Czech J. Genet. Plant Breed., 39, 2003 (Special Issue): 275-278 Fusarium Species Associated with Seeds of Alfalfa Cultivars V. KRNJAJA1*, J. LEV.1.2, M. IVANOVI(3 and Z. TOMI1 'Institute for Animal Husbandry, Belgrade-Zemun, 11080 Zemun; 2Maize Research Institute "Zemun Polje", 11080 Belgrade-Zemun; 3Faculty of Agriculture, 11080 Belgrade-Zemun, Serbia and Montenegro *E-mail: [email protected] Abstract: In Serbia there are little data on certain species of the genus Fusarium parasiting on alfalfa (Medicago sativa L.) as there are a few scientists working on alfalfa diseases, hence outline experience in the identification is insuf- ficient, while only the genus Fusarium, but not the species, is determined in testing of seed health of commercial alfalfa cultivars. Therefore, studies on seed infection of commercial alfalfa cultivars most often grown in Serbia were carried out with the aim to determine the significance of certain species of the genus Fusarium within etiol- ogy of alfalfa seed diseases. Phytopathological investigation of seed of six commercial alfalfa cultivars (L, - K22, L2 - K23, L3 - K28, L4 - ZajeCarska 83, L5 - NS Mediana ZMS V and L6 - NS Banat ZMS II) and twopre-treatments of the seed - rinsing and surface disinfections of seed (T1) and surface disinfections of seed (T2) determined that frequency of species within the genus Fusarium varied from 0 to 20%. In the pre-treatment T1 F. verticillioides was isolated from seeds of the cultivars L, (2%) and L5 (3%), while F. sporotrichioides was isolated from L2 (1%). Fre- quencies of Fusarium species isolated from seeds of all observed cultivars by the application of the pre-treatment T2 ranged from 1% to 20%. F. verticillioides, F. proliferatum, F. subglutinans were isolated from seeds of five (4-20%; L1-L3, L5, L6), four (1-5%; L1-L3, L6) and three cultivars (1-2%, L1, L3, L4), respectively. Keywords: seed; Medicago sativa L.; cultivars; Fusarium verticillioides; Fusarium sporotrichioides; Fusarium proliferatum; Fusarium subglutinans Seed of grown plants represents favourable Published results indicate that suitable seed-treat- substrate for development of numerous phy-ment chemicals partially control seed-rot and damp- topathogene and saprobe fungus. Degree of ing-off in some greenhouse-grown forage legume change, accordingly, depends on type of seed,and grass plantings. Because these diseases can be pathogens and environmental factors (MhoSEvi6 very destructive to forage in greenhouse plantings, et al. 1995). Symptoms expressed on alfalfa seedwidespread belief arose that they occur equally as and seed of other legumes, caused by pathogen destructively in field plantings. It was assumed that organisms, are manifested as seed rot, reduction ofsince some seed-treatment chemicals sometimes seed germination, damping-off, and post emergence improved seedling stands of greenhouse-grown seedling root-rot (TYLER et al. 1956; SCHMITTHENNER forages, they would do likewise in commercial 1964; HANCOCK 1983), and seed coat, cotyledons, farm plantings (TYLER et al. 1956). The agronomic young shoots and leaf primordia of alfalfa andpractice of over seeding, which compensates for red clover could be invaded by hyphae pathogenseedling stand losses, is commonly recommended fungus from the genus Fusarium (CHI et al. 1964). (TESAR & JACKOBS 1972 and PALMER & WYNN-WIL- Alfalfa, red clover, sweetclover, and Ladino white LIAMS 1976 in: HANCOCK 1983) and may be partially clover were extremely susceptible to some spe- responsible for the lack of concern for seedling cies of the fungus when inoculated at the time diseases of alfalfa. of seeding or within 1 day thereafter (HALPIN & WEIMER (1927) in: SCHMITTHENNER (1964) reported HANSON 1958). that Fusarium spp. and Rhizoctonia spp. isolated Proc. 25th EUCARPIA Fodder Crops and Amenity Grasses Section Meeting 275 Czech J. Genet. Plant Breed., 39, 2003 (Special Issue): 275-278 from collar rot and heart rot of alfalfa plants causedOf each cultivar 100 seeds distributed in 10 Petri damping-off of seedlings. F. oxysporum f. medicaginis dishes, 10 grains per dish, were investigated with (Weimer) Snyd. & Hans. was reported pathogenicagar water and incubated on room temperature. to alfalfa seedlings, also (WEIMER 1928, in: SCHMIT- After 10 days colonies developed around seed THENNER 1964). Several species of Fusarium affected were analysed microscopically and for further emergence, and both Pythium debarianum Hesse andidentification sample of colony was transferred on Rhizoctonia solani Kuehn resulted in severe pre-emer- two medias: synthetic low nutrient media (SNA) gence kill (SCHMITTHENNER 1964). Pythium ultimum, with 2 x 4 cm sterile filter paper (NIRENBERG & R. solani and Fusarium roseum (= F. acuminatum and O'DONNELL 1998) in Petri dish of diameter of 9 cm, F. culmorum) were the most common fungi isolatedand on media consisting of 2% agar and 5 sterile from damped-off seedlings. In pathogenicity tests, carnation leaves (CLA) in Petri dish of 6 cm diam- all four of these soilborne fungi caused pre-emer- eter (FISHER et al. 1982). gence and post-emergence