Biological Control of Wood Decay Fungi with Trichoderma Spp

December 2–5, 2015 asca2015 Loews Ventana Canyon • Tucson, AZ

Friday, December 4 Biological Control of Wood Decay Fungi With Trichoderma spp. Members of the Trichoderma genus are known as soil-borne fungi, fast-growing in culture and producing numerous pigmented spores. They occur worldwide and are commonly associated with root, soil, and plant debris. Trichoderma species have long been recognized as biological agents for controlling plant diseases. Past research indicates that Trichoderma can parasitize fungal pathogens and produce antibiotics. More recent research indicates that certain strains of Trichoderma can induce systemic acquired resistance in plants. Several commercial biological products based on Trichoderma species are manufactured and marketed worldwide for use against a wide range of plant pathogens. Currently, there is little knowledge on the effectiveness of biological control agents of wood-decay fungi that colonize urban trees. Studies in Europe, Asia, and Australia indicate that, after careful selection of native and highly antagonistic Trichoderma strains, a range of wood-decay fungi can be used to successfully control or eradicate the casual decay fungus from pruning wounds and wood debris within the soil.

Francis W.M.R. Schwarze, Ph.D., Swiss Federal Laboratories for Materials Science and Technology Dr. Francis Schwarze is an officially appointed and attested expert on wood decay of urban trees, concentrating on research into wood-decay fungi, fungus–host interactions, and the use of fungi for beneficial purposes (e.g., improving the acoustic properties of wood for violins or biological control). He is a member of the British Mycological Society and head of the Research Group Bio-engineered Wood, Applied Wood Materials Laboratory, EMPA, Swiss Federal Laboratories for Materials Science and Technology. Dr. Schwarze has made highly significant contributions to our fundamental under- standing of wood decay by fungi. He has applied the knowledge for the benefit of such important wood science and technology areas as permeability enhancement of heartwoods and refractory timbers, detection of tree rings, performance evaluation of thermo-mechanically densified wood, enhancement of acoustic properties of violins, and biocontrol of decay fungi affecting urban tree health. Many areas of his work have been regarded as path-breaking research and have been published in high impact international journals, such as New Phytologist and Nature Biotechnology, attracting attention of such high profile journals as Nature for commentaries. 12/8/15

Biological control of wood decay fungi with Trichoderma spp.

Prof. Dr. Francis W.M.R. Schwarze Applied Wood Science Bio-engineered Wood

Friday, December 4, 2015

Ma terials Sci ence & Technolog y

Biological control of pests and diseases

Green lacewing fly (Chrysoperla carnea )

Convergent lady beetle (Hippodamia convergens)

Aphidius ervi (Parasitic wasp) Ma terials Sci ence & Technolog y

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Advantages and disadvantages of biological control

Advantages Disadvantages

§ Selectivity, it does not intensify § Control is slow or create new pest problems § It will not exterminate broad § No manufacturing of new spectrum of pathogens chemicals § It is often unpredictable § The pest is unable (or very slow) to develop a resistance § It is difficult and expensive to develop and supply § Control is self perpetuating. § It requires expert supervision.

Ma terials Sci ence & Technolog y

What is Trichoderma?

Trichoderma spp. are free-living fungi that are common in soil and root ecosystems. Recent discoveries show Trichoderma spp. are opportunistic, avirulent plant symbionts, as well as being parasites of other fungi.

Harman, G.E. (2004). Nature Reviews Microbiology 2, 43-56

Ma terials Sci ence & Technolog y

Taxonomy of Trichoderma Mycota

Divison Class Order Family

Ascomycota Sordariomycetes Hypocreales Hypocreaceae

Important species used for biocontrol:

Trichoderma atroviride Trichoderma harzianum T22 Trichoderma hermatum Trichoderma virens

Ma terials Sci ence & Technolog y

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Microscopic features of Trichoderma

Septate hyaline hyphae. Conidiophores are hyaline, branched, Chlamydospores Hyphae phialides are hyaline, flask-shaped, and inflated at the base. The colour of the conidia Conidia is mostly green. Trichoderma spp. may also produce chlamydospores. Conidiophores

Phialides Ma terials Sci ence & Technolog y

Mechanisms by which Trichoderma spp. function

n Mycoparasitism n Antibiosis n Competition for nutrients or space n Tolerance to stress through enhanced root and plant development n Solubilization and sequestration of inorganic nutrients n Systemic aquired resistance (SAR) n Inactivation of the pathogen´s enzymes

Harman G.E. (2004). Plant Disease 84, 377-393

Ma terials Sci ence & Technolog y

Mycoparasitism

Trichoderma spp. grow tropically toward hyphae of other fungi,

§ Einführungcoil about them in a § Materiallectin-mediated & Methoden § Ergebnissereaction, & and degrade Diskussion cell walls of the target § Schlussfolgerungen

§ Ausblickfungi.

Schubert, M., Fink, S., Schwarze, F.W.M.R. (2008). Evaluation of Trichoderma spp. as a biocontrol agent against wood decay fungi in urban trees. Biological Control 45, 111-123. Ma terials Sci ence & Technolog y

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Antibiosis

Trichoderma spp. may release antibiotics (e.g., gliotoxin and viridin) and/or VOCs (volatile organic compounds) that slows down or kills a pathogen in the vicinity of such a product. Growth inhibition of Rhizoctonia solani by the Trichoderma virens–produced antibiotic gliotoxin: A, gliotoxin- amended medium, and B, nonamended medium. Howell (2002). Plant Disease.

Ma terials Sci ence & Technolog y

Competition for nutrients or space

Trichoderma spp. often grow faster or use food sources more efficiently than the pathogen, thereby crowding out the pathogen and taking over. The pathogen can not compete with the biocontrol agent.

Colonisation of root hairs of sweet corn by the highly rhizosphere competent strain of T. harzianum T22. (Harman, G.E. 2000. Plant Disease 84:377-393).

Ma terials Sci ence & Technolog y

Growth rates of Trichoderma and wood decay fungi

Trichoderma spp. Wood decay fungi

24 hrs.

0 hrs.

48 hrs.

5043,2mm1134,2mm2 890,5mm415,3mm2

Ø 5fold stronger growth rate!

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Tolerance to stress through enhanced root and plant development

T22- T22+ T22- T22+

Enhanced root development from field-grown corn and soyabean (Harman G.E. 2000)

T22- T22+ T22- T22+

Improved survival of pepper plants in the field (Harman, G.E. 2000) Ma terials Sci ence & Technolog y

Systemic aquired resistance (SAR)

Some Trichoderma strains clearly are potent inducers of SAR- like responses. When inoculated onto roots or leaves they can provide control of disease caused by Botrytis cinerea on leaves spatially separated from the site of application of the biocontrol agent. Model of induced resistance in tomato.

Harman, G.E. (2004). Nature Reviews Microbiology 2, 43-56

Ma terials Sci ence & Technolog y

Inactivation of the pathogen´s enzymes

When applied to leaves Trichoderma spp. produce a serine protease that is capable of degrading the pathogens plant cell wall degrading enzymes and thereby reducing the ability of the pathogen to infect the plant. Effect of crude protease from cultures of Trichoderma harzianum T39 (■) and NCIM1185 (●) on the germination rate of conidia of Botrytis cinerea (left). Control (◊). Elad & Kapat (1999).

Ma terials Sci ence & Technolog y

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Solubilization and sequestration of inorganic nutrients

Trichoderma spp. can increase iron availability at pH values above 6.5.

Appearance of Cantharanthus cv. Parasol plants grown in planting mix at about pH 7 in the absence and presence of T-22. Courtesy George Elliot.

Ma terials Sci ence & Technolog y

Bumble bees as bee-delivery technique for the dispersal of Trichoderma

• Insertion of Trichoderma conidia into the bee hive • Bees deposit conidia on flowers they visit as they search for pollen and nectar • Bee-delivery is twice as effective as spraying • Strawberry yields improve by 20 -30 %.

Trichoderma is highly effective when applied to blossoms or fruits for control of Botrytis cinerea

Ma terials Sci ence & Technolog y

Treatment of pruning wounds

ü Dujesiefken, 1992; 1995 - treatment with wound sealants not effective. ü Legislation, 1998 - wound treatment with chemicals not always legally permitted. ü Dubos & Ricard, 1974 - curative treatment with Trichoderma spp. against Chondrostereum purpureum. ü BINAB, 1976 is the first company to receive a registration for a biofungicide in the western world. ü Pottle & Shigo, 1975 - treatment of Acer rubrum wounds with Trichoderma viride resulted in a reduction in isolates of Hyphomycetes and Basidiomycetes. ü Pottle et. al., 1977 - treatment of Acer rubrum wounds with Trichoderma harzianum protected colonisation against basidiomycetes for two years. ü Smith, 1981 - Trichoderma spp. counteracted modification of polyphenols by suppressing the growth of Hyphomycetes. ü Mercer & Kirk, 1982 - Wound treatment with Trichoderma spp. partly protected host from infection by basidiomycetes ü Lonsdale, 1992 - Successful wound treatment with Trichoderma spp. against Chondrostereum purpureum. Even five years after treatment Trichoderma was present in the wood.

Ma terials Sci ence & Technolog y

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Field studies to evaluate the efficiency of Trichoderma spp.) for biological control against wound pathogens

§ Treatment of pruning wounds of a range of hosts on different sites with conidial suspensions of T-15603.1 § Reisolation and monitoring of T-15603.1 in the field § Identification of T-15603.1 § Impact of T-15603.1 on wound wood formation and extent of dysfunctional wood.

Schubert, M., Fink, S., Schwarze, F.W.M.R. (2008). In vitro screening of an antagonistic Trichoderma strain against wood decay fungi. Part I. Arboricultural Journal 31, 227-248. Schubert, M., Fink, S., Schwarze, F.W.M.R. (2008). Field experiments to evaluate the application of Trichoderma strain (T-15603.1) for biological control of wood decay fungi in trees. Part II Arboricultural Journal 31, 249-268. Ma terials Sci ence & Technolog y

Objectives of the investigations

§ To evaluate the potential of different Trichoderma spp. as biocontrol agents

§ To identify a competitive species that can be used for the treatment of pruning wounds on urban trees against colonisation by wood decay fungi

§ To establish a method that promotes colonisation and survival of a selected Trichoderma isolate in pruning wounds.

Ma terials Sci ence & Technolog y

Trichoderma isolates and wood decay fungi

Trichoderma spp. Source and origin Isolate-No. Trichoderma atroviride Armillaria mellea - Germany 15603.1¹ Trichoderma atroviride Peel of Citrus aurantium - Israel CBS 351.93² Trichoderma atroviride Forest soil - USA CBS 396.92² Trichoderma fasciculatum Bark of Betula spp. - Netherlands CBS 338.93² Trichoderma virens Decayed wood - Germany CBS 126.65² Trichoderma harzianum BINAB TF WP IMI 206039³ Trichoderma. polysporum BINAB TF WP IMI 206040³ ¹Isolates from the Forest Botany, University of Freiburg. ²Isolates from Centraalbureau voor Schimmelcultures, the Netherlands.

³BINAB Bio-Innovation AB, Sweden.

Wood decay fungi Isolated from Isolate-No. Polyporus squamosus ex Tilia cordata Mill. 291101.2¹ Ganoderma adspersum ex Fagus sylvatica L. 086699.2¹ Ganoderma lipsiense ex Fagus sylvatica L 250593.1¹ Kretzschmaria deusta ex Acer pseudoplatanus L. 271098.1¹ Inonotus hispidus ex Fraxinus excelsior L. 200792.1¹ Inonotus hispidus ex Platanus x hispanica 221105.1 ¹ ¹Isolates from the Forest Botany, University of Freiburg

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Classification of the degree of antagonism of different Trichoderma spp.

In vitro studies Index of dominance (ID) Value

Growth rates ID I 1 – 5

Germination of conidia ID II 1 – 5

Affect of VOCs ID III 1 – 5

Dual culture tests ID IV 1 – 5

Interaction studies in wood ID V 1 – 5

∑IDx ID− Index = ∑Tx ID-Index = Sum of indices IDx / Sum of tests Tx.

Mean growth rate of the Trichoderma isolates under different conditions (mm d-1). ±SE

Temperature MEA LNA °C aw 0.892 aw 0.995 aw 0.998 aw 0.892 aw 0.995 aw 0.998 5 0 0 0 0 0 0 10 0 0 2.9 ±0.18 0 0 2.7 ±0.22 15 0 0 8.9 ±0.21 0 0 7.0 ±0.16 25 0 6.4 ±0.15 18.9 ±0.28 0 7.5 ±0.16 12.7 ±0.25 30 0 5.6 ±0.23 12.9 ±0.17 0 6.9 ±0.21 13.1 ±0.22

Mean germination rate of Trichoderma spp. under different conditions (% d-1). ±SE.

Water Temperature °C activity a w 5 10 15 25 30 0.892 0 0 0 0 0 0.995 0 0 7.7 ±2.8 52.8 ±13.3 48.5 ± 21.8 0.998 0 14.8 ±6.3 52.7 ±17.1 98.8 ±1.5 98.2 ±2.3

Inhibition of radial growth (%) of wood decay fungi by VOCs produced by Trichoderma spp

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Classification of mycelial-interactions in dual culture tests

0 1 Target 2 Target Target fungus fungus fungus

Trichoderma Trichoderma Trichoderma

-1 Target fungus Legend: 0 = No antagonism 1 = Antibiosis 2 = Trichoderma overgrows mycelium of target fungus (mycoparasitism)

-1 = Target fungus overgrows mycelium of Trichoderma Trichoderma [%] = lethal affect (T-MEA) Schubert, M., Fink, S., Schwarze, F.W.M.R. (2008). In vitro screening of an antagonistic Trichoderma strain against wood decay fungi. Part I. Arboricultural Journal 31, 227-248. Ma terials Sci ence & Technolog y

Dual culture tests

Trichoderma / Ganoderma adspersum

Trichoderma / Polyporus squamosus

Classification of the degree of mycoparasitism of different Trichoderma spp. on MEA. ± SE

MEA T-15603.1 T-351.93 T-396.92 T-Binab T-126.65 T-338.93 2.2a ±0.14 2.9 ±0.98 2.4 ± 0.65 2.3 ±0.77 3.0 ±0.89 2.1 ±0.89 I. hispidus [100]b [100] [83] [100] [100] [67] 3.0 ±0.10 2.5 ±0.18 2.9 ±0.12 2.4 ±0.67 2.9 ±0.14 0.7 ±0.09 G. adspersum [100] [100] [100] [100] [100] [17] 2.3 ±0.11 2.4 ±1.23 1.9 ±0.21 1.9 ±0.23 2.3 ±0.54 0 ±0.0 G. lipsiense [83] [100] [67] [83] [100] [0] 3.0 ±0.36 2.8 ±1.31 2.6 ±0.33 2.8 ±0.42 2.9 ±0.56 1.8 ±0.36 K. deusta [100] [100] [100] [100] [100] [67] 2.2 ±0.56 1.8 ±1.05 2.4 ±0.66 1.7 ±0.11 2.9 ±1.45 0 ±0.0 P. squamosus [83] [67] [83] [83] [100] [0] a = Following system was used to classify the rate of mycoparasitism: 0 = no overgrowth; 1 = slow overgrowth; 2 = fast overgrowth; 3 = very fast overgrowth and deadlock of the wood decay fungi within 4 weeks. b = Lethal effect as percent was measured by the ability of Trichoderma spp. to eliminate the wood decay fungi during the incubation time of 4 weeks.

