Alternative Respiration: a Biochemical Mechanism of Resistance to Azoxystrobin (ICIA 5504) in Septoria tritici

Abstract

: The mechanism of resistance to ICIA 5504 (azoxystrobin) in a Septoria tritici mutant raised in the laboratory has been investigated. This mutant was approximately 10 times less sensitive than the wild-type strain in in-vitro tests towards spore germination or fungal growth. Glucose oxidation in whole cells was inhibited in the wild type (80% inhibition at 0É1 kg ml~1), whereas in the resistant mutant, oxygen uptake was stimulated (50% stimulation at 1É0 kg ml~1). Respiration of the wild-type strain was inhibited by antimycin A and cyanide but not that of the mutant. These results indicate the existence of an efficient alternative respiratory pathway in the mutant, which was inhibited by the addition of 2 mM salicylhydroxamate (SHAM). Using mitochondria, antimycin A and ICIA 5504 did not completely inhibit NADH oxidation in either strain. Addition of SHAM inhibited part of the antimycin-and ICIA 5504-insensitive oxygen uptake only in mutant mitochondria. For complete inhibition of oxygen reduction, SHAM and cyanide need to be present. Thus, three systems of electron transfer from exogenous NADH to oxygen are present in S. tritici mitochondria : the cytochrome pathway which is sensitive to ICIA 5504 and antimycin A inhibition in both strains, the system of NADH-cytochrome c reductase which bypasses the methoxyacrylate inhibition at the cytochrome complex, bc 1 and the alternative oxidase which is inhibited by SHAM, and which is partially functioning only in mitochondria isolated from the ICIA 5504-resistant mutant.When the S. tritici isolates were tested for their in-vivo sensitivity to ICIA 5504 on wheat, the resistant strain was controlled better than the wild type. This indicates that the decreased ATP formation by the alternative pathway of respiration was inadequate for efficient parasitic growth on the host.

In vitro toxicity of alternative oxidase inhibitors salicylhydroxamic acid and propyl gallate on Fusicladium effusum

“…However, there are very few examples of such studies that included any biochemical evidence of the suppression of AOX, e.g., measurements of oxygen uptake. Based on direct measurements of oxygen uptake, 2 mM SHAM was sufficient to suppress AOX in Mycosphaerella graminicola (Fuckel) (Ziogas et al 1997 of SHAM (0.653 and 0.2 mM) were used against the same fungal organism Venturia inaequalis ((Cooke) Winter) (Köller et al 2004;Steinfeld et al 2001). Different concentrations of SHAM were used against the different developmental stages of Magnaporthe oryzae ((Hebert) Barr), 0.653 mM SHAM for conidia and 0.98 mM SHAM for the mycelial stage; however, SHAM inhibited conidial germination by 3-14% (Avila-Adame and .…”

“…Due to the inhibition of electron flow through complex III by QoI fungicides, an alternative respiration system located on the mitochondrial membrane can be activated to avoid the fatal effects of the fungicide (Kameko and Ishii 2009;Wood and Hollomon 2003;Ypema and Gold 1999). Therefore, it is a common practice to include alternative oxidase (AOX) inhibitors, e.g., salicylhydroxamic acid (SHAM), in the medium to suppress alternative respiration (Avila-Adame et al 2003b;Avila-Adame and Köller 2003a;Kameko and Ishii 2009;Markoglou et al 2006;Malandrakis et al 2006;Jones et al 2007;Ishii et al 2009;Kim et al 2003;Köller et al 2004;Pasche et al 2004;Steinfeld et al 2001;Walker et al 2009;Wood and Hollomon 2003;Ziogas et al 2002;Ziogas et al 1997).…”

Eficiência de fungicidas sistêmicos para o controle de Cylindrocladium candelabrum em eucalipto

“…A baixa sensibilidade micelial a determinadas estrobilurinas, também, já constatada em outras espécies fúngicas, pode estar associada à indução da respiração alternativa que catalisa a transferência de elétrons para o oxigênio (Ziogas et al, 1997;Olaya & Köller, 1999;Ypema & Gold, 1999). Essa rota alternativa é utilizada por fungos que crescem em meios de ágar, especialmente aqueles ricos em nutrientes (Reuveni & Shelgov, 2002).…”

“…A rota alternativa referenciada também foi induzida quando se utilizaram derivados de estrobilurinas (Mizutani et al, 1995;Ziogas et al, 1997). Carbendazim não reduziu a germinação de conídios e o crescimento micelial de C. candelabrum.…”

Mechanisms and significance of fungicide resistance

“…2b, 3), modifications of plasma membranes causing reduced fungicide permeability, or by synthesis of enzymes that degrade fungicide molecules (13,24,32). Alternatively, overexpression of the gene encoding the fungicide target or utilization of alternative metabolic pathways has been reported (28,34,45,52,57,58). In general, populations consist of individuals with more or less pronounced levels of tolerance to fungicides (Fig.…”