CHAPTER 7 PART - A IN VITRO SCREENING OF SYSTEMIC FUNGICIDES AGAINST PHOMOPSIS AZADIRACHTAE

Transcription

1 122 CHAPTER 7 PART - A IN VITRO SCREENING OF SYSTEMIC FUNGICIDES AGAINST PHOMOPSIS AZADIRACHTAE

2 123 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

3 124 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

4 125 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

5 126 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 ± 2 o C 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 = X 100 C 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.

6 127 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 Beam 75% W.P. Tricyclazole 5-methyl-1,2,4-triazolo [3,4-b] benzothiazole Contaf 5% E.C. Hexaconazole A-butyl-A-(2,4-dichlorophenyl)- 1H-1,2,4-triazole-1-ethanol BASF India Limited, Thane, India. Indofil Chemicals Company, Mumbai, India 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 Roko 70% W.P. Thiophanate-methyl Dimethyl [1,2-phenylene bis (iminocarbonothioyl)] bis [carbamate] Rallis- A TATA enterprise, Mumbai, India Biostadt, Mumbai, India

7 128 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 26 o C 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 70 o C 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 ± 2 o C 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

8 129 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 10 3 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 26 o C 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 thiophanatemethyl 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

9 130 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 ± e 0.00 ± 0.00 a Metalaxyl 8.00 ± d 7.90 ± 0.61 b Isoprothiolane 6.83 ± 0.10 b ± 1.17 d Tricyclazole 7.58 ± 0.10 c ± 1.19 c Carbendazim 0.0 ± 0.00 a ± 0.00 e Hexaconazole 0.0 ± 0.00 a ± 0.00 e Thiophanate-methyl 0.0 ± 0.00 a ± 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.

10 131 Table 18. Effect of hexaconazole, carbendazim and thiophanate-methyl on the mycelial growth of Phomopsis azadirachtae Concentrations of fungicides (ppm) Mycelial Growth (cms) Fungicides Hexaconazole Carbendazim Thiophanate-methyl Growth Inhibition (%) Mycelial Growth (cms) Growth Inhibition (%) Mycelial Growth (cms) Growth Inhibition (%) ± g 0.00 ± 0.00 a 0.00 ± 0.00 a ± 0.00 g 0.00 ± 0.00 a ± 0.00 g ± f 9.97 ± 0.59 b 0.00 ± 0.00 a ± 0.00 g 0.00 ± 0.00 a ± 0.00 g ± e ± 0.77 c 0.00 ± 0.00 a ± 0.00 g 0.00 ± 0.00 a ± 0.00 g ± 0.10 d ± 1.18 d 0.00 ± 0.00 a ± 0.00 g 0.00 ± 0.00 a ± 0.00 g ± c ± 0.69 e 0.00 ± 0.00 a ± 0.00 g 0.00 ± 0.00 a ± 0.00 g ± b ± 0.17 f 0.00 ± 0.00 a ± 0.00 g 0.00 ± 0.00 a ± 0.00 g ± 0.00 a ± 0.00 g 0.00 ± 0.00 a ± 0.00 g 0.00 ± 0.00 a ± 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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12 133 Table 19. Effect of carbendazim and thiophanate-methyl on the mycelial growth of Phomopsis azadirachtae Fungicides Concentrations of fungicides (ppm) Mycelial Growth (cms) Carbendazim Growth Inhibition (%) Thiophanate-methyl Mycelial Growth (cms) Growth Inhibition (%) ± f 0.00 ± 0.00 a 8.72 ± h 0.00 ± 0.00 a ± e 4.79 ± 0.37 b 8.42 ± g 3.39 ± 0.46 b ± d 24.41± 0.56 c 7.63 ± f ± 0.75 c ± c ± 0.77 d 6.40 ± e ± 0.77 d ± b ± 0.45 e 4.46 ± d ± 0.62 e ± 0.00 a ± 0.00 f 3.26 ± c ± 0.89 f ± 0.00 a ± 0.00 f 2.08 ± b ± 0.89 g ± 0.00 a ± 0.00 f 0.00 ± 0.00 a ± 0.00 h ± 0.00 a ± 0.00 f 0.00 ± 0.00 a ± 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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14 135 Table 20. Effect of carbendazim and thiophanate-methyl on the dry weight of Phomopsis azadirachtae Concentrations of fungicides (ppm) Dry weight of Phomopsis azadirachtae (mg ± S.E.) Carbendazim Thiophanate-methyl ± 1.39 f ± 1.39 h ± 0.91 e ± 1.27 g ± 1.02 d ± 1.57 f ± 0.77 c ± 1.28 e ± 0.21 b ± 0.88 d ± 0.00 a ± 0.57 c ± 0.00 a 5.13 ± 0.19 b ± 0.00 a 0.00 ± 0.00 a ± 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 thiophanatemethyl 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.