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Evaluation of antagonistic activity in wood

Mean weight losses of inoculated wood blocks

Wood decay fungus Trichoderma 14.00 a 12.00

10.00

8.00 a

6.00 a b weight losses(%) weight 4.00 b b 2.00

0.00 6 weeks 12 weeks 18 weeks Incubation period

Interaction studies in wood blocks

Southern Bracket

Ganoderma adspersum / Trichoderma Schubert, M., Fink, S., Schwarze, F.W.M.R. (2008). In vitro screening of an antagonistic Trichoderma strain against wood decay fungi. Part I. Arboricultural Journal 31, 227-248. Ma terials Sci ence & Technolog y

Interaction studies in wood blocks

Charcoal stump rot

Kretzschmaria deusta / Trichoderma

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Interaction studies in wood blocks

Dryads saddle

Polyporus squamosus / Trichoderma

Index of dominance ID

T-156031 T-351.93 T-396.92 BINAB TF T-120.65 T-338.93 ID-Indices WP

1 Dual culture 2 3 tests 4 4 4 4 5 2 4 5

1 2 VOC‘s 3 tests 2 2 1 2 1 1 4 5 1 Interactions 2 4 4 3 3 4 3 3 wood 4 5 1 Growth 2 4 4 4 4 5 3 3 rates 4 5 1 Germination 2 3 rates 4 4 3 3 3 2 4 5

Mean ID- 3,6 (a) 3,6 (a) 3,0 (b) 3,2 (b) 3,6 (a) 2,2 (c) value

ID -value 0-1 = no antagonistic potential; ID -value 1-2 = weak antagonistic potential ID –value 2-3 = moderate antagonistic potential; ID -value 3-4 = high antagonistic potential ID -value 4-5 = very high antagonistic potential. Ma terials Sci ence & Technolog y

Field studies I. Wound treatment: Spring / summer 2003 § 9 pruning wounds on 37 trees in Strasbourg and on 81 trees in Ludwigshafen, BASF AG were treated with condial suspensions of T-15603.1

II. Infection trials with T-15603.1 and wood decay fungi: § 9 pruning wounds on 78 trees were treated with T-15603.1 at a site in Freiburg. Three weeks after treatment wounds were additional inoculated with Inonotus hispidus, Ganoderma adspersum and Polyporus squamosus.

Ma terials Sci ence & Technolog y

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Range and number of tree species inoculated with T-15603.1

Platanus x hispanica Münchh. 91 Acer pseudoplatanus L. 40 Tilia cordata L. 24 Populus nigra L. 16

Quercus spp. L. 16

Robinia pseudoacacia L. 9

Total trees: 196 Total wounds: 1764

Schubert, M., Fink, S., Schwarze, F.W.M.R. (2008). Field experiments to evaluate the application of Trichoderma strain (T-15603.1) for biological control of wood decay fungi in trees. Part II Arboricultural Journal 31, 249-268. Ma terials Sci ence & Technolog y

Treatment of pruning wounds with T-15603.1

Method A: Suspension [CFU: 105 conidia/ml] Method B: Suspension [CFU: 105 conidia/ml +0,2% glucose + 0,1 % urea] MEA Method C: Suspension [CFU: 105 condia/ml + 0,2% glucose + 0,1 % urea + 0,4 % T-MEA Luquasorb® (Sodium polyacrylat)] * SNA Method K: Control – no treatment (TSM) *For method C a Hydrogel (Luquasorb® 10 30) developed by BASF AG/ Polymer Department was used as a carrier substance.

At the site in Strasbourg the Hydrogel Luquasorb® was not applied. For this reason a alternative method was used: Schubert (2006) Method CS: Suspension [CFU: 105 conidia/ml + 0,5% glucose + 0,2 % urea] Schubert, M., Fink, S., Schwarze, F.W.M.R. (2008). Field experiments to evaluate the application of Trichoderma strain (T-15603.1) for biological control of wood decay fungi in trees. Part II Arboricultural Journal 31, 249-268. Ma terials Sci ence & Technolog y

Field studies

Experiment I: Treatment of pruning wounds:

Tree spp. Climate data § Einführung

§ Material & Methoden Ratio of sap-, and

§ Ergebnisse & heartwood Diskussion Wound dimensions § Schlussfolgerungen § Ausblick Wound occlusion Discolouration

2 months 8 months 12 months 18 months 24 months 30 months

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Measurement of wound occlusion

Populus nigra Acer pseudoplatanus

h 79,6% 69,2% a bc r b

Quercus rubra Tilia platyphyllos

63,9% 72,7% ( π *h*b) bc ab C% =100 − 4 *100 π *r 2 . a, b, c denote significant differences (P < 0.05) of wound wood formation for tree species. <30%31-60%>60%

I <30% II 31-60% III >60%

T-15603.1 10 [19.2%] 15 [28.8%] 27 [51.9%] WOOD DECAY 15 [26.3%] 26 [45.61%] 22 [38.6%] FUNGI

CONTROL 4 [18.2%] 5 [22.7%] 13 [59.1%] Ma terials Sci ence & Technolog y

Reisolation of Trichoderma atroviride 15603.1 from pruning wounds

Discolouration h a b r b S 1 3 5 Pith

π (Px[%]− B[%])*100 ( 4 * h * b) Wgrad = Kkoeff% = 100 − *100 Px[%] π* r 2

Extent of wood discolouration developing from pruning wounds

Populus Quercus Acer Tilia

T-15603.1 b

P. squamosus b

G. adspersum b

I. hispidus 0 10 20 30 40 50 60 70 80 90 Extension of discolouration [cm2]

a, b denote significant differences (P < 0.05) after contrast analysis (P < 0.05).

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Identification of Trichoderma atroviride 15603.1 with RAPD-PCR

§ Einführung

§ Material & Methoden

§ Ergebnisse & Diskussion

§ Schlussfolgerungen

§ Ausblick

Lane 1 = applied strain T-15603.1 (reference); lanes 2–6 = Trichoderma isolated from the treated pruning wounds after 30 months; lane 7 = Trichoderma virens; lane 8 = Trichoderma fasciculatum.

Reisolation results of T-15603.1 at the site in Ludwigshafen

Control A B C

100 a a a a a a 80

b 60 b

40 b b b b c b Reisolations (%) Reisolations 20 bc bc bc c c c c c c c 0 2 months 8 months 12 months 18 months 24 months 30 months Time after inoculation

Symbols with different letters indicate significant (p≤0,05) differences in reisolation rates according to Ryan-Einot-Gabriel-Welsch-Test (REWGQ).

Reisolation rate of T-15603.1 from sap- and heartwood

Sapwood Ripe/Heartwood

19.2 29.9 26.5 24 37.6 44.9

80.8 70.1 73.5 76 62.4 55.1 Proportion sap- and heartwood (%) heartwood and sap- Proportion 2 months 8 months 12 months 18 months 24 months 30 months Incubation period [m]

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Reisolation rate of T-15603.1 in relation to wound size

35.0

30.0

25.0

20.0

15.0

Reisolations [%] Reisolations 10.0

5.0

0.0 C B bis 5cm Condial bis 10cm A suspensions bis 15cm Wound size >20cm

Reduction of infection rate by wood decay fungi after wound treatment with T-15603.1

100 Infection rate w ithout Trichoderma-treatment Infection rate w ith Trichoderma-treatment

60 § Einführung

§ Material & Methoden 40 § Ergebnisse & * Diskussion Infection rate (%) Infection rate 20 § Schlussfolgerungen ** ** ** § Ausblick 0 I. hispidus G. adspersum P. squamosus Total Fungal spp.

Efficency [%]. Asterices denote significant reduction in the infection rate *=significant (p≤0,05); **=highly significant (p≤0,001).

Trichoderma Research Singapore

Ma terials Sci ence & Technolog y

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Identify causal Host/ fungus Biological fungi interactions control

African mahogany Yellow flame tree Rain tree

Khaya senegalensis Peltophorum pterocarpum Samanea saman

Burcham, DC., Wong, JY, Mohamed A, Mohamed I, Abarrientos, N, Fong, YK, Schwarze, F.W.M.R. (2015) Characterization of host-fungus interactions among wood decay fungi associated with Khaya senegalensis (Desr.) A. Juss (Meliaceae) in Singapore". Forest Pathology DOI: 10.1111/efp.12199. Ma terials Sci ence & Technolog y

Identify causal Host/ fungus Biological fungi interactions control

o Healthy, living trees inoculated with wood decay fungi

o One year incubation period

o To be felled, harvested, and dissected to evaluate infection severity

Identify causal Host/ fungus Biological fungi interactions control

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Re-isolation rates for the respective Trichoderma strains applied to each of the two tree species

Control Hydrogel Water suspension suspension

Rain tree 4 Months 8 Months Liquid Suspension 80.6 69.3 Hydrogel Suspension 82.1 69.4 Control 9.63 12.7 Senegal mahogany Liquid Suspension 46.3 31.95 Hydrogel Suspension 29.2 25.98 Control 14.7 11.78 Ma terials Sci ence & Technolog y

Identify causal Host/ fungus Biological fungi interactions control

1. Treat fresh pruning wounds with Trichoderma conidial suspensions 2. Monitor colonization and persistence of Trichoderma on pruning wounds 3. Evaluate the effect of Trichoderma on wound occlusion and infection rates.

Ma terials Sci ence & Technolog y

Phellinus noxius (Brown root rot)

Oil palm plantation

Schwarze, F.W.M.R., Jauß, F., Spencer, C., Hallam, C., Schubert, M. (2012). Evaluation of an antagonistic Trichoderma strain for reducing the rate of wood decomposition by the white rot fungus Phellinus noxius. Biological Control 61, 160-168. Ma terials Sci ence & Technolog y

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Susceptibility

The USDA-ARS Systematic Botany and Mycology Laboratory currently lists 223 host species. Some of the most notable include mahogany, teak, rubber, oil palm, tea, coffee, and cacao as well as a variety of fruit, nut, and ornamental trees.

Ma terials Sci ence & Technolog y

Hosts of Phellinus noxius

Araucaria bidwillii Delonix regia

Ficus benjamina Jacaranda mimosifolia Ma terials Sci ence & Technolog y

Conditions favoring disease of Phellinus noxius

Chang (1996) found that no Phellinus noxius was recovered from soils containing infested root debris after one month of flooding.

Disease incidence of Phellinus noxius on different soil types

Chang & Yang (1998). Mycol. Res. 102(9): 1085-1088.

Ma terials Sci ence & Technolog y 54

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Isolation of wood decay fungus from infected tree

Cultivation and identification (DNA fingerprint)

Screening of a highly antagonistic Trichoderma strain

Growth rates Dual culture Growth inhibition Mycoparasitism tests in wood Application and impact in the field

Plant growth promotor EradicationMa teri aofls Sci pathogenence & Technolog y

Biocontrol of Phellinus noxius (Brown root rot) in Brisbane, Australia

Keith Foster, Senior Coordinator Arboriculture, Brisbane City Council.

Biocontrol of Phellinus noxius (Brown root rot)

Keith Foster, Brisbane City Council:

“A great outcome, I would like to thank you so much for your help, the trees that we have used the Trichoderma on are all recovering. The Banyan fig at Shorncliffe prior to introducing the Trichoderma had little leaf in the canopy and what leaf was left were yellow, now it has a new canopy of leaf with a dark green color. Amazing.

Once again thank you, without you help, we would not have been in a situation where we can manage the diseased trees, they would have been lost.

Ma terials Sci ence & Technolog y

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Biocontrol of Phellinus noxius in Hong Kong, China

Ribera Regal, J., Tang, A., Schubert, M., Schwarze, F.W.M.R. (2015). Evaluation of an antagonistic Trichoderma strain for eradicating Phellinus noxius in colonised wood. Journal of Tropical Forest Science accepted. Ma terials Sci ence & Technolog y

Wood decay triangle

Total ofENVIRONMENT conditions favouring disease

Amount PATHOGEN of Decay Total of Inoculum Amount of Agrios (1988) Decay Total of conditions favouring susceptibility HOST The more unfavourable the Each side of the triangle represents one conditions that help the of three components. If the three pathogen the shorter the components of the decay triangle could pathogen side would be and be quantified, the area of the triangle the smaller the potential would represent the amount of decay. amount of decay.

Biocontrol of Phellinus torulosa in Italy

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29.9.2014 Two weeks after first treatment

16.10.2014 one month after the first treatment

12.2.2015 Five months after first treatment

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Biocontrol of wood decay fungi

§ Biocontrol methods may be beneficial for veteran trees growing on sites with low microbial activity

§ Circumstantial evidence suggests that carefully selected antagonists can be applied against wood decay fungi that are weak competitors

§ Trichoderma spp., can reduce decomposition rates of opportunistic wood decay fungi in trees and induce SAR.

22 Biological Control xxx (2012) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Biological Control

journal homepage: www.elsevier.com/locate/ybcon

Evaluation of an antagonistic Trichoderma strain for reducing the rate of wood decomposition by the white rot fungus Phellinus noxius ⇑ Francis W.M.R. Schwarze a, , Frederick Jauss a, Chris Spencer b, Craig Hallam b, Mark Schubert a a EMPA, Swiss Federal Laboratories for Materials Science and Technology, Wood Laboratory, Section Wood Protection and Biotechnology, Lerchenfeldstrasse. 5, CH-9014 St. Gallen, Switzerland b ENSPEC, Unit 2/13 Viewtech Place, Rowville, Victoria 3178, Australia h i g h l i g h t s graphical abstract

" Antagonism of Trichoderma species against Phellinus noxius varied in the in vitro studies. " Weight losses by P. noxius were higher in angiospermous than gymnospermous wood. " Biocontrol of P. noxius depends on the speciﬁc Trichoderma strain and its host.

article info a b s t r a c t

Article history: The objective of these in vitro studies was to identify a Trichoderma strain that reduces the rate of wood Received 31 October 2011 decomposition by the white rot fungus Phellinus noxius and Ganoderma australe. For this purpose, dual Accepted 30 January 2012 culture and interaction tests in wood blocks of three hardwoods, Delonix regia, Ficus benjamina, Jacaranda Available online xxxx mimosifolia, and one softwood, Araucaria bidwillii, as well as investigations of fungal growth under different environmental conditions, were performed. The effect of Trichoderma ghanense, two strains of T. har- Keywords: zianum and T. reesei on wood colonization and decomposition by four P. noxius strains and G. australe Biological control were quantitatively analyzed by measuring the dry weight loss of wood. All Trichoderma species and White rot wood-decay fungi showed optimum growth at a mean temperature of 25–35 °C and a high water activity Ganoderma australe ° Dry weight loss (aw) of 0.998. At 35 C and aw 0.928, no growth was recorded for any of the wood-decay fungi after Interaction tests in wood blocks 1 week, whereas most Trichoderma species were still actively growing. The different Trichoderma species all showed an antagonistic potential against P. noxius in the in vitro studies. The species of wood-decay fungi showed significant differences in their sensitivity when challenged by the volatile organic compounds (VOCs) of Trichoderma species. Reduction in the rate of wood decomposition by different Trichoderma species against all wood-decay fungi varied strongly according to the specific plant host. T. harzianum 121009 and T. atroviride 15603.1 showed the highest reduction in weight losses. P. noxius 169 strongly decomposed untreated and pretreated wood of D. regia, whereas weight losses of F. benjamina and J. mimosifolia pretreated with Trichoderma strains were significantly lower. Weight losses by G. australe were significantly reduced for A. bidwillii, D. regia and F. benjamina by all Trichoderma species, but no affect was recorded for J. mimosifolia. The in vitro studies show that only after careful monitoring (i.e. selecting the appropriate strain for the target pathogen and its niche (wood species) can Trichoderma species be used to significantly reduce the growth and rate of wood decomposition by different P. noxius strains. Ó 2012 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Fax: +41 582747694. E-mail address: [email protected] (F.W.M.R. Schwarze).

