IN-VITRO EVALUATION OF ANTIFUNGAL ACTIVITY OF PLANT EXTRACTS AGAINST RHIZOCTONIA ORYZAE-SATIVAE CAUSING AGGREGATED SHEATH SPOT OF RICE
B. Jahan, M. A. Ali, S. Alam, Z. R. Moni and M. A. Alam. 2013. In-vitro evaluation of antifungal activity of plant extracts against Rhizoctonia oryzae-sativae causing aggregated sheath spot of rice. Bangladesh J. Plant Pathol. 29 (1&2):13-18.
In-vitro experiments were conducted to test the antifungal activity of garlic clove (Allium sativam), ginger rhizome (Zingiber officinales), and leaves of henna (Lawsonia inermis), water pepper (Poligonum hydropiper), ivy gourd (Coccinia cordifolia) and neem (Azadirachta indica) against mycelia growth of Rhizoctonia oryzae-sativae, the causal fungus of aggregated sheath spot of rice. Aqueous extracts of all the botanicals were prepared, mixed with liquid potato dextrose agar (PDA) medium at 0, 5, 10, 15, 20 and 25% concentrations and poured into 90 mm Petri dishes at 20 ml per dish. After solidification of PDA, 90 mm glass Petri dishes were inoculated with mycelia blocks of R. oryzae-sativae cut from 5 days old PDA culture of the pathogen at one block per plate. It was found that the plant extract reduced radial colony diameter of the pathogen appreciably at different concentrations of the botanicals compared to control (0%). Among the tested plant extracts, garlic and henna was the most effective material against R. oryzae-sativae showing 50% reduction in colony diameter at 3.25% concentration, which indicated lowest LD (lethal dose) 50 value followed by henna extract at 3.75%. Lowest LD 90 value also showed by garlic extracts at 17.25% followed by henna extract at 19%. Garlic and henna considerably decreased sclerotia germination at all the concen-trations tested in the present study.
Aggregated sheath spot caused by Rhizoctonia oryzae-sativae is usually considered as a minor disease of rice but it can be a very aggressive disease of the crop under favourable conditions (Lanoiselet et al. 2007). It occurs in many of the rice growing countries of the world (Gunnel et al. 1984, 1992, Cedeno et al. 1998, Lanoiselet et al. 2001). In Bangladesh, aggregate sheath spot was reported for the first time in 1988 by Shahjahan et al. (1988). The disease caused yield losses of 20% in Australia, 4 to 9% in Uruguay (Lanoiselet et al. 2005) and in Bangladesh the disease may reduce rice yield by 14.74%.
Chemical, physical and cultural methods are recommended to control aggregated sheath spot of rice. Available reports reveal that extracts of many plant species possess antifungal and antibacterial properties (Hasan et al. 2005, Ogbo and Oyibo 2008, Dubey et al. 2009). Several higher plants and their constituents may be used successfully in plant disease control (Singh et al. 1993). Adityachaudhury (1991) mentioned that use of plant extracts and phytoproducts is gaining attention due to their bio-
2013 Bangladesh Phytopathological Society
degradability, low toxicity and minimum residual toxicity in the ecosystem. Antifungal activities were found in Eucalyptus, Syzygium aromticum, Azadirachta indica, Rosmarinus officinalis against Rhizoctonia solani, R. oryzae, R. oryzae-sativae and Sclrotium hydrophilum (Aye et al. 2011). Cinnamon oil was found efficient plant product that inhibited in vitro colony diameter of R. oryzae-sativae as well as suppression of aggregate sheath spot disease of rice under greenhouse condition. The present piece of research was undertaken to evaluate efficacy of six plant extracts to inihiting invitro vegetative growth and sclerotia germination of R. oryzae-sativae.
MATERIALS AND METHODS
Six locally available plant species namely garlic (Allium sativam), ginger (Zingiber officinales), henna (Lawsonia inermis), water pepper (Poligonum hydropiper), ivy gourd (Coccinia cordifolia) and neem (Azadirachta indica) were collected. Water extracts of garlic clove, ginger rhizome, and leaves of henna, water pepper, ivy gourd and neem were prepared. For preparation of the plant extracts, 100 g of each material was washed in sterile distilled water, 100 ml sterile water was added (1:1 w/v), crushed in a mortar and pestle and passed throught 2-ply cheese cloth. Each plant extract was filtered through filter paper (Whatman no.1), which was considered as standard plant extract of each plant species and tested for antifungal activity.
Poison food technique was followed to test the plant extracts using potato dextrose agar (PDA) as the basic medium (Dhingra and Sinclair 1985, Ali and Archer 2003). The aqueous extracts were thoroughly mixed with warm PDA (40C) at 5, 10, 15, 20 and 25% concentration and poured into sterile Petri plates (90 mm). PDA without any extract served as the control. An isolate (No. MY-1) of R. oryzae-sativae was obtained from Plant Pathology Division, Bangladesh Rice Research Institute (BRRI), Gazipur and multiplied on fresh PDA. Mycelial disc were cut from the actively growing section of 3-day old culture of the pathogen. The disc was placed at the center of each Petri plate containing amended or unamended PDA at one disc per plate. The inoculated plates were incubated at room temperature (25-28C). The plates were arranged on the desks in the Laboratory of Plant Pathology Division, BRRI following completely randomized design. Four replicated plates were used for each treatment. The redial colony diameter was measured in all treatments when the mycelium reached the rim of the Petri plate under control. Percent growth inhibition was calculated based on diameter of colony under control plates.
