ГЛАСНИК ШУМАРСКОГ ФАКУЛТЕТА, БЕОГРАД, 2009, бр. 100, стр. 129-142 BIBLID: 0353 -4537, (2009), 100, p 129-142

Keča N. 2009. In vitro interactions between Armillari­a specie­s and potential biocontrol fungi. Bul­ letin of the Faculty of Forestry 100: 129-142.

Nenad Keča UDK: 630*401.16:582.28 Оригинални научни рад DOI: 10.2298/GSF0900129K

IN VITRO INTERACTIONS BETWEEN ARMILLARI­A SPECIE­S AND POTENTIAL BIOCONTROL FUNGI

Abstract: 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 fasciculare¸ 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. Key words: Armillaria, interaction, biocontrol, antagonism, competition

IN VITRO ИНТЕРАКЦИЈЕ ARMILLARIA ВРСТА И ПОТЕНЦИЈАЛНИХ БИОКОНТРОЛНИХ ГЉИВА Извод: Тестиране су интеракције између Armillaria врста и седам других гљи ­ва in vitro. За оглед су одабране три антагонистичке врсте (Trichoderma viride, Trichotecium roseum и Penicillium sp.) и четири трулежнице (Hypho­ loma fasciculare¸ Hypholoma capnoides, Phlebiopsis gigantea и Pleurotus ostrea­ tus). Најбољи резултати добијени су за Trichoderma viride, пошто је она била способна да убије како мицелију тако и ризоморфе Armillaria врста, док су Hypholoma врсте инхибирале како пораст колонија тако и производњу ри­ зоморфи код Armillaria врста. Кључне речи: Armillaria, интеракција, биокотрола, антагонизам, компетиција

1. INTRODUCTION

Armillaria root disease еxists in most natural forests and plantations around the world (Shaw & K i le, 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 Dr. Nenad Keča, Assistant Professor, University of Belgrade - Faculty of Forestry, Belgrade (e‑ mail: [email protected])

129 Nenad Keča

(Guillaumin 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 iq & Fox , 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 (Chapma n & X ia o, 2000, G al let et al., 1993, R a z iq, 2000, Nicolot 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.

2. MATHERIALS AND METHODS

Isolation was done from fragments of basidiocarps (mushrooms) on selective me­ dium of Hu nt & Cobb (1971). Identification of Armillaria was done according to the keys for identification of carpophores (Te r mor shu i ze n & A r nold s, 1987) and mat­ ing with isolates earlier identified according to the method of G u i l lau m i net 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.

130 IN VITRO INTERACTIONS BETWEEN ARMILLARI­A SPECIE­S & POTENTIAL…

Table 1. Origin of Armillaria isolates Табела 1. Порекло Armillaria изолата Armillari­a Isolate Dipl./ Date № Origin Host Locality spp. label hapl. of isolation 1. А. mellea Mel/Kos Fruit body Quercus petraea Košutnjak D 20.10.2004. 2. A. gallica Gall/Cer Fruitbody Quercus cerris Cer D 30.10.2004. 3. A. ostoyae Ost/Kop Mycelial mat Picea abies Kopaonik D 14.10.2004. 4. A. cepistipes Cep/Gol Fruit body Picea abies Golija D 10.10.2004. 5. A. tabescens Tab/Vrčin Fruit body Quercus farnetto Bojčin D 25.10.2004.

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 (Ke ča , 2005) showed that optimal tempera­ tures range between 20-25°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%).

Table 2. Index of sensitivity (Is) according to the M i r ić (1993) Табела 2. Индекс сензитивности (Is) према М и ри ћу (1993) Good growth and stop before contact with 0 6 Faster growth before and stop at contact other fungi Faster growth before and growth over other 1 Slower growth before and stop after contact 7 fungi mycelium after contact Slower growth before and growth over other 2 8 Slower growth and stop before contact mycelium after contact 3 Good growth before and stop at contact 9 Faster growth and stop before contact Good growth before and overgrowth, above 4 10 Faster production of rhizomorphs other mycelium, after contact Good growth before and grow beneath the 5 colony/mycelium with rhizomorphs after contact

131 Nenad Keča

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 ić, 1993) (Table 2). Reaction types, after hyphal contact with tested fungi, were separated in 5 categories: 1. zone of thicker mycelium - adequate to stop at place of contact; 2. contact with ingrowths of hyphae - adequate to growth over of one fungus; 3. colaps of hyphae - adequate to inhibition of one of the fungi; 4. formation of demarcation line - present in mating of different Armillaria species (Hopk i n et al., 1989); 5. mycoparasitism - reaction characteristic for antagonistic fungi, when hyphae of antagonist use host hyphae as a source of food (D u ma s & Boyonosk i, 1992).

