Fungi isolated from phyllosphere of fodder galega ( Galega orientalis )

The object of the experiment was fodder galega (Galega orientalis Lam.) cultivated in 2001-2003 as field crop on three plots: 1. without fertilization, 2. 40 kg P2O5 × ha -1 and 80 kg K2O × ha -1, 3. 80 kg P2O5 -1 × ha and 160 kg K2O × ha -1. During the dry and warm vegetation season of 2002 almost two times fewer isolates were obtained from the leaves than in 2003 that was the most abundant in fungi. Yeasts-like fungi (30% of the total number of isolates) and saprotrophic fungi with dominated species: Acremonium strictum (8.5%), genus Epicoccum (7.8%), Humicola (9.5%) and Penicillium (18.9%) were the fungi most frequently populating the leaves of galega. The share of pathogens in the total number of isolates obtained from the phyllosphere was 10.6%. They were represented by fungi of Ascochyta spp., Botrytis cinerea, genus Fusarium, Phoma medicaginis and Sclerotinia sclerotiorum. Reduction by 1.9 to 4.6% in the number of fungi isolated from the phyllosphere of galega without fertilization as compared to galega cultivated in combinations with fertilization was recorded. Generally, the smallest number of pathogens was recovered from galega fertilized with 40 kg P2O5 × ha -1 and 80 kg K2O × ha -1. B. cinerea most frequently populated galega in combination without fertilization, genus Fusarium fungi in combination without fertilization and with fertilization with 80 kg P2O5 -1 × ha and 160 kg K2O × ha -1, while Ascochyta spp. were isolated from galega with fertilization only.


INTRODUCTION
Fodder galega, a perennial legume plant with fine seeds, is relatively immune to viral infections as we learn from reports (Kegler, Spaar 1996;Valkonnen 1993).Among fungal pathogens infesting this plant, similar to Fabaceae belonging to the same family, alfalfa and clover, Botrytis cinerea as well as species of Ascochyta and Fusarium should be mentioned (Cwalina-Ambroziak et al. 1999;Gorsen et al. 1994;Leath, Hower 1993;Leath et al. 1994).The above-indicated species of fungi were isolated from the phyllosphere of galega by Cwalina-Ambroziak and Koc (2005); they populated galega cultivated as single crop more frequently than galega cultivated in a mix with brome grass.Mineral fertilization can also influence the composition of fungal community, which was proven during studies on mycological assessment of rhizosphere, rhizoplane and roots of plants belonging to the above-indicated family (Cwalina-Ambroziak, Majchrzak 2000;Deb, Bora 1996).
The laboratory studies conducted aimed at analysis of the fungal community populating the phyllosphere of fodder galega cultivated under conditions of diversified mineral fertilization.