Inhibition of sclerotia germination was determined by the method of Chaizuckam and Davis (2010). PDA medium was amended with the plant extracts at 0, 5, 10, 15, 20 and 25% following the procedures as mentioned earlier. Ten sclerotia of the pathogen (isolate MY-1) were collected from 14 days old PDA culture, soaked in sterile water for 10 min. Water soaked sclerotia were transferred to amended PDA plates. After tree day of incubation at room temperature number of germinated sclerotia was counted. The experiment was repeated once.
Data on radial mycelial growth were subjected to statistical analysis using CropStat (version 7.2) computer software. Paired-t test was performed to assess the effect of plant extract on sclerotial germination comparing with control.
RESULTS AND DISCUSSION
In-vitro mycelial growth of R. oryzae-sativae in the control plates reached the rim within 4 days of incubation. Every plant extract significantly reduced in–vitro radial colony diameter of the fungus at all concentrations compared to control. The degree of reduction corroborated with their concentrations (Table 1).
Percentage of mycelial growth inhibition at different concentrations of five plant extracts over control is shown in Figure 1. In general, the highest reduction was achieved with garlic extract followed by henna, neem and ginger extracts. The reduction in colony growth under the extracts ranged 67.67-100.00%, 33.39-78.33 and 27.22-70.56%, respect-tively. The lowest reduction of 12.22-32.78% was recorded from treatments with water pepper followed by ivy gourd which showed 15.56-61.67% reduction in colony growth. Irrespective of plant extract, growth inhibition percentage increased gradually with the increase of concentration. The relationship between growth inhibition and concentration was linear and positive. The correlation coefficient for each extract was significant (Fig. 1 and Table 2).
All of the plant extracts caused inhibition of sclerotia germination of R. orya sativae. The most effective one was garlic followed by henna. Significant reduction in sclerotia germination was achieved with all concentrations of two plant extracts compared to control. The least effective plant extract was water pepper followed by Ivy gourd (Table 3).
Results of the present investigation reveal that water extracts of garlic, zinger, henna, ivy gourd and neem posses antifungal activity showing cause of inhibition of in-vitro colony growth of R. oryzae-sativae. The most effective botanical is garlic followed by henna. They can reduce mycelial growth of R. oryzae-sativae at least by more than 60% at their lowest concentration. Garlic at 20% concentration and above concentration is effective for complete inhibition of in-vitro growth. Henna extract is able to reduce mycelia growth of the pathogen by 94.44% at 25% concentration. In-vitro antifungal activity of botanicals tested in the present study have also been reported by many other investigators (Chaijuckam and Devis 2010, Aye et al. 2011, Rahman et al. 2012, Chaity et al. 2012).
The factors responsible for antifungal activity of the botanical tested have not been studied in present investigation. However, other investigators reported that sulfur rich protein Ajoene derived from garlic had antifungal activities against Aspergillus niger, Candida albicans (Yoshida et al.1987). Other workers also showed the presences of antifungal properties in A. sativam (Misra and Dixit 1976, Agarwal 1978). Garlic has already been reported to have antifungal activity against R. oryzae-sativae (Chaijuckam et al. 2010). The sensitivity of fungi and even isolates of the same species to plant extracts may vary. Such as, garlic extract at 5% completely inhibited vegetative growth of California isolates of R. oryzae-sativae (Chaijuckam et al. 2010) but in case of Bangladeshi isolate of R. oryzae-sativae, it required 20% concentration.
In the present experiment R. oryzae-sativae was added in the list of sensitivity to the henna extract. The henna leaf extract caused 65.56% – 94.44% inhibition at 5 – 25% concentrations. Leaf extract of henna completely controlled the growth of Drechslera oryzae, Sclerotium oryzae, S. rolfsii and Rhizoctonia solani at 20% (w/v) concentration. The presence of antifungal compound (2- hydroxyl- 1, 4 napthoquinine) in the leaf extract of henna had been identified which might be responsible for microbial growth inhibition (Tripathi et al. 1978).
Neem leaf extract showed 33.89 – 78.33% inhibition at 5 – 25% concentration. The plant extract inhibited the mycelial growth of R. solani, R. oryzae sativae R. oryzae, and Sclerotium hydrophilum by 87.5, 80.0, 92.5 and 49.2% respectively (Aye and Matsumoto 2011). Similarly, findings of the present investigation showed satisfactory reduction in colony growth on R. oryzae-sativae by using neem leaf extract. In present test, ginger showed moderate inhibition against R. oryzae-sativae. Pakrashi (2003) demonstrated that Ginferenone A, a diarylheptenone constituent of ginger, showed strong antifungal action against Pyricularia oryzae and moderate anticoccidium effect in vitro.
Lower inhibition percentage were noted in this investigation with P. hidropiper and Coccinia cordifolia. Hasan et al. (2009) observed that P. hidropiper root extract on chloroform had strong antifungal activities against A. niger, A. fumigatus, A. flavus, C. albicans, Rhizopus oryzae and Tricophyton rubrum. Garlic and henna extracts significantly decreased sclerotia germination at all concentrations while, neem extract at 20% and 25% reduced sclerotia germination significantly compared to the control. The findings of the present investigation reveal that garlic and henna contain effective properties against R. oryzae-sativae (Table 3).
This work was supported by Plant Pathology Division, Bangladesh Rice Research Institute, Gazipur. First author appreciates the Ph.D. grant by University Grant Commission, Bangladesh.
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