1. Introduction Mohd Farid et al., 2009; Sahashi et al., 2010; Wu et al., 2011). The USDA-ARS Systematic Botany and Mycology Laboratory maintains Species of the genus Trichoderma are ubiquitous in soils. Since a website with an updated list of hosts and information on geo- Weindling (1932) recognized the antagonistic effect of Trichoderma graphical distribution; it currently lists 153 host species and some species against plant pathogens, several have been extensively of the most notable include mahogany (Swietenia mahagoni King.) studied as biological control agents against fungal pathogens (Chet, teak (Tectona grandis L.), rubber (Hevea brasiliensis (Willd. ex Adr 1990; Chet et al., 1998; Harman et al., 2004; Howell, 1998). As po- de Juss.) Muell. et Arg.), oil palm (Elaeis guineensis Jacq.), tea Camellia tential biocontrol agents, Trichoderma species have the following sinensis (L.) Kuntze, coffee (Coffea canephora Pierre ex A. Froehner) advantages: they grow on, but do not occur on, most organic mat- and cacao (Theobroma cacao L.), as well as a variety of fruit, nut, ter, sporulate readily in culture and, in natural conditions, can act and ornamental trees. The fungus is believed to be responsible for as either secondary antagonists or primary colonizers (Alabouvette the death of many Hoop Pines (A. cunninghamii), and other trees et al., 2006; Highley, 1997; Holdenreider and Greig, 1998; Scho- throughout Australia (Bolland, 1984). The disease is highly invasive eman et al., 1999). Furthermore, some Trichoderma species survive and has already killed many significant trees throughout the Greater as chlamydospores under unfavourable conditions and are fairly Metropolitan area of Brisbane Queensland, including street and park resistant to common fungicides and herbicides (Sariah, 2003). trees located in the suburbs of Shorncliffe, Taringa, New Farm, Eagle Much of the biological control research in the tropics has fo- Farm, West End, the city’s centre and beside the Brisbane River. cused on the development of strains of Trichoderma species (subdi- As the demand for alternatives to chemical control of plant vision: Ascomycota) that show antagonistic activity against fungal pathogens has become stronger, owing to concerns about the safety root fungal pathogens (Harman et al., 2004; Prasad and Naik, 2002; and environmental impact of chemicals, the application of Tricho- Raziq and Fox, 2006; Sariah, 2003; Sariah et al., 2005; Susanto derma as biological control of P. noxius shows promise. The objective et al., 2005; Widyastuti, 2006). Soepena et al. (2000) successfully of this study was to evaluate the potential of different Trichoderma formulated a biofungicide comprising Trichoderma koningii Oud. species as biocontrol agents and to identify a competitive strain that isolate Marihat (MR14) to manage basal stem rot in Elaeis guineen- can be used against P. noxius. For this purpose, a range of bioassays sis (Jacq.) (oil palm) caused by Ganoderma orbiforme (Fr.) Ryvarden were conducted to evaluate the antagonistic mechanisms of differ- (=G. boninense Pat.). This pathogen is recognized as the single ma- ent Trichoderma species against P. noxius and Ganoderma australe jor disease constraint to sustainable production of oil palm (Fr.) Pat. an important decay fungus on urban trees in Australia. throughout Asia (Ariffin et al., 2000; Durand-Gasselin et al., As a benchmark, we compared the European T. atroviride strain 2005; Flood et al., 2000; Paterson et al., 2000; Singh, 1991; Turner, 15603.1, which has been shown to have high biocontrol efficacy 1981). In in vitro experiments, the growth of two unknown Gano- against several wood-decay fungi (Schubert et al., 2008a–c), with derma species, previously isolated from diseased Acacia mangium four Australian native Trichoderma strains (T. ghanense, T. reesei, in Indonesia, were shown to be strongly inhibited by T. koningii, and two strains of T. harzianum). T. harzianum and T. reesei (Widyastuti, 2006). In field experiments carried out at different locations in France and Germany, a total of 2. Materials and methods 159 angiospermous trees and 1431 wounds on six different species (Platanus Â hispanica Miller ex Münchh., Acer pseudoplatanus L., Til- 2.1. Micro-morphological and molecular identification ia platyphyllos Scop., Populus nigra L., Quercus rubra L., Robinia pseudoacacia L.) were treated with different conidial suspensions All cultures were identified microscopically (Bissett, 1984; of T. atroviride strain T-15603.1 (Schubert et al., 2008a–c). T- 1991a–c, 1992; Gams and Bissett, 1998; Rifai, 1969) and addition- 15603.1 significantly suppressed growth (82.3%) in wounds artifi- ally the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of the cially inoculated with three basidiomycetes Ganoderma adspersum, rDNA was amplified and sequenced for each strain (Schubert, Inonotus hispidus and Polyporus squamosus in comparison with 2006). The origins of the Trichoderma strains and wood-decay growth in untreated control wounds (Schubert et al., 2008b). basidiomycetes are provided in Table 1. All cultures were main- Although these studies show that Trichoderma species can be tained on 2% malt extract agar (MEA) at 4(±1) °C. For further stud- successfully used against wood-decay fungi, to our knowledge lit- ies, Petri dishes with MEA were inoculated with 5 mm diameter tle research on the biological control of the basidiomycete Phellinus agar plug cut from the growing edge of colonies of the strains noxius (Corner) G.H. Cunningham exists (Bolland et al., 1988). This and stored in the dark at 25 (±1) °C and 70% ambient relative white-rot fungus was first described in 1932 as Fomes noxius by humidity. The sequences were deposited in the EMBL Data Bank Corner (1932), who was investigating the cause of a brown root (Table 1). rot disease of trees in Singapore. It was reclassified as P. noxius by Cunningham (1965). The devastating brown root rot disease af- fects a wide variety of important agricultural and forest plant spe- 2.2. Growth of Trichoderma species and wood-decay fungi under cies (Ann et al., 2002). Many early reports of root rot caused by P. different conditions noxius were made without demonstration of pathogenicity. In 1984, Bolland was the first to fulfill Koch’s postulates for the dis- The effects of temperature (20 °C, 25 °C, 30 °C, 35 °C) and water ease on hoop pine (Araucaria cunninghamii Aiton ex D. Don.). An activity (aw: 0.928, 0.955, 0.978, 0.998) on hyphal growth were inoculation technique for pathogenicity tests was described by monitored on 2% MEA. All agar plates (90 mm) were inoculated Ann et al. (2002). centrally with a 5-mm disc of the respective Trichoderma species P. noxius is a basidiomycete with a pan-tropical/subtropical dis- and wood-decay fungi taken from the margin of growing cultures tribution, and has been found in Africa, Asia, Australia and Oceania, and incubated at 25(±1) °C and 70% relative humidity. For each

Central America, and the Caribbean. The pathogen has a wide host experimental treatment (aw and temperature), 10 replicates were range, spanning over 100 genera of Gymnospermae and both classes performed. The growth rate (mm/day) was determined by colony (Monocotyledons and Dicotyledons) within the Angiospermae. diameter measurements carried out along two perpendicular axes

Cross-inoculation studies have shown a lack of host speciﬁcity for after 24 h (Schubert et al., 2009). The aw of the substrate was con- various strains of P. noxius, although varying degrees of resistance trolled by the addition of appropriate weights of the non-ionic sol- are seen in different hosts (Ann et al., 1999; Chang, 1995, 2002; ute, glycerol, prior to autoclaving (Dallyn, 1978).

Table 1 Origin of Trichodermaspeciess and wood-decay fungi used in the present study.

Trichoderma Strain No. EMBL No. Wood-decay fungi Strain No. EMBL No. T. atroviride Karsten T-15603.1a FR178524 Phellinus noxius (Corner) G.H. Cunningham 133f FR821775 T. ghanense Yoshim. Doi, T-4510b FR821772 Y. Abe and Sugiy. P. noxius 144g FR821769 T. harzianum Rifai T-4428c FR821773 P. noxius 169h FR821770 T. reesei E.G. Simmens T-3967d FR821774 P. noxius 178f FR821771 T. harzianum T-121009e FR821767 Ganoderma australe (Fr.) Pat. 101009i FR821768

a From EMPA culture collection, Switzerland; isolated from a basidiocarp of Armillaria mellea (Vahl) P. Kumm., Freiburg, Germany. b From Commonwealth Scientiﬁc and Industrial Research Organisation (CSIRO) culture collection, North Ryde New South Wales (N.S.W.) Australia; isolated from soil, North Carolina, USA. c From CSIRO culture collection, isolated from wooden ﬂooring of shipping container, Sydney, N.S.W. d From CSIRO culture collection; isolated from mutant of ATCC 24449, e From Mt. Coot-tha, Queensland, Australia; isolated from a basidiocarp of Laetiporus portentosus (Berk.) Rajchenb. f Provided by Dr. Geoff Pegg, Department of Employment, Economic Development and Innovation (DEEDI), Brisbane, Queensland, Australia, g From New Farm Park, Queensland Australia; isolated from basidiocarp of Ganoderma australe on Jacaranda mimosifolia D. Don, h From New Farm Park, Queensland Australia; isolated from a basidiocarp of mature Phellinus noxius on a dead Ficus species, i From City Botanic Gardens, Brisbane, Queensland, Australia; isolated from basidiocarp of Ganoderma spp. on mature Ficus microcarpa var. hillii (F.M. Bailey) Corner.

2.3. Inhibitory effects of volatile compounds produced by Trichoderma fungi. The ability of Trichoderma species to eliminate the wood-de- species on wood-decay fungi cay fungi (lethal effect) during the four week incubation period was evaluated by aseptically transferring 5-mm discs from test The effect on wood-decay fungi of volatile organic compounds plates to MEA with 2 mL thiabendazole (2-(40-thiazolyl)- benz- (VOCs) produced by Trichoderma strains was evaluated with the imidazole; Merck, Darmstadt, Germany; 0.46 mg dissolved in lactic following techniques as described by Dennis and Webster (1971). acid). T-MEA suppresses the growth of Trichoderma species but al- Trichoderma strains were centrally inoculated onto 2% MEA by lows growth of wood-decay fungi (Sieber, 1995). The lethal effect placing 5-mm discs taken from the margin of 7-day-old cultures of Trichoderma species was expressed as a percentage of the and then incubating the plates at 25(±1) °C and 70% relative wood-decay fungi which were eliminated. In addition, interaction humidity for 3 weeks. MEA plates were inoculated centrally with tests in sap-wood blocks (Ave. 5 Â 25 Â 40 mm) of three hard- 5-mm discs with the wood-decay fungi and then the top of each woods, Delonix regia (Boj. ex Hook.) Raf., Jacaranda mimosifolia D. plate was replaced with the bottom of a Trichoderma-inoculated Don, and Ficus benjamina L., and one softwood, Araucaria bidwillii plate. Replicates without Trichoderma species were used as the (Molina) K. Koch, were performed as described by Schubert et al. control. Ten replicates were maintained for each treatment. The (2008a). For studies of colonization behaviour, wood blocks were pairs of Petri dishes were fixed and sealed together with Para film inoculated with a conidial suspension of Trichoderma species (col- 5 and incubated at 25(±1) °C and 70% relative humidity. The diame- ony-forming units: 10 /mL + 0.2% D-glucose + 0.1% urea) and ter of the wood-decay fungi colonies was measured after an incu- placed onto Petri dishes with 2% MEA. After the wood blocks were bation period of 7 days and the inhibition of mycelial growth was completely colonized by Trichoderma they were placed with their calculated. cross sections onto 2-week-old cultures of the wood-decay fungi and incubated in the dark at 25(±1) °C for 12 weeks. Untreated 2.4. Dual culture and interaction tests on wood wood blocks served as the control. Twenty replicates were used for each experiment. Before incubation wood blocks were oven- ° Mycoparasitism of all Trichoderma strains against the selected dried at 105 C for 24 h to determine the wood dry weight. The de- ° wood-decay fungi was assessed in dual culture according to the cay tests were run for 12 weeks at 25 C, after which the wood method of Schubert et al. (2006). The agar disc method was carried blocks were removed, cleaned and oven-dried for measuring dry out on 2% MEA. Mycelial discs (5 mm) were removed from 1-week- weight losses (Schwarze and Fink, 1998). old MEA cultures of each of the five wood-decay fungi and placed equidistantly at the margin of Petri dishes (90 mm) containing 2% 2.5. Statistical analysis MEA. These were incubated at 25(±1) °C and 70% relative humidity for 3–4 days. Next, discs (5 mm) were removed from the margins Growth data were log-transformed and data that were ex- of actively growing 1-week-old cultures of the Trichoderma species pressed as percentages, such as the wood weight loss, were arc- and placed at opposite sides of the dish, and incubated in the dark sine-transformed prior to analysis (ANOVA) and back-transformed at 25(±1) °C and 70% relative humidity for 4 weeks. Petri dishes to numerical values for presentation (expressed as mean ± SE). without Trichoderma were used as the control. Twenty replicates Means were separated using Dunett’s test at significance levels of were used for each experiment. Petri dishes were examined at reg- p < 0.05 and p < 0.0001. To compare the performance of the Tricho- ular intervals. The sporulation tufts and pustules of Trichoderma derma isolates with each other a Tukey’s HSD (Honestly Significant Difference) test (P < 0.05) was additionally performed. The statisti- fungi were used as an indication of its activity (Naár and Kecskes, Ò 1998). In order to check whether the antagonist was able to over- cal package used for all analyses was SPSS (Version 17.0, SPSS Inc., grow and parasitize the challenged wood-decay fungus, three agar Chicago, IL, USA). discs (5 mm) were removed from non-sporulating regions of the mycelium of the wood decay fungus and placed on a Tricho- 3. Results derma-selective medium (Askew and Laing, 1993). After 7 days of incubation at room temperature, discs were observed for Tricho- 3.1. Growth under different conditions derma colonies. Competition (mycoparasitism rate) was assessed as follows: 0 = no overgrowth; 1 = slow overgrowth; 2 = fast over- The influence of temperature and aw on the mean growth rates growth; 3 = very fast overgrowth and deadlock of the wood-decay of the Trichoderma species and wood decay fungi is shown in