15 136 Table 21. Effect of carbendazim and thiophanate-methyl on the pycnidial number of Phomopsis azadirachtae Concentrations of fungicides (ppm) Number of pycnidia of Phomopsis azadirachtae (± S.E.) Carbendazim Thiophanate-methyl ± 3.19 f ± 3.19 h ± 2.64 e ± 3.27 g ± 2.97 d ± 3.03 f ± 2.63 c 89.8 ± 2.54 e ± 0.83 b 54.7 ± 1.99 d ± 0.00 a 28.8 ± 1.49 c ± 0.00 a 10.3 ± 0.88 b ± 0.00 a 0.00 ± 0.00 a ± 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

16 137 (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 Concentration of fungicides (ppm) Germ tube length of Phomopsis azadirachtae (µm ± S.E.) Carbendazim Thiophanate-methyl ± 0.61 f ± 0.61 h ± 0.72 e 95.5 ± 0.70 g ± 0.75 d 77.9 ± 0.67 f ± 0.64 c 64.9 ± 0.77 e ± 0.70 b 48.5 ± 0.56 d ± 0.00 a 34.5 ± 0.62 c ± 0.00 a 13.8 ± 0.38 b ± 0.00 a 0.00 ± 0.00 a ± 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]

17 138 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 thiophanatemethyl 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).

18 139 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.

19 140 CHAPTER 7 PART - B BIOLOGICAL CONTROL OF PHOMOPSIS AZADIRACHTAE WITH ANTAGONISTIC FUNGI AND BACTERIA

20 141 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).

21 142 Early in the twentieth century, the ability of a few soil microorganisms to suppress the development of soil-borne plant pathogens was reported. Numerous nonpathogenic 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., 2005 a ; 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;

22 143 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., 2005 a ; 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

23 144 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., 2005 a ).

24 145 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 4 o C. 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 4 o C (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 37 o C for 72 h. Then the cells were harvested by centrifugation (9000 X g for 10 min at 4 o C). The supernatant was collected, the volume of each culture filtrate was made up to 1.5 l with sterile distilled water, filter-

25 146 sterilized using 0.45 µm membrane filter (Sartorius, Goettingen, Germany) and stored at 4 o C. For extraction, the culture filtrates were concentrated to 10% of their original volume by using a flash evaporator at 50 o C (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. (2005 a ). 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 ± 2 o C 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 o C. 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

26 147 (1.0 mg / ml). Sterilized distilled water containing 0.1% Tween-20 served as control solution (Singh et al., 2005 a ). 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 ± 2 o C 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 PI = (C-T) C X 100 Where, C is the colony diameter of the control and the T that of the treated ones.

27 148 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. Effect on mycelial growth of P. azadirachtae earlier. The experiment was carried out employing the same methodology as described 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 26 o C and 25 rpm for 20 days in a

28 149 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 70 o C 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 ± 2 o C 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 10 3 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

29 150 maintained as control. The flasks were incubated aerobically at 26 o C 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

30 151 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) Growth Inhibition (%) Control 8.5 ± f 0.00 ± 0.00 a Trichoderma harzianum 5.58 ± e ± 0.47 b Trichoderma viride 6.02 ± d ± 1.48 c Pseudomonas oleovorans 5.07 ± c ± 0.44 d Bacillus cereus 4.43 ± b ± 0.49 e Bacillus subtilis 0.00 ± 0.00 a ± 0.00 f Pseudomonas aeruginosa 0.00 ± 0.00 a ± 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]

