Agricultural and Food Science 35 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 35–42. © Agricultural and Food Science Manuscript received September 2005 Research note Prochloraz tolerance of Pyrenophora teres population  in Finland Marjo Serenius and Outi Manninen MTT Agrifood Research Finland, Biotechnology and Food Science, Myllytie 10, FI-31600 Jokioinen, Finland, e-mail: marjo.serenius@mtt.fi Barley leaves infected with Pyrenophora teres Drechs. f. teres were collected from farmers’ fields and an experimental field for evaluation of efficacy of fungicides at MTT Agrifood Research Finland (MTT), in 2003. The aim was to test the efficacy of prochloraz to inhibit in vitro growth of P. teres. Potato dextrose agar (PDA) dishes amended with 0.1 and 1.0 μg ml-1 prochloraz were used for testing 364 isolates of P. teres based on prelimenary experiment. Isolates from MTT’s experimental field were growing slower on fungi- cide-amended media than isolates from farmers’ fields. The overall mean inhibition of radial growth was 63 and 86% on media amended with 0.1 and on 1.0 μg ml-1 prochloraz, respectively. Isolates of different origin differed significantly on growth on fungicide-amended media. The isolates capable of growing on increased concentrations of prochloraz were most commonly isolated from fields, where prochloraz was sprayed dur- ing the growing season. Within MTT’s experimental field no effect of fungicide application during the growing season was observed on growth of isolates in vitro. Data from this survey was insufficient for mak- ing further conclusions regarding the effect of agricultural practices on selection of fungicide tolerant P. teres isolates. Fungicides with different types of mode of action are recommended for use together with prochloraz against the net blotch pathogen in Finland. These results are preliminary. Key words: barley, Drechslera teres, fungicide tolerance, population diversity, sterol-biosynthesis inhibitor, net blotch Introduction Prochloraz (1-{N-propyl-N-[2-(2,4,6-trichlorophe noxy)ethyl]carbamoyl}-imidazole; trade names in Finland: Sportak and Prelude) has a wide spectrum of activity and thus can be used on several crops and against several fungal pathogens (Prochloraz: technical information, Hoechst Schering AgrEvo GmbH, Berlin, Germany, 1995). Prochloraz is the most common active ingredient among fungicides used in cereals in Finland (Savela and Hynninen 2004, Plant Production Inspection Centre 2005). It is approved for use against leaf blotch pathogens 36 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Serenius, M. & Manninen, O. Prochloraz tolerance of Pyrenophora teres population before sowing as a seed coating and during the growing season as a foliar application, and on win- ter cereals against pink snow mould before snow- fall. In addition, foliar application during the grow- ing season can be split into two separate spraying times with half the amount of active ingredient, resulting in a maximum of four application times for one field during a growing season. To avoid the establishment of fungicide tolerance fungal geno- types, the same fungicide is not recommended for prolonged use on the same crop. Prochloraz be- longs to the sterol 14α-demethylation inhibitors (DMIs) and more precisely to the imidazole class. DMIs inhibit the cytochrome P450 dependent oxi- dative demethylation of eburicol in filamentous fungi as part of the ergosterol biosynthesis path- way (Steffens et al. 1996). Resistance to prochlo- raz is controlled by a single major gene, and it was acquired after a decade of intensive prochloraz treatments against cereal eyespot pathogen, Tapesia acuformis (Dyer et al. 2000). Prochloraz and imazalil, which belong to the same imidazole class of DMIs are the active ingredients for half of the chemical products approved for seed coating of barley in Finland (Plant Protection Inspection Centre 2005). Prochloraz alone is the leading fun- gicide used for cereals in Finland according to the sales of fungicides (Savela and Hynninen 2004). Pyrenophora teres Drechs. f teres Smedeg. is the cause of net blotch of barley, the most impor- tant barley disease in Finland. The average