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7УДК 633.16:632.421
METHODS AND RESULTS OF INVESTIGATION OF BLUMERIA GRAMINIS F.SP. HORDEI POPULATIONS
A.Dreiseitl
Agricultural Research Institute Kromeriz Ltd., Havlickova 2787, CZ-767 01 Kromeriz, Czech Republic, drei-
seitl@vukrom.cz
The author's activity in the field of studying the domestic (Czech) and other populations of the causal agent of powdery mildew on barley (Blumeria graminis f.sp. hordei fungus) is mentioned. The two methods most often used are described in detail. The reason for the study is outlined and some results obtained in recent years and present objectives of the investigation of the barley-powdery mildew pathosystem in the Czech Republic are presented.
Introduction
In the Czech Republic, barley (Hordeum vul-gare L.) is planted each year on about 500000 ha, of which 30% of winter barley. Powdery mildew is a typical airborne disease. It is caused by the fungus Blumeria graminis f.sp. hordei - B.g.h. (syn. Erysiphe graminis f.sp. hordei). Powdery mildew is the most common disease of barley. An analysis of data of the Central Institute for Supervising and Testing in Agriculture for 1989 to 2001 revealed that 50% of all epidemics in spring barley and 40% in winter barley was caused by this pathogen (Dreiseitl, Jurecka,1996, 1997). Powdery mildew decreases grain yield, its feeding and malting quality and production profitability.
Powdery mildew on barley can be efficiently controlled with fungicide application or growing resistant varieties (Jorgensen,1994; Drei-seitl,j0rgensen, 2000; Dreiseitl,Svacina, 2001). Resistant varieties stabilize yield and quality of production. They allow to avoid losses not only at epidemics but as well as in the case of smaller in-
fection when fungicide treatments are not economically effective. Growing resistant varieties is a low-cost and health-safe control of powdery mildew.
Efficiency of each genetically based resistance is limited by an actual composition of the pathogen population. Therefore, along with breeding and growing resistant varieties, attention should be given to a status and development of the pathogen population (Hovm0ller et al., 2000). We have been conducting a systematic study of the domestic (Czech) population of powdery mildew pathogen since 1992 (Dreiseitl,Schwarzbach, 1994). The present goals of the investigation of the population are 1) to detect virulences to chosen resistance genes that are currently possessed by grown barley varieties, 2) to assess frequencies of chosen virulences and 3) to discover effects of individual adaptation mechanisms on formation of the pathogen population. In addition to the domestic population, we studied some populations of this pathogen in other parts of the world.
We are using several methods of sampling of B.g.h. isolates (Dreiseitl,1998a). Two of them are more often used. The study of any population is based on obtaining representative samples of the pathogen. A mobile spore-sampler is used for
that. If this device is not available (for example, in studying populations abroad), we obtain a sample of the corresponding population by looking for individual colonies of the pathogen in barley stands.
Air conidia-
Sampling of isolates. Samples of the domestic pathogen population are obtained from the air on the territory of the Czech Republic. A car with a spore-sampler (Schwarzbach,1979) fixed to its roof is drived across main barley growing areas.
sampling
Conidia are sampled each year in late spring when barley stands are at GS 30 (late-sown spring barley) up to GS 60 (the earliest winter barley) on the route of ca. 1500 km. Representativeness of the obtained sample of the pathogen population is
given by random sampling conidia from the air and a route length when we pass by a high number of fields planted with various varieties of both barley forms in less or larger distance.
Reproduction of the inoculum. Conidia caught with the spore-sampler settle in a settling tower of this device at the bottom of which there is a Petri dish with cut off leaves of a susceptible variety. The leaves are placed in water agar (6 g/l with addition of 35 ppm benzimidazole). To reproduce conidia, we use fully-developed primary leaves of spring barley variety 'Pallas' (Kolster et al.,1986) or B-3212 (Dreiseitl,Steffenson,1996) that are cultivated under conditions free of infection. Dishes with leaves in the spore-sampler are changed after every ca. 150 km. After one to three days, the leaves with caught conidia are put over with tweezers into other Petri dishes with a fresh agar medium of the identical composition and incubated for other 8-10 days in an incubation room at 18±3°C under indirect natural light or artificial cool-white light (day/night = 8/16 h,
50-100 ^Mol/m2/sec).
Inoculation is performed in a cylindric metal inoculation tower. Conidia of one colony are blown through a hole (10 mm) in the middle of the upper part of the tower. The tower is three times higher than its diameter (diameter of Petri dishes used). For each isolate, a Petri dish with leaf segments of the set of differentials is placed at the bottom of the tower. Conidia of each mildew colony are sucked up with a Varipipette AW 1000 into a tip (the varipipette is to set up to the volume of 0.4 ml). Consequently conidia are blown from the tip into the tower by a syringe (10 ml) connected with a small tube. For each colony, a Petri dish with leaf segments of the set of differentials is placed at the bottom of the tower. To suck up conidia of each colony, an exchangeable tip of a varipipette is always used. The end of a small tube connecting a tip of a varipipette with a syringe is sterilized with spirit after each inoculation. The following parts (differential set, evaluation and designation of isolates) see the method B.
Field <
Sampling of isolates. Leaf segments with individual B.g.h. colonies, each of them hopefully developed from one conidium during a natural infection, are cut and placed in a Petri dish with 0.6% water agar and 35 ppm benzimidazole. The sampling of B.g.h. colonies should be done before the peak of the powdery mildew season. The population of the pathogen should be sampled from a representative area. It is better to collect a less number of colonies (usually up to five) from a higher number of different fields (at least 30).
