly with the soil, watered and left for a week. Pathogen-free sterilized sand

sorghum was mixed at the same rate with the potted soil to serve as control treatment. Pots

were planted with apparently healthy surface sterilized sesame seeds of cv. Giza-32 at the rate

of ten seeds per pot. Three replicated pots were used for each particular treatment. Percentage

of pre-emergence damping-off was calculated 15 days after sowing, while (%) post-

emergence damping-off and (%) healthy survived seedlings were also determined after 45

days. 90 days after sowing, percentages of healthy standing sesame plants were calculated

according to the following formula:

Page 3: Materials and methods...2 hamatum were evaluated for their antagonistic effect against fungal growth (Deshmukh and Raut , 1992;Dinakaran and Marimuthu ,1997). This study was conducted

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(%) Pre-emergence = 100.

edungerminat.x

seedsofplantedNoTotal

seedofNoTotal

(%) Post-emergence = 100xseedssownof.NoTotal

seedlingsrottedof.NoTotal

(%) Survived seedlings = 100xseedssownof.NoTotal

seedlinsurvivedof.NoTotal

Effect of different inducing resistance compounds on the linear growth of M. phaseolina

and F. oxysporum in vitro:

Potassium chloride (KCl) and Hydrogen Peroxide (H2O2) were tested at concentrations of 0.0,

0.25, 0.50, 1.0, 2.0, 4.0 and 8%. In addition ,the Indol acetic acid (IAA) and Indol butyric acid

(IBA) were tested at concentrations of 0.0, 50.0, 100.0, 200.0, 400.0, 800.0 and 1600.0 ppm.

Moreover Tanic acid (TA); Salicylic acid (SA) and Bion were tested at concentrations of 0.0,

0.25, 0.50, 1.0, 2.0, 4.0 and 8.0 mM. The amount required for obtaining a known

concentration of the desired chemical was calculated and added aseptically to a known

amount of sterilized PDA medium immediately before pouring in plates, then plates were

inoculated with the desired isolate of M.phaseolina and F.oxysporum and incubated at 27 o

C

for 5 days. Three plates were used as replicates for each particular concentraion. Diameter of

the linear growth of each isolate was determined .

Effect of 5 plant extracts on the linear growth of M. phaseolina and F. oxysporum in

vitro:

Plant extract of each of Hibiscus sabdariffa (dried sepales), Pimpinella anisum (dried

seeds) Eucalyptus golobulus (dried leaves), Cumin cymium (dried seeds) and Thymus vulgaris

(fresh herb) were obtained to test their inhibitory effects on the linear growth of different

isolates of M. phaseolina and F. oxysporum. Each fungus was represented by four isolates

selected according to the geografical locations and the pathogenicity test. Fifty grams of both

fresh or dried plant parts were used.The fresh parts were weighed, washed under running tap

water, air dried for one hour under natural conditions then used for preparing two kinds of

plant extracts as follows:

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1) Cool extraction and sterilizing by filtration:

The fresh and fine powdered plant parts were used as mentioned by Dubey and

Dwivedi (1988).

2) Hot extractions and sterilizing extracts by autoclaving:

The plant extracts were prepared as reported byYoussef (1991) and Brooker et al.

(2000).

The tested plant extract, i.e. filtrated or autoclaved was added to sterilized PDA

medium at the rate of 10, 25 ,40 and 55% (v/v) immediately before solidification and poured

in plates. Sterilized distilled water was used instead of plant extracts in the control treatment.

Plates were inoculated individually with a disc (5 mm) taken from the 7 days-old culture for

each tested isolate of M. phaseolina or F. oxysporum. Three replicates were used for each

treatment and all plates were incubated at 27 o

C for 7 days.

Linear growth was observed daily

and the diamaters of fungal colonies in “mm” were recorded when the fungal growth in the

plates of any treatment reached the edges of the dish.

Effect of some antagonistic fungi on the linear growth of M. phaseolina and F.oxysporum

in vitro :

Investigating the inhibitory effect of some isolates of antagonistic fungi on the linear

growth of the four tested isolates of each of M. phaseolina and F. oxysporum was carried out

as reported by Elad et al. (1986). In this respect, T. harzianum (4 isolates), T. viride (3

isolates) and T. hamatum (3 isolates) were kindley obtained from Integrated Control

Research Dept. Agric. Res. Center, Giza, Egypt and were used herein. The antagonistic

effects of these fungi were determined by taking two discs (5 mm in diam.) of 7 days-old

culture of both antagonistic and the pathogenic fungi( M. phasseolina and F.oxysporum).

Plates were inoculated simultneously with each opposite the other 1 cm apart from the plate

edge in individual plates (9 cm in diam.) contained 20ml PDA medium. In control treatment,

the plates were inoculated each with one disc bearing the growth of the same isolates of M.

phaseolina and F. oxysporum in individual treatments. Three plates were used for each

particular treatment. All plates were incubated at 27C° for 5 days. Percentage of the fungal

growth reduction (% GR) was calculated by using the following formula (Papavizas ,1985).

% GR = LR1-LR2 x 100

LR1

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Where:

LR1 = linear growth of the pathogen alone.

LR2= linear growth of the pathogen against the Antagonistc fungus.

Statistical Analysies

The experimental design (s) are completely randomized with three replicates, analysis

of variance (ANONA) of the data was performed with MSTAT-C statistical package (A).

Micro computer program was used for the disease mangement and analysis of the

agronomic research experiment. Least significant difference (L.S.D) was used to compare

treatemnt means Gomez and Gomez (1984).

