MORPHOLOGY OF UREDINIA AND UREDINIOSPORES OF THE FUNGUS Melampsora larici-epitea KLEB . A DAMAGING PATHOGEN OF COMMON OSIER ( Salix viminalis L . ) IN POLAND

Rust (Melampsora spp.) is a damaging disease of willows (Salix spp.), including common osier (S. viminalis L.). So far, the pathogens of this species found in Europe were identified as M. larici-epitea Kleb. or M. ribesii-viminalis Kleb. Moreover, a stem infecting form (SIF), deprived of a sexual stage in its life cycle was reported. The aim of this study was to find out which species of the rust fungi cause disease symptoms on common osier in Poland. The isolates from common osier were compared to the ones originating from its putative hybrids with trembling aspen (Populus tremula L.) and Simon’s poplar (P. simonii Carr.). Fungal isolates were obtained in 2008–2010 from 15 different genotypes of willows, including seven varieties of common osier (4 Swedish and 3 Polish), two landraces of common osier and six putative hybrids with poplars. Fungal isolates originated from three experiment sites, including west (Wielkopolska and Lubuskie) and north-east (Warmia) regions of Poland. To ensure the genetic uniformity, the isolates were derived from single uredinia, obtained from natural infection conditions. In all collected samples the position of uredinia was always hypophyllous. The diameter of uredinia was measured by Sigma Scan Pro software, after inoculation of four standard genotypes, including two common osier and two willow hybrids. The studies proved that the main cause of common osier rust is Melampsora larici-epitea f. typica. All studied isolates, including the ones obtained on putative hybrids, were very similar according to the size of uredinia and the size and morphology of urediniospores. The average size of a uredinium was 1.1 mm diameter and slightly differed between the isolates (from 0.9 to 1.3 mm), depending on willow genotype, the quality of plant material used for artificial inoculations and infection conditions. The average size of a typical urediniospore was 12.4 x 10.5 μm, but the sizes varied from 9.8 to 13.2 μm. Urediniospores from common osier were slightly bigger and more oval (12.5 x 10.4 μm) as compared to spores from the putative hybrids Salix MORPHOLOGY OF UREDINIA AND UREDINIOSPORES OF THE FUNGUS Melampsora larici-epitea KLEB. A DAMAGING PATHOGEN OF COMMON OSIER (Salix viminalis L.) IN POLAND Joanna Ciszewska-Marciniak, Małgorzata Jędryczka, Stanisław Jeżowski, Jerzy Przyborowski, Katarzyna Wojciechowicz, Elżbieta Zenkteler 1 Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland 2 Department of Plant Breeding and Seed Production, University of Warmia and Mazury, Olsztyn, Poland 2 Department of General Botany, Adam Mickiewicz University, Poznań, Poland e-mail: jcis@igr.poznan.pl Received: 05.08.2010 x Populus (12.3 x 10.6 μm), but the differences were statistically insignificant. The spores were ovoid, globoid or angular, evenly echinulated. The size of uredinia, as well as the size and morphology of urediniospores were in full agreement with literature data for M. larici-epitea f. typica.

A b s t r a c t Rust (Melampsora spp.) is a damaging disease of willows (Salix spp.), including common osier (S. viminalis L.).So far, the pathogens of this species found in Europe were identified as M. larici-epitea Kleb.or M. ribesii-viminalis Kleb.Moreover, a stem infecting form (SIF), deprived of a sexual stage in its life cycle was reported.The aim of this study was to find out which species of the rust fungi cause disease symptoms on common osier in Poland.The isolates from common osier were compared to the ones originating from its putative hybrids with trembling aspen (Populus tremula L.) and Simon's poplar (P.simonii Carr.).Fungal isolates were obtained in 2008-2010 from 15 different genotypes of willows, including seven varieties of common osier (4 Swedish and 3 Polish), two landraces of common osier and six putative hybrids with poplars.Fungal isolates originated from three experiment sites, including west (Wielkopolska and Lubuskie) and north-east (Warmia) regions of Poland.To ensure the genetic uniformity, the isolates were derived from single uredinia, obtained from natural infection conditions.In all collected samples the position of uredinia was always hypophyllous.The diameter of uredinia was measured by Sigma Scan Pro software, after inoculation of four standard genotypes, including two common osier and two willow hybrids.
The studies proved that the main cause of common osier rust is Melampsora larici-epitea f. typica.All studied isolates, including the ones obtained on putative hybrids, were very similar according to the size of uredinia and the size and morphology of urediniospores.The average size of a uredinium was 1.1 mm diameter and slightly differed between the isolates (from 0.9 to 1.3 mm), depending on willow genotype, the quality of plant material used for artificial inoculations and infection conditions.The average size of a typical urediniospore was 12.4 x 10.5 μm, but the sizes varied from 9.8 to 13.2 μm.Urediniospores from common osier were slightly bigger and more oval (12.5 x 10.4 μm) as compared to spores from the putative hybrids Salix