Reproduction of the inoculum. Leaf segments with individual B.g.h. colonies are incubated for a day in an incubation room. Conidia from each colony are shaken onto one segment, 25 mm in length, excised from the central part of healthy, fully developed primary leaves of a susceptible variety and placed in a Petri dish with water agar. Inoculated leaf segments are incubated under the conditions described above for 10-15 days.
Inoculation is performed in a similar metal inoculation tower (see the method A). For each isolate, a Petri dish with leaf segments of the set of differentials is placed at the bottom of the tower. Inoculum from the leaf segment of an isolate is shaken onto a square piece (40 mm) of black pa-
lidia-sampling
per to check the amount of conidia, and is blown through a side hole (15 mm) into the upper part of the inoculation tower.
Differential set. The set of differentials is usually comprised of 'Pallas' near-isogenic lines (Kolster et al.,1986) and also of other chosen varieties possessing different resistance genes to powdery mildew. We have been using 25-35 differentials during the last several years. A long-term constant core set comprises 12 of them. The order of the first ten differentials carrying appropriate genes is according to the European agreement (Limpert,Dreiseitl,1996). Plants of differentials have to be grown in mildew proof conditions. Leaf segments of 25 mm in length are removed from the central part of healthy and fully-developed primary leaves. At least three leaf segments of each differential line should be used for testing of each isolate. Leaf segments are placed in Petri dishes on water agar and inoculated with an isolate. Inoculum density is 2-5 con-idia.mm-2. The Petri dishes with inoculated leaf segments are placed under the conditions described above for 8 days.
Evaluation. Infection types produced by the response of each barley differential to inoculation
with a corresponding B.g.h. isolate are scored according to the nine-point 0-4 scale including intertypes (Torp et al.,1978).
Designation of isolates is based on their virulence to matching resistance genes in the differentials. Notation of virulences is based on octal
numbers (Limpert et al.,1994). Each of the digits represents virulence or avirulence on three differentials. The resulting number defines the virulence of the isolate and consequently its classification as certain pathotype.
Results an
We have been conducting a systematic study of the B.g.h. population since 1992. A lot of information has been achieved of which a part has been evaluated and published (see references). Studying the domestic (Czech) population, we succeeded, for instance, in clarifying causes of failing experimental growing of spring barley varietal mixtures (Dreiseitl,1998b, 2000b). In addition, we elucidated cases when efficiency of chosen resistances disappeared mostly due to direct selection of virulent pathotypes on varieties with corresponding resistance genes (for instance, resistances controlled by Ml genes a1, a6, a7, a9, a13, g, k1, La, at) (Dreiseitl 2000a) as well as due to indirect selection (Mla12) associated with direct selection of another virulence (Dreiseitl 2000b). We found out that an increasing frequency of the Va3 virulence in the Czech pathogen population was caused by immigration from neighbouring countries (Dreiseitl 2001a). Findings on contribution of the "Czech" virulence Va13 to breaking down corresponding resistance in other European countries resulting from emigration of virulent pathotypes (from the territory of the Czech Republic) have been summarized (Dreiseitl 2001a). A role of recombinations in the studied pathogen population has been assessed (Dreiseitl 2003).
Based on the example of the New Zealand population and its comparison with the Australian one, it was proved that powdery mildew conidia are not able in the viable status to cross the distance between Australia and South Island even under optimum conditions (Dreiseitl,Pickering,
Discussion
1999). High frequency of Va3 and Vp was found in the population from North Dakota, and on the contrary, no isolate exhibited virulence to the resistance gene Mlg (Dreiseitl,Steffenson 2000). Frequences of these virulences does not correspond with our assumption that could be made only on the basis of knowledge of resistance genes present in American and Canadian barley varieties (Dreiseitl,Steffenson, 1996). In Israel, the population of B.g.h. on naturally growing wild barley (Hordeum vulgare ssp. spontaneum) was investigated in details considering both the frequency of virulences to resistance genes that are commonly studied in Europe (Kolster et al.,1986) and to new genes (Jahoor,Fischbeck,1987,1993) that will be present in newly registered European barley varieties in the near future. The isolates obtained during this study in the centre of diversity of both the host (wild barley) and the pathogen (B.g.h.) enabled us to distinguish phenotypic diversity of a large collection of new original resistance sources of wild barley that are fully resistant to the European pathotypes used. That allowed us to continue the rational study of these new genetic resources including currently starting development of molecular markers for detection of individual genes. Now we assess diversity within populations and distance between populations in cooperation with Dr.Kosman (Tel Aviv University, Israel) (Kosman,1996; Kosman, Leonard, 2002). Based on a new project, we should start together with professor Shen Qiuquan a study of barley powdery mildew in one of provinces in China.
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Acknowledgements. This study was conducted within grant no. 522/00/1062 of the Grant Agency of the Czech Republic.
МЕТОДЫ И РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЙ ПОПУЛЯЦИЙ BLUMERIA GRAMINIS f.sp. HORDEI А.Драйсайтл
Описаны два наиболее часто используемые автором метода сбора образцов популяций возбудителя мучнистой росы ячменя (из воздуха и с растений) и их анализ. Проведено сравнение результатов анализа популяций паразита из Европы, Северо-Американского континента, Новой Зеландии и Австралии.
