Zbornik gozdarstva in lesarstva, 39, 1992, s. 5 - 12 FDC: 172.8 Annillaria sp.: 176.1 Quercus sp.: 443.3 ( 436) DETERMINATION OF THE ARMILLARIA SAMPLES FROM OAK FORESTS IN AUSTRIA Alenka MUNDA* Abstract The Armillaria samples from severa! oak stands in lower Austria and in Burgenland were identified in mating with test-samples, after the method of Korhonen. Two species of the genus Armillaria were determined: Armillaria gallica Marx. et Romagnesi and Armillaria ostoyae (Romagnesi) Herink. Key words: Armillaria gallica, A. ostoyae, oak decline DETERMINACIJA VZORCEV ŠTOROVK IZ HRASTOVIH SESTOJEV V AVSTRIJI Izvleček Vzorce štorovk iz hrastovih sestojev z različnih lokacij v Spodnji Avstriji in Burgenlandu smo identificirali s križanjem s testerji po Korhonenovi metodi. Določili smo dve vrsti štorovk: Armillaria gallica Marx. et Romagnesi in Armillaria ostoyae (Romagnesi) Herink. Ključne besede: Armillaria gallica, A. ostoyae, propadanje hrasta Mag. A. M. Dipl. Ing. of Agr.1 Inštitut za gozdno in lesno gospodarstvo, Večna pot 2, 61000 Ljubljana, Slovenija Zbornik gozdarstva in lesarstva, 39 CONTENTS 1 INTROOUCTION 7 2 THE RESEARCH MATERIAL ANO THE METHOOS OF WORK 7 2.1 Isolation of haploid mycelium from spore suspensions 7 2.2 Identification of the Armillarias with test-samples 8 3 4 THE RESULTS ANO CONCLUSIONS LITERATURE 6 11 12 Munda A.: Determination of the Armillaria samples ... 1 INTRODUCTION Among the plenty species of fungi, which are involved in oak decline, Armillaria can always be found. The two species of genus Armillaria, that most often live on roots of the oak trees, are Armillaria mellea (Vahi ex Fr.) Kummer and Armillaria gallica Marx. et Romagnesi (sin. A. bulbosa (Barla) Velen.). A. mellea is an aggressive parasitic fungus, which mainly attacks broad-leaved trees. A. ga/lica is a saprophytic fungus, that lives on tree residues and as a facultative parasite attacks the already weakened trees. In oak forests, the latter species is very frequent. For evaluation of the influence of Armillarias and the root rot upon oak decline, it is in the first place necessary to find out, which Armillaria species actually infected the roots. The morphology of fruitbodies, which develop on infected roots in autumn, is not a reliable sign for determination of the Armillarias. For this purpose, a laboratory method of pairing of Armillaria isolates with standard test-samples is used - the so called mating test, which was introduced by the Finnish scientist K. KORHONEN (1978). With this method, which has already for some years been a routine for determination of Armillarias in Slovenia, the Austrian samples were also identified. 2 THE RESEARCH MATERIAL AND THE METHODS OF WORK In the end of 1990, Dr. Thomas Cech from Federal Forest Research Station (Institute of Forest Protection) in Vienna sent us some Armillaria samples, that were gathered in autumn 1990 on severa! locations in lower Austria and in Burgenland. We got the samples in a form of frozen spore suspensions. There were 13 samples, each in two alternatives (A and B) and severa! parallels. Unfortunately we did not get any data about the origin and the ecology of the samples. 2.1 Isolation of haploid mycelium from spore suspensions For determination of species, haploid isolates, isolated from spore suspensions were needed. The appropriately diluted spore suspension was inoculated with a sterile inoculating needle on the nutrient medium in Petri dishes (1.5 % malt agar). The spores were thinly dispersed ali over the nutrient medium. They were incubated in a dark room by the temperature of 25 degrees C for five to eight days. Afterwards, the germinating spores were isolated under microscope - according to the method of KORHONEN (1980). For the isolation procedure, the Pasteur pipettes were used, which had earlier been curved above a flame, thinned to a calibre of 0.5 mm and adapted for microscopical work. Previous to every isolation, the pipettes were washed in distilled water, disinfected in alcohol and acetone and burned above a flame. Under the microscope, individual germinating spores were cut out of the nutrient medium with a sterile pipette and inoculated each in its own test tube. As the germinative faculty of spores of some samples was rather low, it was - in spite 7 Zbornik gozdarstva in lesarstva, 39 of p1enty repeated experiments with different nutrient media • not possible to isolate a haploid mycelium from samples no. 7, 10, 12 and 13. The haploid, that is single-spore isolates of Armillarias, were kept in a dark room, by a temperature of 4 degrees C, and were every month transplanted to fresh nutrient media. 