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CHAPTER 7

PART - A

IN VITRO SCREENING OF SYSTEMIC FUNGICIDES AGAINST PHOMOPSIS

AZADIRACHTAE

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IN VITRO SCREENING OF SYSTEMIC FUNGICIDES AGAINST PHOMOPSIS AZADIRACHTAE

INTRODUCTION

The ultimate aim of plant pathologists is to manage the plant diseases effectively

and minimize the losses caused by pathogens in field, storage and transport (Meena and

Shah, 2005). Since the plants are very important to man, both aesthetically and

economically, plant disease management is an absolute requirement (Sateesh, 1998).

Chemical control of plant pathogens is one of the common means of controlling plant

diseases in the field, green house and in storage (Agrios, 2004). Most fungal plant

diseases are primarily controlled through fungicide application (Maloy, 1993). Systemic

fungicides are the compounds which are transported over a considerable distance in plant

system after penetration. They kill fungi, which are found remote from the point of

application. Systemic fungicides are very helpful in the control of plant diseases caused

by systemic fungi (Vyas, 1993). Application of systemic fungicides effectively controls

various forest tree diseases (Hudson, 1986).

Die-back of neem is spreading at an alarming rate resulting in the reduction of life

expectancy and flower production. This disease is resulting in almost 100% loss of fruit

production and drastic reduction in evergreen canopy (Bhat et al., 1998). Seeds are the

major commercial product of neem which have many medicinal and biopesticidal

ingredients. Neem with its numerous characteristics is the ‘Tree for solving global

problems’. Many small scale industries depend on the supply of neem products. Neem is

thereby providing employment to large number of people. Thus neem has an important

role in economics of India. Cultivation and establishment of neem plantations is a major

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goal of neem foundation (Anonymous, 2006). The die-back disease is a great obstacle in

this way and presently management of this devastating disease is of prime importance.

Fungicidal protection is one of the effective means to control diseases of forest

trees (Mohanan et al., 2005). Various fungicides including systemic fungicides are

known to suppress many fungal pathogens (Babadoost and Islam, 2003; Gupta et al.,

2000; Iyer, 2000; Johnson, 2006; Matheron and Porchas, 2002; Mocioni et al., 2003;

Peres et al., 2004; Pethybridge and Hay, 2005; Tremblay et al., 2003; Utkhede et al.,

2001). Many die-back diseases are managed by application of fungicides (Ali et al.,

1993; Gill, 1974; Kamanna, 1996; Rolshausen and Gubler, 2005). Chemical control of

many plant diseases incited by Phomopsis spp. was reported (Abbruzzetti et al., 1993;

Hossain et al., 1992; Kliejunas, 1988; Kuropatwa, 1994; Meena and Shah, 2005; Mostert

et al., 2000; Ploper et al., 2000; Todovara and Katerova, 1995; Wang et al., 1995;

Wrather et al., 2004).

Systemic fungicides such as Carbendazim, Thiophanate-methyl, Metalaxyl,

Isoprothiolane, Tricyclazole and Hexaconazole were reported to control many plant

diseases (Barnwal et al., 2003; Biswas and Singh, 2005; Kalim et al., 2000; Kumawat

and Jain, 2003; Lennox and Spotts, 2003; Martinez et al., 2005; Ray, 2003; Sharma and

Gupta, 2004; Singh et al., 2003). Six systemic fungicides viz., Bavistin 50% W.P.,

Bayleton 25% W.P., Baynate 75% W.P., Benlate 50% W.P., Calixin 80% E.C., and

Kitazin 48% E.C., were tried against P. azadirachtae and bavistin provided the best

results (Sateesh, 1998).

In the present study a few other systemic fungicides were screened for their

fungitoxic activity against P. azadirachtae. The in vitro effect on colony diameter was

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initially investigated. Two most effective fungicides were selected and their comparative

effect at different stages of P. azadirachtae growth was studied. Colony diameter,

mycelial dry weight, pycnidial formation and the germ tube length of the pathogen were

the parameters studied. In vitro screening of fungicides has been employed by many

pathologists (Aguin et al., 2006; Ali et al., 1993; Chattopadhyay et al., 2002; Martyniuk

et al., 2006; Mostert et al., 2000; Onsando, 1986; Ponmurugan et al., 2006; Sateesh,

1998).

MATERIALS AND METHODS

Effect of different fungicides on mycelial growth of P. azadirachtae

Six systemic fungicides were selected for screening the fungitoxic activity against

P. azadirachtae under in vitro conditions (Table 16). These fungicides were tested

against the pathogen using poison-food technique (Dhingra and Sinclair, 1995; Nene and

Thapliyal, 2001). All the concentrations of the fungicides are expressed in terms of active

ingredient (a.i.).

Stock solutions of each fungicide were prepared using sterile distilled water.

These solutions were added to potato dextrose agar (PDA, Himedia, Mumbai, India)

separately to obtain final concentrations of fungicides viz., 10, 100, 250, 500, 1000 ppm

(Initial screening), 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 ppm (Second level screening). The PDA

without any fungicide served as control. In initial screening round all the six fungicides

were tested. In the second round carbendazim, hexaconazole and thiophanate-methyl

were screened. The pH of the medium was adjusted to 6.0. About 20 ml of PDA, with

and without fungicide, were poured into separate Petri dishes (9.0 cm diam.). All the Petri

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dishes were inoculated with the five mm mycelial-agar disc drawn from the margin of

mycelial mat of seven-day-old culture of P. azadirachtae. The Petri dishes were

incubated at 26 ± 2oC with 12 h photoperiod for 10 days. All the treatments had four

replications and the experiment was repeated thrice.

Concentration of fungicides required for complete inhibition of the mycelial

growth was noted. Mean colony diameter was found out by measuring linear growth in

three directions at right angles. The colony diameter was compared with the control as a

measure of fungitoxicity. The per cent mycelial growth inhibition (PI) with respect to the

control was computed from the formula

(C-T) PI =

C

X 100

Where, C is the colony diameter of the control and the T that of the treated ones.

The comparative studies of carbendazim and thiophanate-methyl

Carbendazim and thiophanate-methyl controlled the pathogen at lower

concentrations in comparison with the other fungicides screened. Hence, these two

fungicides were tested at much lower concentrations. The two fungicides were compared

for their effect on vegetative growth (mycelial radial growth and dry weight), pycnidial

number and germ tube growth. The different concentrations of these fungicides tried in

all the tests were 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75 and 1.0 ppm. All the treatments

had four replications and the experiments were repeated thrice.

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Table 16. Systemic fungicides used for in vitro screening against Phomopsis azadirachtae

Trade Name Common Name Chemical Name Company

Bavistin 50% W.P. Carbendazim Methyl 1H-benzimidazol-2-yl carbamate

BASF India Limited, Thane, India.

Beam 75% W.P. Tricyclazole 5-methyl-1,2,4-triazolo [3,4-b] benzothiazole

Indofil Chemicals Company, Mumbai, India

Contaf 5% E.C. Hexaconazole A-butyl-A-(2,4-dichlorophenyl)-1H-1,2,4-triazole-1-ethanol

Rallis- A TATA enterprise, Mumbai, India

Downymil 35% W.P.

Metalaxyl Methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate

Contropest, Bangalore, India

Fujione 40% E.C. Isoprothiolane Di- isopropyl 1,3- dithiolan- 2- ylidenemalonate

Rallis- A TATA enterprise, Mumbai, India

Roko 70% W.P. Thiophanate-methyl Dimethyl [1,2-phenylene bis (iminocarbonothioyl)] bis

[carbamate]

Biostadt, Mumbai, India

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Effect on mycelial growth of P. azadirachtae

The experiment was carried out similar to the procedure mentioned earlier.

Effect on mycelial dry weight of P. azadirachtae

50 ml of potato dextrose broth (Himedia, Mumbai, India) was transferred to

250 ml Erlenmeyer flasks. Stock solutions of fungicides were prepared as mentioned

above and added to the medium in different flasks to obtain various required

concentrations (0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75 and 1.0 ppm). Flasks containing

media without fungicides served as control. All the flasks were inoculated with five mm

mycelial-agar disc drawn from the margin of mycelial mat of seven-day-old culture of

P. azadirachtae and incubated aerobically in a controlled environment incubator shaker

at 26oC and 25 rpm for 20 days. After incubation period the mycelial mats were collected

onto a preweighed Whatman No.1 filter paper and dried at 70oC in a hot air oven until a

constant weight is obtained for determination of the mycelial weight.

Effect on pycnidial number of P. azadirachtae

Petri dishes containing 20 ml of PDA amended with different concentrations of

the two fungicides separately (0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75 and 1.0 ppm) were

inoculated with five mm mycelial-agar disc drawn from the margin of mycelial mat of

seven-day-old culture of P. azadirachtae. Petri dishes with media devoid of fungicides

were inoculated and maintained as control. All the Petri dishes were incubated at

26 ± 2oC with 12 h photoperiod for 15 days. After incubation period, the total numbers of

pycnidia present were counted. The base area of Petri dishes was divided into six equal

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parts by diagonally marking the lid with a marking pen. Pycnidia present in each part

were counted and mean value was taken as total count (Sateesh, 1998).

Effect on conidial germ tube growth of P. azadirachtae

Conidial suspension was prepared having 103 conidia per ml of sterile distilled

water. 10 ml of malt extract broth (Himedia, Mumbai, India) with different

concentrations of the two fungicides (0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75 and 1.0 ppm)

taken in separate 100 ml Erlenmeyer flasks was inoculated with one ml of conidial

suspension. Flasks containing media without fungicides and inoculated with conidial

suspension served as control. All the flasks were incubated aerobically in a controlled

environment incubator shaker at 26oC and 25 rpm for 24 h. Then the germ tube growth in

each flask was ceased by adding 2 ml of 1% lactophenol solution. The germ tube length

was measured under microscopic field using micrometer. Only when the germ tube

length was double the conidial length, the conidia were considered as germinated.

RESULTS

Effect on different fungicides on mycelial growth of P. azadirachtae

Among the six fungicides tested, carbendazim, hexaconazole and thiophanate-

methyl showed complete inhibition of mycelial growth of P. azadirachtae at a

concentration of 10 ppm of active ingredients. Other three fungicides viz., metalaxyl,

isoprothiolane, tricyclazole failed to suppress completely the mycelial growth of the

pathogen at 10 ppm (Fig. 25; Table 17). Hexaconazole completely inhibited the mycelial

growth at 10 ppm (Fig. 26; Table 18). Carbendazim and thiophanate-methyl showed

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complete suppression of mycelial growth of P. azadirachtae at a concentration of 0.25

and 0.75 ppm, respectively (Fig. 27 and 28; Table 19).

