Vol. 6 (1997): 25-36. Universally primed polymerase chain reaction analysis of Fusarium avenaceum isolated from wheat and barley in Finland Tapani Yli-Mattila Laboratory ofPlant Physiology and Plant Molecular Biology, Department ofBiology, FIN-20014 University of Turku, Finland, e-mail: tymat@utu.fi Nina V. Mironenko All-Russian Plant Protection Institute, Laboratory ofPlant Immunity to the Pests, St. Petersburg 189620, Russia Irina A. Alekhina Komarov Botanical Institute, Laboratory ofFungal Biochemistry, St. Petersburg 197376, Russia Asko Hannukkala Institute ofPlant Protection, Agricultural Research Centre ofFinland, FIN-31600 Jokioinen, Finland Sergey A. Bulat Petersburg Nuclear Physics Institute (PNPI), Department ofMolecular and Radiation Biophysics, Gatchina 188350, Russia Twenty-two Fusarium avenaceum isolates from Finnish wheat and barley were analysed using the chain reaction with universal primers (UP-PCR). Each isolate could be distinguished from others by UP-PCR products on polyacrylamide gels. The isolates tested were clustered into two main groups and further into several subgroups by UP-PCR profiles and phylogenetic analyses. The phylogenetic relationships of these groups are discussed. No clear correlation was found between the groups and host plant preference or the geographic origin of F. avenaceum isolates. Pathogenicity tests showed differences between F. avenaceum isolates, but two isolates, one from wheat and the other from bar- ley, were the most aggressive in wheat and barley. This fungus, usually known as a weak pathogen of cereals and other crops, has thus probably not evolved in respect to its ability to damage wheat or barley. Key words: Gibberella avenacea, identification, genotyping, parsimony analysis, UP-PCR ntroduction Fusarium avenaceum (Fr.) Sacc. can be regard- ed as a weak pathogen. Under conditions unfa- vourable to its hosts, it may cause damping off. root rot, stalk rot and/or fruit rot (Gerlach and Nirenberg 1982). The sexual state (teleomorph) of F. avenaceum, called Gibberella avenacea (Booth 1971), is quite rare in nature (Booth and Spooner 1984)and has never been reported from Finland. © Agricultural and Food Science inFinland Manuscript received October 1996 25 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=BFVGSytSb1mJ_YtC.d0tePMcpVrDSnn694zuPcg.XZ_niS5k-FMT-pClWrbINsgakWYbfRazi2xWrSRxHTaY-NX5lNMRCx6K-59lPnc1oW6E4BiJPn50MBmQbnjb1b1JFvjH1N3B9GM7SDB3yeMUTg-Aj850jY-ab3jVw3qkJLSE0NkhJwBcR6CtA_b3djOPHyEjThqUO8m-J03T4pKPaePAuv4DQALgQPkKuMMYE0RVqsFW5hXTKfdu38lrB5MWZR9Dyt747Ni9WvqSH3TdExriixED0lvFrE_3IuZ-M9djbZ-4fEw0epxc637T0yLbT_dSSaVODS5iM4x083eencvUr-I6i30KLuOV4gfyOpzg_RLo34zoFgE60aoZIg5V6ozold_MHW7tP5Crakbj Yli-Mattila, T. et al. UP-PCR analysis ofFusarium avenaceum isolatedfrom wheat and barley In Finland, Fusarium avenaceum is a com- mon inhabitant of living and dead organic sub- strates (Ylimäki and Jamalainen 1986). It is fre- quently found on cereal grain, where it causes seedling blight and has potential for mycotoxin production (Ylimäki 1981). Together with other Fusarium species, F. avenaceum is associated with foot and root rot diseases of all cereals grown in Finland (Mäkelä and Parikka 1980). It is considered less destructive than F. culmorum, but a wide range of variation in pathogenicity between isolates has been reported by Uoti (1976). F. avenaceum has also caused consider- able storage losses in potato (Seppänen 1981a). Like those of F. oxysporum, F. avenaceum isolates are very difficult to distinguish from each other on the basis of morphological or physio- logical characters. F. avenaceum isolates have been grouped into three main types by isozyme analysis (Yli-Mattila et al. 1996). Restriction fragment length polymorphism (RFLP) (Nichol- son et al. 1993) and random amplified polymor- phic DNA polymerase chain reaction (RAPD- PCR) analyses (Yli-Mattila et al. 1996, Yli-Mat- tila and Hyvönen 1996) yielded a higher resolu- tion than isozyme analysis, making it possible to distinguish nearly all F. avenaceum isolates from one another. In the present study the less familiar polymer- ase chain reaction with universal primers (Uni- versally Primed Polymerase