Efficacy of Substrate Based Trichoderma Harzianum Bio-Fungicides in Controlling Seedling Disease (Fusarium Oxysporum) of Brinjal in Seed Bed

Efficacy of individual and mixed substrates i.e. rice bran, wheat bran, grass pea bran and their combination with mustard oilcake (MOC), based Trichoderma harzianum bio-fungicides were tested against seedling disease (Fusarium oxysporum) of brinjal in plastic tray and seed bed during 2009 to 2014 in the net house of Bangladesh Agricultural Research Institute (BARI). The results of plastic tray and seed bed experiments revealed that four combinations of substrate based T. harzianum bio-fungicides such as (1) rice bran + wheat bran, (2) rice bran + wheat bran + mustard oil cake (MOC), (3) rice bran + grasspea bran, and (4) rice bran + grasspea bran +MOC were suitable for controlling the soil borne seedling disease (F. oxysporum) of brinjal in tray soil as well as seed bed soil conditions. Keyword: Rice bran, wheat bran, grasspea ban, mustard oilcake, Trichoderma harzianum, Fusarium oxysporum, brinjal. Introduction Bangladesh is yet to be self-sufficient in quality food and nutrition which are the major challenges to achieve a healthy and prosperous country like other developing countries of the world. In the country vegetables play a vital role in everyday diet to a huge population in general. Eggplant vernacularly known as brinjal (Solanum melongena L.) is the major and popular vegetable available round the year in Bangladesh. Brinjal grows round the year in the country and provides vegetable to the consumers and stable financial assistance to the growers. The productivity of brinjal in Bangladesh is low (17.5 t/ha) as compared to that of Japan (32 t/ha), Italy (28.2 t/ha), Turkey (30.2 t//ha) and other countries (FAOSTAT, 2012). About 10% crops are lost worldwide annually due to plant diseases which lead to considerable economic discrepancy to the farmers of underdeveloped countries (Strange and Scott 2005). Among the diseases germination failure and seedling mortality caused by the soil borne pathogen Fusarium oxysporum, are the major constraints of brinjal especially in seed bed (Najar et al., 2011). Conventional disease management with fungicide is less effective to control the soil borne F. oxysporum. On the other hand, application of chemical fungicides is expensive and also hazardous to health and environment (Brown and Hendrix 1980, Punja et. al. 1982). The beneficial microbes such as Trichoderma harzianum has been reported as a bio-control agent that effectively controlled the soil borne pathogens (Elad et al., 1983; Roy, 1989). The native bio-control agents usually remain in low population density in most of the agricultural soil, so upscaling of their density to a higher stability level in soil through artificial inoculation is necessary for successful management of soil borne pathogens in brinjal seed bed. The major limitation is the lack of appropriate mass culturing techniques and inadequate information on the suitable substrate materials of T. harzianum (Harman et al., 1991). The T. harzianum has been formulated as bio-fungicides in various substrates like wheat bran, rice bran, maize bran, sawdust (Das et al., 1997); rice straw, chickpea bran, grass pea bran, rice course powder, black gram bran (Shamsuzzaman et al., 2003); cow dung, poultry manure, ground nut shell, black ash, coir waste, spent straw from mushroom bed, talc, vermiculite (Rettinassababady and Ramadoss, 2000); and jaggery, groundnut cake, neem cake, niger cake, Efficacy of Substrate Based Trichoderma Harzianum Bio-Fungicides in Controlling Seedling Disease (Fusarium Oxysporum) of Brinjal in Seed Bed http://www.ijSciences.com Volume 4 – May 2015 (05) 2 pongamia (Shamarao et al., 1998). All of these substrate materials are available in Bangladesh but their potentialities to use in the formulation of T. harzianum bio-fungicide have not yet been studied in the country. Therefore, the present study was undertaken to find out the effective local substrates to formulate the suitable medium for mass culturing of T. harzianum to be used as effective bio-fungicides against F. oxysporum causing germination failure and seedling mortality of brinjal under seed bed condition. Materials and