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Title: Efficacy of BAU-Biofungicide, Synthetic Fungicides and Plant Extracts on Rice (Oryza sativa L.) Diseases and Yield 
Authors Name: Hyat Mahmud1* and Ismail Hossain2
Affiliation and Address:1Additional Deputy Director(crop), Department of Agricultural Extension, Kushtia and 2Professor, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh.
Corresponding author: Hyat Mahmud, E-mail: HYPERLINK "mailto:mhyat81@gmail.com1*"mhyat81@gmail.com1*, 
                                        Tel: +8801727182615 
Running Title: Eco-friendly rice disease management













Abstract - Extract of garlic (Allium sativum), Neem (Azadirachta indica); BAU-Biofungicide (Trichoderma harzianum) and Bavistin DF (Carbandazim) and Potent 250 EC (Propiconazole) were tested in laboratory and field conditions for eco-friendly management of diseases of rice (Oryza sativa, L.) cv BRRI dhan28. BAU-Biofungicide (2%) was found to have profound effect in inhibiting the mycelial growth of Bipolaris oryzae (brown spot), Cercospora oryzae (narrow brown leaf spot) and Rhizoctonia solani (sheath blight) in vitro and marked reduction of disease incidence of brown spot, narrow brown leaf spot and sheath blight in the field. It was noted that significantly low incidence of narrow brown leaf spot and sheath blight disease was observed in plots sprayed with Carbendazim (0.1%) in the field as well as mycelial growth inhibition of Cercospora oryzae and Rhizoctonia solani was found in Carbendazim (0.1%) under laboratory condition. Highest (20.20%) grain yield was increased in Propiconazole (0.1%), while (17.84%) higher increase grain was obtained with BAU-Biofungicide (3%). No statistical significant difference was observed between yield of BAU-Biofungicide and Propiconazole. Most of the detected seed borne pathogens were controlled by BAU-Biofungicide and Propiconazole. Maximum germination (%) was observed over control when seeds were treated with BAU-Biofungicide (2%). BAU-Biofungicide (2%) exhibited (40.45%) higher increase in vigour index over control. Results indicated that the greatest antagonistic effect of BAU-Biofungicide was found in reducing disease incidence and increased grain yield as an alternative option of synthetic fungicide. 
Keywords: BAU-Biofungicide; Bipolaris oryzae; BRRI dhan28; Cercospora oryzae; Rhizoctonia solani;; Potent; Seed treatment; Yield; 

Introduction
Rice is the most important cereal food grain for more than two billion people living in the rural and urban areas of humid and subhumid Asia. Rice provides household and national food security and generates employment and incomes for the low-income groups in these areas. Rice is also the staple food crop in Bangladesh. Rice sector contributes to one-half of agricultural Gross Domestic Product and one-sixth of national income in Bangladesh. The population of Bangladesh is still growing by two million people every year and may increase by another 30 million over the next 20 years (BRRI 2011). Thus, Bangladesh will require about 27.26 million tons of rice for the year 2020. During this time total rice area will also shrink to 10.28 million hectare. Rice yield therefore, needs to be increased to 3.74 t/ha (BRRI 2011). The cultivation of rice is increasing day by day, while the yield is comparatively lower. The national average yield of husked rice is 3.07 t/ha (BBS 2017). Approximately 70% of the total cultivated land covering about 11.00 million hectares is under rice cultivation (BBS 2017). The total amount of rice production is 33.80 million tons (BBS 2017) in Bangladesh. The future increase in rice production has to come from the same or even reduced land area and the yield must be greatly enhanced by providing additional nutrient input and through effective control of phytopathogens (Sivasakthi et al. 2013). In a study conducted by the International Rice Research Institute (IRRI), average farmers lose 37% of their rice yield to pests and diseases were reported (Sparks et al. 2012). Losses have been estimated about 10-15% due to rice diseases (Kandhari 2005).