31 152

32 153 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 Concentrations of Biofungicides (ppm) Mycelial Growth (cms) Ethyl acetate extracts of culture filtrates Bacillus subtilis Growth Inhibition (%) Pseudomonas aeruginosa Mycelial Growth (cms) Growth Inhibition (%) ± i 0.00 ± 0.00 a 8.9 ± j 0.00 ± 0.00 a ± h 2.97 ± 0.40 b 8.33 ± i 6.40 ± 0.35 b ± g ± 0.47 c 7.50 ± h ± 0.36 c ± f ± 0.53 d 6.73 ± g ± 0.29 d ± e ± 0.38 e 5.85 ± f ± 0.26 e ± d ± 0.36 f 5.20 ± e ± 0.22 f ± c ± 0.32 g 4.26 ± d ± 0.30 g ± b ± 0.43 h 2.30 ± c ± 0.34 h ± a ± 0.29 i 1.29 ± b ± 0.20 i ± 0.00 a ± 0.00 j 0.00 ± 0.00 a ± 0.00 j ± 0.00 a ± 0.00 j 0.00 ± 0.00 a ± 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]

33 154

34 155 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 Concentrations of Bio-fungicides (ppm) Dry weight of Phomopsis azadirachtae (mg ± S.E.) Bacillus subtilis Pseudomonas aeruginosa ± 1.41 j ± 1.41 j ± 1.78 i ± 1.07 i ± 1.41 h ± 1.42 h ± 1.12 g ± 0.68 g ± 0.90 f ± 0.75 f ± 0.60 e 78.8 ± 0.58 e ± 0.51 d 44.7 ± 0.62 d ± 0.41 c 24.4 ± 0.60 c ± 0.15 b 4.1 ± 0.11 b ± 0.00 a 0.00 ± 0.00 a ± ± 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]

35 156 Table 27. Effect of ethyl acetate extracts of Bacillus subtilis and Pseudomonas aeruginosa culture filtrates on the pycnidial number of Phomopsis azadirachtae Concentrations of Bio-fungicides (ppm) Number of pycnidia of Phomopsis azadirachtae (± S.E.) Bacillus subtilis Pseudomonas aeruginosa ± 1.20 j ± 1.20 j ± 1.50 i ± 1.66 i ± 1.17 h ± 1.34 h ± 1.18 g ± 1.35 g ± 1.41 f ± 1.22 f ± 0.99 e ± 0.76 e ± 1.48 d ± 0.99 d ± 1.30 c ± 1.41 c ± 0.40 b ± 1.05 b ± 0.00 a 0.00 ± 0.00 a ± 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.

36 157 Table 28. Effect of ethyl acetate extracts of Bacillus subtilis and Pseudomonas aeruginosa culture filtrates on the germ tube growth of Phomopsis azadirachtae Concentrations of Biofungicides (ppm) Germ tube length of Phomopsis azadirachtae (µm ± S.E.) Bacillus subtilis Pseudomonas aeruginosa ± 0.25 g ± 0.25 g ± 0.51 f 96.3 ± 0.52 f ± 0.57 e 85.9 ± 0.53 e ± 0.41 d 56.2 ± 0.56 d ± 0.38 c 32.0 ± 0.37 c ± 0.41 b 12.4 ± 0.48 b ± 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., 2005 a ). 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

37 158 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

38 159 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

39 160 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).

40 161 CHAPTER 7 PART - C INTEGRATED CONTROL OF PHOMOPSIS AZADIRACHTAE

41 162 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.

42 163 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 biointensive 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

43 164 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

44 165 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 4 o C. 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).

45 166 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 37 o C for 72 h. Then the cells were harvested by centrifugation (9000 X g for 10 min at 4 o C) and the supernatant was collected. The supernatant was concentrated to 10% of the original volume by using flash evaporator at 50 o C (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 g and 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., 2005 a ). 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

46 167 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 thiophanatemethyl 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 ± 2 o C 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.

47 168 The per cent mycelial growth inhibition (PI) with respect to the control was computed from the formula PI = (C-T) 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 26 o C 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 70 o C 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

48 169 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 ± 2 o C 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 10 3 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 26 o C 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.