MATERIALS AND METHODS
Fodder galega was cultivated in 2001-2003 at the Agricultural Experimental Enterprise in Bałcyny as crop field on good wheat complex soil on three plots of 1 ha each: 1. without fertilization 2. 40 kg P 2 O 5 × ha -1 and 80 kg K 2 O × ha -1 (17.46 kg P × ha -1 and 66.45 kg K × ha -1 ) 3. 80 kg P 2 O 5 -1 × ha and 160 kg K 2 O × ha -1 (34.92 kg P × ha -1 and 132.90 kg K × ha -1 ).Fertilization with P 2 O 5 (superphosphate) and K 2 O (potassium salt) was applied before sowing.During fl owering of galega samples of 20 leaves were collect-During flowering of galega samples of 20 leaves were collected from plants growing on individual plots from three locations selected at random.At the laboratory fungi were isolated from the collected plant material according to the methodology by Chruściak (1974).From the basal part of the leave a fragment of 1 cm 2 of leaf blade was cut out and shaken in flasks filled with 200 ml of sterile water.From the suspension of microorganisms prepared in this way 0.2 ml was transferred into Petri dishes and covered with glucose and potato medium PDA with Bengal rose and streptomycin.The fungi growing on the PDA medium were transferred after 5 days of incubation at 22°C on PDA slants for identification of species according to the keys (Arx 1970;Booth 1971;Ellis 1971;Nelson et al. 1983;Skirgiełło et al. 1979).Colonies of yeasts-like fungi were counted.Analysis of variance using the T-Duncan test was carried out to determine the influence of mineral reutilization and sampling time on the numbers of fungi most often isolated from leaves of galega (STATISTICA ® 6 2001 software package).
The structure of fungal community in the phyllosphere of fodder galega formed under the influence of application of mineral fertilizers and weather conditions during the consecutive years of study.The largest numbers of fungi were recovered from the phyllosphere of fodder galega in the combination without fertilization and Ta b l e 1 Fungi isolated from phyllosphere of fodder galega (% of isolates) 1.5 1.9 Alternaria alternata (Fr.)Keissler 2.0 0.7 0.7 6.3 3.9 9.1 8.4 5.0 Ascochyta spp.
1.1 2.9 Helminthosporium sativum Pammel 0.7 Humicola fuscoatra Traaen 7.2 7.4 3.7 1.3 2.0 0.7 6.4 5.0 Humicola grisea Traaen 3.0 4.4 7.9 3.2 6.9 10. the smallest from combination with fertilization with 80 kg P 2 O 5 × ha -1 and 160 kg K 2 O × ha -1 .However, pathogens were the least frequently isolated from the combination with fertilization with 40 kg P 2 O 5 × ha -1 and 80 kg K 2 O × ha -1 .Fusarium spp.and Botrytis cinerea populated leaves of galega cultivated on all plots (Fig. 1a, b, c).B. cinerea populated galega in combination without fertilization most frequently (6.3%), with a significant difference in numbers as compared to both combinations with fertilization (Tab.2).Fungi responsible for wilting of plants colonized the leaves of galega to a similar extent in both plots without fertilization and the one with the highest fertilization rate.Fungi of genus Ascochyta were not recovered from plants cultivated without fertilization while their share in the total number of fungi recovered from combinations with fertilization was 2.5% (lower fertilization) and 4.0% (higher fertilization).
At the same time a larger number of saprotrophic fungi of genera Epicoccum and Penicillium as well as order Mucorales was obtained from plants in combinations with mineral fertilization -39.5% of the total number of isolates (40 kg P 2 O 5 × ha -1 and 80 kg K 2 O × ha -1 ) and 28.4% (80 kg P 2 O 5 × ha -1 and 160 kg K 2 O × ha -1 ) as compared to the control combination -20.7% of isolates.Some authors indicate the stimulating influence of mineral fertilization on development of saprotrophic fungi in communities of soil fungi of galega (Cwalina-Ambroziak, Majchrzak 2000) and other Fabaceae crops (Deb, Bora 1996).
At the same time a larger number of saprotrophic fungi of genera Epicoccum and Penicillium as well as order Mucorales was obtained from plants in combinations with mineral fertilization -39.5% of the total number of isolates (40 kg P 2 O 5 × ha -1 and 80 kg K 2 O × ha -1 ) and 28.4% (80 kg P 2 O 5 × ha -1 and 160 kg K 2 O × ha -1 ) as  1. compared to the control combination -20.7% of isolates.Some authors indicate the stimulating influence of mineral fertilization on development of saprotrophic fungi in communities of soil fungi of galega (Cwalina-Ambroziak, Majchrzak 2000) and other Fabaceae crops (Deb, Bora 1996).
Analyzing the fungal communities of the phyllosphere of fodder galega during individual vegetation seasons the largest number of isolates were recovered in 2001 and 2003 -32.9 and 40% of the total number of colonies respectively.B. cinerea was among the most frequently recovered pathogens during those years.Gorsen et al. (1994) consider vegetation seasons characterized by high precipitations and moderate temperatures as favorable for the causing agent of grey mould and such conditions existed during the above seasons of study.The lowest number of fungi was recovered from leaves during hot and dry summer of 2002 (27.1%).Among the pathogens recovered during that season fungi of genus Fusarium dominated and their number was significantly higher than during the other two years of study.CONCLUSIONS 1. Mineral ferilization reduced the total number of fungi colonizing the phyllosphere of fodder galega.
2. Ferilization had a varied effect on the development of pathogens.The largest numbers of B. cinerea were recovered from galega in treatment without ferilization, in contrast to the genus Fusarium and Ascochyta.