net blotch infection rate between years in Finland de- pends on variety, but reaches 40% on susceptible varieties without fungicide application during the growing season (Kangas et al. 2004). Fungicide treatment during the growing season results in a significant improvement on yield of susceptible spring barley varieties (Kangas et al. 2005). The risk of losing the efficiency of a fungicide against certain pathogens is greatly increased if the same active ingredient is used repeatedly year after year. Therefore the objective of this study was to test the efficiency of prochloraz to inhibit growth of P. teres in vitro, and to find possible differences in prochloraz tolerance among field populations of the net blotch pathogen of barley in Finland. This was a preliminary study. Material and methods Sampling of Pyrenophora teres  populations Leaf samples were collected randomly during the late growing season after milk ripening stage of spring barley in the Lounais-Häme region near MTT Agrifood Research Finland (MTT), Jokioi- nen, in 2003. Samples were collected from three farmers’ fields (sampling area near two hectares) and a MTT’s experimental field (three 16 m2 plots in four replications), where several fungicides were sprayed during the growing season. Details of barley varieties and fungicide applications dur- ing the growing season are given in Table 1. Sam- ples from the same field were considered to repre- sent one population. Single-conidial isolates were established according to McDonald (1967), and kept at –70°C until needed. Isolates were grown on 2.5% V8 medium under near ultra violet light at 18°C, using a 12h light period for two weeks prior to the experiment. Fungicide tolerance assay Prochloraz (trademark: Sportak 45 EC (prochloraz 450 g l-1), Bayer Crop Science, Germany) was ob- tained from MTT Agrifood Research Finland, Evaluation and testing of Fungicides. A radial growth assay was used to determine the tolerance against prochloraz. In preliminary experiment, dif- ferent dilutions of prochloraz in two culture media (2.5% V8 and potato dextrose agar (PDA)) were used to determine a suitable growing media and dilution for testing of the population samples. Ini- tial dilutions used were: 0.1, 0.5, 2.5, 12.5 and 62.5 μg ml-1. Fungicide was added into cooled liquid media prior to pouring into Petri dishes. Medium without fungicide was used as a control since all the dilutions were made in sterile water. A 7 mm diameter mycelium plug was taken from an active- ly growing culture of P. teres for each type of me- dium and then placed in the centre of a Petri dish 37 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 35–42. Table 1. Characteristics of fields and plots, where barley leaves infected with Pyrenophora teres were collected in 2003. Characteristics Farmers’ field MTT’s experimental field1) 1 2 3 Barley variety Scarlett Annabell Rolfi Rolfi Active ingredient of fungicide Prochloraz Prochloraz Azoxystrobin a) Azoxystrobin + Propiconazole, Fenpropidin mixtureFenpropidin mixture b) Prochloraz c) Prochloraz Fungicide rate (l ha-1 of product) 0.5 0.6 Unknown a) 0.4 + 0.4 b) 0.5 c) 1.0 Number of single conidial isolates 52 51 59 a) 54 b) 62 c) 86 1) Samples were collected separately from fungicide treated plots, taken into account in the statistical analysis. containing prochloraz-amended medium. Three replicates were used. Cultures were incubated un- der near ultra violet light at 18°C using the 12 h light period until the fungus in the control Petri dishes reached the edges of the dishes. The diam- eter of radial growth of the fungus, including the inoculum plug, was measured (mm) from all Petri dishes on the same day. Test isolates did not grow on concentrations of 2.5 μg ml-1 prochloraz medi- um or stronger. Therefore concentrations of 0.1 μg ml-1 and 1.0 μg ml-1 prochloraz were chosen as treatments for testing the field populations of P. teres. PDA was chosen as test medium because growth of the fungus was more difficult to measure visually on darker V8 media. P. teres isolates were tested in two successive experiments, both having two replicates per isolate for each treatment. Con- trol was included in all