Abstract

When monitoring powdery mildew populations there is often a problem of a sample size. If the sample size was too small, the results would not be accurate enough. The total of 406 powdery mildew isolates obtained by a mobile spore sampling were tested for virulence frequencies. The sampling was carried out in Slovakia in May and June 2000. Virulence reactions were assessed on a differential set containing twelve resistance genes or gene combinations. Frequency data of pathotypes identified in this way were used for calculation of diversity indices: Simpson index (S), dissimilarity (D) and complexity (C). Standard deviations of the diversity indices were monitored. The results show that 70 isolates of powdery mildew represent the most suitable small sample size, since the curves of standard deviations of diversity indices remain almost steady or only decrease slowly above this sample size.

Introduction

Powdery mildew, caused by Blumeria graminis (DC.) E.O. Speer f.sp. tritici Em. Marchal, syn. Erysiphe graminis, is a fungal disease that has been recognized as a disease problem on wheat for centuries. It is widely distributed in all wheat growing areas throughout the world. Losses up to 45% have been documented (Felsenstein, 1991). Blumeria graminis f.sp. tritici is an obligate parasite specific to wheat. Multiple races of the fungus exist and new ones continue to be formed as a result of genetic recombination. Besides the sexual cycle contributing to diversity, the disease is polycyclic and well adapted for aerial dispersal and long distance transport (Limpert et al., 1999; Limpert et al., 2000).

When monitoring virulence fequencies in powdery mildew populations the frequent problem is that of adequate sample size for accuracy. The monitoring is usually carried out annually, and different numbers of mildew isolates are obtained every year. The results of the virulence analysis could be inaccurate if the sample size was too small.

The aim of this study was to determine the smallest possible sample size for accurate and reproducible virulence analysis.

Materials and Methods

Mildew isolates

The isolates used in this study were collected from the aerial population with a jet spore trap (Schwarzbach, 1979). The sampling was carried out in Slovakia in May and June 2000. Spores were trapped on segments of primary leaves of a susceptible wheat variety 'Košútka' placed in Petri-dishes containing 7% agar and 30 ppm benzimidazole. Petri dishes were changed every 40-140 km, the total sampling route length being 1117 km. The sporulating colonies were scattered amongst the leaves, and each colony was assumed to originate from a single conidium.

Differential set

After 5-7 day incubation, the isolates were transferred onto leaf segments of 'Košútka' for multiplication and tested 10-12 days later on a differential set. Virulence assessments were carried out on a differential set consisting of near-isogenic lines with 'Chancellor' background and other varieties with single gene or combination of resistance genes: 'Axminster' (Pm1) 8xCC*, 'Torysa' (Pm2+Pm6), 'Asosan' (Pm3a) 8xCC, 'Chul' (Pm3b) 8xCC, 'Sonora' (Pm3c) 8xCC, 'Khapli' (Pm4a) 8xCC, 'Armada' (Pm4b), 'Regina' (pm5), 'Timgalen' (Pm6), 'Salzmunde' 14/44 (Pm8), 'Maris Dove' (Pm2+Mld), 'Normandie' (Pm1+Pm2+Pm9) and 'Košútka' as the susceptible control. All varieties except 'Torysa' and 'Regina', were kindly provided by Dr. M. Winzeler from Swiss Federal Research Station of Agronomy, Zürich-Reckenholz.
(* - eight-times backcrossed to 'Chancellor')

Experimental conditions and virulence analysis

The infected leaves were kept under continuous light (2000 lux) at 17-18 °C. Untreated seeds of the differential set varieties were sown in plastic pots. The seedlings were protected against infection inside cellophane bags. Primary leaves of 10-12 days old seedlings were used for the experiments. Fifteen millimetres long leaf segments of each variety were laid out in randomised patterns in Petri-dishes on a medium containing 5% agar and 25 ppm benzimidazole. Each differential set was inoculated with the progeny of the single colony isolate by drawing spores into a pipet and blowing them onto a settling tower. Inoculum density was approximately 250 conidia/cm2. After 10 days of incubation at 18±1 °C under continuous light severity of disease was scored relative to the susceptible control ('Košútka') in the set. Sporulation of more than 50% was considered to be a susceptible reaction and less than 50% was considered to be resistant.

Statistical evaluation

From a total of 406 powdery mildew isolates obtained in 2000, groups of 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360 and 406 isolates were randomly selected. Diversity indices were counted for each group of isolates using a computer program ‚VIRAN'. The complement 1 - lambda was used as the Simpson index of diversity (S):

where ni = the number of isolates of the i-th pathotype, n = the sample size and s = the number of different pathotypes in the sample. A numerical estimate of the standard error of the Simpson index was calculated from 600 bootstrap samples (Efron & Tibshirani, 1991). The mean dissimilarity (D) of a sample was calculated according to the formula:

(Müller et al., 1996). Complexity (C) was defined as a mean number of virulence loci per isolate. Means of the samples and their standard deviations were determined. A graph was constructed from the standard deviations, and the smallest population size giving accurate, reproducible results was determined at the point where curves had a tendency to remain steady.

