Influence of different storage conditions on vitality and virulence of Beauveria bassiana spores

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1 Influence of different storage conditions on vitality and virulence of Beauveria bassiana spores Jana ŠIMKOVÁ University of South Bohemia, Faculty of Agriculture, Department of Plant Protection, Studentská 13, České Budějovice, Czech Republic, Abstract Impact of different carriers and storage temperatures on conidia of the fungus Beauveria bassiana strain I 101 was investigated. The aim was to reach the information on the best storage conditions for the spores shelf-life. The spores were formulated in three types of carriers (one nutritive and two inert carriers that differed in grain size) and the experiment proceeded at temperatures 22, 4 and -20 C. The evaluation was based on vitality bioassays including germination and growth index assessment and the bioassay of virulence based on target organism Tenebrio molitor. The germination differed markedly between temperatures as well as between carriers. Nutritive carrier was found to be the most suitable for storage of B. bassiana conidia in all aspects, especially when kept at both low temperatures. The germination rate was 97.67% after storage at 4 C for 90 days; the initial germination rate was 97.33%. On the contrary, worse results were mostly achieved in unformulated conidia stored at 22 C (germination rate 12.33% after 90 days). Key words: Beauveria bassiana, conidia, carrier type, storage temperature, bioassay Introduction Beauveria bassiana Vuillemin (Deuteromycotina: Hyphomycetes) is an entomopathogenic fungus known to have a wide host range. This fungus has been studied as a microbial control agent of insect pests in various areas (Shimazu et al., 2002). Entomopathogenic fungi have a demonstrated potential to reduce insect pest populations in field, forest, and greenhouse habitats and have great promise as an environmentally safe pest management tool (Parker et al., 1997). Preservation of high viability and virulence (Derakhshan et al., 2008) during storage of conidial formulations of mycoinsecticides is essential for their use in pest control (McClatchie et al., 1994) and is a pre-requisite for success (Hong et al., 1997). A wide range of temperatures where the spores should keep their viability is required and that is why so many bioassays are proceeded. Temperature is an important factor that determines the rate of germination, growth, sporulation and survival of entomopathogenic Hyphomycetes. By varying single or multiple factors, and by using information obtained on fungal tolerances, it should be possible to develop predictive models in the development of entomopathogenic Hyphomycetes as microbial control agents (Goettel and Inglis, 1997). Formulation of mycoinsecticide must be compatible with the agent, enhance its performance and ideally, it must maintain an adequate shelf-life of the agent in order to be successful (Derakhshan et al., 2008). Regarding the finalisation, it is possible to use big amount of carriers. It is essential that the conidia of an isolate selected for a mycopesticide retains a high viability in dry powder form (Consolo et al., 2003). Morley-Davies et al. (1995) determined the effect of six storage temperatures in the range from -10 to 50 C on dry conidia and oil formulations, Hong et al. (2001) proved features of different isolates of B. bassiana spores at five temperatures, Hidalgo et al. (1998) assessed viability through time of formulated conidia stored at two temperatures. It is clear that the relevant questions concerning storage conditions are very important for the development of new products. As much information as possible on the effects of storage temperature on shelf-life should be given (Jenkins and Grzywacz, 2000). It is very helpful that models used for M. anisopliae have been applied to data for conidia of B. bassiana (Hong et al., 2001) showing that, although conidia of different fungal species can vary in their absolute longevity, the relative effect of temperature on the life span of conidia is similar across species. 75