Ten of the 32 diseases of rice have the potentiality to cause economic damage and considered as major in Bangladesh (Haq et al. 2008). Brown spot, sheath blight, sheath rot, bacterial leaf blight, blast, ufra, bakanae, bacterial leaf streak, tungro and false smut are the major constraints for rice production in Bangladesh. In particular, brown spot disease caused by Bipolaris oryzae, that affects both yield and grain quality (Savary et al. 2000), and caused loss from 12 to 45% yield (IRRI 2012), caused in 1943 the Bengal famine (Padamanabhan 1973) and yield loss upto 90% in certain areas (Gangopadhyay and Padamanabhan 1987). Application of fungicides for the control of brown spot is the most effective management option, but under high disease pressure effective control is difficult to achieve (Lore et al. 2007). Narrow brown leaf spot (C. oryzae) affects rice at various crop growth stages, from seedling to panicle formation but is most severe at heading. It causes a premature ripening of grains, reduces yield quantity and grain milling quality (Groth and Hollier 2010), and yield losses more than 40% (Texas, 2011). Sheath blight is the most common and economically important rice field disease and caused 10-30% yield loss (Xie et al. 2008) and may reach up to 50% (Kumar et al. 2011). Good quality seed possess major characteristics such as high yielding potentiality, viability, purity, free from varietal mixtures and infection by pathogens (Fakir and Mia 2004). Twenty seven of the 43 diseases are reported to seed-borne in Bangladesh. Seed borne diseases caused by, brown spot (Bipolaris oryzae), blast (Pyricularia grisea), sheath rot (Sarocladium oryzae), seed rot and seedling blight (Bipolaris oryzae, Sclerotium rolfsii and Fusarium spp.), grain spots (Bipolaris oryzae, Curvularia lunata, Nigrospora oryzae, Phoma glumarum and Cladosporiumsp.); bacterial leaf blight (Xanthomonas oryzaepv. oryzae), bacterial leaf streak (Xanthomonas oryzae pv. oryzicola), and white tip (Aphelenochoides besseyi) are inflicting diseases in seed bed as well as in field (Fakir 2004).
Persistent, indiscriminate and injudicious use of chemicals has toxic effects on non-target organisms and can cause undesirable impact on environment and human health. These chemicals are too expensive. Large-scale and long-term use of resistant cultivars exerts strongly selection pressure that may result in significant shifts in the virulence characteristics of pathogens, as a result resistance breakdown. Research is still concentrated on the identification, evaluation and formulation of potential biocontrol agents for deployment and it makes the potential option for rice disease management through the use of biocontrol agent (Abdel-Fattah 2007 and Rasmy 1991). Trichoderma compete for space and nutrients, through the secretion of antibiotic volatiles and/or diffusible metabolites, which modify promoting growth and plant defense mechanisms. So, mycoparasitism is considered a direct biocontrol mechanism (Howell 2003 and Benitez et al. 2004). Plant extracts and biofungicide play a vital role to treat seed (Koike et al. 2011). Treating of seeds with BAU-Biofungicide (a Trichoderma based preparation) resulted strong barrier on the seed surface to protect the seed from seed borne pathogens as well as it protects the seed from soil borne pathogens when treated seeds are sown in the field or in the bed (Hossain 2011).The aim of the present study is to test plant extracts and biocontrol agents as alternative means for the control major fungal disease in order to increase yield of rice by avoiding environmental pollution.
Materials and Methods
Preparation of plant extracts 
	Healthy leaves of neem (Azadirachta indica) was collected from medicinal garden (botanical garden), Bangladesh Agricultural University, Mymensingh and garlic cloves (Allium sativum) grown in Spices research centre, Gazipur, were collected and the samples were washed thoroughly under running tap water followed by sterile distilled water (SDW). The extracts were prepared by homogenizing 5 g of plant sample in 50 ml of SDW using a blender and the extracts were then prepared at 1% and 2% concentration by dilution with water and kept in conical flasks separately before use.