Table 17. Effect of different systemic fungicides on the mycelial growth of Phomopsis azadirachtae at 10 ppm concentration

Systemic Fungicides Mycelial Growth (cms) Growth Inhibition (%)

Control 8.69 ± 0.057 e 0.00 ± 0.00 a

Metalaxyl 8.00 ± 0.048 d 7.90 ± 0.61 b

Isoprothiolane 6.83 ± 0.10 b 21.34 ± 1.17 d

Tricyclazole 7.58 ± 0.10 c 12.70 ± 1.19 c

Carbendazim 0.0 ± 0.00 a 100.00 ± 0.00 e

Hexaconazole 0.0 ± 0.00 a 100.00 ± 0.00 e

Thiophanate-methyl 0.0 ± 0.00 a 100.00 ± 0.00 e

Values are means of three experiments and each with four replications ± S.E. Figures followed by different superscript letters differ significantly when subjected to Tukey’s HSD (Honestly Significant Differences) [ α = 0.05]


The mycelial growth of P. azadirachtae was totally inhibited at 0.25 ppm of

carbendazim while thiophanate-methyl completely suppressed the mycelial growth at

0.75 ppm. Effect of different concentrations of these two fungicides on mycelial dry

weight of the pathogen is mentioned in the table 20.

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Table 18. Effect of hexaconazole, carbendazim and thiophanate-methyl on the mycelial growth of Phomopsis azadirachtae

Fungicides

Hexaconazole Carbendazim Thiophanate-methyl

Concentrations of fungicides

(ppm) Mycelial Growth (cms)

Growth Inhibition (%)

Mycelial Growth (cms)




0.00 8.58 ± 0.065 g 0.00 ± 0.00 a 0.00 ± 0.00 a 100.00 ± 0.00 g 0.00 ± 0.00 a 100.00 ± 0.00 g

1.0 7.72 ± 0.051 f 9.97 ± 0.59 b 0.00 ± 0.00 a 100.00 ± 0.00 g 0.00 ± 0.00 a 100.00 ± 0.00 g

2.0 7.05 ± 0.070 e 17.84 ± 0.77 c 0.00 ± 0.00 a 100.00 ± 0.00 g 0.00 ± 0.00 a 100.00 ± 0.00 g

4.0 5.59 ± 0.10 d 35.00 ± 1.18 d 0.00 ± 0.00 a 100.00 ± 0.00 g 0.00 ± 0.00 a 100.00 ± 0.00 g

6.0 4.36 ± 0.060 c 49.21 ± 0.69 e 0.00 ± 0.00 a 100.00 ± 0.00 g 0.00 ± 0.00 a 100.00 ± 0.00 g

8.0 2.14 ± 0.014 b 75.04 ± 0.17 f 0.00 ± 0.00 a 100.00 ± 0.00 g 0.00 ± 0.00 a 100.00 ± 0.00 g

10.0 0.00 ± 0.00 a 100.00 ± 0.00 g 0.00 ± 0.00 a 100.00 ± 0.00 g 0.00 ± 0.00 a 100.00 ± 0.00 g


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Table 19. Effect of carbendazim and thiophanate-methyl on the mycelial growth of Phomopsis azadirachtae

Fungicides

Carbendazim Thiophanate-methyl

Concentra-tions of

fungicides (ppm)


Growth

Inhibition (%)



0.00 8.72 ± 0.022 f 0.00 ± 0.00 a 8.72 ± 0.022 h 0.00 ± 0.00 a

0.025 8.30 ± 0.032 e 4.79 ± 0.37 b 8.42 ± 0.040 g 3.39 ± 0.46 b

0.050 6.59 ± 0.049 d 24.41± 0.56 c 7.63 ± 0.066 f 12.50 ± 0.75 c

0.075 3.50 ± 0.067 c 59.84 ± 0.77 d 6.40 ± 0.068 e 26.61 ± 0.77 d

0.10 1.69 ± 0.039 b 80.57 ± 0.45 e 4.46 ± 0.054 d 48.87 ± 0.62 e

0.25 0.00 ± 0.00 a 100.00 ± 0.00 f 3.26 ± 0.078 c 62.61 ± 0.89 f

0.50 0.00 ± 0.00 a 100.00 ± 0.00 f 2.08 ± 0.078 b 76.11 ± 0.89 g

0.75 0.00 ± 0.00 a 100.00 ± 0.00 f 0.00 ± 0.00 a 100.00 ± 0.00 h

1.0 0.00 ± 0.00 a 100.00 ± 0.00 f 0.00 ± 0.00 a 100.00 ± 0.00 h



Carbendazim completely suppressed the pycnidial formation at 0.25 ppm when no

pycnidia were observed on five mm diameter mycelial disc. At 0.1 ppm concentration

very few pycnidia without conidial cirrhi, were observed. Thiophanate-methyl produced

similar results, but at 0.75 and 0.5 ppm respectively (Table 21).

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Table 20. Effect of carbendazim and thiophanate-methyl on the dry weight of Phomopsis azadirachtae

Dry weight of Phomopsis azadirachtae (mg ± S.E.) Concentrations of fungicides (ppm)


0.00 304.80 ± 1.39 f 304.80 ± 1.39 h

0.025 235.68 ± 0.91 e 266.35 ± 1.27 g

0.050 138.93 ± 1.02 d 181.05 ± 1.57 f

0.075 62.85 ± 0.77 c 120.65 ± 1.28 e

0.10 8.75 ± 0.21 b 50.15 ± 0.88 d

0.25 0.00 ± 0.00 a 22.82 ± 0.57 c

0.50 0.00 ± 0.00 a 5.13 ± 0.19 b

0.75 0.00 ± 0.00 a 0.00 ± 0.00 a

1.0 0.00 ± 0.00 a 0.00 ± 0.00 a



Carbendazim totally checked the germ tube growth at 0.25 ppm and thiophanate-

methyl showed complete suppression of germ tube growth at 0.75 ppm (Table 22). In the

presence of 0.25 ppm of carbendazim and 0.75 ppm of thiophanate methyl conidia lost

their fusiform nature and got converted to non-germinable oval-shaped structure.

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Table 21. Effect of carbendazim and thiophanate-methyl on the pycnidial number of Phomopsis azadirachtae

Number of pycnidia of Phomopsis azadirachtae (± S.E.) Concentrations of fungicides (ppm)


0.00 185.2 ± 3.19 f 185.2 ± 3.19 h

0.025 150. 2 ± 2.64 e 156.2 ± 3.27 g

0.050 97.7 ± 2.97 d 123.2 ± 3.03 f

0.075 48.8 ± 2.63 c 89.8 ± 2.54 e

0.10 12.2 ± 0.83 b 54.7 ± 1.99 d

0.25 0.00 ± 0.00 a 28.8 ± 1.49 c

0.50 0.00 ± 0.00 a 10.3 ± 0.88 b

0.75 0.00 ± 0.00 a 0.00 ± 0.00 a

1.0 0.00 ± 0.00 a 0.00 ± 0.00 a


DISCUSSION

Systemic fungicides were evaluated in vitro for their potential to control

P. azadirachtae. Use of chemical fungicides cannot be avoided until there is development

of a better method of disease management (Sateesh, 1998). When thought of cost

effectiveness, fungicides provide a cheaper and reliable source for the control of plant

pathogenic fungi. In spite of known environmental hazardous effect, Norman Borlaug,

father of green revolution, argued for the use of synthetic chemical control methods

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(Nigam et al., 1994). In general, before subjecting any fungicide for field trials, they will

be screened in the lab against the plant pathogen. The poison-food technique and spore

germination test in shaker flasks are a few common methods employed to test the

efficacy of fungicides under lab conditions (Dhingra and Sinclair, 1995). In vitro

screening helps to identify fungicides that are effective against plant pathogens by

maintaining a protective barrier (Sbragia, 1975).

Table 22. Effect of carbendazim and thiophanate-methyl on the germ tube growth of Phomopsis azadirachtae

Germ tube length of Phomopsis azadirachtae (µm ± S.E.) Concentration of fungicides (ppm)


0.00 107.3 ± 0.61 f 107.3 ± 0.61 h

0.025 91.0 ± 0.72 e 95.5 ± 0.70 g

0.050 75.2 ± 0.75 d 77.9 ± 0.67 f

0.075 50.0 ± 0.64 c 64.9 ± 0.77 e

0.10 19.8 ± 0.70 b 48.5 ± 0.56 d

0.25 0.00 ± 0.00 a 34.5 ± 0.62 c

0.50 0.00 ± 0.00 a 13.8 ± 0.38 b

0.75 0.00 ± 0.00 a 0.00 ± 0.00 a

1.0 0.00 ± 0.00 a 0.00 ± 0.00 a


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Among the six fungicides screened against P. azadirachtae, carbendazim was

highly effective at very low concentration, in comparison with all other fungicides tested,

and followed by thiophanate-methyl. Both these fungicides belong to benzimidazole

group (Brent, 1995) and have similar mode of activity. They interfere with nuclear or cell

division through inhibition of spindle formation during mitosis (Kalim et al., 2000;

Richmond and Phillips, 1975; Sharma and Gupta, 2004) and other biosynthetic processes

(Vyas, 1993). Carbendazim increases production of phenolic compounds (Sharma and

Gupta, 2004).

Comparative study of fresh weight, pycnidial number and germ tube length

showed that both carbendazim and thiophanate-methyl are effective in inhibiting the

growth of pathogen and carbendazim was more effective than thiophanate-methyl.

Progressive decrease in the colony diameter, dry weight, pycnidial number and germ tube

length with reduced germ tube branching was observed with the increase in the

concentration of both the fungicides. Carbendazim completely suppressed the growth of

P. azadirachtae at 0.25 ppm and thiophanate-methyl showed the same result at 0.75 ppm.

Thus the two fungicides being effective against the pathogen at very low concentrations

are cost effective. This is in agreement with the results reported by Sateesh (1998) on the

sensitivity of P. azadirachtae to bavistin (carbendazim). Carbendazim and thiophanate-

methyl are effective against Phomopsis spp. (Meena and Shah, 2005; Ploper et al., 2000).