RESULTS

Isolation and identification of sesame seed borne fungi

This study was conducted to reveal the best method of isolation of seasame seed-borne

fungi, i.e. agar plate and blotter methods. The isolated fungi were identified as mentioned

before. Data in Table (1) illustrate that the blotter method was the most convenient for

isolation. Alternaria sesami recorded the highest incidence (4.5 & 19.0%) followed by A.

Alternata (3.5&17.5%) from apparently healthy and infected seeds, respectively. Also,

Fusarium roseum recorded (2.5 & 16.5%) for AH & INF sesame seeds, respectively. Isolation

on PDA medium from sesame seeds yielded different fungal species,i.e. A. alternata, A.

sesami, Aspergillus flavus. Drechslera sesame, F. solani, F. oxysporum and M. phaseolina

which resulted 14.5, 18.0, 11.0, 16.5, 12.0, 4.6 and 8.5% for INF sesame seeds, respectively.

On the other hand, the A.H. sesame seeds yielded A. alternata., A. sesami and A. flavus, being

(1.5, 2.5 and 5.0 %, respectively).

Table (1) Incidence of seed-borne fungi in samples, apparently healthy and

infected sesame seeds cv. Giza - 32

Isolated fungi

Method of isolation

Blotter PDA

*A..H. **INF A..H. INF Alternaria alternata 3.5 17.5 1.5 14.5 Alternaria sesami 4.5 19.0 2.5 18.0 Aspergillus flavus 0.0 14.5 5.0 11.0 Drechslera sesame 6.0 11.0 0.0 16.5 Fusarium oxysporum 0.0 13.5 0.0 4.6 Fusarium solani 0.0 0.0 0.0 12.0 Fusarium roseum 2.5 16.0 0.0 0.0

Macrophomina phaseolina 0.0 6.5 0.0 8.5 Mean 2.1 12.3 1.1 10.6 *A.H.% = apparently healthy sesame seeds.

**INF% = infected sesame seeds.

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Pathogenicity tests:

Data in Table (2) indicate that all tested isolates of M. phaseolina recorded different

percentage of pre-and post-infection at seedling stage. M. phaseolina (M1) recorded the

lowest percentage of pre-infection (16.6%), while (M9) recorded 20.0% followed by (M8) that

recorded 30%. For survival plants, it is clear from the same data that significant differences

were recorded between the survival of sesame plants. The highest percentage of survived

plants, being 80% was recorded with isolate (M8), while the lowest percentage of survived

plants was recorded with isolate M1. All the tested F. oxysporum isolates were differed in

their reaction from one isolate to another. The lowest percentage of wilted plants (13.4%) was

recorded by isolate and isolate F9 (16.8%) without significant, while isolate F3 recorded the

highest percentage of healthy mature plants (43.4%).The lowest percentage of healthy mature

plants (16.4%) was recorded with isolate F6 Giza isolate showed the highest percentage of

survived plants, whreas the lowest percentage of survived plants (36.4 %) was recorded in

case of F6 (Ben Sweif isolate).

Table (2): Pathogencity of 5 isolates of M. phaseolina and 6 isolates of F. oxysporum on

sesame cv. Giza 32 under greenhouse conditions

M. phaseolina

isolates Location

(%) Disease incidence

Seedling stage Mature stage

% pre %post %

Servival %

mortality Healthy

% mean

(M1) Giza 26.6 20.0 53.4 16.6 36.8 30.68

(M5) Ismailia 23.3 13.3 63.4 40.0 23.4 37.36 (M8) Bensweif 6.6 13.4 80.0 30.0 50.0 36.0

(M9) Sharkyia 20.0 20.0 60.0 20.0 40.0 40.0

(M10) Sohag 13.3 10.7 76.0 40.0 36.0 35.2 Control 0.0 100.0 0.0 0.0 100.1 40

L.S.D 4.3 4.2 6.3 7.1 8.2 -

F. oxysporium

Location

% Disease incidence

Seedling stage Mature stage

%pre %post %

Servival %

mortality %

Healthy Mean

F3 Giza 6.6 20.0 73.4 30.0 43.4 34.68 F4 Ismailia 10.0 30.0 60.0 20.0 40.0 32.0

(F6) Bensweif 30.0 33.3 36.4 20.0 16.4 27.2

(F8) Sharkyia 13.3 16.6 70.7 40.1 30.7 34.16

(F9) Giza 26.6 26.6 46.8 16.8 30.0 29.36 (F10) Sohag 16.6 40.0 43.4 13.4 30.0 34.86

L.S.D 5.8 4.2 5.1 5.4 7.2 -

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3- Effect of different concentrations of some resistance inducing agents on the linear

growth in (mm) of 4 isolates of M. phaseolina and 4 isolates of F. oxysporum:

A) Effect on the linear growth of M. phaseolina isolates:

Data shown in Table (3 a) demonstrate that the linear growth of the 4 tested isolates of

M. phaseolina was singificantly decreased due to the effect of all the tested chemical

inducers. The highest decrease in linear growth was noticed with KCl at concentration 4.0%

for all isolates.

Table(3a) Effect of some KCl and H2O2 concentration as chemical inducers on the linear

growth of four isolates of M. phaseolina.

