Sensitivity of Phytophthora infestans (Mont.) de bary Isolates to Fluazinam, Fosetyl-Al and Propamocarb-hydrochloride

A survey of in vitro sensitivity of twelve isolates of the Phytophthora infestans to the fluazinam, fosetyl-Al and propamocarb-hydrochloride was conducted. The isolates were isolated from infeceted potato leaves collected from eight different localities in Serbia during 2005-2007. All P. infestans isolates were sensitive to tested fungicides. The obtained values of resistance factor were in the range from 1.0 to 2.8. The EC50 values of fluazinam were from 0.14 to 0.27 mg l-1, fosetyl-Al from 30.2 to 85.8 mg l-1, propamocarb-hydrochloride between 12.1 and 31.1 mg l-1, respectively.


INTRODUCTION
Potato late blight, caused by Phytophthora infestans (Mont.)de Bary, is the economically most important potato disease in Serbia.The asexual life cycle of P. infestans is short; and sporulating foliar lesions develop three to seven days after successful infection under conducive conditions resulting in a polycyclic epidemic (Stein and Kirk, 2002).
In recent years, the severity of late blight increased in many parts of the world.This is frequently associated with migrations that have introduced new, more aggressive, populations of the pathogen (Fry and Goodwin, 1997).The late blight epidemic in 1999 was devastating in Serbia.The new genotypes of P. infestans are being detected in the countries from which a large quantity of potato seed is imported to Serbia for its own potato production, in particular from Holland, Germany and Hungary.Ivanović (2005) reported that the A2 mating type occurs in Serbia.The appearance of more aggressive strains of P. infestans, containing both A1 and A2 mating types makes this problem even more complicated.Control of P. infestans in Serbia relies on intensive use of fungicides often without any appropriate program.Discovery and development of the first systemic fungicides from phenylamides class was a significant improvement and one of the most important contributions to agrochemical industry.However, the intensive use of metalaxyl led to the rapid selection of metalaxyl-resistant strains of P. infestans in Europe within one year from its introduction (Parra and Ristaino, 2001).
Knowledge of the sensitivity of P. infestans isolates to fungicides with different mode of action, is very important for the development of late blight management strategies in Serbia.
The objective of this study was to test the sensitivity of P. infestans isolates collected in Serbia over the period 2005-2007, to the fungicides fluazinam, fosetyl-Al and propamocarb-hydrochloride.

Samples collection and isolation
Isolates were collected from the major potato growing regions in Serbia during 2005 and 2007 growing season.The host variety on which late blight was detected, plot size, fungicides used, and late blight incidence and severity were also recorded.P. infestans was isolated from the infected potato leaves collected from eight different locations according to the methods described by Mukalazi et al. (2001) and Zhu et al. (2008).Fragments of infected leaf tissue were placed under a thin, surface disinfected slice of potato tuber of Desiree variety and incubated at 18 o C for seven days to allow mycelial growth through the potato slice.Mycelia was picked from the surface of the slice using a sterile needle, and then put on rye B medium (200 g rye, 20 g glucose, 17 g agar, 1 l deionized water) amended with rifampicin 20 μg ml -1 , ampicillin 200 μg ml -1 , and nystatin 100 μg ml -1 , and incubated at 18°C in darkness for 7 days.After two transfers of hyphal tips on media containing antibiotics, twelve pure isolates were obtained (Table 1).The identity of isolates of P. infestans was confirmed by polymerase chain reaction (PCR) using species-specific primers (Tooley, 1998) and their morphological traits according to Erwin and Ribeiro (2005).The isolates were kept on potato dextrose agar (PDA) at 5 o C in the Culture Collection of the Institute of Pesticides and Environmental Protection, Belgrade.

Fungicides
Commercial formulations of fungicides were used as active ingredients (a.i.) respectively: fluazinam provided by Syngenta Agro Service, Serbia, (Shirlan 500-SC, 500 g l -1 ), fosetyl-Al (Aliette 80-WP, 800 g kg -1 , Bayer CropScience), and propamocarb-hydrochloride (Previcur 607-SL, 722 g l -1 , Bayer CropScience).Each fungicide was diluted into a set of stock solutions with sterile distilled water.Freshly-made stock solutions were prepared to give specific concentrations of active ingredient in ml l -1 .Volumes of stock solution were added to molten (50 o C) sterile culture media prior to pouring, thereby, producing active ingredient concentrations ranging from 0.01 to 100.0 mg l -1 (Locher and Lorenz, 1991).