Methods One experiment was conducted in the plastic tray and three experiments were conducted under seed bed conditions in the net house of Plant Pathology Division, Bangladesh Agricultural Research Institute (BARI), Gazipur during the period 2009 to 2014 to find out the suitable substrate material for mass culturing of T. harzianum and thereby formulation of an effective bio-fungicides to control soil borne seedling disease (F. oxysporum) of brinjal. A pure culture of T. harzianum (TM7) was grown in potato dextrose agar (PDA) medium which was used as inoculum of bio-fungicide. Three substrates such as rice bran, wheat bran, grass pea bran and their combination with mustard oilcake (MOC) were used in the experiment for mass multiplication of T. harzianum. Thus the treatments (substrates) of the experiment were T1 = Rice bran, T2 = Wheat bran, T3= Grasspea bran, T4= Rice bran + Wheat bran (1:1), T5 = Rice bran + Grasspea bran (1:1), T6 = Rice bran + Mustard oilcake (1:1), T7 = Rice bran + Wheat bran + MOC (1:1:1), T8 = Rice bran + Grasspea bran + MOC (1:1:1), T9 = Wheat bran + Grasspea bran + MOC (1:1:1), T10 = Rice bran + Wheat bran + Grass pea bran+ MOC(1:1:1:1), T11 = Seed treatment with Provax and T12 = Untreated control. According to the treatment combinations 600 g of individual or combination of substrate materials were taken separately in 1000 ml Erlenmeyer flask. The flask with substrate materials were sterilized in an autoclave at 121C for 15 minutes and cooled down to make it ready for inoculation. The sterilized substrate was inoculated individually with 5 mm diameter mycelia disc of five-day old culture of T. harzianum grown on PDA and then incubated at room temperature (25±2 C) for 15 days. After incubation the colonized substrates were removed from the flasks and air dried and finally preserved in refrigerator at 10 C. The tray soils as well as seed bed soils were inoculated with Fusarium oxysporum to make it sick soil. The inoculum of T. harzianum, colonized on different substrates, were incorporated with the F. oxysporum sick soils @100 g/m soil and kept for 7 days with proper soil moisture to establish T. harzianum in the soils. The untreated control did not receive any colonized substrate of T. harzianum except the inoculum of F. oxysporum. The seeds of BARI Bagun-7 were sown both in the plastic tray and seed bed experiments using 200 seeds per treatment. The experiments were laid out in completely randomized design (CRD) with four replications. Proper weeding, irrigation and intercultural operations were done in time to raise the brinjal seedlings. Data were collected on percent seedling emergence, seedling mortality, height and weight of shoot, length and weight of root of brinjal seedlings. The data were analyzed statistically by using the MSTATC program. The treatment effects were compared by applying the least significant different (LSD) test at p=0.05 level. Results and Discussions A. Efficacy of different substrate based T. harzianum bio-fungicides in the plastic tray


Introduction
Bangladesh is yet to be self-sufficient in quality food and nutrition which are the major challenges to achieve a healthy and prosperous country like other developing countries of the world. In the country vegetables play a vital role in everyday diet to a huge population in general. Eggplant vernacularly known as brinjal (Solanum melongena L.) is the major and popular vegetable available round the year in Bangladesh. Brinjal grows round the year in the country and provides vegetable to the consumers and stable financial assistance to the growers. The productivity of brinjal in Bangladesh is low (17.5 t/ha) as compared to that of Japan (32 t/ha), Italy (28.2 t/ha), Turkey (30.2 t//ha) and other countries (FAOSTAT, 2012). About 10% crops are lost worldwide annually due to plant diseases which lead to considerable economic discrepancy to the farmers of underdeveloped countries (Strange and Scott 2005). Among the diseases germination failure and seedling mortality caused by the soil borne pathogen Fusarium oxysporum, are the major constraints of brinjal especially in seed bed (Najar et al., 2011). Conventional disease management with fungicide is less effective to control the soil borne F. oxysporum.
