Elucidation of underlying mechanisms of pathogenesis in the entomologenous fungus Metarhizium anisopliae

Ibrahim, Ludmilla M (2003). Elucidation of underlying mechanisms of pathogenesis in the entomologenous fungus Metarhizium anisopliae. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f735

Abstract

In order to determine the effects of nutrition on underlying mechanisms of attenuation of the entomogenous fungus Metarhizium anisopliae, a series of in vitro and in vivo studies were undertaken. Nutritional composition of culture media influenced germination, growth, conidiation, yield, and accumulation of endogenous reserves, conidial attributes and virulence. Addition of KCl to SDA media (SDAM) significantly reduced germination of the three isolates in vitro and lowered germination and appressorial development on the cuticles of potato-peach aphids and pollen beetles. Conidia grown on SDAM and minimal medium (MM) were more aggressive than conidia derived from SDA or Yeast Extract Agar (YEA). Results also identified that culture media influenced virulence of M. anisopliae isolates by influencing the fungal physiological qualities such as presence or absence of erythritol and arabitol, and the balance of carbon and nitrogen (C:N ratio) in conidia. Dark, pigmented conidia from aphid cuticles or nutrient-limited MM media bound less lectins and calcofluor, and accumulated higher concentration of polyols and proteins, had higher germination potential when placed on insect cuticles, formed appressoria more frequently and were highly efficacious against aphids and pollen beetles than conidia from nutrient-rich SDA media. The level of virulence was also affected by the ability of conidia to adhere to the cuticle of aphids and beetles. A strong linear relationship between fluorescence intensity of calcofluor treated conidia and the number of conidia adhered to the insect cuticles, where increasing spore fluorescence corresponded to increased spore adhesion, suggested an important role of β-glucans in pathogen-host interaction mechanism.

Further studies were conducted to determine the effects of repeated sub-culturing on nutrient-poor and -rich media on morphology, phenotype, pathogenicity and physiology of resultant inocula. Results revealed that eleven consecutive passages of the three M anisopliae isolates on SDA media significantly changed colony colour, increased sectoring and production of sterile colonies, reduced sporulation and germination potential of harvested conidia. In contrast, passaging on MM eleven times in succession had no effect on the morphology or phenotype of the pathogen. Continuous sub-culturing on both SDA and MM media influenced the ability of M. anisopliae isolates to absorb various carbohydrates including glucose. This in turn, influenced the accumulation of reserves carbohydrates such as glycerol, mannitol and affected the expression of surface carbohydrates such as β-glucans and surface proteins such as hydrophobins. Increased concentration of endogenous carbon, polyols, surface carbohydrates and hydrophobins in attenuated inocula were associated with increased adherence to and reduced germination on aphid cuticles. Reduced abilities of attenuated inocula to germinate faster and failure to produce appressoria resulted in increased LT50 values and thus reduced virulence.

There was no link established between virulence, as measured by LT50 values, and Prl production. Prl gene expression was observed in both attenuated and non-attenuated subcultures of M. anisopliae isolates, suggesting that repeated sub-culturing had no effect on gene expression. Although pathogenicity of the pathogen was not affected by repeated sub-culturing on both SDA and MM media, the key attributes of virulence such as LT50 and LC50 were significantly affected, where increased time and dose taken to initiate and complete disease development corresponded with increased LT50 and LC50 values.