Chain Reaction - UP-PCR) was used (Bulat et al. 1992, Bulat et al. 1994, Naumov et al. 1997). The main differ- ence between UP-PCR and RAPD-PCR (Wil- liams et al. 1990) and arbitrarily primed PCR (AP-PCR) (Welsh and McClelland 1990) is that the single primers of UP-PCR (ca 16nucleotides) consist of random 3’ end and “natural” minisat- ellite-like sequences (which can be found in any genome) at the 5’ end, which along with a spe- cific PCR protocol using Tsp polymerase as well as high ramping thermal cycler, makes it less sensitive to reaction conditions (minor changes in annealing temperature, Mg2+ concentration etc.) than AP- PCR and RAPD-PCR. The prim- er extention at the 5’ end by minisatellite-like sequence makes the primer hybridization more stable. UP-PCR banding patterns are fully repro- ducible, even between different cyclers, provid- ed that exactly the same protocol including ram- ping rate is used (S.A. Bulat, unpublished data). The purpose of the present study was to as- sess identification and phylogenetic relationships in F. avenaceum isolated from wheat and barley in Finland. The UP-PCR technique was em- ployed to test the correlation between the genome structure of the isolates and their host preference. Such a correlation has previously been demon- strated for the fungus Cochliobolus sativus (an- amorph Bipolaris sorokiniana), which is also a pathogen of wheat and barley and of which the sexual stage is rare in nature (Bulat and Miro- nenko 1993). Another objective was to compare the isolaterelationships obtained here with those obtained previously by RAPD-PCR analysis (Yli-Mattila et al. 1996,Yli-Mattila and Hyvönen 1996). In addition, the species uniqueness of F. avenaceum at the genome level as compared with other Fusarium fungi was studied by means of cross dot blot hybridization ofUP-PCR products. Preliminary results of UP-PCR analysis have been presented (Yli-Mattila et al. 1997). Material and methods Fungal isolates, growth conditions and DNA extraction Twenty-three isolates of Fusarium avenaceum and six isolates from other Fusarium species were isolated from barley, wheat or oats collect- ed in different locations in Finland (Table 1, Fig. 1) as described by Yli-Mattila et al. (1996, 1997). Each isolate was grown for 4-7 days on a cello- phane membrane on the surface of potato-dex- trose agar at 25°C. DNA was extracted from fresh mycelium with a chloroform/octanol mixture (Yli-Mattila et al. 1997) to eliminate proteins, lipids and polysaccharides, which are potential inhibitors of PCR reaction. 26 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 6 (1997): 25-36. Table 1.List of Fusarium isolates. Map numbers refer to the Figure 1. Species Host Geographic Year of Code No. (isolated origin isolation (Isolate No.*) from) (map number) F. avenaceum 15 (93015) barley (stem base) Apukka(l) 1992 28 (93014) barley (stem base) Apukka(l) 1992 51 (92003) barley (root) Honkajoki (2) 1986 40 (92004) barley (root) Honkajoki (2) 1986 14 (92016) barley (root) Kihniö (3) 1986 25 (92006) barley (root) Nousiainen (4) 1986 17 (92013) barley (stem base) Kankaanpää (5) 1986 19 (92020) barley (root) Rautalampi (7) 1986 20 (92024) barley (root) Harjavalta (8) 1986 47 (92026) barley (root) Leppävirta (9) 1986 50 (92009) barley (root) Parkano (10) 1986 37 (92014) wheat (root) Janakkala (11) 1986 38 (92015) wheat (root) Nummi (12) 1986 39 (93084) wheat (stem base) Pälkäne (6) 1992 46 (93071) wheat (stem base) Pälkäne (6) 1992 26 (93095) wheat (stem base) Kokemäki (13) 1992 27 (93093) wheat (stem base) Kokemäki (13) 1992 41 (93088) wheat (stem base) Kokemäki (13) 1992 42 (93096) wheat (stem base) Kokemäki (13) 1992 45 (93094) wheat (stem base) Kokemäki (13) 1992 21 (92005) wheat (root) Vihti (14) 1986 23 (92007) wheat (root) Karjaa (15) 1986 43 (93101) oats (stem base) Kokemäki (13) 1992 F. graminearum 2 (92029) barley (root) Espoo (16) 1986 F culmorum 35 (93004) oats (stem base) Pälkäne (6) 1992 F. equiseti 9 (92011) barley (root) Kruunupyy (19) 1986 F. redolens II (93152) barley (root) Strömfors (18) 1986 F. oxysporum 33 (93138) barley (root) Espoo (16) 1986 /■.' poae 53 (93146) barley (root) Siuntio (17) 1985 ■"lsolate