On the other hand, application of chemical fungicides is expensive and also hazardous to health and environment (Brown andHendrix 1980, Punja et. al. 1982). The beneficial microbes such as Trichoderma harzianum has been reported as a bio-control agent that effectively controlled the soil borne pathogens (Elad et al., 1983;Roy, 1989). The native bio-control agents usually remain in low population density in most of the agricultural soil, so up-scaling of their density to a higher stability level in soil through artificial inoculation is necessary for successful management of soil borne pathogens in brinjal seed bed. The major limitation is the lack of appropriate mass culturing techniques and inadequate information on the suitable substrate materials of T. harzianum (Harman et al., 1991). The T. harzianum has been formulated as bio-fungicides in various substrates like wheat bran, rice bran, maize bran, sawdust (Das et al., 1997); rice straw, chickpea bran, grass pea bran, rice course powder, black gram bran (Shamsuzzaman et al., 2003); cow dung, poultry manure, ground nut shell, black ash, coir waste, spent straw from mushroom bed, talc, vermiculite (Rettinassababady and Ramadoss, 2000); and jaggery, groundnut cake, neem cake, niger cake, pongamia (Shamarao et al., 1998). All of these substrate materials are available in Bangladesh but their potentialities to use in the formulation of T. harzianum bio-fungicide have not yet been studied in the country. Therefore, the present study was undertaken to find out the effective local substrates to formulate the suitable medium for mass culturing of T. harzianum to be used as effective bio-fungicides against F. oxysporum causing germination failure and seedling mortality of brinjal under seed bed condition.

Materials and Methods
One experiment was conducted in the plastic tray and three experiments were conducted under seed bed conditions in the net house of Plant Pathology Division, Bangladesh Agricultural Research Institute (BARI), Gazipur during the period 2009 to 2014 to find out the suitable substrate material for mass culturing of T. harzianum and thereby formulation of an effective bio-fungicides to control soil borne seedling disease (F. oxysporum) of brinjal.
According to the treatment combinations 600 g of individual or combination of substrate materials were taken separately in 1000 ml Erlenmeyer flask. The flask with substrate materials were sterilized in an autoclave at 121 0 C for 15 minutes and cooled down to make it ready for inoculation. The sterilized substrate was inoculated individually with 5 mm diameter mycelia disc of five-day old culture of T. harzianum grown on PDA and then incubated at room temperature (25±2 0 C) for 15 days. After incubation the colonized substrates were removed from the flasks and air dried and finally preserved in refrigerator at 10 0 C. The tray soils as well as seed bed soils were inoculated with Fusarium oxysporum to make it sick soil. The inoculum of T. harzianum, colonized on different substrates, were incorporated with the F. oxysporum sick soils @100 g/m 2 soil and kept for 7 days with proper soil moisture to establish T. harzianum in the soils. The untreated control did not receive any colonized substrate of T. harzianum except the inoculum of F. oxysporum.
The seeds of BARI Bagun-7 were sown both in the plastic tray and seed bed experiments using 200 seeds per treatment. The experiments were laid out in completely randomized design (CRD) with four replications. Proper weeding, irrigation and intercultural operations were done in time to raise the brinjal seedlings. Data were collected on percent seedling emergence, seedling mortality, height and weight of shoot, length and weight of root of brinjal seedlings. The data were analyzed statistically by using the MSTATC program. The treatment effects were compared by applying the least significant different (LSD) test at p=0.05 level.

Results and Discussions A. Efficacy of different substrate based T. harzianum bio-fungicides in the plastic tray experiment
The efficacy of T. harzianum bio-fungicides against F. oxysporum causing germination failure and mortality of brinjal seedlings was tested in a plastic tray experiment where the soil was artificially inoculated with the pathogen F. oxysporum and then treated with the different substrates based T. harzianum bio-fungicides. Percent seedling emergence as well as seedling mortality was varied among the substrate based T. harzianum biofungicides (Table1). The seedling emergence of brinjal was ranged from 82% (wheat bran) to 98% (rice bran + grasspea bran) due to T. harzianum biofungicide treatments whereas untreated control tray gave 75% seedling emergence. The amount of both pre-emergence as well as post-emergence seedling mortality of brinjal varied among the T. harzianum bio-fungicides resulting total seedling mortality of 10% to 31%. The highest total seedling mortality (51%) was recorded from the untreated control tray. Thus soil treatment with T. harzianum reduced seedling mortality up to 20% as compared to untreated control (Table 1). The result showed that T. harzianum treated tray soil gave higher amount of healthy seedlings (69-90%) while untreated control tray soil produced only 49% healthy seedling. Considering the overall performance of biofungicides with respect to reduction of seedling disease and thereby producing healthy seedlings of brinjal under F. oxysporum sick soils, the rice bran + grasspea bran, rice bran + wheat bran, rice bran + wheat bran MOC, grasspea bran and rice bran based T. harzianum bio-fungicides were seemed to be better ones (Table 1).

