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Viability of Beauveria bassiana isolates after storage under several preservation methods

Viability of Beauveria bassiana isolates after storage under several preservation methods Ann Microbiol (2011) 61:339–344 DOI 10.1007/s13213-010-0147-8 ORIGINAL ARTICLE Viability of Beauveria bassiana isolates after storage under several preservation methods Ivo Oliveira & José Alberto Pereira & Albino Bento & Paula Baptista Received: 22 July 2010 /Accepted: 7 October 2010 /Published online: 10 November 2010 Springer-Verlag and the University of Milan 2010 Abstract Beauveria bassiana is a worldwide distributed et al. 2007; Akello et al. 2009). In addition to being a entomopathogenic fungus, which is used nowadays as an necrotrophic parasite, it presents the capability to inhibit the alternative to chemical pesticides in the control of several growth of several phytopathogenic fungi (Ownley et al. agriculture pests. Therefore, a preservation method must be 2008), and no known phytopathogenic activity is associated established in order to maintain fungal culture and stocks, to this fungus (Reddy et al. 2009). Considering all these while fungal characteristics, like morphological features, facts, B. bassiana has become an alternative to the use of spore production and viability, are kept well preserved. chemical pesticides under several conditions (Uribe and Although a large number of different storage methods are Khachatourians 2004). available and described, each fungus presents different The establishment of entomopathogenic fungal collec- characteristics, and therefore a new challenge for its tions is very important for biotechnology and agricultural preservation. In this work, we evaluate the effect of 1-year research. Without them, there would be no stocks to support preservation by lyophilization, glycerol-freeze at −20°C and research on fungi for potential biocontrol of insects. Thus, sub-culturing on growth, production and viability of spores several methods of cultivation and preservation are required and macro- and micro-morphology of three isolates of B. to ensure the viability and morphological, physiological, bassiana. Overall, results indicate that sub-culturing is the and genetic integrity of the fungi cultures over time. best method for conservation of this fungal species. Many methods for the preservation of fungal isolates However, if long-term storage is required, glycerol-freeze have been developed. According to Gallo et al. (2008), they proved to be the most capable method of preservation. can be divided in two groups, according to the continued or suspend metabolism of the fungus. The methods that allow . . Keywords Beauveria bassiana Preservation Fungus continued metabolism of the fungus include storage in morphology Viability sterile water, sub-cultures in agar, cool storage at temper- atures ranging from 5 to 8°C and deep freeze at −20°C. Although storage in sterile water has been proven to be an Introduction easy and inexpensive method for preservation of most fungi, it can lead to a loss in the sporulation rates of fungi, Beauveria bassiana is a ubiquitous entomopathogenic as well as of changes in morphological characteristics, fungus, from the class Deuteromycetes, currently under and should always be complemented with other methods intensive study as a promising biocontrol agent against a (Borman et al. 2006). Sub-culturing fungal isolates in agar large number of agricultural insect pests (Quesada-Moraga is a routinely used method to preserve fungi due to its simplicity. When it comes to storage of a large number of specimens, it becomes time-consuming, contaminations are : : : I. Oliveira J. A. Pereira A. Bento P. Baptista (*) more likely to occur, and genetic and physiological changes CIMO / School of Agriculture, Polytechnic Institute of Bragança, are not prevented (Homolka et al. 2007). Cool storage and Campus de Santa Apolónia, Apartado 1172, deep freezing storage have demonstrated success in 5301-854 Bragança, Portugal preserving long-term fungal isolates. However, even these e-mail: pbaptista@ipb.pt 340 Ann Microbiol (2011) 61:339–344 methods can prove to be inadequate, due to some of the Materials and methods variabilities, biological or innate, of some organisms (Pasarell and McGinnis 1992). Fungal isolates To suspend the fungal metabolism, preservation through drying, silica gel, lyophilization, liquid nitrogen or cryo- Autochthonous isolates of Beauveria bassiana were genic freezer beads at −70°C can be used. Although obtained during 2007 from field-collected pupae of Prays lyophilization and cryopreservation are considered to be oleae from several localities in the Trás-os-Montes region the storage methods that reduce the possibility of mutation (northeast Portugal). Fungal isolation was performed on (Borman et al. 2006), both methods require specialized and potato-dextrose agar (PDA) medium containing 0.01% (w/v) expensive equipment, not available in many laboratories chloramphenicol (Oxoid). Pure cultures were obtained by (Nakasone et al. 2004). sub-culture in the same medium, and the different isolates Several methods of preservation of B. bassiana have been were identified by amplification and sequence of the internal proposed and studied. Most of them analyzed the effect of transcribed spacer region (ITS), using the universal primers storage temperatures on the viability of both formulated and ITS1 and ITS4 (White et al. 1990). The isolates obtained unformulated conidia. The temperatures tested ranged from were deposited in the culture collection of Escola Superior ambient and refrigerator temperatures (Sandhu et al. 1993; Agrária of Instituto Politécnico de Bragança (Portugal), with Alves et al. 1996) to freezer temperatures at −10 to −7°C the designations Bb 1T/07, Bb 2T/07 and Bb 3T/07. (Alves et al. 1996; Marques et al. 2000). In all these studies, Colonies produced from single spores