IN VITRO INTERACTIONS BETWEEN ARMILLARI A SPECIE S AND POTENTIAL BIOCONTROL FUNGI

Interaction between Armillaria species and seven other fungi were test­ ed in vitro. Tree antagonistic (Trichoderma viride, Trichotecium roseum and Pen­ icillium sp.) and four decaying (Hypholoma fascicularȩ Hypholoma capnoides, Phlebiopsis gigantea, and Pleurotus ostreatus) fungi were chosen for this study. The best results were noted for Trichoderma viride, because fungus was able to kill both mycelia and rhizomorphs of Armillaria species, while Hypholoma spp. inhib­ ited both growth of Armillaria colonies and rhizomorph production.


INTRODUCTION
Armillaria root disease еxists in most natural forests and plantations around the world (S h a w & K i l e , 1991).Besides, in forests Armillaria species cause damage in orchards, vineyards and on horticultural plants, in urban areas.Six Armillaria spe cies of different pathogenicity live in natural forests and plantations throughout Europe ГЛАСНИК ШУМАРСКОГ ФАКУЛТЕТА, БЕОГРАД, 2009, бр. 100, стр. 129142 BIBLID: 03534537, (2009), 100, p 129142 UDK: 630*401.16:582.28Оригинални научни рад DOI: 10.2298/GSF0900129K (G u i l l a u m i n et al., 2005).This fact makes control of Armillaria root disease even more difficult.
Traditional control methods are inadequate, ineffective or impractical, and com plete eradication of the pathogen is almost impossible (R a z i q & Fo x , 2004).Chemi cal treatments are usable in urban areas on solitary trees, but even there eradication is incomplete.
Biological control of Heterobasidion annosum (Fr.)Bref.with Phlebiopsis gigan tean (Fr.: Fr.) Kummer motivated researchers to find similar biological agent that could be used against Armillaria.
Armillaria species have quite complex life cycle.They cause butt rot, necrosis of cambial tissues, and decline of trees.The fungus produces pseudosclerotial plate around mycelium, and different antibiotics that protect it from other microorganisms.Formation of rhizomorphs is another way to fight back antagonists and competitors.Different an tagonistic fungi like Trichoderma spp., but also many decaying fungi (Hypholoma fas ciculare (Huds.ex Fr.) Kummer, Gymnoilus spectabilis (Fr.)A.H. Smith and Bjerkandera adusta (Willd.)P. Karst.) were tested as possible control agents against Armillaria spe cies in vitro and in vivo (C h a p m a n & X i a o , 2000, G a l l e t et al., 1993, R a z i q , 2000, N i c o l o t t i et al., 1993).
The aim of this study was to check interactions between seven antagonistic fun gi and five Armillaria species that exist in natural forest ecosystems and plantations in Serbia.

MATHERIALS AND METHODS
Isolation was done from fragments of basidiocarps (mushrooms) on selective me dium of H u n t & C o b b (1971).Identification of Armillaria was done according to the keys for identification of carpophores (Te r m o r s h u i z e n & A r n o l d s , 1987) and mat ing with isolates earlier identified according to the method of G u i l l a u m i n et al. (1991).All isolates with localities, hosts and period of isolation are presented in Table 1.
During collection of Armillaria species many fruit bodies of different mushrooms were found.Most often Hypholoma capnoides (Fr.:Fr.)Kummer has been found on co nifer stumps and Hypholoma fasciculare (Huds.ex Fr.) Kummer in broadleaved forests.Isolation was performed on MEA (2%), and presence of mycelial cords confirmed iso lation of Hypholoma spp.Culture of Phlebiopsis gigantea (Fr.)Jülich (syn.Peniophora gigantea (Fr.)Massee) was produced from Rotstop® according to the manufacturer instructions.
Isolates of Pleurotus ostreatus (Jacq.ex Fr.) Kumm.; Champ.Jura.Vosg., Tricho derma viride Pers.and Trichotecium roseum (Pers.)Link were kindly donated by Prof. Dr. D. Karadžić.One Penicillium species was isolated during Armillaria isolation.Isolate was very successful in suppression of growth of Armillaria isolates on MEA.All tested isolates that were tested were grown on MEA (2%) in Petri dishes.The rate of growth speed, during solitary growth, was measured for all species.
Earlier studies of Armillaria growth (K e č a , 2005) showed that optimal tempera tures range between 2025°C.Based on that results all tests were performed on 22°C.
Interaction between Armillaria and seven antagonistic and decaying fungi, were evaluated in Petri dishes on MEA containing 20 g (Merck, Darmstadt, Germany) of malt and 20 g of agar (Torlak, Beograd, Serbia).All mating were performed in 10 replicates.Fragments (10×10 mm) with mycelium from the end of well developed fungus colony were set about 2 cm apart.Because of faster growth antagonistic fungi were plated two weeks later, than Armillaria isolates.Growth of Armillaria in first two weeks was not included in calculation of growth in column mixed (Table 4).Petri dishes were sealed with Para film®, and incubated at 22°C, in dark.Interactions were tested in a week intervals for seven weeks (approx.45 days).
Evaluation of growth inhibition was calculated as a ratio of growth in mixed cul ture (r) and growth in pure culture (R) on MEA (2%).Changes in linear growth of mycelium in pure and mixed cultures were measured and interactions between Armillaria spp.and antagonistic fungi were evaluated with In dex of sensitivity (Is), according to the method of Ekstein & Liese (1970, ref. M i r i ć , 1993)