INTRODUCTION
The diversity of willows (Salix spp.) worldwide is enormous.According to different sources, the number of willow species varies between 300 and 500, with about 270 willow species in China, 120 in the former Soviet Union, over 100 in North America and some 65 willow species in Europe (A r g u s , 1997; P e i , 2005).Moreover, the genetic diversity within one willow species can be also tremendous.Immense diversity present between and within willow species influences its main pathogen -willow rust (Melampsora spp.).This important fungal genus, gathering numerous pathogens of various plant species, coevolves with hosts, what also results in its great diversity (S a v i l e , 1976; R a m s t e d t , 1999; S a m i l s , 2005).The populations of this pathogen greatly depend on the composition of host-plant populations, in respect to their resistance (S a m i l s et al.  , 1972).
By now, there were no thorough studies of willow rust and its causal agent in Poland.The aim of this study was to find out which species of the rust fungi cause disease symptoms on common osier in Poland.The isolates from common osier were compared to the ones originating from its putative hybrids with trembling aspen (Populus tremula L.) and Simon's poplar (P.simonii Carr.).

Plant material
Fungal isolates originated from nine genotypes of common osier (S. viminalis) and its five putative hybrids with trembling aspen (P.tremula) and one putative hybrid with Simon's poplar (P.simonii).The detailed description of the methods used to obtain the hybrids was presented elsewhere, by Z e n k t e l e r et al. (2005).Two genotypes of common osier were growing at both locations in west Poland, and the hybrids were cultivated in west and north-east Poland (Table 1).The oldest field originated from Marzęcin and it was planted in the spring of 1997, whereas the common osier willow genotypes from Poznań started their growth in the spring of 2002 and these from Bałdy -in the spring of 2006.All hybrids were planted in the autumn of 2007.

Fungal material
Samples of leaves with the symptoms of willow rust were collected at each time of disease assessment.The leaves were dried for a few days at room temperature.Then, the samples were placed in Kristal semi-transparent glassine smooth bags (Brian Springfield, Bristol, UK) and stored at -20 o C. The propagation of urediniospores was done using young leaves of a hybrid S. aurita x viminalis x caprea var.Stipularis, grown for 6-8 weeks in glasshouse conditions.Plant material used for the propagation was each time obtained from cuttings originating from the previous season and stored at +4 o C. Cultivation in a glasshouse was done using 16/8 hour photoperiod, at 18-20 o C and 16-18 o C, respectively at daytime and night (darkness).Freshly collected young and soft willow leaves were placed in 90 mm diameter Petrie dishes on discs of filter paper soaked with sterile distilled water.Each leaf was covered with urediniospores originating from one chosen uredinium, obtained from the material collected in field conditions, at particular genotypes, years and experiment sites.The spores were uniformly spread with a brush, previously sterilized in 99,8% ethanol.Petrie dishes with leaves bearing spores were transferred to controlled environment (CE) chambers (Mytron WB1500, Bio-und Solartechnik GmbH, Heilbad Heiligenstadt, Germany) with 16/8 hour photoperiod, at a constant temperature of 16 o C. Each cycle of propagation of urediniospores lasted 13 days.The second propagation cycle was also started using one selected uredinium, whereas the following cycles used a mixture of all uredinia obtained from the previous propagation.In such way, the finally obtained urediniospores were numerous and they were all genetically identical.The propagation of a sufficient number of spores lasted at least four cycles, i.e. two months of subculture in CE conditions.

