Optimization of growth conditions and medium composition for improved conidiation of newly isolated Beauveria bassiana strains

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1 Indian Journal of Experimental Biology Vol. 54, October 2016, pp Optimization of growth conditions and medium composition for improved conidiation of newly isolated Beauveria bassiana strains Sushant Dhar*, Vikas Jindal & VK Gupta Insect Molecular Biology Laboratory, Punjab Agricultural University, Ludhiana , Punjab, India. Received 06 February 2015; revised 07 June 2015 Beauveria bassiana is an entomopathogenic fungus with high potential in controlling insect pests. In this study, we propose optimum cultural conditions and culture media for better growth of various B. bassiana strains. B. bassiana strains achieved their maximum growth during optimal incubation period of seven days. The optimum ph and temperature for maximal growth of B. bassiana strains was found to be 6-7 and 25-30ºC, respectively. All the tested carbon and nitrogen sources supported growth and development of the B. bassiana strains. Starch and peptone as carbon and nitrogen sources supported maximum radial growth ( cm) and conidiospore count in both solid state culture ( conidia/ml) and liquid state culture ( conidia/ml). Strain BbR2 was the fastest growing strain on almost all nutrient sources studied and possessed commendable growth rate and sporulation potential. Wheat bran (WB) and rice bran (RB) in the proportion of 3:1 supported maximum conidiospores yields ( conidia/ml) for strain BbR2 in solid state fermentation conditions. Keywords: Conidiospore, IPM, Pest control, Wheat bran, Rice bran Entomopathogenic fungi are effective natural enemies to insect pests of agricultural importance causing significant mortality. Beauveria bassiana appears to have significant potential in controlling such insect pests for improved agricultural production. Mass production of B. bassiana can be achieved using different methodologies as solid state and liquid state fermentations. Environmental conditions, especially temperature and humidity, play a significant role in the incidence of epizootics of insect pathogens 1. Daoust and Pereira 2 demonstrated that temperature and humidity affect both survival and germination rates in B. bassiana. Medium ph also plays a vital role in growth, development and metabolism of fungi. It can affect growth rate, conidia formation 3,4 germination 5, pigment production 6, inhibition by antimycotics 7 and toxin formation 8. Some studies made with B. bassiana reveal that the carbon sources used for production are closely related with the spore production 9 and also with the spore-type produced 10. In this study, we evaluated the effect of different carbon and nitrogen sources on the radial growth, conidiospore yield of some newly isolated Beauveria *Correspondence: dharsushants@gmail.com bassiana strains (Data on isolation not shown.) Mainly, we optimized the growth conditions on low economic substrates (wheat bran and rice bran) with respect to incubation period ph, temperature for achieving mass production of B. bassiana under solid state fermentation conditions. Material and Methods Beauveria bassiana strains Standard culture of Beauveria bassiana (BbM1) MTCC 2028 was procured from Microbial Type culture collection, CSIR-Institute of Microbial Technology (IMTECH), Chandigarh India. The standard culture of B. bassiana strain MTCC-2028 was originally isolated from sugarcane defoliator Phytoscaphus sp. (Coleoptera: curculionidae) from IISR, Lucknow 11 and is being maintained by Microbial Type Culture Collection, IMTECH, Chandigarh as MTCC Besides, Beauveria related species were isolated from different field soils using selective media 12 for Beauveria species. Effect of incubation period, medium ph and temperature on growth and conidiation of B. bassiana strains The physiological characteristics of B. bassiana strains were studied by testing the ability of the strains to grow in medium with different initial ph, incubation temperatures and different incubation periods by

