Persian Gulf Crop Protection

Persian Gulf Crop Protection, 3(1): 69-78 69

Persian Gulf Crop Protection Available online on: www.cropprotection.ir

ISSN: 2251-9343 (online) Volume 3 Issue 1, March 2014

Pages 69-78

Influence Of Antagonistic Fungaion The Root Infection Caused By

Macrophomina phaseolina On Okra.

Anam Mehwish Khanzada1*, Dr. Abdul Mubeen Lodhi2, Nadil Shah3, Shagufta Rani Khanzada4 and Muhammad Siddique Khanzada5.

Department of Plant Pathology, Sindh Agriculture University, Tandojam (*Corresponding author e-mail: [email protected]).

Abstract: Macrophomina phaseolina was isolated as root infecting fungi from okra plants showing stunted growth, necrosis and severe plant mortality. The pathogenecity test, has confirmed that M. phaseolina is an aggressive pathogen of okra plant. Bio-control agents including Trichoderma spp., Paecilomyces spp. and Gliocladium virens successfully checked the mycelial growth of the M. phaseolina in dual assay test; however P. variotii followed by T. harzianum, T. pseudokoningii and T. polysporum were the most effective bio-control agents in inhibiting the colony growth of test pathogen. In pot experiment, application of bio-control agents greatly reduced the root infection in plants grown in soil, artificially infested with M. phaseolina. Among six bio-control agents, T. harzianum and P. variotii appeared as highly effective in reducing the root infection by test pathogen. The highest root infection was found in plants treated with T. pseudokoningii followed by P. lilacinus. The application of these bio-control agents also caused positive impact on plant growth. Moreover, these bio-control agents also checked the pathogen infection and thus increased seed germination and decreased plant mortality.

Key Words: Macrophomina phaseolina, Antagonistic fungi, Okra.


Introduction Root rot caused by Macrophomina phaseolina is considered as one of the most destructive diseases of okra (Hafiz, 1986). The fungus can also cause hallow stem, root rot, pre-emergence and post-emergence damping-off (Reuveni et al., 1983). M. phaseolina is most often seen during summer weather (Tosi and Zazzerini, 1990 and Gulya et al., 2002). About 5-100% yield losses due to this disease have been reported (Vyas, 1981). M. phaseolina does not survive more than seven days in the mycelial form but it sclerotia can survive over ten months in soil (Ghaffar, 1968). It usually develops when soil temperatures are 80-95oF (27-35oC) for 2 to 3 weeks (Yang and Navi, 2003). Use of chemicals is common practice for controlling the plant diseases. During the past few decades various approaches involving non-chemical means of control were tested to bring down inoculum densities to a level where cultivation of high value crops has become profitable. Therefore, in present study bio-control agents will be evaluated against M. phaseolina causing root rot of okra. Materials and Methods Isolation and Identification of Macrophomina phaseolina: Isolation was carried out from the infected okra plants collected from Agriculture Research Institute, Tandojam. The infected roots and stems were washed with the tape water thoroughly. Washed roots and stems were cut into small pieces, these pieces were then surface sterilized with 5% Sodium hypochlorite (commercial bleach) for 1-1.5 minutes. The sterilized pieces dried onto the tissue paper and then transferred on petridishes containing PDA (Potato Dextrose Agar) medium at five pieces per petridish. These petridishes were incubated at 30±1°C temperature for 7 days in incubator. However, after every 24 hours petridishes were observed for fungal growth. The different fungal colonies appeared were purified and identified on the basis of colony characteristics as well