damping-off, whereas Determination of Fusarium species was carried only P. ultimum incited a significant amount of theout by colony inspection in situ and native prepa- forked-root symptoms (HANCOCK 1983). P. deba- rations prepared from fungus cultures grown on ryanum, P. irregulare, P. splendes, and P. ultimum SNA and CLA nutrient media (NELSON et al. 1983; were most pathogenic and had similar effects on BURGESS et al. 1994). alfalfa, sweetclover and Ladino clover seedlings. P. debaryanum and P. ultimum caused somewhat RESULTS more seed rotting but all these isolates killed all the alfalfa and sweetclover seedlings and nearly Microscopic inspection of fungus colonies isolated all the Ladino clover seedlings prior to emergence from alfalfa seed has determined smaller differences (HALPIN et al. 1954). Fusarium spp. are associated in regard to infection of seed between investigated with alfalfa root tissues from early in the seedlingcultivars than in different pre-treatments (Table 1). stage throughout the life of the plants (O'RouRRE In pre-treatment T1 Fusarium species were present & MILLAR 1968). Experiments in which inoculum of in low percentage (1-3%) on seed of three cultivars Stagonospora meliloti was placed over alfalfa seeds (L2, L4 and L5). Species Fusarium sporotrichioides Sherb have shown that seedling infection may occur was determined on seed of cultivar L2 (1%) and without wounding (ERwIN 1954). F. verticillioides (Sacc.) Nirenberg (syn. F. month:forme Considering the importance of use of healthy Sheldon) on seed of cultivars L4 (2%) and L5 (3%). seeding material, and in order to determine the Besides Fusarium species, by application of significance of certain species of the Fusarium genus pre-treatment T1 presence of Alternaria spp. was it etiology of alfalfa seed disease, in this paper in- determined in relatively high percentage on seed vestigation of the nature of infection of the seed ofof cultivar L2 (5%) and in low percentage on seed commercial alfalfa cultivar most frequently grown of cultivars L5 (2%) and L3, L4, L6 (1%). Low per- in Serbia was carried out. centage in regard to presence of Cladosporium spp. and Stemphylium botryosum Wallr. (syn. Pleospora MATERIAL AND METHODS herbarum (Pers. ex Fr.) Rab. was established on seed of cultivar L4 (1%). Seed of cultivar L1 was free of Investigation was carried out in Laboratory for infection. Phytopathological Research in the Maize Research Fusarium species were isolated from seed of all Institute in Zemun Polje. As initial material in this investigated cultivars in frequency 1 to 20% by research seed of six alfalfa cultivars was used (L1 application of pre-treatment T2. Species Fusarium - K22, L2 - K23, L3 - K28, L4 - Zajaarska 83, L5 - NS verticillioides was determined in high percentage Mediana ZMS V and L6 - NS Banat ZMS II). on seed of cultivars L1 (20%) and L6 (10%) and in For isolation of fungi from alfalfa seed method ofrelatively high percentage on seed of cultivars L3 nutrient media was used with seed pre-treatment. In (7%), L2(6%) and L5 (4%). Presence of F. prolifera- first pre-treatment (T1) seed was rinsed for 4 hourstum (Matsushima) Nirenberg was determined in in running tap water, surface was disinfected forrelatively high percentage on seed of cultivar L1 5 min in 1% sodium hypochlorite solution (NaOC1), (5%) and in low percentage on seed of cultivars rinsed several times in sterile water and dried be- L3, L6 (2%) and L2 (1%). Low percentage of F. sub- tween two filter papers. In second pre-treatmentglutinans (Wollenw. & Reinking) Nelson, Toussoun (T2) only surface disinfection of seed was carried & Marasas was established on seed of cultivars L1, out without 4 hour rinsing in running tap water. L3 (2%) and L4(1 %). 276 Czech J. Genet. Plant Breed., 39, 2003 (Special Issue): 275-278 Table 1. Frequency ( %) of Fusarium species and other pathogen fungi on seed of various alfalfa cultivars Intensity of seed infection (%) Cultivar Pre-treatment T1 Pre-treatment T2 fungus type fungus type % * 0 Aspergillus spp. 2 Alternaria spp. 9 Chaetomium spp. 1 Cladosporium spp. 1 L1- K22 Gliocladium spp. 1 Fusarium verticillioides 20 Fusarium proliferatum 5 Fusarium subglutinans 2 Alternaria spp. 5 Alternaria spp. 26 Fusarium sporotrichioides 1 Aspergillus spp. 1 Cladosporium spp. 6 L2- K23 Fusarium verticillioides 6 Fusarium proliferatum 1 Penicillium spp. 1 Alternaria spp. 1 Alternaria spp. 1 Cladosporium spp. 7 L3- K28 Fusarium verticillioides 7 Fusarium proliferatum 7 Fusarium subglutinans 2 Alternaria spp. 1 Alternaria spp. 7 Cladosporium spp. 1 Aspergillus spp. 1 L4- ZajeCarska 83 Fusarium verticillioides 2 Cladosporium spp. 8 Stemphylium botryosum 1 Fusarium subglutinans 1 Alternaria spp. 2 Acremonium spp. 1 3 Alternaria spp. 22 L5- NS Mediana ZMS V Cladosporium spp. 2 Fusarium verticillioides 4 Penicillium spp. 1 Alternaria spp. 1 Alternaria spp. 11 Cladosporium spp. 12 L6- NS Banat ZMS II Fusarium verticillioides 10 Fusarium proliferatum 2 Stemphylium botryosum 2 *non-infected seed Besides Fusarium species, by application of pre- DISCUSSION treatment T2, presence of Alternaria spp.