numbers are stock numbers of isolates in the collection of the Agricultural Research Centre of Finland. Pathogenicity testing All F. avenaceum isolates except isolate 50 were studied for pathogenicity. They were transferred to 9 cm plastic petri dishes on potato dextrose agar (PDA, Difco) and incubated for 2 weeks at 18-20°C under continuous light toenhance spore production. The pathogenicity of F. avenaceum isolates to potato tubers was tested on cv. Bintje. The tuber material was certified basic seed that had been multiplied from meristem culture for two 27 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Yli-Mattila, T. et al. UP-PCR analysis ofFusarium avenaceum isolatedfrom wheat and barley generations in the greenhouse and for two gen- erations in the field. It was health tested against major viral, bacterial and fungal pathogens. Tu- bers uniform in shape within the size fraction of 40-45 mm were selected for the test. They were washed under tap water and their surface was sterilized with 10% natriumhypochlorite (NaO- HCI) for 10 min. The tubers were then washed with sterile water and dried for 12 h. Five tubers per isolate were inoculated using the method described by Seppänen (1981b). Each set of five tubers was placed in a separate plas- tic box, where they were incubated for 3 weeks in darkness at 6-B°C. After inoculation they were cut, and the depth and width of the rotten tissue were measured. The tubertest was repeated twice for each isolate. Pathogenicity to barley and wheat seedlings was studied in a greenhouse test on health-in- spected commercial certified seed of barley cv. Pokko and wheat cv. Tapio. Twenty-five seeds were sown in 0.5-1 plastic pots in sterile sand. The seeds were inoculated by pouring 10 ml of spore suspension of F. avenaceum (100 000 spores/ml) onto them, after which they were cov- ered with a 2-cm layer of sterile sand. Four rep- licates of each isolate were inoculated and pots were organized by randomized block design in the greenhouse. Tests on wheat were carried out twice. The plants were grownfor 4 weeks at 18- 20°C under 12-h light periods and were then re- moved carefully from the sand. Stem bases were rated into four categories: 0= healthy, 1= stem lesions, 2= totally brown stem bases and 3= dead plants. To compare the pathogenicity ofF. aven- aceum isolates to wheat, barley and potato the different disease ratings of the cereals and pota- to were scaled to 0-100. The highest average dis- ease score, 100 was given to the isolate causing the most severe disease symptoms. The average disease scores of other isolates were expressed as percentages of the highest rating. A simple regression model for pathogenicity of F. avenaceum isolates to barley, wheat and potato was calculated using the SAS GLM pro- cedure (Littel et al. 1991). UP-PCR amplification UP-PCR was performed using the thermal cy- cler TC-1000M (PNPI, St. Petersburg, Russia) for 30 cycles as described by Yli-Mattila et al. (1997). The rate of temperature change was about 4°C s l . The sequences of universal primers de- signed and synthesized in PNPI were as follows: 0.3-2 (16mer)5'-TGAGGACAACGGTTCC-3(Bulatetal. 1992) AA2M2 (16 mer) s'-GAGCGACCCAGAGCGG-3' (this work) HE 45 (16 mer) S'-GTAAAACGAGGCCAGT-S' (this work) Of these, 0.3-2 and AA2M2 primers were used to generate UP-PCR products to be analysed Fig. 1. Geographical origin of Fusarium fungi collected in Finland. Map numbers refer to those given in Table 1. 28 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 6 (1997): 25-36. Table 2. Binary character matrix of F. avenaceum isolates analysed by UP-PCR with two universal primers. OTU Primer AA2M2 Ch=3o + (2)** Primer 0.3-2 Ch=2B + (7)** A* 000000000000000000000000000000 0000000000000000000000000000 M H G F E D CB 38 010000000001011110101100000111 (01) 10 10 110 11110 1000101100000001 (0000001) 50 010000000001011110101100000111 (01) 1010110010100100101100000001 (0000110) 17 000001000001011110101100000111 (01) 10 10 10 1111100100101101001001 (0000000) 19 010001100001011110101100000111 (00) 00 10 10 1111101000101001001001 (0001000) 37 001100000000010011001000111001 (01) 10010100000001010001110010 10 (0000000) 