Inducer Isolate Concentration (%)

Mean Grand mean 0.0 0.25 0.50 1.0 2.0 4.0 8.0

KCl

M1 90.0 81.5 68.5 56.2 13.2 0.0 0.0 43.5

40.08 M5 90.0 65.7 57.3 37.4 18.4 0.0 0.0 37.8 M8 90.0 74.8 61.6 42.3 12.8 0.0 0.0 39.5

M10 90.0 67.2 66.5 46.8 11.4 0.0 0.0 39.5

H2O2

M1 90.0 75.4 72.8 56.6 17.7 5.9 0.0 44.7

41.78 M5 90.0 66.7 56.3 46.8 24.5 10.4 0.0 41.4 M8 90.0 77.0 65.7 30.6 8.5 2.3 0.0 38.5

M10 90.0 80.2 73.9 34.5 18.7 5.4 0.0 42.5 LSD. at 5% for:

Isolates (Iso) = n.s. Inducers (Ind) =1.23 Conc. ( C ) =1.23 Iso x Ind =2.33

Iso x C = n.s. Ind x C = 1.92 Iso x Ind x C = 3.90

Data in Table (3b) show that IAA caused a noticeable decrease in the linear growth

(Ranged from 40.9 to 45.0 mm) compared to control (90.0 mm), while IBA caused a decrease

in linear growth of the 4 tested isolates of M. phaseolina, ranged from 40.3 to 43.4mm.

Table(3b) Effect of some growth regulators on the linear growth of five isolates of M.

phaseolina.

Inducer Isolate Concentration (%)

Mean Grand mean 0.0 0.25 0.50 1.0 2.0 4.0 8.0

IAA

M1 90.0 85.0 73.9 30.2 6.8 5.2 0.0 40.8

43.68 M5 90.0 85.0 80.3 63.4 7.5 0.0 0.0 45.9

M8 90.0 85.0 65.7 39.2 16.3 9.5 0.0 42.9 M10 90.0 85.0 80.4 48.6 11.5 4.6 0.0 45.1

IBA

M1 90.0 85.0 72.3 31.4 6.5 1.5 0.0 40.3

41.95 M5 90.0 85.0 72.4 56.6 4.1 0.0 0.0 43.4

M8 90.0 85.0 81.9 46.8 3.8 0.0 0.0 43.2 M10 90.0 85.0 74.0 40.0 2.5 0.0 0.0 40.9

LSD. at 5% for:

Isolates (Iso) = n.s. Inducers (Ind) =1.61 Conc. ( C ) =1.61 Iso x Ind =2.11

Iso x C = n.s. Ind x C = 2.13 Iso x Ind x C = 3.22

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Data in Table (3c) show that tanic acid caused the highest decrease in the linear

growth recorded with isolate M8 (being 37.1mm). Salicylic acid SA, and bion decreased the

linear growth completely for the 4 tested isolates at 8.0mM.Also, bion suppressed the linear

growth for all M. phaseolina isolates at 8.0 mM..

Table(3c) Effect of some chemical inducers on the linear growth of four isolates of M.

phaseolina.

Inducer Isolate Concentration (%)

Mean Grand mean 0.0 0.25 0.50 1.0 2.0 4.0 8.0

Tanic acid

M1 90.0 82.9 76.8 22.7 6.3 0.0 0.0 39.2

39.85 M5 90.0 76.5 66.3 36.5 8.3 0.0 0.0 38.9 M8 90.0 66.5 61.5 37.4 8.9 0.0 0.0 37.1 M10 90.0 80.4 73.9 56.2 11.2 2.5 0.0 44.2

SA

M1 90.0 85.0 81.9 41.2 19.8 5.6 0.0 45.6

42.10 M5 90.0 85.0 72.4 22.8 9.5 2.5 0.0 39.9 M8 90.0 85.0 74.3 37.8 11.3 4.2 0.0 42.5 M10 90.0 85.0 65.7 25.1 13.5 8.1 0.0 40.4

Bion

M1 90.0 85.0 72.3 30.8 15.6 4.3 0.0 41.8

42.30 M5 90.0 85.0 81.9 21.4 9.8 2.1 0.0 40.7 M8 90.0 85.0 75.4 67.5 12.4 3.2 0.0 46.9 M10 90.0 85.0 65.2 28.2 11.5 4.2 0.0 39.8

LSD. at 5% for:

Isolates (Iso) = n.s. Inducers (Ind) =1.82 Conc. ( C ) =1.82 Iso x Ind =2.77

Iso x C = n.s. Ind x C = 2.60 Iso x Ind x C =3.11

B) Effect on linear growth of F.oxysporum isolates:

Data in the Table (4a) illustrate that the linear growth of the 4 tested isolates of

F.oxysporum was significantly decreased due to the tested chemical inducers. The linear growth

was compeletly suppressed at conc. 2.0% with H2O2. Generaly, the linear growth (mm) was

decreased at coc.of 4.0% with KCl and H2O2.

Table(4a) Effect of some KCl and H2O2 as chemical inducers on the linear growth of

four isolates of F.oxysporum.