2 DHAR et al.: OPTIMIZED MEDIUM FOR IMPROVED CONIDIATION OF BEAUVERIA BASSIANA 635 measuring radial growth and conidiospore production by different B. bassiana strains on PDA medium in Petri plates. Effect of ph In order to determine the effect of initial ph on the growth of B. bassiana strains, PDA medium was prepared with different initial ph values such as 4.0, 5.0, 6.0, 7.0 and 8.0 as adjusted using 0.1N HCl or 10% Na 2 CO 3 before solidifying the same with required amount of Agar (1.6%). The sterilized media were poured into petri dishes and allowed to solidify. For each strain and initial medium ph, three plates were single spot inoculated at the centre with 5 μl ( spores/ml) of the standard inoculum of respective fungal strain. All the inoculated plates were incubated at 25 C for 7 days. The radial growth (colony diameter) of individual strains were measured with the help of a centimeter scale and recorded for all individual strains. Effect of temperature Effect of different incubation temperature on the growth of B. bassiana was determined essentially as in case of ph, except that radial growth and conidiospore yield on normal PDA medium were recorded at different incubation temperatures (20, 25, 30, 35 and 40 C). Effect of incubation period Effect of different incubation periods on the growth of B. bassiana was determined essentially as in case of temperature, except that radial growth was recorded in normal PDA medium after different incubation periods (2-7 days) at incubation temperatures of 25 C. Effect of carbon and nitrogen sources on the radial growth of different B. bassiana strains In order to determine the effect of different carbon and nitrogen sources on radial growth by different B. bassiana strains, basal medium (0.2% yeast extract) were developed with various combinations of three different carbon (starch, sucrose and glucose) and nitrogen sources (peptone, urea and potassium nitrate) each in the basal medium. Whereas, carbon sources were used at concentration to provide 0.8 % carbon, the nitrogen sources were used at approximate concentrations to provide a fixed C:N ratio of 5:1. For this purpose, the basal medium with yeast extract and agar (1.6%) was separately amended with respective carbon and nitrogen sources. The ph of the medium was adjusted to 7.0 (with 1N HCl or 1N NaOH). The sterilized media were cooled to room temperature at 37 C and poured into petri plates. For each strain and each carbon-nitrogen combination, three plates were single spot inoculated at the centre with 5 μl ( spores/ml) of the standard inoculum of respective fungal strain. All the inoculated plates were incubated at 25 C. After seven days, the radial growth (colony diameter) of individual strains were measured with the help of a centimeter scale and recorded for all individual strains. Effect of different nitrogen sources on conidiospore count of different B. bassiana strains on agar solidified medium The basal medium containing starch as sole sources of carbon (2 % level, 0.80 % carbon) was supplemented with different nitrogen sources (peptone, potassium nitrate, urea) in concentrations (equivalent to 0.16 % N) to provide a uniform C:N ratio of 5:1. For this purpose, the basal medium with yeast extract and agar (1.6%) was separately amended with respective carbon and nitrogen sources. The ph of the medium was adjusted to 7.0 (with 1N HCl or 1N NaOH). The sterilized media were cooled to room temperature and poured into petri plates. For each strain and each carbon-nitrogen combination, three plates were single spot inoculated at the centre with 5 μl of the standard inoculum of respective fungal strain. All the inoculated plates were incubated at 25 C. Besides, conidiospore production was also determined by suspending agar plug (press cut with a plastic tube having inner diameter of 1.0 cm) in 0.3% Tween-20 solution (by vortexing). The resultant suspension was filtered through four layers of muslin cloth to remove any fungal mycelium and/or suspended particles and its conidiospore count determined with the help of a hemocytometer under a phase contrast microscope (Zeiss). Effect of different nitrogen sources on growth of different B. bassiana strains under liquid state cultures The basal medium containing starch as sole source of carbon (2 % level, 0.80 % carbon) was supplemented with different nitrogen sources (peptone, urea, potassium nitrate) in concentrations (equivalent to 0.16 % N) to provide a uniform C:N ratio of 5:1. The sterilized medium was inoculated with one ml of standard conidial suspension for each B. bassiana strain and incubated under stationary conditions at 25 C. After incubation for seven days, besides some suspended fungal mycelium, mycelial growth also