45 001100000000010001001000111001 (01) 1001101000010 10100011000 10 10 (0000000) 23 001100000000000011000000111000 (01) 1001101000000111000011111010 (0000000) 20 001100000000000011000000110000 (01) 10010010000001110000 111110 10 (1000000) 26 001100000000000011000000110000 (01) 0001101000000111010011001110 (0000000) 39 000000101010100011011001110011 (01) 1001101000000101000010101010 (0100000) 27 000000101010100011001010110011 (01) 110 1110001000101000001011010 (0000000) 25 000000101010100011001010110011 (01) 110 1110000001101000000011010 (0000000) 40 000000101000100011001010110011 (01) 1101101000010101000000101110 (0000000) 14 000000010100010001000000111001 (01) 1001101001000101000011011010 (0000000) 15 000000010100000011000000111001 (11) 1101110001000101010001001010 (0000000) 47 000000010100010001000000110011 (01) 010 110 1000000001000011011010 (0000000) 21 100000101010100011111001110011 (01) 1001101001000101010111011010 (0000000) 28 100010001100110001000000110000 (01) 1100101000000101000001001110 (0010000) 46 000000010100010001001000111001 (01) 1001101000000101000011011110 (0000000) 41 0(KXKXK)1010001001 1001000111001 (01) 110 110 1000000101000001011110 (0000000) 42 000000010100010011001000111001 (01) 1001101000000101000011001100 (0000000) 51 0000100001(XX) KMH) 1000000110000 (01) 1001101000000101000111001010 (0000000) OTU - Operative Taxonomic Unit (isolate); A* - Artificial Ancestor; Ch - character (only phylogenetically significant and autapomorphic characters shown; **Numberof autapomorphic characters; M -position of molecular weight markers (B -0.25kb; C -0.34kb; D-0.45 kb; E -0.47 kb; F -0.51 kb; G -0.80, H -1.16 kb) of Fig. 3. by gel electrophoresis. The HE 45 primer was used only in dot blot hybridization experiments. Several amounts (0.1-1.0 \i\) of template DNA were tested in UP-PCR and run on 1.7% agarose (Sigma A-7431) gel at 150V with cooled TBE buffer to be sure of the reliability of elec- trophoretic banding profiles. In all cases it was possible to obtain reproducible results on agar- ose gel by choosing appropriate amounts oftem- plate DNA and primer. The efficiency of DNA amplification was also estimated on agarose gel, and equal amounts of the amplification products with the sharpest bands were run on 6% poly- acrylamide gel (thickness 0.8 mm, length 20 cm) at 160 V for 10-14 h with cold (ca 12°C) TBE buffer in order to obtain better resolution than with agarose gel. Both agarose and polyacryla- mide gels were stained with ethidium bromide and photographed in UV light. Dot blot hybridization analysis Amplification products of three F. avenaceum isolates (17, 28 and 14), which represented the two main groups of RAPD-PCR (Yli-Mattila et al. 1996, Yli-Mattila and Hyvönen 1996) pro- files, were compared with those of F. gramine- arum (isolate 2), F. culmorum (isolate 35), F. equiseti (isolate 9), F. redolens (isolate 11), F. oxysporum (isolate 33) and F. poae (isolate 53) and also with isolate 43, which, according to 29 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Yli-Mattila, T et al. UP-PCR analysis ofFusarium avenaceum isolatedfrom wheat and barley previous RAPD-PCR and isozyme analyses (Yli- Mattila et al. 1996,Yli-Mattila and Hyvönen 1996), is only distantly related to other F. aven- aceum isolates. Total UP-PCR products from all these iso- lates generated with primers HE 45 and AA2M2 were dotted onto nylon filters (Hybond N + , Am- ersham) according to the manufacturer's instruc- tions. The filter-bound fixed PCR products from these isolates were hybridized with the corre- sponding amplification products ofF. avenaceum isolate 28, labelled with (alpha- ,2P) dCTP (Izo- top, St. Petersburg), at 68°C for up to 16 h, as described by Sambrook et al. (1989). The filters were washed and exposed to x-ray film as de- scribed by Yli-Mattila et al. (1997). Data analysis The negatives of the polyacrylamide gels were scanned using an original scanning device (V. Zenin, Institute of Cytology, St. Petersburg, Rus- sia) and the images obtained were processed with original image analysis software (N. Klopov, V. Patzekin, PNPI, Russia). High-resolution image analysis allowed us to study all bands in the