Use of BAU-Biofungicide and fungicide
BAU-Biofungicide was used @ 2% and 3% in this experiment. BAU-Biofungicide is a Trichoderma harzianum preparation (Hossain 2011). BavistinDF (Carbendazim), and Potent 250EC (Propiconazole) were used @ 0.1% and 0.05% concentrations. 
Field experiments
	Field experiments were conducted with rice cv. BRRI dhan28 during two Boro seasons in two successive years 2012 and 2013. BRRI dhan28 is the first mega variety during this season covering about 40% used as fine rice. The experiments were conducted in the Field Laboratory of the Department of Agronomy, Bangladesh Agricultural University (BAU), Mymensingh. The experiment was carried out by using Randomized Complete Block Design (RCBD) having three replications. The field was fertilized as per recommendation of Bangladesh Rice Research Institute, Gazipur (BRRI 2004). The individual plot size was 10 m2. Block to block and plot to plot distances were 2.0 m and 1.5 m, respectively. Thirty five day old seedlings were uprooted from seed bed and three seedlings per hill were transplanted on January 21 in two successive years 2012 and 2013. Hill to hill and row to row distances were respectively 15 cm and 20 cm, respectively. The spray schedule was started just after commencement of disease symptoms at least 65 days after transplantation and two sprays were followed at 15 days interval (as per label of fungicide). Symptoms have been assessed at 65, 80, 90 days after transplantation. Disease incidence of each plot was assessed following the procedure of Standard Evaluation System for Rice (IRRI 1996). Grain yield and thousand grain weight of each treatment were recorded.
Laboratory experiments
Seed health test was carried out following blotter method to detect seed borne pathogens associated with seed sample (ISTA 1996). Three layers of blotting paper (Whatman filter No.1) soaked in water and were kept at the bottom of a 9.0 cm dia. plastic petri dish and there after 25 seeds were kept on filter paper. Four hundred harvested seeds of each treatment were taken randomly from each sample of each year. The experiment was conducted in the nethouse of the Seed Pathology Centre, BAU, Mymensingh. The petri dishes containing seeds were incubated at 20 � 2 �C under alternating cycles of 12 hours near Ultra Violet light and darkness for 7 days. Incubated seeds were examined under stereo-binocular microscope to record the incidence of different seed borne fungi. Each seed borne infection was recorded and expressed in percentage (Agarwal et al. 1989).


Isolation of Bipolaris oryzae, Cercospora oryzae and Rhizoctonia solani
	Bipolaris oryzae and Cercospora oryzae were isolated from infected leaf and seed collected from the field. Rhizoctonia solani infected sheath of rice plants were used. Isolation of fungi from seed was done following ISTA rules (1996). After 72 hours of incubation, the growths of fungi were studied under stereoscopic microscope. The single conidium of each fungus from the infected leaf piece was transferred to PDA plate for incubation at 25 ( 1 �C for 10-15 days. The 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.
Seed treatment and plantlet vigour
Seeds of rice cv BRRI dhan28 of control plot were treated with each plant extracts, separately over time (witin one hour) by weight basis @ 1% and 2%; BAU-Biofungicide @ 2% and 3%;as well as with Bavistin and Potent @ 0.1% and 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. Formalin-sterilized sand, as described by Dashgupta (1988) was used as substrate for filling plastic trays. The experiment was conducted in Complete Randomized Design with three replications. Three hundred seeds of each treatment were sown in plastic trays (100 seed/plastic tray) maintaining equal distances among the seeds. Randomly 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 Baki and Anderson (1973).
Bioassay of plant extracts, BAU-Biofungicide, Bavistin and Potent on collected fungi
	Potato dextrose agar medium was used. 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 was put into each hole and the plates were stored overnight. Next day, the plates were inoculated at the centre with 6 mm blocks of 15 days old culture of fungus separately and incubated at 24 � 1 0C. Linear mycelial growth of fungi was measured (Nene and Thaplial 1993) and percent inhibition of growth was calculated using formula of Sundar et al. (1995).
