The objective of this work was to verify if there was correlation between sensitivity (EC50) values generated with the recommended methodology using ASR populations sampled from field trials where Azoxystrobin-based fungicide did not perform as expected against ASR.
Autores: KOGA, L.J.1; CAMPOS, J.B.1; FEREZIN, D.F.2; GENET, J-L.1; NGUGI, H.K1
A drop of efficacy of all site-specific fungicides, QoIs (Quinone outside Inhibitors), DMIs (DeMethylation Inhibitors) and SDHIs (Succinate DeHydrogenase Inhibitors), in controlling Asian Soybean Rust (ASR), caused by Phakopsora pachyrhizi Syd. & P. Syd. is a major concern for all soybean (Glycine max (L.) Merr.) producing chain (Godoy et al., 2017).
ASR is one of the most important diseases in the soybean with yield losses ranging from 10% to 90% in geographical regions where it was reported (Sinclair & Hartman, 1999). In South America, ASR was described in 2001, first detected in Paraguay (Paiva & Yorinori, 2002) and soon afterwards in Brazil (Yorinori & Lazzarotto, 2004). QoI fungicides have been used in the control of ASR since 2002/03 crop season and are included in many mixture products currently registered in Brazil (Brasil, 2018). Field performance of QoI fungicides was observed to decline in 2013/14 crop season (e.g. Azoxystrobin dropped 66% in control compared to 2012/13) (Godoy et al., 2014; Godoy et al., 2013). Resistance to QoI fungicides associated with a F129L which confers differential sensitivity to QoI fungicides in P. pachyrhizi was documented in 2016 (Klosowski et al., 2016).
Resistance monitoring is crucial for understanding what changes the population may be undergoing. When growers observe a marked lack of control from previously efficacious products, diseased samples can be taken from these fields to confirm a reduction in sensitivity of the pathogen under controlled conditions in the laboratory. This confirmation step is crucial as there are many other reasons for reduced efficacy (e.g. application timing, inadequate fungicides rates, incomplete application coverage, poor crop management and climate favoring the disease progress, interfering with fungicide applications, etc) (FRAC, 2018a).
The recommended FRAC method for QoI resistance monitoring in ASR is a spore germination test in agar plate (FRAC, 2018b). The objective of this work was to verify if there was correlation between sensitivity (EC50) values generated with the recommended methodology using ASR populations sampled from field trials where Azoxystrobin-based fungicide did not perform as expected against ASR.
The assays were performed at DuPont´s Plant Pathology Laboratory conditions, Paulínia, SP, during 2014/15 crop season. The first step was to validate the spore germination methodology, ensuring that the range of concentration covered between 0 and 100% inhibition of spore germination. We used 3.5-cm Petri-plates with 2% Agar-water media amended with 10ug/mL of SHAM (salicylhydroxamic acid) – 1.5 g SHAM diluted in 15 mL of methanol (0.1%) – with our ASR-Paulínia-GH-reference isolate. SHAM minimizes the effect of the alternative oxidative pathway that some fungi use to evade QoI fungicide toxicity in in vitro fungicide sensitivity assay (Owati et al., 2017). Urediniospores concentration were calibrated to 10.000/mL, after sporulating soybean leaflets originating from the field were immersed in dionized water with 1% Tween20 to dislodge the spores. Each Petri-plate received 100 uL of spore suspension. Plates were then placed in growth-chambers for 6-hrs in the dark, at 25°C. After this period, 50% acetone was added in all Petri-plates to paralyze the germination of spores. Plates were then wrapped in plastic-film to preserve the media humidity and stored at 4°C until evaluated for spore germination. Spore germination was determined by counting the numbers of germinated/non-germinated spores out of 100, using 10X magnification microscope. The number of germinated/non-germinated spores were used to calculate the % of inhibition and EC50 values were determined.
Assay repeatability and reproducibility were validated for Azoxystrobin SC, 25% with the doses: 0, 0.003125, 0.03125, 0.125, 0.25, 1, 10 ppm using our reference ASR population. Coefficient of variance (CV) among the six independent tests and among the four repetitions of each test conducted with the reference isolate were below 15%. The mean EC50 value was 0.1170 ppm (Table 1) and the average dose-response curve is shown in Figure 1. The frequency of F129L mutation of the reference isolate was 21%, (internal results) determined by a quantitative next generation sequencing method.
Figure 1. Average dose-response curve for validation of Azoxystrobin SC (25%) with ASR-PaulíniaGH-reference isolate at DuPont´s Plant Pathology Laboratory. Average of 6 tests with 4 replicates.
Table 1. Repeatability and reproducibility validated with Azoxystrobin SC (25%) with ASR-PaulíniaGH-reference isolate at DuPont´s Plant Pathology Laboratory. EC50 values (ppm) of each four repetitions from all six tests; average EC50 values of each test and of all six tests with their respective coefficient of variance (CV). 2014/15 crop season.
After spore germination methodology was validated in our laboratory conditions, we made the necessary alignment to receive ASR sporulating leaflets samples from the main soybean producing regions of Brazil. In 2014/15, we processed 141 samples, but only 59 of them presented a sufficient number of germinated spores (above 25) in the untreated plates (ranged from 27 to 99). From the 59 populations, only 43 showed a reliable dose-response curve from which the EC50 values were calculated. Mean EC50 values from the 43 samples was 0.1873 with an average F129L mutation frequency of 88% (Table 2).
Table 2. State, cities, sampling month and F129L frequency of P. Pachyrhizi samples with respective EC50 median, EC50 lower and upper values (ppm) for Azoxystrobin SC (25%), 2014/15 crop season.
Results from efficacy trials with straight applications of Azoxystrobin product from internal protocols (22 trials with 3 sequential application at 14-18 days intervals), and from ASR net-trials (31 trials with 2 to 4 sequential applications at 10 to 26 days interval) (Godoy et al., 2015) were compiled and summarized (Table 3).
Table 3. Treatments, DuPont internal and net-trials protocols (number of trial compiled), rate, g.a.i./ha, formulation, average severity (%) and average % control calculated considering untreated severity (%) values as 0% control. 2014/15 crop season.
Analyzing generated and public available data, it was possible to conclude that there was no correlation between the frequency of the F129L mutation spore germination EC50 values generated with ASR samples from the main soybean producing regions in Brazil (r = 0.145; p = 0.353) and the field performance of Azoxystrobin (Tables 2 and 3).
As FRAC-international is the reference for resistance monitoring methods, not just for industry, but also for universities and research institutions – private and public – where future scientists are trained, it is desirable to develop and validate a methodology that can show a correlation between the field efficacy of QoI-based fungicides in controlling ASR, the frequency of F129L mutation in the sampled population and sensitivity monitoring data.
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Informações dos autores:
1Corteva Agriscience™ Divisão Agrícola DowDuPont™, Mogi-Mirim, SP;
2Destak – Treinamento e Desenvolvimento Empresarial, São Paulo, SP. .
Disponível em: Anais do VIII Congresso Brasileiro de Soja. Goiânia – GO, Brasil.