2 The effect of long-term storage has been solved at different temperatures and formulations but a general conclusion has not been made yet. The aim of this work was to carry out the experiments in wider range, it means the viability tests together with virulence bioassay and to find the most suitable formulation and storage temperature for longterm storage of the spores of B. bassiana strain I101. Material and Methods Production and storage of spores of B. bassiana strain I101 Fungus B. bassiana strain I101 was originally isolated from bark beetle in 2004 in the National Park Šumava (SW Bohemia) and belongs to the Collection of fungi in the Department of Plant Protection, Faculty of Agriculture, University of South Bohemia. On purpose of this bioassay the fungus was maintained on pot barley for two weeks at 23 C. The substrate overgrown with conidia was airdried and conidia were obtained by taking off directly into one of the following carriers nutritive carrier (NC), inert carrier no.1 (IC1, grain size µm), inert carrier no.2 (IC2, grain size µm) or separately as unformulated conidia (UC). All formulations as well as UC were stored in closed plastic containers in dark under controlled temperatures 22, 4 and -20 C. Experiment proceeded through 90-day long period. Persistency of vitality of B. bassiana spores upon storage Conidial suspension was prepared by mixing of dry formulated or unformulated conidia with sterile 0.05% Tween 80. The number of conidia determined in a counting chamber was adjusted to the final concentration of 1.0 x 10 7 conidia/ml before the bioassay was initiated. The viability of conidia was verified before the bioassay in a standard germination test (Landa et al., 1994). For this purpose, sterile slides coated with a thin layer of 2% water agar were prepared. Ten drops of inoculum were placed on the top of agar using an inoculation loop and incubated for 24h in a humid chambre (90 mm Petri dish containing filter paper wetted with 0.5 ml of sterile distilled water) at 23 C. Germination was considered to have occurred when a germ-tip was observed. Percentage germination was determined by viewing a minimum of 100 conidia under light microscope. The demonstrated results are mean values from triplicates. Together with germination, growth index (GI) was evaluated according to the scale from 0 to 3 (Table 1). Each half-point represents one part of the fungal development cycle. Evaluation of B. bassiana spores virulence Evaluation of the virulence was tested on target organism Tenebrio molitor. Larvae of the same instar (the same size) were selected for the bioassay. The larvae surface was sterilised by washing in 0.05% NaClO and rinsing three times in sterile distilled water and then larvae were let to dry on sterile filter paper. Conidial suspension obtained and adjusted (1.0 x 10 7 spores/ml) as described above was poured into a sterile glass beaker. The larvae were dipped singly into a suspension for 1s and surplus suspension was quickly drained off by suction with the filter paper (Goettel and Inglis, 1997). The treated larvae were incubated singly in humid chambers (60 mm Petri dish with filter paper wetted with 0.3 ml of sterile distilled water). Untreated control was prepared by dipping larvae into 0.05% Tween 80. Bioassay was repeated in 30-day intervals during storage (total time 90 days). In evaluation of Fungal Development Index (FDI), each larva was assessed individually under a binocular and rated according to the modified index scale (Table 2) originally described in Horňák (2004) where Galleria mellonella larvae were used as target organism. The final outcome represents a mean value of 30 larvae from each treatment after 6 days of the bioassay. For