Abstract: For mass production of V. lecanii, three types of cultivation methods including liquid, solid and diphasic production systems were investigated. In the liquid state of production, six media were tested in stationary culture conditions. Among the six media tested, Molasses Yeast Broth (MYB) supported maximum sporulation (8.33x108 spores mL-1) and biomass production (746 mg/100 mL). In the MYB, 4% molasses concentration was found to produce highest spore count (8.56x108 spores mL-1) and biomass (776 mg/100 mL) followed by 5 and 6% molasses. Among the six solid substrates tested, rice grains supported highest spore production (1.14 g/100 g). In diphasic state of production, combination of MYB and rice grains produced the greatest amount of spores, (1.70 g/100 g). Results of this study indicated that diphasic method using MYB as liquid medium and rice as solid substrate are the best method and media for mass production of Vl-7 isolate of V. lecanii.
INTRODUCTION
Verticillium lecanii has been recognized as an enthomopathogen with high potential in biological control of aphids. Many isolates of this fungus demonstrate high pathogenicity to several species of aphids (Askary et al., 1998; Derakhshan et al., 2007; Kim et al., 2007). A successful microbial insecticide should be able to produce high quantities of inoculums (Goettle and Roberts, 1992). The method of culturing largely depends on fungal species and the type of propagule required for formulation and method of application.
The type of growing medium affects conidial production of entomopathogenic fungi (El Damir, 2006; Pandey and Kanaujia, 2006). Variety of substrates have been used for mass production of V. lecanii viz., sorghum, pearl millet and maize (Lakshmi et al., 2001), almond mesocarb (Lopez-Liorca and Carbonell, 1998), rice, rice bran, rice husk and the mixture of these components (Feng et al., 2000), sugar beet molasses (Farsi et al., 2005). The majority of industrial production systems of fungi utilize a two stages system in which fungal inoculum of mycelium or hyphal bodies is produced in liquid culture and transferred to a solid substrate for production of aerial conidia (Vimala Devi, 1994). Despite the many attempts to screen commercially available, low-cost ingredients of industrialized biological pesticides, the research on nutritional requirements of fungal agents were overlooked and a systematic investigation of fungal nutrition utilization is much needed to improve mass production and accelerate commercialization (Sun and Liu, 2006).
In a previous study (Derakhshan et al., 2007) a total of 25 isolates of fungal pathogens consisting of Beauveria bassiana, Metarhizium anisopliae, Verticillium lecanii, Paecilomyces fumosoroseus, Nomuraea rileyi were tested on the cabbage aphid. Among the isolates, V.l-7 isolate of V. lecanii was selected for mass production, as it was found to be the most virulent isolate to B. brassicae. In this study we have evaluated different types of cultivation methods with different liquid and solid substrates for mass culturing of V. lecanii.
MATERIALS AND METHODS
This study was conducted at the Project Directorate of Biological Control (PDBC), Bangalore, India during 2005-2006. Isolate vl-7 of V. lecanii originally isolated from Bemisia tabaci, was obtained from microbial repository of PDBC. For mass production, three types of cultivation methods including liquid, solid and diphasic states were investigated.
Mass production on liquid media: In liquid state production, six liquid media viz., Molasses Yeast Broth (MYB), Potato Dextrose Broth (PDB), Potato Carrot Broth (PCB), Jaggery Yeast Broth (JYB), Sucrose Yeast Broth (SYB) and Potato Sucrose Broth (PSB) were tested. Conical flasks containing 100 mL sterilized liquid media were inoculated with 1 mL of spore suspension (1x107 spores mL-1) with three replications and incubated in a growth chamber at 25 ± 1 °C, 90 ± 3 RH and 12:12 photoperiod for 14 days. To quantify the spore and biomass production, cultures were agitated vigorously and filtered through Whatman No. 2 filter paper. The fungal mat was oven-dried at 40-45 °C until constant weight was achieved. The filtrate was diluted in known quality of water and the total of spores mL-1 was assessed with the help of Naeubauer improved haemocytometer. To determine spore germination, spore suspension (0.1 mL of 1x105 spores mL-1) was spread on PDA Petri plates and incubated at 25 ± 1 °C, 90 ± 3 RH and 12:12 photoperiod. After 24 h, per cent germination was estimated by counting 100 spores for each plate using microscope at 400x magnification. A spore was considered germinated when the germ tube was at least equal to its width. Three plates were used per treatment and each plate served as a replicate.
Effect of molasses concentration on biomass and spore production of V. lecanii: As MYB medium produced maximum biomass and spores, the effect of different concentrations of molasses viz., 1, 2, 3, 4, 5, 6 and 7% were evaluated to determine the optimum concentration of molasses in MYB. Experiment conditions were the same as mentioned earlier.
