THE OCCURRENCE OF Fusarium poae (Peck) Wollenw. ON OAT (Avena sativa L.) PANICLES AND ITS HARMFULNESS

Field observations of oat panicles carried out in the fields of Danko Plant Breeding Company in the period 2006–2007 and in the fields of Strzelce Plant Breeding Company in 2008 showed the occurrence of panicles with Fusarium head blight symptoms in each growing season. In 2006 the percentage of such panicles ranged from 0.25 to 1.5%, in 2007 from 2.0 to 9.0%, whereas in 2008 from 0.5 to 8.0%. The species Fusarium poae was the main causal agent of Fusarium head blight. A study on inoculation of panicles of 12 genotypes of oats with Fusarium poae strain no. 35, which was conducted in 2008 in experimental fields near the city of Zamość, determined the number of kernels per panicle, grain yield from 40 panicles (4×10 panicles), and 1000-kernels weight (TKW) after the harvest of the crop at full grain maturity. Compared to the control, the lowest reduction in the number of kernels per panicle was found in the case of the cultivar ‘Krezus’ (88.69% of the control), while the highest one in ‘Szakal’ (22.46% of the control). As a result of inoculation of panicles with F. poae, the breeding line STH 8107 was characterized by the lowest decrease in kernels yield (69.76% of the control), whereas the highest decrease was found in the breeding line CHD 1430/02 (14.26% of the control). Compared to the control, the lowest reduction in TKW was observed in the breeding line STH 8107 (96.46% of the control), whereas the highest one in the breeding line CHD 1430/02 (45.06% of the control). The presence of secondary metabolites of F. poae and group A trichothecene compounds: HT-2 toxins (from 0 to 0.013 mg × kg), diacetoxyscirpenol (DAS) (from 0 to 0.002 mg × kg), T-2 tetraol (from 0.001 to 0.014 mg x g), and scirpentriol (from 0.008 to 0.074 mg × kg), was found in infected oat kernels. Group B trichothecenes: nivalenol (from 0 to 0.157 mg × g), deoxynivalenol (DON) (from 0 to 0.127 mg × kg) as well as its acetylated derivatives: 3-AcDON (from 0 to 0.059 mg × kg) and 15-Ac DON (from 0 to 0.288 mg × kg), were also present in oat kernels obtained from panicles artificially infected with Fusarium poae.


INTRODUCTION
The first reports on the occurrence of Fusarium head blight in oats come from the 1920's and they relate to crops in the state of Indiana in the United States of America, but the authors did not mention the species responsible for this disease (M a i n s et al. 1929, according to P a r r y et al. 1995). At the beginning of the 20th century, Fusarium graminearum Schwabe was considered to be the main cause of In oat grain infected by Fusarium poae, the occurrence of the following trichothecene compounds has been found: nivalenol (NIV), deoxynivalenol (DON), diacetoxyscirpenol (DAS), monoacetoxyscirpenol (MAS), scirpentriol (STO), fusarenon-X (FUS--X), T-2 toxins, HT-2 toxins, and neosolaniol (NEO) (L a n g s e t h et al. 1997; K i e c a n a and P e r k o ws k i , 1998; K i e c a n a et al. 2005; P a r i k k a et al. 2010). Moreover, F. poae can produce metabolites such as: beauvericin, butenolide, culmorin, cyclonerodiol, enniatins, and fusarin C (F a b e r and A n d e r s , 1986a; T h r a n e , 1989; 2001; D e N i j s et al. 1996; L o g r i e c o et al. 1998b; 2002a, according to D es j a r d i n s , 2006).
Due to an increase in the percentage of Fusarium poae in the species composition of fungi colonizing cereal grain, a study was undertaken to determine the contribution of F. poae to infection of panicles of oats grown in two Polish regions and to assess the harmfulness of the above-mentioned species to some genotypes of this cereal.

MATERIALS AND METHODS
Field observations of Fusarium head blight in oats Avena sativa L.were carried out in the period 2006-2007. In the years 2006 and 2007, the study was conducted in the fields of Danko Plant Breeding Company (located in Wielkopolska Voivodeship (region)) where the genotypes were grown on podzolic soil, while in 2008 it was carried out in the fields of Strzelce Plant Breeding Company (located in Łódź Voivodeship) with typical brown soil. A list of cultivars and breeding lines is shown in Fig. 1. Each year, the occurrence of Fusarium head blight was studied at full grain maturity (92 in the Zadoks scale according to Tottman, 1987). The study material consisted of 100 successive panicles collected from four places of the field, that is, a total of 400 panicles of each genotype were examined, among which the number of panicles with Fusarium head blight symptoms was determined. 20 panicles from each cultivar and breeding line were sampled for laboratory analysis. In the laboratory, kernels and chaff were separated from the sampled panicles in order to isolate fungi by the method described earlier by K i e c a n a and M i e l n i c z u k (2010) and using mineral medium (M i e l n i c z u k et al. 2010). 50 kernels and 50 chaff (chaff) from the panicles collected from a single study area were analysed.