were used as pure both refrigerating and freezing were proven to be good isolates. methods for storing pure conidia for, respectively, 24 (Sandhu et al. 1993) and 80 months (Marques et al. 2000). Storage methods In formulated conidia, the preservation of viability occurred for longer periods of time, for at least 7 years (Alves et al. The three B. bassiana isolates were produced in 9-cm Petri 1996). Conidia of B. bassiana cryopreserved at low temper- dishes containing PDA medium, for 15 days at 25±1°C in atures and in liquid nitrogen have also been subject to the dark, in order to provide mycelium and conidia for the viability tests. Cultures have been satisfactorily maintained different storage methods. After that, fungal isolates were on sawdust medium containing 10% glycerol at −85°C, for stored using three different methods: (1) freeze at -20°C in 20 months (Kitamoto et al. 2002), and on potato dextrose an aqueous glycerol solution (30%, v/v); (2) freeze-dried agar medium at −70°C, for periods ranging from 6 months to (lyophilized) and maintained at ambient temperature; and 13 years (Pasarell and McGinnis 1992). Storage in liquid (3) by continual sub-culture in PDA medium. In the first nitrogen, using 10% glycerol as cryprotectant, also seems to two methods, mycelium and conidia of each isolate were be an effective way to preserve B. bassiana, for up to removed from the agar surface by scraping with a sterile 84 months (Faria et al. 1999). Only one study has been scalpel and placing in sterile 2-ml cryovials tubes contain- performed to evaluate the effect of lyophilization on B. ing 1 mL of 30% (v/v) glycerol (freeze method) or in sterile bassiana viability (Faria et al. 1999). In the lyophilization of 15-mL Falcon tubes (freeze-dried method). In the first conidia, a mixture of 3% glucose and 3% gelatin was used method, the tubes were stored at −20°C, and in the second and a loss of viability was verified, ranging from 41 to 94%, method, the fungal cultures are frozen at −20°C for 24 h after 49 months of storage at 4°C. and subsequently dried under vacuum in a freeze-dryer The lack of information regarding the storage of B. model Ly-8-FM-ULE (Snijders) for 24 h. In the continuous bassiana as well as the complexity of some of the sub-culture method, Petri dishes containing PDA medium conservation methods described until now suggest that a were centrallyinoculatedwithasingle circular 5-mm specific protocol must be selected having regard to the mycelial plug removed from the edge of an actively growing characteristics of the fungus. Thus, the objective of the colony. After sealed with Parafilm, the Petri dishes were present work was to estimate the viability and macro and stored in the dark at 25±1°C and sub-culture every micro-morphology changes of three autochthonous B. month. bassiana isolates after being stored for 1 year at −20°C in glycerol (30%, v/v), freeze-dried (lyophilized) and contin- Viability assessment ually sub-cultured in potato-dextrose agar (PDA) medium. This work will allow a direct comparison of storage The viability of each isolate was assessed after 1 year of methods that may be applied to unformulated conidia of storage. Growth assessment was achieved by inoculating B. bassiana, and introduces, for the first time, the use of the centre of a 9-cm Petri dishes containing PDA medium lyophilization without the use of cryoprotective and with 2 μL of stored spores’ suspension containing 2×10 lyoprotective agents. spores/mL. Five replicate of each isolate per storage Ann Microbiol (2011) 61:339–344 341 15 Bb 1T/07 method were performed. The plates were incubated at 25± Lyophilized Freezing 1°C in the dark, and the radial growth of fungal cultures was measured during 18 days. Mycelium growth indicated that the culture was viable. Conidia production 0 3 6 9 12 15 18 -5 The conidia produced by each fungal strain were evaluated -10 after 18 days of incubation. For this, a conidia suspension was Bb 2T/07 retrieved from PDA cultures, obtained in the viability * * assessment, to 500 μL of an aqueous solution of Tween 80 (0.02%, v/v). The number of conidia was counted in a Thoma counting chamber. Results were expressed in conidia per mL. Spores’ viability 0 3 6 9 12 15 18 -5 The spores’ viability was measured by quantifying the -10 percentage of germinated conidia. Therefore, 1 mL of Bb 3T/07 stored spore suspension, containing approximately 10 spores/mL, was spread in 9-cm Petri dishes containing agar 0 3 6 9 12 15 18 -20 medium (15 g/L agar-agar). Five replicates of each isolate * * * * * * * * * * per storage method were performed. After incubation, at -40 25±1°C in the dark for 24 h, the percentage of germina- tion was evaluated by counting the number of germinated -60 and non-germinated spores, from a total of 300 spores per -80 Petri dish. * * * * * * * * * * * * * * * * * * -100 Macroscopic and microscopic characterization Incubation time (days) of the colonies Fig. 1 Differences on radial growth (%) of Beauveria bassiana strains (Bb 1T/07, Bb 2T/07 and Bb 3T/07) maintained by sub-culture Macroscopic characteristics of the colonies were registered and preserved by two methods (lyophilization and freezing on 30% during 18 days of fungal culture and include mycelium glycerol), for 18 days. Statistical significance: **p<0.01, ***p<0.001 texture, color and border appearance of the colony, medium coloration and exudates production. Microscopic character- Subculture Lyophilized Freezing ization was evaluated under bright-field conditions after 1.4 18 days of culture with a Leica model CTR 5000 aa microscope, using lactophenol cotton blue staining. 