In vivo sensitivity of Phakopsora pachyrhizi to DMI and QoI fungicides

Sensibilidade de Phakopsora pachyrhizi a fungicidas triazóis e estrobilurina in vivo

Abstracts

In in vivo experiments the sensitivity of 18 isolates of Phakopsora pachyrhizi from several regions of Brazil to IDM fungicides (cyproconazole, epoxiconazole and tebuconazole and an IQE (pyraclostrobin) were evaluated. The assessments were based on leaflet uredia density. Inhibitory concentration (IC50) and sensitivity reduction factor were determined for all fungicide x strain interactions. Tebuconazole sensitivity reduction was detected for most fungus isolates. In contrast, there was no fungicide shift in sensitivity of the fungus to pyraclostrobin. We conclude that the control failure of soybean rust found in some farms is due to the reduced sensitivity of the fungus to the IDM fungicide and that it remains sensitive to pyraclostrobin.

Soybean rust; fungitoxicity; resistance; triazol and strobilurin fungicides

Em experimentos conduzidos in vivo avaliou-se a sensibilidade de 15 isolados de Phakopsora pachyrhizi procedentes de várias regiões do Brasil. Foram testados fungicidas IDMs (ciproconazol, epoxiconazol e tebuconazol e um IQe (piraclostrobina). As avaliações foram baseadas na densidade foliolar de urédias. Determinou-se a concentração inibitória (CI50) e o fator de redução da sensibilidade para todos os isolados. Demonstrou-se a ocorrência de redução da sensibilidade de P. pachyrhizi ao fungicida tebuconazol. Contrariamente, não se detectou alteração na sensibilidade do fungo à piraclostrobina. Conclui-se que a falha de controle da ferrugem observadas em algumas lavouras de soja se deve a redução da sensibilidade do fungo ao fungicida IDM.

Ferrugem da soja; fungitoxicidade; resistência; fungicidas IDM e IQE

In the 2013/14 growing season, soybean acreage in Brazil reached 30.17 million ha, 86.1 million tons production, and mean yield of 2.8 t/ha (³).

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi Sydow. was first reported in South America during the 2001 growing season in Paraguay (¹²). Chemical control of ASR in Brazil started as earlier as 2002/03 growing season and in the next season, an area of about 20 million hectares was sprayed with fungicides. Mean number of sprays per hectare has been three (⁴). As early as the 2007/08 growing seasons, at five seasons after the beginning of fungicide use, initially in Mato Grosso state, farmers started to complaine about the control efficiency of demethylation inhibitors (DMI) fungicides (⁸, ¹⁸). Tebuconazole and flutriafol fungicides were the most efficient fungicides and largely used at that time (⁴). Flutriafol was even adopted as effectiveness pattern in fungicide trials.

A large number of methods have been described to measure the fungitoxicity of a chemical (¹, ², ⁵, ⁶, ¹¹, ¹⁵, ¹⁶), or the sensitivity of a fungus to a given fungicide, or even to monitor the reduction or loss.

In vivo assays are needed for biotrophic pathogens, in which in vitro procedures are not compatible with their objectives. In vivo tests can also be used for necrotrophic pathogens, when the in vitro techniques are considered inappropriate (¹, ¹⁵, and ¹⁶). Several methodologies are available and their choice will depend on the target pathogen and the properties of the fungicide. In vivo tests typically include detached plant parts, mostly leaves, leaf discs or segments deposited on a culture medium containing the fungicide in suspension or solution, or even whole seedlings (¹, ², ⁶, ¹⁵, and ¹⁷).

Scherb and Mehl (¹⁷) suggested a similar methodology for sensitivity tests of P. pachyrhizi to fungicides, especially for DMIs.

Parameters such as ED50 (the effective dose that promotes a desired effect in 50% of microorganism subjected to the test), LD50 (lethal dose), LC50 (lethal concentration), EC50 (effective concentration), GI50 (growth inhibition), IC50 (inhibitory concentration) or MIC (minimum inhibitory concentration) have been used to define the fungitoxicity of a chemical (⁶, ⁹, ¹⁰, ¹⁵). IC50 values determined in vivo for different fungicides, specifically against P. pachyrhizi on soybean plants, are scarce in the literature, although they are useful in studies to monitor the sensitivity of the fungus, especially in regions where fungicides are largely used in soybean crops.