3 636 INDIAN J EXP BIOL, OCTOBER 2016 appeared in the form of fungal mats at the surface of the medium. Whole of the mycelial biomass for all the strains was filtered through whatman1 filter paper. The fungal biomass on filter paper (oven dried and pre-weighed) was washed two times with distilled water in conical flasks so as to remove any medium component. The mycelial biomass now on filter paper was ovendried (60 C, 24 h) and its weight recorded in an electronic balance. Effect of nitrogen sources on conidiospore count of different B. bassiana strains under liquid state cultures The basal medium containing starch as sole source of carbon (2 % level, 0.80 % carbon) was supplemented with different nitrogen sources (peptone, urea, potassium nitrate) in concentrations (equivalent to 0.16 % N) to provide a uniform C:N ratio of 5:1. The sterilized medium was inoculated with one ml of standard conidial suspension for each B. bassiana strain and incubated under stationary conditions at 25 C. After incubation for 7 days, besides some suspended fungal mycelium, mycelial growth also appeared in the form of fungal mats at the surface of the medium. The mycelium (whole suspension) was filtered through four layers of muslin cloth to remove any fungal mycelium and/or suspended particles and its conidiospore count determined in the filtrate with the help of a hemocytometer under a phase contrast microscope (Zeiss). Standardizing cultural conditions for maximum conidiation by different B. bassiana strains on wheat bran based semisolid medium About 10 g of wheat bran (WB)-rice bran (RB) mixtures in different ratios (1:0, 1:1, 2:1, 3:1) in a 50 ml conical flask was moistened with moistened with 23 ml of aqueous solution of 2 % starch and 1% peptone. This produced SSF (sold state fermentation) media having 70 % moisture with a uniform C:N ratio of 5:1 (optimized level of C and N sources), while ignoring C and N contributions from wheat bran and rice bran. The steam sterilized media (10 psi, 30 min) were inoculated with one ml of standard conidial suspension of each B. bassiana strain and incubated at 25 C. After 7 days of incubation, whole of biomass was thoroughly suspended in 50 ml of sterile aqueous solution of 0.01% Triton-X-100 to release conidiospores. The suspension was filtered through four layers of muslin cloth to remove all fungal mycelium and/ or suspended particles. The residual mass on muslin cloth was washed two times with 25 ml of the Triton X-100 solution and all washings pooled with the filtrate. The conidiospore count in the filtrate was quantified in a hemocytometer for each strain and calculated as conidiospore yield ml -1 of suspension. Conidiospore germination efficiency Germination efficiency of conidiospores is directly related to bioefficiency of entomopathogenic fungal preparations. Agar slide technique was used for studying the rate of conidiospore germination. Petri plates containing three glass slides on a blotting paper disc were autoclaved. One ml liquefied PDA medium was evenly spread as a thin film on each of the glass slides using a micropipette. A 100 μl of fresh conidial suspension (10 7 conidia ml -1 ) prepared from 7 days grown cultures was evenly spread on media coated slides. The inoculated slides were placed back in the respective Petri plates and the blotting paper discs were moistened with sterilized water. The Petri plates were incubated at 25 C. The slides were observed under phase contrast microscope (40X) for count of germinating conidia at every 2 h intervals. A conidium was considered germinated when a distinct germ tube projected from it, and was at least twice the diameter of the conidium 13. Approximately, 300 conidia were scored per replicate for each of the different fungal strains and germination efficiency calculated as per cent of total conidia observed. Results and Discussion Physiological conditions such as medium ph, incubation period and incubation temperature have significant effect on growth of different fungal species. Effect of ph on radial growth of B. bassiana strains Medium ph plays a vital role in growth, development and metabolism of fungi. It can affect growth rates and conidia formation 3,4 germination 5, pigment production 3,4,6, inhibition by antimycotics 7 and toxin formation 7,8. Therefore, the radial growth for all the B. bassiana strains was measured after incubating in solidified PDA medium raised with different initial medium ph. The radial diameter of the fungal colony was taken after 7 days with a scale (Table 1). Results have shown that all the three B. bassiana strains could grow over a wider medium ph range of However, these strains showed maximum radial growth at a medium ph of 6-7, though a ph of

4 DHAR et al.: OPTIMIZED MEDIUM FOR IMPROVED CONIDIATION OF BEAUVERIA BASSIANA 637 Table 1 Effect of medium ph, Incubation period and Temperature on the radial growth of B. bassiana strains in PDA medium [Data is mean±sd of triplicate values each] Parameters ph Incubation period (days) Temperature Levels *Colony diameter was recorded after seven days of growth 7.0 was optimum. Outside of this medium ph range, radial growth was significantly retarded. Strain BbR2 showed maximum radial growth i.e cm and 2.65 cm at this medium ph followed by BbR1 (2.61 cm) and BbR3 (2.46 cm). Whereas, at the optimum ph of 7.0, the standard BbM1 strain showed a maximum radial growth of 2.20 cm, the same in case of BbR1, BbR2 and BbR3 was 2.42, 2.80 and 