statistical evaluation of all experiments one-way ANOVA and Tukey`s HSD test were used. Results and Discussion Vitality of stored spores of B. bassiana depended on the temperature as well as on the formulation. Initial germination of B. bassiana spores did not differ markedly. The values moved in range from to 97.33% in different variants. Generally, it is possible to say that germination through the 90-day storage period had decreasing trend but the acquired values differed among the formulations and especially among the temperatures. The viability of samples stored at both low temperatures remained fairly constant. Combined effect of low temperature and nutrient carrier is obvious because of the highest germination found out at all temperatures in this formulation (Table 3). The highest percentage of germinating conidia (98.67% after 90 days of storage) was observed at -20 C. It supports reported findings 76

3 that decrease in temperature increases the longevity of conidia of B. bassiana (Stathers et al., 1993, Morley-Davies et al., 1995, Hong et al., 1997). Morley-Davies et al. (1995) (working with M. anisopliae and B. bassiana) concluded that too low temperature can harm the conidia and the temperature of long-term storage should be preferably between 0 C and 20 C. Nevertheless, this opinion was not proved in our work because of good results obtained even under storage at -20 C. The germination rate remained above 90% irrespective of the formulation through the whole testing period. Development of growth index had similar trend at temperatures 4 C and -20 C, except of IC2 at 4 C. Data showing the development of GI are summarised in Table 4. The highest GI throughout the testing period was attained using NC at all temperatures. Conidia stored at 22 C reached the highest values at the very beginning of the bioassay and in NC formulation, namely after 30 and 60 days of storage (1.84 and 1.55, respectively). At low temperatures (4 and -20 C), the lowest GI showed UC almost throughout the storage period but the bottom value (0.90) was achieved in conidia formulated in IC2. The data indicate that low temperature induce faster development of the fungus. Mortality of T. molitor larvae caused by B. bassiana had also a downward trend (Table 5). The assay system of fungus virulence was described by Goettel and Inglis (1997) who had tested it successfully with B. bassiana and some other fungi. The time of the evaluation in our work (after 6 days of the bioassay) was supported by Lekimme et al. (2006) who found 100% of the mites in the bioassay covered with the fungus when exposed to 1.0 x 10 7 conidia/ml after this time. In our experiment, UC and conidia formulated in IC2 stored at 22 C caused almost no mortality after 90 days of storage. Mortality in other variants was higher than 50% after the same time. The variants at low temperatures showed statistically insignificant differences except of conidia formulated in IC2 after 60 days of storage at 4 C. At -20 C, there was a significant difference in case of IC1 as well as IC2 after 60 days of storage. Table 6 presents FDI values obtained from virulence biassay. FDI had as well as previous bioassays a downward trend. The colonisation of host with fungus is defined by value of 1.5 when the fungal hyphae in the body is present what confirms the entomopathogenic activity of B. bassiana (Lekimme et al., 2006). This phase is irreversible and the host insects do not recover from the infection (Landa et al., 1994). In our bioassay there were apparent differences in effectiveness between conidia stored at 22 C and other two temperatures. Especially UC did not reach the value of 1.5 in any control period. This is connected with larval mortality that was very low or was not induced at all. Particularly all variants stored at 4 C attained FDI above 1.5 after 60 days of storage. This finding is very important in the assessment of the ability