1.2 1.0 Data analysis 0.8 Data from radial growth of colonies, spore germination (%) 0.6 and number of spores (conidia/mL) were presented as the mean of five independent experiments displaying the 0.4 respective standard error (SE) bars or standard deviation 0.2 (SD) values. Differences among means were done by analysis of variance (ANOVA), using SPSS v.17 software, 0.0 and averages were compared using Tukey test (p<0.05). Bb 1T/07 Bb 2T/07 Bb 3T/07 Strain Fig. 2 Growth rates (mean ± SE, n=5) of Beauveria bassiana strains Results and discussion (Bb 1T/07, Bb 2T/07 and Bb 3T/07) preserved by sub-culture, lyophilized and freezing (30% glycerol) methods, on PDA medium, at The preservation of fungal strains as reference stocks for day 18. Bars with different letters indicate values with significant differences (p<0.05), within each strain ongoing research requires that the stored cultures remain Growth rate (mm/day) Radial growth (%) Radial growth (%) Radial growth (%) 342 Ann Microbiol (2011) 61:339–344 Table 1 Spore germination and spore production (mean ± SD, n=5) of Beauveria bassiana strains after 1-year preservation by sub-culture (Sb), lyophilization (Ly) and freeze in aqueous solution of glycerol (30%, v/v) (Fr) Strain Spore germination (%) No. spores (conidia/mL) Sb Ly Fr Sb Ly Fr 6 6 6 Bb 1T/07 100±0 a 100±0 a 100±0 a 20×10 ±9.7 a 25×10 ±10.7 a 149×10 ±76.3 b 6 6 6 Bb 2T/07 66.7±57.7 a 66.7±57.7 a 100±0 a 53×10 ±18.0 a 36×10 ±20.7 a 530×10 ±307.6 b 6 6 Bb 3T/07 100±0 a n.d. 100±0 a 26×10 ±10.3 a n.d. 28×10 ±4.3 a Means within a row with different letters differ significantly (p<0.05.) If there was a significant difference between strains in the spore germination and number, then means were compared by Tukey’s test, since equal variances could be assumed (p>0.05 by means of Levene test). n.d. Not determined viable for long time periods without any morphological or glycerol frozen and lyophilized, presented, in the first physiological alterations. The entomopathogenic fungal 3 days of culture, minor growth when compared with sub- (EF) collection, of the School of Agriculture–Polytechnique culture in PDA medium. After that, the radial growth of Institute of Bragança, has been established by us since fungi frozen and lyophilized increased as compared to 2007, especially with the aim to support biocontrol research control, leading to significantly statistical differences (p< involving these fungi. Considering the lack of available 0.01) after 12 days of culture. By contrast, isolate Bb 3T/07 information regarding the storage of entomopathogenic showed a significantly decrease on radial growth in the fungi, we have decided to study the effect of several freeze and freeze-dried methods when compared to control preservation methods on the viability of autochthones B. culture. It was inclusively verified, for this isolate, that bassiana isolates. Therefore, in a first attempt, we were lyophilized conidia were not able to grow. interested in studying the viability over short periods of Similar results were obtained for fungal growth rates time (up to 1 year) in order to: (1) prevent loss of (Fig. 2). In the strain Bb 1T/07, similar growth rates were autochthones B. bassiana isolates, since this is the only observed, with no differences between control assay (PDA EF collection in Portugal; and (2) provide fungal material, sub-cultures) and lyophilization or glycerol freeze storage. to perform laboratory bioassays, with the aim to selected For strain Bb 2T/07, higher growth rates were observed for the most virulent strains. We also intended, in a future lyophilization and glycerol freeze storage, with no signif- work, to proceed to the evaluation of the viability of B. icant differences between both methods, while sub-cultures bassiana storage over long periods. Taking this into presented a slower rate of growth, with significant differ- consideration, we decided to include the lyophilization ences (p<0.05). Results for strain Bb 3T/07 showed method in the present study. differences between all preservation methods. For this The results obtained from the radial growth shown strain, PDA sub-cultures present higher growth rates, while considerable differences in the fungal isolate response to glycerol freeze led to a decrease on this parameter. storage method (Fig. 1). Isolate Bb 1T/07, although The number of spores produced by the different isolates presenting smaller radial growth when frozen in glycerol showed differences related to the storage method (Table 1). and slightly higher growth when lyophilized, did not While sub-cultures and lyophilization presented a similar present significant differences to the sub-culture method result for two of the three isolates, the colonies obtained (considered as control assay). Isolate Bb 2 T/07, when after the glycerol freeze storage produced a much higher ab c Fig. 3 Macroscopic (a) and microscopic (b, c) morphology of Beauveria bassiana isolate Bb 3T/07, after 1-year preservation by glycerol-freeze at −20°C. Colony (a), hyphae (b) and conidia (c)of B. bassiana grown in PDA medium. Bar 10 μm Ann Microbiol (2011) 61:339–344 343 number of spores, with significant statistical differences. et al. 2003), which may account for the promising results Isolate Bb 3T/07 was the exception, presenting analogous achieved with this method of conservation. Overall, results results between subcultures and glycerol freeze, but in our work indicate that continual sub-culturing in PDA nevertheless a higher production of spores was observed medium remains the preservation method in which fungal for the glycerol-conserved isolate. In the lyophilized isolate, isolates can be stored, while maintain high viability and due to the lack of germinated spores, it was not possible to production of spores, as well as growth rate. Nevertheless, evaluate the number of spores produced by the colonies. It and due to the characteristics of this method, referred to is known that repeated sub-cultures can lead to changes in before, as being time consuming, prone to contaminations some of the characteristics of fungi, such as a decrease on and not preventing genetic and physiological changes in the the ability to sporulate (Nakasone et al. 2004), and some long term, there is always