gel. In this way we could detect weak bands, which are not clearly visible in the photo in Fig. 3. The sizes of the bands were determined according to the peaks of the densitograms of the UP-PCR profiles. All bands detected in image analysis were recorded in a binary matrix irrespective of their intensity. The dataproduced by the image analysis (the binary matrix shown in Table 2 and the Dice dis- tance matrix derived from it using the program of N. Klopov, PNPI, Russia) were analysed by the Wagner parsimony (branch and bound algo- rithm), Neighbor-Joining (NJ) and unweighted pair group method with arithmetic mean (UPG- MA) methods ofPH YLIP 3.5 (Felsenstein 1993). These analyses included only phylogenetically informative characters, which were present in at least two isolates. To root phylogenetic trees, an Artificial Ancestor (A) taxon (Hennig 1966, Pav- linov 1989) withall characters “0” was invoked. The reliability of the phylogenetic trees was explored by a bootstrapping procedure using the PHYLIP package. This procedure was adapted for parsimonious trees using SEQBOOT to gen- erate 3 x 100 different data sets (jumble options 5,81 and 97), which were analysed by the branch and bound method followed by the CONSENSE program. For the UPGMA and NJ methods the same 300 binary data sets were converted to Dice distance ones before analysis by the NEIGHBOR program followed by CONSENSE. In addition to the branch and bound algo- rithm, Maximum parsimony analysis was per- formed with the heuristic search mode of PAUP 3.1.1 by random addition and tree bisection-re- connection (TBR) swapping and by collapsing zero-length branches (Swofford 1993). Results Dot blot hybridization analysis of Fusarium isolates F. avenaceum isolates 17, 28 and 14, which were chosen as representatives ofall isolates by UP- PCR profiles, showed a strong positive signal Fig. 2. Dot blot hybridization of UP-PCR products gener- ated with primers HE 45 (A) and AA2M2 (B) in F. aven- aceum (isolates 17, 28, 14 and 43), F. poae (isolate 53), F. oxysporum (isolate 33), F. redolens (isolate \\),F. equiseti (isolate 9), F. culmorum (isolate 35) and F. graminearum (isolate 2). Dotted samples were hybridized with labelled total UP-PCR products generated with primers HE 45 (A) or AA2M2 (B) for isolate 28. 30 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 6 (1997): 25-36. when UP-PCR products of isolate 28, generated with two UP-PCR primers, were used as a label, whereas amplified DNA from other Fusarium species showed no hybridization (Fig. 2). The hybridization signal from isolate 43 was some- what weaker than that from other F. avenaceum isolates. UP-PCR analysis of F. avenaceum isolates All F. avenaceum isolates collected from wheat and barley could be distinguished from each oth- er by the UP-PCR technique using two primers (Fig. 3, Table 2). The primers produced UP-PCR profiles consisting of7-16 phylogenetic and au- tapomorphic bands per primer (Table 2). The siz- es ofbands ranged from 200 to 2000 bp (Fig. 3). The isolates studied were not derived from single spores, but the clonal origin of four se- lected isolates of F. avenaceum was confirmed by UP-PCR analysis. Single-spore colonies were isolated from original isolates 20, 28, 39 and 51, which were characterized by the most distin- guishable UP-PCR patterns on agarose gel. UP- PCR products from two single-spore colonies of each of these isolates, together with the original DNA sample, were analysed by polyacrylamide gel electrophoresis. No intrastrain differences were found, and all four isolates could be dis- Fig. 3. UP-PCR banding profiles of Fusarium avenaceum isolates with primer 0.3-2. Molecular weight markers (M) - lambda DNA digested by Pst 1.The sizes (kb) of six molecular weight markers are indicated (modified from the proceedings paper of T.Yli-Mattilaetal. 1997). 