of the fungus to initiate infection of insect and complete its own life cycle. Faster establishment of fungus on the host was recorded at formulations with NC. It supports a report of Landa et al. (1994) who found out that most nutrients increased the FDI. The acquired data show the importance of the effects of formulation and storage temperature for surviving of B. bassiana spores. It is necessary to know how the spores are influenced with the storage conditions because of the seasonal application of many mycopesticides. Acknowledgement I thank Professor Zdeněk Landa for his professional guidance and assistance and all the staff of the Department of Plant Protection for their help. This work was financially supported by the project MSM of the Czech Ministry of Education, Health and Sports. References Consolo V.F., Salerno G.L., Beron C.M. (2003). Pathogenicity, formulation and storage of insect pathogenic hyphomycetous fungi tested against Diabrotica speciosa. BioControl 48: Derakhshan A., Rabindra R.J., Ramanujam B. (2008). Effect of storage conditions of formulations on viability of Verticillium lecanii (Zimmerman) Viegas and its virulence to Brevicoryne brassicae (L.). J. Biol. Sci. 8: Goettel M.S., Inglis G.D. (1997). Fungi: Hyphomycetes. In Manual of techniques in insect pathology. Lacey L.A. (ed.). San Diego, Academic Press, pp Hidalgo E., Moore D., Le Patourel G. (1998). The effect of different formulations of Beauveria bassiana on Sitophilus zeamais in stored maize. J. Stored Prod. Res. 34: Hong T.D., Ellis R.H., Moore D. (1997). Development of a model to predict the effect of temperature and moisture on fungal spore longevity. Ann. Bot. 79:

4 Hong T.D., Gunn J., Ellis R.H., Jenkins N.E., Moore D. (2001). The effect of storage environment n the longevity of conidia of Beauveria bassiana. Mycol. Res. 105: Horňák P. (2004). Monitoring přirozeného výskytu entomopatogenních hub v půdních ekosystémech. Disertační práce, Jihočeská univerzita v Českých Budějovicích, Zemědělská fakulta. Jenkins N.E., Grzywacz D. (2000). Quality control of fungal and viral biocontrol agentsassurance of product performance. Biocontrol Sci. Technol. 10: Landa Z., Osborne L., Lopez F., Eyal J. (1994). A bioassay for determining pathogenicity of entomogenous fungi on whiteflies. Biol. Control 4: Lekimme M., Mignon B., Tombeux S., Focant C., Maréchal F., Losson B. (2006). In vitro entomopathogenic activity of Beauveria bassiana against Psoroptes spp. (Acari: Psoroptidae). Vet. Parasitol. 139: McClatchie G.V., Moore D., Bateman R.P., Prior C. (1994). Effects of temperature on the viability of the conidia of Metarhizium flavoviride in oil formulations. Mycol. Res. 98: Morley-Davies J., Mooore D., Prior C. (1995). Screening of Metarhizium and Beauveria spp. conidia with exposure to simulated sunlight and a range of temperatures. Mycol. Res. 100: Parker B.L., Skinner M., Gouli V., Brownbridge M. (1997). Impact of soil applications of Beauveria bassiana and Mariannaea sp. on nontarget forest arthropods. Biol. Control 8: Shimazu M., Sato H., Maehara N. (2002). Density of the entomopathogenic fungus, Beauveria bassiana Vuillemin (Deuteromycotina : Hyphomycetes) in forest air and soil. Appl. Entomol. Zool. 37(1): Stathers T.E., Moore D., Prior C. (1993). The effect of different temperatures on the viability of Metarhizium flavoviride conidia sored in vegetable and mineral oils. J. Invertebr. Patol. 62(2):