Evaluation of the size of uredinia
The size of uredinia was measured using the subset of internationally recognized standard geno- types, including two common osier cultivars (Jorr and Mullatin) and two willow hybrids: S. viminalis x S. schwerinii cv.Beagle and S. aurita x viminalis x caprea var.Stipularis.Uredinia used for the measurement were obtained on young and fresh leaves of the above mentioned genotypes, after spraying them with a suspension of urediniospores.The concentration of spores used for inoculation was each time adjusted to 2 x 10 5 urediniospores per 1 ml of sterile distilled water (P e i et al. 1996).Each time the viability of spores was checked after 24 hours inoculation at 15 o C, in 60 mm Petrie dishes on 5% water technical agar medium.The experiment was repeated if the germination of urediniospores was less than 60%.Leaf inoculation was done using 95 mm 2 leaf discs freshly cut from tested cultivars and placed with the abaxial side up, on 1,5 cm wide and 4 cm long stripes, prepared from Whatmann 3MM filter paper and formed in the shape of a bridge.Paper stripes supporting leaf discs were placed in 25-well plastic square boxes (Sterilin Ltd., Caerphilly, UK) and then soaked with sterile distilled water, applied by a laboratory sprayer, carefully avoiding moistening of tested willow leaves.The measurement of the size of uredinia was done with Sigma Scan Pro computer software, version 5 (Systat Software Inc., Chicago, USA).The data were subjected to statistical calculations of variance analysis, using Statistica Package version 9 (StatSoft Polska).

Evaluation of the morphology of urediniospores
The morphology of urediniospores was checked on freshly propagated materials, obtained from the spores stored at -20 o C. The spores were placed on specimen stubs covered with double sided adhesive tapes and sprayed with gold particles (99,9%) in sputter coater Bal-Tec SCD050 (Balzers, Lichtenstein).The spores were photographed using Scanning Electron Microscope (SEM) Zeiss EVO40 (Carl Zeiss AG, Oberkochen, Germany).Each time two longest measurements were taken at right angles.The mean size of urediniospores was a result of 10 measurements.

RESULTS
Uredinia of rust were formed on all tested genotypes of willow, including two common osier cultivars and two hybrids with the other willow species.They were always produced on the abaxial leaf side (Figs 1-4).The color was bright yellow with little variation of its shading and intensity.At the propagation cycles the number of uredinia was usually between 250 and 2500 per leaf.The highest yields were obtained on cv.Stipularis.
On average the smallest uredinia were produced on the Swedish cultivar Jorr; the mean diameter of a uredinium formed at the abaxial side of leaves of this cultivar was 0.99 mm and varied from 0.89 to 1.17 mm (Table 2).The uredinia formed on cultivar Mullatin were also rather small -1.11 mm on average, with size variation between 0.98 and 1.20 mm.Uredinia formed on the hybrids between common osier and other willow species were bigger.They were 1.17 and 1.24 mm diameter, respectively for the cultivar Beagle (S. viminalis x S. schwerinii) and cv.Stipularis (S. aurita x viminalis x caprea).The respective ranges of uredinia diameters were 1.07-1.24mm and 1.17-1.31mm.On average the mean uredinium size was 1.13 mm, with variation between 0.89 and 1.31 mm.There were no differences between the mean diameters of uredinia produced by isolates obtained from common osier willow and its putative hybrids with poplars.
The mean diameter or a urediniospore was 11.52 μm and ranged from 9.79 to 13.16 μm.The spores were usually ovoid or globoid (Figs 5-8).Some urediniospores were angular, when too dry, old or changed by the SEM photography preparation   8).
The size of uredinia was negatively correlated with the size of urediniospores.In case of osier willow cultivars as well as its hybrid with S. schwerinii (cv.Beagle) the Pearson correlation coefficient ranged between -0.519 and -0.611.In case of cv.Stipularis this correlation was smaller (-0.223).

DISCUSSION
All isolates studied in this experiment produced bright yellow uredinia.They were formed only on abaxial leaf side.There was slight variation between the size of uredinia (0.89-1.31 mm) with the average of 1.13 mm.Such size and location of uredinia is typical for M. larici-epitea.According to the data shown by P e i ( 2005) the position of uredinia of this species is hypophyllous and the size range varies from 0.25 to 1.5 mm.The identical range of uredinia size of this species was also presented by B a g y a n a r a y an a (2005).Uredinia formed by M. ribesii-viminalis are much smaller; their typical diameter is 0.25 mm.It is very difficult to identify the alternate host of a rust species, and in such case the indirect character linked to this property is in demand.The result of this work suggests that the alternate host of the studied isolates is larch (Larix spp.), both in case of the isolates obtained from common osier and its hybrids with poplars.No significant differences were found between the isolates from both plant sources and the variation in uredinia size was nearly identical for both groups.
The size of uredinia of M. larici-epitea is bigger than of M. ribesii-viminalis, but the spore size is reversed -bigger for the latter species.According to P e i ( 2005), the range of urediniospore size for the common osier rust species alternating on current varies from 14 to 19 μm, and according to B a g y a n a r a ya n a (2005) the typical length of an urediniospore can reach 22 μm.In the present study the mean urediniospore size was 10.48 x 12.41 μm, and the measurements ranged from 9.79 to 13.16 μm.Such spore sizes are typical for M. epitea f. sp.larici-epitea typica also referred to as M. larici-epitea f. typica.Hence, the size of urediniospores was an additional proof the isolates in study were willow rusts alternating on larch.