Inducer Isolate Concentration (%)

Mean Grand mean 0.0 0.25 0.50 1.0 2.0 4.0 8.0

KCl

F6 90.0 85.0 68.3 37.3 0.0 0.0 0.0 39.4

40.1 F8 90.0 85.0 57.2 56.3 0.0 0.0 0.0 40.5 F9 90.0 85.0 61.5 36.5 0.0 0.0 0.0 38.3 F10 90.0 85.0 76.8 48.2 0.0 0.0 0.0 42.2

H2O2

F6 90.0 85.0 73.9 48.4 14.6 0.0 0.0 43.8

41.8 F8 90.0 85.0 67.3 41.3 13.8 0.0 0.0 41.7 F9 90.0 85.0 65.8 22.8 13.8 0.0 0.0 38.8 F10 90.0 85.0 70.5 48.4 11.9 0.0 0.0 42.9

LSD. at 5% for:

Isolates (Iso) = 1.50 Inducer (Ind) =1.60 Conc. ( C ) =1.60 Iso x Ind =2.41

Iso x C = 3.10 Ind x C = 2.60 Iso x Ind x C =3.90

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Data in Table (4b) show that IAA and IBA also decreased the linear growth for all the

tested isolates, F10 was the least affected by IAA and IBA and recorded 12.9&17.9 mm,

respectively at conc.1600ppm with IAA and IBA, respectively .

Table(4b) Effect of some growth regulators on the linear growth of four isolates of

F.oxysporum.

Inducer Isolate Concentration (%)

Mean Grand mean 0.0 0.25 0.50 1.0 2.0 4.0 8.0

IAA

F6 90.0 81.6 66.7 40.0 23.5 12.8 6.8 45.2

48.3 F8 90.0 85.0 80.3 56.6 24.2 12.4 7.9 50.2 F9 90.0 65.7 65.7 46.8 40.8 20.2 11.6 47.9 F10 90.0 85.0 85.0 37.8 22.6 21.7 12.9 50.0

IBA

F6 90.0 74.9 73.9 30.6 19.4 17.7 7.5 44.5

45.3 F8 90.0 73.9 65.8 36.0 24.1 12.8 6.9 43.6 F9 90.0 66.6 73.9 34.6 27.2 20.1 16.3 46.4 F10 90.0 75.4 66.6 31.4 27.5 23.0 17.9 46.7

LSD. at 5% for:

Isolates (Iso) = 1.50 Inducer (Ind) =1.90 Conc. ( C ) = 1.93 Iso x Ind =3.10

Iso x C = 4.29 Ind x C = 2.90 Iso x Ind x C = 4.81

Data in Table (4c) show that tanic acid caused a noticeable reduction in the linear

growth (mm) for all the tested isolates by using the aforementioned chemical inducers. Isolate

F9 recorded the least reduction, being 53.8mm on the average. whereas, the highest inhibition

was recorded with isolate F6 (44.3 mm). As for SA, all the tested concentration caused a

reduction in the linear growth at 4.0mM for all tested isolates. Regarding bion, the reduction

was noticed at 8.0 mM for all the tested isolates, whereas, the highest inhibition was recorded

with isolate F8 (43.9 mm) and the least was recorded with F9 (60.6 mm).

Table(4c) Effect of some chemical inducers on the linear growth of four isolates of

F.oxysporum.

Inducer Isolate Concentration (%)

Mean Grand mean 0.0 0.25 0.50 1.0 2.0 4.0 8.0

Tanic acid

F6 90.0 85.0 73.9 30.2 16.2 12.4 7.3 44.3

49.7 F8 90.0 85.0 70.7 48.6 43.5 17.8 11.6 51.7 F9 90.0 85.0 73.9 47.1 43.2 24.5 17.5 53.8 F10 90.0 85.0 75.4 39.2 17.2 27.0 13.0 48.8

SA

F6 90.0 85.0 74.3 70.1 42.0 0.0 0.0 50.9

51.8 F8 90.0 85.0 73.9 72.4 70.8 0.0 0.0 55.4 F9 90.0 85.0 72.3 63.4 59.8 0.0 0.0 52.21 F10 90.0 85.0 85.0 58.6 28.2 0.0 0.0 48.8

Bion

F6 90.0 85.0 81.9 48.3 23.2 14.2 0.0 48.3

49.9 F8 90.0 85.0 72.4 25.3 21.4 18.7 0.0 43.9 F9 90.0 85.0 85.0 79.3 67.5 22.4 0.0 60.6 F10 90.0 85.0 74.3 31.3 27.2 23.8 0.0 46.7

LSD. at 5% for:

Isolates (Iso) = 1.50 Inducer (Ind) =2.11 Conc. ( C ) = 2.13 Iso x Ind =3.59

Iso x C = 1.90 Ind x C = 3.10 Iso x Ind x C = 4.65

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5- (A) Effect of different concentrations of filtered and autoclaved plant extracts on the

linear growth of M. phaseolina in vitro:

Five different aqueous plant extracts were used after sterilizing by filtratrion or

autoclaving for evaluating their effect on the linear growth of four isolates of M. phaseolina

i.e., M1, M5, M8 and M10.

Concerning the aqueous filtered extracts, the recorded data in Table(5) show that the

filtered extract of thyme caused a complete growth inhibition of the isolate (M1) at the

concetration of (40%). Also, the filtered extract of each of eucalyptus,cumin and thyme

caused complete growth inhibition at (40%) for isolate (M5). While, the filtered cumin and

thyme extracts caused a complete inhibition to the growth of isolate (M8) at conc. 40% and

55%, respectively. The aqueous filtered extract of anise at 55%, eucalyptus and thyme at

40% completely inhibited the growth of isolate (M10).

Regarding the autoclaved plant extracts, the obtained data indicate , generally that all

tested plant extracts caused a noticeable inhibition to the linear growth of the four isolates of

M. phaseolina. The highest inhibition was increased by increasing concentrations of the plant

extracts. The autoclaved extract of anise caused a complete growth inhibition at conc. 40%,

and the extract of eucalyptus and thyme at conc.55% in case of isolate (M1).While, all the

tested plant extracts caused complete inhibition for the linear growth at conc.55% with isolate

(M5). However, the autoclaved plant extract of anise, eucalyptus,cumin and thyme caused a

noticealbe reduction in the linear growth of isolate M8 at conc.55% while, the autoclaved

plant extract of cumin and thyme completely inhibited the linear growth of isolate (M10) at

55%.