the need to maintain isolates by fungal species have their sporulation ability decreased when other methods of preservation. storage with glycerol freeze, when compared to storage in Freeze-drying and especially glycerol freezing could be medium at 4°C (Mota et al. 2003). good methods for permanent preservation of B. bassiana. Regarding spore viability, there are no significant differ- The possibility of storing lyophilized B. bassiana cultures ences (p<0.05) between the tested storage methods for B. at room temperature (20–25°C), without addition of bassiana (Table 1). Although some studies showed that protective agents, was verified for the first time. Although lyophilization can lead to a decrease on spore viability, the results demonstrated intra-specific sensitivity to the either by structural damage in spores, due to the formation lyophilization process, it could be a promising method for of ice crystals on the freezing process prior to lyophilization long-term conservation of this fungus. This hypothesis must (Nakasone et al. 2004), or in the drying process (Horaczek be confirmed through the evaluation of the viability of B. and Viernstein 2004), two of our B. bassiana isolates bassiana storage over long periods. Glycerol freeze seems seemed to tolerate this kind of preservation. Isolate Bb 3T/ to be the best preservation method. Higher spore production 07 proved to be an exception, presenting 0% of germination and 100% viability of spores were achieved by this method, of spores. The obtained results showed that none of the and although presenting some significant differences in preservation methods induced morphological alterations, growth rate and radial growth when compared to sub- either macroscopic or microscopic, in the different tested culturing, results were more promising when compared to isolates of B. bassiana. In the macroscopic observation, all lyophilization. Furthermore, glycerol-freeze is a rapid the colonies presented a whitish-yellow mycelium, with method for preserving cultures, with no need of expensive aerial growth and cotton-like aspect, and regular borders equipment, and which allows a quick retrieval of conidia (Fig. 3a). The back of the colony also presented with a for use in laboratory assays. white color, although slightly more yellow in the center. Those characteristics are in accordance with those de- Acknowledgments Authors are grateful to Fundação para a Ciência e Tecnologia (FCT) for financial support (Project PTCD/AGR-AAM/ scribed for B. bassiana (Varela and Morales 1996; 102600/2008), and Ivo Oliveira for the PhD grant (SFRH/BD/44265/ Fernandes et al. 2006). Microscopic characteristics of B. 2008). bassiana are similar to those previously described for this fungus, either for hyphae or conidia (Liu et al. 2003). The hyphae were septated, with hyaline aspect and thin walls References (Fig. 3b), and conidia showed a rounded or ovoid shape, also with a hyaline aspect, and about 4 μm in diameter Akello J, Dubois T, Coyne D, Kyamanywa S (2009) The effects of (Fig. 3c). Beauveria bassiana dose and exposure duration on colonization Although no differences were observed in the spore and growth of tissue cultured banana (Musa sp.) plants. Biol Control 49:6–10 viability and fungal macro- and microscopic characteristics, Alves S, Pereira R, Stimac J, Vieira A (1996) Delayed germination of some differences were found in the growth and production Beauveria bassiana conidia after prolonged storage at low, of spores of the tested isolates. It is known that response to above-freezing temperatures. Biocontrol Sci Technol 6:575–581 stressful conditions, either induced on the conservation Borman A, Szekely A, Campbell C, Johnson E (2006) Evaluation of the viability of pathogenic filamentous fungi after prolonged process or in the recovery process, may differ considerably storage in sterile water and review of recent published studies on among different taxonomic groups, or even between strains storage methods. Mycopathologia 161:361–368 of some species (Kirsop and Doyle 1991). Some factors Faria M, Martins I, Mello R, Tigano M (1999) Entomopathogenic may account for the differences observed between the three fungal (Hyphomycetes) collection: assessment of conidial viabil- ity. Pesqui Agropecu Bras 34:1497–1503 tested B. bassiana strains, such as the rate of cooling, size Fernandes E, Costa G, Moraes Á, Zahner V, Bittencourt V (2006) of fungal propagules or thickness of the cell wall (Faria et Study on morphology, pathogenicity, and genetic variability of al. 1999). Furthermore, studies prove that glycerol is the Beauveria bassiana isolates obtained from Boophilus microplus cryoprotectant most suitable for almost all strains (Homolka tick. Parasitol Res 98:324–332 344 Ann Microbiol (2011) 61:339–344 Gallo M, Guimarães D, Momesso L, Pupo M (2008) Natural products MS (eds) Biodiversity of fungi: inventory and monitoring from endophytic fungi. In: Jain S (ed) Microbial Biotechnology, methods. Elsevier, Amsterdam, pp 37–47 Volume 4. Publishing Agency, New Delhi, pp 139–147 Ownley B, Griffin M, Klingeman W, Gwinn K, Moulton J, Pereira R Homolka L, Lisá L, Nerud F (2003) Viability of basidiomycete strains (2008) Beauveria bassiana: Endophytic colonization and plant after cryopreservation: comparison of two different freezing disease control. J Invertebr Pathol 98:267–270 protocols. Folia Microbiol 48:219–226 Pasarell L, McGinnis M (1992) Viability of fungal cultures maintained Homolka L, Lisá L, Kubátová A, Váňova M, Janderová B, Nerud F at -70°C. J Clin Microbiol 30:1000–1004 (2007) Cryopreservation of filamentous micromycetes and yeasts Quesada-Moraga E, Maranhao E, Valverde-García P, Santiago- using perlite. Folia Microbiol 52:153–157 Álvarez C (2007) Selection of Beauveria bassiana isolates for Horaczek A, Viernstein H (2004) Comparison of three commonly control of the whiteflies Bemisia tabaci and Trialeurodes vapor- used drying technologies with respect