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
Evaluation of Disease incidence 
Starting from 80 days after transplanting, all treatments significantly reduced the incidence of brown spot, narrow brown leaf spot and sheath blight with respect to control. Highest reduction (86.82%, 87.65%) in incidence of brown spot disease of rice was recorded at 95 DAT with foliar spray of Potent (0.1%) followed by 86.08, 86.71% with BAU-Biofungicide (2%) in two successive years 2012 and 2013, respectively (Table 1). Maximum reduction in incidence of narrow brown leaf spot disease (78.82% and 77.28%) was observed in BAU-Biofungicide (2%) followed by Potent (0.1%& 0.05%) in 2012 and 2013, respectively. Incidence of sheath blight disease was not found with Potent (0.1%) at 95 DAT in 2012 and 2013, respectively. In this experiment, Potent and BAU-Biofungicide had significant effect in the reduction of the incidence of all the diseases, while Bavistin was found to have good effect in reducing the incidence of diseases. Extract of garlic 2% had good effect in controlling the diseases between two plant extracts. Maximum increase of grain yield (20.20%) over control was found in Potent (0.1%) followed by (17.65%) with BAU-Biofungicide (2%) in 2012. Bavistin and garlic also caused an increase in grain weight and yield (Table 2).
Incidence of seed borne pathogen evaluation
Highest reduction of seed-borne infection of Bipolaris oryzae (69.23%), Curvularia lunata (72.41%), Fusariummoniliforme (60.00%), Sarocladium oryzae (100%) and penicillium sp (77.78%) were recorded in BAU-Biofungicide (2%) over control (Table 3). Trichoderma harzianum was found to remain viable in the harvested seeds of sprayed plot with BAU-Biofungicide as observed with incubated seeds.
Seed quality and plantlet vigour evaluation
Highest increase of seed germination (13.79%), vigour index (40.45%) and normal seedlings (34.14%) obtained from treated seeds were observed with BAU-Biofungicide (2%). Maximum reduction of diseased seedling (72.39%) was found in BAU-Biofungicide (2%) over control. Highest shoot length (20.82%) and root length (25.21%) were observed with BAU-Biofungicide (2%). BAU-Biofungicide (2%) also resulted in the highest shoot weight (50.00 mg) and root weight (38.00mg) (Table 4).
Determination of mycelial growth inhibition 
Lowest (18.0 mm) mycelial growth of B. oryzae was found with BAU-Biofungicide (2%), and minimum mycelial growth of C. oryzae (11.00 mm) and R. solani (22.33 mm) were observed with BAU-Biofungicide (3%) followed by Potent (0.1%) as shown in Fig. 1.
Discussion
BAU-Biofungicide was found as an excellent means of rice disease control. Joshi et al. (2007) evaluated the efficacy of Trichoderma harzianum in talc- and oil-based formulations against brown spot (caused by Helminthosporium oryzae of rice cultivars PR116 and Basmati rice 386 under field conditions. Gomathinayagam et al. (2012) also reported that spraying of spore suspension of T. harzianum significantly reduced the severity of brown spot disease. BAU-Biofungicide (2%) and Tilt 250 EC (0.1%) resulted in significant reduction of narrow brown leaf spot disease as reported by Razu and Hossain (2015). Zhou and Uppala (2015) reported that the fluxappyroxad and propiconazole provided greatest reductions in severity of narrow brown leaf spot, resulting in significant increases in yield under field conditions.This finding was also supported by Groth and Hollier (2010). Costa et al. (2008) reported that rice sheath blight was significantly reduced by application of antagonists. Kandhari (2007) tested seven fungicides and three botanicals at different concentrations against sheath blight (R. solani) of rice cv. Pusa Basmati-1 under field conditions. Bavistin used as standard fungicide at 1000 ppm was the most effective with disease incidence. Abdel-Fattah et al. (2007) reported that four times spraying of 1�108 spore suspension of T. harzianum at 15 days interval significantly increased the grain yield (15.63%) of rice over control under field conditions. Khan and Sinha (2007) also reported that Talc + CMC based formulation of T. harzianum was found to have increasing grain yield (27.3%) over control. Under the present study no significant effect of neem leaf extract (A. indica) was found although garlic clove (A. sativum) showed good effect to increase grain yield which is supported by Razu and Hossain (2015).