2.46 being higher than BbM1. This showed that all the three B. bassiana strains isolated in this study possessed a higher growth rate than the standard culture (BbM1) at optimum ph. Different B. bassiana strains have been reported to show a broad ph tolerance range, such as , as well as 10 and above 12. In general, fungal growth is favoured when ph of the medium is around However, B. bassiana strains have a preference for a near neutral ph with many strains growing well in media with neutral to moderately alkaline ph. Obviously, this property of entomopathogenic B. bassiana strains is related to the ph of their natural host (insect) surface, which in case of M. anisopliaeinfected insect cuticle has been reported to rise from about 6.3 to 7.7 at which most of the extracellular enzymes are produced by this fungus 17. In view of the significance of ph in the growth and development of fungi, few investigators have studied the role of ph on the physiology and growth of EPFs (Entomopathogenic Colony diameter (cm)* BbR1 BbR2 BbR3 BbM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± C 1.46 ± ± ± ± C 2.78 ± ± ± ± C 2.72 ± ± ± ± C 1.06 ± ± ± ± C Nil Nil Nil Nil fungi) 12 and the toxin production by B. bassiana 15, biomass production and trehalose content of the fungal propagules 18. The role of ph in the acquisition of the nutrients by the fungal hyphae is well documented phenomenon 19,20. Effect of incubation period The radial growth for all the B. bassiana strains was measured from 2 nd day up to 7 th day to observe the effect of incubation period. After 48 h of incubation, only limited radial/mycelial growth was observed, being 0.90, 0.95, 0.81 and 0.75 cm for BbR1, BbR2, BbR3 and BbM1strains, respectively (Table 1). Maximum radial growth (2.78 cm) was observed at the end of 7 days in strain BbR2 followed by BbR1 (2.35 cm) and BbR3 (2.20 cm). However for strain BbR2, radial growth of 2.45 cm was recorded after 6 days of incubation, which was higher than that of BbR1, BbR3 and BbM1 strains. In comparison, standard strain BbM1 showed least radial growth (2.15 cm) amongst all the strains. Thus, incubation period of 7 days was found to be optimum for the growth of all B. bassiana strains. Effect of incubation temperature In this study, B. bassiana strains were allowed to grow in PDA medium of ph 7.0 and incubated for 7 days at different temperatures from C before measuring the radial growth of developed colonies.

5 638 INDIAN J EXP BIOL, OCTOBER 2016 All B. bassiana strains showed their ability to grow over this wider range of temperatures, but optimally at 25 C and 30 C (Table 1). The growth was particularly affected more at 35 C than 20 C, and none of these strains grew at 40 C. The radial growth of B. bassiana strains was observed least at the temperature of 35 C being 1.06, 0.70, 0.42 and 0.56 cm for BbR1, BbR2, BbR3, and BbM1 strains. At the optimum temperature of 25 C, B. bassiana strain BbR2 showed maximum radial growth (2.80 cm) followed by BbR1 (2.78 cm) and BbR3 (2.29 cm), and in this respect, BbM1 strain (2.04 cm) was poorest in radial growth. This range of temperature is similar to that reported by other researchers 21,22. Environmental factors such as temperature, light radiation and humidity are equally important in determining fungal entomopathogenecity 21. Temperature is an abiotic limiting factor that directly affects the rates of sporulation, germination, mycelial growth and the survival entomopathogenic fungi and continually fluctuates throughout the day 22. Gauging the thermal tolerance range of fungal strains, thus makes up part of the criteria upon which researchers select fungal biological control agents prior to bioassays designed to challenge insect pests during evaluating the ability of entomopathogenic fungi to control insect populations under different temperatures 21,22. Hallsworth and Magan 21 have used growth optima at 25 C during evaluation of strains of B. bassiana against different insect species. The observed optimum temperature between 25 and 30 C for growth of B. bassiana strains is also in agreement with other published reports 9,22,23. However, the optimal range of temperature for production and germination of conidia (15-28 C) is also the range in which in vitro dry conidia lose viability faster Thus, synchronization between germination, faster development of the disease, and production of infective units at moderate temperatures may be an evolutionary strategy for which the fungus will remain dormant until optimum temperature is reached, therefore, aiding in the survival and spread of the fungus. Effect of carbon and nitrogen sources The different carbon and nitrogen sources showed significant differences in mycelial growth of different B. bassiana strains. Analysis of