31 AGRICULTURAL AND FOOD SCIENCE IN FINLAND 1 Yli-Mattila, T. et al. UP-PCR analysis ofFusarium avenaceum isolatedfrom wheatand barley including the smallest ones, for each data set. Neither the consensus tree topology nor the boot- strap values actually differedfor the two settings (100 and 1000). In addition, the topologies of the Neigbor-Joining and UPGMA consensus trees made of 300 trees (results not shown) were the same as in the Wagner parsimony consensus tree (Fig. 4), allowing us to use these 7672 trees in calculating bootstrap values. In the Wagner parsimony consensus tree (Fig. 4) of the UP-PCR matrix (Table 2) F. avenaceum isolates could be divided into two main groups with bootstrap values higher than 89%. Group II consisted of isolates 17, 19, 38 and 50. These two groups are also clearly visible in Fig. 3, al- though group I is fairly heterogeneous. In addi- tion, group I could be divided into three sub- groups, of which Ic (isolates 27, 25, 40, 21 and 39) was supported by a bootstrap value higher than 50%. The 50% majority rule consensus tree ob- tained by the heuristic search mode of PAUP 3.1.1 showed a topology identical to the Wagner tree of PHYLIP 3.5 (Fig. 4), except for slight changes in the large subgroup lb (results not shown). All parsimony and distance methods highlighted subgroup Ha as the most distantly related. tinguished from each other (results not shown). Since the isolation methods were the same in all F. avenaceum isolates, the rest of the isolates were also probably genetically homogeneous. This is important, because a mixture of geno- types can give confused UP-PCR profiles. Phylogenetic relationships among F. avenaceum isolates In each of the 300 data sets analysed by the branch and bound method, the search broke off when the upper limit was set at 100 or 1000 (the “how many” option), but we succeeded in ob- taining 7672 trees characterized by quite differ- ent Cls (= consistency index, Felsenstein 1993), Pathogenicity The relative pathogenicity of F. avenaceum iso- lates in wheat was between 42 and 100, in bar- ley 16-100 and in potato 63-100 (Table 3). The isolates least pathogenic in wheat were 51 and 25 and the most pathogenic 38, 17, 47 and 14. In barley the least pathogenic isolates were 40, 25 and 51 and the most pathogenic 38 and 17; in potato the least pathogenic isolates were 27, 45 and 21 and the most pathogenic 17, 51 and 38. The best correlation was found between path- ogenicity to wheat and barley (pathogenicity to barley = 11.3 + 0.55 x pathogenicity to wheat; R-square 0.33, F-value 18.42 and p 0.00012). There was no statistically significant correlation between pathogenicity to barley and potato Fig. 4. Phylogenetic relationships amongF. avenaceum iso- lates as obtained by Wagner parsimony method of PH YLIP 3.5, Majority rule and strict Wagner consensus tree. Only bootstrap values greater than 50% are shown. 32 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 6 (1997): 25-36. Table 3. Relative pathogenicity of F. avenaceum isolates to barley, wheat and potato. Isolate Phylogenetic Wheat Barley Potato 37 la 74 83 81 2b Ia 82 84 68 45 la 80 85 65 gg gg 74 23 Ia 87 89 68 51 lb 42 26 95 47 lb 95 83 82 41 lb 87 84 84 4() Ib 75 84 88 4 9[ gg 20 27 jc 34 8 3 31 40 Id 88 16 77 25 Ic 64 21 70 Ic 83 86 63 21 Ic 85 87 67 39 Ic 90 88 88 jj g] g 0 79 j 7 jla 96 9 q jqq 33 H a 100 100 89 (R-square 0.07, F-value 3.09, p 0.087) or wheat and potato (R-square 0.12, F-value 5.27, p 0.027), but isolates 17 and 38 were among the 2-3 most pathogenic isolates in all three crops. DiSCUSSIOn All isolates of F. avenaceum could be separated into two main groups and distinguished from one another by UP-PCR analysis, which produces more characters per primer than does RAPD- PCR (Bulat et al. 1995, Yli-Mattila et al. 1996, Yli-Mattila and Hyvönen 1996). Image analysis of polyacrylamide gel negatives enabled us to clearly separate bands from each other and to compare bands of equal size more accurately than was possible in the previous RAPD-PCR work (Yli-Mattila et al. 1996, Yli-Mattila and Hyvönen 1996), in which only visual analysis 0f photos from agarose gel was used. The UP-PCR products from other Fusarium „ , ,fungi were completely nonhomologous with those from F. avenaceum in the dot blot hybrid- ization analysis, and so we could not infer the phylogeny of F. avenaceum isolates from those V,u r ■ • . ITDof other Fusarium species by comparing UP- PCR banding profiles. Therefore, in F. aven- aceum, phylogenetic analyses