Results and Discussion

The total of 406 powdery mildew isolates obtained from the air population were analysed. The standard deviations of the Simpson index (S), dissimilarity (D) and complexity (C) for different numbers of isolates are given in Table 1, and are plotted in two different ways in Figure 1. All indices show a steeply decreasing curve from 20 to 70 isolates, followed by a levelling off to 406 isolates. They indicate that a group of 70 powdery mildew isolates is the minimum practical size small set of isolates which would give accurate and reproducible virulence analysis.

It is not easy to obtain a group of powdery mildew isolates of this size in years when an infection pressure is low and few spores are trapped in the jet spore sampler. Unfavourable climatic conditions with extremes such as drought or extremely rainy weather can substantially influence the sample size. The survival chance of spores can be seriously reduced by UV light, and their probability of long distance dispersal can be considerably increased under cloudy conditions (Limpert et al., 2000). Furthermore, spore release with these pathogens benefits from dry conditions (Hau & de Valavieille-Pope, 1998).

The sampling method used can play a very important role as well. Mobile sampling using a high throughput jet trap (Schwarzbach, 1979) makes it possible to collect about 50 colonies per Petri dish, and Petri dishes may be changed every 50-100 km while driving through the regions of interest. Stationary nurseries also enable isolation of sufficient numbers of colonies, but infected leaves taken directly from a field do not usually give enough isolates. For instance, Pasquini (1992) analysed mildew spores from the infected leaves in a field, and the method enabled her to obtain up to 75 isolates in 1990, but only 23 in 1984. Winzeller et al. (1991) analysed a total of 919 mildew isolates within 1980-1989, and their sample size varied from 27 to 162. Stationary nursery made it possible for Hovmøller & Østergård (1991) to obtain about 100 barley powdery mildew isolates from each locality, except for the year 1985 when they obtained only 52 isolates. Müller et al. (1996) made use of the mobile sampling and analysed a total of 3624 colonies from across most of Europe, the smallest number of colonies being collected in the former Czechoslovakia (85) and Italy (99). From 27 up to 196 barley powdery mildew isolates were obtained by Caffier et al. (1996) using the mobile sampler. Numbers of powdery mildew isolates tested in 10-11 European countries participating in the work of the Wheat Mildew Sub-group of COST 817 in 1996-1998 varied conciderably between 17 and 618 (Clarkson, 2000). In Slovakia, we were able to collect from 20 to 52 wheat powdery mildew colonies from 40-140 km long sampling routes.

Different authors use different sample size, therefore, it is not possible to find out which sample size is the most suitable one. However, from our results we recommend analysis of a set of at least 70 isolates, but use of more than 70 isolates will obviously increase accuracy.

Acknowledgements

The work was supported by the Slovak Grant Agency VEGA (Project VEGA 1/7251/2000). We thank Ms Iveta Cajkovicová and Ms Marta Hladká for technical assistance.

References

Caffier V, Hoffstadt T, Leconte M, de Vallavieille-Pope C, 1996. Seasonal changes in pathotype complexity in French populations of barley mildew. Plant Pathology 45, 454-468.

Clarkson JDS, 2000. Virulence survey report for wheat powdery mildew in Europe, 1996-1998. Cereal Rusts and Powdery Mildews Bulletin [www.crpmb.org/] 2000/1204clarkson.

Efron B, Tibshirani R, 1991. Statistical data analysis in the computer age. Science 253, 390-395.

Felsenstein FG, 1991. Virulenz und Fungizidsensitivität des Weisenmehltaus (Erysiphe graminis DC. f.sp.tritici Marchal) in Europa. Munich, Germany: Technical University Munich, PhD thesis.

Hau B, de Vallavieille-Pope C, 1998. Wind-dispersed diseases. In: Jones DG, eds. The Epidemiology of Plant Diseases. Dordrecht: Kluwer Publishers, 323-347.

Hovmøller MS, Østergård H, 1991. Gametic disequilibria between virulence genes in barley powdery mildew populations in relation to selection and recombination. II. Danish observations. Plant Pathology 40, 178-189.

Limpert E, Godet F, Müller K, 1999. Dispersal of cereal mildews across Europe. Agricultural Forest Meteorology 97, 293-308.

Limpert E, Bartoš P, Graber WK, Müller K, Fuchs JG, 2000. Increase of Virulence Complexity of Nomadic Airborne Pathogens from West to East Across Europe. Acta Phytopathologica et Entomologica Hungarica 35, 317-322.

Müller K, McDermott JM, Wolfe MS, Limpert E, 1996. Analysis of diversity in populations of plant pathogens: the barley powdery mildew pathogen across Europe. European Journal of Plant Pathology 102, 385-395.

Pasquini M, 1992. Occurence and virulence of Erysiphe graminis tritici in Italy. Pflanzenzüchtung 24, 178-180.

Schwarzbach E, 1979. A high throughput jet trap for collecting mildew spores on living leaves. Phytopathologische Zeitschrift 94, 165-171.

Winzeler M, Streckeisen Ph, Fried PM, 1991. Virulence analysis of the wheat powdery mildew population in Switzerland between 1980 and 1989. In: Jørgensen JH, eds. Integrated Control of Cereal Mildews: Virulence Patterns and their Change. Roskilde, Denmark: Risø National Laboratory, 15-21.