In-vitro Tests to Determine the Efficacy of Plant Extracts , BAU-Biofungicide and Fungicides on the Inhibitory Effects on Some Important Rice Pathogen

Extract of garlic (Allium sativum), Neem (Azadirachta indica), BAU-Biofungicide (Trichoderma based preparation), Bavistin DF (Carbandazim) and Potent 250 EC (Propiconazole) were studied in in-vitro test to evaluate the efficacy of plant extracts, BAU-Biofungicide and fungicides on the inhibitory effects on some selected rice pathogen. BAU-Biofungicide (2 & 3%) showed significant effect in reducing mycelial growth of Bipolaris oryzae, Cercospora oryzae, Rhizoctonia solani and Ustilaginoidea virens. Potent (0.1%) showed excellent effect in controlling mycelial growth of Ustilaginoidea virens, Sarocladium oryzae, Rhizoctonia solani, Bipolaris oryzae and Cercospora oryzae. Bavistin (0.1%) was found to have better result in inhibiting the mycelial growth of Cercospora oryzae, Sarocladium oryzae and Rhizoctonia solani. Highest increase (16.67%) in seed germination was recorded over control when seeds were treated with BAU-Biofungicide (3%). BAU-Biofungicide (3%) also resulted in higher increase vigour index compared to control.


INTRODUCTION
Rice is the staple food crop in Bangladesh.As a rice producer the country is occupying 4 th position in the world (USDA, 2016).It provides nearly 48% of rural employment, about two-third of total calorie supply and about one-half of the total protein intakes of an average person in the country.The cultivation of rice is increasing day by day, while the yield is comparatively lower.The national average yield of husked rice is 3.05 t/ha (BBS, 2016).There are many factors responsible for low yield of rice in Bangladesh.Among these, diseases are considered as major and the productivity yield must be greatly enhanced by providing additional nutrient input and through effective control of phytopathogens (Sivasakthi et al., 2013).
Rice disease management strategies mainly aim at preventing epidemics through the use of host plant resistance and chemical pesticides.The use of chemicals, no doubt, has been found very effective in controlling fungal diseases of plant, but some major problems threaten to limit the continuous use of fungicides.Many researchers tried to find out safe and economical control of plant diseases by using extracts of different plant parts (Akhter et al., 2006;Bdliya and Alkali, 2008).Control of plant disease by biological means instead of using chemicals has drawn special attention all over the world.Out of different biotic stresses which influence the performance of rice crop, brown spot of rice caused by (Bipolaris oryzae) is disease that impairs grain quality and results in about vary widely from 4 to 52% yield losses (Barnwal et al., 2013).Narrow brown leaf spot (Cercospora oryzae) causes a great loss both in the storage and field with reduced the seed viability (Arunyanrat et al., 1981).Rice Sheath blight disease causes 30% yield loss in very susceptible rice fields with artificial inoculation (Groth, 2008) and may reach up to 50% during prevalent years (Meng et al., 2001).Naeimi et al. (2003) reported that sheath rot of rice occurred in most rice growing areas of the world.It usually causes 20-85% yield losses.The rice false smut has already changed from a minor disease to a major disease in all rice-growing countries in Asia since 1970.The highest disease incidence (61.20%) and yield loss (14.18%) were reported (Singh et al., 2012) and yield losses caused by RFS (rice false smut) disease is attributed to both smut balls as well as chaffiness, reduction in grain weight and infertility of the spikelet near the smut balls (Rani, 2014).The rice false smut disease not only reduces yield but also affects grain quality and imposes health hazards significantly in all rice producing areas.Research is still concentrated on the identification, evaluation and formulation of potential biocontrol agents for deployment.Some species of Trichoderma produce antibiotics.Trichoderma hamatum and Trichoderma harzianum produce hydrolytic enzymes (chitinases and glucanases) that attack the hyphae and sclerotia of the pathogen (Gajera et al., 2013).Plant extracts and biofungicide are available to treat seed (Koike et al., 2011).BAU-Biofungicide has its strong potentiality as a seed treating bioagent (Hossain, 2011).It also increases the plant growth and plant stand of rice (Mahmud and Hossain, 2016).The present study has been designed to control major fungal diseases of rice by using plant extracts and biocontrol agent as an alternative option avoiding environmental pollution.