2.1. Statistical analysis

Was performed with analysis of variance and Duncan test (p<0.05). Data were analysed in Statistica ver. 6 (StatSoft, Inc. (2001) Tulsa, OK, USA) and Excel 2002 from Windows Office (Microsoft Corporation 1985-2001).

3. 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 63-81%. 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 31-40%. The results of four decaying fungi, shaw that observed values are between 17 and 52% (Table 3).

132 IN VITRO INTERACTIONS BETWEEN ARMILLARI­A SPECIE­S & POTENTIAL…

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.

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) Tricho­ Tricho­ Hy­ Hypholo­ Phle­ Pleuro­ Competitive Penicil­ derma tecium pholoma ma fas­ biopsis tus os­ p fungi lium sp. viride roseum capnoides ciculare gigantea treatus Single 4.2 culture A. cepistipes Mixed 1.2 a 2.8 b 2.9 b 3.1 bc 3.0 b 2.8 b 3.4 c <0.01a % 24 67 69 74 71 66 71 Single 4.1 culture A. gallica Mixed 1.5 a 2.8 bc 2.6 b 2.8 bc 2.5 b 2.9 bc 3.1 c <0.01 % 37 68 63 68 60 70 75 Single 4.8 culture A. mellea Mixed 1.0 a 1.9 c 1.5 b 2.2 cd 2.0 c 2.7 d 2.9 d <0.01 % 25 49 38 56 51 69 74 Single 4.2 culture A. ostoyae Mixed 0.8 a 2.2 bc 1.8 b 2.5 c 2.5 c 3.4 d 3.0 cd <0.01 % 19 47 43 48 60 71 71 Single 4.3 culture A. tabescens Mixed 1.4 a 2.6 b 2.6 b 3.3 c 2.9 bc 3.3 c 3.6 d <0.01 % 33 60 60 76 67 77 83 a - Average values in the rows with different letters are significant at (p<0.05) according to the Duncan test

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.

133 Nenad Keča

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 -1 2 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.

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 Tricho­ Hypho­ Hypho­ Phle­ Competi- Trichoder­ Penicil­ Pleurotus tecium loma loma biopsis tion fungi ma viride lium sp. ostreatus roseum capnoides fasciculare gigantea A. cepistipes 1 7 8 0 1 3 1 2 1 2 1 3 5 2 A. gallica 1 7 8 0 5 3 1 2 1 2 1 3 5 2 A. mellea 1 7 8 0 1 3 1 2 1 2 1 3 1 4 A. ostoyae 1 7 8 0 1 3 1 2 1 2 1 3 1 4 A. tabescens 1 7 8 0 1 3 1 2 1 2 1 3 1 2

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

134 IN VITRO INTERACTIONS BETWEEN ARMILLARI­A SPECIE­S & POTENTIAL…

­ os . п ­ t A - os oyae reaction . ; H ; ре­ - A interaction Hypholoma ; E ; - паразитиране ; C ostoyae . A viride

Growth of . - T ирање ризоморфи ­ м ­ m ostoyaemixedincultures with

. + Ar mellea illaria A Trichoteciumroseum colony; H colony; ' оморфе ­ з ostoyae

. р A ање раста и фор ostoyae& ­ в . - A приконтакту мицелијомса инхибициона зонаформирана између - с врста; B врста; и - ; D ; ; F прeча after contactwith Armillaria rhizomoprhs parasited with mellea *

Armillaria . . gallica - A A & Hypholomacapnoides inhibition zone between Armillariamellea sp. и - colony; B ; D ; ea ­ l преко колоније преко inhibitiongrowth rhizomorphandof formation of - преко колоније Penicillium Hypholomacapnoides ија мицелијеија ; F ; Armillaria ­ ц viride р over mellea - Armillariamel . ) A Trichoteciumroseum сa viride sp.; G sp.; еак Trichoderma gallica

s % & p. . A Hypholoma fasciculare reaction of mycelium of of myceliumreaction of - Penicilium реакцијона зона између интеракција Trichoderma - - Penicillium ; Е ; C ; s G p.; Trichoteciumroseum & viride roseum . . Growth of T прерастањемицелије T - - и A растање мицелије од стране toyaeод од од fasciculare over colony zone betweenzone  А between Penicillum 

$ ( Figure 1. Слика 1.

135 Nenad Keča

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.