experiments with two rep- licates for each isolate. Cultures were incubated for 7 days prior to measuring colony growth as de- scribed above. Data analysis The radius of the inoculum plug (7 mm) was sub- stracted from the growth measures before statisti- cal analysis. Radial growth of each isolate on fun- gicide-amended media was calculated as a propor- tion of the growth on the control medium. This corrects for differences in growth rates between isolates (Peever and Milgroom 1993). Analyses of variances were performed using PROC GLM in SAS® Proprietary Software Release 8.2 (SAS In- stitute Inc., Cary, NC, USA). The residuals of pro- portional growth were close to a normal distribu- tion on data from 0.1 μg ml-1 prochloraz medium, whereas the residual distribution for data from the 1.0 μg ml-1 prochloraz medium was wider than but close to the normal distribution. Transformations on data were considered unnecessary. Firstly, we tested the hypothesis that isolates of P. teres from each plot within MTT’s experimental field were homogenous. Secondly, we hypothesised that there were no differences between populations in pro- portional growth on fungicide-amended medium. Three sources of variation were included in analy- sis: repetition of experiment, field (or plots within MTT’s field) and isolate (within field/plot varia- tion). Results Isolates from MTT’s experimental field Growth differed significantly from zero for both concentrations of prochloraz (P < 0.0001). The 38 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Serenius, M. & Manninen, O. Prochloraz tolerance of Pyrenophora teres population sampling plot of an isolate did not have marked effect on growth differences. However, an interac- tion between repetition of experiment and plot was found for both concentrations of prochloraz (F = 101.48, P < 0.0001 and F = 25.64, P < 0.0001 on 0.1 and on 1.0 μg ml-1 medium, respectively). These significant interactions were mainly due to high variation between the two experiments among growth results for isolates originating from plot b, which were growing weakly on the first experi- ment. The overall mean radial growth of P. teres isolates originating from MTT’s experimental field was 7 and 1 mm, while the proportional growth was 0.18 and 0.03 on 0.1 μg ml-1 prochloraz me- dium and on 1.0 μg ml-1 medium, respectively. The distribution of isolates (sum of two experiments) growing on fungicide-amended media is shown in Figure 1 and 2 (MTT’s field). Combined data Data from both the experimental and farmer’s fields were combined to test whether the isolates from different fields differed in their ability to grow on fungicide-amended media. Two of the farmers’ fields and two plots within the MTT’s ex- perimental field had prochloraz applied during the growing season, whereas the third farmer’s field and a plot of the MTT’s experimental field had an azoxystrobin-based fungicide applied (Table 1). The effect of field of P. teres isolates on propor- Fig. 1. Distribution of radial growth of P. teres isolates (frequency) growing on 0.1 μg ml-1 prochloraz-amended medium as proportional growth of the control media. Farm 3 0 5 10 15 20 25 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Farm 1 0 5 10 15 20 25 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Frequency of isolates Radial growth as proportion of the control MTT's field 0 5 10 15 20 25 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Farm 2 0 5 10 15 20 25 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 10 39 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 35–42. tional growth was significant on both media (F = 212, P < 0.0001 and F = 267, P < 0.0001 on 0.1 and on 1.0 μg ml-1 prochloraz medium, respectively). Most of the variation was between fields, namely 38.0 and 43.5% on 0.1 and 1.0 μg ml-1 prochloraz medium, respectively. Less than 1% of the total variation was due to different fungicide treatments during the growing season (i.e. the effect of fields with different fungicide regimes). No significant effects were found among plots within MTT’s ex- perimental field. In addition, difference of normal- ised growth was high between farmers’ fields and MTT’s