RESULTS
Decrease in mycelia growth in mixed cultures was noted for all Armillaria species.Differences can be clasified in three groups: 1. greatest slow down, observed for three antagonistic fungi (Trichoderma viride, Trichotecium roseum and Penicillium sp.); 2. me dium slow down observed for Hypholoma spp.; 3. minor changes in growth observed for decaying fungi Phlebiopsis gigantea and Pleurotus ostreatus.
Trichoderma viride caused greatest decrease of growth, after plating on medium.Growth decreas ranged between 6381%.The greatest decrease was observed for A. os toyae, following with A. mellea and A. cepistipes (Table 3).Compared with other six fungi T. viride had much more intensive and lethal effect on all five Armillaria species.
Trichotecium roseum also caused decrease of Armillaria growth between 32% for A. gallica and 53% for A. ostoyae.Greatest and statistically significant (p<0.05)decrease was observed for A. mellea and A. ostoyae.
Growth decrease observed in competition with Penicillium sp. was higher than for T. roseum, but lower than for T. viride.Greatest decrease was for A. mellea (62%) and A. ostoyae (57%), while for other three Armillaria species ranged between 3140%.
The results of four decaying fungi, shaw that observed values are between 17 and 52% (Table 3).
The greatest sensitivity on presence of Hypholoma capnoides was observed for A. ostoyae, where growth was decelerated for 52%.For A. cepistipes and A. tabescens growth decrease was lower than for other three species.H. fasciculare was placed after T. viride, but had better results than T. roseum and Penicillium sp.(p<0.01) in interaction with A. gallica.Even interaction of Phlebiopsis gigantea and Pleurotus ostreatus was modest, this phenomena could not be neglected.In interaction with A. cepistipes decrease of mycelial growth was around 34%, while for A. mellea it was 31%.In some Petri dishes decrease of growth for A. cepistipes in interaction with P. ostreatus was almost the same as for T. roseum, but usually P. ostreatus had the weakest influence on Armillaria spp., among 7 tested decaying fungi.
Evaluation of reactions based on Index of sensitivity showed clear difference for decaying and antagonistic fungi.Nine different types of reactions was observed (Table 4) Trichoderma viride in mixed cultures showed Is 7.After plating T. viride started to grow six times faster than Armillaria spp.Imediately after contact between Trichoderma and Armillaria, hyphae of Trichoderma began to grow over Armillaria mycelium.Myco parasitism of Trichoderma against Armillaria was evidenced after several days.This is very important characteristic because Armillaria produce rhizomorphs that are usually resistant to attack of antagonistic fungi.Overgrown Armillaria colonies stoped to grow and two weeks later mycelium and rhizomorphs lost vitality.
For Trichotecium roseum reaction was evaluated as Is 0. Armillaria cultures start ed to decelerate growth two days after T. roseum was plated.Total absence of growth of Armillaria was notified when mycelium came 12 mm apart from T. roseum colony.In the same time on the other side opposite to antagonistic fungi, colony continues to grow.Clear line of medium, about 1 mm wide, separated two colonies.
Penicillium sp.formed thick layer of mycelium on the contact with Armillaria colony.Beneath the surface MEA changed colour into yellowish.Rhizomorphs were not produced anymore on culture side where Penicillium was present.In only one Petri dish, with A. gallica, rhizomorphs were produced normally.