Fungi of the genus Fusarium were identified using the keys and monographs referenced in the paper by K i e c a n a and M i e l n i c z u k (2010).
A strictly controlled experiment with panicle inoculation was conducted in 2008 in experimental fields near the city of Zamość on leached brown soil derived from loess deposits. The study included 12 genotypes of oats (Table 2) and Fusarium poae strain no. 35, with its pathogenicity proved by the method of M i s h r a and B e h r (1976). The harmfulness of F. poae to panicles of 12 genotypes of oats was determined on the basis of a field experiment with artificial inoculation of panicles with F. poae no. 35 during flowering. Infectious material was prepared following M e s t e r h á z y (1978), but with a modification since the fungus was cultured on SNA medium based on a decoction of oat leaves (K i e c a n a et al. 2002). The method of inoculation of panicles in field was the same as in the case of barley (K i e c a n a , 1994). The infectious material consisted of a suspension of coinidia of F. poae no. 35 with a density of 5 × 10 5 × 1ml -1 . Plants whose panicles were inoculated and control plants were grown in 10 m 2 plots. There were 1 m wide buffer strips and 50 cm wide paths between the plots. Panicles were inoculated at the flowering stage, which was between 27 June and 2 July 2008. 80 panicles of each genotype were inoculated with the infectious fungal material and 20 panicles were considered to be one replication. Inoculation was performed using a garden sprayer and 4 ml of infectious material per 1 panicle. Panicles sprayed only with sterile distilled water were the control. After inoculation, the panicles were covered with plastic bags and in this way the infectious material was protected against air currents and drying for 48 hours.
At full grain maturity (92 in the Zadoks scale according to T o t t m a n , 1987), inoculated and control panicles were cut off and kernels were separated; subsequently, the number of kernels per panicle was determined in 40 panicles (4×10 panicles) as well as kernels yield obtained from them and 1000 kernels weight. The obtained results were statistically analysed using analysis of variance and Tukey's multiple confidence intervals. Tukey's least significant differences were calculated for a significance level of 0.05 (Ż u k , 1989).
In the grain samples obtained from panicles artificially infected with F. poae, an analysis of the content of Fusarium toxins was performed at the Department of Chemistry of the Poznań University of Life Sciences.
To determine the content of group A and B trichothecenes, the grain samples were extracted with a mixture of acetonitrile/water (82:18, v/v) and subsequently they were purified using columns filled with 5 cm 3 of a mixture of activated carbon (Draco G 60, 100 mesh), celite (Celite 545), and neutral aluminium oxide (70-230 mesh), mixed at a weight ratio of 1:1:1.
To confirm the presence of the identified toxins in the sample, a full mass range (from 100 to 700 amu) analysis was performed which produced a mass spectrum that was compared to the reference spectrum obtained in the same way. This spectrum, together with a comparison of retention times of the compound in question with the reference standard, is the basis for the identification of toxins. In addition to the qualitative analysis, the concentrations of the examined toxins were determined by comparing the relative heights of selected ions. The obtained results were processed using Chem Station software and the quantitative content of toxins in the oat samples assayed was determined.
Information on weather patterns in the study area was obtained from the Zamość-based Faculty of Agricultural Sciences of the University of Life Sciences in Lublin.

RESULTS
The field observations carried out at full grain maturity in the fields of Danko Plant Breeding Company and Strzelce Plant Breeding Company showed that plants with ears exhibiting Fusarium head blight symptoms occurred each year. In 2006 the percentage of panicles with Fusarium head blight symptoms ranged from 0.25 to 1.5%, in 2007 from 2.0 to 9.0%, whereas in 2008 from 0.5 to 8.0% (Fig. 1). As a result of mycological analysis of kernels and chaff collected from the panicles with Fusarium head blight symptoms, 2383 fungal isolates were isolated (Table 1). Depending on the growing period, colonies of fungi of the genus Fusarium isolated from kernels and chaff accounted for, respectively: 24.5% (2006) (Fig. 2).