as morphological characteristics by using keys of (Barnet and Hunter, 1972 and 1996). Multiplication of M. phaseolina Inoculum: In order to prepare large quantity of M. phaseolina inoculum, its sclerotia were obtained by growing the test pathogen on sand+wheat meal substrate. For this purpose 95 gm of sand and 5 gm of wheat meal mixed together thoroughly and then moistened with 10 ml sterilized water. The substrate was transferred into 250 ml conical flask and sterilized in the autoclave at 15Lbs for 20 minutes. Leaved it 24 hours for cooling then added 5 mm disc from actively growing M. phaseolina pure culture in the conical flask and incubated at room temperature for 4-6 weeks and shake the conical flask daily. After 4-6 weeks the colour of the substrate in the conical flask turned black due to the sclerotial formation. The contents of the conical flash were pored onto the 15µm sieve and black tiny sclerotia present on the surface of the sieve were collected in the sterilized glass beaker for further use. Pathogenicity Test of M. phaseolina: The pathogenicity test of M. phaseolina, the most frequent fungus was carried out on local variety Sabz Pari. Effect of Bio-Control Agents on Mycelial Growth of M. Phaseolina: Different bio-control agent’s viz., Trichoderma harzianum, T. polysporidm and T. pseudokoningii, Paecilomyces lilacinus, Paecilomyces variotii and, Gliocladium virens were evaluated against M. phaseolina by dual assay method. For this purpose, a 5 mm disc of M. phaseolina was cut from freshly growing colony with the help of cork borer and placed at one side of PDA plate, on its opposite side placed 5mm disc of test bio-control agents and tapped the petridish with tape. Then lined the back side of petridish with the help of marker and incubated it at 30±1ºC. The growths of pathogen as well as bio-control agents were measured daily, till the colonies of both met. There were five replications of each treatment.


Effect of Bio-Control Agents on M. Phaseolina and Okra: A pot experiment was conducted on local variety Sabz Pari at Department of Plant Pathology, Sindh Agriculture University Tandojam in the month of December 2010, to evaluate the effect of bio-control agents on plant growth and disease development on okra. The seeds of commonly growing okra variety Sabz Pari were surface sterilized with commercial bleach and sown into thermopol glasses of 7 cm diameter filled with the mixture of 190 gm sterilized sandy soils in 1 cm depth. Prior to sowing, the soil is artificially infested with the pathogen inoculum at 40 sclerotia/gm of soil. The took sterilized sandy loam soil 6700 scelrotia per 190 g soil and mixed 0.38g rice gain of Trichoderma spp. each Trichoderma spp. mixed separately and other bio-control agents, Paecilomyces lilacinus, Paecilomyces variotii and, Gliocladium virens inoculums of each mixed two petridishes of pure culture per 3 thermopol glasses and without bio-control agents glasses served as a control. The uninoculated soil served as control. The experiment was conducted complete randomized block design with 3 replications. Seeds germinations were recorded after 10 days of sowing, while seedling mortality recorded after 30 days. Plants were uprooted after one month of sowing and data of plant growth as well as root infection were recorded. Results Isolation and Identification: Mcrophomina phaseolina was predominantly found from the root of okra plant collected from Tando jam. The effected plant showed stunted growth, die-back and pre-mature mortality. Due to high disease severity large number of seedling were died due to post-emergence damping-off (Fig. 1). M. Phaseolina produced black colour colony on PDA its produced tiny black sclerotia under favorable environments (Fig. 1). Microsclerotia are black, spherical to oblong, and occasionally irregular in shape.