Pleurotus ostreatus
Hypholoma capnoides and H. fasciculare grow in the same ecological niche as Armillaria species.Hypholoma species in mixed cultures with Armillaria produced less mycelial cords.Armillaria spp.produced less or did not produced rhizomorphs at all.Af ter arrest in growth, Hypholoma colonies begin to accelerted growth till contact with Ar millaria colony.Few days after contact Hypholoma spp.started to grow over Armillaria colonies.This type of reaction was marked as Is 2, while reaction of Armillaria was Is 1. Hypholoma spp.seems to be stimulated by presence of Armillaria species.
Phlebiopsis gigantea is very often used for biological control of root rot Hetero basidion spp., but its reaction with Armillaria species is still not clear.Growth in mixed cultures was smooth and very intense, and was marked as Is 3.After contact with Ar millaria colonies Phlebiopsis stopped to grow and formed thick layer of mycelium.For mation of dark brown line suggested that of hyphas in both colonies seem to lose vital ity.According to the observation there was no influence of Phlebopsis on growth of rhizomorphs.
Reaction of Pleurotus ostreatus was marked with Is 2 and 4. Three Armillaria species had the same growth as in pure cultures, while A. cepistipes and A. gallica de celerated growth.Mycelium of P. ostreatus grows 5 to 6 times faster than Armillaria's.After contact thick layer of mycelium was formed, but 2 or 3 days later mycelium started to overgrow Armillaria colony.Rhizomorphs were produced regularly even colony was covered with mycelium of P. ostreatus.
Among seven tested species, the best results are obtained for Trichoderma viride, Hypholoma capnoides and Hypholoma fasciculare.Trichoderma demonstrated its my coparasitism and Hypholoma, possibility of inhibition of mycelial growth and decline of rhizomorph production.Regarding to the presented results final solution for control of Armillaria species should be build on combination of organisms with different mode of action, in fact combination of antagonists and competitors.