We hypothesized that such reduction in ASR control noticed in several fields (2007/08 growing season) was due to the reduction in the fungus sensitivity to the DMI fungicides used for six seasons (from 2002/03 to 207/08 seasons).

The aimof this study was to determine in vivo the fungitoxicity of DMI and QoI fungicides for suspected isolates of P. pachyrhizi, in samples taken from several locations in Brazil and one from Paraguay.

MATERIAL AND METHODS

The experiments to quantify the in vivo sensitivity of P. pachyrhizi to fungicide, were conducted in a growth chamber in the laboratory of Plant Pathology (Mycology), Faculty of Agronomy and Veterinary Medicine, University of Passo Fundo - UPF in 2008/09.

Soybean rust inoculum originated from uredospore samples obtained from naturally infected leaves, collected from several farms in the country and one farm in Paraguay in 2007/08 growing season (Table 1). The initial inocula, as uredospores, were removed by manually shaking soybean leaflets into an erlenmeyer containing sterile-distilled water and two-drops/L water of tensoactive polyoxyethylene sorbitane monolaurate (Tween 20 Synth Laboratory).

Table 1.
Soybeans samples with Phakopsora pachyrhizi maintained in growth chamber at the Universidade de Passo Fundo. 2007 2008

Isolate	Collected date	Sender	Location
1	01/23/2007	Tiago/Elaine	Passo Fundo/RS
2	03/03/2008	Elder Diniz	Rio Verde/GO
3	03/03/2008	Miguel	Santa Helena/GO
4	03/03/2008	Miguel	CEFET/GO
7	03/03/2008	Miguel	Jataí/GO
9	-/- /08	Tatiana Dalla Nora	Primavera do Leste/MT
19	03/25/08	Nilceli F. Buzzerio	Holambra/SP
20	03//3108	Weber Barrinha	Rio Verde/GO
21	-/- /08	Rafael R. Gonçalves	Chapadão do Sul/MS
22	04/04/2008	Marco T. Fujino	Aral Moreira/MS
24	-/- /08	Jairo dos Santos	Rondonópolis/MT
26	04/12/2008	Erlei M. Reis	Paraguai
27	04/14/08	Vitor T. Igarashi	Rio Verde/GO
29	04/15/08	João Cason	Mogi Mirim/SP
31	06/05/2008	Nilda Santos	Paulínia/SP
35	05/28//08	Márcia K.Pala	Sorocaba/SP
36	05/27/08	Reinaldo Bonnecarrere	Santo A. de Posse/SP
37	-/-/08	Nilda Santos	Paulínia/SP

Each inoculum sample was continuously maintained and multiplied in soybean plants grown in for 1L-pots (CD 219 soybean cultivars RR, low susceptible to powdery mildew, Erysiphe diffusa Cooke & Peck), protected inside individual plastic acrylic boxes (30 x 40 x 60 cm high) under controlled environment (22 ± 2°C and 14 h photoperiod) to avoid mixture of isolates.

Plant inoculation. Spores were removed from the surface of leaves by introducing leaflets in a plastic bottle (500 mL volume) containing 200 mL distilled water added of two drops of polyoxyethylene sorbitane monolaurate (Tween 20, Synth Laboratory). The bottle was manually shaken for three minutes for spore removal and passed through a two layeres of cheesecloth. The inoculum was sprayed on the leaves in V3 growth stage and plants were kept in a moisture chamber for 24 h, in the dark at 22oC.

Fungicide formulations: The used commercial formulation were: Pyraclostrobin - Comet (250), Tebuconazole - Folicur (200 EC), Flutriafol - Impact (125 SC), Epoxiconazole - Opus (125 SC) and Cyproconazole - Alto 100 (100 CE).

