Vol.18_No.2 - Pesticide Alternatives Lab - Michigan State University

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