49 170 Table 29. Combinations of fungicides (Carbendazim and Thiophanate-methyl) and microbial ethyl acetate extracts (Bacillus subtilis and Pseudomonas aeruginosa) Combinations (%) Concentrations of Fungicides and Ethyl acetate extracts of microbial culture filtrates Combination of Carbendazim with Combination of Thiophanate-methyl with Bacillus subtilis Pseudomonas aeruginosa Bacillus subtilis Pseudomonas aeruginosa 100F: 0E 0.25 ppm: ppm: ppm: 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 ppm:12.5 ppm ppm:12.5 ppm ppm:12.5 ppm 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).

50 171 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

51 172 treatment they were plated on PDA at the rate of five seeds per plate and incubated for seven days at 26 ± 2 o C 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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53 174

54 175 Table 30. Effect of different combinations of fungicides and microbial ethyl acetate extracts on the mycelial growth of Phomopsis azadirachtae Concentrations Mycelial Growth (cms) Combination of fungicides and ethyl acetate extracts of microbial culture filtrates Carbendazim with Thiophanate-methyl with Bacillus subtilis Pseudomonas aeruginosa Bacillus subtilis Pseudomonas aeruginosa Growth Inhibition (%) Mycelial Growth (cms) Growth Inhibition (%) Mycelial Growth (cms) Growth Inhibition (%) Mycelial Growth (cms) Growth Inhibition (%) ± c 0.00 ± 0.00 a 8.57 ± c 0.00 ± 0.00 a 8.57 ± d 0.00 ± 0.00 a 8.57 ± d 0.00 ± 0.00 a 100F: 0E 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 d 0.00 ± 0.00 a ± 0.00 d 80F: 20E 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 d 0.00 ± 0.00 a ± 0.00 d 60F: 40E 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 d 0.00 ± 0.00 a ± 0.00 d 50F: 50E 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 d 0.00 ± 0.00 a ± 0.00 d 40F: 60E 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 c 0.94 ± b ± 0.54 c 0.74 ± b ± 0.34 c 20F: 80E 1.15 ± b ± 0.30 b 1.24 ± b ± 0.43 b 2.03 ± c ± 0.99 b 1.88 ± c ± 0.63 b 0F: 100E 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 c 0.00 ± 0.00 a ± 0.00 d 0.00 ± 0.00 a ± 0.00 d 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]

55 176 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.) Concentrations Combination of fungicides and ethyl acetate extracts of microbial culture filtrates Carbendazim with Thiophanate-methyl with Bacillus subtilis Pseudomonas aeruginosa Bacillus subtilis Pseudomonas aeruginosa ± 2.89 c ± 2.89 c ± 2.89 c ± 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 ± 0.76 b ± 0.58 b ± 0.61 b ± 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

56 177 against the root length, shoot length and per cent germination of neem seeds (P 0.142, and 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 Concentrations 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 Bacillus subtilis Pseudomonas aeruginosa Bacillus subtilis Pseudomonas aeruginosa ± 0.49 c ± 0.49 c ± 0.49 c ± 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 ± 0.48 b ± 0.82 b ± 0.52 b ± 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]

57 178 Table 33. Effect of 50F: 50E concentration of different combinations of fungicides and microbial ethyl acetate extracts on the germination of neem seeds Combinations Concentrations Root Length (cm) Shoot Length (cm) Percentage Germination Vigour Index Control ± ± ± ± Carbendazim : Bacillus subtilis (50F: 50E) A Carbendazim : Pseudomonas aeruginosa (50F: 50E) B Thiophanatemethyl : Bacillus subtilis (50F: 50E) C Thiophanatemethyl : Pseudomonas aeruginosa (50F: 50E) D A X ± ± ± ± A X ± ± ± ± A X ± ± ± ± A X ± ± ± ± A X ± ± ± ± 8.91 B X ± ± ± ± B X ± ± ± ± B X ± ± ± ± 9.78 B X ± ± ± ± 6.54 B X ± ± ± ± 5.09 C X ± ± ± ± C X ± ± ± ± 8.25 C X ± ± ± ± 6.85 C X ± ± ± ± 2.74 C X ± ± ± ± 7.45 D X ± ± ± ± 6.92 D X ± ± ± ± 8.11 D X ± ± ± ± 7.10 D X ± ± ± ± 6.40 D X ± ± ± ± 4.61 F value Significance Values are means of four replications ± S.E. The data was subjected to Tukey s HSD (Honestly Significant Differences) [α = 0.05].

58 179