the results showed that all carbon and nitrogen sources supported growth and development of the B. bassiana strains. However, significant differences amongst different B. bassiana strains were observed in radial growth amongst different combinations of carbon and nitrogen sources (Table 2). Maximum radial growth (3 cm) was observed in case of BbR2 strain in medium constituted with starch and peptone as sole sources of carbon and nitrogen, respectively. The above combination of carbon and nitrogen source also proved most favoring for growth of all the three other B. bassiana strains. Though sucrose in combination with peptone also provided good growth of these B. bassiana strains, the same was declined by the presence of potassium nitrate as nitrogen source, irrespective of carbon source. The presence of urea, the third nitrogen source could also support growth of these B. bassiana strains but Table 2 Effect of carbon and nitrogen sources on the radial growth of the B. bassiana strains in PDA medium [Data is mean ± SD of triplicate values each after 7 days] Strains BbR1 BbR2 BbR3 BbM1 Carbon/Nitrogen source *Colony diameter was recorded after seven days of growth Colony diameter (cm)* Peptone Urea Pot. Nitrate Starch 2.54 ± ± ± 0.07 Sucrose 2.21 ± ± ± 0.05 Dextrose 2.15 ± ± ± 0.10 Starch 3.00 ± ± ± 0.06 Sucrose 2.74 ± ± ± 0.13 Dextrose 2.68 ± ± ± 0.15 Starch 2.75 ± ± ± 0.15 Sucrose 2.63 ± ± ± 0.14 Dextrose 2.60 ± ± ± 0.07 Starch 2.13 ± ± ± 0.12 Sucrose 2.02 ± ± ± 0.05 Dextrose 2.00 ± ± ± 0.09

6 DHAR et al.: OPTIMIZED MEDIUM FOR IMPROVED CONIDIATION OF BEAUVERIA BASSIANA 639 the same was highly reduced suggesting the same to be an unsuitable N source for maximum growth. In general, starch-peptone (2.54, 3.00, 2.75, 2.13 cm) followed by sucrose-peptone (2.21, 2.74, 2.63 and 2.02 cm) were found to be the best C-N combinations for efficient growth of BbR1, BbR2, BbR3 and BbM1strains, respectively. Amongst the B. bassiana strains, maximum growth was observed in the starchpeptone combination, the same was highest in case of BbR2 (3 cm) followed by BbR3 (2.75 cm) and BbR1 (2.54 cm) with the standard BbM1 strain showing least growth of 2.13 cm only. The results established that whereas starch-peptone is the best C-N combination for maximum growth of these B. bassiana strains under study, and BbR2 is the relatively fastest growing strain. The observed variability in carbon requirements among different Beauveria species and different B. bassiana strains has been variously reported, which establish the maximum suitability of glucose, starch and sucrose for growth and sporulation of B. bassiana 27. Radial growth of fungal strains has also been reported to vary not only with fungal species and strains but also with the C:N ratio of the media used for production of conidia 28. Effect of nitrogen sources on the conidiospore yield of B. bassiana strains The different nitrogen sources showed significant differences (P 0.05) in conidiospore yields by different B. bassiana strains. The results (Fig. 1a) revealed that peptone was most suitable nitrogen source yielding maximum mean conidiospore count ( conidia/ml) that was significantly higher than the corresponding yields with potassium nitrate ( conidia/ml) and urea ( conidia/ml) for different B. bassiana strains i.e. BbR1, BbR2, BbR3 and BbM1. The medium containing starch as sole carbon source at 2.0% level (0.80% carbon) was supplemented with different nitrogen sources (equivalent to 0.16% N) to provide a uniform C:N ratio of 5:1. The same combination of starch with peptone yielded in maximum radial growth of different B. bassiana strains. This suggested that the conidiospore yield by a specific B. bassiana strain is directly related to its growth. However, amongst the B. bassiana strains, BbR1 strain was most potent in maximum conidiospore yield ( conidia/ ml) followed by BbR2 ( conidia/ml) though the yields were significantly lowered. Standard strain BbM1 showed least conidial yields among all the B. bassiana strains. For all the treatments used, SP (starch and peptone) showed maximum mean conidial count ( conidia/ml, mean conidial count of strains BbR1, BbR2, BbR3 and BbM1). The above results hold significance as the production of conidia and their viability is dependent upon the culture medium with carbon and nitrogen Fig. 1 Effect of nitrogen sources in (a) solidified medium; and (b) liquid medium on the conidiospore yields by different B. bassiana strains. [Data is mean of triplicate values each after 7 days]