Carbendazim is known to be functional against many species of Phomopsis pathogenic to

crop plants (Grewal and Jhooty, 1987; Islam and Pan, 1993 and 1989; Ponmurugan et al.,

2006; Singh and Chakraborthy, 1982) and trees (Hossain et al., 1992; Jamalludin et al.,

1988; Otta, 1974). Both these fungicides increased the fruit yield in Mango (Ray, 2003).

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Studies on the effect of fungicides against spore germination are a quick, initial

screening for antifungal activity (Prom and Isakeit, 2003). In this study, inhibition of

germination and changes in morphology were observed in the spores of P. azadirachtae

when treated with carbendazim and thiophanate-methyl revealing the effective antifungal

activity of these two fungicides against P. azadirachate. Fungicides are known to inhibit

spore germination (Montag et al., 2006; Smilanick et al., 2005). Treatment with

fungicides reduces the ability of spores to attach and penetrate the host tissue (Vicedo et

al, 2006). At low concentration carbendazim causes abnormalities in germ tube (Wang et

al., 1995).

P. azadirachtae is seed-borne (Sateesh and Bhat, 1999; Sateesh, 1998).

Application of carbendazim and thiophanate-methyl could help to overcome this

problem. Carbendazim is efficient in controlling the seed microflora of neem (Punam

Singh et al., 1999; Sateesh, 1998). Benzimidazoles can be applied as foliar spray,

seedling dip, soil drench or seed dressing (Sateesh, 1998) and they exhibit systemic

action (Maloy, 1993). Benzimidazole fungicides have great penetrating capacity and last

long in host tissue, and thereby are capable of eradicating many latent infections (Dekker,

1977). They are widely used to control plant pathogens owing to their broad spectrum

activity (Russel, 1995).

Both carbendazim and thiophanate-methyl which effectively control the growth of

P. azadirachtae under in vitro conditions could be utilized for the control of die-back of

neem. In all the tests conducted the concentration required was comparatively more with

thiophanate-methyl than carbendazim suggesting the preference of carbendazim over

thiophanate-methyl for the control of P. azadirachtae.

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CHAPTER 7

PART - B

BIOLOGICAL CONTROL OF PHOMOPSIS AZADIRACHTAE WITH ANTAGONISTIC

FUNGI AND BACTERIA

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BIOLOGICAL CONTROL OF PHOMOPSIS AZADIRACHTAE WITH ANTAGONISTIC FUNGI AND BACTERIA

INTRODUCTION

Biological measures for the control of plant diseases are gaining popularity in the

recent years (Sateesh, 1998). Biocontrol agents provide disease management supplements

with different mechanisms of action than chemical pesticides (Fravel, 2005). Biocontrol

of plant diseases using microorganisms provides a possible alternative to decrease the

input of agrochemicals in agriculture (Lugtenberg and Bloemberg, 2004).

With time there is continuing loss of appropriate, effective pesticides available for

plant disease control and the concern over potential toxicity of pesticides is increasing

(Agrios, 2004). Fungicides are biohazardous and adversely affect the components of

ecosystems (Rathmell, 1984). Fungicides lead to residue problems and accumulation of

toxic pollutants in the soil or underground water. They are deleterious for associated soil

microbiota (Bunker and Mathur, 2001). Carcinogenic, teratogenic, oncogenic and

genotoxic properties of synthetic fungicides were reported (Anonymous, 1986; Carter et

al., 1984; Dalvi and Whittaker, 1995; Hellman and Laryea, 1990). Plant pathogens

develop resistance to the synthetic fungicides with continuous exposure (Brent, 1995;

Geordopoulos, 1987; Nigam et al., 1994). This has resulted in the development of some

new management practices for plant diseases (Agrios, 2004). Management of plant

diseases based on ecofriendly alternative approaches is highly recommended (Ahmed et

al., 1999; Cook and Baker, 1983; Lyon et al., 1995; Parveen and Kumar, 2004).

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Early in the twentieth century, the ability of a few soil microorganisms to

suppress the development of soil-borne plant pathogens was reported. Numerous non-

pathogenic fungi and bacteria antagonizing various plant pathogenic fungi, bacteria and

nematodes were reported and exploited for plant disease control (Agrios, 2004).

Microbial communities in natural ecosystems, through competition, predation or by

antibiosis control the abundance of other microorganisms (Arras and Arru, 1997;

Bolwerk et al., 2005; Chin-A-Woeng et al., 2003; Harman et al., 2004; Maloy, 1993).

They also induce local or systemic resistance mechanisms in plants (Iavicoli et al., 2003;

Siddiqui and Shaukat, 2004; Singh et al., 2005a; van Loon et al., 1998). Many

commercially available biological agents to control pathogenic fungi, bacteria, viruses

and nematodes, in different countries, were listed by Maloy (1993).

Various microorganisms including both bacteria (Hajra et al., 1992; Johri et al.,

1997; Perdomo et al., 1995; Podile, 2000; Srinivasan, 2003) and fungi (Bari et al., 2000;

Bettiol, 1996; Bohra et al., 2005; Dhingra and Sinclair, 1995; Narain and Behera., 2000)

were reported to be antagonistic to plant pathogens. Many microorganisms were

employed against phytopathogens occurring on the aerial parts of plants (Arya and

Parashar, 1997; Elad, 2003; Quesada-Chanto and Jimenez-Ulate, 1996).

Bacillus spp. are used as biological control agent against many plant pathogens

(Girija and Jubina, 2000; Sharifi-Tehrani and Ramezani, 2003; Sharifi-Tehrani et al.,

2004). Antagonistic activity of Trichoderma harzianum and Trichoderma viride against

plant pathogens were reported (Ahmed et al, 1999; Parakhia and Akbari, 2004; Parveen

and Kumar, 2004; Patel and Anahosur, 2001; Singh and Singh, 2000). Biocontrol of

many plant pathogens was tried employing Pseudomonas spp. (Cazorla et al., 2006;

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Godfrey et al., 2000; Laha et al., 1998; Parke et al., 1991; Steddom et al., 2002; Walker

et al., 1996).

Antifungal activity of Bacillus subtilis against many plant pathogenic fungi were

reported (Asaka and Shoda, 1996; Eshita et al., 1995; Phookan and Chaliha, 2000; Podile

and Prakash, 1996). B. subtilis produces many antibiotics having antagonistic activity

such as iturin A and surfactin (Asaka and Shoda, 1996; Tsuge et al., 1995), bacillopeptins

and bacillomycin (Eshita et al., 1995; Kajimura et al., 1995), mycosubtilin (Leclere et al.,

2005). Pseudomonas aeruginosa was employed as active biocontrol agent against many

plant pathogens (Anjaiah et al., 2003; Brathwaite and Cunningham, 1982; Krishna

Kishore et al., 2005a; Siddique and Ehteshamul-Haque, 2001). Pseudomonas spp.

including Ps. aeruginosa are known to produce high affinity siderophores, hydrogen

cyanide, lytic enzymes, phenazine-1-caboxylic acid (PCA), oxychlorophine (OCP),

anthranilite, ramnolipids, 2,4-diacetyl phlorglucinol having antifungal activity (Anjaiah et

al., 1998; Audenaert et al., 2001; Buysens et al., 1996; Castric, 1975; Dwivedi and Johri,

2003; Krishna Kishore et al., 2006; Sunish Kumar et al., 2005). Ps. aeruginosa was

reported to induce systemic resistance to provide control against pathogen and to increase

plant growth (Audenaert et al., 2002; De Meyer and Hofte, 1997; Siddiqui and

Ehteshamul-Haque, 2001; Siddiqui and Shaukat, 2002). With all these characteristics

both B. subtilis and Ps. aeruginosa serve as promising biocontrol agents against plant

pathogens.

Many tree diseases are managed employing antagonistic microorganisms (Gupta

and Sarkar, 2000; Gyenis et al., 2003; Okigbo and Osuinde, 2003; Pitt et al., 1999;

Srinivasan, 2003). Biological control of die-back diseases using antagonistic

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microorganisms were reported (Adejumo, 2005; Killgore et al., 1998; Schmidt et al.,

2001). Many plant diseases caused by Phomopsis spp. are controlled utilizing

antagonistic microorganisms (Al-Rashid and Hentschel, 1988; Anderson and

Gnanamanickam, 2002; Fuchs and Defago, 1991; Gangadharaswamy et al., 1997; Kita et

al., 2005; Moody and Gindrat, 1977).

Die-back of neem incited by P. azadirachtae can be controlled by the application

of bavistin, a systemic fungicide. Since the biohazardous nature of fungicides is known,

and phytopathogenic fungi tend to develop resistance against benzimidazole fungicides

on continuous exposure (Brent and Hollomon, 1998; Davidse and Ishii, 1995),

development of alternative strategies for management of this disease are required.

Control of die-back of neem by biological means is preferred because this provides an

economically sustainable method resulting in pollution free environment (Tate, 1995). In

the present study, screening of a few bacterial and fungal antagonists against

P. azadirachtae was carried out under in vitro conditions.

Microorganisms secrete unique secondary metabolites with prominent

antagonistic effects (Lange et al., 1993). There is considerable progress towards the

understanding of the role of antifungal metabolites and their production. Isolation of

antimycotic secondary metabolites using ethyl acetate and in vitro studies on the effect of

ethyl acetate fraction against pathogenic fungi were reported (Jackson et al., 1994;

Lavermicocca et al., 2000; Singh et al., 2005a).

145


Antagonistic microorganisms

The bacterial and fungal antagonists selected for in vitro screening against

P. azadirachtae were Bacillus cereus (MTCC 430), Bacillus subtilis (MTCC 619),

Pseudomonas aeruginosa (MTCC 2581), Pseudomonas oleovorans (MTCC 617),

Trichoderma harzianum (MTCC 792) and Trichoderma viride (MTCC 800). All the

cultures were procured from Microbial Type Culture Collection (MTCC), Institute of

Microbial Technology (IMTECH), Chandigarh, India. All the bacterial cultures, except

Ps. aeruginosa were first streaked on nutrient agar (NA, Himedia, Mumbai, India) in

Petri dishes and single cell colony was isolated from the culture. The single cell colony of

each bacterium was grown on NA slant and was maintained at 4oC. Ps. aeruginosa was

isolated and maintained on King’s B medium (Himedia, Mumbai, India). The fungal

isolates were sub cultured and maintained on malt extract agar (Himedia, Mumbai, India)

slants and plates at 4oC (Dhingra and Sinclair, 1995; Sateesh, 1998; Tuite, 1969).

