Abstract: In the present study, the pathogenic fusaria viz., Fusarium solani f. sp. melongena and F. oxysporum f. sp. lycopersici causing brinjal and tomato wilt were isolated from soil as well as from the infected plant parts. In vitro efficacy of three medicinal plants viz., Azadirachta indica (leaf extract), Psidium guajava (leaf extract), Eucalyptus camaldulensis (bark extract) and three fungal antagonists viz., Trichoderma harzianum, T. atroviride and T. longibrachiatum were tested at 25, 50 and 75% (v/v) by poisoned food technique against both the pathogens. The assessment of fungitoxicity was carried out in terms of percent mycelial growth inhibition against the test fungi. Among different medicinal plant extracts, Azadirachta indica (leaf) was found significantly superior to the rest in suppressing the growth of F. oxysporum f. sp. lycopersici as 100% inhibition was recorded at 50 and 75% concentration followed by Psidium guajava and Eucalyptus camaldulensis on 7th day of inoculation. On the other hand, among different microbial antagonists, T. longibrachiatum against both the test fungi was highly effective and there was 100% inhibition of mycelial growth at 50 and 75% concentration, while T. harzianum was effective against F. oxysporum f. sp. lycopersici followed by T. atroviride as it completely inhibited the mycelial growth at 75% concentration.
INTRODUCTION
The Fusarium wilt of tomato (Lycopersicion esculantum Mill) caused by Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder and Hansen (FOL) and Brinjal wilt (Solanum melongena L.) caused by Fusarium solani f. sp. melongena is recognized as a devastating disease in tomato and brinjal growing regions worldwide (Beckman, 1987; Bondad-Reantaso et al., 2005) besides in different regions of India in severe to moderate (50-60%) form (Sherf and Macnab, 1986; Jiskani et al., 2007; Chakraborty and Chatterjee, 2009). In Kanpur (U. P.), the vegetable growers suffer more than 25.14-47.94% crop losses due to Fusarium wilt of tomato and 18.61-33.37% due to Fusarium wilt of brinjal in heavily infested fields. Kapoor (2008) has reported that most of the common varieties of tomato and brinjal are susceptible and fungicides are frequently used to control the disease. However, the soil-borne disease is very difficult and uneconomical to control with chemicals alone. In this context, biological control is an alternative and eco-friendly strategy for disease management (Bowers and Locke, 2000; Momin and Nair, 2001; Eziashi et al., 2007).
The extract of medicinal plants (Daferera et al., 2000; Manczinger et al., 2002; Sridhar et al., 2003; Dwivedi and Enespa, 2012) as well as antimicrobial agents (Haggag and Mohamed, 2007; Dwivedi and Enespa, 2013) have been found effective as antifungal agent. The leaf extract of Azadirachta indica showed in vitro antifungal effect against Fusarium solani and Fusarium oxysporum f. sp. lycopersici (Jarvis and Morgan, 2000; Dai et al., 2001; Verma and Kharwar, 2006; Agbenin and Marley, 2006). The antifungal effect (bark extract) of Eucalyptus camaldulensis (Pattanaik et al., 2002), leaf extract of Psidium guajava (Khayungarnnawee et al., 2004) against F. solani and F. oxysporum f. sp. lycopersici have also been studied.
On the other hand, the antagonistic fungi especially Trichoderma spp. viz., T. harzianum, T. atroviride and T. longibrachiatum have been widely used against F. solani and F. oxysporum f. sp. lycopersici (Ahmed, 2011; Hend and Perveen, 2012). Christopher et al. (2010) have reported successful reduction of fusarial wilt in many crops with application of different species of Trichoderma. Rini and Sulochana (2007) and Kapoor (2008) have found Trichoderma spp. to be effective biocontrol agents against F. solani and F. oxysporum. However, it is also reported that all the isolates of Trichoderma spp. are not equally effective in the control of the pathogens in vitro and in vivo (Moussa et al., 2006; Morsy et al., 2009). Therefore, a specific effective native Trichoderma isolate has to be identified for successful control of a particular pathogen. The purpose of this study was to evaluate the efficacy of medicinal plants as well as fungal antagonists (Trichoderma spp.) against F. solani and F. oxysporum f. sp. lycopersici.