were performed only on isolates whose UP-PCR products gave a strong hybridization signal showing that these isolates produced homologous bands and indi- cating that they belonged to the same species (Bulat and Mironenko 1992, Bulat et al. 1995). On these criteria isolate 43 did not belong to F. avenaceum and was excluded from UP-PCR analysis. The phylogenetic analyses of UP-PCR prod- ucts run on polyacrylamide gel revealed two .. , , , T,quite different groups: a large one (I), compns- ing the majority of isolates, and a smaller one (II), with isolates differing greatly in genome structure from the others. The parsimonious trees obtainedfor the same F. avenaceum isolates with UP-PCR and RAPD data (Yli-Mattila and Hy- vönen 1996) were found to be almost the same at main group level. In the RAPD-PCR analy- sis, group II also included isolate 37 instead of 38, which constituted its own RAPD group. At subgroup level, the differencesbetween UP-PCR and RAPD-PCR trees were much greater. The resolution and the number of branches with a bootstrap value higher than 50% were superior in the UP-PCR parsimony tree. However, there were still branches in the UP-PCR parsimony tree with a bootstrap value lower than 50%, which may have been due to the lack of a hierar- chic signal within the main groups of isolates collected from a comparatively small area, as was suggested by Yli-Mattila and Hyvönen (1996). The differences between the trees for UP- PCR and RAPD-PCR data are probably due to the fact that the polyacrylamide gel used in the UP-PCR analysis had a better resolution than the agarose gel used in RAPD analysis. Differences in primer sequences and in protocols cannot, 33 Yli-Mattila, T. et al. UP-PCR analysis ofFusarium avenaceum isolatedfrom wheat and barley however, be excluded. There were also some dif- ferences in the algorithms used for phylogeny inference. We are now studying PCR products generated with the same RAPD-PCR primers in a polyacrylamide gel in the hope that we shall be able to compare UP- and RAPD-primer based data with greater accuracy in the future. The reliability and suitability of RAPD-PCR results for phylogenetic analyses have been ques- tioned, especially above the intraspecific level, due to the uncertain homology of the bands (e.g. Rieseberg 1996), their non-codominant inherit- ance, asymmetrical transformation features and possible GCpriming bias, which altogether make current models of parsimony inappropriate (Backeljeau et al. 1995). In UP-PCR the phylo- genetic analysis is performed only at taxon lev- el, where the amplification products are cross- homologous, the primers are not GC-rich and the better resolution obtainedby polyacrylamide gel causes fewer errors in finding homologous bands; the two other features of bands (non-co- dominant inheritanceand asymmetrical transfor- mation), however, remain. UP-PCR data may thus be more appropriate for parsimony analy- sis than RAPD-PCR data. In addition, the parsi- mony tree of the present study was supported by NJ and UPGMA trees, and previous studies deal- ing with different fungi (S.A. Bulat, personal communication) show that nearly all bands of UP-PCR profiles are independent of each other and derived from numerous locations dispersed throughout the genome. It should also be noted that it is practically impossible to test all bands in a profile on cross homology by Southern hy- bridization and that the findings of some studies of phylogenetic relationships based on RAPD- PCR or AP-PCR data are in accordance with morphological (Millan et al. 1996) and isozyme (Castagnone-Sereno et al. 1994) results and the history of strains (Canzian et al. 1995). The use of an artificial ancestor (Hennig 1966, Pavlinov 1989), which is one kind of in- groupcomparison, has been criticized (Watrous and Wheeler 1981). It nevertheless offered the only way to calculate all the bootstrap values and still obtain a reasonable tree, since PHYLIP’s CONSENSE program produces trees of differ- ent topology when different taxa are allocated as an outgroup. We furthermore found that the Artificial Ancestor created does not affect the branching order of other taxa, since its