Isolation of ethyl acetate fractions from bacterial culture filtrates (BCF)

The extraction of antifungal ethyl acetate fraction from BCF was carried out as

per Lavermicocca et al. (2000). A loopful of 24 h old culture was used to inoculate

100 ml of nutrient broth (Himedia, Mumbai, India) taken in 500 ml Erlenmeyer flask.

Totally 1500 ml of medium was inoculated and all the bacterial cultures were inoculated

separately. The inoculated flasks were incubated at 37oC for 72 h. Then the cells were

harvested by centrifugation (9000 X g for 10 min at 4oC). The supernatant was collected,

the volume of each culture filtrate was made up to 1.5 l with sterile distilled water, filter-

146

sterilized using 0.45 µm membrane filter (Sartorius, Goettingen, Germany) and stored at

4oC. For extraction, the culture filtrates were concentrated to 10% of their original

volume by using a flash evaporator at 50oC (Zhang and Watson, 2000) and the pH of the

BCF (150 ml) was adjusted to 3.6 using 1.0 N HCl. Then the BCF was extracted with

equal volume of ethyl acetate for three times. The aqueous fraction was discarded and the

organic extracts of culture filtrates were pooled and evaporated at RT to obtain brownish,

semi-solid crude extract.

Isolation of ethyl acetate fractions from fungal culture filtrates (FCF)

This was carried out according to Singh et al. (2005a). 100 ml of potato dextrose

broth (PDB, Himedia, Mumbai, India) medium in 500 ml conical flask was inoculated

with mycelial agar discs taken from the margin of seven-day-old culture of both the

fungal antagonists separately. Totally 1500 ml of medium was inoculated and all the

inoculated flasks were incubated at 26 ± 2oC for 25 days. Then the mycelial mats were

filtered through Whatman No.1 filter paper, culture filtrates of each fungus were

collected separately and concentrated to 10% of their original volume by using a flash

evaporator at 50 oC. For extraction the volume was made to 300 ml using sterile distilled

water and the culture filtrates were fractionated thrice with equal volume of ethyl acetate.

The ethyl acetate extracts were combined and evaporated at RT to obtain a dark brown,

semi-solid crude material.

Bioassay of antifungal activity

Stock solutions (1000 ppm) of each microbial ethyl acetate fraction were prepared

by dissolving fractionated material in sterile distilled water containing 0.1% Tween-20

147

(1.0 mg / ml). Sterilized distilled water containing 0.1% Tween-20 served as control

solution (Singh et al., 2005a).

Effect of ethyl acetate fractions of different antagonistic microbial culture filtrates

on mycelial growth of P. azadirachtae

The ethyl acetate fractions were tested against the pathogen using poison-food

technique (Dhingra and Sinclair, 1995). Stock solutions of all the ethyl acetate fractions

were added separately to sterile potato dextrose agar (PDA, Himedia, Mumbai, India) to

obtain different concentrations such as 25, 50, 100, 250, and 500 ppm. The PDA with the

control solution (500 ppm) served as control. About 20 ml of all the treated and untreated

PDA media were poured into separate Petri dishes (9.0 mm diam.). All the Petri dishes

were inoculated with the five mm mycelial-agar disc drawn from the margin of mycelial

mat of seven-day-old culture of P. azadirachtae and were incubated at 26 ± 2oC with

12 h photoperiod for 10 days. All the treatments had four replications and the experiment

was repeated thrice.

Concentration of ethyl acetate fractions required for complete inhibition of the

mycelial growth was noted. Mean colony diameter was found out by measuring linear

growth in three directions at right angles. The colony diameter was compared with the

control as a measure of fungitoxicity. The per cent mycelial growth inhibition (PI) with

respect to the control was computed from the formula

(C-T) PI = C

X 100


148

The comparative study of ethyl acetate fractions of culture filtrates of B. subtilis and

Ps. aeruginosa

The ethyl acetate fractions of culture filtrates of B. subtilis and Ps. aeruginosa

inhibited the mycelial growth of P. azadirachtae at lower concentrations in comparison

with the ethyl acetate fractions of the other antagonistic microorganisms. Thus these two

ethyl acetate fractions were screened at much lower concentrations viz., 2.5, 5.0, 7.5,

10.0, 12.5, 15.0, 17.5, 20.0, 22.5 and 25.0 ppm for all the tests such as the effect on

vegetative growth (mycelial radial growth and dry weight), pycnidial number. For the

study of their effect on germ tube length, 2.5, 5.0, 10.0, 15.0, 20.0 and 25.0 ppm

concentrations were tested. All the treatments had four replications and the experiments

were repeated thrice.


The experiment was carried out employing the same methodology as described

earlier.


50 ml of PDB amended with ethyl acetate fractions at 2.5, 5.0, 7.5, 10.0, 12.5,

15.0, 17.5, 20.0, 22.5 and 25.0 ppm concentrations were taken in separate 250 ml

Erlenmeyer flasks. Flasks containing medium with the control solution (25 ppm) served

as control and all the flasks were inoculated with the five mm mycelial-agar disc drawn

from the margin of mycelial mat of seven-day-old culture of P. azadirachtae. The

inoculated flasks were incubated aerobically at 26oC and 25 rpm for 20 days in a

149

controlled environment incubator shaker. Then the mycelial dry weight was determined

using dried mycelial mats with constant weight, which were collected on to a preweighed

Whatman No.1 filter paper and dried at 70oC in a hot air oven until a constant weight was

obtained.


PDA amended with different concentrations of the two ethyl acetate extracts (2.5,

5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5 and 25.0 ppm) were poured to separate Petri

dishes (20 ml / Petri dish). The Petri dishes contained PDA amended with the control

solution (25 ppm) served as control. All the Petri dishes were inoculated with the five

mm mycelial-agar disc drawn from the margin of mycelial mat of seven-day-old culture

of P. azadirachtae. The Petri dishes were incubated at 26 ± 2oC with 12 h photoperiod.

The pycnidial number was counted after 15 days of incubation. The base area of Petri

dishes was divided into six equal parts by diagonally marking the lid with a marking pen.

Pycnidia present in each part were counted and mean value was taken as total count

(Sateesh, 1998).


Conidial suspension having 103 conidia per ml of sterile distilled water was

prepared and 1.0 ml of this suspension was inoculated to 10 ml of malt extract broth

(Himedia, Mumbai, India) containing various concentrations of ethyl acetate extracts

(2.5, 5.0, 10.0, 15.0, 20.0 and 25.0 ppm) taken in different 100 ml Erlenmeyer flasks.

Flasks containing medium with control solution (25 ppm) were inoculated and

150

maintained as control. The flasks were incubated aerobically at 26oC and 25 rpm for 24 h

in a controlled environment incubator shaker. Then the germ tube growth in each flask

was ceased by adding 2.0 ml of 1% lactophenol solution. The germ tube length was

measured under microscopic field using micrometer. Only when the germ tube length

was double the conidial length, the conidia were considered as germinated.

RESULTS

Isolation of ethyl acetate fractions from microbial culture filtrates

The amount of ethyl acetate fractions obtained from the culture filtrates of

different antagonistic microorganisms are mentioned in table 23.

Table 23. Amount of ethyl acetate fractions obtained from culture filtrates of different antagonistic microorganisms

Microorganisms Ethyl acetate fraction (mg)

Bacillus cereus 501

Bacillus subtilis 477

Pseudomonas aeruginosa 445

Pseudomonas oleovorans 396

Trichoderma harzianum 522

Trichoderma viride 558

151

Effect of ethyl acetate fractions of different antagonistic microbial culture filtrates

on mycelial growth of P. azadirachtae

Of the six microbes tested, only two bacterial species viz., Bacillus subtilis and

Pseudomonas aeruginosa exhibited complete suppression of mycelial growth of the

pathogen at 25 ppm concentration of their ethyl acetate fraction. All the other four

microorganisms including Bacillus cereus, Pseudomonas oleovorans, Trichoderma

harzianum and T. viride were unable to completely inhibit the mycelial growth of

P. azadirachtae at 25 ppm of ethyl acetate fraction of their culture filtrates (Fig. 29;

Table 24). The ethyl acetate fractions of B. subtilis and Ps. aeruginosa showed fungistatic

effect at 22.5 ppm and fungicidal effect at 25 ppm (Fig. 30 and 31; Table 25).

Table 24. Effect of ethyl acetate extracts of different microbial culture filtrates on the mycelial growth of Phomopsis azadirachtae at 25 ppm concentration

Microorganisms Mycelial Growth (cms)


Control 8.5 ± 0.018 f 0.00 ± 0.00 a

Trichoderma harzianum 5.58 ± 0.040 e 35.63 ± 0.47 b

Trichoderma viride 6.02 ± 0.058 d 29.64 ± 1.48 c

Pseudomonas oleovorans 5.07 ± 0.038 c 41.68 ± 0.44 d

Bacillus cereus 4.43 ± 0.042 b 49.12 ± 0.49 e

Bacillus subtilis 0.00 ± 0.00 a 100.00 ± 0.00 f

Pseudomonas aeruginosa 0.00 ± 0.00 a 100.00 ± 0.00 f

Values are means of three experiments and each with four replications ± S.E. Figures followed by different superscript letters differ significantly (P ≤ 0.000) when subjected to Tukey’s HSD (Honestly Significant Differences) [α = 0.05]

153


The ethyl acetate extract of both B. subtilis and Ps. aeruginosa totally checked the

mycelial growth of P. azadirachtae in liquid media at 22.5 (fungistatic) and 25 ppm

(fungicidal). Inhibition of the mycelial growth of the pathogen at different concentrations

of the two ethyl acetate extracts is shown in the table 26.