7 640 INDIAN J EXP BIOL, OCTOBER 2016 sources playing an important role in determining the quantity and the quality of conidia in terms of spore germination and infection 29. Effect of nitrogen sources on growth of B. bassiana strains in liquid cultures Different nitrogen sources showed significant differences (P 0.05) on mycelial growth of different B. bassiana strains. Peptone (1.137 g) followed by potassium nitrate (0.995 g) was found to be the best nitrogen source supporting maximum mycelial growth in case of BbR1 strain amongst all B. bassiana strains (Fig. 2). In this respect, mycelial growth in case BbR2 (0.875 g), BbR3 (0.873 g) and BbM1 (0.630 g) strains remained at par with each other, but significantly lower than growth in case of BbR1. In comparison, whereas potassium nitrate could support only non-significantly lower growth, the same in case of all the strains was significantly reduced. The results suggested that peptone followed by potassium nitrate is the most suitable nitrogen sources for growth of all the B. bassiana strains in liquid cultures. These observations are in accordance with the observations on effect of carbon and nitrogen on radial growth of different B. bassiana strains. Liu and Holdom 30 also reported that potassium nitrate supports better mycelium growth and sporulation of Beauveria bassiana. Fig. 2 Effect of nitrogen sources on the mycelial growth of the different B. bassiana strains in liquid cultures. [Data is mean of triplicate values each after 7 days. The medium containing starch as sole source of carbon (2 % level, 0.80 % carbon) was supplemented with different nitrogen sources in concentrations (equivalent to 0.16 % N) to provide a uniform C:N ratio of 5:1] Effect of nitrogen sources on the conidial yields by B. bassiana strains in liquid state In order to determine the effect of different nitrogen sources on conidiospore count in liquid medium, the conidiospore counts in the culture filtrates from the previous experiments were determined and results on the same are given in Fig. 1b. Presence of different nitrogen sources in the medium resulted in significant differences (P 0.05) in the conidiospore yields by different B. bassiana strains. The maximum conidial count was observed in BbR1 ( conidia/ml) with peptone as nitrogen source, which was significantly higher than the conidiospore yield with strains using potassium nitrate and urea as nitrogen sources. The mean conidial count over different treatments with all the B. bassiana strains was highest for peptone ( conidia/ml) followed by potassium nitrate ( conidia/ml and urea ( conidia/ml). Peptone and potassium nitrate as nitrogen sources were evaluated to be the best treatments over the strains and significantly superior than other treatments. Petalmul and Prasertsan 29,31 have also reported high concentration of B. bassiana spores in medium supplemented with potassium nitrate and peptone in liquid and solid state cultures. Standardizing cultural conditions of B. bassiana strains for maximum conidiation in wheat bran: rice bran based semisolid media Effect of different concentrations of wheat bran and rice bran based media on conidiospore production by different B. bassiana strains was studied as explained in M & M section. Availability of different mixtures of wheat bran and rice bran resulted in significant differences (P 0.05) in the conidiospore yield of B. bassiana strains (Fig. 3). The entire WB:RB combinations supported conidiospore production by all the four B. bassiana strains including BbM1. The mean conidiospore yield for different WB:RB ratios suggested increasing yields with progressively increase in WB:RB contents from 1:1 ( /ml) to 3:1 ( /ml) irrespective of the fungal strain. Thus, WB:RB::3:1 medium supported maximum conidiospore count ( conidia/ml) by strain BbR2, which was statistically at par with strain BbR1 ( conidia/ml) at 3:1 proportion and significantly superior over other strains. For different ratios of WB:RB used, highest mean conidial count ( conidia/ml) was observed for 3:1(WB:RB conc.) followed by 2:1 ( conidia/ml), 1:1( conidia/ml) and 1:0 ( conidia/ml).

8 DHAR et al.: OPTIMIZED MEDIUM FOR IMPROVED CONIDIATION OF BEAUVERIA BASSIANA 641 Fig. 3 Effect of relative concentrations of wheat bran and rice bran based media on conidiospore production by different B. bassiana strains under semi-solid medium fermentation conditions. [Each figure represents an average of 3 replicates at different conc. of wheat bran and rice bran. About 10 g of wheat bran (WB)-rice bran (RB) mixture was moistened with 23.0 ml of aqueous solution of 2.0% starch and 1% peptone to provide a uniform C:N ratio of 5:1 while ignoring C and N contributions from WB and RB] Surprisingly, as against the increased conidiospore yields with increase in WB contents, WB alone (WB:RB::1:0) supported lowest yields of conidiospores irrespective of fungal strain. However, above results could be viewed in terms of the relative texture and nutrient contents of WB (coarse, amorphous, high