THE OCCURRENCE OF Fusarium spp . ON OAT ( Avena sativa L . ) AND SUSCEPTIBILITY OF SEEDLINGS OF SELECTED GENOTYPES TO INFECTION WITH Fusarium graminearum SCHWABE

The present study was carried out in the years 2010–2012 in the fields of the Strzelce Plant Breeding Company Ltd., belonging to the Plant Breeding and Acclimatization Institute in Radzików, and it included 39 oat genotypes. At the six-week seedling stage, the percentage of plants with root and leaf sheath necrosis symptoms was evaluated. In 2010 the percentage of seedlings with disease symptoms ranged from 6.5% to 25%, in 2011 it ranged from 17% to 34.5%, whereas in 2012 from 10% to 25%. In 2010 the disease index ranged from 1.4 to 5.7, in 2011 from 4.5 to 8.8, while in 2012 it was between 2.0 and 5.4. Mycological analysis showed that large numbers of Fusarium spp. colonies were obtained both from the roots and leaf sheaths. Isolates of these fungi accounted for 63.48% of the total fungi isolated from seedlings. Seedlings grown under the conditions of central Poland were damaged by the species F. culmorum, F. avenaceum, and F. solani. The investigation of the susceptibility of 15 oat genotypes to infection with two Fusarium graminearum strains – Tz 56 and Tk 235 – was carried out under growth chamber conditions at a temperature of 22–23C and relative air humidity of 85%. The F. graminearum strain Tz 56 proved to be the most pathogenic to seedlings of the breeding lines STH 0.9403 and POB 1316/08, for which the disease index was 80.5 and 75.5, respectively. The lowest pathogenicity of the a.m. strain was recorded in the case of the genotype DC 1832/05, for which the disease index was 26.5. The F. graminearum strain Tk 235 proved to be the most pathogenic to the genotypes STH 0.9403 and STH 0.9423, for which the disease index was 70.5 and 70.0, respectively, whereas this strain was least pathogenic to the breeding line DC 2112/05, in the case of which the disease index was 25.5.


INTRODUCTION
Due to the introduction of reduced tillage systems, the problem of infection of seedlings as well as of the roots and stem base of cereals with fungi of the genus Fusarium is gaining special importance [1,2,3,4,5].The oat is considered to be a species that produces good yields in crop rotations with a large proportion of cereals, but it is infected by fungi of the genus Fusarium, one of the components causing root and stem rot diseases [6,1].F. culmorum and F. avenaceum have been recognized as the main cause of these diseases in oat crops [7,1,2].These fungi are distinguished by abundant conidial sporulation.Macroconidia of these fungi are formed in sporodochia developing in crop residue on the soil surface [8].The above-mentioned Fusarium species are also characterized by high tolerance to temperature and humidity [1,9].Moreover, seedlings and the stem base of cereals are damaged by the species F. graminearum; seed material is thought to be the main source of primary infection of cereal seedlings with this fungus [10,11,12,13].The species F. avenaceum (teleomorph: Gibberella avenacea) and F. graminearum (teleomorph: G. zeae) form on cereal crop residue and on the lower nodes of the stem base perithecia containing ascospores, which are carried by the wind for long distances [12,14,15].
The aim of the present study was to determine the contribution of fungi of the genus Fusarium to the infection of oat seedlings grown under the conditions of central Poland as well as to determine the pathogenicity of F. graminearum to seedlings of several oat genotypes under controlled temperature and humidity conditions.