Table 25. Effect of ethyl acetate extracts of Bacillus subtilis and Pseudomonas aeruginosa culture filtrates on the mycelial growth of Phomopsis azadirachtae

Ethyl acetate extracts of culture filtrates

Bacillus subtilis Pseudomonas aeruginosa

Concen-

trations of Bio-

fungicides (ppm)





0.00 8.9 ± 0.037 i 0.00 ± 0.00 a 8.9 ± 0.037 j 0.00 ± 0.00 a

2.5 8.64 ± 0.035 h 2.97 ± 0.40 b 8.33 ± 0.032 i 6.40 ± 0.35 b

5.0 7.92 ± 0.039 g 11.08 ± 0.47 c 7.50 ± 0.032 h 15.73 ± 0.36 c

7.5 6.86 ± 0.047 f 22.95 ± 0.53 d 6.73 ± 0.026 g 24.35 ± 0.29 d

10.0 6.17 ± 0.034 e 30.69 ± 0.38 e 5.85 ± 0.023 f 34.27 ± 0.26 e

12.5 5.49 ± 0.032 d 38.33 ± 0.36 f 5.20 ± 0.019 e 41.55 ± 0.22 f

15.0 4.50 ± 0.029 c 49.47 ± 0.32 g 4.26 ± 0.027 d 52.13 ± 0.30 g

17.5 2.66 ± 0.031 b 69.99 ± 0.43 h 2.30 ± 0.031 c 74.19 ± 0.34 h

20.0 1.72 ± 0.025 a 80.62 ± 0.29 i 1.29 ± 0.018 b 85.55 ± 0.20 i

22.5 0.00 ± 0.00 a 100.00 ± 0.00 j 0.00 ± 0.00 a 100.00 ± 0.00 j

25.0 0.00 ± 0.00 a 100.00 ± 0.00 j 0.00 ± 0.00 a 100.00 ± 0.00 j


155


The pycnidial formation of P. azadirachtae was completely suppressed at 22.5

and 25 ppm of the ethyl acetate fractions of both B. subtilis and Ps. aeruginosa. At

20 ppm concentration a few pycnidia that were devoid of conidial cirrhi were produced in

both the cases. Effect of different concentrations of these two ethyl acetate extracts on

pycnidial production of the pathogen is mentioned in the table 27.

Table 26. Effect of ethyl acetate extracts of Bacillus subtilis and Pseudomonas aeruginosa culture filtrates on the mycelial dry weight of Phomopsis azadirachtae

Dry weight of Phomopsis azadirachtae

(mg ± S.E.) Concentrations

of Bio-fungicides (ppm)


0.00 315.1 ± 1.41 j 315.1 ± 1.41 j

2.5 279.5 ± 1.78 i 285.6 ± 1.07 i

5.0 218.0 ± 1.41 h 224.5 ± 1.42 h

7.5 163.2 ± 1.12 g 178.2 ± 0.68 g

10.0 133.2 ± 0.90 f 123.1 ± 0.75 f

12.5 90.4 ± 0.60 e 78.8 ± 0.58 e

15.0 53.7 ± 0.51 d 44.7 ± 0.62 d

17.5 28.9 ± 0.41 c 24.4 ± 0.60 c

20.0 4.5 ± 0.15 b 4.1 ± 0.11 b

22.5 0.00 ± 0.00 a 0.00 ± 0.00 a

25.0 0.00 ± 0.00 0.00 ± 0.00 a


156

Table 27. Effect of ethyl acetate extracts of Bacillus subtilis and Pseudomonas aeruginosa culture filtrates on the pycnidial number of Phomopsis azadirachtae

Number of pycnidia of Phomopsis azadirachtae

(± S.E.)

Concentrations of Bio-fungicides (ppm)


0.00 220.33 ± 1.20 j 220.33 ± 1.20 j

2.5 196.67 ± 1.50 i 203.17 ± 1.66 i

5.0 162.33 ± 1.17 h 182.50 ± 1.34 h

7.5 141.00 ± 1.18 g 169.83 ± 1.35 g

10.0 112.67 ± 1.41 f 142.17 ± 1.22 f

12.5 97.33 ± 0.99 e 110.33 ± 0.76 e

15.0 65.50 ± 1.48 d 85.50 ± 0.99 d

17.5 33.17 ± 1.30 c 51.00 ± 1.41 c

20.0 8.83 ± 0.40 b 17.83 ± 1.05 b

22.5 0.00 ± 0.00 a 0.00 ± 0.00 a

25.0 0.00 ± 0.00 a 0.00 ± 0.00 a



Ethyl acetate extracts of both B. subtilis and Ps. aeruginosa completely inhibited

the germ tube growth at 25 ppm. The suppression of germ tube growth of the conidia at

different concentrations is presented in the table 28. Conidia lost their fusiform shape and

turned into non-germinable oval-shaped structures on exposure to 25 ppm of ethyl acetate

fractions of both the bacterial culture filtrates.

157

Table 28. Effect of ethyl acetate extracts of Bacillus subtilis and Pseudomonas aeruginosa culture filtrates on the germ tube growth of Phomopsis azadirachtae

Germ tube length of Phomopsis azadirachtae

(µm ± S.E.)

Concentrations of Bio-

fungicides (ppm)


0.00 106.1 ± 0.25 g 106.1 ± 0.25 g

2.5 91.9 ± 0.51 f 96.3 ± 0.52 f

5.0 82.3 ± 0.57 e 85.9 ± 0.53 e

10.0 61.8 ± 0.41 d 56.2 ± 0.56 d

15.0 38.1 ± 0.38 c 32.0 ± 0.37 c

20.0 18.4 ± 0.41 b 12.4 ± 0.48 b

25.0 0.00 ± 0.00 a 0.00 ± 0.00 a


DISCUSSION

As biological control methods are compatible with sustainable agriculture they are

becoming popular (Singh et al., 2005a). In the present experiment six antagonistic

microorganisms including four bacterial species and two fungal species were screened for

their ability to produce antagonistic secondary metabolites effective against

P. azadirachtae. Both bacteria and fungi have proved to be potential antagonists (Hajra et

al., 1992). The secondary metabolites were extracted from culture filtrate using ethyl

158

acetate. Several microbial agents produce and secrete distinct secondary metabolites with

significant antagonistic activity (Lange et al., 1993).

Among the six antagonistic microorganisms screened, secondary metabolites of

B. subtilis and Ps. aeruginosa were highly effective against P. azadirachtae at

comparatively low concentrations. Biological control activity of B. subtilis against

Phomopsis spp. was reported (Al-Rashid and Hentschel, 1988; Cubeta et al., 1985; Kita

et al., 2005; Sateesh, 1998). Cubeta et al. (1985) reported the antagonistic activity of

B. subtilis against Phomopsis spp. on soybean. Al-Rashid and Hentschel (1988) and Kita

et al. (2005) reported the same against Phomopsis sclerotioides on cucumber. B. subtilis

strain MTCC 441 exhibited significant antagonistic activity against P. azadirachtae

(Sateesh, 1998). Pseudomonas spp. are successful biocontrol agents against Phomopsis

spp. (Fuchs and Defago, 1991; Maurhofer et al., 1992; Sharifi-Tehrani et al., 1998)

especially against P. sclerotioides.

The results revealed the production of antibiotics and other antifungal secondary

metabolites by both B. subtilis and Ps. aeruginosa effective against P. azadirachtae.

B. subtilis secretes antifungal antibiotics (Eshita et al., 1995; Leclere et al., 2005;

Phookan and Chaliha, 2000). The antibiotics produced by Bacillus spp. have broad

spectrum activity (Cavaglieri et al., 2005). Bacillus spp. produce many other volatile and

non volatile antifungal metabolites (Sharifi-Tehrani and Ramezani, 2003; Sharifi-Tehrani

et al., 2005; Tsuge et al., 1995). In several strains of fluorescent pseudomonads

production of antibiotics is recognized as a major factor in suppression of plant pathogens

(Maurhofer et al., 1992). These antibiotics are highly potent broad spectrum antifungal

molecules (Dwivedi and Johri, 2003; Haas and Keel, 2003). Pseudomonas also produces

159

other antifungal secondary metabolites (Dowling and O’Gara, 1994; Srinivasan, 2003;

Thomashow and Weller, 1996). Microorganisms that produce antibiotics are very

important in the plant disease management because the antibiotics produced by them play

a major role in the significant reduction of diseases in the field (Fravel, 1988).

Studies on the effect of ethyl acetate extract on mycelial weight, pycnidial number

and germ tube length of the pathogen revealed that both the bacterial extracts were highly

effective in suppressing the growth of the pathogen. With the increase in the

concentration of solvent extract of culture filtrate progressive decrease in the colony

diameter, dry weight, pycnidial number and germ tube length were observed owing to the

exposure of the pathogen to increasing concentrations of antibiotics or other antimycotic

secondary metabolites produced by the antagonistic bacteria. Growth rate of pathogen

decreases on exposure to increasing concentrations of antibiotics from the antagonistic

microorganisms (Sateesh, 1998).

The other organisms viz., B. cereus, Trichoderma harzianum and T. viride have

antagonistic activity against many plant pathogens (Huang et al., 2005; Kaur et al., 2006;

Sharifi-Tehrani et al., 2004; Sharma et al., 2003; Wani, 2005). T. harzianum was reported

to control black root rot pathogen of cucumber, Phomopsis sclerotioides (Thinggaard,

1988) and Phomopsis vexans (Gangadharaswamy et al., 1997). But these microorganisms

failed to exhibit considerable inhibitory effects against P. azadirachtae. This may be due

to the inability of these microorganisms to produce potent toxic secondary metabolites

that are lethal to P. azadirachtae.

Singh et al. (2005) reported significant effect of ethyl acetate fraction of

Leptoxyphium axillatum on per cent spore inhibition of plant pathogenic and saprophytic

160

fungi. B. subtilis induces morphological abnormalities in the phytopathogenic fungi such

as mycelial and conidial deviations (Chaurasia et al., 2005). Similar effects were

observed in the present study on the germination and morphology of P. azadirachtae

conidia by the ethyl acetate extracts of both the bacterial culture filtrates. This result

revealed the effective antifungal activity of the ethyl acetate extracts of both the bacterial

culture filtrates against P. azadirachtae.

The present isolates of B. subtilis and Ps. aeruginosa produce agar diffusible,

solvent soluble, antimycotic substance antagonistic against P. azadirachtae. The efficacy

of the fractions from both B. Subtilis and Ps. aeruginosa at low concentrations obviously

indicates a possibility of their use as safe alternative to chemical fungicides for the

effective management of die-back of neem under field conditions and for seed treatment.

Bacteria serve as promising bioinoculants for agricultural system to control plant

diseases and to increase productivity since the action of such bacteria and their

compounds is highly specific, ecofriendly and cost-effective (Dwivedi and Johri, 2003).

With the discovery of many effective biocontrol agents and development of better

formulations and methods of application, in future the biological control method will be

the highly accepted and preferred, effective method for plant disease management

(Agrios, 2004).