experimental field. The overall mean radial growth of isolates originating from farmers’ fields was 19 mm and 8 mm, and the proportional growth 0.50 and 0.21 on 0.1 and 1.0 μg ml-1 prochloraz medium, respectively. The growth of isolates orig- inating form MTT’s experimental field was ap- proximately one third of that. Isolates originating from farmers’ field 1 and 2 had the highest growth rates on fungicide-amended media (Figure 1 and 2). Efficiency of prochloraz The percentage of all isolates, in which growth was inhibited less than 50% was 27.1% on 0.1 μg Farm 3 0 10 20 30 40 50 60 70 80 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 M TT's fie ld 0 10 20 30 40 50 60 70 80 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Farm 1 0 10 20 30 40 50 60 70 80 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Farm 2 0 10 20 30 40 50 60 70 80 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Frequency of isolates Radial growth as proportion of the control Fig. 2. Distribution of radial growth of P. teres isolates (frequency) growing on 1 μg ml-1 prochloraz-amended medium as proportional growth of the control media. 40 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Serenius, M. & Manninen, O. Prochloraz tolerance of Pyrenophora teres population ml-1 prochloraz medium and 10.8% on 1.0 μg ml-1 medium. The percentage of all isolates growing as well or better on 0.1 μg ml-1 prochloraz medium compared with the control medium was 3%. Most of these isolates were collected from fields that had been sprayed with prochloraz during the growing season (Farms 1, 2 in Figure 1). Only one isolate (from Farm 1) had similar growth rates on 1.0 μg ml-1 prochloraz medium as compared to the con- trol medium. In contrast, the number of all isolates with less than 5% of the growth of the control when cultured on the 0.1 μg ml-1 prochloraz medi- um was 11.6% and 53.5% on 1.0 μg ml-1 medium. These isolates mostly originated from farmer’s field 3 and from MTT’s experimental field (Figure 1 and 2). Discussion The radial growth assay experiments were per- formed during the mid-winter in growth chambers in laboratory, where the effect of environmental factors was minimised. Still results demonstrated that experiment had an effect on growth of isolates. The variation in growth was marked within iso- lates and among experiments. In spite of the effect of experiment, results indicated that field origin of P. teres isolates had an effect on radial growth in vitro. The EC50 value for P. teres was 0.026 μg ml -1 of prochloraz in Sweden in 1982 before the fungi- cide Sportak 45 EC was registered for use in there (Olvång 1988). Concentrations of 1 μg ml-1 of prochloraz resulted in 90% growth inhibition of P. teres isolates from Sweden (Olvång 1988). In this study the overall mean growth inhibition of P. teres isolates was 86% (79% for farmer’s fields, and 69% for Farms 1 and 2) with the same concen- tration. Since earlier results from Finland are lack- ing, we cannot conclude that the effectiveness of prochloraz has changed during the years. Howev- er, P. teres isolates originating from farmers’ fields, in which prochloraz was sprayed during the grow- ing season had increased growth on fungicide- amended media. It is possible that isolates from farmers’ fields have been under stronger selection pressure. Prochloraz can be used in the same field sev- eral times, while the net blotch pathogen popula- tion can be maintained in the field for several years. P. teres survives over the winter in infected stubble (Jordan 1981) and Finnish P. teres popula- tions seem to be genetically differentiated between fields (Serenius et al. 2005). At least one of the farmers’ fields had barley cultivation before the study year, while another field was not ploughed before barley was sown. Continuous barley culti- vation and reduced tillage are factors that can in- crease the net blotch infection risk on barley (Jor- dan 1981). However, this survey was not estab- lished to test the effect of fungicide application during the growing season or the effect of other agricultural factors, such as variety or ploughing on natural selection of fungicide tolerant P. teres isolates. Therefore