MATERIALS AND METHODS
Test fungal strains: The pathogenic fusaria viz., Fusarium solani and Fusarium oxysporum f. sp. lycopersici and fungal antagonists viz., Trichoderma harzianum, T. atroviride and T. longibrachiatum were isolated from the wilt affected (soil+plant parts) and healthy tomato and brinjal crop fields of Kanpur, Uttar Pradesh, India, using serial dilution technique as well as direct inoculation of plant parts. The cultures of the fungi were purified by single spore isolation technique on Czapek’s Dox Agar medium (Chauhan et al., 2002). The isolated pathogenic and biocontrol mycoflora were identified on the basis of cultural and morphological characters (Gilman, 1957; Barnett and Hunter, 1972; Booth, 1985). The fungitoxicity was studied by poisoned food technique (Grover and Moore, 1962).
Preparation of crude plant extracts: Fresh plant materials of Azadirachta indica (leaves), Psidium guajava (leaves), Eucalyptus camaldulensis (bark) were washed thoroughly under running tap water followed by sterilized distilled water. The leaves were air dried and then grinded with the help of pestle and mortar. One mL of extract was added in 1ml distilled water (1:1 v/v) separately for each plant extract; filtered through muslin cloth and 100% plant extract solution was prepared. The extracts were poured in the flasks and further diluted to different concentrations (25, 50 and 75%) by adding sterile distilled water for further studies.
Determination of mycelial growth inhibition
Dry weight method: One hundred
ml of broth amended with different filtrate of antagonistic fungi individually
were inoculated with 6.0 mm agar discs in 250 mL Erlenmeyer flask. The flasks
were then incubated at 25±2°C for 10 days. The fungal mats were removed
by filtration (Whatman Filter Study 1 and then 42 Nos.), dried at 60±3°C
for 24 h and dry weight (g) was recorded.
Linear growth method: The filtrates of the antagonistic Trichoderma spp. viz., T. harzianum, T. atroviride and T. longibrachiatum and extracts of medicinal plant viz., Azadirachta indica (leaves), Psidium guajava (leaves), Eucalyptus camaldulensis (bark) were added to autoclaved CZA medium to give fungal concentration of 25, 50 and 75% (v/v). The plates were inoculated with 6.0 mm disc of F. solani and F. oxysporum f. sp. lycopersici individually in the centre of each of the Petri plates and incubated at 25±2°C for 7 days. Three replicates were maintained for each treatment. The growth of fungus was measured on 7th day and mean of colony growth dia. (mm) was recorded and percentage of mycelial growth inhibition was calculated as compared to control (Gaspar et al., 2004).
Statistical analysis: The Data recorded during the course of investigation was subjected to two-way ANOVA without replication. The conclusion was drawn on the basis of analysis of variance. The calculated value of F was compared with Table value of F at 5% levels of significance for an appropriate degree of freedom.
RESULTS AND DISCUSSION
Evaluation of medicinal plants against fungal pathogens: The mean fungal radial growth (mm) for each treatment is presented in (Table 1) indicate the inhibition zone of different extracts. All the medicinal plant extracts showed significant inhibition in the mycelial growth of the test pathogens. The A. indica leaf extract against F. oxysporum f. sp. lycopersici was most effective as it completely inhibited the mycelial growth at the concentration of 50 and 75%. A. indica leaf extract at 75% concentration was also most effective against F. solani on 7th day of inoculation.
| Table 1: | Efficacy of medicinal plant extracts against Fusarium solani f. sp. melongena and Fusarium oxysporum f. sp. lycopersici at different concentration on 7th day of inoculation |
| Values shown are the Mean±SE of 3 replicates, significant at p≤0.05. Application of A. indica on F. oxysporum f. sp. lycopersici at 50 and 75% concentration had highest inhibiting effect followed by 75% conc. in A. indica on F. solani. These biopesticides checked fungal growth by 100, 100 and 90.10%, respectively | |
| Fig. 1: | Effect of medicinal plant extracts on radial growth of F. solani and F. oxysporum f. sp. lycopersici on 7th day of incubation |
The growth of Fusarium solani was inhibited at 25% concentration of Azadirachta indica (leaves), Psidium guajava (leaves) and Eucalyptus camaldulensis (bark) by 63.48, 26.62 and 4.44%, respectively. On the other hand, at 50% concentration of the foresaid medicinal plants inhibited the mycelial growth of Fusarium solani by 74.40, 35.84 and 22.87%, respectively. However, maximum inhibition i.e., 90.10, 39.59 and 78.84% was recorded at 75% concentration. The foresaid medicinal plants at 25% concentration inhibited the mycelia growth of Fusarium oxysporum f. sp. lycopersici by 84.25, 19.86 and 1.72%, respectively. On the other hand, at 50% concentration of these plants inhibited mycelial growth by 100.00, 34.59 and 9.93%, respectively. Maximum percentage inhibition i.e., 100.00, 37.67 and 14.04% was recorded at 75% concentration of Azadirachta indica (leaves) against F. oxysporum f. sp. lycopersici as indicated in Fig. 1.