161

CHAPTER 7

PART - C

INTEGRATED CONTROL OF PHOMOPSIS

AZADIRACHTAE

162

INTEGRATED CONTROL OF PHOMOPSIS AZADIRACHTAE

INTRODUCTION

A plant disease results because of an interaction between a host plant, a pathogen

and the environment. When a susceptible host comes in contact with virulent pathogen

under suitable environmental conditions then a plant disease develops and symptoms

become evident. Disease control strategies must therefore focus on the host, the pathogen

and the environment. “Integrated Disease Management (IDM)” involves the selection and

application of a harmonious range of disease control strategies that minimize losses and

maximize returns. The objective of integrated control programmes is to achieve a level of

disease control that is acceptable in economic terms to farmers and at the same time

causes minimal disturbance to the environments of non-target individuals.

The most common mode of fungal disease control is through the application of

fungicides (Maloy, 1993). Extensive utilization of fungicides has resulted in many

problems (Rathmell, 1984) including the risk of development of fungicide resistance by

the pathogen (Geordopoulos, 1987), and this has led to the development of alternative

disease control methods wherein biological control being the most preferred control

measure (Baker, 1986). Biocontrol agents may not function well under certain conditions

such as low temperatures, etc. (Omar et al., 2006). Chalutz and Droby (1997) reported

that the lack of consistency is a major drawback of the biocontrol. These problems with

extensive fungicide application and inefficiency of biocontrol agent can be overcome by

‘Integrated Disease Management’ strategy that provides more stable disease control.

163

Integrated management has potential to increase the durability of resistance

through reduction of pathogen population size and imposition of disruptive selection

(Mundt et al., 2002). IDM strategies are at the forefront of ecologically based or bio-

intensive pest management (Jacobsen, 1997). Agronomic practices, crop health

surveillance, resistant varieties and biological control are the elements of IDM. IDM

provides many procedures that help us to reduce the usage of chemical pesticides

(Paroda, 2000). Jacobsen et al. (2004) stated that, “IDM is a sustainable approach to

managing pests by combining biological, cultural, physical and chemical in a way that

minimizes economic, health and environmental risks”.

Integrated management approaches involving the combinations of many of the

above mentioned measures were reported by various workers, that include integration of -

chemicals and antagonistic microorganisms (Conway et al., 1997; Deepak and Dubey,

2001; Harman et al., 1996; Kiewnick et al., 2001), chemicals and botanicals (Shobita

Devi, 2000), two or more antagonistic microorganisms, or two or more botanicals

(Bunker and Mathur, 2001; Guetsky et al., 2002; Jatav and Mathur, 2005; Landa et al.,

2004; Lourenco Junior et al., 2006; Szczech and Shoda, 2004; Zewian et al., 2005), soil

amendments, chemicals and biocontrol agents (Baby and Manibhushanrao, 1993;

Clarkson et al., 2006; Isabel Trillas et al., 2006) resistant varieties and other methods

(Filippi and Prabhu, 1997; Jimenez-Diaz and Trapero-Casas, 1985; Kraft and Papavizas,

1983; Landa et al., 2004; Thakur, 2000), cultural practices and other measures (Edelstein

et al., 1999; Elad and Shtienberg, 1995; Landa et al., 2004; Shtienberg and Elad, 1997).

Among these, combination of chemicals and antagonistic microorganisms has received

major preference. World wide, with an ecological and economic point of view many

164

integrated control techniques including chemical and biological control have been

developed recently for various plant diseases (Budge and Whipps, 2001).

Integration of biocontrol agent with chemical fungicides helps to overcome

biocontrol limitations (Elad, 2003; Khattabi et al., 2001). This also reduces the amount of

fungicides to be applied and thus avoids associated residual problems. The advantage of

integrating a biological control agent with a fungicide is that it reduces the risk of

development of fungicide resistance by the pathogen and also provides a reliable disease

control that cannot be provided by the biocontrol agent alone (Omar et al., 2006).

Fungicides can be applied simultaneously with a biocontrol agent (Budge and Whipps,

2001; Conway et al., 1997; Harman et al., 1996) or alternative applications of chemicals

and biocontrol agents can be done (Budge and Whipps, 2001; Elad et al., 1993; Mondal,

2004). The efficiency of the biocontrol agent improves when combined with a fungicide

at a lower concentration (Elad, 2003; Govender et al., 2005; Khattabi et al., 2001;

Someya et al., 2006; van der Boogert and Luttikholt, 2004).

Systemic fungicides carbendazim and thiophanate-methyl are known to control

many plant diseases (Biswas and Singh, 2005; Bowen et al., 2000; Meena and Shah,

2005; Ponmurugan et al., 2006). Bacillus subtilis and Pseudomonas aeruginosa are

utilized as biocontrol agents to manage many plant diseases (Anjaiah et al., 2003; Asaka

and Shoda, 1996; Leifert et al., 1995; Sunish Kumar et al., 2005). Bacillus spp. and

Pseudomonas spp. are combined with chemicals to control many plant pathogens

(Errampalli and Brubacher, 2006; Govender et al., 2005; Kiewnick et al., 2001; Mondal,

2004; Omar et al., 2006). Bacillus provides a potential biological control agent that could

be exploited for Integrated Pest Management (IPM) (Jacobsen et al., 2004). There are

165

reports of integration of B. subtilis and Ps. aeruginosa with fungicides for the

management of plant diseases (Hwang and Chakravarty, 1992; Kondoh et al., 2001;

Korsten et al., 1997; Krishna Kishore et al., 2005 a & b).

In the present investigations the ethyl acetate extracts of culture filtrates of

B. subtilis and Ps. aeruginosa were combined with carbendazim and thiophanate-methyl

and the effect of these combinations on the growth of P. azadirachtae was studied. The

effect of these combinations on neem seed germination and growth of seed-borne

pathogen was also studied.


The bacterial antagonists and fungicides

Two bacterial isolates used in this study, Bacillus subtilis (MTCC 619) and

Pseudomonas aeruginosa (MTCC 2581) were procured from Microbial Type Culture

Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India.

B. subtilis was maintained on nutrient agar (Himedia, Mumbai, India) and Ps. aeruginosa

was maintained on King’s B medium (Himedia, Mumbai, India), as single cell cultures,

at 4oC. The systemic fungicides tested were two benzimidazole fungicides, carbendazim

(50% W.P.) and thiophanate-methyl (75% W.P.). These fungicides and biocontrol agents

were selected based on the results obtained from the studies on their effect on growth of

P. azadirachtae. Carbendazim at 0.25 ppm and thiophanate-methyl at 0.75 ppm

completely suppressed the growth of the pathogen. The ethyl acetate extracts from

culture filtrates of both B. subtilis and Ps. aeruginosa inhibited the growth of

P. azadirachtae at 25 ppm (Chapter seven - Part A and B).

166

Isolation of ethyl acetate fractions from bacterial culture filtrates (BCF)

The extraction of antifungal ethyl acetate fraction from BCF was carried out as

per Lavermicocca et al. (2000). A loopful of 24 h old culture of both the bacteria was

inoculated separately to 100 ml of nutrient broth (Himedia, Mumbai, India) taken in

500 ml of Erlenmeyer flask. In each case totally 10 l of medium was inoculated. All the

inoculated flasks were incubated at 37oC for 72 h. Then the cells were harvested by

centrifugation (9000 X g for 10 min at 4oC) and the supernatant was collected. The

supernatant was concentrated to 10% of the original volume by using flash evaporator at

50oC (Zhang and Watson, 2000) and filter-sterilized using 0.45 µm membrane filter

(Sartorius, Goettingen, Germany). The pH of the BCF (1000 ml) was adjusted to 3.6

using 1.0 N HCl and was extracted with equal volume of ethyl acetate for three times.

The organic extracts were pooled and evaporated at RT to obtain 2.718 g and 2.579 g of

brownish, semi-solid crude extract from B. subtilis and Ps. aeruginosa respectively. The

ethyl acetate fraction of each bacterium was dissolved in sterile distilled water containing

0.1% Tween-20 to obtain stock solution (10000 ppm). Sterilized distilled water

containing 0.1% Tween-20 was used as control solution (Singh et al., 2005a).

Effect of combinations of fungicides and ethyl acetate fractions of the bacteria on

the growth of P. azadirachtae

The tests were carried out using poison-food technique (Dhingra and Sinclair,

1995). The stock solutions of each fungicide were prepared using sterile distilled water.

All the concentrations of the fungicides are expressed in terms of active ingredient (a.i.).

Each fungicide was combined with ethyl acetate extract of each bacterium separately as

167

mentioned in Table 29 to obtain different concentrations viz., 100F: 0E, 80F: 20E,

60F: 40E, 50F: 50E, 40F: 60E, 20F: 80E, 0F:100E. Based on the results of the previous

chapters the 0.25 ppm and 0.75 ppm concentrations of carbendazim and thiophanate-

methyl respectively were taken as 100%. Similarly 25 ppm concentration was considered

as 100% for ethyl acetate extracts of both B. subtilis and Ps. aeruginosa.


The solutions of fungicides and ethyl acetate extracts were added in combinations

to potato dextrose agar (PDA, Himedia, Mumbai, India) to obtain final concentrations

viz., 100F: 0E, 80F: 20E, 60F: 40E, 50F: 50E, 40F: 60E, 20F: 80E, 0F:100E. PDA

amended with control solution but no fungicides served as control. About 20 ml of the

treated and untreated PDA were poured into separate Petri-dishes (9.0 mm diam.). All

the Petri-dishes were inoculated with the five mm mycelial-agar disc drawn from the

margin of mycelial mat of seven-day-old culture of P. azadirachtae and were incubated

at 26 ± 2oC with 12 h photoperiod for 10 days. All the treatments had four replications

and the experiment was repeated thrice.

Concentration of combinations of fungicides with ethyl acetate fractions of the

bacteria required for complete inhibition of the mycelial growth was noted. Mean colony

diameter was found out by measuring linear growth in three directions at right angles.

The colony diameter was compared with the control as a measure of fungitoxicity.