Fungicide concentrations. Seven concentrations of DMIs: were used in the tests 0.0; 0.02; 0.2; 2.0; 20.0; 50.0 and 100.0 mg/L, as well as six concentrations of QoI: 0.001; 0.01; 0.1; 1; 10.0 mg/L of active ingredient were used in the tests.

Fungicide application. Fungicide suspensions were prepared in distilled water added of 6.0 μL/L of Tween 20 in a 250 mL-volume Becker. Central leaflets detached from soybean in V2 - V3 growing stage were immersed for three seconds in each suspension by holding the petiole with a tweezers and shaken three times to eliminate excess suspension. After soaking, the leaflets were placed inside the boxes, with the adaxial side down, and distributed four leaflets per box totalizing sixteen leaflets per treatment.

Leaflets inoculation. On the followingd day when fungicide suspension had dried, boxes were open and inoculated by spraying a spore suspension containing > 2 x 104 spores/mL. The boxes were covered and kept in a growth chamber, initially under dark for 24 hs for spore germination and penetration, and later at 22oC and 12 h photoperiod, until to fungus sporulation.

During the incubation period, care was taken to keep the filter paper saturated with distilled water.

Disease assessment. The disease was evaluated at 15 to 20 days after inoculation by counting the uredinium/cm2. Counts were done in a selected area of the lealet with uniform uredia density in a 0.9 mm circle diameter marked with a hole borer. Data were presented as uredinium density per square centimeter.

A complete randomized block design with four replicates was used, adopting as experimental units a plastic box with four soybean leaflets.

IC50 and IC90 (inhibitory concentration) were calculated based on Weilbull's model, using the equation (y = d exp{- exp [b(log x - e]}), described by Knezevic et al. (¹²).

The sensitivity reduction factor (SRF) was calculated by dividing the actual IC50 value for the isolate by that for the sensitive fungal isolate. Baseline values were taken from Blum (¹). This shift indicates the amount of sensitivity reduction for a fungicide (¹⁰, ¹⁵).

IC50 and the SRF are shown in Table 1; for two times the experiments were conducted in relation to the number of uredia/cm2 of Pp.

Scherb and Mehl (¹⁷) described the methodology proposed by FRAC, in which the disease is measured by based on the estimated severity (visual assessment using a scale).

Each experiment, for every isolate, was replicated twice per concentration of the fungicide.

RESULTS AND DISCUSSION

Sampling was directed to those farms where fungicides had been sprayed for several growing seasons. We received and mantained 18 samples here called isolates. No monosporic isolation was done (Table 1).

The in vivo toxicity of the fungicide is shown in Tables 2 to 6; for two times the experiments were conducted assessed as the number of lesions/cm2 of P. pachyrhizi is shown in Tables 2 to 6.

Blum (1) showed that either lesions or uredia density may be used to assess P. pachyrhizi sensitivity.

There was a large variation of P. pachyrhizi sensitivity for tebuconazole among the isolates from samples collected in several regions of Brazil (Table 2). The magnitude of in sensitivity can be calculated by the SRF. A value < 1.0 indicates lower sensitivity than the baseline, and value > 1.0 indicates reduction in the isolate sensitivity (10). Seven isolates showed SRF < 2.0 while eight showed SRF > 2.0 mg/L. The greatest shift occurred for isolates 7, 26, 27, 31 and 37 (Table 2). In this experiment, the baseline mean values of the IC50s ≤ 0.11 mg/L (mean = 0.053) were adopted to calcularte SRF. The sensitivity reduction was not general for all samples. Only five out of 18 isolates showed sensitivity reduction.