Mahmud et al. (2017) who evaluated the efficacy of T. harzianum against B. oryzae and recorded inhibited growth of rice brown spot pathogen. Similar findings was also reported by Khalili et al. (2012). Farid et al. (2002) evaluated four fungicides (Bavistin, Hinosan, Tilt 250 EC and Dithane M-45) against B. oryzae and reported highest mycelial growth inhibition by (95.58%) with Tilt 250 EC at 500 ppm. T. harzianum also inhibited pathogenic fungus C. lunata to a great extent in rice seed as reported by Mahmud and Hossain (2016). These findings were also supported by Jat and Agalave (2013). Sagar et al. (2005) and Ahmad and Mukesh (2002) reported that T. harzianum was effective in reducing seed borne infection of F. moniliforme and increased seedling vigour and seed germination. Maximum inhibition of mycelial growth was observed in hexaconozole at 1000 ppm. Torres et al. (2013) tested antagonic activity of T. harzianum against S. oryzae and observed significant mycelial growth inhibition. Three species of Trichoderma showed antagonistic activity against Penecillium notatum as reported by Patale and Mukadam (2011). These findings were in accordance with the observation of Jat and Agalave (2013). T. harzianum was associated with Bipolaris oryzae infected seeds and when kept for few days for incubation it overgrew B. oryzae. T. harzianum was associated even one year preservation of seeds of BAU-Biofungicide sprayed plot which is reported by Mahmud and Hossain (2016). 
Mahmud and Hossain (2016) reported that 50.07% and 33.76% vigour index were increased over control when rice seeds of BRRI dhan40 variety were treated with BAU-Biofungicide (3%) and Bavistin (0.1%), respectively at 14 days after sowing. Mahmud and Hossain (2017) also reported that highest shoot weight (40.0) mg, maximum root weight (44.3) and highest (90%) of normal seedling were observed when seeds were treated with BAU-Biofungicide (3%). The results can be correlated with the work of by Mahmud et al. (2011) and Hossain et al. (2015). 
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, T. harzianum. The findings were also supported by Manimegalai et al. (2011). Mahmud et al. (2017) tested antagonism ability of endophytic fungi to control C. oryzae in vitro test. They observed the highest 59.03% inhibition of mycelial growth. The efficacy of 78 strains of Trichoderma against R. solani was studied by Naeimi et al. (2010) and recorded superior effect of Trichoderma on R. solani. Mahmud et al. (2017) also reported that T. harzianum reduced R. solani significantly. 	 
In this experiment, BAU-Biofungicide (2%) was found highly effective in reducing the disease incidence of brown spot, narrow brown leaf spot and sheath blight as well as inhibiting mycelial growth of B. oryzae, C. oryzae and R. solani under field conditions and in-vitro test, respectively. Evidently, BAU-Biofungicide (2%) produced highest (99.00%) germination, seedling growth and increased 40.45% vigour index over control of treated seeds. Therefore, BAU-Biofungicide (2%) can be used as foliar application for controlling diseases, seed borne pathogens and increasing grain yield of rice by avoiding environmental pollution. 
Acknowledgments
I would gratefully acknowledge the funding authority, Director of National Agricultural Technology Project, Department of Agricultuaral Extension, Dhaka, Bangladesh.
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