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Table (5): Effect of different concentrations of filtered watery and autoclaved plant

extrats on the linear growth of 4 M. phaseolina isolates in vitro.

Plant

extract Isolate

Linear growth,mm

Mean Grand

mean

Linear growth,mm

Mean Grand

mean

Filtered extract

concetration

Autoclaved extract

concetration

10% 25% 40% 55% 10 20 30 40

Roselle M1 79.0 65.3 51.7 40.7 59.1

63.3

85.0 80.1 79.3 60.2 76.2

64.16

M5 67.8 39.0 26.8 13.5 36.8 85.1 22.8 18.3 0.0 34.1

M8 82.6 71.4 66.7 30.5 62.8 72.8 66.3 43.2 21.3 50.9

M10 80 77.3 69.1 44.3 67.8 85.0 80.2 67.3 46.5 69.7

Control 90 90 90 90 90 90 90 90 90 90

Anise M1 81.5 64.2 50.3 33.1 57.2

55.6

82.5 42.8 0.0 0.0 31.3

40.3

M5 69.0 47.5 36.1 12.7 41.3 39.8 12.8 0.0 0.0 13.2

M8 65.8 67.2 55.6 33.4 55.6 85.0 38.2 0.0 0.0 30.8

M10 67.2 31.4 18.5 0.0 34.3 56.8 48.6 26.1 13.2 36.2

Control 90 90 90 90 90 90 90 90 90 90

Eucalyptus M1 60.7 44.5 33.6 13.7 38.6

48.72

67.8 49.3 23.7 0.0 35.2

49.62

M5 79.0 62.3 0.0 0.0 35.3 77.3 62.5 12.6 0.0 38.1

M8 65.1 44.5 31.3 29.1 42.5 79.0 70.9 52.0 0.0 50.3

M10 81.3 67.3 0.0 0.0 37.2 56.8 39.2 28.2 13.8 34.5

Control 90 90 90 90 90 90 90 90 90 90

Cumin M1 78.3 66.8 39.1 26.5 52.7

53.54

85.0 35.0 85.0 85.0 85

54.7

M5 39.0 27.3 0.0 0.0 16.6 85.0 60.5 54.6 0.0 50.2

M8 67.2 37.6 16.8 0.0 39.5 64.5 27.3 0.0 0.0 22.9

M10 81.2 77.3 67.5 79.5 68.9 26.7 18.8 11.3 0.0 25.4

Control 90 90 90 90 90 90 90 90 90 90

Thyme M1 69.3 53.2 0.0 0.0 30.6

55.94

59.3 39.7 18.9 0.0 29.5

73.66

M5 43.5 32.4 0.0 0.0 18.9 81.6 79.3 26.9 0.0 76.9

M8 81.8 76.3 0.0 0.0 39.5 69 38.4 19.5 0.0 31.9

M10 29.4 13.5 0.0 0.0 10.7 44 38.3 14.9 0.0 85

Control 90 90 90 90 90 90 90 90 90 90

L.S.D at 5% for.

Isolate (I) =1.61

Extract (E) = 2.10

Concent (C) =1.91

IxE = 1.30

IxC =2.19

L.S.D at 5% for.

Isolate (I) = 1.61

Extract (E) = 2.19

Concent (C) =1.80

IxE = 1.20

IxC =1.90

IxExC = 3.11 IxExC = 3.20

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6- (B) Effect of different concentrations of filtered and autoclaved plant extracts on the

linear growth of F. oxysporum in vitro :

Concerning the effect of the filtered plant extracts on the four isolates of F. oxysporum

i.e.,F6, F8, F9 and F10, data shown in Table (6) indicate that the filtered plant extract of

eucalyptus completely inhibited the fungal growth at conc. of 55%, and by cumin plant

extract at conc. 40 % for isolate (F6). Meanwhile, the filtered roselle plant extract caused

complete growth inhibition for isolate (F8) at conc. 55%, and eucalyptus extract at conc. 55

%. The filtered plant extract of each of roselle,anise,cumin and thyme caused inhibition for

isolate F9 at conc. 55%. As for isolate F10 the filtered roselle, eucalyptus and cumin extracts

caused a complete inhibition at conc. 55%.

Regarding to the autoclaved plant extracts, the recorded data (Table 6) show that, in

most cases,tested plant extracts caused an inhibition in the linear growth of F. oxysporum

isolates F6, F8, F9 and F10, respectively. The highest fungal growth inhibition was recorded

when the highest concentrations of plant extracts, i.e 40% & 55%, respectively were used.

The autoclaved extract of roselle caused a complete inhibition at conc. of 40% & 55%,

respectively for F. oxysporum isolate (F6), while the highest inhibition for isolate F8 of F.

oxysporum was recorded with autoclaved plant extract of roselle and cumin (18.8 &

9.7mm,respectively). The autoclaved plant extract of roselle at concentration 55%

completely inhibited the growth of isolate (F9),while autoclaved plant extract of cumin

caused a complete inhibition of isolate (F10) compared to control.

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Table (6) : Effect of different concentrations of filtered watery and autoclaved plant

extracts on the linear growth (mm) of 4 F. oxysporum isolates in vitro.