MATERIALS AND METHODS
Isolation: Bipolaris oryzae and Cercospora oryzae were isolated from leaf and seed collected from the rice field.Rhizoctonia solani and Sarocladium oryzae from infected sheath of rice plants and U. virens were isolated from the collected seed following the method of Ming-Xia et al. (2009).Isolation of fungi from seed was done following ISTA rules (1996).The single conidium of each fungi from the infected leaf piece was transferred to PDA plate for incubation at 25  1 °C for 10-15 days.Pure culture of the pathogen was preserved in PDA with the help of hyphal tip culture method aseptically and stored in a refrigerator at 4 °C for further study.

Preparation of plant extracts:
Healthy leaves of neem and garlic cloves were collected and washed thoroughly under running tap water followed by Sterile Distilled Water (SDW).The extracts were prepared by homogenizing plant parts using a blender and prepared at 1 and 2% concentration by dilution with water and kept in conical flasks separately before use.
Bioassay of plant extracts, BAU-Biofungicide, bavistin and potent on collected fungi: Potato dextrose agar medium was prepared and poured into 9 cm Petri plates at 20 mL/plate.After solidification, three 5 mm discs of the medium were scooped from three places maintaining equal distance of 4 cm from the centre using a sterilized disc cutter.One milliliter of each of plant extracts, suspension of BAU-Biofungicide, Bavistin DF and Potent 250 EC were put into each hole and the plates were stored over night.Next day, the plates were inoculated at the center with 6 mm blocks of 15 days old culture of fungi and incubated at 24 ± 1 °C.Each treatment was replicated thrice and only water was used for control treatment.

Collection of data and statistical analysis:
Observation was made regularly to record the mycelial growth.After 4, 6, 8, 10 and 12 days of inoculation, Linear mycelial growth of fungi was measured (Nene and Thaplial, 1993) and percent inhibition of growth was calculated using the following formula as suggested by Sundar et al. (1995): where, X = Mean mycelial growth (radial) of pathogen in control plate Y = Mean mycelial growth (radial) of pathogen in treatment

Seed treatment and vigour index determination:
Three hundred harvested seeds of rice susceptible variety BRRI dhan40 of control plot were treated with each plant extracts, separately over time by weight basis at 1 and 2%, and also treated with BAU-Biofungicide at 2 and 3% and both with Bavistin and Potent at 0.1, 0.05% of seed weight.The experiment was conducted in the nethouse of the Seed Pathology Centre, BAU, Mymensingh.Sand was collected from Brahmaputra river, Mymensingh.Collected sand was sterilized with formalin (40%) at the rate of 5 mL formalin diluted with 20 mL of water for 4 kg sand (Dasgupta, 1988).Formalin treated soil was covered with polythene sheet for 48 h and then exposed for 48 h for aeration before setting experiment.The plastic trays (128) were filled with sand.The experiment was carried out in CRD with three replications.Three hundred seeds of each treatment were sown in plastic trays (100 seeds/tray) maintaining equal distances among the seeds.Plants were watered for maintaining proper moisture.Randomly selected 10 seedlings were uprooted carefully from each tray and washed thoroughly with running tap water.Data was recorded for each treatment at 14 days after sowing on different parameters.Vigour index (VI) was computed using formula of Abdul-Baki and Anderson (1973): Vigor index = (mean shoot length + mean root length) × % germination

Statistical analyses:
All the recorded data on different parameters were analysed statistically using MSTAT-C computer program to find out the significance of variation resulting from experimental treatments.The difference between the treatment means were evaluated for significance using Duncan's Multiple Range Test (DMRT) following the procedure as described by Gomez and Gomez (1984).

