Entomopathogenic fungal (Hyphomycetes) collection: assessment of conidial viability

Coleção de fungos entomopatogênicos (Hyphomycetes): avaliação da viabilidade conidial

NOTAS CIENTÍFICAS

ENTOMOPATHOGENIC FUNGAL (HYPHOMYCETES) COLLECTION:

ASSESSMENT OF CONIDIAL VIABILITY¹

MARCOS RODRIGUES DE FARIA², IRENE MARTINS³, RAQUEL MELLO⁴ AND MYRIAN SILVANA TIGANO⁵

ABSTRACT - Twenty four strains of the entomopathogenic fungi (Hyphomycetes) Beauveria bassiana, Metarrhizium anisopliae, Nomuraea rileyi, Paecilomyces farinosus, P. fumosoroseus and P. lilacinus, maintained in the culture collection of Embrapa-Centro Nacional de Pesquisa de Recursos Genéticos e Biotecnologia (Cenargen) and preserved by lyophilization and in liquid nitrogen, had their conidial viability assessed. Germination rates of 16- to 84-month-old cultures stored in liquid nitrogen decreased, on average, less than 13.3%. For 29- to 49-month-old cultures preserved by lyophilization, the viability loss ranged, on average, from 28.6 to 94.5%. The results demonstrated the efficiency of the tested methods, especially liquid nitrogen, in preserving the viability of entomopathogenic fungi.

COLEÇÃO DE FUNGOS ENTOMOPATOGÊNICOS (HYPHOMYCETES): AVALIAÇÃO DA VIABILIDADE CONIDIAL

RESUMO - Vinte e quatro isolados dos fungos entomopatogênicos (Hyphomycetes) Beauveria bassiana, Metarrhizium anisopliae, Nomuraea rileyi, Paecilomyces farinosus, P. fumosoroseus e P. lilacinus, mantidos na coleção de culturas da Embrapa-Centro Nacional de Pesquisa de Recursos Genéticos e Biotecnologia (Cenargen) e preservados em nitrogênio líquido e liofilização, tiverama viabilidade conidial avaliada. As taxas de germinação de conídios preservados em nitrogênio líquido diminuíram, em média, menos de 13,3% após 16 a 84 meses de armazenamento. A perda de viabilidade de isolados liofilizados, após 29 a 49 meses de armazenamento, variou de 28,6 a 94,5%. Os resultados demonstraram a eficiência dos métodos testados, especialmente nitrogênio líquido, na preservação da viabilidade de fungos entomopatogênicos.

In Brazil, the occurrence of over 20 genera of entomopathogenic fungi has been reported (Alves, 1992). Embrapa-Centro Nacional de Pesquisa de Recursos Genéticos e Biotecnologia (Cenargen), in Brasília, Brazil, is engaged in establishing an entomopathogenic fungal culture collection (Embrapa, 1996), to support biocontrol research involving these microorganisms. Activities such as acquisition, preservation, identification and distribution of strains have been carried out during the last eight years. Currently, this collection holds approximately 600 strains belonging to 16 species and eight genera with potential for use in biocontrol programs.

This study was carried out to quantitatively determine the viability of entomopathogenic fungi after long-term preservation, considering the lack of information regarding the storage of this group of natural enemies. Fungal strains belonging to species Beauveria bassiana, Metarrhizium anisopliae, Nomuraea rileyi, Paecilomyces farinosus, P. fumosoroseus and P. lilacinus were used (Table 1). Conidial viability of tested strains was not determined months before, when the preservation methods were applied. Therefore, germination rates of both fresh conidia and 24 hours following preservation procedures were estimated to allow assess the loss of viability over time. In order to do that, cultures originally stored in liquid nitrogen using 10% glycerol as protectant were thawed, and a conidial suspension plated on complete medium (FeSO₄ 0.001 g; KCl 0.5 g; KH₂PO₄ 1.5 g; NaNO₃ 6 g; ZnSO₄ 0.001g; hydrolyzed casein 1.5 g; yeast extract 0.5 g; glucose 10 g; peptone 2g; agar 20g, per 1 liter), followed by incubation at 28°C in darkness. N. rileyi strains were cultivated on SMAY (Sabouraud Maltose Agar with yeast extract 10g; maltose 40 g; neopeptone 10 g; agar 20 g; per 1 liter) at 25°C. For each strain, conidia produced on the surface of culture media after 10 days incubation were harvested with a spatula and added to an aqueous suspension (0.1% Tween 80).

To determine viability before applying the preservation procedure, 100 mL of the conidial suspension containing approximately 8x10⁶ conidia mL^-1 were spread on solid culture medium. Viability of cultures was estimated by microscopically assessing the conidial germination percentage between 18 and 48 hours following incubation, depending on tested strain. One hundred conidia were counted in each of four microscopic fields.