Microsclerotia vary widely in size and number, depending upon culture and host material on which they are growing with diameters of 60-200 µm common. Effect of Bio-Control Agents on Mycelial Growth of M. Phaseolina: All bio-control agents significantly checked the mycelial growth of the M. phaseolina is dual assay test, as compared to control (Fig. 2). Among six bio-control agents Paecilomyces variotii was appeared as most effective antagonist, in which pathogen can produce only 32.0 mm colony growth followed by T. harzianum (43 mm), T. pseudokoningii, (45 mm) and T. polysporum (45.5 mm). The maximum colony growth of test pathogen was recorded in plats of P. lilacinus (70.20 mm) followed by G. virens (48.0 mm) (Fig.2). Effect of Bio-Control Agents on M. Phaseolina and Okra: Maximum plant length was recorded in plants treated with P. variotii (133.78 mm) and T. harzianum (133.38 mm) followed by T. polysporum (110.35 mm) and G. virens (99.67 mm). Whereas, minimum plant length was recorded in untreated plants (control) (59.87 mm) followed by plants treated with T. pseudokoningii (72.08mm) (Fig. 3). Similar, trend was also observed in shoot weight, where plants treated with T. harzianum and P. variotii produce maximum plant weight (53.890 mg and 51.540 mg). followed by P. lilacinus T. polysporum and G. virens (30.410 mg, 28.767mg and 28.010 mg).The minimum plant weight was recorded in untreated plants followed by T. pseudokoningii (13.112 mg) (Fig 4). All bio-control agents increased seed germination as compared to control. (Fig. 5). Maximum seed germination were observed in T. harzianum treated soil (97.56%) followed by P. variotii (95.83%). Whereas, minimum germination was observed in untreated soil followed by soil treated with either T. pseudokoningii or T. polysporum (83.33%) (Fig. 5). Among six bio-control agents minimum plant mortality occurred


in plants treated with T. harzianum (13.0%) and P. variotii (16.6%) followed by G. virens (51.0%) and T. polysporum (53.430%) (Fig. 6). The maximum plant mortality was found in untreated (control) plants followed by plants treated with T. pseudokoningii (83.0%) followed by P. lilacinus (79.16%) (Fig. 6). Similarly, application of bio-control agents greatly reduce the root infection of okra plants grown in soil artificially infested with M. phaseolina. However, among bio-control agents T. harzianum and P. variotii appeared as highly effective in reducing the root infection of test pathogen as only 14.0 and 20.0 % infection were recorded in T. harzianum and P. variotii treated plant, respectively. The highest root infection was found in untreated (control) plants followed by plants treated with T. pseudokoningii (50%) and P. lilacinus (44.0 %) (Fig. 7). Similarly, application of bio-control agents greatly reduce the root infection of okra plants grown in soil artificially infested with M. phaseolina. However, among bio-control agents T. harzianum and P. variotii appeared as highly effective in reducing the root infection of test pathogen as only 14.0 and 20.0 % infection were recorded in T. harzianum and P. variotii treated plant, respectively. The highest root infection was found in untreated (control) plants followed by plants treated with T. pseudokoningii (50%) and P. lilacinus (44.0 %) (Fig.6g). Discussion The pathogenicity test, carried out during present study on commonly growing okra variety has confirmed that Macrophomina phaseolina is an aggressive pathogen of the okra. Its inoculation on test plants significantly reduced seed germination and plant growth, as well as increased the plant mortality in okraIn present study, all antagonistic fungi viz., Trichoderma harzianum, T. polysporum and T. pseudokoningii, Paecilomyces lilacinus, P.

variotii and Gliocladium virens successfully checked the mycelial growth of the M. phaseolina in dual essay test. However, P. variotii followed by T. harzianum T. pseudokoningii and T. polysporum were the most effective bio-control agents in inhibiting the colony growth of the test pathogen. In pot experiment, the application of these bio-control agents also caused positive impact on plant growth. Our result indicates that plants treated with P. variotii and T. harzianum followed by G. virens produced more plant growth as compared to others. Moreover, these bio-control agents also checked the pathogen infection and thus increased seed germination and decreased plant mortality. The antagonistic effects of different biological control agent against M. phaseolina were well documented. Our result were in confirmation to those reported by (Sandoval, 2000) who observed that T. harzianum showed marked antagonistic and hyperparasitic effect against M. phaseolina and other pathogens, as well its application reduced root infection and incidence of charcoal rot in soybean. Similarly, (El-Mohommedy, 2004) reported that T. harzianum brought 86% reduction in the growth of M. phaseolina which caused damping-off and root rot disease in okra. Similarly (Gurjar et al., 2004) and (Dawar et al., 2008) tested many antagonistic fungi against M. phaseolina and other pathogens and observed that most of the antagonistic fungi were effective against all pathogens. Their application increased the seed germination and plant growth as well as reduced the disease severity in treated plants. Malathi and (Doraisamy, 2004) also observed that Trichoderma spp. significantly inhabited the colony growth of M. phaseolina. They also reported that the application of T. harzianum brought maximum reduction in pathogen infection and enhanced the plant growth parameters.