Plant

extract Isolate

Filtered extract Mean

Grand

mean

Autoclaved extract Mean

Grand

mean 10% 25% 40% 55% 10 25 40 55

Roselle M6 56.3 44.2 18.3 9.7 32.1

24.92

38.2 18.5 0.0 0.0 14.2

46.10

M8 75.8 48.3 16.7 0.0 35.2 78.3 67.2 51.2 18.8 53.9

M9 67.3 48.2 19.5 0.0 33.6 65.0 54.8 15.6 0.0 33.7

M10 44.3 32.2 18.5 0.0 23.7 67.8 37.8 29.3 19.8 38.7

Control 90 90 90 90 90 90 90 90 90 90

Anise M6 78.0 63.0 52.3 6.3 52.3

51.06

85.3 81.8 79.7 34.8 61.6

66.50

M8 77.8 60.5 44.1 18.7 50.3 85 80.7 79.3 67.2 78.0

M9 69.6 45.5 18.1 0.0 78.0 78.6 60.3 44.5 15.6 49.8

M10 85.6 85.0 85.0 85 85.0 79.3 65.2 48.3 19.3 53.1

Control 90 90 90 90 90 90 90 90 90 90

Eucalyptus M6 68.2 40.1 15.2 0.0 30.9

33.46

72.0 53.0 37.0 24.2 46.6

71.94

M8 69 48.3 19.6 0.0 34.2 85 85 85.0 85 85

M9 85.8 81.2 79.2 66.7 78.0 85 85 85 85 85

M10 68.7 49.3 18.7 0.0 34.2 79.3 65.2 48.3 19.3 53.1

Control 90 90 70 90 90 90 90 90 90 90

Cumin M6 42.2 23.2 0.0 0.0 17.6

46.90

42 32.8 27.9 19.7 30.6

44.48

M8 78.8 75.8 54.3 57.3 67.7 58.8 32.2 18.9 9.7 29.9

M9 46.2 42.8 12.8 0.0 30.2 59.6 47.2 22.8 11.5 35.3

M10 58.5 39.4 0.0 0.0 29.5 85 61.5 0.0 0.0 36.6

Control 90 90 90 90 90 90 90 90 90 90

Thyme M6 81.0 63.1 50.4 18.2 53.2

40.40

75.0 43.7 22.3 13.2 38.5

53.18

M8 67.2 79.8 54.3 34.1 58.9 68.0 59.7 48.3 32.7 52.2

M9 67.2 32.5 27.5 0.0 31.8 46.0 31.7 18.3 9.7 26.4

M10 79.4 65.1 53.1 43.8 60.4 85.0 80 41.8 23.0 58.8

Control 90 90 90 90 90 90 90 90 90 90

L.S.D at 5% for:

Isolate (I) = 1.90

Extract (E) = 1.10

Concent (C) =2.33

IxE = 3.50

IxC =1.82

L.S.D at 5% for:

Isolate (I) = 1.90

Extract (E) = 2.30

Concent (C) =2.18

IxE = 3.61

IxC =1.10

IxExC = 3.60 IxExC =4.60

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7- (A) Antagonistic effect of some Trichoderma spp. against four isolates of M. phaseolina:

The aim of this experiment is to investigate the antagonistic effect of three species of

Trichoderma i.e., T. harzianum, T. viridi and T.hamatum on the linear growth of M.

phaseolina isolates.

Data presented in Table (7) indicate that the tested antagonistic Trichoderma spp.

reduced significantly the mycelial growth of the four tested M. phaseolina isolates, i.e. M1,

M5, M8 and M10. The four isolates of T. harzianum, i.e. T1, T2, T3, and T4, reduced the

fungal growth of M. phaseolina isolates, i.e. M1, M5, M8 and M10, being 57.9, 59.8, 62.3

and 59.6 % on the average, respectively. The highest reduction was recorded in case of T.

harzianum (T4) against M. phaseolina (M1) followed by (T2) and (T1),respectively. Data in

the same Table also show that T. viride isolates, i.e.T5, T6 and T7 caused a considerable

reduction to the linear growth of the four M. phaseolina isolates, being 60.2 , 50.6 & 59.4%,

respectively.The highest effective isolate was T.viride (T7) followed by T5 against M.

phaseolina (M1). For T. hamatum isolates; T8, T9 and T10 recorded reduction in the linear

growth, being 61.4, 57.7 and 51.7 % on the average, respectively as compared to control.

As for the three tested Trichoderma spp., T. harzianum (T3) followed by T. hamatum

(T8) and T. viridi (T5) were the best effective isolates in reducing the growth of M.

phaseolina isolates.

Table(7): Antagonistic effect of Tricholderma spp. on growth reduction (mm)of 4 M.

phaseolina isolates in vitro at 25o for 7 days.

Antagonistic fungi

Isolate/ No.