Tables 2 and 3, respectively. Generally, the growth rates of all Table 3 Mean growth rate (mm/day)of the Phellinus noxius strains and Ganoderma australe Trichoderma strains increased with increasing aw (Table 2). T- under different conditions. 4510 showed the strongest growth at a mean temperature of ° 35 °C and at the highest aw of 0.998 (Table 2). T-3967 and T- Temperature ( C) Water activity (aw) 121009 showed an optimum growth at a mean temperature of 0.998 0.978 0.995 0.928 ° 30 C and at the highest aw of 0.998, whereas the European strains P. noxius 133 T-15603.1 and T-121009 showed optimum growth at a mean tem- 20 5.5 ± 0.9 4.2 ± 0.8 0.5 ± 0.2 0.0 ± 0.0 perature of 25 °C and the highest aw of 0.998. Even at 35 °C and the 25 7.7 ± 1.0 7.1 ± 1.0 1.9 ± 0.4 0.0 ± 0.0 30 7.8 ± 1.0 9.1 ± 1.2 2.9 ± 0.5 0.0 ± 0.0 lowest water activity (aw 0.928), growth was recorded for some Trichoderma species. P. noxius strains 144, 169 and 178 showed 35 1.9 ± 0.8 3.7 ± 1.0 1.2 ± 0.3 0.0 ± 0.0 optimum growth at a mean temperature of 25–30 °C and a high P. noxius 144 a of 0.998 (Table 3). By contrast, P. noxius 133 showed optimum 20 5.0 ± 1.1 2.9 ± 1.0 0.5 ± 0.2 0.0 ± 0.0 w 25 7.1 ± 1.6 5.6 ± 1.3 1.4 ± 0.6 0.0 ± 0.0 ° growth at a mean temperature of 30 C and moderate aw of 30 7.9 ± 1.6 7.0 ± 1.7 2.6 ± 0.5 0.1 ± 0.13 0.978. At the lowest aw (0.928) and at 25 °C and 30 °C, growth 35 3.1 ± 1.6 6.3 ± 0.7 1.4 ± 0.3 0.0 ± 0.0 ° was only limited, and at 35 C growth of the P. noxius strains failed P. noxius 169 completely. G. australe showed optimum growth at a mean tem- 20 4.8 ± 0.8 3.7 ± 0.5 0.4 ± 0.2 0.0 ± 0.0 perature of 30 °C and high aw of 0.998, but no growth was recorded 25 7.2 ± 0.7 6.0 ± 0.7 1.3 ± 0.4 0.0 ± 0.1 30 9.3 ± 0.5 7.2 ± 0.8 2.2 ± 0.7 0.0 ± 0.1 at any temperature at the lowest aw (0.928). 35 2.8 ± 0.7 3.5 ± 1.0 0.4 ± 0.3 0.0 ± 0.0 P. noxius 178 3.2. Evaluation of antagonistic activity in dual cultures 20 5.4 ± 1.4 4.1 ± 0.7 0.5 ± 0.2 0.0 ± 0.0 25 8.4 ± 1.7 6.5 ± 0.9 1.5 ± 0.3 0.0 ± 0.0 During initial screening of the Trichoderma strains, a variety of 30 9.2 ± 1.8 7.6 ± 1.0 2.6 ± 0.5 0.0 ± 0.1 reactions were recorded as a result of antagonism. Growth of all 35 3.4 ± 0.7 5.6 ± 1.0 0.8 ± 0.3 0.0 ± 0.0 wood-decay basidiomycetes was inhibited by at least one of the G. australe Trichoderma strains. Contact between wood-decay fungi and Trich- 20 2.5 ± 0.5 0.6 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 oderma species occurred, but the ability to overgrow and parasitize 25 4.1 ± 0.5 1.0 ± 0.6 0.0 ± 0.0 0.0 ± 0.0 30 6.3 ± 0.8 1.8 ± 0.4 0.0 ± 0.1 0.0 ± 0.0 the mycelia of the wood-decay fungi was highly dependent on the 35 3.5 ± 0.6 1.4 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 antagonistic potential of each Trichoderma strain and the resistance of the challenged wood-decay fungus to antagonism (Table 4). The For each experimental treatment (water activity aw and temperature), 10 replicates were performed. The growth rate (mm/day) was determined by colony diameter lethal effect of mycoparasitism by Trichoderma species was most measurements carried out along two perpendicular axes after 24 h. The aw of the prevalent for T. harzianum 121009. It showed a high antagonistic substrate was controlled by the addition of appropriate weights of the non-ionic solute, glycerol, prior to autoclaving. Data were analyzed by analysis of variance

(ANOVA) to assess the effect of aw and temperature on growth rate (mm/day). ±, Table 2 Standard deviation. Mean growth rate (mm/day) of the Trichoderma species under different conditions.

Temperature (°C) Water activity (aw) potential against three P. noxius strains and G. australe. P. noxius 178 showed moderate resistance to T. harzianum 121009. Tricho- 0.998 0.978 0.995 0.928 derma reesei 3967 revealed the weakest effect against all wood-de- T. atroviride 15603.1 cay fungi. The highest resistance of P. noxius to antagonism of 20 8.3 ± 1.3 4.6 ± 0.3 2.3 ± 0.2 0.2 ± 0.1 25 10.6 ± 0.7 6.1 ± 0.5 2.9 ± 0.3 0.4 ± 0.1 Trichoderma species was recorded for strain 133. The highest sus- 30 8.8 ± 0.5 6.2 ± 0.4 2.1 ± 0.4 0.0 ± 0.0 ceptibility of P. noxius to antagonism of Trichoderma species was re- 35 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 corded for strain 169 (Table 4). T. ghanense 4510 20 9.0 ± 1.3 5.2 ± 0.4 1.9 ± 0.6 0.1 ± 0.1 3.3. Inhibitory effects of volatile compounds produced by Trichoderma 25 11.8 ± 2.9 7.8 ± 1.1 3.5 ± 0.6 0.6 ± 0.9 30 16.1 ± 1.6 8.9 ± 0.5 3.9 ± 0.4 0.9 ± 0.2 species on wood-decay fungi 35 18.1 ± 1.6 9.1 ± 0.5 3.5 ± 0.4 0.3 ± 0.1 T. harzianum 4428 The results revealed that after 6 days’ incubation, the VOCs pro- 20 8.6 ± 0.7 5.4 ± 0.7 2.2 ± 0.3 0.3 ± 0.1 duced by T. atroviride 15603.1 caused a signiﬁcant (p < 0.0001) inhi- 25 12.0 ± 0.6 7.6 ± 1.2 2.9 ± 0.3 0.7 ± 0.2 bition of growth of all P. noxius strains (Table 5). None of the VOCs 30 8.1 ± 0.5 6.4 ± 0.5 2.7 ± 0.5 0.2 ± 0.2 from the Australian Trichoderma strains were effective in reducing 35 0.0 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.00 ± 0.0 pathogen growth except T-4428 that inhibited P. noxius 178. T. reesei 3967 The weakest effect against the P. noxius strains was recorded for 20 10.7 ± 0.4 3.5 ± 0.4 1.1 ± 0.2 0.1 ± 0.1 T. harzianum 121009. In the presence of VOCs, the growth of P. nox- 25 11.7 ± 3.1 7.3 ± 0.4 1.7 ± 0.3 0.5 ± 0.2 30 13.7 ± 0.4 9.7 ± 0.5 3.1 ± 0.5 0.3 ± 0.2 ious strains was often enhanced. By contrast, the VOCs of T. atrovi- 35 11.7 ± 0.4 9.7 ± 0.5 3.1 ± 0.5 0.3 ± 0.2 ride 15603.1 and T. harzianum 121009 markedly inhibited the T. harzianum 121009 growth of G. australe. Among the Trichoderma species, with regard 20 13.0 ± 1.7 4.5 ± 0.3 1.5 ± 0.2 0.1 ± 0.1 to the production and effect of VOCs, the wood-decay fungi dif- 25 11.8 ± 1.9 6.7 ± 0.5 2.6 ± 0.2 0.2 ± 0.2 fered signiﬁcantly in their reaction. P. noxius strains 169 and 133 30 14.4 ± 1.4 8.7 ± 0.5 3.5 ± 01.0 0.2 ± 0.2 showed a strong sensitivity to the VOCs, whereas P. noxius 144 35 6.3 ± 0.8 4.1 ± 0.7 1.1 ± 0.2 0.0 ± 0.0 showed a lower sensitivity (Table 5). For each experimental treatment (water activity aw and temperature), 10 replicates In summary, the effect of VOCs against P. noxious strains re- were performed. The growth rate (mm/day) was determined by colony diameter sulted in the inhibition of growth of approximately 19–25%, measurements carried out along two perpendicular axes after 24 h. The aw of the substrate was controlled by the addition of appropriate weights of the non-ionic whereas growth of G. australe was inhibited by approximately solute, glycerol, prior to autoclaving.Data were analyzed by analysis of variance 15% (Table 5). Trichoderma atroviride 15603.1 inhibited the growth

(ANOVA) to assess the effect of aw and temperature on growth rate (mm/day). ±, of P. noxius strains and G. australe by 70–85% and 46%, respectively Standard deviation. (Table 5).

Table 4 Classiﬁcation of the degree of mycoparasitism of different Trichoderma species against different Phellinusnoxiusstrains and Ganodermaaustrale on malt extract agar.

Trichoderma species T. atrovirde 15603.1 T. ghanense 4510 T. harzianum 4428 T. reesei 3967 T. harzianum 121009 Phellinus noxius 133 1.1a ± 0.1 [80]b 1.2 ± 1.0 [60] 2.9 ± 0.7 [60] 1.8 ± 0.8 [30] 3.0 ± 0.9 [100] Phellinus noxius 178 1.6 ± 0.1 [70] 1.4 ± 0.2 [50] 3.0 ± 0.1 [100] 1.1 ± 0.7 [60] 2.8 ± 0.1 [60] Phellinus noxius 144 2.5 ± 0.1 [100] 2.2 ± 1.2 [60] 2.9 ± 0.2 [60] 1.9 ± 0.2 [30] 3.0 ± 0.5 [100] Phellinus noxius 169 2.0 ± 0.4 [100] 1.5 ± 1.3 [60] 2.8 ± 0.3 [100] 1.8 ± 0.4 [60] 2.6 ± 0.6 [100] Ganoderma australe 3.0 ± 0.6 [100] 2.3 ± 1.1 [60] 3.0 ± 0.7 [60] 1.7 ± 0.2 [0] 2.9 ± 1.5 [100]

For each experimental treatment (interactions and lethal effect) in dual cultures, 10 replicates were performed. a Following system was used to classify the rate of mycoparasitism in dual cultures: 0 = no overgrowth; 1 = slow overgrowth; 2 = fast overgrowth; 3 = very fast overgrowth and deadlock of the wood decay fungi within 4 weeks. ±, Standard deviation. b The ability of Trichoderma species to eliminate the wood-decay fungi (lethal effect in %) during the four week incubation period was evaluated by aseptically transferring 5-mm discs from test plates to MEA with 2 mL thiabendazole (2-(40-thiazolyl)- benzimidazole; dissolved in lactic acid).

Table 5 Inhibition of radial growth (in %) of wood decay fungi by volatile organic compounds (VOCs) produced by Trichoderma species.

Phellinus noxius strain Ganoderma australe 133 144 169 178 Control 9.3 ± 2.0 6.5 ± 1.3 6.9 ± 2.6 7.7 ± 2.6 4.5 ± 1.4 T. atrovirde15603.1 2.2 ± 1.4** 1.9 ± 2.0** 1.0 ± 1.2** 1.4 ± 1.5** 2.4 ± 0.7** T. ghanense 4510 11.0 ± 2.4n.s. 6.3 ± 1.5n.s. 6.1 ± 2.4n.s. 8.4 ± 3.0n.s. 5.8 ± 2.2n.s. T. harzianum 4428 5.3 ± 0.5n.s. 5.3 ± 0.5n.s. 4.4 ± 1.1n.s. 3.6 ± 1.1** 3.8 ± 1.8n.s. T. harzianum121009 10.8 ± 2.7n.s. 6.9 ± 1.5n.s. 8.1 ± 3.4n.s. 8.8 ± 3.4n.s. 3.4 ± 1.8n.s. T. reesei 3967 5.8 ± 0.5n.s. 5.8 ± 0.5n.s. 6.28 ± 2.7n.s. 7.1 ± 1.7n.s. 4.4 ± 1.4n.s.

For assessing the inhibition of radial growth (in %) 10 replicates were performed for each combination of wood decay fungus and Trichoderma species.*Signiﬁcant inﬂuence on growth rate by VOCs. Dunnett’s test: ±, Standard deviation. * p < 0.05. ** p < 0.0001. n.s. p P 0.05.

3.4. Interaction tests in wood et al., 1998; Hjeljord and Tronsmo, 1998). Physical as well as chemical factors influence growth and germination, therefore knowl- All wood-decay fungi had completely colonized the control edge of the optimal conditions for growth, as well as the wood samples, but showed distinctive differences in their potential influence of ecological factors on the antagonist and the target to decompose the wood of D. regia, J. mimosifolia, F. benjamina and pathogen, is essential for successful application in the field (Hjelj- A. bidwillii. In untreated wood blocks of A. bidwillii the wood-decay ord and Tronsmo, 1998; Kredics et al., 2000, 2003). fungi only caused negligible weight losses (Table 6). In untreated In this study, the growth of the Trichoderma strains was affected wood blocks of D. regia and F. benjamina, G. australe caused greater by the environmental factors tested. Most Trichoderma strains weight losses than all four P. noxius strains (Table 6). By contrast, in showed optimum growth at a mean temperature of 25–30 °C and untreated wood blocks of J. mimosifolia the weight losses caused by a high aw of 0.998. Even at 35 °C and the lowest aw (0.928) growth G. australe were always lower than those of the P. noxius strains. was recorded for some strains. The results have to be interpreted Interactions among the wood-decay fungi, Trichoderma species carefully because predicting the behaviour of Trichoderma species and the wood substrate resulted in interesting differences in under specific conditions is complicated by the mutual effect of weight losses (Table 6). A significant reduction in weight losses environmental parameters (Harman, 2006). in wood by G. australe was recorded for A. bidwillii, D. regia and F. Growth of all P. noxius strains at different temperatures did not benjamina pretreated with Trichoderma species (Table 6). By con- differ significantly. Optimum growth for most strains was recorded trast, in J. mimosifolia wood the weight losses caused by G. australe at 30 °C, which is in good agreement with the experimental find- were not reduced by the Trichoderma strains. ings of Ann et al. (2002), who showed that the optimum growth In wood of D. regia and F. benjamina pretreated with T-15603.1 of P. noxius is at 30 °C, and that of Albrecht and Venette (2008), and T-3967, the weight losses caused by P. noxius 133 were signif- who defined the optimum growth range as approximately 25– icantly reduced (Table 6). In J. mimosifolia wood pretreated with 31 °C. All of the P. noxius strains in this study were isolated from Trichoderma species, the weight losses by P. noxious 144 and 178 urban sites in Brisbane, Queensland, Australia. Interestingly, the were also significantly reduced (Table 6). Wood blocks of F. benj- optimum growth of the P. noxius strains correlates closely with a amina and D. regia pretreated with Trichoderma species showed recent study on the local soil temperature. Prangnell and McGowan the greatest reduction in weight loss. By contrast, wood blocks of (2009) used a soil temperature calculation equation to calculate A. bidwillii and J. mimosifolia pretreated with Trichoderma species soil temperature at various depths in a cemetery located in Bris- only showed a weak reduction in weight losses. bane. Their study revealed that throughout the year the mean temperature at a soil depth of 1 m was approximately 28 °C. In the 4. Discussion absence of vegetation cover, extreme temperatures of approximately 17 °C and 40 °C were measured. In soils with vegetation 4.1. Growth under different conditions cover, only minor temperature fluctuations were reported (Prang- nell and McGowan, 2009). The present study showed that the The competitiveness of Trichoderma species is based on rapid growth of different P. noxius strains does not appear to be strongly growth (i.e. a decisive feature for antagonism) (Chet, 1990; Chet affected by different temperatures.