a more precise experimental design and a sufficient number of samples are needed to study factors affecting the selection of fungicide tolerant P. teres isolates. The current study did not indicate significant differences be- tween plots within MTT’s experimental field. Our earlier studies have identified little or no differen- tiation in P. teres populations within fields based on AFLP markers (unpublished data), but high dif- ferentiation between fields in Finland (Serenius et al. 2005). Short distance spore dispersal may help to distribute genotypes of P. teres resulting in more uniform within field populations with similar fun- gicide sensitivities. While the potential for spore dispersal over long distances is unclear, it may be limited, which may result in greater differentiation between fields. However, seed-borne infection with P. teres may help to move novel genotypes of P. teres over long distances. Olvång (1988) found high variation in sensitiv- ity of P. teres isolates to prochloraz, and the popu- lation was divided into two groups: sensitive and less sensitive. We identified a marked effect of field of isolates on growth on prochloraz-amended media. In the two farmers’ fields, in which prochlo- raz spraying took place, the mean growth rates on prochloraz-amended media were similar and were higher compared with other P. teres populations. 41 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Vol. 15 (2006): 35–42. The high variation between experiments within isolates originating from MTT’s experimental field precluded the detection of differences among iso- lates taken from plots with and without the appli- cation of prochloraz. Implications To maintain the efficiency of fungicides, evalua- tion of long-term effectiveness of fungicides on cereal plant pathogens is important. Fungicides with different modes of action should be used on cereals according to official recommendations. Sales of fungicides in Finland (Savela and Hyn- ninen 2004, Plant Production Inspection Centre 2005) show that fungicides with two different ac- tive ingredients and modes of action are beginning to replace fungicides with only one active ingredi- ent, which is a positive change. However, different active ingredients may still have the same type of mode of action, e.g. fungicides belonging to ima- dazole class of DMIs (imazalil and prochloraz). In addition, the occurrence of cross-resistance has been observed in P. teres among imazalil-propico- nazole and fenarimol-triadimenol, which are all sterol-inhibiting fungicides, but more importantly belong to different chemical groups among the DMIs (Peever and Milgroom 1993). Under pro- longed use of the same type of fungicides, less sen- sitive isolates will survive in the pathogen popula- tion residing in a particular field (Olvång 1988). Sexual reproduction can combine genes effective- ly and potentially produce more tolerant isolates. This risk must be taken into account since sexual reproduction of P. teres is possible in Finland (Se- renius et al. 2005). However, to fully understand the population biology of the net blotch pathogen and the potential for development and spread of fungicide tolerant isolates more research is need- ed. Acknowledgements. Tarja Hovivuori is thanked for her technical assistance. The authors want to acknowledge the help and co-operation of people that work in the Evalua- tion and testing of plant production products at MTT Agri- food Research Finland. This work was supported by grants from the Finnish Cultural Foundation and Finnish Minis- try of Agriculture and Forestry. References Dyer, P.S., Hansen, J., Delaney, A. & Lucas, J.A. 2000. Ge- netic control of resistance to sterol 14α-demethylase inhibitor fungicide prochloraz in the cereal eyespot pathogen Tapesia yallundae. Applied and Environmen- tal Microbiology 66: 4599–4604. Jordan, V.W.L. 1981. Aetiology of barley net blotch caused by Pyrenophora teres and some effects on yield. Plant Pathology 30: 77–87. Kangas, A., Kedonperä, A., Laine, A., Lindroos, M., Niska- nen, M., Salo, Y., Vuorinen, M., Jauhiainen, L. & Ram- stadius, E. 2004. Viljalajikkeiden herkkyys tautitartun- noille virallisissa lajikekokeissa 1997–2004. Abstract: Disease susceptibility of cereal varieties in Finnish of- ficial variety trials 1997–2004. Agrifood Research Working papers 77. 