position has been defined so as to coincide with the mid- point root in a tree. According to the hybridization analysis per- formed here and the previous results of RAPD- PCR and isozyme studies (Yli-Mattila et al. 1996), isolate 43 is more distantly related to other F. avenaceum strains than any other strain under study. Further molecular and morphological studies are required to clarify the status of this isolate and the isolates of group II which dif- fered from all other isolates of F. avenaceum. The latest morphological studies (H. Nirenberg, personal communication) have confirmed that the isolates 28 and 37 of group I and the isolate 17of group II really do belong to F. avenaceum, but isolate 43 was identified as F. tricinctum. RFLP and sequence analyses of ribosomal DNA in F. avenaceum isolates are now in progress. No clear correlation was found between iso- late clustering and their host plant preference or geographic origin, except for two subgroups de- tected in all trees inferred. In one group (Ha) three of the four isolates (50, 17 and 19) were from barley and in another (la) four of the five isolates (45, 37, 23 and 26) were from wheat. Of the five isolates collected from one field at Kokemäki, isolates 41 and 42 were clustered to subgroup lb and isolates 45 and 26 to subgroup la. The lack of a clear correlation between F. avenaceum groups and geographic origin and the host habits of isolates is in agreement with RAPD-PCR and isozyme analyses of the same isolates (Yli-Mattila et al. 1996). This fungus, known as a weak pathogen of cereals and other crops, thus apparently exists under field condi- tions in the form of numerous vegetative clones and very probably does not evolve in its ability to damage wheat and barley. Pathogenicity testing of F. avenaceum iso- lates on wheat and barley shows no clear corre- lation between the F avenaceum isolate group- ing and its pathogenicity. Moreover, the most 34 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 6 (1997): 25-36. pathogenic isolates against potato (17 and 51) were positioned quite far from each other in the phylogenetic trees. However, the same two iso- lates (38 and 17) were among the most patho- genic isolates in wheat and barley, which sug- gests that the strains of F avenaceum have not specialized for wheat or barley. In addition, it can be noted that these two most pathogenic iso- lates against wheat and barley belong to the same phylogenetic group(Ha) and that they were also among the most pathogenic isolates against po- tato. Acknowledgments. We are grateful toProfessor J. Hyvönen (Dept of Plant Ecology and Systematics, University of Turku) for critically reading an earlier version of the man- uscript and to G. Häkli for linguistic revision. This investi- gation was supported by the University of Turku, the Com- mission for Scientific and Technological Co-Operation be- tween Finland and Russia, The Finnish Academy, and the Russian State Program “Frontiers in Genetics” (in part). References Backeljeau, T., De Bruyn, L, De Wolf, H., Jordaens, «., Van Dongen, S., Verhagen, R. & Winnepenninckx, B. 1995. Random amplified polymorphic DNA (RAPD) and parsimony methods. Cladistics 11: 119-130. Booth, C. 1971. The Genus Fusarium. 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Maatalouden tutkimuskeskus ja Petersburg Nuclear Physics Institute Suomalaisia vehnästä ja ohrasta eristettyjä punaho- mekantoja (Fusarium avenaceum) tutkittiin UP-PCR (Universally Primed Polymerase Chain Reaction) -menetelmällä. F. avenaceum -kannat voitiin jakaa erilaisilla foneettisilla ja fylogeneettisillä tietokone- ohjelmilla kahteen pääryhmään, jotka vielä jakautui- vat useaan alaryhmään. Fylogeneettisten ryhmien ja kantojen maantieteellisen jakauman välillä ei voitu havaita selvää riippuvuutta. Kannat erosivat toisistaan patogeenisuustesteissä, mutta samat ohrasta ja veh- nästä eristetyt isolaatit olivat patogeenisimpiä sekä vehnällä että ohralla. Tulosten perusteella vaikuttaa siltä, että F. avenaceum, jonka tiedetään olevan suh- teellisen heikko patogeeni viljakasveilla, ei ilmeisesti ole erikoistunut kumpaankaan isäntäkasviin. 36 AGRICULTURAL AND FOOD SCIENCE IN FINLAND