Table 6 Dry weight losses (in %) caused by Phellinus noxius and Ganoderma australein untreated controls and in wood blocks pretreated with different Trichoderma strains.

P. noxius 133 P. noxius144 P. noxius169 P. noxius178 G. australe Araucaria bidwillii Control 0.94 ± 0.82 1.60 ± 0.98 1.38 ± 1.40 3.43 ± 0.33 4.42 ± 4.38 T. atrovirde15603.1 1.71 ± 3.07n.s. 0.32 ± 0.65n.s. 0.33 ± 0.67n.s. 1.12 ± 0.63* 0.55 ± 1.24* T. ghanense 4510 0.00 ± 0.00n.s. 1.36 ± 1.34n.s. 0.00 ± 0.00* 0.29 ± 0.59** 0.06 ± 0.13** T. harzianum 4428 0.62 ± 0.83n.s. 1.69 ± 1.80n.s. 0.05 ± 0.12* 0.00 ± 0.00** 0.00 ± 0.00** T. reesei 3967 1.80 ± 2.78n.s. 1.16 ± 0.72n.s. 0.00 ± 0.00* 0.00±0.00** 0.00±0.00** T. harzianum121009 2.75±3.54n.s. 1.90±2.53n.s. 3.04±2.79n.s. 2.99±4.10* 1.03±2.31* Delonix regia Control 34.78±17.02 21.34±3.36 22.03±5.44 18.43±9.74 59.89±4.19 T. atrovirde15603.1 13.01±5.17* 14.65±7.94n.s. 23.95±9.29n.s. 12.31±3.66n.s. 18.97±7.60** T. ghanense 4510 23.05±7.40n.s. 18.07±8.24n.s. 23.45±6.37n.s. 20.97±9.82n.s. 16.65±8.49** T. harzianum 4428 25.43±3.69n.s. 20.82±5.76n.s. 21.44±9.18n.s. 21.07±5.23n.s. 18.08±6.58** T. reesei 3967 13.61±4.35* 20.15±2.04n.s. 22.20±5.35n.s. 22.61±3.73n.s. 26.15±7.12** T. harzianum121009 24.05±10.42n.s. 23.62±5.71n.s. 22.83±2.71n.s. 19.96±5.61n.s. 28.59±5.99** Ficus benjamini Control 23.30±8.74 22.40±4.60 9.83±3.98 18.00±2.60 36.91±10.02 T. atrovirde15603.1 7.58±1.19* 6.70±2.42** 0.78±1.68** 5.89±1.31** 6.76±1.92** T. ghanense 4510 7.33±2.17* 7.70±3.15** 5.02±2.85n.s. 5.02±2.85** 7.23±2.61** T. harzianum 4428 7.28±1.69* 7.59±3.00** 9.77±2.23n.s. 5.39±1.91** 7.45±3.07** T. reesei 3967 8.49±2.75* 8.23±1.91** 5.39±1.91n.s. 8.42±3.58* 11.19±2.41** T. harzianum121009 6.51±6.02* 16.32±11.51n.s. 10.17±6.31n.s. 7.84±4.51** 5.70±5.24** Jacaranda mimosifolia Control 11.82±3.32 20.62±3.10 9.50±4.71 14.04±0.97 4.46±2.39 T. atrovirde15603.1 9.79±2.38n.s. 12.66±5.20n.s. 8.54±1.16n.s. 7.96±1.58** 9.07±2.14n.s. T. ghanense 4510 9.10±1.17n.s. 10.81±2.22* 7.03±4.19n.s. 9.41±1.98* 6.60±2.24n.s. T. harzianum 4428 8.25±1.70n.s. 10.33±3.32* 11.16±2.34n.s. 0.68±1.93* 8.98±1.89n.s. T. reesei 3967 8.46±1.30n.s. 8.27±1.45* 7.24±7.79n.s. 9.58±2.36* 8.10±2.95n.s. T. harzianum121009 11.93±13.75n.s. 11.86±7.88* 12.74±12.96n.s. 15.16±10.47n.s. 8.66±5.12n.s.

Untreated wood blocks served as the control. Twenty replicates were used for each experiment. Before incubation wood blocks were oven-dried at 105 °C for 24 h to determine the wood dry weight. The decay tests were run for 12 weeks at 25 °C, after which the wood blocks were removed, cleaned and oven-dried for measuring dry weight losses. Significant reduction of wood decay (wood weight loss in %). Dunnett’s test: ±, Standard deviation. * Significant p < 0.05. ** Highly significant p < 0.0001. n.s. Not significant p P 0.05.

In comparison with the P. noxius strains, growth of most Trich- Pathogen and strain resistance to mycoparasitism by Tricho- oderma species was more rapid at the highest and lowest water derma species is well documentated (Sivan and Chet, 1989; Cortés activity values (0.998 and 0.928). At the lowest aw (0.928) and at et al., 1998; Zeilinger et al., 1999). In vitro tests conducted by Sivan 25 °C and 30 °C, growth was minimal and at 35 °C the growth of and Chet, 1989 showed that two strains of Trichoderma harzianum the P. noxius strains failed completely. failed to parasitize colonies of Fusarium oxysporum f. vasinfectum The European species, T. atroviride 15603.1, that was used as a (G.F. Atk.) W.C. Snyder and H.N. Hansen and Fusarium oxysporum ‘‘bench mark’’ failed to grow at 35 °C, which may indicate its limited f. melonis W.C. Snyder and H.N. Hansen. However, these strains adaptation to the subtropical climate of Brisbane where soil temper- were strongly mycoparasitic on Rhizoctonia solani J.G. Kühn and ature can exceed 35 °C. This finding demonstrates the importance of Pythium aphanidermatum (Edson) Fitzp. (Sivan and Chet, 1989). screening Trichoderma species for the specific niche where they are The studies suggest that proteins in the cell walls of F. oxysporum envisaged to be applied (i.e. increasing target specificity). may make these walls more resistant than those of R. solani to degradation by extracellular enzymes of T. harzianum. Savoie et al. 4.2. Evaluation of antagonistic activity in dual culture (2000) studied interactions between Lentinula edodes (Berk.) Pegle- r and Trichoderma spp. and observed that there were great differ- The direct mycoparasitic activity of Trichoderma species is one ences between two modified substrates for each strain. Similary, of the major mechanisms proposed to explain their antagonistic Tokimoto and Komatsu (1979) observed that carbon rich medium activity against soil-borne plant-pathogenic fungi. favours L. edodes while a nitrogen rich medium favours Tricho- In the dual culture tests, hyphal contact between Trichoderma derma and that mycoparasitism could be reduced by controlling species and wood-decay fungi was observed for all host–pathogen nutritional conditions. Giovannini et al. (2004) selected resistant combinations. However, not all strains of Trichoderma were able strains of the wood decay fungi Grifola frondosa (Dicks.) Gray and to overgrow and parasitize the mycelia of the wood-decay fungi. Fomitopsis pincola (Sw.) P. Karst. with a Teflon tubes confrontation In this study three lethal effect of mycoparasitism by Trichoderma method. Results showed great variations in behaviour between species was most prevalent for T. harzianum 121009 which was iso- strains, but also for a given strain growing on different substrates, lated from a basidiocarp of Laetiporus portentosus (Berk.) Rajchenb. indicating the importance of trophic factors (Giovannini et al., collected at Mt. Coot-tha, west of Brisbane, Queensland. It showed a 2004). Interestingly in their experiments with F. pinicola they dem- high antagonistic potential against three P. noxius strains and G. onstrated that the substrate with the lower nitrogen content was australe. The European strain T-15603.1 and T4428 showed a high more favourable for Trichoderma (Giovannini et al., 2004). In a antagonistic potential against all wood-decay fungi, whereas the previous study on mycoparasitism of a range of Trichoderma spe- antagonistic potential of T-4510 and T-3967 was limited. cies against wood decay fungi, the white rot fungus Polyporus

Please cite this article in press as: Schwarze, F.W.M.R., et al. Evaluation of an antagonistic Trichoderma strain for reducing the rate of wood decomposition by the white rot fungus Phellinus noxius. Biological Control (2012), doi:10.1016/j.biocontrol.2012.01.016 F.W.M.R. Schwarze et al. / Biological Control xxx (2012) xxx–xxx 7 squamosus was not only able to circumvent parasitism but also The European strain, T. atroviride 15603.1, showed a high antago- adapted its hyphal structure, to overgrow the mycelia of the Trich- nistic potential against all wood-decay fungi and its VOCs greatly oderma isolates (Schubert et al., 2008a). inhibited their growth. However, its failure to grow at 35 °C indicates its limited adaptation to the subtropical climate of Brisbane 4.3. Inhibitory effects of volatile compounds produced by Trichoderma where temperatures can exceed 35 °C. Even at high temperatures species on wood-decay fungi and low aw, T-4510 was a very competitive species and mycoparasitism in wood resulted in a reduction in wood weight losses, but Antibiosis was recognized and initially described by Weindling its VOCs actually stimulated the growth of some P. noxius strains. (1932) and is defined as the production of secondary metabolites These results demonstrate that one and the same Trichoderma spe- that have an antimicrobial effect, even at low concentrations (Coo- cies may possess positive and negative attributes for biological ney et al., 1997a,b; Galindo et al., 2004; Howell, 1998; Scarselletti control. As one Trichoderma species rarely has only positive attri- and Faull, 1994; Schirmböck et al., 1994; Wheatley et al., 1997). butes, the selection of one potential Trichoderma species is not a The VOCs produced by T. atroviride 15603.1 strongly inhibited trivial exercise and will require further screening studies. For bio- growth of all P. noxius strains used in this study. Interestingly, logical control a number of Trichoderma species with different the VOCs produced by T. harzianum 121009 and T. ghanense 4510 antagonistic properties may have to be combined to successfully appeared to stimulate the growth of P. noxius strains, however inhibit the growth of the target pathogen in the field. the effect was not significant. The VOCs studies also indicated that In conclusion, the present study shows that the success of bio- the antagonistic potential of Trichoderma species against P. noxius logical control of P. noxius depends on the Trichoderma species, the varies from strain to strain, so for successful selection of a Tricho- strain of wood-decay fungus, the specific wood species and the derma species for biological control of P. noxius these variations prevailing environmental conditions (temperature/water activity). in strain resistance have to be considered. The in vitro screening of antagonistic potential used in this study allowed a systematic investigation of several Trichoderma strains, 4.4. Interaction tests on wood as well as specific ecological factors, and thus identification of effective strains. The study shows that Trichoderma species can In order to assess the antagonistic potential of Trichoderma spe- be used to significantly inhibit the growth of different P. noxius cies on its natural substrate, interaction studies were performed on strains, and have a lethal effect on some basidiomycota species wood blocks. After 12 weeks’ incubation, the Trichoderma species in controlled growing conditions. The study also showed that Trich- failed to completely inhibit decomposition, as measured by dry oderma species can be used to reduce the rate of decomposition by weight losses. This may be partly explained by the degradation P. noxius and Ganoderma australe in the wood of the species studied of readily accessible carbohydrates by Trichoderma species within here. However, positive results obtained from in vitro studies are parenchymal cells and pits (Kubicek-Pranz, 1998). The inoculum only indicative, as experimental conditions do not take all ecolog- potential in turn is crucial for the invasiveness of pathogens (Red- ical and endemic factors into account. For this reason, field studies fern and Filip, 1991). Nevertheless, a significant reduction in dry are essential to test the selected competitive biocontrol agent un- weight loss was induced after pretreatment of the wood with a der field conditions (Schubert et al., 2008b). conidial suspension of Trichoderma species. Significant differences between the species and strains of Trichoderma were evident. Acknowledgments Interestingly the antagonistic potential of different Trichoderma species against P. noxius strains varied according to the specific We thank Brisbane City Council for assisting in the preparation wood substrate. Thus, P. noxius strains showed a different degree of wood materials and facilities, and Keith Foster for technical and of adaptation to the wood. Phellinus noxius 169 strongly decom- project support. Finally, the financial and technical support (i.e. posed the wood of D. regia, whereas weight losses on F. benjamina data collection and study design) provided by ENSPEC and Brisbane and J. mimosifolia were significantly lower. By contrast, only negli- City Council is gratefully acknowledged. gible weight losses were recorded from A. bidwillii. The variations in weight loss may be explained by the specific lignin composition of the different wood species (Schwarze, 2007). 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(Eds.), Trichoderma and Gliocladium: Basic Biology, Schubert, M., Fink, S., Schwarze, F.W.M.R., 2008c. Evaluation of Trichoderma spp. as a Taxonomy and Genetics. Taylor & Francis, London, pp. 3–34. biocontrol agent against wood decay fungi in urban trees. Biological Control 45, Giovannini, I.S., Job, D., Hmamda, A., 2004. Selection of Grifola frondosa and 111–123. Fomitopsis pinicola resistant strains in teflon tubes confrontation method. Schubert, M., Mourad, S., Fink, S., Schwarze, F.W.M.R., 2009. Ecophysiological Mycological Progress 3, 329–336. responses of the biocontrol agent Trichoderma atroviride (T-15603.1) to Harman, G.E., 2006. Overview of mechanisms and uses of Trichoderma species. combined environmental parameters. Biological Control 49, 84–90. Phytopathology 96, 190–194. Schwarze, F.W.M.R., 2007. Wood decay under the microscope. Fungal Biology Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., Lorito, M., 2004. 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Enhancing biological control of basal enzyme secretion and in vitro enzyme activities of Trichoderma harzianum at stem rot disease (Ganoderma boninense) in oil palm plantations. different temperatures. Current Microbiology 40, 310–414. Mycopathologia 159, 153–157. Kredics, L., Antal, Z., Manczinger, L., Szekeres, A., Kevei, F., Nagy, E., 2003. Influence Tokimoto, K., Komatsu, M., 1979. Effect of carbon and nitrogen sources in media on of environmental parameters on Trichoderma strains with biocontrol potential. the hyphal interference between Lentinus edodes and some species of Food Technology and Biotechnology 41, 37–42. Trichoderma. Annals of the Phytopathological Society of Japan 45, 261–264. Kubicek-Pranz, E.M., 1998. Nutrition, cellular structure and basic metabolic Turner, P.D., 1981. Oil Palm Diseases and Disorders. Oxford University Press, Kuala pathways in Trichoderma and Gliocladium. In: Harman, G.E., Kubicek, C.P. Lumpur, Malaysia. (Eds.), Trichoderma and Gliocladium: Basic Biology, Taxonomy and Genetics. Weindling, R., 1932. Trichoderma lignorum as a parasite of other soil fungi. Taylor & Francis, London, pp. 95–119. Phytopathology 22, 837–845. Mohd Farid, A., Lee, S.S., Maziah, Z., Patahayah, M., 2009. Pathogenicity of Wheatley, R., Hackett, C., Bruce, A., Kundzewicz, A., 1997. Effect of substrate Rigidoporus microporus and Phellinus noxius against four major plantation tree composition on the production of volatile organic compounds from Trichoderma species in peninsular Malaysia. Journal of Tropical Forest Science 21, 289–298. species inhibitory to wood decay fungi. International Biodeterioration and Naár, Z., Kecskes, M., 1998. Factors influencing the competitive saprophytic ability Biodegradation 39, 199–205. of Trichoderma species. Microbiological Research 53, 119–129. Whetten, R., Sederoff, R., 1995. Lignin biosynthesis. Plant Cell 7, 1001–1013. 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Indonesia, February 7–9, 2006). ACIAR Proceedings No. 124, Australian Centre Zeilinger, S., Galhaup, C., Payer, K., Woo, S.L., Mach, R.L., Fekete, C., Lorito, M., for International Research, Canberra, pp. 67–74. Kubicek, C.P., 1999. Chitinase gene expression during mycoparasitic interaction Wu, J., Peng, S.L., Zhao, H.B., Tang, M.H., Li, F.R., Chen, B.M., 2011. Selection of species of Trichoderma harzianum with its host. Fungal Genetics and Biology 26, 131– resistant to the wood rot fungus Phellinus noxius. European Journal Plant 140. Pathology 130, 463–467.

IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN AGAINST WOOD DECAY FUNGI

Mark Schubert1,2, Siegfried Fink2, Francis W.M.R. Schwarze1,2

The objective of the in vitro studies was to identify a Trichoderma strain with a high antagonistic potential against the basidiomycetes Ganoderma adspersum, Ganoderma lipsiense, Inonotus hispidus, Polyporus squamosus and the ascomycete Kretzschmaria deusta. For this purpose dual culture and interaction tests in wood blocks as well as investigations on fungal growth and germination behavior of conidia under different conditions were performed. Hyphal interactions were observed by scanning electron microscopy (SEM). The effect of Trichoderma spp. on wood colonization and degradation of wood decay fungi were quantitatively analyzed by means of dry weight loss measurements of wood and qualitatively by histological studies. The different Trichoderma species all showed an antagonistic potential against wood decay fungi in the in vitro studies. However, significant differences between the species and strains were found (P<0.001). Trichoderma atroviride (T-15603.1) showed the highest competitive activity against most wood decay fungi. An influence of physical and chemical parameters, in particular temperature and water potential on growth and germination behavior of conidia was evident. The species of wood decay fungi showed significant differences in their sensitivity when challenged by Trichoderma. Polyporus squamosus showed an extensive resistance in most laboratory tests indicating that target specificity of the antagonist needs consideration.

Introduction

Species of the genus Trichoderma are ubiquitous in the environment and especially in the soil. Since Weindling (1932) recognized the antagonistic effect of Trichoderma species against plant pathogens, several species of Trichoderma have been extensively studied as biological control agents against fungal pathogens (Chet, 1990; Chet et al., 1998; HOWELL, 1998). The demand for alternatives to chemical control of plant pathogens has

1 Swiss Federal Laboratories for Materials Testing and Research (EMPA), Wood Laboratory, Lerchenfeldstrasse. 5, CH-9014 St. Gallen, Switzerland 2 Albert-Ludwigs-Universität Freiburg, Professur für Forstbotanik, Bertoldstrasse 17, D-79085 Freiburg i.Br., Germany

227 228 ARBORICULTURAL JOURNAL

become stronger owing to concerns about the safety and environmental impacts of chemicals. Today Trichoderma species are used in a wide range of commercial applications including the biological control of plant diseases (Hjeljord & Tronsmo, 1998; Harman, 2006). Characterization of the antagonistic potential of Trichoderma spp. is the first step in utilizing the full potential of Trichoderma species for specific applications. In vitro screening with different bioassays is an effective and rapid method for identifying strains with antagonistic potential. For the evaluation of the antagonistic potential of different Trichoderma species a range of mechanisms have to be considered.

• Production of antibiotic, volatile and non-volatile chemicals. These substances influence the permeability of cell membranes and result in an efflux of the cytoplasm (Howell, 1998). • Mycoparasitism and excretion of lytic enzymes. The antifungal enzyme system of Trichoderma spp. plays an important role for detection and destroying the host cell wall (Schirmböck et al., 1994). • Competitiveness is based on rapid growth and the production of various asexual generated conidia and chlamydospores (Chet, 1990; Chet et al., 1998). • The ability to promote growth and induce resistance in plants is a mechanism which has also been described for members of this genus (Harman, 2006).

The objective of this investigation was to evaluate the potential of different Trichoderma species as biocontrol agents and to identify a competitive strain that can be used for the treatment of pruning wounds of urban trees against colonization by wood decay fungi. Successful infection and colonization of pruning wounds depends on the ability to overcome host barriers in the wood and to circumvent and/or degrade phenolic compounds (Schwarze et al., 1999, Schwarze & Ferner, 2003). Inonotus hispidus and Polyporus squamosus are both classified as wound parasites and are able to infect and colonize small wounds (McCracken & Toole, 1974, Schwarze et al., 1999). The ability of Ganoderma. adspersum to degrade polyphenolic deposits in reaction zones was recently demonstrated by Schwarze & Ferner (2003). In addition to in vitro studies field experiments were performed with a highly antagonistic Trichoderma strain to enhance and to complete the in vitro investigations (Schubert et al., 2008a).

Materials and Methods

The origin of the Trichoderma isolates and wood decay fungi are provided in Table 1. All cultures were maintained on 2% malt extract agar (MEA) IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN 229 1 1 1 1 1 Isolat-N° 291101.2 086699.2 250593.1 200792.1 271098.1 (S. Schulz.) Donk (Hud.:Fr.) (Hud.:Fr.) Fr. Ganoderma adspersum Polyporus Polyporus squamosus Ganoderma lipsiense (Batsch) Atk. Inonotus hispidus (Bull.:Fr.) Karsten Kretzschmaria deusta (Hoffm.) P.M.D. Mar. Wood Wood decay fungi 3

2 2 2 2

1 IMI 206039/40 15603.1 CBS 351.93 CBS 396.92 CBS 338.93 CBS 126.65 Isolate-N°

) (DRUZHININA (DRUZHININA & KUBICEK, 2005)

T. T. strictipile Karsten Trichoderma isolates and wood decay fungi used in the present study T. T. harzianum/T. polysporum Origin of 1. T. T. fasciculatum synonym ABLE T Trichoderma atroviride Trichoderma atroviride Karsten Trichoderma atroviride Karsten Trichoderma fasciculatum (strictipile) Bissett* Trichoderma virens Miller, Giddens & Foster BINAB TF WP ( ¹ = Isolates from Forest Botany, University of Freiburg ² = Isolates from Centraalbureau voor Schimmelcultures – Netherland ³ = BINAB Bio-Innovation AB, Sweden * = Trichoderma 230 ARBORICULTURAL JOURNAL

at 4(±1)°C. For further studies Petri dishes containing the respective media were inoculated with 0.5cm diameter agar plug, cut from the growing edge of colonies of the isolates and incubated in the dark at 25(±1)°C and 70% relative humidity.

Bioassays for growth and germination rate

The effect of temperature (5, 10, 15, 25, 30°C) and water activity (aw 0.998, 0.955, 0.892) on the growth were detected on two different media types, 2% malt extract agar (MEA) and a modified low nutrient medium (LNA) (Huttermann & Volger, 1973 as cited in Freitag, 1989). The LNA- medium was selected because of its low C:N ratio which is more representative for the nutritional status of wood (Srinivasan et al., 1992). One litre contained

H2O: L-asparagine, 0.013g; KH2PO4, 1g; MgSO4, 0.3g; KCL, 0.5g; FeSO4, 0.01g; MnSO4 4H2O, 0.008g; ZnSO4 6H2O, 0.002g; CaNO3 4H2O, 0.05g; CuSO4, 0.002g; NH4NO3, 0.008g; D-glucose, 5g; and agar, 10g. All Petri dishes (90mm) were inoculated centrally with one 5mm disc of the respective Trichoderma isolate taken from the margin of actively growing cultures and incubated at 25(±1)°C and 70% relative humidity. For each experimental treatment (agar type, aw and temperature) 3 replicates were performed. The growth rate was determined after 24h (mm d–1) by colony diameter measurements, carried out along two perpendicular axes. The water activity of the substrate was controlled by the addition of appropriate weights of the non-ionic solute glycerol prior to autoclaving (Dallyn, 1978). For determination of the germination rate under the specific conditions mentioned above a slight nutrient agar (SNA) was used (Nirenberg, 1981)

which contained H2O: KH2PO4, 1g; KNO3, 1g; MgSO4, 0.5g; KCL, 0.5g; D-glucose, 0.2g; saccharose 0.2g; and agar, 17g per liter. After extracting agar plugs from the growth media a direct observation of conidial behaviour under the light microscope was possible after 6h, 16h, 24h and 48h. To obtain defined conidial suspensions, cultures were flooded with sterile water and filtered twice. Conidia were pelleted by centrifugation (300 -1 revmin ) and resuspended in sterile distilled water to eliminate leached metabolites and nutrients (Naár & Kecskés, 1998). Concentrations of the conidial suspensions were determined and adjusted to approx. 105 cfu per ml.

Inhibitory effects of volatile compounds produced by Trichoderma spp. on wood decay fungi

The effect of the production of volatile organic compounds (VOCs) by Trichoderma isolates was evaluated with the following techniques as described by Dennis & Webster (1971). Trichoderma isolates were centrally IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN 231 inoculated by placing 5mm discs on the two different growth media taken from the margin of 7 days old cultures and incubated at 25(±1)°C and 70% relative humidity for 3 weeks. The top of each Petri dish was replaced with the bottom of the MEA plates and than inoculated centrally (5mm discs) with the wood decay fungi. Plates without Trichoderma spp. were used as control. Eight replicates were maintained for each treatment. The pairs of each Petri dish were fixed and sealed together with paraffin tape and incubated at 25(±1)°C and 70% relative humidity. Colony diameter of the wood decay fungi was measured after an incubation period of 7 days and the inhibition of mycelial growth was calculated.

Dual culture and interaction tests on wood

Mycoparasitism of all Trichoderma isolates against the selected wood decay fungi was assessed in dual culture according to Schubert et al. (2008b). The agar disc method was carried out on two different media types, 2% malt extract agar (MEA) and a modified low nutrient medium (LNA) The LNA- medium was selected because of its low C:N ratio which is more representative of the nutritional status of wood (Srinivasan et al., 1992). Mycelial discs (5mm) were removed from fresh MEA cultures of each of the 5 wood decay fungi and were placed equidistantly at the margin of Petri dishes (90mm) containing the two media types and then incubated at 25(±1)°C and 70% relative humidity for 3-4 days. Thereafter, discs (5mm) were removed from the margins of actively growing 1-week-old cultures of the Trichoderma isolates and placed at opposite sides of the dish, and incubated in the dark at 25(±1)°C and 70% relative humidity for 4 weeks. Petri dishes without antagonistic fungi were used as controls. Six replicates were used for each experiment. Mycoparasitism was observed in samples removed from the interaction zones according to Moussa (2002). Finally the samples were sputter-coated with gold (Cressington Sputter Coater 108auto) and analyzed with a scanning electron microscope (zeiss DSM 940a). In addition interaction tests in wood blocks of Platanus x hispanica were performed as described by Schubert et al. (2008b). For studies of the colonization behaviour, wood blocks were inoculated with two types of conidial suspensions (suspension 1 without additives, suspension 2 with 0.2% glucose and 0.1% urea), placed onto 2-weeks old cultures of the wood decay fungi and incubated in the dark at 25 (±1)°C for 6, 12, 18 weeks. Untreated wood blocks served as controls. Ten replicates were used for each experiment. Analysis of dry weight losses of wood and histological studies of selected wood blocks were performed as described by Schwarze & Fink (1998). 232 ARBORICULTURAL JOURNAL

Statistical analysis

The results of viable counts are expressed as mean ± SE after log transformation. Mean values among treatments were compared by ANOVA and contrast analysis at 5% (P<0.05) and 0.1% (P<0.001) level of significance. Correlations were tested using Spearmen’s correlation coefficient . Non parametric variables were measured using the Kruskall-Wallis test at 5% (P<0.05). All statistical analyses were performed with SPSS 14 statistical software.

Results

Growth and germination rate under different conditions

The influence of temperature, water activity and growth media on mean growth rate and the germination of Trichoderma spp. is provided in Tables 2 and 3. Growth rates of all Trichoderma isolates increased with nutritional status of the media (LNA

was measured at aw 0.892 within one week and at aw 0.955 the growth and germination of all Trichoderma isolates was greatly enhanced. The highest temperature supporting growth was recorded on MEA at 25(±1)°C and on LNA at 30(±1)°C. All Trichoderma isolates showed a

growth and germination optimum at the highest water activity of aw 0.998 and at 25(±1)°C. Significant differences between the Trichoderma isolates were measured. The highest growth rate was measured for T-126.65 (5.6mm d–1), followed by T-Binab (4.6mm d–1) and T-15603.1 (4.3mm d–1) whereas the highest germination rate was measured for T-15603.1 (37.6%), followed by T-351.93 (37.4%) and T-126.65 (32.8%). The lowest growth and germination rates were observed by T-338.93 (P<0.001).

Effect of volatile compounds

The results revealed that after 7 days incubation volatile compounds produced by Trichoderma spp. caused a significant inhibition of growth as indicated in Figure 2 (P<0.05). No influence of the type of growth media on the mean production and effect of VOCs was detected (P<0.05). In addition only three of the Trichoderma isolates (T-15603.1 32.8%; T-Binab 28.3%; T352.93 25.7%) were able to significantly inhibit the growth of the wood decay fungi. The weakest effect was recorded for T.338.93 (8.7%). Among varieties of Trichoderma spp. concerning the production and effect of VOCs, the wood decay fungi differed significantly in their reaction to the VOCs (P<0.001). I. hispidus and G. adspersum showed a strong sensitivity to the VOCs followed by P. squamosus. IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN 233 0 0.998 w a 2.7 ± 0.22 7.0 ± 0.16

12.7 ± 0.25 13.1 ± 0.22 0 30 98.2 ±2.3 48.5 ± 21.8 LNA 0 0 0 0.995 w a ). ± SE. 7.5 ± 0.16 6.9 ± 0.21 –1 0 25 98.8 ±1.5 52.8 ±13.3 ). ± SE 0 0 0 0 0 –1 0.892 w a 0 15 7.7 ±2.8 52.7 ±17.1 Temperature Temperature °C 0 0.998 w a 2.9 ± 0.18 8.9 ± 0.21 0 0

18.9 ± 0.28 12.9 ± 0.17 10 14.8 ±6.3 Trichoderma spp. under different conditions (% d 5 0 0 0 0 0 0 MEA 0.995 w a 6.4 ± 0.15 5.6 ± 0.23 Trichoderma spp. under different conditions (mm d Mean germination rate of 3. w a ABLE Water Water activity 0.892 0.995 T 0.998 0 0 0 0 0 0.892 w a Mean growth rate of the 2.