5 Table 1. Scale used for the evaluation of Growth Index of Beauveria bassiana spores. 0 spore without any morfological changes 0.5 spore elongation, small germination tip germination hypha on one side of the spore, approximately the same 1 length as the spore germination hypha on one side of the spore, 2-3 fold longer than the 1.5 spore two germination hyphae 2 long hypha, side branches, hyphae on both sides of the spore 2.5 first new conidia on the mycelium, 1-4 new formed conidia on conidiophor 3 more than 4 conidia on conidiophore, full sporulation Table 2. Scale used for the evaluation of Fungal Development Index of Beauveria bassiana spores on the surface of Tenebrio molitor larvae. 0 living larva, no signs of infection on the body 0.5 small melanic spots on larval surface, big amount is possible 1 big melanic spots on larval surface, could join together beginning of mycelial growth, individual hypha on softer parts of 1.5 larval body 2 compact mycelium on 1/3 of the body 2.5 beginning of sporulation, single spores on conidiophore 3 fully sporulating mycelium on cadaver Table 3. Germination (%) of Beauveria bassiana spores after storage in different conditions. Formulation Unformulated conidia Nutritive carrier Inert carrier no.1 Inert carrier no.2 Temperature Day 22 C Aa Aa Aa Aa Bc Aa Aa Bb Cc Ba Bb Cb Dd Ca Bb Dc 4 C Aa Aa Aa Aa Bc Aa Bb Bb Cd Aa Bb Cc Bb Aa Cb Dc -20 C Aa ABa Aa Aa Bb ABa Aab Bab Bc Ba ABb Bb Bb Aa Bb Bb * Means in the same row followed by the same lower-case letter and means in the same column followed by the same capital letter are not significantly different as determined by ANOVA and the Tukey HSD test. 79

6 Table 4. Growth index of Beauveria bassiana spores after storage at different temperatures and formulations, after 24h bioassay. Formulation Unformulated conidia Nutritive carrier Inert carrier no.1 Inert carrier no.2 Temperature Day 22 C ±0.58 Aa 1.78±0.48 Ba 1.74±0.50 Aa 1.74±0.52 Aa ±0.73 Bd 1.84±0.45 Aa 1.23±0.71 Bb 1.11±0.86 Bc ±0.79 Cc 1.55±0.73 Ca 0.66±0.81 Cb 0.55±0.74 Cc ±0.44 Dc 0.59±0.70 Da 0.57±0.73 Da 0.19±0.42 Db 4 C ±0.58 Aa 1.78±0.48 Ca 1.74±0.50 Ba 1.74±0.52 Aa ±0.72 Bd 1.89±0.39 Ba 1.84±0.51 Ab 1.76±0.57 Ac ±0.77 Cd 2.06±0.41 Aa 1.79±0.54 ABb 1.71±0.63 Ac ±0.65 Ab 1.92±0.40 Ba 1.44±0.73 Cc 0.90±0.87 Bd -20 C ±0.58 Aa 1.78±0.48 Ca 1.74±0.50 Ba 1.74±0.52 Ba ±0.70 Bc 1.89±0.40 Ba 1.76±0.59 Bb 1.84±0.51 Aa ±0.78 Ac 2.10±0.47 Aa 1.82±0.53 Abc 1.87±0.49 Ab ±0.65 Ab 2.05±0.57 Aa 1.63±0.70 Cc 1.76±0.59 Bb * Means in the same row followed by the same lower-case letter and means in the same column followed by the same capital letter are not significantly different as determined by ANOVA and the Tukey HSD test. Table 5. Mortality of Tenebrio molitor larvae caused by Beauveria bassiana spores stored at different temperatures and formulations. Results acquired after 6-day bioassay. Formulation Untreated control Unformulated conidia Nutritive carrier Inert carrier no.1 Inert carrier no.2 Temperature Day 22 C Ac 100 Aa Aab 100 Aa Ab Ab Ba Ba Ba Ba Aa 3.33 Ca Ca 6.68 Ca 3.33 Ca 4 C Ac 100 Aa Aab 100 Aa Ab Ac Aa Aa ABa Ab Ab Aa Aa Ba Aa -20 C Ac 100 Aa Aab Aa Ab Ac Aa Aa Bb Ab Ab Ba Ba Ba Aa * Means in the same row followed by the same lower-case letter and means in the same column followed by the same capital letter are not significantly different as determined by ANOVA and the Tukey HSD test. 80

7 Table 6. Fungal development index of Beauveria bassiana spores stored at different temperatures and formulations after 6 days of the bioassay on Tenebrio molitor larvae. Formulation Untreated control Unformulated conidia Nutritive carrier Inert carrier no.1 Inert carrier no.2 Temperature Day 22 C Ac 2.85±0.37 Ab 1.90±0.87 Aa 2.98±0.05 Ab 2.63±0.76 Aa Ac 0.58±0.56 Bb 0.83±0.16 Bb 1.62±0.53 Ba 1.37±0.79 Ba Ab 0.15±0.23 Cab 0.42±0.47 Ba 0.22±0.43 Ca 0.38±0.50 Cab 4 C Ac 2.85±0.37 Ab 1.90±0.87 Aa 2.98±0.05 Ab 2.63±0.76 Aa Ac 2.55±0.70 Aa 1.90±0.15 Ab 2.63±0.53 Aa 1.98±0.89 ABb Ab 1.87±0.69 Ba 1.60±0.96 Aa 1.58±0.88 Ba 1.33±0.91 Ba -20 C Ac 2.85±0.37 Ab 1.90±0.87 Aa 2.98±0.05 Ab 2.63±0.76 Aa Ac 1.55±0.78 Bb 1.88±0.93 Aab 2.38±0.59 Ba 1.58±0.95 Bb Ac 0.98±0.72 Cb 1.62±0.99 Aa 1.22±0.83 Cab 1.32±0.97 Bab * Means in the same row followed by the same lower-case letter and means in the same column followed by the same capital letter are not significantly different as determined by ANOVA and the Tukey HSD test. 81

8 Journal of Agrobiology, 26 (2): 82,