4. DISCUSSION

Biological control represents both the oldest and the younges technologies for the control of plant diseases (Cook , 1991). In its wide sense, it involves all the practice which can enhance activity of non-pathogenic microorganisms in the rhizosphere. As conse­ quence of activity decrease of appearance of plant diseases is obtained (Campbell, 1989, ref. R a z iq, 2000). Biological agents that could be used for biological control of Armillar­ ia should colonize woody substrate or be rhizosphere-component (R a z iq, 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 (Hagle & Shaw, 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 iq, 2000). Opposite there are characteristics which posses Armillaria. Antibiotic production, formation of pseudoscle­ roctial plate (Campbell, 1934, ref. R a z iq, 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 ishbet , 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 Kwa śn 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 (Kwa śn a et al., 2002), and most of them

136 IN VITRO INTERACTIONS BETWEEN ARMILLARI­A SPECIE­S & POTENTIAL… 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 (Kwa śn 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 (1985, ref. R a z iq, 2000) different enzymes are involved these proces. Slower but successful destroy of rhizomorphs was noticed by D u­ ma s & Boyon sk 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 ma s & Boyon sk i, 1992). Nicolot t i et al. (1994) were test­ ing efficiency ofTrichoderma 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. (Kwa śn a et al., 2002) should be checked in vivo. Competition with other wood decaying fungi is very important in control of root rotting fungi. Armillaria can not stay infinitely in woody substrate in saprophytic phase (Hagel & Shaw, 1991). The whole group of wood decayers Lenzites saepiaria Fr., Pe­ niophora gigantea (Fr.) Massee, further Fomitopsis pinicola (Swartz:Fr.) Karst., Pleuro­ tus ostreatus (Jacq.:Fr.) P. Kumm., Coriolus versicolor (L.:Fr.) Quel; and Stereum hir­ sutum (Willd.:Fr.) S.F. Gray, should be checked for control of Armillaria species. How­ ever, different studies showed that decaying fungi that can form mycelial cords, and are colonizing the same niche as Armillaria, could be successful competitor with Armillaria. Beside Hypholoma fasciculare and H. capnoides, there are several others like: Phanero­ chaete velitina (DC per Pers.:Fr.) Karst and Steccherinum fimbriatum (Pers.: Fr.) J. Erikss (Hagel & Shaw, 1991). This study showed that Hypholoma spp. reduces growth of Armillaria spp., but they also suppress or in some cases even interrupt development of rhizomorphs. Chap ­ ma n & X ia 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 let 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 withH . fascicu­ lare vs. experimental field tests without inoculation (control), for the period of five years (Chapma n & X ia o, 2000).

137 Nenad Keča

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 al let et al. (1993) concluded that P. gigantea is not able to replace mycelium of A. os­ toyae. Contrary Nicolot t i et al. (1994) showed efficiency ofHypholoma 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 (Ła komy, 2004a, b). This is not too long for natural stands, but for plantations, this is not acceptable. According to St a n iv u kov ić (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 iq & Fox , 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, Mu r r ay & Woodwa rd , 2003). Use of Trichoderma and Hypholoma species for biocontrol with implement of good management practice and selection of appropriate tree species could be base for ob­ taining good results in eradication of species from Armillaria complex.

5. CONCLUSIONS

– Trichoderma viride caused decrease of growth of Armillaria species in range 63 -81%; – Trichotecium roseum reduced growth of mycelium from 32% for A. gallica to 57% for A. ostoyae; – Penicillium sp. caused growth reduction of 62% for A. mellea to 31-40% for sap­ rophytic Armillaria species; – Hypholoma capnoides reduced growth of A. ostoyae culture for 52%; – The weakest influence on Armillaria development was noticed for Phlebiopsis gigantea, Pleurotus ostreatus;

138 IN VITRO INTERACTIONS BETWEEN ARMILLARI­A SPECIE­S & POTENTIAL…

– Observed Index of sensitivity for tested species are: Trichoderma viride Is 7;

Trichotecium roseum Is 0; Penicillium sp. Is 3; Hypholoma spp. Is 2; Phlebiopsis gigantea Is 3; Pleurotus ostreatus Is 2 & 4; – Armillaria mycelium and rhizomorphs die 2-3 weeks after contact with Tricho­ derma viride mycelium; – No mycoparasitism was noticed for Trichotecium roseum, but inhibition of my­ celial growth wsa notified; – By contacts with Penicillium sp. Armillaria cultures stopped production of rhizomorphs; – Hypholoma species produced less mycelial cords in mixed cultures, but on the other hand Armillaria stopped production of rhizomorphs too; – Phlebiopsis gigantea inhibited rhizomorphs production, but has not stopped it; – Pleurotus ostreatus had not influence on development of rhizomorphs on artifi­ cial medium (MEA); – Reactions in mixed cultures showed that Trichoderma viride has mycoparasit­ ic activity, while Hypholoma capnoides and Hypholoma fasciculare inhibited growth of mycelium and stopped rhizomoprh development.