ABLE 5 10 15 25

T Temperature ºC 30 234 ARBORICULTURAL JOURNAL

Figure 1: A: Oval conidia of T. fasciculatum (bar, 5µm). B: Conidia of T. atroviride FIGUREare spherical1: A: Oval (bar, conidia 5µm). ofC: T. Thick fasciculatum -walled chlamydospore (bar, 5µm). of B : T.Conidia atroviride of 352.93T. atroviride are (bar, spherical 5µm). D: (bar, During 5µm). the C process: Thick-walled of germination chlamydospore conidia absorbed of water T. atroviride and swelled 352.93 (bar,1,5 5µm). fold to D their: During normal dimension the process (bar, 2µm).of germination E: Germination conidia [arrow] absorbed of an inactive water and swelledoccurs 1,5 via fold a germ to their tube resultingnormal indimension the formation (bar, of 2µm). a hypha E: (bar, Germination 2µm). [arrow] of an inactive occurs via a germ tube resulting in the formation of a hypha (bar, 2µm).

I. hispidus G. adspersum G. lipsiense K. deusta P. squamosus Mean

50.0

40.0

30.0 p≤0.05 20.0

10.0

0.0

Radial growth inhibition [%] T-15603.1 T-351.93 T-396.92 T-Binab T-126.65 T-338.93 0 1 2 3 4 5 6 7 -10.0

-20.0

FIGURE 2: Inhibition of radial growth [%] of wood decay fungi by volatile organic compounds (VOC) produced by Trichoderma spp. IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN 235

Evaluation of antagonistic activity on different media

During initial screening of the Trichoderma isolates a variety of reactions were recorded as a result of antagonism. Growth of all wood decay fungi, except P. squamosus, was inhibited by the Trichoderma isolates, although no inhibition zone was observed. Contact between wood decay fungi and Trichoderma isolates occurred but the ability to overgrow and to parasitise the mycelia of the wood decay fungi was highly dependent on the antagonistic potential of each Trichoderma isolate, their nutritional condition and the resistance of the challenged wood decay fungus to antagonism (Table 4 & 5). The growth medium used had a significant effect on the antagonistic activity (P<0.05). The lethal effect of Trichoderma spp. was more prevalent on MEA (85.2%) than on the lower nutrient medium (63.7%). The isolates T-126.65 and T-15603.1 showed the strongest antagonistic potential with a statistically similar performance (P<0.05). T-338.93, however, had the weakest effect (35%). The highest resistance of wood decay fungi to antagonism of Trichoderma spp. was recorded for P. squamosus. Trichoderma isolates were able to parasitise the mycelia of P. squamosus in only 43% of the cases. P. squamosus was not only able to circumvent parasitism but also adapted its hyphal structure, to overgrow the mycelia of the Trichoderma isolates (Figure 3A). During parasitism Trichoderma spp. showed a target-directed growth towards the mycelia of its hosts and an increased formation of conidiophores, phialides and conidia. Formation of apressoria-like structures enabled the hyphae of Trichoderma spp. to attach firmly to the surface of its host mycelia (Figure 3 F&G). Penetration of the mycelia occurred with fine hyphae. The secretion of lytic enzymes and fungicidal substances lead to complete cell wall degradation and efflux of cytoplasm.

Evaluation of antagonistic activity in wood

All wood decay fungi had completely colonized the control wood samples but showed distinctive differences in their potential to decompose the wood. Kretzschmaria deusta caused the highest mean dry weight losses (11.7%) followed by the Ganoderma species (8.2%), whereas P. squamosus (5%) and I. hispidus (3.6%) caused the lowest mean weight losses. Only negligible weight losses were recorded from wood samples that were only treated with Trichoderma spp. (1.6%). Analysis of variance showed that the pre-treatment of wood samples with conidial suspensions of Trichoderma spp. significantly reduced the mean dry weight losses of all wood decay fungi. When data from treatments with conidial suspension 1 and 2 were compared with the untreated control, significant differences (P<0.05) were observed after six weeks 236 ARBORICULTURAL JOURNAL [0] [0] [67] [17] [67] 0 ± 0.0 0 ± 0.0 7 ± 0.09 T-338.93 2.1 ± 0.89 1.8 ± 0.36 [100] [100] [100] [100] [100] T-126.65 3.0 ± 0.89 2.3 ± 0.54 2.9 ± 0.56 2.9 ± 1.45 2.9 ± 0.14 0 [83] [83] [100] [100] [100] T-Binab 2.3 ±0.77 2.4 ± 0.67 1.9 ± 0.23 2.8 ± 0.42 1.7 ± 0.11 MEA [83] [67] [83] [100] [100] T-396.92 2.4 ± 0.65 2.9 ± 0.12 1.9 ± 0.21 2.6 ± 0.33 2.4 ± 0.66 Trichoderma spp. on MEA. ± SE. Trichoderma spp. to eliminate the wood decay fungi during the incubation time of 4 weeks. [67] [100] [100] [100] [100] 2.4 1.23 T-351.93 2.9 ± 0.98 2.5 ± 0.18 2.8 ± 1.31 1.8 ± 1.05 b ±0.10 ±0.11 ±0.36 ±0.56 ± 0.14 [83] [83] a [100] [100] [100] -15603.1 3.0 2.3 3.0 2.2 T 2.2 Classification of the degree of mycoparasitism of different 4. ABLE = Lethal effect as percent was measured by the ability of = Following system was used to classify the rate of mycoparasitism: 0 = no overgrowth; 1 = slow overgrowth; 2 = fast overgrowth; 3 = very fast over-

G. adspersum T I. hispidus G. lipsiense K. deusta P. squamosus a b

growth growth and deadlock of the wood decay fungi within 4 weeks.

IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN 237 [0] [0] [67] [17] [33] 0 ± 0.0 0 ± 0.0 T-338.93 1.9 ± 0.10 0.8 ± 0.14 1.3 ± 0.12 [83] [83] [83] [100] [100] T-126.65 1.9 ± 1.32 1.8 ± 0.89 1.8 ± 0.14 3.0 ± 1.20 2.3 ± 1.01 [0] [0] [100] [100] [100] T-Binab 0 ± 0.0 0 ± 0.0 1.9 ±0.09 2.1 ± 0.17 2.7 ± 0.19 A LN [83] [17] [17] [100] [100] T-396.92 2.2 ± 0.73 2.4 ± 0.44 0.9 ± 0.69 2.3 ± 0.74 0.6 ± 0.75 Trichoderma spp. on LNA. ± SE. Trichoderma spp. to eliminate the wood decay fungi during the incubation time of 4 weeks. [0] [33] [100] [100] [100] 0 ± 0.0 T-351.93 2.4 ± 0.89 2.2 ± 0.82 1.3 ± 0.07 2.3 ± 0.11 b ± 0.07 ± 0.33 ± 0.33 [0] ± 0.0 ± 0.43 [17] [83] a [100] [100] -15603.1 0 T 2.3 1.1 2.9 1.9 Classification of the degree of mycoparasitism of different 5. ABLE = Lethal effect as percent was measured by the ability of = Following system was used to classify the rate of mycoparasitism: 0 = no overgrowth; 1 = slow overgrowth; 2 = fast overgrowth; 3 = very fast over-

G. adspersum T I. hispidus G. lipsiense K. deusta P. squamosus a b

growth growth and deadlock of the wood decay fungi within 4 weeks.

238 ARBORICULTURAL JOURNAL

FIGURE 3: A: Polyporus squamosus was not only able to circumvent parasitism but also formed mycelial strands to overgrow the mycelia of Trichoderma isolates B: Specific features e.g. clamp connections, typical for basidiomycetes, served to distinguish the mycelia of the wood decay fungi from that of Trichoderma (bar, 1µm). C: The hyphae of T-396.92 grew target-oriented and branched to increase the contact area with the host mycelium (bar, 5µm). D + E: After initial contact an increase in conidiophore (bar, 10µm) and conidial formation (bar, 2µm) by Trichoderma spp. was observed. F + G: Adhesion of mycelia of wood decay fungi occurred with appressoria-like structures (<1 µm) of Trichoderma spp. (bar, 1 µm). The process of parasitism was completed after cell wall degradation and efflux of cytoplasm by secretion of lytic enzymes and fungicidal substances. of incubation. After 12 and 18 weeks the differences increased and were highly significant (P<0.001). The additives used in conidial suspension 2 enhanced significantly the establishment of Trichoderma spp. on wood and the protective effect (P<0.05). The reduction of wood decay by the Trichoderma isolates is illustrated in Table 6 & 7. Contrast analysis of Trichoderma isolates revealed significant P ( <0.05) differences between the species and strains. T-15603.1 induced the greatest reduction in dry weight losses followed by isolates T-351.93 and T-126.65. The isolate T-396.92 and Binab were less effective during the three incubation periods (P<0.05). T-338.93 induced the least reduction in weight losses (P<0.05). IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN 239

FIGURE 4: A-C: Wood samples were completely colonized by Ganoderma adspersum (A), Kretzschmaria deusta (B) and Polyporus squamosus (C) D: T-15603.1 inhibited colonization by Ganoderma adspersum after 18 weeks of incubation. E: Wood pretreated with T-15603 completely inhibited colonization by Kretzschmaria deusta. F: Despite pre-treatment with Trichoderma spp., Polyporus squamosus revealed a high resistance to antagonism and was able to colonize and degrade the wood.

Despite the treatment of wood samples with conidial suspensions of Trichoderma spp., P. squamosus showed a high resistance to antagonism and caused substantial dry weight losses. All other fungi showed similar performance (P<0.05) and sensitivity against Trichoderma spp. Histological analysis supported the results of the macroscopic observations and dry weight loss measurements (Figure 5). High dry weight losses were recorded from control samples by all wood decay fungi, but samples pretreated with Trichoderma spp. did not reveal typical signs of cell wall degradation. Ganoderma spp. and P. squamosus caused a typical white rot i.e. simultaneous rot and selective delignification. Inonotus hispidus showed dual modes of action, i.e. a simultaneous rot and a soft rot, whereas K. deusta exclusively caused a soft rot. An alternative degradation pattern was observed for P. squamosus on wood pre-treated with Trichoderma. Hyphae predominantly grew within intercellular spaces and subsequently degraded the cell wall in close proximity to the hyphae. In wood specimens exclusively inoculated with Trichoderma spp. no signs of cell wall degradation were apparent. Hyphae grew predominantly within the parenchyma cells and growth to adjacent cells occurred exclusively via pits. 240 ARBORICULTURAL JOURNAL n.s n.s n.s n.s n.s 2.55 1.79 18 w –6.64 –5.36 75.35** –0.51

n.s 12 w 43.29* 43.29* 67.97* 58.01* 56.93* –21.65

Polyporus n.s 6 w 41.46* 39.84n.s 55.69* 28.46 55.28* 43.09*

n.s 18 w 78.82** 78.11** 76.19** 30.54 78.76** 78.98**

n.s 12 w 83.10** 83.10** 56.62* 69.45** 71.49** –11.20 squamosus n.s Kretzschmaria Trichoderma spp. 6 w 80.88** 81.17** 74.60** 29.64 75.91** 75.77** P < 0.05); **=high significant ( P < 0.001); n.s = not significant 18 w 73.51** 75.72** 83.66** 80.72** 86.24** 85.80** Suspension 1 85.04** 85.47** 70.66** 72.79** 88.75** 88.18** 12 w deusta Conidial Ganoderma lipsiense 6 w 84.68** 83.83** 68.30* 71.91** 85.53** 86.81** 18 w 74.31** 74.98** 84.29** 81.46** 88.40** 88.27**

12 w 60.33* 60.16* 69.27** 69.76** 75.77** 76.59** 6 w Ganoderma adspersum 60.82* 60.82* 57.39* 49.14* 59.79* 60.82* 18 w 19.75n.s 17.59n.s 42.73* 49.01* 61.58* 62.30*

n.s hispidus

54.78* 53.33* 48.12* 34.49 58.84* 60.00* 12 w

n.s n.s n.s n.s Inonotus w 6 10.78 62.28* 64.07* 22.75 20.96 14.37 Reduction (%) of the wood decay (wood weight loss) by applying conidial suspension 1 of 6. 0.05) ≥ ABLE -338.93 T

T T-351.93 T-396.92 T-Binab T-126.65 Significant reduction of thewood decay (wood weight loss) is indicated by * = significant ( T-15603.1 ( P

IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN 241 n.s n.s n.s n.s n.s 5.75 18 w 17.75 17.11 71.21** 16.09 14.81

n.s 12 w 46.75* 47.19* 48.37* –6.06 61.04* 61.04* = not significant

n.s Polyporus n.s squamosus 6 w 33.433 62.20* 62.60* 70.33* 63.41* 64.23*

n.s P < 0.001); 18 w 35.30 78.22** 78.27** 62.83** 82.05** 82.38**

n.s 12 w 83.30** 83.50** 53.14* 76.78** 76.78** –7.33 deusta * Kretzschmaria Trichoderma spp. 6 w 89.93** 89.34** 74.16** 44.38 85.69** 85.69** 18 w P < 0.05); ** = high significant ( 79.40** 79.62** 72.22** 83.96** 86.98** 87.12**

Suspension 2 90.17** 89.60** 68.94** 77.64** 90.17** 89.03** 12 w Conidial Ganoderma lipsiense 6 w 86.81** 86.91** 81.49* 73.19** 87.23** 86.38** 18 w 81.25** 81.29** 66.83** 88.81** 91.10** 90.96**

12 w 61.46* 61.46* 37.46** 77.89** 86.83** 87.64** Ganoderma adspersum 6 w 61.51* 61.86* 67.70* 68.73* 78.35* 78.35* 18 w 52.42* 50.27* 53.62* 69.48* 73.25* 73.43*

n.s hispidus

59.71* 59.13* 19.16* 59.13 67.25* 68.12* 12 w

n.s n.s n.s n.s Inonotus 6 w 52.10 63.47* 64.67* 25.15 25.75 43.71 Reduction (%) of the wood decay (wood weight loss) by applying conidial suspension 2 of

7. ABLE T-338.93

T T-15603.1 T-351.93 T-396.92 T-Binab T-126.65 Significant reduction of thewood decay (wood weight loss) is indicated by * = significant (

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FIGURE 5. A: Progressive degradation (simultaneous rot) of the secondary wall by Ganoderma adspersum (bar, 5µm). B: Wood pre-treated with Trichoderma spp. did not show typical signs of cell wall degradation (bar, 50µm). C: In pre-treated wood the hyphae of Polyporus squamosus predominantly grew within intercellular spaces (yellow arrow) and were rarely observed in the cell lumina (red arrow, bar, 2 µm). D: Hyphae of Trichoderma spp. were generally located in the parenchyma cells and growth to adjacent cells occurred exclusively via pits (bar, 5µm).