Our findings are in conformity with Joseph and Priya (2010) with respect to efficacy of guava leaf extract against Aspergillus niger and Aspergillus aculeatus. Similar results were reported (Mossini et al., 2004; Niaz and Kazmi, 2005) with the increase in concentration of the medicinal plants, the greater inhibition of mycelial growth of Fusarium spp. and Aspergillus alternata at 0.15 and 0.1% concentration. Antifungal activities and chemical properties of Azadirachta indica oil and leaf extracts on the growth of Aspergillus, Rhizopus, Alternaria solani and Alternaria brassicae have been also reported (Mondali et al., 2009; Suleiman, 2010; Sasode et al., 2012). Bajwa and Ifftikhar (2005) evaluated Eucalyptus camaldulensis and Datura tetramera against fungal pathogens causing wilt in vegetable crops.
Evaluation of Trichoderma spp. against fungal pathogens: The percentage reduction of F. solani and F. oxysporum at different concentration of culture filtrates of T. harzianum, T. atroviride and T. longibrachiatum is presented in Table 2. The data revealed a significance increase (p<0.001) in colony growth reduction of F. solani and F. oxysporum f. sp. lycopersici with increasing the concentration of both the fungal filtrates. T. harzianum, T. atroviride and T. longibrachiatum filtrates inhibited the growth of F. solani by 84.01, 48.91 and 100% and F. oxysporum f. sp. lycopersici by 100.00, 73.67 and 100.00%, respectively at 75% concentration. At 50% concentration, the foresaid antagonists inhibited the growth of F. solani by 74.88, 44.06 and 86.87% and F. oxysporum f. sp. lycopersici by 92.35, 65.74 and 100.00%, respectively.
| Table 2: | Effect of Trichoderma spp. against Fusarium solani (FS) and Fusarium oxysporum f. sp. lycopersici (FOL) at different concentration on 7th day of inoculation |
| Values shown are the Mean±SD of 3 replicates, significant at p≤0.05. Application of T. longibrachiatum at 50 and 75% concentration had highest inhibiting effect on both the pathogens followed by T. harzianum at 75% concentration on Fusarium oxysporum. These fungal antagonists checked fungal growth by 100, 100 and 100%, respectively | |
| Fig. 2: | Percentage inhibition of F. solani f. sp. melongena and F. oxysporum f. sp. lycopersici at different concentration of Trichoderma spp. on 7th day of inoculation |
At lower concentration (25%), T. harzianum, T. atroviride and T. longibrachiatum were least effective and inhibited the growth of F. solani by 47.20, 42.06 and 75.17% and F. oxysporum f. sp. lycopersici by 77.63, 57.25 and 78.76%, respectively compared to control on 7th day of inoculation (Fig. 2)
Our findings are in conformity with Devi and Singh (2012) with respect to in vitro efficacy of Trichoderma spp. against Fusarium oxysporum f. sp. lycopersici causing tomato wilt. Antagonistic activity of three isolates of Trichoderma spp. against F. solani and F. oxysporum f. sp. lycopersici due to parasitism, competition and antibiosis was reported by Elad (2000); Freeman et al. (2004); Dubey et al. (2007); Anwar et al. (2008); coiling and penetration of antagonistic hyphae of T. virens and T. harzianum around the hyphae of F. solani and their lysis (Kumar and Dubey, 2001; Zaldivar et al., 2001) and production of organic metabolites (Haggag and Mohamed, 2007). It was observed that with the increase in concentration of the culture filtrates of Trichoderma spp. there was greater inhibition of the mycelial growth of Rhizoctonia solani, Sclerotinia, Fusarium, Pythium (Ashrafizadeh et al., 2005; Yigit and Dikilitas, 2007; Khan and Sinha, 2007).
CONCLUSION
In the present study, the medicinal plant (Azadirachta indica) leaf extract and fungal antagonists viz., T. longibrachiatum and T. harzianum were most effective against both the pathogens. Therefore, these bio-pesticides (botanicals and bio-agents) can be used as an alternative to pesticides to minimize the wilt disease of these crops besides improving the yield as they are environmental safe.