168

The per cent mycelial growth inhibition (PI) with respect to the control was

computed from the formula

(C-T) PI = C

X 100



50 ml of potato dextrose broth (Himedia, Mumbai, India) amended with various

combinations of fungicides and ethyl acetate fractions at 100F: 0E, 80F: 20E, 60F: 40E,

50F: 50E, 40F: 60E, 20F: 80E, 0F: 100E concentrations were transferred to separate

250 ml Erlenmeyer flasks. Flasks containing medium with control solution and without

fungicides were maintained as control and all the flasks were inoculated with the five mm

mycelial-agar disc drawn from the margin of mycelial mat of seven-day-old culture of

P. azadirachtae. The inoculated flasks were incubated aerobically in a controlled

environment incubator shaker at 26oC and 25 rpm for 20 days. Then the mycelial dry

weight was determined using dried mycelial mats with constant weight, which were

collected on to a preweighed Whatman No.1 filter paper and dried at 70oC in a hot air

oven until a constant weight was obtained.


PDA amended with different combinations of fungicides and ethyl acetate

extracts (100F: 0E, 80F: 20E, 60F: 40E, 50F: 50E, 40F: 60E, 20F: 80E, 0F: 100E) were

poured to separate Petri dishes (20 ml / Petri dish). The Petri dishes having media

amended with control solution, but no fungicides served as control. All the Petri dishes

169

were inoculated with the five mm mycelial-agar disc drawn from the margin of mycelial

mat of seven-day-old culture of P. azadirachtae and were incubated at 26 ± 2oC with

12 h photoperiod. The pycnidial number was counted after 15 days of incubation. The

base area of Petri dishes was divided into six equal parts by diagonally marking the lid

with a marking pen. Pycnidia present in each part were counted and mean value was

taken as total count (Sateesh, 1998). All the treatments had four replications and the

experiment was repeated thrice.


Conidial suspension having 103 conidia per ml of sterile distilled water was

prepared and 1.0 ml of this suspension was inoculated to 10 ml of malt extract broth

(Himedia, Mumbai, India) containing various combinations of fungicides and ethyl

acetate extracts (100F: 0E, 80F: 20E, 60F: 40E, 50F: 50E, 40F: 60E, 20F: 80E,

0F: 100E) taken in different 100 ml Erlenmeyer flasks. Flasks containing medium with

control solution and without fungicides were inoculated and maintained as control. The

flasks were incubated aerobically in a controlled environment incubator shaker at 26oC

and 25 rpm for 24 h. Then the germ tube growth in each flask was ceased by adding 2.0

ml of 1% lactophenol solution. The germ tube length was measured under microscopic

field using micrometer. Only when the germ tube length was double the conidial length,

the conidia were considered as germinated. All the treatments had four replications and

the experiment was repeated thrice.

170

Table 29. Combinations of fungicides (Carbendazim and Thiophanate-methyl) and microbial ethyl acetate extracts (Bacillus subtilis and Pseudomonas aeruginosa)

Concentrations of Fungicides and Ethyl acetate extracts of microbial culture filtrates

Combination of Carbendazim with Combination of Thiophanate-methyl with

Combinations

(%)



100F: 0E 0.25 ppm: 0 0.25 ppm: 0 0.75 ppm: 0 0.75 ppm: 0

80F: 20E 0.20 ppm: 5 ppm 0.20 ppm: 5 ppm 0.60 ppm: 5 ppm 0.60 ppm: 5 ppm

60F: 40E 0.15 ppm: 10 ppm 0.15 ppm: 10 ppm 0.45ppm: 10 ppm 0.45ppm: 10 ppm

50F: 50E 0.125 ppm:12.5 ppm 0.125 ppm:12.5 ppm 0.375 ppm:12.5 ppm 0.375 ppm:12.5 ppm



0F: 100E 0 : 25 ppm 0 : 25 ppm 0 : 25 ppm 0 : 25 ppm

E: Ethyl acetate extract of microbial culture filtrate; F: Fungicide (Based on the results of the previous chapters the 0.25 ppm and 0.75 ppm concentrations of carbendazim and

thiophanate-methyl respectively were taken as 100%. Similarly 25 ppm concentration was considered as 100% for ethyl acetate extracts of both B. subtilis and Ps. aeruginosa).

171

Effect on germination of neem seeds

The 50F: 50E concentration of each combination of fungicides and ethyl acetate

extracts and its multiple concentrations viz., 50F: 50E X 10, 50F: 50E X 50, 50F: 50E X

100, 50F: 50E X 500, were prepared in 100 ml of sterile distilled water. Healthy neem

seeds were freshly harvested, hard endocarp was dissected out, thoroughly washed, and

surface-sterilized using sodium hypochlorite solution (with 5% available chlorine) for

15 min. Then the seeds were rinsed well in sterile distilled water for five times. 100 seeds

were placed in 25 ml of each solution taken in separate 100 ml beakers and were exposed

to the solutions for 24 h. Seeds treated only with distilled water served as control. After

treatment the 100 seeds were germinated by blotter paper and paper towel methods

(ISTA, 1993), incubating for 15 days at RT with natural alternate day and night

photoperiod. Each treatment had four replications. Then root length, shoot length and

percentage germination were recorded and the vigour index was calculated using the

formula given by Abdul-Baki and Anderson (1973).

Effect on seed-borne P. azadirachtae

The 50F: 50E concentration of each combination of fungicides and ethyl acetate

extracts and its multiple concentrations viz., 50F: 50E X 10, 50F: 50E X 50, 50F: 50E X

100, 50F: 50E X 500, were prepared in 100 ml of sterile distilled water. Die-back

affected neem seeds were thoroughly washed and surface-sterilized as above. 100 seeds

were placed in 25 ml of each solution taken in separate 100 ml beakers and were exposed

to the solutions for 24 h. Seeds treated with only distilled water served as control. After

172

treatment they were plated on PDA at the rate of five seeds per plate and incubated for

seven days at 26 ± 2oC with 12 h photoperiod. Each treatment had four replications.

RESULTS

Effect on mycelial growth, pycnidial number and conidial germ tube growth of

P. azadirachtae

The mycelial growth of P. azadirachtae on solid medium (Fig. 32, 33, 34 and 35)

and in liquid medium, pycnidial formation and germ tube growth were completely

suppressed at all the combinations of fungicides and ethyl acetate extracts except

20F: 80E wherein little mycelial radial growth, formation of a few pycnidia and germ

tube growth were observed. The pycnidia formed were devoid of conidial cirrhi. Mycelial

growth on solid media was also observed at 40F: 60E concentrations of combinations of

thiophanate-methyl and ethyl acetate extracts of B. subtilis and Ps. aeruginosa (Fig. 34

and 35). Even in the 20F: 80E and 40F: 60E concentrations of all the combinations,

mycelial growth in liquid media were completely suppressed. In all the treatments except

20F: 80E, conidia lost their fusiform shape and turned into non-germinable oval-shaped

structures. Effect of different concentrations of each combination of fungicides and ethyl

acetate extracts on mycelial growth, pycnidial formation and germ tube growth of the

pathogen is mentioned in the table 30, 31 and 32 respectively.

175

Table 30. Effect of different combinations of fungicides and microbial ethyl acetate extracts on the mycelial growth of Phomopsis azadirachtae

Values are means of three experiments and each with four replications ± S.E. Figures followed by different superscript letters differ significantly when subjected to Tukey’s HSD (Honestly Significant Differences) [α = 0.05]

Combination of fungicides and ethyl acetate extracts of microbial culture filtrates

Carbendazim with Thiophanate-methyl with

Bacillus subtilis Pseudomonas aeruginosa Bacillus subtilis Pseudomonas aeruginosa

Concen-trations


Growth Inhibition

(%)


Growth Inhibition

(%)


Growth Inhibition

(%)


Growth Inhibition

(%) 0 8.57 ± 0.040 c 0.00 ± 0.00 a 8.57 ± 0.040 c 0.00 ± 0.00 a 8.57 ± 0.040 d 0.00 ± 0.00 a 8.57 ± 0.040 d 0.00 ± 0.00 a

100F: 0E 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 d 0.00 ± 0.00 a 100.00 ± 0.00 d

80F: 20E 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 d 0.00 ± 0.00 a 100.00 ± 0.00 d

60F: 40E 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 d 0.00 ± 0.00 a 100.00 ± 0.00 d

50F: 50E 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 d 0.00 ± 0.00 a 100.00 ± 0.00 d

40F: 60E 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 c 0.94 ± 0.047 b 89.24 ± 0.54 c 0.74 ± 0.030 b 91.55 ± 0.34 c

20F: 80E 1.15 ± 0.021 b 86.33 ± 0.30 b 1.24 ± 0.038 b 85.67 ± 0.43 b 2.03 ± 0.086 c 76.62 ± 0.99 b 1.88 ± 0.043 c 78.37 ± 0.63 b

0F: 100E 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 c 0.00 ± 0.00 a 100.00 ± 0.00 d 0.00 ± 0.00 a 100.00 ± 0.00 d

176

Table 31. Effect of different combinations of fungicides and microbial ethyl acetate extracts on the pycnidial number of Phomopsis azadirachtae

Number of pycnidia of Phomopsis azadirachtae (± S.E.)



Concen- trations

Bacillus subtilis

Pseudomonas aeruginosa


0 200.17 ± 2.89 c 200.17 ± 2.89 c 200.17 ± 2.89 c 200.17 ± 2.89 c

100F: 0E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

80F: 20E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

60F: 40E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

50F: 50E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

40F: 60E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

20F: 80E 10.33 ± 0.76 b 12.00 ± 0.58 b 18.67 ± 0.61 b 21.50 ± 0.88 b

0F: 100E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a


Effect on germination of neem seeds

Neem seeds treated with the 50F: 50E X 1, 50F: 50E X 10, 50F: 50E X 50, 50F:

50E X 100, 50F: 50E X 500 concentrations of all the combinations for 24 h germinated

normally similar to that of control wherein the seeds were only treated with distilled

water. There was no significant effect of different concentrations of the treatments

177

against the root length, shoot length and per cent germination of neem seeds (P ≤ 0.142,

0.641 and 0.957 respectively) (Fig. 36; Table 33).

Effect on seed-borne P. azadirachtae

In all the treatments the growth of P. azadirachtae was completely inhibited

whereas the untreated control seeds showed almost 90% incidence of P. azadirachtae.

Few treated seeds even showed a little germination (Fig. 37).

Table 32. Effect of different combinations of fungicides and microbial ethyl acetate extracts on the germ tube growth of Phomopsis azadirachtae

Germ tube length of Phomopsis azadirachtae (µm ± S.E.)