Table 2.
In vivo inhibitory concentrations (IC50) (mg/L) and sensitivity reduction factor (SRF) for tebuconazole to different isolates of Phakopsora pachyrhizi related to the number of uredias/cm2

Isolates (no)	IC50	Error	SRF
Isolate 2	0.076	0.0205	1.43z
Isolate 3	0.025	0.0198	0.47
Isolate7	0.725	0.2369	13.67
Isolate 9	0.11	0.0384	2.07
Isolate 19	0.114	0.0052	2.15
Isolate 20	0.054	0.0873	1.01
Isolate 21	0.099	0.0314	1.86
Isolate 22	0.207	0.0500	3.9
Isolate 26	5.102	66.700	96.26
Isolate 27	0.455	0.2370	8.58
Isolate 29	0.093	0.0582	1.75
Isolate 31	0.343	0.1251	6.47
Isolate 35	0.013	0.0057	0.27
Isolate 36	0.011	0.0074	0.207
Isolado 37	29/04/1910	0.9048	71.16

Several genes command sensitivity shift for DMIs fungicides and the response is dose-dependent (¹⁰).

Blum (¹) determined in vitro and in vivo, the IC50 of DMI and QoI fungicides for a sensitive isolate of P. pachyrhizi. For tebuconazole, IC50 was 0.61 and in the present study, we used the IC50 mean of 0.053 mg/L. This difference may be due to sensitivity difference for the tested isolates.

For the isolate 1 (Table 1), considered sensitive to P. pachyrhizi, 0.61 for cyproconazole, 2.16, for cyproconazol., 0.87 for epoxyconazole, 2.50 for metconazole , and 0.192 mg/L for pyraclostrobin.

Regarding the CI50s values obtained in the present study, the tested DMI fungicides had a different behavior. In addition, SFR was not similar among them. The greatest shift in value was found for tebuconazole. Although they have been reported to have the same biochemical mode of action, i.e., demethylation inhibitors (DMI), CI50 values greatly differ among them (Tables 2 to 6). For instance,SRF for tebuconazole was 96.26 (Table 2) and for cyproconazole SRF 1.24 (Table 4). This may be due to ingredients of commercial formulation as pointed out by Blum (¹) and Furlan and Scherb (⁹).

Table 3.
In vivo inhibitory concentrations (IC50) for fungicides to isolate 01 (Sensitive) of Phakopsora pachyrhizi related to the number of uredia/cm2

Fungicides	IC50	Error
Tebuconazole	0.33	0.23
Cyproconazole	1.27	0.69
Epoxiconazole.	0.20	0.47
Pyraclostrobina	0.03	0.06
Prothioconazole	0.11	0.17

Table 4.
In vivo inhibitory concentrations (IC50) for fungicides to isolate 21 (Chapadão do Sul) of Phakopsora pachyrhizi related to the number of urredia/cm2

Fungicides	IC50	Error	Baselinez	SRF
Tebuconazole	4.65	5.80	0.61	6.62
Cyproconazole	6.14	2.90	2.16	2.84
Epoxiconazole.	1.43	1.81	0.87	1.67
Pyraclostrobina	0.20	0.03	0.192	1.01
Prothioconazole	0.18	0.04	z	z

• (x) Baseline IC50 values for each fungicide taken from Blum (2009).
• (z) Baseline not determined.

Testing a sensitive isolate, the lowest IC50 was 0.03 mg/L for pyraclostrobin and 1.27mg/L for cyproconazole.

Isolate 21 (Table 1) showed sensitivity shift to DMI fungicides tested. This fact did not occur with pyraclostrobin (Table 4). The largest change was for tebuconazole with SRF of 7.62.

O isolate 24 (Table 1) showed sensitivity shift to the tested DMI fungicides. This did not occur with pyraclostrobin (Table 5). The greatest changes was for the tebconazole with SRF of 20.44.

Table 5.
In vivo inhibitory concentrations (IC50) and sensitivity reduction factor (SRF) for fungicides to isolate 24 (Rondonópolis, MT 2008) of Phakopsora pachyrhizi related to the number of uredia/cm2

Fungicides	IC50	Error	Baselinex	SRF
Tebuconazole	12.47	2.85	0.61	20.44
Cyproconazole	2.68	0.60	2.16	1.24
Epoxiconazole	1.14	0.80	0.87	1.42
Pyraclostrobin	0.11	0.13	0.192	0.57
Prothioconazole	0.27	0.33	z	z

• (x) Baseline IC50 values for each fungicide taken from Blum (2009).
• (z) Baseline not determined.