to activity and longevity of ariorum on the basis of their virulence, thermal requirements, and aerial conidia of Beauveria brongniartii and Metarhizium toxicogenic activity. Biol Control 36:274–287 anisopliae. Biol Control 31:65–71 Reddy NP, Ali Khan AP, Devi UK, Sharma HC, Reineke A (2009) Kirsop B, Doyle A (1991) Maintenance of microoganisms and Treatment of millet crop plant (Sorghum bicolor) with the cultured cells – a manual of laboratory methods. Academic, entomopathogenic fungus (Beauveria bassiana) to combat London infestation by the stem borer, Chilo partellus Swinhoe. J Asia Kitamoto Y, Suzuki A, Shimada S, Yamanaka K (2002) A new Pac Entomol 12:221–226 method for the preservation of fungus stock cultures by deep- Sandhu S, Rajak R, Agarwal G (1993) Studies on prolonged storage of freezing. Mycoscience 43:143–149 Beauveria bassiana conidia: effects of temperature and relative Liu H, Skinner M, Brownbridge M, Parker BL (2003) Characterization humidity on conidial viability and virulence against Chickpea of Beauveria bassiana and Metarhizium anisopliae isolates for Borer, Helicoverpa armigera. Biocontrol Sci Technol 3:47–53 management of tarnished plant bug, Lygus lineolaris (Hemiptera: Uribe D, Khachatourians G (2004) Restriction fragment length Miridae). J Invertebr Pathol 82:139–147 polymorphism of mitochondrial genome of the entomopatho- Marques E, Alves S, Marques I (2000) Virulência de Beauveria genic fungus Beauveria bassiana reveals high intraspecific bassiana (Bals.) Vuill. a Diatraea saccharalis (F.) (Lepidoptera: variation. Mycol Res 108:1070–1078 Crambidae) após Armazenamento de Conídios em Baixa Tem- Varela A, Morales E (1996) Characterization of some Beauveria peratura. An Soc Entomol Bras 29:303–307 bassiana isolates and their virulence toward the Coffee Berry Mota M, Campos A, Araújo J (2003) Sporulation, radial growth and Borer Hypothenemus hampei. J Invertebr Pathol 67:147–152 biomass production of A. robusta and M. thaumasium submitted White Tj, Bruns T, Lee S, Taylor J (1990) Amplification and direct to different methods of preservation. Braz J Microbiol 34:157– sequencing of fungal ribosomal RNA genes for phylogenetics. 160 In: Innis M, Gelfand D, Shinsky J, White Tj (eds) PCR protocols: Nakasone KK, Peterson SW, Jong S-C (2004) Preservation and a guide to methods and applications. Academic, San Diego, distribution of fungal cultures. In: Mueller GM, Bills GF, Foster pp 315–322 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Viability of Beauveria bassiana isolates after storage under several preservation methods

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Copyright © 2010 by Springer-Verlag and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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Abstract

Ann Microbiol (2011) 61:339–344 DOI 10.1007/s13213-010-0147-8 ORIGINAL ARTICLE Viability of Beauveria bassiana isolates after storage under several preservation methods Ivo Oliveira & José Alberto Pereira & Albino Bento & Paula Baptista Received: 22 July 2010 /Accepted: 7 October 2010 /Published online: 10 November 2010 Springer-Verlag and the University of Milan 2010 Abstract Beauveria bassiana is a worldwide distributed et al. 2007; Akello et al. 2009). In addition to being a entomopathogenic fungus, which is used nowadays as an necrotrophic parasite, it presents the capability to inhibit the alternative to chemical pesticides in the control of several growth of several phytopathogenic fungi (Ownley et al. agriculture pests. Therefore, a preservation method must be 2008), and no known phytopathogenic activity is associated established in order to maintain fungal culture and stocks, to this fungus (Reddy et al. 2009). Considering all these while fungal characteristics, like morphological features, facts, B. bassiana has become an alternative to the use of spore production and viability, are kept well preserved. chemical pesticides under several conditions (Uribe and Although a large number of different storage methods are Khachatourians 2004). available and described, each fungus presents different The establishment of entomopathogenic fungal collec- characteristics, and therefore a new challenge for its tions is very important for biotechnology and agricultural preservation. In this work, we evaluate the effect of 1-year research. Without them, there would be no stocks to support preservation by lyophilization, glycerol-freeze at −20°C and research on fungi for potential biocontrol of insects. Thus, sub-culturing on growth, production and viability of spores several methods of cultivation and preservation are required and macro- and micro-morphology of three isolates of B. to ensure the viability and morphological, physiological, bassiana. Overall, results indicate that sub-culturing is the and genetic integrity of the fungi cultures over time. best method for conservation of this fungal species. Many methods for the preservation of fungal isolates However, if long-term storage is required, glycerol-freeze have been developed. According to Gallo et al. (2008), they proved to be the most capable method of preservation. can be divided in two groups, according to the continued or suspend metabolism of the fungus. The methods that allow . . Keywords Beauveria bassiana Preservation Fungus continued metabolism of the fungus include storage in morphology Viability sterile water, sub-cultures in agar, cool storage at temper- atures ranging from 5 to 8°C and deep freeze at −20°C. Although storage in sterile water has been proven to be an Introduction easy and inexpensive method for preservation of most fungi, it can lead to a loss in the sporulation rates of fungi, Beauveria bassiana is a ubiquitous entomopathogenic as well as of changes in morphological characteristics, fungus, from the class Deuteromycetes, currently under and should always be complemented with other methods intensive study as a promising biocontrol agent