Vol.18_No.2 - Pesticide Alternatives Lab - Michigan State University
Vol.18_No.2 - Pesticide Alternatives Lab - Michigan State University
Vol.18_No.2 - Pesticide Alternatives Lab - Michigan State University
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Spring 2009 Resistant Pest Management Newsletter Vol. 18, No. 2<br />
The genotype (C to T mutation) of the<br />
boscalid-resistant isolates was confirmed using As-<br />
PCR analysis. DNAs extracted from mutants carrying<br />
this mutation were amplified while no products were<br />
obtained after amplification of the DNA from boscalidsensitive<br />
isolates and mutants without this mutation.<br />
The PCR-RFLP method was successfully used to<br />
diagnose the mutants with the A to G mutation. The<br />
primer pair amplified an expected 300-bp DNA<br />
fragment for all boscalid-resistant and –sensitive<br />
isolates tested. The PCR products were then treated<br />
using a mutation specific enzyme AciI. The PCR<br />
products from resistant mutants carrying the mutated<br />
sequence CGC at position 997 were digested into two<br />
fragments 210 and 90-bp in length on agarose gels,<br />
whereas products from sensitive isolates and other<br />
mutants remained undigested.<br />
DISCUSSION<br />
The sampled A. alternata isolates were<br />
divided into two populations based on their response in<br />
boscalid amended medium. These results showed that<br />
the use of Pristine® can promote the selection of A.<br />
alternata boscalid resistant subpopulations that could<br />
compromise its use.<br />
Resistant strains are frequently cross resistant<br />
to structurally related chemicals or to chemicals with<br />
similar mode of action (Delp, 1980). In this study, we<br />
have established the existence of a cross resistance<br />
relationship between boscalid and carboxin in A.<br />
alternata. Cross resistance relationship has been<br />
demonstrated in the carboxamide group for flutolanil<br />
and carboxin (Ito et al., 2004).<br />
The rapid appearance of boscalid resistant<br />
populations of A. alternata in pistachio orchards, as<br />
was the case in the development of resistance to Q o Is,<br />
indicates that it is imperative to monitor boscalid<br />
resistance in A. alternata in pistachio orchards when<br />
boscalid-containing fungicides are used extensively to<br />
combat Alternaria late blight. The partitioning in two<br />
distinct groups of sensitivity to boscalid suggests that<br />
the resistance to boscalid occurred as a result of<br />
disruptive selection and is likely to be monogenic in<br />
nature, as expected for single-site inhibitor fungicides.<br />
This type of resistance is known to be one of the most<br />
important factors contributing to the rapid appearance<br />
and spread of fungicide resistant alleles (Milgroom,<br />
1990) and a continual increase in the frequency of<br />
isolation of the boscalid-resistant isolates, in other<br />
pistachio orchards where resistance has not been<br />
detected, is thus predictable.<br />
An important step in establishing resistance<br />
management strategies consists at elucidating the<br />
molecular basis of resistance which will enable to<br />
develop a quick method for these monitoring studies.<br />
Studies on the molecular mechanisms responsible for<br />
the acquisition of resistance to boscalid analogfungicide<br />
in resistant populations of some<br />
Basidiomycete and Ascomycete fungi and bacteria<br />
have shown that mutations in conserved regions of the<br />
genes SdhB, SdhC and SdhD encoding their molecular<br />
targets result in reduced sensitivity (Broomfield &<br />
Hargreaves, 1992; Ito et al., 2004; Matsson et al., 1998;<br />
Skinner et al., 1998). Given the cross resistance<br />
relationship between boscalid and carboxin, we implied<br />
that boscalid resistance in A. alternata could be<br />
governed by similar mutations in the target genes.<br />
In this study we targeted the SDHB gene of<br />
this fungus and showed that a substitution of a<br />
conserved histidine residue within the third Cys-rich<br />
cluster that takes part in binding of iron-sulfur centers<br />
correlated with the phenotype of resistance as it was<br />
described in other fungal species. Other resistant<br />
isolates did not have the mutation in the histidine<br />
codon. This finding suggests that other loci could<br />
confer the resistant phenotype to such mutants. The<br />
molecular characterization of boscalid resistance in A.<br />
alternata isolates will allow the development of full<br />
methods that will facilitate sensitivity monitoring<br />
20