Isolate 26 (Table 2) showed sensitivity shift to the tesgted DMI fungicides. This did not occur for pyraclostrobin. The greatest changes were for the tebuconazole and epoxiconazole, SRF of 14.59 and 5.16 respectively (Table 6).

Table 6.
In vivo Inhibitory concentrations (IC50) and sensitivity reduction factor (SRF) for fungicides to isolate 26 (Paraguay) of Phakopsora pachyrhizi related to the number of uredia/cm2

Fungicides	IC50	Error	Baseliney	SRF
Tebuconazole	8.90	2.11	0.61	14.59
Cyproconazole	5.91	2.16	2.16	2.73
Epoxiconazole	4.49	2.14	0.87	5.16
Pyraclostrobin	0.16	0.04	0.192	0.83
Prothioconazole	0.10	0.04	z	z

• (y) Baseline IC50 values for each fungicide taken from Blum (2009).
• (z) Baseline not determined.

Sensitivity reduction was shown for P. pachyrhizi isolates towards DMIs fungicides. It was also shown that the fungus is still sensitivity to pyraclostrobin (2007/08 growing season). In some farms, rust control has been achieved by the QoI fungicides and therefore DMIs should not be used alone to prevent control failure. On the other hand, QoI should not be used alone to prevent selection pressure towards shift in their sensitivity loss.

Junqueira (¹¹), working on the chemical control of P. pachyrhizi (latter determined as P. meibomiae) obtained in vivo CI50 (number of lesions/cm2 in non-detached leaflets), for benomyl (7.5 mg/L), triadimefon (38.3 mg/L), triforine (18.3 mg/L), copper oxychloride (296.2 mg/L), chlorothalonil (5.7 mg/L) and for maneb (0.75 mg/L).

Buzzerio et al. (²) monitored in vivo the sensitivity of P. pachyrhizi for cyproconazole fungicide, and reported IC90s in the range of 0.0934 to 0.5007 mg/L. However, the methodology used by Buzzerio et al. (²), differs from the methodology used in our work, which could explain the variations in results, depending on the sensitivity of pathometric method. The authors used FRAC International and Brazil methodology, i.e. visual assessment of the disease using a grading scale.

Furlan and Scherb (⁹) determined the IC50 for four commercial formulations of tebuconazole in Brazil for P. pachyrhizi. IC50 values varied from 0.54 for Folicur (200 CE), 0.81 for Orius (250 CE), 1.5 for Rival (200 EC) and 1.6 mg/L for Tebuconazole Nortox, demonstrating what Russell (15) warned about possible variations in IC50 for different commercial formulations of a given fungicide. The values reported for the fungicide tebuconazole, in our experiments (Folicur 200 EC formulation), considerim uredinium number/cm2 of P. pachyrhizi are similar and confirm the values reported in the literature, IC50 of 0.32 and 0.77 mg/L,for Experiments 1 and 2, respectively.

The CI50 determined by Blum (¹) can be used as a baseline for future studies monitoring the sensitivity of P. pachyrhizi to fungicides tebuconazole in soybean plants.

