In vivo sensitivity reduction of Puccinia triticina races, causal agent of wheat leaf rust, to DMI and QoI fungicides

Palavras-chave adicionais: IC50, fungicida resistência, fator de redução da sensibilidade. Experimentos foram conduzidos para determinar, in vivo a IC50 e CI 90 para fungicidas inibidores da demetilação (IDM)(triazois) e inibidores da quinona externa (IQe) (estrobilurinas) a cinco raças de Puccinia triticina mais frequentes na safra 2007, no Sul do Brasil. Os experimentos foram realizados em câmara de crescimento com plântulas de tr igo. Os fungicidas IDMs foram testados nas concentrações em mg/L de 0,0; 0,02; 0,2; 2,0; 20,0; 100,0 e 200,0 e os IQes nas concentrações de 0,0; 0,0001; 0,001; 0,01; 0,1, 1 e 10,0 mg de ingrediente activo/L de água. Os fungicidas foram aplicados preventivamente 24 horas antes da inoculação das plântulas com os Arduim, F.S.; Reis, E.M.; Barcellos, A.L.; Turra, C. Redução da sensibilidade de raças de Puccinia triticina, agente causal da ferrugem da folha do trigo, aos fungicidas DMI e QoI, in vivo. Summa Phytopathologica, v.38, n.4, p.306-311, 2012.

In vivo sensitivity reduction of Puccinia triticina races, causal agent of wheat leaf rust, to DMI and QoI fungicides Additional keywords: IC 50 , fungicide resistance, sensitivity reduction factor.

ABSTRACT
Diseases are among the main factors that have limited wheat production in Brazil. Most of the fungal diseases are favored by weather conditions with predominance of high temperature and frequent rainfall (2). Among the main fungal diseases of wheat, leaf rust, caused by Puccinia triticina Eriks, is considered one of the most important crop diseases causing damage of up to 63% (2, 5).
The main strategies for leaf rust control have been the development and the cultivation of cultivars with genetic resistance and the application of fungicides to above-ground plant parts (26,27). The genetic resistance of wheat to P. triticina is not durable due to the high variability of this fungus, resulting in the occurrence of at least one new virulent race in almost each wheat growing season (6). The earliest identification of leaf rust physiologic races in Brazil dates from 1949 and started in the former "Instituto Agronômico do Sul" (IPEAS -Pelotas-RS). There are reports of 26 races identified in samples collected from 1949 to 1952, with the use of an international differential set. In the period from 1952 to 1957, using a group of additional cultivars to the international set, 24 P. triticina races were determined (2). Until 2007 growing season, 60 races of the fungus were identified in Brazil (Barcellos & Chaves 2003). In this pathosystem, genetic variability (vertical resistant genes) is well known for the host and the fungus concerning virulence (2, 3, 7, 8, 24).
In 2006/2007 growing season, after 16 years of use of the fungicide tebuconazole (23), 14 years of cyproconazole (24) and seven years of epoxiconazole (25), complaints arose from farmers and technicians about the low efficiency of leaf rust control in commercial wheat fields, to where demethylation inhibitor (DMI) fungicides were applied alone. This fungicide group was most efficient in controlling wheat leaf rust, especially tebuconazole, which as widely used.
A hypothesis for the control failure observed in several wheat fields was that such inefficiency could be attributed to the reduced sensitivity of the fungus to the DMI fungicides used for 16 years and to new P. triticina races. To test this hypothesis, the present study was conducted to compare, based on the IC 50 and IC 90 values, the sensitivity of P. triticina races to demethylation inhibitors (DMI) and quinone outside inhibitors (QoI).

MATERIAL AND METHODS
The experiment was conducted at OR -Seed Improvement Ltd. and at the Laboratory of Plant Pathology -Mycology, University of Passo Fundo, Brazil, from July 2007 to July 2008.
The sensitivity of current races (including the dominant ones) was compared to an older race MCG-MN (B34), isolated from Ônix cultivar. This race was first detected in 1989 wheat growing season, which in preliminary tests showed sensitivity to DMI fungicides.
Wheat cultivars: Wheat cultivars Ônix, Abalone, Fundacep 52 and CD 104; genetic domain of OR -Seed Improvement Ltd. -RS FUNDACEP -RS, and COODETEC -PR, respectively, were used. Seedlings were grown in 300-mL pots containing vermiculite and compost at 3:1 proportion; five wheat seedlings were grown in each pot as an experimental unit.
IC 50 determination: The method to quantify the sensitivity of biotrophic fungi to fungicides described in FRAC (Frac 1991;Frac 1992) protocol was used throughout this study. The tool used to measure the sensitivity of races was the IC 50 (inhibitory concentration of fungicide that reduces by 50% the number of uredia/cm 2 ) and the IC 90 determined in in vivo tests.
The inoculum multiplication of each individual race was performed according to the methodology described by McIntosh et al. (19) and Reis (24). For inoculation, uredospores of P. triticina races were suspended in mineral oil Soltrol and sprayed with a compressed air device. Subsequently, water was sprayed [Tween 20, two drops/L water (Polyoxy ethylene sorbitane monolaurate -Synth)] prior to runoff. After 24 hours, in a moist chamber, in the absence of light, with 100% relative humidity at 20°C, seedlings were transferred to a greenhouse under natural light and partial control of temperature and relative humidity, where they were maintained for approximately 15 days until evaluation.
The effect of treatments was assessed by counting the uredia number/cm 2 on the leaf surface with more uniform uredinium distribution. A hand lens (10 x magnification) with a delimited marked area of 1.0 cm 2 was positioned on the leaf blade.
Experimental design was in completely randomized blocks with five replicates. Each fungal race (5) tested with all fungicide (8) at their concentrations (7) constituted one experiment. To increase data reliability, all tests were repeated twice.
Weilbull's model equation [where (y = concentration of active ingredient (mg/L) and x = mycelial growth], described by Knezevic et al. (17), was used to determined IC 50 and IC 90 .
Sensitivity reduction factor (SRF): A useful tool to quantify the shift in the sensitivity of a fungus to a fungicide is the sensitivity reduction factor. SRF = IC 50 of suspected race/IC 50 of the sensitive race (race MCG-MN). When there was no shift in sensitivity, the SRF value for that race was 1, and when there was shift >1 with different magnitudes (12).  Inhibitory concentrations for DMI. (Tables 1, 2, 3, 4 and 5) For the race MCG-MN, considered a standard for sensitivity, IC 50 overall mean for all fungicides was 0.69 mg/L (Table 6), which shows the sensitivity of this race to DMI fungicides. Among the tested races, suspected of having reduction in sensitivity, there were differences in IC 50 . The lowest IC 50 value was obtained for MFT-HT race, 16.84 mg/ L. The highest value was found for MDT-MR 55.08, MDK-MR 55.56 and MDP-MR, 70.28 mg/L. Metconazole showed in the overall mean the highest fungitoxicity, IC 50 of 26.79 mg/L (Table 3).