2.2 ldentification of the Armillarias with test-samples Single-spore isolates of Annillarias were identified in mating tests with test-samples. The test-samples are haploid isolates of all the European species of genus Armillaria, which are as reference isolates used for identification of Armillarias. Two years ago, they were sent to us by Dr. K Korhonen. As it is not possible for the haploid isolates of Armillaria to be kept for a longer tirne, because they are inclined to degeneration and are afterwards no longer suitable for mating experiments, the Finnish test-samples were partially replaced by the test-samples from our own collection (in TABLE 1, these test-sam ples are marked by an S). Every Austrian sample was paired with 24 testers • four for each Armillaria species. Beside the species with a veil (A. borealis, A. cepistipes, A. gallica, A. mellea and A. ostoyae), the ringless species A. tabescens, which often Iives parasitically on oak trees in warmer climates, was used in mating tests as well. Table 1: The list of test-samples, used for identification of the haploid Armillaria isolates A. borealis: 1 - S900930.1.2 2 · S890930 .1.1 3 • 880800.2.2/5 4 - 901002.1.2/1 A. cepistipes 5 • S891019.3.1 6 S901028.3.1 7 810912.1.2n 8 - 880902.2.1/2 A. gallica 9 - S891107.J.2 10 - S901006.1.2 11 - $891014.1.2 12 • 861024.1.1/1 A. mellea 13 - S891019.2.2 14 - S881025.1.2 15 - S891112.1.1 16 - S901013.4.l A. ostoyae 17 - 870919.2.2/3 18 $901006.2.l 19 - 870923.4.2/2 20 - S891021.2.2 A. tabescens 21 - 871104.8.1/2 22 - 871104.8.1/5 23 - 871111.1.1/3 24 - 871111.1.1/5 Both of the partners (the test-sample and the isolate, which was to be identified), were inoculated in Petri dishes with a diarneter of 9 cm, on a nutrient medium with 1.5 % of malt agar. The isolates were inoculated near to each other (at a distance of 3 - 5mm), so that the results of mating would be as clear as possible. There were four pairings in every Petri dish. The Petri dishes were incubated in dark, by room temperature. The results of mating tests were first evaluated after three weeks, and after six weeks for the second tirne. When the studied sample was of the same species as the tester, diploid mycelium developed after mating, which was easily recognized by its morphological characteris- tics. The differences betwen haploid and diploid mycelium by genus Armillaria are very evident: the haploid mycelium is white and airy, while the diploid mycelium is dark and crusty. 8 Munda A.: Dc tcrmin a tion of thc Armillaria sa mples ... Figure 1: Pairing of haploicl Armillaria isolates with test-sam ples (10 days after inoculation) Figure 2: The haploicl (left) and the cliploicl (right) mycclium of Armillaria 9 Zbornik gozda rstva in lesarstva, 39 Figure 3: Compatible mating (20 days after inoculation) Figure 4: Incompatible mating (20 days after inoculation) . Ali the photos: documentation of IGLG. The photographer: Dušan Jurc. 10 Munda A.: Determination of the Armillaria samples ... The cornpatible reaction, resulting from pairing of fungi of the same species, was recognized by the following characteristical signs (KORHONEN 1978, GUILLAU- MIN, BERTHELA Y 1981): 15 - 20 days after the inoculation, the isolates grew together in a homogenous diploid colony; the separating line, which in the beginning appeared between the mycelia, disappeared completely; the growth of mycelia was slowed down; the exuberant airy mycelium darkened gradually, became thicker and grew into the agar, finally it was covered by a crusty pseudostroma, the rhizomorphs appeared. For pairing of isolates, not belonging to the same species it was typical, that the mycelia did not grow together and their morphology did not change significantly they remained white and airy, and a separating black demarcation line appeared in the fusion area. Often, the signs of antagonism between both isolates were also noticed. 3 THE RESULTS ANO CONCLUSIONS Table 2: Identification of haploid Armillaria isolates with test-sam ples Number of the tester ~:•e 1 2 ~ tl ~ 7 8 : ~ ~! ~ 13 14 15 16 l7 18 19 20 21 22 ~3 2• :p::::" 1-l_B_l -+-+-+-+- ..:.. l...J...u_·_-1-+-l-+-+--+-l-+-+-+--+--+-+--l--+-+--l--+-+--l--+-A-'. g'-al_Jic_a--1 2A2 [ ~T~ ) + +. + + A. ostoyae 3A4 • + + + A. gallica : . m : : : : . . ::: 4A4* ttt-+--t--t-+--1---t-?-++-?-+---t-++-?-+-+--t-+-? +-? -+---t-+--+---t-?~A'"".os-,o-~a-,-t 1-4:.:.;Ac;,_1 '-+-+--i---i--l--_ m. '! 1 + '! ? 1 SA3 + ? A. ga/lica A. ga/lica - ++ t-'-A5-+-+-+-+--t _++ + + + ~ ? F5::,.B1:..,_+-+-+-+--+--+-+- --+-+-+-+-'-· -l--+-l-----l--+--t---+--+-l-----+--+--1-----1--"-- i-;6;.;;A4.;__,1-,1-.;....+-+-+-+- --l-+--+---+-+--+---+-·-++-l-+--+_+ __ +-1---+-+--+---+---' A. gallic:a A. ostoyM 8A2 8B1 8B2 9A4 9B5 11A1 llAS 11B5 o o . . . ffl: +I+ + + + compatible mating O contamination + + + + + * = the sample number 4 is badly contaminated by bacteria + + ? + A. ostoyae . + + +? + ~ A. rutoyae + + 1 + A, ostoyat + . A. gallica + A. ga/lica + A. gallit:a + A.gallici1 + A. ,:allica - = incompatible mating ? = the rcsults of mating tests are unclear 11 Zbornik gozdarstva in lesarstva, 39 Among the studied Armillaria samples, the prevailing species was A. gallica. A. gallica normally lives saprophytically, but in spite of this, it sometimes also causes root rot of weakened trees. It is characteristical for this species, that it first infects the roots superficially and penetrates inwards, when the tree has already been sufficiently weakened by some other causes. It is rather surprising, that among the samples, not even one of A. mellea was found, although this very species is a very frequent oak parasite. Surprisingly as well, the A. ostoyae was present, although this species is normally found in coniferous forests. A. ostoyae also grows in mixed forests, together with A. cepistipes and A. gallica, yet its frequency there depends upon the history of a stand (LEGRAND, 1990). In the saprophytic phase of this, otherwise very pathogenic fungus, an oak tree is even a better substratum for its growth and development asa conifer (GUILLAUMIN, LUNG 1985). The mentioned species (A. ostoyae) has already been found on oaks in Slovenia too, yet never in unmixed oak stands. On the mere basis of taxonomical