B. Efficacy of different substrate based T.
harzianum bio-fungicides in the seedbed experiment a) Emergence and pre-emergence mortality of brinjal seedling Seedling emergence of brinjal was enhanced in all the three years due to application of different substrate material based T. harzianum bio-fungicide in the F. oxysporum sick soils of seed bed. The seedling emergence varied from 53-62% among the bio-fungicide treated beds whereas untreated control bed gave comparatively low emergence (52%) of brinjal seedling in 1 st year trial (Table 2). Similarly, the bio-fungicides gave higher seedling emergence in 2 nd year (85-89%) and 3 rd year (58-70%) while control seed beds showed 70% and 50% seedling emergence, respectively.
The T. harzianum bio-fungicide treated seed beds showed lower pre-emergence mortality of brinjal seedlings over untreated control beds in all the years ( Table 2). The percentage of pre-emergence seedling mortality was lower among the bio-fungicide treated beds in 1 st year (38-47%), 2 nd year (11-15%) and also in 3 rd year (30-42%). Besides, the pre-emergence seedling mortality of brinjal in control beds was 48, 30, and 50 percent, respectively in the consecutive three years. The results indicated that the effect of single and mixed substrate based T. harzianum biofungicides were almost similar among themselves in respect of seedling emergence as well as preemergence seedling mortality of brinjal in F. oxysporum sick soils in seed bed.

b) Post-emergence mortality of brinjal seedling
Post-emergence mortality of brinjal seedling in F. oxysporum sick soils in seed bed was sharply reduced in all the years by the different substrate based T. harzianum bio-fungicides and Provax as compared to the untreated control. The seedling mortality in 1 st year trial was 18% in the untreated control bed while it ranged from 6 to 8% among the bio-fungicides treated seed beds (Table 3). As compared to untreated control bed, the reduction of seedling mortality was ranged from 57 to 68% among the biofungicide treated beds. In the 2 nd year trial, 11 to 14% seedling mortality was observed in the bio-fungicides treated seed beds where control bed showed 35% seedling mortality. Thus the seedling mortality was reduced from 65 to 69% by the application of different bio-fungicides. The reduction of seedling mortality in the bio-fungicides treated seed beds and untreated control bed in the 3 rd year trial were also showed similar trend of results. The Provax treated seed bed showed similar reduction of seedling mortality as observed in the bio-fungicides treated beds in all the years. The individual and mixed substrate material based T. harzianum bio-fungicides were equally effective on the post-emergence mortality of brinjal seedling in F. oxysporum sick soils of seed beds.

c) Shoot growth of brinjal seedling
The shoot growth of brinjal seedling was significantly accelerated by the application of various substrate based T. harzianum bio-fungicides and Provax in F. oxysporum sick soils of seed bed in all the years. In the 1 st year trial, the shoot length of brinjal seedling was 15.9 to 17.3 cm among the T. harzianum bio-fungicide treated beds whereas it was 13 cm in the untreated control seed bed (Table 4). Higher shoot length of brinjal seedling in 2 nd year trial, ranging from 8.07 to 10.1 cm, was recorded from the bio-fungicides treatments and lowest length of 5.67 cm was observed in control. Similarly, longer shoots (7.93 to 10.87 cm) was recorded from the biofungicides treated seed beds in the 3 rd trial, shortest shoot (6.27 cm) was in the control bed.