• ⁰¹
  Blum, M.M.C. Sensibilidade de Phakopsora pachyrhizi a fungicidas. 2009. 174 f. Tese de doutorado em fitopatologia, Universidade de Passo Fundo.
• ⁰²
  Buzzerio, N. F.; Basso, M. E.; Lachi, G. B.; Lovato, B. V.; Gonçalves, F. A.; Paiva, S. B. Determinação de metodologia para produção de inóculo de Phakopsora pachyrhizi em condições de laboratório para fins de estu dos de sensibilidade. Summa Phytopathologica, Botucatu, v. 33, supl., p. 61, 2007.
• ⁰³
  CONAB. Soja. Safra 2013-2014. Disponível em: <http://www.conab.gov.br/conabweb/download/safra/3graos_08.09.pdf>. Acesso em 10 de outubro de 2014.
  » http://www.conab.gov.br/conabweb/download/safra/3graos_08.09.pdf
• ⁰⁴
  CONSÓRCIO ANTIFERRUGEM: Monitorando a dispersão da ferrugem asiática no Brasil. Disponível em: <http://www.consorcioantiferrugem.net/?O_mapa_de_dispers%E3o>. Acesso em: 16 fev. 2009.
  » http://www.consorcioantiferrugem.net/?O_mapa_de_dispers%E3o
• ⁰⁵
  Edgington, L. V.; Klew, K. L.; Barron, G. L. Fungitoxic spectrum of benz imidazole compounds. Phytophathology, St. Paul, v.61, p.42-44, 1971.
• ⁰⁶
  Fungicide Resistance Action Committee. FRAC methods for monitoring fungicides resistance. EPPO Bulletin, West Sussex, v. 21, p. 291-354, 1991.
• ⁰⁷
  Fungicide Resistance Action Committee. Global Crop Protection Organiza tion. Brussels (www.frac.info).
  » www.frac.info
• ⁰⁸
  Fundação MT em campo. Boletim Informativo. v. 5, n. 24, Abril/Maio, 2008.
• ⁰⁹
  Furlan, S. H.; Scherb, C. Formulações de tebuconazole quanto à eficiência no controle da ferrugem asiática da soja., Summa Phytopathologica Botucatu, v. 33, supl., p. 29, 2007.
• ¹⁰
  Ghini, R.; Kimati, H. Resistência de fungos a fungicidas. Jaguariúna, SP: Embrapa Meio Ambiente, 2000. 78 p.
• ¹¹
  Junqueira, N. T. V. Controle químico da ferrugem (Phakopsora pachyrhizi Syd.) da soja. 1982. Dissertação (Mestrado em Fitopatologia) - Universi dade Federal de Viçosa, Viçosa, 1982.
• ¹²
  Knezevic, S.Z.; Streibig, J.C.; Ritz, C. Utilizing R software package for dose-response studies: the concept and data analysis. Weed Technology, Champaign v. 21, p. 840-848, 2007.
• ¹³
  Morel Paiva, W. Roya de la soja. Itapúa: Ministerio de Agricultura y Gana deria, Subsecretaria de Agricultura, Dirección de Investigación Agrícola: Centro de Investigación Agrícola, 2001. (Comunicado Técnico - Reporte Oficial, Série Fitopatologia, 1).
• ¹⁴
  Reis, E. M.; Reis, A. C.; Carmona, M. A. Manual de fungicidas: guia para o controle de doenças de plantas. 6. ed. rev. e ampl. Passo Fundo: Ed. Universidade de Passo Fundo, 2010. 226 p.:il.
• ¹⁵
  Russel, P. E. Sensitivity baselines in fungicide resistance research and management, Cambridge: Edit., (FRAC Monograph, 3). CB2 5AN, UK, 2004.
• ¹⁶
  Sharvelle, E. G. The nature and uses of modern fungicides. Minneapolis: Burgess Publishing Company, 1961. 308 p.
• ¹⁷
  Scherb, C. T; Mehl, A. Fungicide Resistance Action Committee. - Phako psora pachyrhizi: detached leaf test. Disponível em: <http://www.frac.info/frac/Monitoring_Methods/anhang/PHAKPA_detached_leaf_monito ring_method_BCS_2006_V1.pdf>. Acesso em : 10 out. 2014.
  » http://www.frac.info/frac/Monitoring_Methods/anhang/PHAKPA_detached_leaf_monito ring_method_BCS_2006_V1.pdf
• ¹⁸
  Silva, L. H. C. P.; Campos, H. D.; Silva, J. R.; Ribeiro, G. C.; Rocha. R. R.; Moraes, D. G. Eficácia reduzida de triazóis no controle da ferrugem asiá tica, Fitopatologia Brasileira, Lavras, v. 33, p. 228, 2008. (Suplemento).

Publication Dates

• Publication in this collection
  Jan-Mar 2015

History

• Received
  06 Feb 2014
• Accepted
  16 Jan 2015