|DE CRECY, EUDES - Evolugate, Llc|
|LYONS, BENJAMIN - Evolugate, Llc|
|LYONS, THOMAS - Evolugate, Llc|
|KEYHANI, NEMAT - University Of Florida|
Submitted to: BMC Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2009
Publication Date: 8/26/2009
Citation: De Crecy, E., Jaronski, S., Lyons, B., Lyons, T.J., Keyhani, N. 2009. Directed Evolution of a Filamentous Fungus for Thermotolerance. BioMed Central (BMC)Biotechnology. 9:74 doi:10.1186/1472-6750-9-74.
Interpretive Summary: Using a machine assisted, automated, continuous culture machine we selected an entomopathogenic fungus for greater heat tolerance than it normally displays. Whereas the fungus, Metarhizium anisopliae Strain ARSEF 2575, normally does not grow above 33-34 degrees Celsius, the selected clones were able to grow readily at 37 C. But there were tradeoffs. One heat tolerant clone partially lost ability to sporulate (reproduce), the other almost completely. Both lost a degree of infectivity at room temperature (28 C.) for insects, specifically for the grasshopper, Melanoplus sanguinipes; the Median Lethal Dose (LD50) of both heat-tolerant clones was significantly greater than the original strain. Virulence, as measured by the speed of kill or Median Survival Time, was decreased in one selected clone but not the other, compared to the parent fungus. When grasshoppers were treated with either of the two clones or the parent fungus, and held at 36 C (rather than the normal 28 C), none died of fungal infection; the higher temperature evidently blocked fungus growth within the insects and pathogenesis, even though the heat tolerant strains could grow in vitro. Our data indicate that complex phenotypic consequences can occur during selection but that pathogenicity can be maintained during adaptation. Further, in vitro phenomena, such as ability to grow at a higher temperature in artificial media, may not be reflected in vivo such as within the body of an insect.
Technical Abstract: Filamentous fungi represent the most widely used eukaryotic biocatalysts in industrial and chemical applications. Metarhizium anisopliae is a broad-host-range entomopathogenic fungus currently under intensive investigation as a biologically based alternative to chemical pesticides. One of the most pressing problems regarding the application of fungi for pest control is the relatively low tolerance of these organisms to abiotic stresses such as heat, with most strains displaying little to no growth at temperatures near 35-37oC. In this study we have used a natural selection-adaptation strategy to select for thermotolerant isolates of M. anisopliae that display robust growth at 37oC. M. anisopliae strain 2575 was adapted for growth at 37oC using a novel automated continuous culture machine that can be used in either turbidostat- or chemostat-like modes to actively select fast-growing variants. The use of flexible tubing as growth chamber allowed for the continuous culture of fungal cells in a closed system without common problems such as wall growth. Selection of thermotolerant strains proceeded via 22 dilution cycles of the machine over a 4 month time course of culture growth. Two strains EVG016 and EVG017 were isolated and their thermal growth characteristics were measured. Both strains displayed robust growth at 37oC, whereas EVG017 but not EVG016 also was able to grow at 37.5oC. Neither the mutant strains nor the wild-type isolate were virulent at 37oC. Insect bioassays performed at 28oC using Melanoplus sanguinipes (grasshoppers) revealed increased lethal dose responses (LD50) for EVG016 and EVG017g (a sporulating variant of EVG017) as compared to the wild-type parent strain. In contrast to the LD50, the median survival time (MST50) was significantly reduced for EVG017g, whereas EVG016 and the wild-type gave similar values. We report the first experimental evolution of a filamentous fungus using a novel continuous culture device. Temperature adapted strains of the insect-pathogenic, filamentous fungus M. anisopliae were isolated and demonstrated to show robust growth at a temperature that is inhibitory for the parent strain. Insect virulence assays revealed differential consequences with regards to virulence parameters. EVG016 displayed a decreased virulence attributed due a larger LD50, whereas EVG017g displayed increased virulence due to a smaller MST50. These data indicate that complex phenotypic consequences can occur during selection but that pathogenicity can be maintained during adaptation. In principle, this technology can be used to adapt fungal strains to virtually any environmental condition including abiotic stress and growth substrate utilization.