Concen-trations

Bacillus subtilis

Pseudomonas aeruginosa


0 108.03 ± 0.49 c 108.03 ± 0.49 c 108.03 ± 0.49 c 108.03 ± 0.49 c

100F: 0E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

80F: 20E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

60F: 40E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

50F: 50E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

40F: 60E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a

20F: 80E 13.68 ± 0.48 b 16.52 ± 0.82 b 20.22 ± 0.52 b 21. 57 ± 0.80 b

0F: 100E 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a


178

Table 33. Effect of 50F: 50E concentration of different combinations of fungicides and microbial ethyl acetate extracts on the germination of neem seeds

Combinations Concentr-

ations Root Length

(cm) Shoot

Length (cm) Percentage

Germination Vigour Index

Control 0 11.01 ± 0.058 3.81 ± 0.080 89.38 ± 0.46 1330.76 ± 12.96

A X 1 10.84 ± 0.046 3.76 ± 0.038 88.00 ± 0.65 1274.85 ± 11.25

A X 10 10.55 ± 0.042 3.64 ± 0.032 87.38 ± 0.46 1239.71 ± 10.12

A X 50 10.15 ± 0.060 3.49 ± 0.040 86.50 ± 0.33 1179.74 ± 10.50

A X 100 10.00 ± 0.060 3.28 ± 0.037 85.50 ± 0.42 1135.08 ± 10.69

Carbendazim :

Bacillus subtilis

(50F: 50E) A

A X 500 9.80 ± 0.057 3.11 ± 0.030 84.75 ± 0.31 1094.43 ± 8.91

B X 1 11.00 ± 0.060 3.80 ± 0.057 88.75 ± 0.59 1313.85 ± 16.54

B X 10 10.85 ± 0.042 3.75 ± 0.046 87.88 ± 0.40 1281.96 ± 10.66

B X 50 10.49 ± 0.030 3.65 ± 0.042 87.38 ± 0.38 1235.36 ± 9.78

B X 100 10.15 ± 0.050 3.49 ± 0.030 86.38 ± 0.42 1177.91 ± 6.54

Carbendazim : Pseudomonas

aeruginosa (50F: 50E)

B B X 500 9.88 ± 0.031 3.24 ± 0.046 85.63 ± 0.26 1122.73 ± 5.09

C X 1 10.93 ± 0.045 3.83 ± 0.037 88.00 ± 0.68 1299.94 ± 11.25

C X 10 10.69 ± 0.030 3.75 ± 0.042 87.50 ± 0.42 1263.34 ± 8.25

C X 50 10.33 ± 0.037 3.58 ± 0.031 87.00 ± 0.46 1209.30 ± 6.85

C X 100 10.11 ± 0.035 3.46 ± 0.042 85.88 ± 0.35 1165.66 ± 2.74

Thiophanate- methyl : Bacillus subtilis

(50F: 50E) C

C X 500 9.79 ± 0.035 3.31 ± 0.030 85.38 ± 0.38 1118.46 ± 7.45

D X 1 10.88 ± 0.031 3.78 ± 0.031 87.75 ± 0.31 1285.55 ± 6.92

D X 10 10.59 ± 0.030 3.65 ± 0.042 87.25 ± 0.45 1242.20 ± 8.11

D X 50 10.21 ± 0.030 3.53 ± 0.045 85.63 ± 0.38 1176.25 ± 7.10

D X 100 9.94 ± 0.050 3.45 ± 0.042 84.88 ± 0.30 1136.21 ± 6.40

Thiophanate- methyl :

Pseudomonas aeruginosa (50F: 50E)

D D X 500 9.69 ± 0.040 3.19 ± 0.040 84.38 ± 0.46 1086.19 ± 4.61

F value 1.776 0.832 0.461 1.079 Significance 0.142 0.641 0.957 0.380

Values are means of four replications ± S.E. The data was subjected to Tukey’s HSD (Honestly Significant Differences) [α = 0.05].

180

DISCUSSION

Integrated Disease Management (IDM) is a flexible, multidimensional approach

to disease control utilizing a range of control components such as cultural, biological and

chemical strategies needed to hold diseases below damaging economic threshold without

damaging the agrosystem (Papavizas and Lewis, 1988).

In the present study, systemic fungicides carbendazim and thiophanate-methyl

were combined with ethyl acetate extracts of culture filtrates of B. subtilis and

Ps. aeruginosa and tested in vitro against P. azadirachtae. Integration of biological

control agents and chemical fungicides is the widely accepted and practiced IDM strategy

(Budge and Whipps, 2001). In vitro agar plate or nutrient broth based experiments are

often used as test systems to determine potential tolerance of fungi to pesticides

(Fernando and Linderman, 1994). Carbendazim is very effective for the control of die-

back of neem (Sateesh, 1998). Although benzimidazoles can provide a significant level of

disease control they are prone to commercial failure owing to the evolution of resistant

genotypes (Brent and Hollomon, 1998). Benzimidazoles bind to microtubules assembly

in cell wall and a mutation in the β-tubulin in fungal cells results in resistance to these

fungicides (Davidse and Ishii, 1995). In this context it becomes important to develop an

alternative control measure and for which the present investigations were carried out.

The combinations of each chemical with each biocontrol extract were

significantly effective against the growth of P. azadirachtae. These combinations in all

the concentrations tested, totally suppressed the sporulation and germination of spores of

the pathogen, and except 20F: 80E and 40F: 60E completely inhibited the vegetative

growth. The results of present studies are in conformity with the previous findings. Omar

181

et al. (2006) found the combinations of carbendazim (1µg ml-1) with Burkholderia

cepacia c91 and Bacillus megaterium c96 effective against the Fusarium crown and root

rot of tomato pathogen, Fusarium oxysporum f. sp. radicis-lycopersici. Trichoderma

viride and carbendazim combination along with soil amendments provided maximum

reduction in severity of rice sheath blight (Surulirajan and Kandhari, 2005). Severity of

avocado black spot caused by Pseudocercospora purpurea was consistentantly reduced

by the preharvest applications of Bacillus subtilis field sprays integrated with copper

oxychloride or benomyl (Korsten et al., 1997). Damping-off of tomato caused by

Rhizoctonia solani was successfully controlled by the treatment with combination of

Bacillus subtilis RB 14-C and flutonil (Kondoh et al., 2001). Pseudomonas aeruginosa

GSE18 in association with thiram controlled collar rot disease of groundnut caused by

Aspergillus niger (Krishna Kishore et al., 2005a). Combination of Ps. aeruginosa GSE18

and chlorothalonil resulted in significant reduction of late leaf spot of groundnut disease

severity (Krishna Kishore et al., 2005b).

The 20F: 80E concentration of all the combinations and 40F: 60E concentration

of combinations of thiophanate-methyl and ethyl acetate extracts of B. subtilis and

Ps. aeruginosa showed higher toxicity rate against P. azadirachtae in broth medium than

agar medium. These results were in agreement with that of Ko et al. (1976). They

reported that fungicides generally were more effective against fungal growth in liquid

than in agar medium. Sateesh (1998) made similar observations with bavistin and baynate

treatment against P. azadirachtae. At low concentration carbendazim causes

abnormalities in germ tube (Wang et al., 1995). Morphological abnormalities such as

mycelial and conidial deviations are induced by B. subtilis in phytopathogenic fungi

182

(Chaurasia et al., 2005). In the present study, the combinations of fungicides and ethyl

acetate extracts produced similar effects on the germination and morphology of

P. azadirachtae conidia revealing the efficient antifungal activity of the combinations

evaluated against P. azadirachtae.

In the integrated management strategies employing combinations of chemicals

and biocontrol agents, the incompatibility between chemical pesticides and microbial

antagonists may be a major setback (Omar et al., 2006). Thus for the success of IDM

compatibility between these two is highly required. The survival and effective activity of

a microbe at an environment in the presence of a chemical is very important. Isolation of

secondary metabolites having antagonistic activity from biocontrol microorganisms and

combining them in a known concentration with low concentrations of fungicides would

help to overcome this problem. The control effect of these combinations can be attributed

to the synergistic effect of the combined treatments. Such synergistic effect was observed

between fungicides and ethyl acetate extracts of microbes in this study which resulted in

effective control of the pathogen in vitro.

Germination of seeds is used as bioassay to demonstrate the toxic effect of

fungicides or biocontrol extracts on the host plant. Dalvi et al. (1972) studied germination

of wheat and mung bean seeds to demonstrate toxic effects of menazon, disulfoton and

Gs-14254. Chauhan et al. (1997) reported the toxicity of culture filtrates of Exserohilum

turcicum on maize seed germination. Systemic and non-systemic fungicides influence the

germination of seeds (Maude, 1996). Some fungicides adversely affect the seed

germination (Devaki, 1991) and reduce the yield. Thus knowledge of phytotoxicity of

fungicides or any combinations on host plants is necessary before utilized for field

application. In the present study, neem seeds treated with the combinations of carbendazim

183

and thiophanate-methyl with ethyl acetate extracts of B. subtilis and Ps. aeruginosa

showed significant germination in comparison with control. Exposure to higher

concentration (50F: 50E X 100 and above) did not inhibit the germination but only

delayed the initiation of germination wherein germination occurred after five days. This

shows that these combinations are non-toxic to the neem tissues at concentrations that

may be used in the field.

P. azadirachtae is seed-borne (Sateesh and Bhat, 1999) and the pathogen

transmission and disease spread can occur through seeds. Seed treatment is the best

method to suppress the pathogen in seeds. Seed mycoflora of some forest trees including

Azadirachta indica (neem) were effectively controlled by the seed treatment with

carbendazim (Punam Singh et al., 1999). Seed treatment with a combination of

Pseudomonas aureofaciens (Agat-25K) and iprodione (Rovral) increased the disease

resistance of sunflower plants against Phomopsis sp. (Diaporthe helianthi) which in turn

improved plant growth and increased yield (Begunov et al., 2000). Similarly, the

combinations of fungicides and biocontrol extracts used in the present study completely

inhibited the growth of the P. azadirachtae in neem seeds and thus could be used for

neem seed treatments. These results are in accordance with those of Sateesh (1998)

wherein the neem seeds were treated with bavistin and evaluated for germination and

growth of P. azadirachtae from seeds.

Owing to the results of present investigations, the treatments with 50F: 50E

concentrations of combinations of carbendazim and thiophanate-methyl with culture

filtrate extracts of B. subtilis and Ps. aeruginosa could be potential integrated control

measure against P. azadirachtae.

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