Mass production on solid substrates: For solid state of production, five cereal grains viz., rice, wheat, maize, Sorghum and ragi were evaluated. Raw grains were taken in autoclavable polypropylene bags (20x28 cm) at 100 g/bag and were soaked with tap water for 12 h and the excess water was drained completely. To each of these bags, 2 g of calcium carbonate and 2 g of calcium sulphate were added and mixed thoroughly to get uniform coating of salts over grains. This process helped in preventing the grain particles sticking together and thereby providing more surface area for the growth of fungus. The bags were then sterilized by autoclaving twice at 121 °C for 20 min. The grain media was inoculated with 1 mL of fungal suspension (1x107 spores mL-1) under aseptic condition. The bags were once again sealed manually and incubated for 14 days at 25 ± 1 °C, 90 ± 3% RH and 12-12 photoperiod for the production of aerial conidia. Three replications were used for each substrate. After 14 days of incubation, grains with fungal growth were dried under aseptic conditions at 30 °C until the moisture content was reduced to 8%. After drying, the spore production of each grain was estimated by Naubauer`s improved haemocytometer. Then, the grains with fungal growth were sieved through a sterile coffee filter with vigorous agitation and the coarse dust thus collected was further sieved through a sterile 105 μ sieve to get fine spore dust. The quantity of spores obtained per 100 g of each substrate was estimated.
Mass production in diphasic system: For diphasic state of production, MYB, PDB and PSB as liquid media and broken rice, broken corn and broken sorghum as solid substrates were tested. One hundred milliliter of each above mentioned broths prepared in 250 mL conical flask and inoculated with 1 mL of fungal suspension (1x107 spores mL-1) in a shaker maintained at 120 rpm for 3 days. Fifteen milliliter of 3 day-old shake cultures inoculated to each of the bag containing 100 g grain and mixed well with the grains. The inoculated bags incubated as mentioned earlier. Production of spore, spore dust and viable spore count (cfu g-1) for each treatment were estimated.
All data were subjected to analysis variance (ANOVA) and the means were separated by using Duncan`s multiple range test (Duncan, 1955).
RESULTS AND DISCUSSION
Liquid media: Biomass and spore production and viable spore count are significantly affected by type of media (Table 1). Among six liquid media tested, MYB produced maximum biomass (746 mg/100) followed by PSB and PCB, 573 and 563 mg/100 mL of medium respectively. Also MYB supported maximum sporulation (8.33x108 spores mL-1) followed by PCB and PSB, 60.50 and 6.30x108 spores mL-1, respectively. The results indicated that MYB medium is a suitable liquid medium as it produced maximum biomass and spores. Sugarcane molasses concentrations in MYB medium had significant
| Table 1: | Effect of different liquid media on biomass, spore production and spore germination |
| Means followed by the similar letter(s) in the columns are not significantly different at 5% by Duncan`s Multiple Range Test (DMRT), *: Non significant | |
effect on biomass and spore production (Table 2). Of the different molasses concentrations tested, 4% molasses supported higher biomass and spore production followed by 5 and 6% molasses.
It was found that there is no significant difference in spore germination of the spores produced in different media although PCB and MYB medium were higher than other media (Table 1). The findings are in accordance with those of Farsi et al. (2005) who reported that among different media for mass production of V. lecanii, sugar beet molasses had the highest rates of sporulation, Tincilley et al. (2004) evaluated different concentration of sugar cane molasses for mass production of Nomuraea rileyi and found that maximum biomass and spore production was observed at 6% molasses.
Solid media: The spore count recorded on rice, sorghum and corn were on par and significantly higher than ragi and wheat. Maximum spore dust was harvested from rice grain followed by corn. It was found that viable spore counts in different grains are not significantly different (Table 3). These finding are in agreement with Nelson et al. (1996) who found that for spore production of Beauveria and Metarhizium, these fungi produced more spores on rice over other growing substrates. Lakshmi et al. (2001) studying the mass culturing of V. lecanii on three grain media found that broken sorghum grain with a spore load of 1.5x109 spores g-1 was the best followed by pearl millet broken grain medium with 1.3x109 spores g-1.
Diphasic system: Combinations of MYB + rice and PSB + rice supported significantly higher spore production than other treatments yielding 2.28 and 2.21x109 spores g-1, respectively. Other combinations were on par. Results indicated that MYB + rice produced highest amount of spore dust and MYB + corn and PCB + sorghum and PCB + corn were on par and poorest media to produce spore dust. Results on viable spore count showed that there were no significant differences among treatments (Table 4). These findings are in accordance with those of Nirmala et al. (2006) who reported that spore production of four isolates of V. lecanii in diphasic system (PDB and rice) after 10 days is ranges from 0.23 to 1.75x109 spores mL-1.
Mass production is an important component of a successful microbial insecticide program. Present results of mass production of V. lecanii revealed that among liquid media, MYB medium with 4% sugar cane molasses and among solid substrates, rice grains and in diphasic system, combination of MYB+rice supported highest
| Table 2: | Effect of different concentrations of molasses on spore and biomass production |
| Means followed by the similar letter(s) in the columns are not significantly different at 5% by DMRT | |
| Table 3: | Effect of different solid media on spore production and spore viability |
| Means followed by the similar letter(s) in the columns are not significantly different at 5% by DMRT.*: Non significant | |
| Table 4: | Effect of media on spore production and spore viability in diphasic system of mass production |
| Means followed by the similar letter(s) in the columns are not significantly different at 5% by DMRT. *: Non significant | |
spore production and resulted in 2.43 and 1.16 times more spores than MYB and rice in liquid and solid states respectively. Such differences can have significant effects on the economical production of V. lecanii for field application.
ACKNOWLEDGMENTS
The first author is grateful to the Project Directorate of Biological Control, Bangalore, India for providing necessary cultures of entomopathogenic fungi and research facilities to conduct the above research.