fibre, low proteins) and RB (fine sticky, low fibre, high protein) with WB allowing better penetration of air into medium. Thus, whereas sufficient aeration could support better growth of the fungus in amorphous WB medium, the reduced conidiospore yield was most likely due to lack of additional nutrients. Parallel to above, the presence of high levels of RB (50% in WB: RB: 1:1) also proved limiting due to its interference with appropriate availability of aeration in this medium. This possibility is supported by a parallel increase in conidiospore yields with a progressive decrease in RB contents as WB:RB ratios increased from 1:1 (50% RB) to 3:1 (25% RB). It suggested that though WB alone could not support active growth and hence sporulation, the same was enhanced by presence of RB in lower amount that are just sufficient to meet optimal growth and hence sporulation of these B. bassiana strains. Alternatively, higher levels of RB might prove inhibitory due to its interference with appropriate aeration and oxygen availability for Fig. 4 Conidiospore germination efficiency of B. bassiana strains. [Each value represents the average (±SD) of three replicates. Under each replicate 300 conidiospores were observed for germination. There was no germination during initial 2 h] growth and penetration of growing mycelia into substrate. A direct relation of oxygen availability and conidiospore yields by B. bassiana on agro-industrial wastes (refused potatoes, coffee husks and sugarcane bagasse) has already been established by Santa et al. 32. Conidiospore germination efficiency of B. bassiana strains Conidial germination is an important factor in pest management with entomopathogenic fungi, because the beginning of epizootics is conditioned to the capacity of these structures to germinate on the host insect. It has been reported that nutrient source is an integral determinant of growth and virulence of entomopathogenic fungi 31. Temperature and humidity affect both survival and germination rates in B. bassiana. Also, B. bassiana conidia have been shown to germinate even at a low humidity of 2:92% RH 25. This study was performed at optimal temperature of 25 C and adequate moisture in enclosed Petri plates through moist filter papers. A conidium was considered germinated when a distinct germ tube projected from it, and was at least twice the diameter of the conidium 13. The results demonstrated that the conidiospore did not germinate during first two hours (Fig. 4). First germination was observed only after 4 h when it ranged between (BbR3) and (BbR1). The conidiospore germination improved after this period when it increased progressively with the increase in incubation period up to 8 h reaching between 81.00% (BbR3) and 92.33% (BbR1). All

9 642 INDIAN J EXP BIOL, OCTOBER 2016 the strains showed unipolar germination type of conidiospores. However, subsequently it improved after 8 h when it was maximum with BbR1 (92.33%) followed by BbR2 (88.33%), BbM1 (83.00%) and BbR3 (81.00%). In further extended incubations, the conidiospore germination could not be quantified as extensive mycelial growth of previously germinated conidiospores interfered with the observations on new germinations. In relative terms, the newly isolated strains particularly BbR1 and BbR2 expressed highest germination potential suggesting their better utility as potential biocontrol agents. Investigations under present study resulted in isolation of three B. bassiana strains from the soils of Punjab. These were characterized with respect to physiological conditions and nutritional parameters for optimum growth and conidiospore production in microbiological media. Optimized wheat bran (WB) and rice bran (RB) based mixed medium and cultivation condition have been optimized for conidiospore production in SSF conditions. This study assessed the effects of culture medium ph, carbon sources, nitrogen sources, C:N ratios and medium supplements on the sporulation of Beauveria bassiana and evaluated the best conditions for growth and development of B. bassiana strains. Acknowledgement This work was supported by the Department of Entomology, Punjab Agricultural University, Ludhiana. References 1 Benz G, Environment, In: Epizootiology of Insect Diseases. (Eds. Fuxa & Y Tanada; Wiley, New York), 1987, Daoust RA & Pereira RM, Stability of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae on beetle-attracting tubers and cowpea foliage in Brazil. Environ Entomol, 15 (1986) Hamsa TAP & Ayre JE, A differential medium for the isolation of Aspergillus flavus from cotton seed. J Food Sci, 42 (1977) Carels M & Shepherd D, The effect of ph and amino acids on conidiation and pigment production of Monascus major ATCC in submerged shaken culture. Can J Microbiol, 