31 p. Kangas, A., Laine, A., Niskanen, M., Salo, Y., Vuorinen, M., Jauhiainen, L. & Nikander, H. 2005. Virallisten lajike- kokeiden tulokset 1997–2004. Research report in Finn- ish with English subtitles: Results of official variety tri- als 1997–2004. Agrifood Research Working papers 83. 193 p. McDonald, W.C. 1967. Variability and inheritance of mor- phological mutants in Pyrenophora teres. Phytopathol- ogy 57: 747–755. Olvång, H. 1988. Sensitivity of Drechslera teres and Septo- ria nodorum to sterol-biosynthesis inhibitors. Nether- lands Journal of Plant Pathology 94: 57–68. Peever, T.L. & Milgroom, M.G. 1993. Genetic correlation in resistance to sterol biosynthesis-inhibiting fungicides in Pyrenophora teres. Phytopathology 83: 1076–1082. Plant Production Inspection Centre 2005. Torjunta-aineet 2005. Luettelo rekisterissä olevista torjunta-aineista ja niiden käyttöä koskevista ehdoista. (Pesticides 2005, In Finnish, Active substances in English), Helsinki, Fin- land. 128 p. Savela, M.-L. & Hynninen, E.-L. 2004. Slower growth in pes- ticide sales. Kemia-Kemi 31, 6: 57–59. Serenius, M., Mironenko, N. & Manninen, O. 2005. Genetic variation, occurrence of mating types and different forms of Pyrenophora teres causing net blotch of bar- ley in Finland. Mycological Research 109, 7: 809–817. Steffens, J.J., Pell, E.J. & Tien, M. 1996. Mechanisms of fungicide resistance in phytopathogenic fungi. Current Opinion in Biotechnology 7: 348–355. 42 A G R I C U L T U R A L   A N D   F O O D   S C I E N C E Serenius, M. & Manninen, O. Prochloraz tolerance of Pyrenophora teres population Ohranverkkolaikku on tärkein taudinaiheuttaja suoma- laisessa ohranviljelyssä, ja se aiheuttaa aroilla ohralajik- keilla keskimäärin 40 % tautisuuden ja merkittäviä sato- tappioita. Tautia torjutaan siemenpeittauksella tai kas- vustoruiskutuksilla kasvukauden aikana. Prokloratsi (kauppavalmisteet Sportak ja Predule) on yleinen ohran kasvinsuojeluaine. Suomessa myydyistä peittausaineista puolet sisältää vaikuttavana aineena joko prokloratsia tai imatsaliilia tai näiden yhdistelmiä. Molemmat aineet kuuluvat samaan kemialliseen ryhmään ja vaikuttavat samalla tavalla. Tämän tutkimuksen tarkoituksena oli selvittää prokloratsia kestävien ohranverkkolaikkua ai- heuttavien tautikantojen esiintymistä lounaishämäläisil- lä pelloilla. Näytteitä kerättiin yhteensä 364 kpl vuonna 2003 kolmelta viljelijän pellolta ja yhdeltä MTT:n (Maa- ja elintarviketalouden tutkimuskeskus) koealalta. Viljeli- jöiden pelloilta kerätyt tautikannat, jotka olivat saaneet Sportak-käsittelyn kasvukauden aikana, kasvoivat labo- ratoriotesteissä paremmin kasvitautiainetta sisältävillä kasvatusmaljoilla kuin koealalta kerätyt tautikannat. Viljelijöiden pelloilta peräisin olevat tautikannat kasvoi- vat parhaiten sekä laimeammalla että vahvemmalla tes- tialustalla verrattuna tautikantoihin, joilla oli käytetty muita kasvinsuojeluaineita. Vastaavaa testiä ei ole tehty aiemmin, joten ei voida osoittaa tautiaineen kestävyy- den muuttuneen. Tulokset osoittavat, että tautikantojen kasvinsuojeluaineen kestävyys voi vaihdella eri pelloil- la. Tämä alustava aineisto oli kuitenkin riittämätön yleistettävien johtopäätösten tekoon. Aineiden tehoa kannattaa seurata. Suositeltavinta olisi käyttää aineseoksia ohranverkkolaikun torjunnassa ja välttää samalla tavalla vaikuttavien kemiallisten kas- vinsuojeluaineiden pitkäaikaista käyttöä samalla pelto- lohkolla. Tautiaineiden tehon turvaamiseksi tautia pitäisi pyrkiä vähentämään myös muilla keinoilla, kuten vilje- lykierrolla, kasvijätteiden muokkauksella ja taudinkes- tävien ohralajikkeiden viljelyllä. Tämä alustava tutki- mus osoitti, että ohranverkkolaikun aiheuttajan proklo- ratsin kestävyydessä on eroa peltojen välillä. SELOSTUS Ohranverkkolaikkutaudin aiheuttajan prokloratsin kestävyys Suomessa Marjo Serenius ja Outi Manninen Maa- ja elintarviketalouden tutkimuskeskus Prochloraz tolerance of Pyrenophora teres population in Finland Introduction Material and methods Results Discussion Implications References SELOSTUS