Detection and characterisation of pr1 virulent gene deficiencies in the insect pathogenic fungus Metarhizium anisopliae

Abstract

In an initial attempt to characterise single-spore isolates from Metarhizium anisopliae wild-type strain V275, three genetically identical spontaneous pr1A-and pr1B-deficient mutants were detected. These mutants were identified by Nested-PCR amplification and verified by Southern hybridisation. Sequencing and comparison of the highly conserved sequences of 28S rDNA domains 9^11 confirmed that the mutant isolates were Metarhizium anisopliae, but each had both morphological and genetic differences from the wild-type parent. RAPD analyses indicated that the overall genetic similarity between wild-type strain and the mutant was less than 70%. Enzyme assays proved that the mutant isolates had significantly lower Pr1 and elastase activities. Bioassays showed reduced lethal activity of the mutants (20%) on Tenebrio molitor, but lethal activities were similar to those of the wild-type parent on the larvae of Galleria mellonella. This paper shows for the first time that a stable mutant strain lacking the most important pr1 genes is still able to infect its respective hosts. Our observations suggest that it is not uncommon for Metarhizium anisopliae to lose its virulence genes during maintenance on artificial medium. ß

Basic Leucine Zipper (bZIP) Domain Transcription Factor MBZ1 Regulates Cell Wall Integrity, Spore Adherence, and Virulence in Metarhizium robertsii

“…For liquid incubation, fungi were grown in Sabouraud dextrose broth (SDB; Difco) with or without 50 g/ml Congo red (Bio Basic Inc.) at 25°C for 18 h in a rotary shaker. To induce protease expression, fungal cultures were grown in a minimum medium containing 1% silkworm pupa homogenate at 25°C in a rotary shaker (27). For mycotoxin production assays, the WT and mutants were grown in Czapek Dox broth at 25°C for 1 week in a rotary shaker (28).…”

“…The mycelia were then washed twice with sterile-distilled water, transferred to a basal salt medium supplemented with 1% (w/v) homogenized silkworm pupae, and incubated at 25°C for 48 h at 180 rpm (27). The alkaline protease activities were assayed using the substrate N-succinyl-Ala 2 -Pro-Phe p-nitroanilide (Sigma) as described previously (38).…”

A Novel Role for an Insect Apolipoprotein (Apolipophorin III) in β-1,3-Glucan Pattern Recognition and Cellular Encapsulation Reactions

“…Conidia were prepared as previously described by Wang et al (37), and aliquots of 80 l of conidia (1 ϫ 10 7 ml Ϫ1 ) were suspended in GPS containing 0.05% Tween 80. To obtain supernatant, conidia were centrifuged at 15,000 ϫ g. Sodium chloride elution of bound apoLp-III was not performed, as too many other proteins would have been coeluted from the conidial surface.…”

Optimization of the Chitinase Production by Different <i>Metarhizium anisopliae</i> Strains under Solid-State Fermentation with Silkworm Chrysalis as Substrate Using CCRD

“…On the other hand, the use of low cost substrates has a significant advantage under economic view, reducing the production costs in the industry. Many works showing chitinase production have used colloidal chitin or crab and shrimp shell powder [22][23][24][25] as the main substrate/carbon source. Chitinase production using arthropods cuticle was also demonstrated by Da [4].…”