%Reduction in linear growth Mean

Grand mean

M. phaseolina isolates

M1 M5 M8 M10

T.harzianum

T 1 70.7 44.2 53.3 63.4 57.9

59.9 T 2 70.8 54.8 52.2 61.3 59.8 T3 66.9 61.0 59.2 62.0 62.3 T 4 71.5 45.7 59.2 62.0 59.6

T. viridi T5 70.7 49.4 60.8 59.8 60.20

56.6 T6 46.8 46.6 48.3 60.6 50.6 T7 72.3 44.3 58.3 62.7 59.4

T. hamatum T 8 75.4 43.4 63.4 63.5 61.4

52.6 T 9 68.9 46.5 58.3 57.3 57.7 T10 74.6 37.8 55.0 39.3 51.7

Control 0.0 0.0 0.0 0.0 0.00 0.00

LSD.at 5% for:

Isolates of the pathogen ( I so ) = 1.31 Antagonistic fungi (A ) =2.82

I so x A = 3.87 Isolates of the Antagonistic fungi (B)=1.90 Iso x A x B =4.11

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8- (B) Antagonistic effect of some Trichoderma spp. against 4 isolates of F. oxysporum:

Data shown in Table(8) indicate that all the tested antagonistic Trichoderma spp.

caused a significant reduction in the fungal linear growth of the four tested isolates of F.

oxysporum, i.e. F6, F8, F9 and F10. The four isolates of T. harzianum , i.e. T1, T2, T3 and T4,

supressed the growth of F. oxysporum isolates which recorded 63.3, 62.2, 64.5 and 62.6%, on

the average, respectively. Whereas, T. viride isolates , i.e. T5, T6 and T7 caused a rdeuction to

the linear growth, being 58.4 , 60.7 and 57.5 %, respectively on the average, for the tested F.

oxysporum isolates, i.e. F6, F8, F9 and F10. Regarding to T. hamatum isolates, T8, T9 and

T10 gave a reduction in the linear growth, being 63.8, 50.9 & 61.6 % on the avertage,

respectively with the tested isolates of F.oxysporum.

Table(8):Antagonistic effect of Tricholderma spp. on growth reduction of 4

F. oxysporum isolates in vitro.

Antagonistic fungus

Isolate/ No.

%Reduction in thelinear growth

Mean Grand Mean F. oxysporum isolates

F6 F8 F9 F10

T.harzianum

T 1 50.0 58.1 73.8 70.8 63.30

63.20 T 2 51.4 63.2 59.3 75.5 62.30

T3 55.7 62.6 70.8 68.9 64.50

T 4 53.2 52.6 71.3 73.9 62.70

T. viridi

T5 49.9 61.8 50.5 71.3 58.40

58.73 T6 47.9 59.8 61.0 74.6 60.70

T7 52.0 64.1 54.8 59.3 57.50

T. hamatum

T 8 57.6 67.5 63.4 66.6 63.80

58.70 T 9 50.1 49.6 60.0 43.7 50.90

T10 62.0 63.4 57.3 63.0 61.60

Control 0.0 0.0 0.0 0.0 0.00 -

LSD.at 5% for:

Isolates of the pathogen ( I so ) = 3.12 Antagonistic fungi (A ) =3.90

I so x A = 4.58 Isolates of the Antagonistic fungi (B) =2.80 Iso x A x B = 6.11

DISCUSSION

Using different methods for isolation from sesame seeds yielded different

numbers of fungi from both infected seed samples and apparantly healthy ones. Blotter

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16

method proved to be the most favorable for fungal isolation for both apparantly

healthy and infected sesame seeds, followed by PDA medium.

Pathogenicity tests were conducted on sesame cv. Giza 32 using 10 isolates of

M. phaseolina and another 10 isolates of F. oxysporum, revealed that these isolates

varied in their pathogenicity on sesame plants.

M. phaseolina isolates i.e., M1, M5, M8 and M10 which were isolated from

different locations expressed different levels of infection. These results are in

agreement with those reported by Pereira et al., (1995) and Zhang et al.(2001) who

attributed the root-rot and wilt diseases of sesame to M. phaseolina and F. oxysporum

and several species of Fusarium, R. solani, S. rolfsii, and Pythium spp.

Studying the effect of different concentrations of some reducing agents on the linear

growth of 4 isolates of M. phaseolina showed that the linear growth of the tested isolates (M1,

M5, M8 and M10) was significantly suppressed by all chemical inducers i.e., KCl, H2O2,

IAA, TBA, tanic acid and Bion, respectively. The highest decrease was induced by KCl at

con. 4.0 % for all isolates . All chemical inducess showed noticieable decrease at 4.0% in the

linear growth of F. oxysporum isolates. IBA suppressed the linear growth of f. oxysporum at

can 1600 ppm. Also, IAA and IBA decreased the linear growth.

The present work was carried out to study the effect of some plant extracts on mycelial

growth of some isolates of M. phaseolina and F. oxysporum. The obtained data showed that,

generally all the tested extracts decreased the linear growth of the tested fungi.Increasing

the concentration dose resulted in more suppression of linear growth.Concentration of 55 %

gave the highest decrease in fungal growth of isolates of M.phaseolina (M1, M5, M8 & M10)

and isolates of F. oxysporum (F6, F8, F9 & F10).On the other hand,conc. 10% was the least

effective on the linear growth for M. phaseolina and F. oxysporum isolates.

Application of the plant extracts,even as crude,would be probably show antifungal

activities. Phenolic compounds, steroidal alkaloids like saponins, also the compounds ceneole

(Eucalyptol) and Terpinol of Eucalyptus species extracts (Shalaby et al.,2001), may affect on

the fungal growth of the tested isolates of M. phaseolina and F. oxysporum (Hilal et al.,2002).

As for the principle toxic compounds of anise (anethole & methyl chavicol),which are present

in the essential oil of anise were found to be responsible for their effect on fungal activity

(Shukla &Tripathi,1987). Whereas,the fungicidal activity of thyme was attributable to

thymol and carvacol which are found in its essential oils (Maruzzela & Balter, 1959). Also,

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the fungicidal activity of the cumin extract was attributed to cuminic aldehyde which is

secreted from glandular ducts (vittea) of the enternal secretor strucrure in deep tissues of

cumin seeds as reported on the antifungal action of various volatile substances bearing from

different plant tissues (Ali,1999).

