Screening and characterization of resistance to succinate dehydrogenase inhibitors in Alternaria solani

Abstract

Field isolates of Alternaria solani, which causes early blight of potato in Idaho, USA were evaluated in vitro for their sensitivity towards the succinate dehydrogenase inhibitor (SDHI) fungicides boscalid, fluopyram and penthiopyrad. A total of 20 isolates were collected from foliar-infected tissue in 2009, 26 in 2010 and 49 in 2011. Fungicide sensitivity was tested using the spiral-gradient end point dilution method. The frequency of boscalid-resistant isolates (>50% relative growth when using a spiral dilution gradient starting at 507 mg L À1 ) drastically increased over the duration of this study (15% in 2009, 62% in 2010 and 80% in 2011). Increasing resistance to fluopyram and penthiopyrad was observed. However, cross-resistance was only observed between boscalid and penthiopyrad. The target site of this fungicide class is the succinate dehydrogenase (SDH) enzyme complex, which is vital for fungal respiration. Sequence analysis of the SDH complex revealed mutations in the subunits B and D that were correlated with the emergence of boscalid resistance in potato fields in Idaho. In particular, H277R and H133R were identified in SDH subunits B and D, respectively. The presence of restriction sites in the gene sequences allowed the development of a rapid PCR-RFLP method to assess boscalid sensitivity in A. solani populations.

Sensitivity of Alternaria alternata from Citrus to Boscalid and Polymorphism in Iron-Sulfur and in Anchored Membrane Subunits of Succinate Dehydrogenase

“…This is in agreement with our results, where the highest polymorphism was observed in the SDHD subunit. The occurrence of multiple mutations in the SDH complex of individual isolates has been previously reported in resistant isolates of A. solani (32) and Mycosphaerella graminicola (19). In contrast, multiple mutations in individual isolates of A. alternata from pistachio were not identified (5,8).…”

“…However, Hu et al (24) demonstrated that YBA did not support mycelial growth of Monilinia fructicola isolates and, therefore, could not be used to test fungicide sensitivity for this species. Furthermore, the fungal growth inhibition for SDHIs has been performed using conventional media such as PDA (3,5,9,32,54), MM (3,24), and Sabouraud dextrose (19). Due to the inconsistent and sometimes contradictory information published, the validation of different nutrient media and methods to be used for boscalid sensitivity tests is necessary before screening a large set of isolates of the target population in a system that has never been previously tested.…”

“…Additionally, analysis of the SDHC and SDHD subunits of SDHI-resistant isolates showed the substitution of histidine by arginine in SDHC (H134R) and SDHD (H133R) subunits, both of them implicated in the heme b ligation, as well as the substitution of aspartate by glutamic acid at position 123 (D123E) in SDHD (5). More recently, Miles et al (32) reported that, in addition to the mutations mentioned above, mutations T28A and A47T in the SDHD subunit were also observed in some moderately resistant isolates of A. solani. Thus far, more than 27 mutations conferring resistance to SDHIs have been reported in field and laboratory isolates of different plant pathogens (40).…”

“…Recently, different methods to measure the antifungal activity at different fungal stages have been proposed for testing sensitivity to SDHIs, such as a microtiter test (41,44) and a spiral-gradient endpoint dilution method (3,32). In addition to the standard techniques such as fungicide-amended agar plates used to measure the inhibition of mycelial growth or conidial germination, the microtiter test could be used for a rapid phenotypic characterization using automated equipment that quantitatively measures the fungicide activity.…”

Toxicity of mancozeb, chlorothalonil, captan, fluopyram, boscalid, and difenoconazole toDidymella applanataisolates from Serbia

“…Increasing resistance to fluopyram was recorded in Alternaria solani isolates originating from Idaho, but contrary to our results, no crossresistance between fluopyram and boscalid was observed. [31] These opposed findings indicate that additional research is indisputably needed to provide the crucial information for successful disease managementwhether cross-resistance between these two SDHI fungicides exists. However, it should be emphasized that different levels of sensitivity to SDHIs were found in closely related species D. bryoniae.…”