Figure 1. Scerotia of Macrophomina phaseolina.

(a)

(b)


(c)

Figure 1. (i). Effect of different bio-control agents (a) P. variotii (b) P. lilacinus and (c) T. harzianum on mycelial growth of M. Phaseolina.

(d)

(e)

(f)

Figure 2. (ii) Effect of different bio-control agents (d) T. pseudokoningii (e) T. polysporum and (f) G. virens on

mycilial growth of M. Phaseolina.


Table 1. Effect of different bio-control agents on mycelial growth of M. phaseolina.

S. NO Bio-control agents Incubationdays

Diameter of pathogen in interaction

(mm)

Diameter of bio-control agent

(mm)

1 Paecilomyces variotii 3 32.0 d

58

2 Paecilomyces lilacinus 4 70.2 a 19.8

3 Gliocladium virens 4 48.0 b 42.0

4 Trichoderma pseudokoningii 5 43.0 c

47.0

5 Trichoderma polysporum 4 45.42 c

44.6

6 Trichoderma harzianum 4 45.0 c

45.0

7 Macrophomina phaseolina (Control) 3 90.0

d

bc

a a

c

e

020406080

100120140

T. pse

udok

oning

ii T. p

olysp

orum

P. lilac

inus

P. vari

otii

T. harz

anium

G. v

irens

contr

ol

Plan

t Len

gth

(mm

)

T. pseudokoningii T. polysporum P. lilacinus P. variotii T. harzanium G. virens control

Figure 3. Effect of different bio-control agents on Plant length and of okra plants inoculated with M. phaseolina.


d

b

aa

bb

c

0

20

40

60

T. pse

udok

oning

ii T. p

olysp

orum

P. lilac

inus

P. vari

otii

T. harz

anium

G. v

irens

contr

ol

Plan

t Wei

ght (

mg)


Figure 4. Effect of different bio-control agents on Plant weight and of okra plants inoculated with M. phaseolina.

dd c b a c

e

0

20

40

60

80

100

T. pse

udok

oning

ii

T. poly

sporu

m

P. li

lacinu

s

P. vari

otii

T. harz

anium

G. v

irens

contr

ol

Plan

t ger

min

atio

n(%

)

Figure 5. Effect of different bio-control agents on Plant germination and of okra plants inoculated with M.

phaseolina.

b

c

b

e f

d

a

0

20

40

60

80

100

T. pse

udok

oning

ii T. p

olysp

orum

P. lilac

inus

P. vari

otii

T. harz

anium

G. v

irens

contr

ol

Plan

t Mor

talit

y (%

)


Figure 6. Effect of different bio-control agents on Plant mortality and of okra plants inoculated with M. phaseolina.


b

dc

ef

d

a

0

20

40

60

80

T. pse

udok

oning

ii T. p

olysp

orum

P. lilac

inus

P. vari

otii

T. harz

anium

G. v

irens

contr

ol

Plan

t Inf

ectio

n (%

)


Figure 7. Effect of different bio-control agents on Plant root infection and of okra plants inoculated with M. phaseolina.

dd

b

ed c

a

0

20

40

60

80

100

T. pse

udok

oning

ii

T. poly

sporu

m

P. lilac

inus

P. vari

otii

T. harz

anium

G. v

irens

contr

ol

Inha

bitio

n (%

)


Figure 8. Effect of different bio-control agents on inhabitation percentage of M. phaseolina on PDA plates.

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