The fungicidal activities of the tested plant extracts might be due to their high

capabilities to damage structure and function of the fungal cell wall as well as disturbing the

physiology of the enzymatic bioactivity.In this respect,fungal growth damages caused by the

essential oils might be due to their capabilities to penetrate into the fungal cells (Wilson et

al.,1987). The effect of roselle sepals extract may be attributed to the active compounds;

hibicin hydrochloride and calcium oxalate;which are found in a high concentrations in the

extract and may play an important role together in suppresion the fungal growth of the isolates

studied herein.

Studying the efficacy of the biocontrol agents, Trichoderma isolates under lab.

conditions showed that the the mode of action by which the antagonistic fungi suppressed the

activity of plant pathogens, is that the biocontrol agent contact their host,either by

appressorium-like bodies or coiling around host hyphae,then enzymatically digest host cell

walls. Antagonistic actions of the tested antagonistic fungi against the different isolates of

M.phaseolina and F.oxysporum are in agreement with those reported by (Dinakaran &

Marimuthu,1997 and Hemeda & Rasmy, 2003) .

REFERENCES

Ali, A.A. (1999). Studies on some medicinal plants as a source of antifungal substances in

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Bankole, S. A. and Adebanjo, A. (1995). Inhibition of growth of some plant pathogenic

fungi using leaf extracts from some Nigerian plants. International Journal of Tropical

Plant Diseases, 13,(1)91-95.

Barnett, H.L. and Hunter, B.B. (1998). Illustrated Genera of Imperfect Fungi. Minesotta:

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Brooker, N.L. Long, J.H. and Stephan, S.M. (2000). Field assessment of plant derivative

compounds for managing fungal soybean diseases. Biochemical Society Transaction,

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Datar,V.V. (1999). Bioefficacy of plant extracts against Macrophomina phaseolina (Tassi.)

Goid., the incitant of charcoal-rot of sorghum. Journal of Mycology and Plant

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Elad, Y.; Zvieli, Y. and Chet, I. (1986). Biological control of Macrophomina phaseolina

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Maruzzella, J.C. and Balter, J. (1959). The action of essential oils on phytopathogenic

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Orober, M.; Siegrist, J. and Buchenauer, H. (1998). Induction of systemic aquired

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Pereira,Y.; Gutherrez, Z. and Subero,L.J.(1995).Comparizon between morphological and

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لفطرى ماكروفومينافاسيوليناالمضادة في تثبيط النمو الطولى الفطرياتو المواد المحثة للمقاومةبعض تأثير تحت ظروف المعملالمعزولة من بذور السمسم و فيوزاريم أوكسيسبورم

**،عبد الفتاح عبد الحميد الوكيل*نوال عبد المنعم عيسى ،*محمد مهدى عبدة مهدى

**ضياء عبد الفتاح الوكيل ،*فاتن محمود عبد اللطيف

.جامعة بنها-كلية الزراعة بمشتهر-قسم النبات الزراعى* .مصر-مركزالبحوث الزراعية-معهد بحوث أمراض النباتات**

الفطريات الماكروفومينا شملت ( blotter)و (plate agar)سمسم بطريقتيتم عزل عدد من الفطريات من بذور ال

اتببالمس هىوكانت 23التى تم دراسة قدرتها االمراضية على السمسم صنف جيزة أوكسيسبورم الفيوزاريموفاسيولينا

الختبار تأثير بعض أجريت هذه الدراسة لقدو .الرئيسية ألهم أمراض السمسم وهى العفن الفحمى والذبول على التوالى

ألربع عزالت ى مترلوتأثيرها على النمو الطولى بالموالفطريات المستخدمة في المقاومة الحيوية المواد المحثة للمقاومة

.أوكسيسبورممن الماكروفومينا فاسيولينا وأربعة عزالت من الفيوزاريم

، ومنها كلوريدد البوتاسديوم النمو الطولى للعزالت المختبرة أمكن تثبيطه بجميع المواد المختبرةأن أظهرت النتائج

التركيدزات وان .فوق أكسيد األيددروجين ، واننددول أسديتس أسديد ، واننددول بيدوترس أسديد، والتانيدس أسديد علدى التدوالى

فدى النمدو الطدولى لعدزالت الفطريدات تحدت تثبيطلمرشدحة أظهدرأو ا بدالحرارة لنباتية سواًء المعقمةاالمختلفة للمستخلصات

الكددافور ، والكمددون ، والزعتدر سدداعدت علددى تثبدديط النمددو مستخلصدات كددل مددن أشددارت النتددائج أن ، كمدداالدراسدة معمليدداً

% .55الطولى وكان أفضل المعامالت عند تركيز

المختبرة وهى ترايكوديرما هارزيانم ، وترايكوديرمدا فيدردى التأثير التضادى ألنواع التريكوديرما الثالثة تم اختبار

أن ظهدرم وي،وأربع عزالت مدن الفيدوزاريم أكسيسدبورولينا ي، وتريكوديرما هاماتم ضد أربع عزالت من الماكروفومينا فاس

. سيسبوريمو الفيوزاريم أكمن تثبيط النمو الطولى لعزالت الماكروفومينا فاسولينا تتمكن T1, T2, T3, T4عزالت