MATERIALS AND METHODS
The study on the occurrence of Fusarium spp. on oat was conducted in the years 2010-2012 in the fields of the Strzelce Plant Breeding Company Ltd., belonging to the Plant Breeding and Acclimatization Institute in Radzików.15 oat genotypes were included in the study each year and a total of 39 genotypes were investigated (a list of cultivars and breeding lines is shown in Table 1).At the six-week seedling stage, 200 (4x50) seedlings of each genotype were sampled.The sampling method was the same as the one used in the study of oat conducted by K i e c a n a et al. [1].
The percentage of plants with root and leaf sheath necrosis symptoms was evaluated in a laboratory using a five-point scale and the disease index was calculated in the same way as in the study of oat conducted by K i e c a n a et al. [2].The obtained results were statistically analyzed using T-Tukey's confidence half-intervals [16].
Subsequently, a mycological analysis of the diseased plants was performed.The number of pieces of seedlings collected for analysis and the analysis method were the same as in the study by K i e c a n a and M i e l n i c z u k [7].The cultures of Fusarium spp.were identified to the species level using monographs and keys by K w a n a et al. [17] and L e s l i e and S u m m e r e l l [18].
The infection experiment to evaluate the susceptibility of seedlings of 15 oat genotypes to infection by Fusarium graminearum No. Tz 56 obtained from kernels and with F. graminearum No. Tk 235 isolated from the roots was conducted in a growth chamber at a temperature of 23-24 o C and relative air humidity of 85%.The strains whose pathogenicity had been earlier tested in the laboratory by the method of M i s h r a and B e h r [19] were used in the investigation.14-day-old cultures of F. graminearum grown on PDA medium in Petri dishes at 22 o C were used as fungal inoculum.
Kernels of the analyzed oat genotypes whose sprouts had reached a length of 10 mm and were normally developed were selected for the investigation of susceptibility.The selected material was placed on a PDA layer with the studied fungal strains, which were in plastic pots with a diameter of 10 cm and a height of 15 cm filled with all-purpose growing medium with an addition of sand at a 2:1 ratio and a pH 6.5, previously sterilized twice in an autoclave for two hours at 121°C and then covered with the medium [20].Pots in which sprouted kernels were placed on layers of PDA medium without fungus were the control.The experiment was established on 4 May 2012 and was performed in four replicates with 25 plants per replicate.Plants grew for 24 days and then the degree of infection of seedlings was determined using a four-point scale and the disease index was calculated in the same way as in the study by K i e c a n a and K o c y a k [21].The results were statistically analyzed in the same way as in the case of plants collected from plantations.
Ten seedlings with disease symptoms from each treatment of the growth chamber experiment were designated for mycological analysis, which was carried out in the same way as in the case of plants grown under field conditions.The cultures of fungi were identified to the species using monographs and keys [22,23,24,25,26,27].
The study in the growth chamber showed that plants with disease symptoms occurred in the experimental treatments with oat kernels infected with both Fusarium graminearum Tz 56 and F. graminearum Tk 235.Plant losses caused by pre-and post-emergence damping-off were recorded in the case of both investigated F. graminearum strains.Infected plants were characterized by root and hypocotyl necrosis and quite often by hypocotyl contraction and root reduction.Furthermore, infected oat seedlings showed inhi-bited growth and yellowing leaves.Control seedlings of the tested genotypes did not exhibit distinct disease symptoms; they had a well-developed root system and root necrosis was observed sporadically.On the basis of the statistical analysis of the disease indices, seedlings of the oat genotypes in question were found to show varying susceptibility to infection by the studied strains of Fusarium graminearum.The statistical analysis of the disease indices demonstrated that artificial inoculation of the medium with the F. graminearum strain Tz 56 had a significant effect on the health of investigated seedlings compared to the control treatment in the case of all studied oat genotypes (Table 3).
The F. graminearum strain Tz 56 proved to be most pathogenic to seedlings of the breeding lines STH 0.9403 and POB 1316/08, for which the disease index was 80.5 and 75.5, respectively.The lowest pathogenicity of the a.m.strain was found in the case of the genotypes DC 1832/05 and STH 0.9322, for which the disease index was 26.5 and 32.0, respectively (Table 3).
The present study showed that inoculation of the medium with the F. graminearum strain Tk 235 also had a significant effect on the infection of oat seedlings in all tested genotypes compared to the control (Table 3).
The F. graminearum strain Tk 235 proved to be most pathogenic to the genotypes STH 0.9403 and STH 0.9423, for which the disease index was 70.5 and 70.0, respectively, while it was least pathogenic to the breeding lines DC 2112/05, POB 4129-4416/11, DC 1832/05 and DC 239/06, in the case of which the disease index was, respectively, 25.5, 26.75, 28.25, and 29.25 (Table 3).
The disease index for seedlings in the control treatment ranged from 0.5 to 2.25 (Table 3).
The mycological analysis of infected oat seedlings indicates that the F. graminearum was the cause of pre-and post-emergence damping-off (Table 4).