against a (Borman et al. 2006). Sub-culturing fungal isolates in agar large number of agricultural insect pests (Quesada-Moraga is a routinely used method to preserve fungi due to its simplicity. When it comes to storage of a large number of specimens, it becomes time-consuming, contaminations are : : : I. Oliveira J. A. Pereira A. Bento P. Baptista (*) more likely to occur, and genetic and physiological changes CIMO / School of Agriculture, Polytechnic Institute of Bragança, are not prevented (Homolka et al. 2007). Cool storage and Campus de Santa Apolónia, Apartado 1172, deep freezing storage have demonstrated success in 5301-854 Bragança, Portugal preserving long-term fungal isolates. However, even these e-mail: pbaptista@ipb.pt 340 Ann Microbiol (2011) 61:339–344 methods can prove to be inadequate, due to some of the Materials and methods variabilities, biological or innate, of some organisms (Pasarell and McGinnis 1992). Fungal isolates To suspend the fungal metabolism, preservation through drying, silica gel, lyophilization, liquid nitrogen or cryo- Autochthonous isolates of Beauveria bassiana were genic freezer beads at −70°C can be used. Although obtained during 2007 from field-collected pupae of Prays lyophilization and cryopreservation are considered to be oleae from several localities in the Trás-os-Montes region the storage methods that reduce the possibility of mutation (northeast Portugal). Fungal isolation was performed on (Borman et al. 2006), both methods require specialized and potato-dextrose agar (PDA) medium containing 0.01% (w/v) expensive equipment, not available in many laboratories chloramphenicol (Oxoid). Pure cultures were obtained by (Nakasone et al. 2004). sub-culture in the same medium, and the different isolates Several methods of preservation of B. bassiana have been were identified by amplification and sequence of the internal proposed and studied. Most of them analyzed the effect of transcribed spacer region (ITS), using the universal primers storage temperatures on the viability of both formulated and ITS1 and ITS4 (White et al. 1990). The isolates obtained unformulated conidia. The temperatures tested ranged from were deposited in the culture collection of Escola Superior ambient and refrigerator temperatures (Sandhu et al. 1993; Agrária of Instituto Politécnico de Bragança (Portugal), with Alves et al. 1996) to freezer temperatures at −10 to −7°C the designations Bb 1T/07, Bb 2T/07 and Bb 3T/07. (Alves et al. 1996; Marques et al. 2000). In all these studies, Colonies produced from single spores were used as pure both refrigerating and freezing were proven to be good isolates. methods for storing pure conidia for, respectively, 24 (Sandhu et al. 1993) and 80 months (Marques et al. 2000). Storage methods In formulated conidia, the preservation of viability occurred for longer periods of time, for at least 7 years (Alves et al. The three B. bassiana isolates were produced in 9-cm Petri 1996). Conidia of B. bassiana cryopreserved at low temper- dishes containing PDA medium, for 15 days at 25±1°C in atures and in liquid nitrogen have also been subject to the dark, in order to provide mycelium and conidia for the viability tests. Cultures have been satisfactorily maintained different storage methods. After that, fungal isolates were on sawdust medium containing 10% glycerol at −85°C, for stored using three different methods: (1) freeze at -20°C in 20 months (Kitamoto et al. 2002), and on potato dextrose an aqueous glycerol solution (30%, v/v); (2) freeze-dried agar medium at −70°C, for periods ranging from 6 months to (lyophilized) and maintained at ambient temperature; and 13 years (Pasarell and McGinnis 1992). Storage in liquid (3) by continual sub-culture in PDA medium. In the first nitrogen, using 10% glycerol as cryprotectant, also seems to two methods, mycelium and conidia of each isolate were be an effective way to preserve B. bassiana, for up to removed from the agar surface by scraping with a sterile 84 months (Faria et al. 1999). Only one study has been scalpel and placing in sterile 2-ml cryovials tubes contain- performed to evaluate the effect of lyophilization on B. ing 1 mL of 30% (v/v) glycerol (freeze method) or in sterile bassiana viability (Faria et al. 1999). In the lyophilization of 15-mL Falcon tubes (freeze-dried method). In the first conidia, a mixture of 3% glucose and 3% gelatin was used method, the tubes were stored at −20°C, and in the second and a loss of viability was verified, ranging from 41 to 94%, method, the fungal cultures are frozen at −20°C for 24 h after 49 months of storage at 4°C. and subsequently dried under vacuum in a freeze-dryer The lack of information regarding the storage of B. model Ly-8-FM-ULE (Snijders) for 24 h. In the continuous bassiana as well as the complexity of some of the sub-culture method, Petri dishes containing PDA medium conservation methods described until now suggest that a were centrallyinoculatedwithasingle circular 5-mm specific protocol must be selected having regard to the mycelial plug removed from the edge of an actively growing characteristics of the fungus. Thus, the objective of the colony. After sealed with Parafilm, the Petri dishes were present work was to estimate the viability and macro and stored in the dark at 25±1°C and sub-culture every micro-morphology changes of three autochthonous B. month. bassiana isolates after being stored for 1 year at −20°C in glycerol (30%, v/v), freeze-dried (lyophilized) and contin- Viability assessment ually sub-cultured in potato-dextrose agar (PDA) medium. This work will allow a direct comparison of storage The viability of each isolate was assessed after 1 year of methods that may be applied to unformulated conidia of storage. Growth assessment was achieved by inoculating B. bassiana, and introduces, for the first time, the use of the centre of a 9-cm Petri dishes containing PDA medium lyophilization without the use of cryoprotective