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Evenly echinulated surface of urediniospores allowed to exclude one more potential rust candidate species -M.larici-populina, commonly found on P. simonii.Urediniospores of this pathogen are smooth at the apex and much bigger that these found on common osier.Their size may reach as much as 50 μm (P e i and S h a n g , 2005).Obviously this species is not expected on common osier, but it could attack its hybrid with Simon's poplar.However, this situation was not observed on the studied plant material.In turn, the hybrids of common osier with trembling aspen could result in the susceptibility of hybrids to M. larici-tremulae.Urediniospores of this species are also spiky and echinulated evenly on the whole surface of the spore.In this case however the uredinia are smaller (0,5 mm) and the urediniospores are bigger (up to 22 μm) than the observed ones.
There were no uredinia or urediniospore groups on the stems of tested willows, what suggested that a stem infecting form could be also excluded from this consideration.
The first uredinia to be produced in the pathogen's life cycle develop on mycelium resulting from the germination of aeciospores.Uredinia are produced on a dikaryotic mycelium, and the urediniospores also produce dikaryotic mycelium when they germinate (C u m m i n s and H i r a t s u k a , 2003).The mycelium resulting from the germination of urediniospores usually produces more uredinia and this character was used to obtain genetically identical urediniospores for the present study.The most efficient propagation was obtained on a hybrid of common osier with two other willow species (S. aurita x viminalis x caprea var.Stipularis).The effectiveness of this process was most probably a result of the correlation between bigger uredinia and numerous small urediniospores produced by rust isolates on this genotype.Out of four standards used in our study, cv.Stipularis proved to be the most effective, probably also due to its production of much broader and longer leaves than traditional common osier genotypes.
There were no thorough studies of willow rust and its causal pathogens in Poland by now.Our work ensured proper taxonomical classification of the rust fungi.Moreover, the studies were also done using unique plant materials of putative hybrids between willows and poplars.The first cytological analyses of these intergeneric crosses showed numerous abnormalities of hybrid embryos, what indirectly proved the success in creating hybrids between these two major genera of Salicaceae (B a g n i e w s k a -Z a d w o r n a et al. 2010).These plant materials are currently subjected to molecular studies, that can directly visualise the genetic content of the offspring resulting from the interbreeding between these two related taxa.Whatever is the result of launching such hybrid production, it is already shown that the studied materials are susceptible to willow rust caused by M. larici-epitea.

CONCLUSIONS
1.The size and morphology of uredinia and urediniospores of rust isolates obtained from common osier grown in different parts of Poland was typical for Melampsora larici-epitea f. typica.2. No significant differences were found between the rust isolates obtained from common osier and its putative hybrids with trembling aspen and Simon's poplar.3. The hybrid of S. aurita x viminalis x caprea var.

Morfologia urediniów i urediniospor grzyba
Recent interest in the common osier (Salix viminalis L.), a potential source of biomass production for renewable energy (K e o l e i a n and V o l k , 2005; S z c z u k o w s k i et al. 2005), showed great genetic differences between the genotypes (P r z y b o r o w s k i and S u l i m a , 2010).The variation was also observed for numerous traits, including disease resistance (J ę d r y c z k a et al. 2008; R ö n n b e r g -W ä s t l j u n g et al. 2008).

Table 1
The characterization of studied plant materials and localization of experiment sites * presence of the genotype is marked with "+" and absence is marked with "-"

Table 2
The mean size of uredinia and urediniospores of Melampsora larici-epitea isolates obtained on leaves of different willow genotypes (Salix spp.)The spores were 9.79 to 11.47 μm wide and 11.79 to 13.16 μm long (Table2).The average spore was 10.48 x 12.41 μm.The typical spore of a fungal isolate obtained from common osier willow was 10.40 x 12.50 μm.The spores of the isolates obtained from putative hybrids were slightly less ovoid, with mean width of 10.60 μm and mean length of 12.28 μm.All spores were spiny, spiky and evenly echinulated on the whole surface (Figs 5- 2 S. viminalix x viminalis 3 S. viminalis x schwerinii 4 S. aurita x viminalis x caprea 5 urediniospores were produced in controlled environment at 16/8 photoperiod and constant temperature of 16 o C on the willow hybrid S. aurita x viminalis x caprea cv.Stipularis process.