LITERATURE

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Ненад Кеча

IN VITRO ИНТЕРАКЦИЈЕ ARMILLARIA ВРСТА И ПОТЕНЦИЈАЛНИХ БИОКОНТРОЛНИХ ГЉИВА

Ре зи ме Успешна контрола Heterobasidion annosum (Fr.) Bref. уз помоћ трулежнице Phlebi­ opsis giganteа (Fr.:Fr.) Kummer, навела је истраживаче широм света да потраже организам који би се могао применити у биоконтроли Armillaria врста. Током ових истраживања кон­ тролисане су ин витро интеракције између пет Armillaria врста присутних у шумским еко­ системима у Србији и седам антагонистичких гљива. Коришћене су три антагонистичке гљи ­ве (Trichoderma viride Pers., Тrichotecium roseum (Pers.) Link и Penicillium sp.) и четири ком ­петитора (Pleurotus ostreatus (Jacq. ex Fr.) Kumm.; Champ. Jura. Vosg., Hypholoma cap­ noides (Fr.:Fr.) Kummer, Hypholoma fasciculare (Huds. ex Fr.) Kummer и Phlebiopsis gigantea (Fr.) Jülich.). Пет врста из рода Armillaria (А. mellea, А. gallica, А. ostoyae, А. cepistipes и А. tabe­ scens) супротстављене су на истој хранљивој подлози МЕА (2%) са седам наведених вр­ ста. Праћене су промене у порасту (убрзавање, успоравање и др.), као и интеракције на

хран­ ­љивој подлози. Оцене су вршене на основу индекса сензитивности (Is) Ekstein и Liese (1970, према M i r ić, 1993) (табела 1), као и на основу типа реакција мицелије после њиховог кон­ ­такта. Промена у брзини пораста рачуната је као однос између самосталног пораста на хран­ ­љивој подлози и пораста у смешаним културама. Због споријег пораста Armillaria вр­ сте су две недеље раније засејане на МЕА подлогу, а тај период раста је изузет из рачунања пораста. Смањење пораста мицелије уочено је код свих изолата и свих Armillaria врста. Уоче­ не промене могу се сврстати у три групе: 1. значајно успоравање које је карактерисало присуство антагонистичких гљива (Trichoderma viride, Trichotecium roseum и Penicilli­ um sp.); 2. средње успоравање пораста уочено је код Hypholoma spp.; и мање промене које се јављало у присуству трулежница Phlebiopsis gigantea и Pleurotus ostreatus. Trichoderma viride је проузроковала смањење пораста Armillaria врста у интервалу 63 -81%, док је код Trichotecium roseum уочено смањење од 32% за A. gallica до 57% за A. os­ toyae. Penicillium sp. је највеће смањење пораста показао у реакцијама са A. mellea (62%), а нешто мање са сапрофитским Armillaria врстама. Hypholoma capnoides је, такође, утицала на смањење пораста у просеку 52%, док је нај ­мањи утицај уочен за трулежнице Phlebiopsis gigantea, Pleurotus ostreatus.

Уочени су следећи индекси сензитивности: Trichoderma viride Is 7, Trichotecium rose­ um Is 0, Penicillium sp. Is 3, Hypholoma spp. Is 2, Phlebiopsis gigantea Is 3; Pleurotus ostreatus Is 2 и 4. 141 Nenad Keča

Микопаразитизам, као и заустављање у порасту Armillaria врста уочен је код Tricho­ derma viride. Одмах после контакта са мицелијом Trichoderma колоније, Armillaria врста престају са порастом. А већ 2-3 недеље касније колонија изумире заједно са ризоморфама. За T. roseum није уочено паразитирање, али су колоније Armillaria престајале са растом када су биле на растојању од 1-2 mm од Trichotecium колонија. Вероватно је присуство антибиотских материја у подлози зауставило раст Armillaria врста. На контакту са мицелијом Penicillium врста Armillaria су престале са порстом ризоморфи, али је њихов раст на супротној страни колоније био несметан. Hypholoma врсте су у смешаним културама производиле мање мицелијских врпци, али је истовремено код Armillaria скоро потпуно престала производња ризоморфи. Слично претходној врсти, Ph. gigantea је инхибирала пораст ризоморфи, али њихова производња није потпуно престала. Најслабију активност показала је P. ostreatus, која није утицала на про ­мену у настајању ризоморфи, на вештачкој подлози у контролисаним условима. Наведени резултати показују да контрола Armillaria врста представља сложен про­ блем и да се решење мора тражити у интегралним мерама борбе, које би уз примену одго­ варајућих антагонистичких и компетиторских гљива укључиле и све доступне шумско-уз­ гојне мере.

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