The shoot weight of brinjal seedling was significantly increased by the T. harzianum biofungicides over untreated control bed. The individual shoot weight was increased up to 4.63 g by the application of T. harzianum bio-fungicide while shoot weight was 2.99 g in control bed of the 1 st year trial ( Table 4). The bio-fungicide treated beds gave accelerated shoot weights of 6.17 to 8.8 g in the 2 nd year and 6.37 to 7.8 g in the 3 rd year trial when the untreated beds gave 3.87 and 3.6 g shoot weights, respectively. The inferior shoot growth was noticed in the Provax treated beds as compared to untreated control beds in all the years. The results indicated that different substrate based T. harzianum biofungicides possessed the ability to enhance shoot growth of brinjal seedling in addition to seedling disease reduction caused by F. oxysporum under seed bed conditions.

d) Root growth of brinjal seedling
The root growth of brinjal seedling was accelerated significantly by different substrate based T. harzianum bio-fungicides as compared to the untreated control in F. oxysporum sick soils of seed beds. In the 1 st year experiment, the root length of brinjal seedling was ranged from 6.27 cm to 6.97 cm among the bio-fungicides treated beds which was minimum (4.63 cm) in the untreated control bed (Table 5). Similarly, the root length varied from 6.50 cm to 8.60 cm in the 2 nd year and 6.33 cm to 8.80 cm in the 3 rd year experiment due T. harzianum biofungicides. Minimum root lengths of 4.20 cm and 3.73 cm were recorded from the untreated control beds in the 2 nd and 3 rd year experiments, respectively. Seed treatment with Provax also gave comparatively inferior root length in all the years.
The root weight of individual brinjal seedling was enhanced significantly by different substrate based T. harzianum bio-fungicides while inferior root weights were recorded from the Provax treated as well as untreated control seed beds in all the years (Table 5).
The root weights of individual brinjal seedling grown in bio-fungicide treated seed beds was varied from 385 to 410 mg, 410 to 480 mg and 550 to 700 mg during 1 st year, 2 nd year and 3 rd year trials, respectively. Inferior root weights of brinjal seedling were recorded from the Provax (370-510 mg) and untreated control beds (330-450 mg) in all the years.
The results of consecutive three years experiments revealed that both individual as well as mixed substrate based T. harzianum bio-fungicides effectively reduced the seedling mortality caused by F. oxysporum and also enhanced shoot and root growth of brinjal seedling under seed bed conditions.
The results of the present study revealed that rice bran and wheat bran based T. harzianum biofungicide gave better seed germination, reduced seedling mortality and increased vegetative growth of brinjal seedling in seed bed. Similar observation with wheat and rice bran for the formulation of T. harzianum bio-fungicide was reported by Sangeetha et at. (1993). Enhanced seed germination due to Trichoderma species was also reported by Mukhtar (2008). It was noticed by Tjamos et al. (1992) that T. harzianum controls F. oxysporum by competing for both rhizosphere colonization and nutrients. Disease incidence of brinjal, water melon and cotton was reported to be reduced considerably by the application of T harzianum (Sivan and Chet, 1986). Shoresh et al. (2005) stated that Trichoderma spp. were effective bio-control agents for a number of soil borne plant pathogens.
In this trial there was significant increase in emergence, shoot and root growth of brinjal seedling due to T. harzianum bio-fungicides which was supported by the findings of many investigators (Prasad and Anes, 2008;Mishra and Sinha, 2000;Chaur-Tsuen and Chien-Yih, 2002). Harman, (2006) and Manju and Mall, (2008) also reported positive role of Trichoderma species in increasing plant growth and productivity. Enhanced the root growth of brinjal seedling in F. oxysporum sick soils was observed in the present experiment which was supported by the findings of (John et al., 2010). The study confirmed the reports of other researchers regarding the role of T. harzianum to enhanced seedling emergence as well as root and shoot growth of seedlings (Dubey et al., 2007) and thereby increasing the frequency of healthy plants (Rojoa et al., 2007).