RESULTS AND DISCUSSION
Maximum (56.80%) reduction of average radial mycelial growth of B. oryzae was observed in BAU-Biofungicide (2%) followed by Potent (0.1%) over control and highest mycelial growth was recorded in control.Garlic (2%) and neem (2%) were found to have good effect in controlling mycelial growth (Table 1).Khalili et al. (2012) reported that mycelial growth of B. oryzae was reduced by up to 100% by producing volatile and nonvolatile metabolites of antagonistic fungi, such as T. harzianum.These findings were in accordance with the observation of Manimegalai et al. (2011) and Abdel-Fattah et al. (2007).Ahmed et al. (2002) reported that four fungicides viz., Bavistin, Hinosan, Tilt 250 EC and Dithane M-45 were evaluated against B. oryzae.Tilt 250 EC inhibited highest (95.58%) mycelial growth inhibition at concentration of 500 ppm.
Maximum 60.65%, 55.16%, 57.14%, 52.22% and 50.19% reduction of mycelial growth of Rhizoctonia solani were found with Potent (0.1%) at 4, 6, 8, 10 and 12 DAI, respectively over control followed by BAU-Biofungicide (3%) as shown in Table 3. Mayo et al. (2015) evaluated Trichoderma isolates for their potential to antagonize R. solani by dual culture and Trichoderma spp. to over grew the pathogen and found growth inhibition of R. solani between 86 and 58%.The findings were also supported by Naeimi et al. (2010).Rahman (2007) tested that T. harzianum showed linear over growth against R. solani and significantly reduced the pathogen.
In this study no good effect of the extract of Azadirachta indica was observed, while Rajappan et al. (1999) reported that formation of neem oil had significant inhibitory effect against S. oryzae even at the lowest concentration (0.2%) although extracts of neem (2%) and garlic (2%) were found to have good effect in reducing the mycelial growth of C. oryzae.

CONCLUSION
From this study, BAU-Biofungicide (Trichoderma harzianum) may be recommended for treating seeds as biological agent to improve seed health, plant stand as well as controlling mycelial growth of seed borne fungi of rice in-vitro test by avoiding chemicals as an alternative option in controlling diseases of rice.
Besides this, by using of agricultural chemicals in our water, soil and food affect the health, safety and environment.As a result, the use of biological control needs to be emphasized strongly.

Table 1 :
In -vitro evaluation of extracts of Garlic and Neem; BAU-Biofungicide, Bavistin and Potent on radial mycelial growth of Bipolaris oryzae

Table 2 :
, figures having same letter(s) do not differ significantly at 5% level of significance by DMRT; DAI = Days After Inoculation; Data represent the means of three replications; Data in parentheses indicate % growth inhibition over control In-vitro evaluation of extracts of Garlic and Neem; BAU-Biofungicide, Bavistin and Potent on radial mycelial growth of Cercospora oryzae

Table 3 :
, figures having same letter(s) do not differ significantly at 5% level of significance by DMRT; DAI = Days after inoculation; Data represent the means of three replications; Data in parentheses indicate % growth inhibition over control In-vitro evaluation of extracts of Garlic and Neem; BAU-Biofungicide, Bavistin and Potent on radial mycelial growth of Rhizoctonia solani

Table 4 :
, figures having same letter(s) do not differ significantly at 5% level of significance by DMRT; DAI = Days after inoculation; Data represent the means of three replications; Data in parentheses indicate % growth inhibition over control In-vitro evaluation of extracts of Garlic and Neem; BAU-Biofungicide, Bavistin and Potent on radial mycelial growth of Sarocladium oryzae

Table 5 :
, figures having same letter(s) do not differ significantly at 5% level of significance by DMRT; DAI = Days after inoculation; Data represent the means of three replications; Data in parentheses indicate % growth inhibition over control In-vitro evaluation of extracts of Garlic and Neem; BAU-Biofungicide, Bavistin and Potent on radial mycelial growth of Ustilaginoidea virens

Table 6 :
Effect of seed treatments with extracts of Garlic and Neem; BAU-Biofungicide, Bavistin and Potent on germination (%) and vigour index at 14 days after sowing of seeds of cv BRRI dhan40 following Tray method , figures having same letter (s) do not differ significantly at 5% level of significance by DMRT; Data represent t he means of three replications; Data in parentheses indicate % increased (+) and % decreased (-) over control; DAS = Days after sowing