Identification of races.
Inhibitory concentrations for QoI. (Tables 7, 8 and 9) IC 50 values for strobilurin ranged from 0.021 for trifloxystrobin to 0.0053 mg/L for pyraclostrobin, and from 0.01 for MDK-MR to 0.0059 mg/ L for MFT-HT race (Table 10). Trifloxystrobin was the least toxic while pyraclostrobin, the most toxic fungicide.
Sensitivity reduction factor. Another procedure to show shift in a fungus sensitivity is SRF determination. Regarding DMIs, the lowest SRF was found for race MHF-HT, requiring a 31.0-fold higher concentration, while the highest SRF required a 110.66-fold higher concentration, for race MDP-MR. Regarding fungitoxicity, tebuconazole (B) was the most toxic fungicide, requiring a 80.7fold higher concentration, while the least toxic was cyproconazole which required a 187.8-fold higher concentration (Table 11). Regarding QoIs, the most sensitive race was MHF-HT, 0.45-fold higher, and the least sensitive race was MDP-MR, 1.11-fold higher. Among fungicides, trifloxystrobin showed the lowest fungitoxicity, 1.25-fold higher (

Survey of Puccinia triticina races
No data on the sensitivity of P. triticina races to fungicides was found in the available literature to compare with our findings. The frequent fungus variability has resulted in the occurrence of one new race per growing season (5, 8). Race MCG-MN (B34) was identified for the first time in 2000 growing season, before the control failure.
When the IC 50 values for the sensitive race were compared with those for other races, the shift in the sensitivity of P. triticina races to DMI fungicides was clear, confirming the hypothesis that the observed control failure in wheat crops in 2006 and 2007 seasons could be attributed to the reduced sensitivity of those races to DMI fungicides used for 16 years and in a large acreage.
Inhibitory concentration. Among races, the most sensitive one was MFT-HT and there was cross-sensitivity reduction for DMI fungicides. The least fungitoxic QoI was trifloxistrobin, while the most potent ones were azoxystrobin and pyraclostrobin.
Sensitivity reduction factor. SRF for P. triticina races was calculated by considering race MCG-MN values the standard sensitivity (SRF = 1.0).
DMI fungicides: The use of SRF allows data comparison to analyze the occurrence and the magnitude of the reduction in sensitivity to a fungus. SRF shows how many times the IC 50 value increases for a sensitive race (baseline) compared to the IC 50 of the suspected race. Therefore, the determined IC 50 values reinforce the statement that DMI fungicides should not be used alone for the economic control of wheat leaf rust.
The comparison between the IC 50 values of the two fungicide groups leads us to infer that all tested races are still sensitive to QoIs (Table 3) and that the control efficacy obtained in commercial fields is due to the use of QoIs mixed with DMIs.
The DMI fungicide that was most used alone was tebuconazole. The shift in sensitivity of fungi to DMI fungicides is a quantitative phenomenon (9, 12, 16); hence dose-dependent. Our data showed the occurrence of cross-reduction sensitivity to all DMIs used by growers to control wheat leaf rust.
Up to 2005 wheat growing season, the virulence variability of P. triticina was known and well documented, with numerous published studies (3). Moreover, the reduction in sensitivity to fungicides can be considered a new fact here reported by the first time. We have not found any references in the literature about reduction in P. triticina sensitivity to DMI fungicides. However, sensitivity reduction has been reported for Drechslera teres (25), Blumeria graminis f. sp. hordei (14), Blumeria graminis f.sp. tritici (14), Pyrenophora teres (26) and Septoria tritici (16) to DMI fungicides, and for Blumeria graminis f.sp. tritici (1) to strobilurins.
According to the present study, under field conditions growers cannot obtain economic control of wheat leaf rust by spraying DMI fungicides alone. The lowest SRF shifted 8.8 times and the highest one, 238.8 times. Even with 8.8 times, control was not satisfactory and became the target of farmers' complaints.