Fungicide sensitivity and EC 50 assays
P. infestans isolates grown on rye B medium amended with the fungicides: fluazinam, fosetyl-Al, and propamocarb-hydrochloride, were used for sensitivity tests.Based on the preliminarily obtained results, the selected concentrations of fluazinam for further study was: 0.01, 0.1, 1.0 and 10.0 mg l -1 ; fosetyl-Al: 0.1, 1.0, 10.0 and 100.0 mg l -1 ; propamocarb-hydrochloride 0.1, 1.0, 10.0 and 100.0 mg l -1 .Control plates were not amended with fungicides.Tests for each isolate were replicated three times per each concentration of each fungicide.Mycelial plugs (10 mm in diameter) were removed from the margins of colonies grown on rye B medium, placed upside down on the fungicide-amended and fungicide-free rye B medium in Petri dishes, and incubated at 18 o C.After 10 days, the colony diameter of each isolate was measured in two directions (minus the diameter of the inoculation plug) and the percent inhibition (PI) values of each of the fungicide rates were calculated using the formula given below: percent inhibition = (a -b) / a x 100 where a = the colony diameter of the control plate and b = the colony diameter of the fungicide-amended plate PI values were subjected to regression analysis against the logarithmic values of the fungicide rates.The EC 50 (fungicide concentration which inhibits mycelial growth by 50%) was determined for each isolate and data on fungicide concentration and relative inhibition were analysed using probit analysis, according to Finney (1971).

RESULTS
Sensitivity of P. infestans isolates to the fungicides tested are shown in Tables 2, 3 and 4.Among twelve P. infestans isolates tested for sensitivity to fluazinam, the PR1 isolate showed greatest sensitivity.The EC 50 value of this isolate was 0.14 mg l -1 .The DO7, KT2 and KV1 isolates had the highest EC 50 value (0.27 mg l -1 ).
The EC 50 values of the remaining tested isolates were between 0.15 and 0.24 mg l -1 (Table 2).the control of potato late blight in Serbia and worldwide.The biological mode of action is uncoupling of oxidative-phosphorilation in both true fungi and pseudofungi (Stein, 2002).The obtained EC 50 values were in a very narrow range (0.14 to 0.27 mg l -1 ), and were similar to the values established by Matheron and Porchas (2000) for Phytophthora parasitica isolates (0.1 mg l -1 ) originating from the United States.All isolates were sensitive to fluazinam (RF=1.0-1.9)according to criteria established by Gouot (1994).FRAC(b) (2011) also classified fluazinam as fungicide with low risk of resistance.
As a member of carbamate fungicides, propamocarbhydrochloride is highly specific for fungi from order Peronosporales.The biological mode of action has not been elucidated but disruption of cellular membrane and/or function was observed.The biological activity of propamocarb-hydrochloride is relatively low compared to other semi-systemic late blight fungicides in the terms of rate comparisons, and large amounts have to be applied for comparable activity (Stein, 2002).Widest range of EC 50 values of tested isolates was observed in tests conducted with this carbamate fungicde; they varied from 12.1 mg l -1 (GU6) to 31.1 mg l -1 (PR1).The obtained values of RF indicate that all isolates were sensitive to propamocarb-hydrochloride. The results were similar to those of Hu et al. (2007) for Phytophthora nicotianae (EC 50 = 2.2-90.1 mg l -1 ).FRAC(b) (2011) states that propamocarb-hydrochloride, in terms of resistance, is a low to medium risk fungicide.However, as propamocarb-hydrochloride since its introduction to the Serbian market was combined with contact or systemic fungicides (mancozeb, chlorothalonil, fosetyl-Al and fenamidone), the intensity of selection pressure of P. infestans was relatively lower.
Fosetyl-Al showed the lowest toxicity among all tested fungicides (30.2 to 85.8 mg l -1 ).EC 50 values obtained in our experiments were similar to the values obtained by the Farih et al. (1981).
However, the values of RF indicate that most isolates obtained from infected potato leaves in Serbia are sensitive to fosetyl-Al.Although fosetyl-Al and its breakdown product, phosphorous acid are not always more active against P. infestans in vitro, they are much more selective for this pathogen in vivo (Erwin and Ribeiro, 2005).Fosetyl-Al cause disturbance at several metabolic sites in the mycelial phase of the life-cycle, and inhibit sporulation at low concentrations, without affecting mycelial growth.Even at high concentrations (1-10 mM), fosetyl-Al is fungistatic rather than fungitoxic (Erwin and Ribeiro, 2005).The probability of high levels of resistance emergence in the field is reduced by the multi-site action of fosetyl-Al in the matebolism of P. infestans (Erwin and Ribeiro, 2005;FRAC(b), 2011) Management of potato blight will continue to rely on chemical control.Low-risk fungicides (fluazinam, fosetyl-Al and propamocarb-hydrochloride) with different modes of action will be needed in problematic fields.Our study suggests that continuous monitoring of P. infestans field populations with respect to fungicide resistance is very important for the development of late blight management strategies.

Table 1 .
Phytophthora infestans isolates and their origin

Table 2 .
Sensitivity of isolates of P. infestans to fluazinam EC 50 -Fungicide concentration which inhibits mycelial growth by 50%; RF -The resistance factor was expressed as the ratio of the EC 50 and the lowest EC 50 for the isolates tested; b -Regression coefficient; *95% confidence interval (P=0.05)