For lyophilization of conidia, a mixture of3% glucose (Merck, Rio de Janeiro, Brazil) and 3% gelatin (Sigma, St. Louis, MO, USA) was used as the suspending medium. Glass ampoules (100 x 6 mm) containing cultures were kept at -80°C for 30 minutes before transferring to a Lyph-Lock 18 lyophilization apparatus (Labconco, Missouri, USA). After overnight lyophilization, ampoules were sealed under vacuum and stored at 4°C. For cryopreservation, a conidial suspension was prepared in 10% glycerol and transferred to 1.0 mL Nunc cryotubes. The cryotubes were kept at -20°C for approximately 20 minutes, before transferring to liquid nitrogen (-196°C).

Following storage for 24 hours in liquid nitrogen and by lyophilization, cryotubes and ampoules, respectively, had their contents resuspended in a 0.1% Tween 80 solution. Samples kept in liquid nitrogen were directly removed to room temperature. The percentage of conidial germination was then assessed as indicated above. To estimate the efficiency of the two methods after long-term storage, the same procedure was used for cultures kept in liquid nitrogen for periods varying from 16 to 84 months, and lyophilized cultures stored for 29 to 49 months. There was only one experimental replicate per strain because a limited number of vials, usually two, were stored months earlier.

A small reduction in conidial viability was observed 24 hours following storage in liquid nitrogen. The average reduction of germination ranged from 0.1 to 10.3%, with best results being observed for B. bassiana and Paecilomyces spp. (Table 2). For long-term storage in liquid nitrogen, the average reduction of germination varied from 0.2 to 5.9%, with the exception of N. rileyi. Considerable intraspecific variation was recorded for N. rileyi, for which the estimated total viability reduction ranged from 0 to 29.9%. For B. bassiana, P. farinosus and P. fumosoroseus the reduction of viability was more severe during the storage period, when compared to M. anisopliae and most strains of N. rileyi, for which the loss of viability seemed to be greater during the storage procedure itself.

Following lyophilization, B. bassiana and Paecilomyces spp. showed low loss of viability after 24 hours. Reduction in germination rates ranged from 3.1% for P. lilacinus to 42.9% for M. anisopliae. After long-term storage, the estimated loss of viability varied from 28.6% for P. lilacinus to 94.5% for M. anisopliae. In the present study, total loss of viability for P. lilacinus varied from 17.9 to 37.5% after four years storage. Recently, Rybnikár (1995) reported losses in viability during storage ranging from 30.4 to 96.6% for 26 lyophilized cultures; for instance, the loss of viability for P. lilacinus was 56.1% after 11 years.

Our results demonstrated an evident reduction of viability of M. anisopliae and N. rileyi due to the storage procedure, especially when lyophilized. For both liquid nitrogen storage and lyophilization, it was observed that for some strains the viability reduction due to the storage procedure was slightly greater than the estimated total loss of viability. The use of one replicate for each strain and the utilization of viability loss based on conidial germination of the strains stored at different times may explain these results.

With the exception of a few reports (Bunse & Steigleder, 1991; Rybnikár, 1995), in which the reduction in viability of lyophilized cultures was quantified by recording the number of Colony Forming Units (CFUs), previous publications dealing with the efficiency of long-term preservation of fungi considered only the capability of growth on synthetic media. According to this criterion, all strains used in the present study were viable following lyophilization, although the germination rates were extremely low in certain cases. For example, the germination rate of strain CG334 (P. fumosoroseus) was 0.5% after 47 months storage.

For the six strains stored in both liquid nitrogen and lyophilization, viability 24 hours after storage and following long-term storage (16 to 84 months) was considerably higher when cultures were preserved in liquid nitrogen. For cultures maintained in liquid nitrogen, loss of conidial viability following preservation for 24 hours and long periods varied from 0 to 8.5% and from 0 to 23.5%, respectively. In contrast, losses for lyophilized cultures ranged from 2.2 to 35.9% and 28.5 to 96.9%, respectively. The satisfactory results obtained with cryopreservation in this study support the idea that liquid nitrogen is the most reliable method for long-term preservation of fungi (Goos et al., 1967; Stalpers et al., 1987).

Although attempts to improve conidial viability oflyophilized fungal cultures were not successful, such as the combinations of protectants [skim milk (10%), glutamic acid (5%) and raffinose (10%)] suggested by Berny & Hennebert (1991) and the use of another lyophilization apparatus (Supermodulyo 12K Freeze Dryer, Edwards, USA), all strains tested showed some degree of viability.

According to Kirsop & Doyle (1991), different taxonomic groups and even strains within a given species can exhibit considerable differences in response to stresses imposed by the storage procedure itself and/or recovery methods used. Rate of cooling, size of propagules, and thickness of cell wall are important parameters not considered in this study (Berny & Hennebert, 1991; Schoenlein-Crusius et al., 1993; Tan et al., 1995), and they may explain the observed intra and interspecific variability of fungal cultures as measured by conidial viability after different storage periods.

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Publication Dates

• Publication in this collection
  03 Jan 2006
• Date of issue
  Aug 1999

History

• Received
  16 Nov 1998
• Accepted
  16 Nov 1998