Inoculation of panicles with F. poae strain proved to be effective. The panicles exhibited symptoms of premature whitening of heads. Kernels derived from inoculated panicles were small, light, and soft, while kernels obtained at that time from control panicles were properly developed. Artificial infection of panicles with F. poae had a significant effect on reducing the number of kernels per panicle in 10 oat genotypes, compared to the control; 'Chwat' and 'Krezus' were an exception. The lowest reduction in the number of kernels per panicle, compared to the control, was found in the case of 'Krezus' (88.69% of the control), whereas the highest one in 'Szakal' (22.46% of the control) ( Table 2). Significant differences in kernels yield, compared to the control, were observed in all genotypes tested; the breeding line STH 8107, in the case of which kernels yield accounted for 69.76% of the control, was characterized by the lowest decrease in grain yield as a result of inoculation of panicles with F. poae, while the highest decrease was recorded in the breeding line CHD 1430/02 -14.26% of the control ( Table 2). The statistical analysis of TKW found that artificial inoculation of panicles with F. poae resulted in a significant decrease in 1000 kernels weight in 9 oat genotypes. Compared to the control, the lowest reduction in TKW was found in the case of the breeding line STH 8107 (96.46% of the control) and the highest one in the breeding line CHD 1430/02 (45.06% of the control) ( Table 2).
The presence of group B trichothecenes -nivalenol, deoxynivalenol and its acetylated derivatives (3-Ac DON, 15-Ac DON), was also found in grain derived from panicles artificially infected with Fusarium poae. The presence of deoxynivalenol (DON) was detected in the case of seven oat genotypes at a level from 0.036 ('Zuch') to 0.127 mg × kg -1 ('Szakal'), while the presence of 15-Ac DON was observed in the case of eight genotypes at a concentration from 0.017 ('Zuch') to 0.288 (STH 8107). 3-Ac DON was found to be present only in 'Szakal' at a level of 0.059 mg × kg -1 . Oat grain contamination with nivalenol was observed in most of the genotypes assayed, except for cv. 'Krezus' and the breeding line STH 8107. The highest amounts of this compound (0.157 mg × kg -1 ) were found in grain of the breeding line CHD 1430/02 (Table 3).
In Zamość, the 2008 growing season was characterized by temperature higher than the long-term mean in April, May, June, July, and August by +2.2 o C up to +3.5 o C. The percent of normal rainfall, as compared to the long-term mean, was higher in April, May, July, and August by 11.3% up to 91.7%, whereas in June 2008 it was 64.8% of normal rainfall (Table 4).      According to P a r r y et al. (1995), optimal temperature for infection of ears by F. poae is 25 o C, though they can also be infected at a temperature of 15 o C under the conditions of increased humidity. In the case of the present study, which was carried out under the conditions of a strictly controlled field experiment, temperature in excess of 20 o C and increased humidity after inoculation of oat panicles seemed to be beneficial for the growth of F. poae in these organs.
To study different aspects of Fusarium head blight, the most effective inoculation methods are selected (T a k e d a and H e t a , 1989; M c C a l l u m and T e k a u z , 2002; M i e l n i c z u k et al. 2004; K i ec a n a et al. 2006). In the present study, oat panicles were sprayed with a suspension of F. poae coinidia, because this method was considered to be the closest to natural infection.
Taking into account the varying virulence of strains within the population of the tested species (M a ń k a , 1989; K i e c a n a and K o c y ł a k , 1999; K i e c a n a et al. 2005), the infectious material was prepared from F. poae strain with its pathogenicity proved by the method of M i s h r a and B e h r (1976). The present strictly controlled field experiment demonstrates that artificial inoculation of panicles with F. poae strain no. 35 using the method applied proved to be effective, since it had an effect on reducing the number of kernels per panicle and inhibited their growth, but typical etiological signs were not observed on the panicle heads, similarly as in the case of wheat and oat kernels obtained from ears and panicles inoculated with F. poae (S c h i p i l o v a and G a k a e v a , 1997; K i e c a n a et al. 2005).
The present study shows that infection of panicles with F. poae in 2008 had a similar effect on reducing kernels yield as infection of these organs of oats by F. culmorum in the period 1996-1998 (M i e l n ic z u k , 1999).
The results of quantitative and qualitative analysis of mycotoxins in oat grain infected with F. poae no. 35 has proved the ability of this species to produce group A and B trichothecene compounds (L a n g s e t h et al. In the present study, the presence of diacetoxyscirpenol (DAS) and HT-2 toxins from group A trichothecenes and nivalenol from type B trichothecenes was found in the case of most of the oat samples assayed. The above-mentioned metabolites are typical of the profile of mycotoxins produced by F. poae strains, both under laboratory and field conditions (K i e c a n a and P On the other hand, the investigated F. poae strain no. 35 produced STO and T-2 tetraol in infected oat grain. The former compound was also detected in kernels obtained from panicles inoculated with F. poae strain no. 37 in 2001 (K i e c a n a et al. 2005). The occurrence of T-2 tetraol was found in oat grain harvested from panicles naturally infected by this fungal species in plantations of Lubelskie and Podlaskie Voivodeships (K i e c a n a and P e r k o ws k i , 1998). poae (Peck) Wollenw. oraz zawartość mikotoksyn w