and with 2 μL of stored spores’ suspension containing 2×10 lyoprotective agents. spores/mL. Five replicate of each isolate per storage Ann Microbiol (2011) 61:339–344 341 15 Bb 1T/07 method were performed. The plates were incubated at 25± Lyophilized Freezing 1°C in the dark, and the radial growth of fungal cultures was measured during 18 days. Mycelium growth indicated that the culture was viable. Conidia production 0 3 6 9 12 15 18 -5 The conidia produced by each fungal strain were evaluated -10 after 18 days of incubation. For this, a conidia suspension was Bb 2T/07 retrieved from PDA cultures, obtained in the viability * * assessment, to 500 μL of an aqueous solution of Tween 80 (0.02%, v/v). The number of conidia was counted in a Thoma counting chamber. Results were expressed in conidia per mL. Spores’ viability 0 3 6 9 12 15 18 -5 The spores’ viability was measured by quantifying the -10 percentage of germinated conidia. Therefore, 1 mL of Bb 3T/07 stored spore suspension, containing approximately 10 spores/mL, was spread in 9-cm Petri dishes containing agar 0 3 6 9 12 15 18 -20 medium (15 g/L agar-agar). Five replicates of each isolate * * * * * * * * * * per storage method were performed. After incubation, at -40 25±1°C in the dark for 24 h, the percentage of germina- tion was evaluated by counting the number of germinated -60 and non-germinated spores, from a total of 300 spores per -80 Petri dish. * * * * * * * * * * * * * * * * * * -100 Macroscopic and microscopic characterization Incubation time (days) of the colonies Fig. 1 Differences on radial growth (%) of Beauveria bassiana strains (Bb 1T/07, Bb 2T/07 and Bb 3T/07) maintained by sub-culture Macroscopic characteristics of the colonies were registered and preserved by two methods (lyophilization and freezing on 30% during 18 days of fungal culture and include mycelium glycerol), for 18 days. Statistical significance: **p<0.01, ***p<0.001 texture, color and border appearance of the colony, medium coloration and exudates production. Microscopic character- Subculture Lyophilized Freezing ization was evaluated under bright-field conditions after 1.4 18 days of culture with a Leica model CTR 5000 aa microscope, using lactophenol cotton blue staining. 1.2 1.0 Data analysis 0.8 Data from radial growth of colonies, spore germination (%) 0.6 and number of spores (conidia/mL) were presented as the mean of five independent experiments displaying the 0.4 respective standard error (SE) bars or standard deviation 0.2 (SD) values. Differences among means were done by analysis of variance (ANOVA), using SPSS v.17 software, 0.0 and averages were compared using Tukey test (p<0.05). Bb 1T/07 Bb 2T/07 Bb 3T/07 Strain Fig. 2 Growth rates (mean ± SE, n=5) of Beauveria bassiana strains Results and discussion (Bb 1T/07, Bb 2T/07 and Bb 3T/07) preserved by sub-culture, lyophilized and freezing (30% glycerol) methods, on PDA medium, at The preservation of fungal strains as reference stocks for day 18. Bars with different letters indicate values with significant differences (p<0.05), within each strain ongoing research requires that the stored cultures remain Growth rate (mm/day) Radial growth (%) Radial growth (%) Radial growth (%) 342 Ann Microbiol (2011) 61:339–344 Table 1 Spore germination and spore production (mean ± SD, n=5) of Beauveria bassiana strains after 1-year preservation by sub-culture (Sb), lyophilization (Ly) and freeze in aqueous solution of glycerol (30%, v/v) (Fr) Strain Spore germination (%) No. spores (conidia/mL) Sb Ly Fr Sb Ly Fr 6 6 6 Bb 1T/07 100±0 a 100±0 a 100±0 a 20×10 ±9.7 a 25×10 ±10.7 a 149×10 ±76.3 b 6 6 6 Bb 2T/07 66.7±57.7 a 66.7±57.7 a 100±0 a 53×10 ±18.0 a 36×10 ±20.7 a 530×10 ±307.6 b 6 6 Bb 3T/07 100±0 a n.d. 100±0 a 26×10 ±10.3 a n.d. 28×10 ±4.3 a Means within a row with different letters differ significantly (p<0.05.) If there was a significant difference between strains in the spore germination and number, then means were compared by Tukey’s test, since equal variances could be assumed (p>0.05 by means of Levene test). n.d. Not determined viable for long time periods without any morphological or glycerol frozen and lyophilized, presented, in the first physiological alterations. The entomopathogenic fungal 3 days of culture, minor growth when compared with sub- (EF) collection, of the School of Agriculture–Polytechnique culture in PDA medium. After that, the radial growth of Institute of Bragança, has been established by us since fungi frozen and lyophilized increased as compared to 2007, especially with the aim to support biocontrol research control, leading to significantly statistical differences (p< involving these fungi. Considering the lack of available 0.01) after 12 days of culture. By contrast, isolate Bb 3T/07 information regarding the storage of entomopathogenic showed a significantly decrease on radial growth in the fungi, we have decided to study the effect of several freeze and freeze-dried methods when compared to control preservation methods on the viability of autochthones B. culture. It was inclusively verified, for this isolate, that bassiana isolates. Therefore, in a first attempt, we were lyophilized conidia were not able to grow. interested in studying the viability over short periods of Similar results were obtained for fungal growth rates time (up to 1 year) in order to: (1) prevent loss of (Fig. 2). In the strain Bb 1T/07, similar growth rates were autochthones B. bassiana isolates, since this is the only observed, with no differences between control assay (PDA EF collection in Portugal; and (2) provide fungal material, sub-cultures) and lyophilization or glycerol freeze storage. to perform laboratory bioassays, with the aim to selected For strain Bb 2T/07, higher growth rates were observed for the most virulent strains. We also intended, in a future lyophilization and glycerol freeze storage, with no signif- work, to proceed to the evaluation of the viability of