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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]
Effect on mycelial dry weight of P. azadirachtae
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)
Growth Inhibition (%)
Mycelial Growth (cms)
Growth Inhibition (%)
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
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]
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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)
Mycelial Growth (cms)
Growth
Inhibition (%)
Mycelial Growth (cms)
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
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]
Effect on pycnidial number of P. azadirachtae
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)
Carbendazim Thiophanate-methyl
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
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]
Effect on conidial germ tube growth of P. azadirachtae
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)
Carbendazim Thiophanate-methyl
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
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]
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)
Carbendazim Thiophanate-methyl
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
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]
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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
144
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
MATERIALS AND METHODS
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
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(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
Where, C is the colony diameter of the control and the T that of the treated ones.
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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.
Effect on mycelial growth of P. azadirachtae
The experiment was carried out employing the same methodology as described
earlier.
Effect on mycelial dry weight of P. azadirachtae
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.
Effect on pycnidial number of P. azadirachtae
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).
Effect on conidial germ tube growth of P. azadirachtae
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
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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)
Growth Inhibition (%)
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]
152
153
Effect on mycelial dry weight of P. azadirachtae
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)
Mycelial Growth (cms)
Growth Inhibition (%)
Mycelial Growth (cms)
Growth Inhibition (%)
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
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]
154
155
Effect on pycnidial number of P. azadirachtae
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)
Bacillus subtilis Pseudomonas aeruginosa
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
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]
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)
Bacillus subtilis Pseudomonas aeruginosa
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
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]
Effect on conidial germ tube growth of P. azadirachtae
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)
Bacillus subtilis Pseudomonas aeruginosa
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
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]
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
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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).
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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.
MATERIALS AND METHODS
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).
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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.
Effect on mycelial growth of P. azadirachtae
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.
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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.
Effect on mycelial dry weight of P. azadirachtae
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.
Effect on pycnidial number of P. azadirachtae
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
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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.
Effect on conidial germ tube growth of P. azadirachtae
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.
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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
(%)
Bacillus subtilis Pseudomonas aeruginosa
Bacillus subtilis Pseudomonas aeruginosa
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
40F: 60E 0.10 ppm: 15 ppm 0.10 ppm: 15 ppm 0.30 ppm: 15 ppm 0.30 ppm: 15 ppm
20F: 80E 0.05 ppm: 20 ppm 0.05 ppm: 20 ppm 0.15 ppm: 20 ppm 0.15 ppm: 20 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
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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.
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174
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
Mycelial Growth (cms)
Growth Inhibition
(%)
Mycelial Growth (cms)
Growth Inhibition
(%)
Mycelial Growth (cms)
Growth Inhibition
(%)
Mycelial Growth (cms)
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
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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.)
Combination of fungicides and ethyl acetate extracts of microbial culture filtrates
Carbendazim with Thiophanate-methyl with
Concen- trations
Bacillus subtilis
Pseudomonas aeruginosa
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
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]
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
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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.)
Combination of fungicides and ethyl acetate extracts of microbial culture filtrates
Carbendazim with Thiophanate-methyl with
Concen-trations
Bacillus subtilis
Pseudomonas aeruginosa
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
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]
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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].
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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
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(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
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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.