24 (1978) Cochrane VW, Physiology of Fungi. (Wiley, New York), 1958, 19, 55; Wolf FA & Wolf FT, The Fungi. Vol. 2. (Wiley, London), 1947, Holmquist GU, Walker HW & Stahr HM, Influence of temperature, ph, water activity and antifungal agents on growth of Aspergillus flavus and Aspergillus parasiticus. J Food Sci, 48 (1983) Joffe AZ & Lisker N, Effect of light, temperature, and ph value on aflatoxin production in vitro. Appl Microbiol, 18 (1969) Thomas KC, Khachatourians GG & Ingledew WM, Production and properties of Beauveria bassiana conidia in submerged culture. Can J Microbiol, 33 (1987) Hegedus DD, Bidochka MJ & Khachatourians GG, Beauveria bassiana submerged conidia production in a defined medium containing chitin, two hexosamines or glucose. Appl Microbiol Biotechnol, 33 (1990) Varma A, Tandan BK & Singh K, First record of Beauveria bassiana (Balsamo) Vuillemen, an entomogenous fungus from the sugarcane defoliator Phytoscaphus sp. (Coleoptera: curculionidae) from India. J Curr Sci, 57 (1988) Shimazu M & Sato H, Media for selective isolation of an entomogenous fungus, Beauveria bassiana (Deuteromycotina: Hyphomycetes). Appl Entomol Zool, 31 (1996) Milner RJ, Huppatz RJ & Swaris SC, A new method for assessment of germination of Metarhizium conidia. J Invertebr Pathol, 57 (1991) Sanzhimitupova RD, Effect of the ph of the medium on the growth and development of the causal agent of mycosis of the seabuckthorn moth (Gelecjia hippophaella Schrk). Izvestiya-Sibirskogo-Otdeleniya-Akademii-Nauk-SSSR- Biology, 15 (1980) Galani G, Cultivation of some entomopathogenic fungi in liquid media with various initial ph values. Analel- Institutului-de-Cercetari-pentru-Protecta-Plantelor, 21 (1988) Goettel MS & Inglis GD, Fungi: Hyphomycetes. In: Manual of Techniques in Insect Pathology (Ed. LA Lacey; San Diego, USA), 1997, St. Leger RJ, Joshi L & Roberts DW, Ambient ph is a major determinant in the expression of cuticle-degrading enzymes and hydrophobin by Metarhizium anisopliae. Appl Environ Microbiol, 64 (1998) Hallsworth, JE & Magan N, Culture age, temperature, and ph affect the polyod and trehalose contents of fungal propagules. Appl Environ Microbiol, 62 (1996) Griffin DH, Chemical requirements for growth. In: Fungal physiology. (Wiley-Liss, New York), 1994, Olsson S, Evidence of diffusion being the mechanism of translocation in the hyphae of three molds. Exp Mycol, 15 (2000) Hallsworth JE & Magan N, Water and temperature relations of growth of the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus. J Invertebr Pathol, 74 (1999) Dimbi S, Maniania NK, Lux SA & Mueke JM, Effect of constant temperatures on germination, radial growth and virulence of Metarhizium anisopliae to three species of African tephritid fruit flies. Biocont, 49 (2004) Ekesi S, Maniania NK & Ampong-Nyarko K, Effect of temperature on germination, radial growth and virulence of Metarhizium anisopliae and Beauveria bassiana on Megalurpthrups sjostedti. Biocont Sci Technol, 9 (1999) Walstad JD, Anderson RF & Stambaugh WJ, Effects of environmental conditions on two species of muscardine fungi (Beauveria bassiana and Metarrhizium anisopliae). J Invertebr Pathol, 16 (1970) Jayaramaiah M & Veeresh GK, Influence of temperature and humidity on the survival of spores of the fungus Beauveria brongniartii (Sacc.) Petch. J Soil Biol Ecol, 4 (1984) 82.

10 DHAR et al.: OPTIMIZED MEDIUM FOR IMPROVED CONIDIATION OF BEAUVERIA BASSIANA Alves SB, Pereira RM, Stimac JL & Vieira SA, Delayed germination of Beauveria bassiana conidia after prolonged storage at low and above freezing temperatures. Biocont Sci Technol, 6 (1996) Campbell RK, Barnes GL, Cartwright BO & Eikenbary RD, Growth and sporulation of Beauveria bassiana and Metarhizium anisopliae in a basal medium containing various carbohydrate sources. J Invertebr Pathol, 41 (1983) Safavi SA, Shah FA, Pakdel AK, Rasoulian GR, Bandani AR & Butt TM, Effect of Nutrition on Growth and Virulence of the Entomopathogenic Fungus Beauveria bassiana. FEMS Microbiol Lett, 270 (2007) Petlamul W & Prasertsan P, Spore production of entomopathogenic fungus Beauveria bassiana BNBCRC for biocontrol: Response surface optimization of medium using decanter cake from palm oil mill. J Kor Soc Appl Biol Chem, 57 (2014) Liu DP & Holdom DG, Effects of nutrients on colony formation, growth, and sporulation of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes). J Invertebr Pathol, 65 (1995) Petlamul W & Prasertsan P, Medium Optimization for Production of Beauveria bassiana BNBCRC Spores from Biohydrogen Effluent of Palm Oil Mill Using Taguchi Design. Int J Biosc, Biochem & Bioinfo, 4 (2014). 32 Santa HS, Brand D & Luciana PV, Spore production of Beauveria bassiana from agro-industrial residues. Braz Arch Biol Technol, 48 (2005) 121.