DISCUSSION
Field observations carried out in central Poland revealed that oat plants with root and leaf sheath necrosis symptoms occurred at a higher percentage than in the case of oat grown in the eastern area of the Lublin region [1].
The species Fusarium culmorum, F. avenaceum and F. solani proved to be the cause of damage of oat seedlings in the spring.The pathogenicity of F. avenaceum and F.culmorum to oat seedlings has also been confirmed by the research of K i e c a n a and K o c ya k [21] as well as of M a k a [20].Fusarium culmorum has been found to show high pathogenicity to seedlings of various cereal species [20,28,29].Secondary metabolites, including deoxynivalenol (DON), participate in the disease process caused by Fusarium culmorum.This metabolite exhibits phytotoxic activity in particular against the coleoptile, shoots and callus tissue of wheat [30].Moreover, DON reduces the germination capacity of seeds and causes a reduction in the number and length of radicles of wheat seedlings.Reduced germination capacity and growth inhibition in seedlings occur already at a DON concentration of 4 g/ml, whereas the use of a concentration of 25 g/ml completely inhibits root growth and mitotic activity in the cells of the apical meristems of DON-treated roots.This mycotoxin leads to disturbances in the mitotic division of the meristematic cells of wheat roots [31].In wheat roots subjected to the effect of DON, these authors observed the occurrence of chromatin bridges in the chromosomes and they also found the occurrence of lagging chromosomes in the anaphase.According to P a c k a [32], DON leads to excessive condensation of chromosomes in the metaphase and anaphase and disturbs the synthesis of proteins making up the microtubules of the karyokinetic spindle.Other secondary metabolites can also participate in the pathogenesis of diseases caused by F. culmorum, including culmorin, which can inhibit wheat coleoptile elongation at a concentration from 100 M to 1 mM [33].
Fusarium avenaceum is considered to be a serious pathogen of cereals, including oat [1,34,35].Moniliformin participates in the pathogenesis of diseases caused by F. avenaceum; this mycotoxin affects the permeability of the cytoplasmic membranes and causes disturbances in mitotic cell division [32].
Fusarium solani has contributed to root infection in single cropped oat and in oat mixtures with spring barley and spring triticale as well as in triticale grown with oat.This fungus was the main cause of root damage in winter wheat grown after previous crops such as single cropped oat and oat grown in mixtures [35].This species is considered to be an important producer of fusaric acid (5-(butyl)-2-pyridinecarboxylic acid) [36], a phytotoxin that destroys the vascular bundles in plants.The strong phytotoxicity of this metabolite to rice seedlings has been proved [33].
Fusarium equiseti belonged to the species isolated from infected oat roots and leaf sheaths.In the study by M a j c h r z a k et al. [37], this fungus proved to be the main causal agent of 190 infections of wheat roots and had a significant contribution to stem base damage in this plant grown in the conditions of the north-eastern regions of Poland.Fusarium oxysporum, which is not considered to be a pathogen of cereal plants [38], was a species that was isolated in each year of the study.
The results obtained in the growth chamber experiment confirmed the high phytotoxicity of Fusarium graminearum to oat seedlings.Under the studied conditions in which 14-day cultures of the tested F. graminearum strains grown on dextrose-potato medium were used as an inoculum, this method proved effective, since there were seedlings with pre-and post-emergence damping-off in the studied oat genotypes in the experimental treatments with both F. graminearum Tz 56 obtained from grass seed and with F. graminearum Tk 235 obtained from grass roots.The present growth chamber study and previous information in the literature indicate the contribution of F. graminearum to the damage of cereal seedlings [10,11].

Fig. 1 .
Fig. 1.Percentage of plants with root and leaf sheath necrosis symptoms in 2010.

Fig. 2 .
Fig. 2. Percentage of plants with root and leaf sheath necrosis symptoms in 2011.

Fig. 3 .
Fig. 3. Percentage of plants with root and leaf sheath necrosis symptoms in 2012.

Table 1
Mean values of the disease index for seedlings of the tested oat genotypes grown under field conditions in 2010-2012

Table 3
Mean values of the disease index for seedlings of the tested oat genotypes in the experimental treatment with Fusarium graminearum No. Tz 56, F. graminearum, No. Tk 235 and in the control treatment under growth chamber conditions.
*Means in lines are significantly different compared to the control at 0 0.05 -Means in columns followed by the same letter are not significantly different at 0 0.05 n -naked oat

Table 4
Fungi isolated from oat seedlings grown in the growth chamber experiment with inoculation of the medium with the Fusarium graminearum strain No. Tz 56 and the F. graminearum strain No. Tk 235 and in the control treatment.

1 .
2he species F. culmorum and F. avenaceum are a threat to oat seedlings grown in central Poland.2.Due to the high pathogenicity of these species, these pathogens should be taken into account in the breeding of new oat cultivars.3.The breeding line DC 1832/05 seems to be useful in breeding oat for resistance to F. graminearum.Fusarium Paul E. Nelson Memorial Symposium.APS Press The American Phytopathological Society St.Paul, Minnesota: 360-392.37.M a j c h r z a k B , K u r o w s k i T P, O k o r s k i A .2008.Fungi isolated from the roots and stem bases of spring wheat grown after different cruciferous plants as forecrops.Pol.