B. icant differences between both methods, while sub-cultures bassiana storage over long periods. Taking this into presented a slower rate of growth, with significant differ- consideration, we decided to include the lyophilization ences (p<0.05). Results for strain Bb 3T/07 showed method in the present study. differences between all preservation methods. For this The results obtained from the radial growth shown strain, PDA sub-cultures present higher growth rates, while considerable differences in the fungal isolate response to glycerol freeze led to a decrease on this parameter. storage method (Fig. 1). Isolate Bb 1T/07, although The number of spores produced by the different isolates presenting smaller radial growth when frozen in glycerol showed differences related to the storage method (Table 1). and slightly higher growth when lyophilized, did not While sub-cultures and lyophilization presented a similar present significant differences to the sub-culture method result for two of the three isolates, the colonies obtained (considered as control assay). Isolate Bb 2 T/07, when after the glycerol freeze storage produced a much higher ab c Fig. 3 Macroscopic (a) and microscopic (b, c) morphology of Beauveria bassiana isolate Bb 3T/07, after 1-year preservation by glycerol-freeze at −20°C. Colony (a), hyphae (b) and conidia (c)of B. bassiana grown in PDA medium. Bar 10 μm Ann Microbiol (2011) 61:339–344 343 number of spores, with significant statistical differences. et al. 2003), which may account for the promising results Isolate Bb 3T/07 was the exception, presenting analogous achieved with this method of conservation. Overall, results results between subcultures and glycerol freeze, but in our work indicate that continual sub-culturing in PDA nevertheless a higher production of spores was observed medium remains the preservation method in which fungal for the glycerol-conserved isolate. In the lyophilized isolate, isolates can be stored, while maintain high viability and due to the lack of germinated spores, it was not possible to production of spores, as well as growth rate. Nevertheless, evaluate the number of spores produced by the colonies. It and due to the characteristics of this method, referred to is known that repeated sub-cultures can lead to changes in before, as being time consuming, prone to contaminations some of the characteristics of fungi, such as a decrease on and not preventing genetic and physiological changes in the the ability to sporulate (Nakasone et al. 2004), and some long term, there is always the need to maintain isolates by fungal species have their sporulation ability decreased when other methods of preservation. storage with glycerol freeze, when compared to storage in Freeze-drying and especially glycerol freezing could be medium at 4°C (Mota et al. 2003). good methods for permanent preservation of B. bassiana. Regarding spore viability, there are no significant differ- The possibility of storing lyophilized B. bassiana cultures ences (p<0.05) between the tested storage methods for B. at room temperature (20–25°C), without addition of bassiana (Table 1). Although some studies showed that protective agents, was verified for the first time. Although lyophilization can lead to a decrease on spore viability, the results demonstrated intra-specific sensitivity to the either by structural damage in spores, due to the formation lyophilization process, it could be a promising method for of ice crystals on the freezing process prior to lyophilization long-term conservation of this fungus. This hypothesis must (Nakasone et al. 2004), or in the drying process (Horaczek be confirmed through the evaluation of the viability of B. and Viernstein 2004), two of our B. bassiana isolates bassiana storage over long periods. Glycerol freeze seems seemed to tolerate this kind of preservation. Isolate Bb 3T/ to be the best preservation method. Higher spore production 07 proved to be an exception, presenting 0% of germination and 100% viability of spores were achieved by this method, of spores. The obtained results showed that none of the and although presenting some significant differences in preservation methods induced morphological alterations, growth rate and radial growth when compared to sub- either macroscopic or microscopic, in the different tested culturing, results were more promising when compared to isolates of B. bassiana. In the macroscopic observation, all lyophilization. Furthermore, glycerol-freeze is a rapid the colonies presented a whitish-yellow mycelium, with method for preserving cultures, with no need of expensive aerial growth and cotton-like aspect, and regular borders equipment, and which allows a quick retrieval of conidia (Fig. 3a). The back of the colony also presented with a for use in laboratory assays. white color, although slightly more yellow in the center. Those characteristics are in accordance with those de- Acknowledgments Authors are grateful to Fundação para a Ciência e Tecnologia (FCT) for financial support (Project PTCD/AGR-AAM/ scribed for B. bassiana (Varela and Morales 1996; 102600/2008), and Ivo Oliveira for the PhD grant (SFRH/BD/44265/ Fernandes et al. 2006). Microscopic characteristics of B. 2008). bassiana are similar to those previously described for this fungus, either for hyphae or conidia (Liu et al. 2003). The hyphae were septated, with hyaline aspect and thin walls References (Fig. 3b), and conidia showed a rounded or ovoid shape, also with a hyaline aspect, and about 4 μm in diameter Akello J, Dubois T, Coyne D, Kyamanywa S (2009) The effects of (Fig. 3c). Beauveria bassiana dose and exposure duration on colonization Although no differences were observed in the spore and growth of tissue cultured banana (Musa sp.) plants. 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Journal

Annals of Microbiology – Springer Journals

Published: Nov 10, 2010

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