DISCUSSION
Biological control represents both the oldest and the younges technologies for the control of plant diseases (C o o k , 1991).In its wide sense, it involves all the practice which can enhance activity of nonpathogenic microorganisms in the rhizosphere.As conse quence of activity decrease of appearance of plant diseases is obtained (Campbell, 1989(Campbell, , ref. R a z i q , 2000)).Biological agents that could be used for biological control of Armillar ia should colonize woody substrate or be rhizospherecomponent (R a z i q , 2000).They should act by inhibiting or preventing rhizomorphs and mycelium development in sub strates already colonized.Also should actively colonize substrate adequate for pathogen development and should eliminate Armillaria from substrates which are already occupied (H a g l e & S h a w, 1991).Ideal antagonist should produce more inoculum than pathogen; resist, escape, or tolerate other antagonists; germinate and grow rapidly; and invade and occupy orgnic supstrates (Baker & Cook, 1974, ref. R a z i q , 2000).Opposite there are characteristics which posses Armillaria.Antibiotic production, formation of pseudoscle roctial plate (Campbell, 1934, ref.R a z i q , 2000) can protect Armillaria against invasion of antagonists.It should be stressed also that Armillaria can spread intensively in the cam bial tissues of recently dyed or freshly cut trees (R i s h b e t , 1976).Such a defend demand that organism has both antagonistic and competition abilities, and such is not found yet.
Communities present on stump roots of Quercus robur has been investigated by Kw a ś n a et al. (2002,2003,2004), during last few years.Studies have shown that after death of tree great number of microfungi colonize root tissues.About 162 species colo nize fine and thicker roots of Pedunculate oak (Kw a ś n a et al., 2002), and most of them act as antagonists.But some of them (Penicillium lanosum, P. notatum, P. spinulosum, Cylindrocarpon destructans and Mycelium radicis atrovirens) stimulate production of rhizomorphs of A. ostoyae (in vitro).Rhizomorph formation was inhibited by Trichoder ma hamatum and T. viride (Kw a ś n a et al., 2004).Presented information implies that all results obtained in laboratory should be tested in field experiment as well.
Three antagonists and four decaying fungi were chosen because of its presence in ecosystems and according to the results of earlier studies.Trichoderma viride is proved antagonists for different fungi.Produces antibiotics, which change MEA medium into yellowish and according to the Papavizas (1985Papavizas ( , ref. R a z i q , 2000) ) different enzymes are involved these proces.Slower but successful destroy of rhizomorphs was noticed by D u m a s & B o y o n s k i (1992) too.Hyphae of Trichoderma were able to attack rhizomorphs of A. gallica after 1 week in mixed cultures.Further, after just one week all hyphae inside rhizomorph were dead (D u m a s & B o y o n s k i , 1992).N i c o l o t t i et al. (1994) were test ing efficiency of Trichoderma hartianum on wood in vitro.Their results, similar as ours, showed that T. hartianum on MEA can inhibit all Armillaria on great range of tempera tures, but its activity on wood is slower because lack of lignolitic enzymes.
Trichotecium roseum and Penicillium sp. had lower antagonistic activity.They have shown good antibiotic activity and intensive occupation of MEA, but mycoparasitic activity was not observed.These results indicate that this fungi could not be efficient on substrates that are already colonized by Armillaria.Idea of stimulative activity of Penicil lium spp.(Kw a ś n a et al., 2002) should be checked in vivo.
This study showed that Hypholoma spp.reduces growth of Armillaria spp., but they also suppress or in some cases even interrupt development of rhizomorphs.C h a p m a n & X i a o (2000) noticed that hyphae of Armillaria became rounded and swell after contact with Hypholoma sp.Soon after swelling, they die or become decomposed.G a l l e t et al. (1993) also noticed that Hypholoma fasciculare, but also Gymnoilus spectabi lis and Bjerkandera adusta, showed great aggressiveness towards A. ostoyae isolates in experiment of wood fragments (in vitro).In the field experiment, A. ostoyae was statisti cally significant less common in the fields where stumps were inoculated with H. fascicu lare vs. experimental field tests without inoculation (control), for the period of five years (C h a p m a n & X i a o , 2000).
Results obtained for Phlebiopsis gigantea and Pleurotus ostreatus are not satis factory in laboratory conditions.They can colonize substrate, but do not have influence on rhizomorph development (in vitro).During study of interactions on wood fragments, G a l l e t et al. (1993) concluded that P. gigantea is not able to replace mycelium of A. os toyae.Contrary N i c o l o t t i et al. (1994) showed efficiency of Hypholoma fasciculare and Phlebiopsis gigantea in replacement of Armillaria on woody fragments.According to au thors, these species can even kill rhizomorphs of A. mellea.Same authors obtained suc cessful antagonism for Gleophyllum trabeum (Pers.:Fr.)Murril., Creerna unicolor (Fr.)Murr.and Trametes odorata Wulf.
We have presented the whole set of decaying fungi, from phylum Basidiomycota that could be useful for control against Armillaria species.However, they need period of 4-5 years to show results (Ł a k o m y, 2004a, b).This is not too long for natural stands, but for plantations, this is not acceptable.According to S t a n i v u k o v i ć ( 2004) decline of young trees, starts already in next vegetation, and in period of four year could be significant.
Because of Trichoderma species efficiency in mycoparasitism different species like T. lignorum, T. hamatum, T. harziantum should be tested.In addition, efficiency of several decaying fungi (Hypholoma fascisulare, H. capnoides, H. sublateraturm, P. os treatus) should be tested in the field (in vivo) in natural forests and plantations.Use of mix of antagonistic species originated from the same order (i.e.Trichoderma) that could have synergistic action could improve efficiency of biocontrol (R a z i q & Fo x , 2004).In addition, there are results which indicate that some other organisms from rhizosphere like Pseudomonas spp., Bacillus spp., Eubacter spp., Serratia spp.and Agrobacterium radiobacter could influence spread of Armillaria species (D u m a s , 1992, M u r r a y & Wo o d w a r d , 2003).

Table 3 .
Decrease of growth of Armillaria species in mixed cultures (mm per 7 days).Табела 3. Успоравање пораста Аrmillaria врста у смешаним културама (mm за 7 дана) a Average values in the rows with different letters are significant at (p<0.05) according to the Duncan test

Table 4 .
Indexes of sensitivity of Armillaria species and seven tested fungi in mixed cultures Табела 4. Индекси сензитивности Armillaria врста са седам тестираних гљива у смешаним културама Figure 1.А Growth of Trichoderma viride over Armillaria colony; B Armillaria rhizomoprhs parasited with T. viride; C interaction between Trichotecium roseum & Armillaria mel lea; D inhibition zone between A. ostoyae & Trichotecium roseum; E reaction zone between Penicillium sp.& A. mellea; F inhibition of growth and rhizomorph formation of A. ostoyae in mixed cultures with Penicillum sp.; G reaction of mycelium of Hypholoma capnoides after contact with A. ostoyae colony; H Growth of Hypholoma fasciculare colony over A.