Table 1.
Isolates of Sclerotinia sclerotiorum used in the present experiment.
Table 2.
Concentration (ppm) of fungicide stock solutions and resultant concentration gradient when depositeda onto a 150 mm PDA plate in a concentric spiral pattern.
Fig 1.
Method for identification of 50–100% growth inhibition zone of Sclerotinia sclerotiorum isolates grown on a fungicide concentration gradient.
Mycelial growth perpendicular to the pre-inoculated filter strip after 40 hours on three replicated control plates were used to determine 100% growth (A), where A/2 is equal to 50% growth. At the same time point, mycelial growth on the fungicide-deposited gradient equivalent to distance A/2 was identified in both directions perpendicular to the pre-inoculated filter strip. Mycelium was harvested from the 50–100% inhibition zone in this experiment. To calculate the fungicide concentration at the 50% growth inhibition (EC50), an average of the two radial distances at which 50% inhibition was observed perpendicular to the filter strips was input into the SGE software. This example in the photos shows S. sclerotiorum growth on boscalid fungicide deposited in a concentric concentration gradient of 9.05 to 0.05 ppm from center to periphery of plate, with EC50 estimated as 0.14 ppm.
Fig 2.
Change in EC50 values of S. sclerotiorum isolates exposed to sublethal doses of boscalid over 12 generations for experiment 1 and 2; G1 to G12 correspond to each generation.
Fig 3.
Change in fungicide sensitivity (EC50) of eight isolates after twelve generations of growth on a fungicide gradient of the respective commercial fungicide: pyraclostrobin, iprodione, azoxystrobin, thiophanate methyl, and boscalid.
EC50 estimates were determined as the average of four replicates, where each point above represents the relative fold change in EC50 on an individual isolate basis, calculated as difference in EC50 at G12 and G0, divided by the EC50 at G0. Circles correspond with values from experiment 1 and triangles correspond to experiment 2, where solid filled shapes indicate EC50 values of G12 that were significantly different to G0, determined using a Student’s t-test (P ≤ 0.05).
Table 3.
Stepwise mutations at six microsatellite loci observed in eight Sclerotinia sclerotiorum isolates independently exposed for 12 generations to sublethal doses (50–100% inhibition) of five fungicides.
Fig 4.
Number of AFLP alleles detected in Sclerotinia sclerotiorum isolates exposed to sublethal doses of fungicides as well as non-exposed controls.
Isolates were exposed to fungicides for 12 generations before molecular characterization. Control isolates were characterized at the beginning (G0) of the experiment as well as after transferring for 12 generations (G12) on PDA without fungicide. Fungicides included boscalid (Bos), iprodione (Ip), thiophanate methyl (TM), azoxystrobin (Az), and pyraclostrobin (Py). Naming convention for each fungicide-exposed isolate is the fungicide used, isolate identification number, and experiment number; the experiment was repeated. All control isolates were from the first experiment and depicted as ‘Con’ followed by isolate name and generation (G0 or G12). 4A) Original number of alleles detected for each isolate. 4B) Number of alleles present after censoring AFLP data by removing loci polymorphic from G0 to G12 in the control for each isolate, resulting in the same multilocus genotype for the non-exposed control of each isolate.
Fig 5.
Neighbor joining (NJ) tree constructed from AFLP data of Sclerotinia sclerotiorum isolates exposed to sublethal doses of fungicides and non-exposed controls.
Roger’s similarity coefficient was used to calculate pairwise distances between isolates. Isolates prior to fungicide-exposure at the beginning (G0) of the experiment and after fungicide exposure for 12 generations (G12) were genotyped using AFLP markers. Non-exposed, negative control isolates were simultaneously grown for 12 generations and included in the analysis; the experiment was repeated. Eight isolates were independently exposed to four fungicides: boscalid (Bos), iprodione (Ip), thiophanate methyl (TM), azoxystrobin (Az), and pyraclostrobin (Py). Fungicide used, isolate identification number, and experiment number are given for each taxon. Control isolates are depicted as Con followed by isolates name and their generation (either G0 or G12). All control isolates were used from the first experiment. Bootstrap values above 50% are shown at the beginning of clusters. A) NJ tree for uncensored data. B) NJ tree constructed from censored AFLP data, where loci polymorphic from G0 and G12 in control isolates were removed and corresponding loci of fungicide-exposed isolates also removed (see Fig 4).
Fig 6.
Principal coordinate analysis (PCoA) of AFLP fragments of Sclerotinia sclerotiorum isolates before and after exposure to sublethal doses of fungicides.
Control isolates (both G0 and G12) are shown with a ‘C’ and fungicide-exposed isolates are denoted ‘T’ at the center of the respective groups, with each circle representing 95% of the variation associated with each group. Isolates Ip_467_Exp1, Az_646_Exp1, and Con_467_G0 have less than 90% membership probability to their respective treatment group; also shown in Fig 7.
Fig 7.
Heatmap of membership probabilities of Sclerotinia sclerotiorum isolates belonging to fungicide-exposed and non-exposed control clusters as shown in Fig 6.
Probability of 100% is shown in red and 0% probability is white. Blue crosses represent the prior cluster assignment for isolates. 4A) Membership of isolates pre-assigned to fungicide-exposed and non-exposed control groups, where isolates Az_646_Exp1, Con_467_G0, and Ip_467 had membership probability <90% to their respective group. 4B) Membership probabilities of isolates after censoring AFLP data by removing loci polymorphic from G0 to G12 in the control isolates to result in the same multilocus genotype for each non-exposed control isolate; all isolates show 100% membership probability.