Induction of Laccase Activity in Rhizoctonia solani by Antagonistic Pseudomonas fluorescens Strains and a Range of Chemical Treatments

Abstract

Fungi often produce the phenoloxidase enzyme laccase during interactions with other organisms, an observation relevant to the development of biocontrols. By incorporating the laccase substrate 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) into agar, we analyzed laccase induction in the plant-pathogenic fungus Rhizoctonia solani when paired against isolates of the soil bacterium Pseudomonas fluorescens. Substantial induction of R. solani laccase was seen only in pairings with strains of P. fluorescens known to produce antifungal metabolites. To study laccase induction further, a range of chemical treatments was applied to R. solani liquid cultures. p-Anisidine, copper(II), manganese(II), calcium ionophore A23187, lithium chloride, calcium chloride, cyclic AMP (cAMP), caffeine, amphotericin B, paraquat, ethanol, and isopropanol were all found to induce laccase; however, the P. fluorescens metabolite viscosinamide did not do so at the concentrations tested. The stress caused by these treatments was assessed by measuring changes in lipid peroxidation levels and dry weight. The results indicated that the laccase induction seen in pairing plate experiments was most likely due to calcium or heat shock signaling in response to the effects of bacterial metabolites, but that heavy metal and cAMP-driven laccase induction was involved in sclerotization.Rhizoctonia solani is a soil-living plant-pathogenic fungus which attacks a wide range of crop plants, including sugarbeet, potato, and rice. Seeds and seedlings are particularly susceptible to this fungus, which persists in soil in the form of resistant sclerotia. Fungicides can be used to control R. solani, but recently attention has focused on the development of biocontrol techniques. To this end, strains of the bacterium Pseudomonas fluorescens that are antagonistic to R. solani have been isolated and are currently being evaluated as biocontrol agents in our department. The most promising strain is P. fluorescens DR54, which produces the antifungal depsipeptide metabolite viscosinamide (18).Inevitably, most fungi will encounter competitive or antagonistic organisms, whether bacterial, fungal or animal, during their life cycles. Fungi engaged in such competition frequently produce secondary metabolites, extracellular phenol-oxidizing enzymes, and differentiated structures in the zone of conflict (2,4,8). These responses may be critical in determining the outcome of a biocontrol treatment.Our first aim was to study the induction pattern of the fungal phenol-oxidizing enzyme laccase in interactions between R. solani and P. fluorescens and how this related to the characteristics of different P. fluorescens strains. Laccase, ubiquitous in fungi and flexible in function (28), is often induced during antagonistic interactions, and R. solani has been shown to possess four laccase genes (29). Our second aim was to determine the pathways of laccase induction in R. solani and relate these to the effects of antagonistic bacteria. We believed that this work would aid the...

Insights into the development of fungal biomarkers for metal ecotoxicity assessment: Case of Trametes versicolor exposed to copper

“…Although fungi have mechanisms for metal tolerance [12], the activity of their extracellular enzymes has been shown to be modulated during the metal exposures in liquid cultures. Copper increases the extracellular laccase in different filamentous fungi [13][14][15]. It is also known that Cu induces the laccase transcription in Trametes versicolor [16].…”

Expression of the Laccase Gene from a White Rot Fungus in Pichia pastoris Can Enhance the Resistance of This Yeast to H 2 O 2 -Mediated Oxidative Stress by Stimulating the Glutathione-Based Antioxidative System

“…One study on the plant-pathogenic fun- As mentioned above, fungal laccase may be involved in the adaptive response to oxidative stress. However, the hypothesis that laccase plays an important role in defense against oxidative stress is mainly based on the phenomenon that laccase activity can be induced by various oxidative stress factors (2,12,16,20,(32)(33)(34). To our knowledge, there is little direct evidence supporting the involvement of laccase in the defense against oxidative stress.…”

“…One study on the plant-pathogenic fun-gus Rhizoctonia solani has revealed that copper, paraquat, and alcohol treatments, which are known to cause oxidative stress by promoting the formation of free radicals, can induce laccase activity and increase the level of lipid peroxidation. A straightforward link between oxidative stress and laccase induction was found in the case of paraquat treatment (20). A genetic study on another plant-pathogenic fungus, Fusarium oxysporum, has also suggested that laccase may have a role in protection against oxidative stress.…”

Melanogenesis in dermatophyte species in vitro and during infection

“…A qualitative laccase assay in dermatophytes was modified from Srinivasan et al (1995) and Crowe & Olsson (2001) by evaluating the oxidation of 2,29-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Sigma). The assay buffer used was a mixture of 0.1 M boric acid, 0.1 M acetic acid and 0.1 M phosphoric acid (Britton-Robinson buffer) adjusted to pH 5.0 with NaOH (Wahleithner et al, 1996).…”