Discussion

Growth and germination under specific conditions

Competitiveness of Trichoderma spp. is based on rapid growth and germination i.e. a decisive feature for antagonism (Chet, 1990; Chet et al., 1998; Hjeljord & Tronsmo, 1998). Physical as well as chemical factors influence growth and germination, therefore knowledge of the optimal conditions for growth as well as the influence of suboptimal ecological factors on the antagonist is essential for a successful application in field (Papavizas, 1985; Hjeljord & Tronsmo, 1998; Kredics et al. 2003). In this study, growth of the Trichoderma isolates corresponded strongly to the ecological factors tested. All Trichoderma isolates showed an optimum growth and germination under an optimized nutritional status, at a mean temperature of

20-25°C and a high water activity of aw 0.998. At lower temperatures and water activity the growth and germination was significantly reduced to such IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN 243 a point that at 5°C and aw 0.892 no growth and germination was recorded after one week. These observations confirm results obtained by Kredics et al. (2000; 2003) and Lupo et al. (2002), who classified Trichoderma spp. as a mesophilic organism with a low xerotolerance. The prognosis of the behaviour of Trichoderma spp. under specific conditions is complicated, however, due to the mutual effect of the environmental parameters (Harman, 2006).

Inhibitory effect of volatile organic compounds

Antibiosis in Trichoderma was recognized and initially described by Weindling (1934) and is defined as the production of secondary metabolites, that have an antimicrobial effect even at low concentrations (Howell, 1998). In addition to several other substances (aldehydes, ketones, peptides, etc.), 6-pentyl-α-pyrone (6-PP) is basically responsible for the antifungal effect of the volatile organic compounds (Scarselletti & Faull, 1994; Wheatley et al., 1997; Cooney et al., 1997a,b; Galindo et al., 2004). Srinivasan et al. (1992) reported that the composition of the growth media had a significant influence on the production of VOCs and thereby on the levels of inhibition of wood decay fungi by Trichoderma spp. However, the results of the present work contrast with these observations, because no significant influence of the growth media type on the mean production and effect of the VOCs could be measured. Significant differences were only detected between different Trichoderma isolates. The mean inhibition of 21.4% was low and additionally only 3 of the Trichoderma isolates were able to achieve a significant inhibitory effect. This could be an indication for a sub-item of antibiosis concerning the antagonism of Trichoderma against wood decay fungi.

Dual culture and interaction tests on wood

In the dual culture tests, hyphal contact between Trichoderma spp. and the wood decay fungi was observed for all host/pathogen combinations. However, not all strains of Trichoderma were able to overgrow and parasitize the mycelia of wood decay fungi. The antagonistic potential of Trichoderma isolates was determined by the nutritional condition of the antagonists and the susceptibility of the wood decay fungi. Previous studies have demonstrated that before mycelia of fungi interact, Trichoderma spp. produces low quantities of extracellular exochitinases (Kullnig et al., 2000; Brunner et al., 2003). The diffusion of these enzymes dissolves cell fragments of host cells. These cell fragments in turn induce the production of further enzymes and trigger a cascade of physiological changes, stimulating rapid and directed growth of Trichoderma spp. (Zeilinger et al., 1999). In 244 ARBORICULTURAL JOURNAL

the present, work not only directed growth, but also an induced hyphal branching of Trichoderma spp. was observed. Previous in vitro studies have demonstrated that due to chemotropism hyphae of Trichoderma harzianum can grow and branch directly towards the host (Chet, 1987). In order to increase the antagonistic potential of Trichoderma spp. for in vitro tests, interaction studies were performed on wood samples. After 18 weeks incubation, treatment with Trichoderma spp. failed to completely inhibit decomposition, as measured by dry weight loss. This may partly be explained by the degradation of readily accessible carbohydrates by Trichoderma spp. within parenchyma cells and pits (Kubicek-Pranz, 1998). A further explanation may be related to the experimental design. Thus wood samples were treated with conidial suspensions of Trichoderma and then inoculated with an artificially high inoculum of wood decay fungi. The inoculum potential in turn is crucial for the invasiveness of pathogens (Redfern & Filip, 1991). Nevertheless a significant reduction in dry weight losses was induced after pre-treatment of the wood with different conidial suspensions of Trichoderma spp. The additives (glucose, urea) stimulated rapid colonization of the wood samples by Trichoderma spp. and in their presence the protective effect was increased (Hjeljord et al., 2001). In dual culture tests as well as in interaction tests, significant differences between the species and strains of Trichoderma spp. were evident. Thus, T-15603.1, T-351.93 and T-126.65 showed a high antagonistic potential. By contrast, the antagonistic potential of T-396.92, the commercial product Binab and especially, T-338.93 was limited. The different antagonistic activities of the Trichoderma strains and the fixed test conditions and the challenged wood decay fungi proved to be decisive factors for the laboratory studies. In vitro tests showed that Polyporus squamosus is resistant to Trichoderma spp. Former studies by Shield & Atwell (1963) and Highley (1997) demonstrated, without further explanation, that Trichoderma spp. have a limited effect on Polyporus adustus (Wflld.) Fr. and Gleophyllum trabeum (Pers. ex Fr.) Murr. The mechanism that allowed P. squamosus to circumvent parasitism in dual culture tests has not been previously described. Formation of hyphal strands by P. squamosus was observed after initial contact with hyphae of Trichoderma spp. The individual hyphae merged to form compact strands. Thus the surface size was reduced and subsequently the area of hyphae exposed to parasitism. Hyphal strands appeared to be more resistant and enabled P. squamosus to readily overgrow the mycelium of Trichoderma spp. The resistance of P. squamosus hyphae could be due to increased melanin content within the cell wall. Duffy et al. (2003) described melanin as a primary defence system in all organisms and that resistance of pathogenic fungi to microbial lysis is positively correlated with the melanin content in hyphae. During the interaction studies, P. squamosus showed specific growth behaviour. Hyphae IN VITRO SCREENING OF AN ANTAGONISTIC TRICHODERMA STRAIN 245 of P. squamosus were predominantly located within the intercellular spaces escaping mycoparasitism by Trichoderma spp. The latter growth pattern has been previously described for Meripilus giganteus (Pers. ex Fr.) Karsten (Schwarze and Fink, 1998). Thus the basidiomycete was apparently able to circumvent polyphenolic impedances within the reaction zone of beech, Fagus sylvatica L. by growing through intercellular spaces. The limited effect of the commercial product Binab TF WP and the differences in resistance among wood decay fungi in the present study demonstrates the importance of screening Trichoderma species for the specific niche where they are envisaged to be applied i.e. increasing target specificity. The in vitro screening of the antagonistic potential used in this work allowed a systematic investigation of several Trichoderma isolates including specific ecological factors and a selection of one effective strain. However, positive results obtained from in vitro studies are only indicative, as experimental conditions do not take all ecological and endemic factors into account. For this reason field studies are essential to test the selected competitive biocontrol agent under field conditions. The observations and results of field studies with the selected Trichoderma strain 15603.1 are reported in Schubert et al. (2008a).

Acknowledgments

We would like to thank Mrs. Gack, (Institute for Biology I, University of Freiburg, Germany) and Mr. Kiesel (Institute for Forest zoological Institute; University of Freiburg) for their assistance in scanning electron microscopy (SEM). We would also like to thank Mr. Robert Dietrich and Mr. Karl Merz (Institute for Forest Botany, University of Freiburg) for preparing the wood samples and for technical support. Finally the financial support from the Ev. Studienwerk Villigst e.V. is gratefully acknowledged.

References

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Schubert, M., Fink, S. & Schwarze, F.W.M.R. (2008a) Field experiments to evaluate the application of Trichoderma strain (T-15603.1) for biological control of wood decay fungi in trees. Part II. Arboric. Journal, 31, 249– 268. Schubert, M., Fink, S. & Schwarze, F.W.M.R. (2008b) Evaluation of Trichoderma spp. as a biocontrol agent against wood decay fungi in urban trees. Biological control 45, 111–123. Schwarze, F.W.M.R. & Ferner, D. (2003) Ganoderma on trees – Differentiation of species and studies of invasiveness. Arboricultural Journal, 27, 59–77. Schwarze, F.W.M.R. & Fink, S. (1998) Host and cell type affect the mode of degradation by Meripilus giganteus. New Phytologist, 139, 721–731. Schwarze, F.W.M.R., Engels, J. & Mattheck, C. (1999) Fungal strategies of wood decay in trees. Springer Verlag, Berlin, Heidelberg, New-York. Shields, J.K. & Atwell, E.A. (1963) Effect of mold, Trichoderma viride on decay of birch by four storage rot fungi. For. Prod. J., 13, 262–265. Srinivasan, U., Staines, H. J. & Bruce, A. (1992) Influence of media type on antagonistic modes of Trichoderma spp. against wood decay basidiomycetes. Material und Organismen, 27, 301321. Weindling, R. (1932) Trichoderma lignorum as a parasite of other soil fungi. Phytopathol., 22, 837-845. Weindling, R. (1934) Studies on lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other soil fungi. Phytopathology, 24, 1153–1179. Wheatley, R.E., Hackett, C., Bruce, A. & Kundzewicz, A. (1997) Effect of substrate composition on the production of volatile organic compounds from Trichoderma spp. inhibitory to wood decay fungi. Intern. Biodet. & Biodeg., 39, 199–205. Zeilinger, S., Galhaup, C., Payer, K., Woo, S.L., Mach, R.L.; Fekete, C., Lorito, M. & Kubicek, C.P. (1999) Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host. Fungal Genet. Biol., 26, 131–140. Arboricultural Journal 2008, Vol. 31, pp. 249–268 © AB Academic Publishers 2008 Printed in Great Britain

FIELD EXPERIMENTS TO EVALUATE THE APPLICATION OF TRICHODERMA STRAIN (T-15603.1) FOR BIOLOGICAL CONTROL OF WOOD DECAY FUNGI IN TREES

Mark Schubert1,2 Siegfried Fink2, Francis W.M.R. Schwarze1,2

Field experiments were carried out at different locations and on hosts with T-15603.1, a Trichoderma strain for biological control of wood decay fungi. The objective of the studies was to monitor and optimize conditions for colonization of the antagonist, its survival in time and space and to improve its effectiveness as wound treatment method. A total of 159 angiosperm trees and 1431 wounds from six different species (Platanus x hispanica, Acer pseudoplatanus, Tilia platyphyllos, Populus nigra, Quercus rubra, Robinia pseudoacacia) were treated with different conidial suspensions of T-15603.1. In comparison to untreated control wounds, T-15603.1 significantly suppressed growth (82.3%) of wounds colonised by three basidiomycetes Ganoderma adspersum, Inonotus hispidus and Polyporus squamosus (P<0.001). Monitoring results with RAPD-PCR showed that spore suspensions applied in a humidity storing gel as a carrier suspension significantly increased the germination rate and therefore colonization of the wound surface by T-15603.1 (P<0.001). Interpretation and characterization of the isolated microorganisms such as diversity and succession were analyzed using diversity indices. The results demonstrate that T-15603.1 can be successfully applied as a biological wound treatment against wood decay fungi of urban trees.

Introduction

Since the pioneering work of RISHBETH (1961), interest in the genus Trichoderma as competitive antagonists against important pathogens of forest trees such as Heterobasidion annosum (Fr.:Fr.) Bref. and Armillaria spp. has increased steadily (HOLDENRIEDER, 1984; NICOLOTTI et al., 1999; FOX, 2003). GROSCLAUDE et al. (1973) and CORKE (1980) successfully applied T. viride Pers. Ex S.F. Grey as wound treatment method for fruit trees against the wound parasite Chondrostereum purpureum (Pers. ex Fr.) Fr.

1Swiss Federal Laboratories for Materials Testing and Research (EMPA), Wood Laboratory, Lerchenfeldstrasse 5, CH-9014 St. Gallen, Switzerland 2Albert-Ludwigs-Universität Freiburg, Professur für Forstbotanik, Bertoldstrasse 17, D-79085

249 250 ARBORICULTURAL JOURNAL

In studies by POTTLE & SHIGO (1975) and POTTLE et al. (1977) Trichoderma harzianum Rifai treatment of wounds of Acer rubrum L. with Trichoderma inhibited colonization even after 21 months. Currently there is little knowledge on the effectiveness of biological control of wood decay fungi which colonize wounds of urban trees. On urban sites air temperature is usually higher than in forests. Moreover, soil permeability is often low due to compaction causing additional stress to the root system of trees. These factors, together with repeated pruning operations, can, alone or in combination, suppress tree health and in the presence of pathogens, trigger disease. It is also interesting to note that most wood decay fungi that colonize urban trees via pruning wounds rarely occur on forest trees. The unique ecological conditions of urban sites i.e. the number of pruning wounds inflicted and the low incidence of antagonists due to lack of organic material on root plates may promote infection by wound decay fungi. In vitro screening is the first step for utilizing the full potential of Trichoderma species. In a previous study we identified and selected a highly competitive Trichoderma atroviride strain (T-15603.1) in several in vitro tests against five basidiomycetes Ganoderma adspersum (S. Schulz.) Donk, Ganoderma lipsiense (Batsch) Atk., Inonotus hispidus (Bull.:Fr.), Polyporus squamosus (Hud.:Fr.) Fr. and one ascomycete Kretzschmaria deusta (Hoffm.) P.M.D. Martin (SCHUBERT et al., 2008). The objective of the present work was to evaluate the potential of the highly competitive strain (T-15603.1) in field experiments for a biological treatment method of pruning wounds on urban trees against colonization by wound decay fungi.

Materials and Methods

Field experiments were undertaken on two urban sites in Freiburg-Lehen, Baden-Württemberg (278m above sea level, 10°C; precipitation approx 1000mm per annum), Germany and Ludwigshafen, Rheinland-Pfalz (96m above sea level, 9.2°C; precipitation approx 500mm per annum), Germany. A total of 159 trees were pruned (Table 1). A total of 1431 wounds with a mean diameter of 6.4cm were treated in July and August 2003 with three different types of conidial suspensions of T-15603.1:

(1) Suspension (CFU: 105/ml) (2) Suspension (CFU: 105/ml + 0.2% D-glucose + 0.1% urea) (3) Suspension (CFU: 105/ml + 0.2% D-glucose + 0.1% urea + 0.4% sodiumpolyacrylate, a component of the product Luquasorb 1030, BASF AG, Ludwigshafen, Germany. FIELD EXPERIMENTS TO EVALUATE THE APPLICATION OF TRICHODERMA STRAIN 251

Conidial suspensions 1 and 2 were sprayed onto the wound surface; the third was applied with a conventional brush (Figure 1). Untreated wounds served as controls. In addition, artificial inoculation tests were performed on selected trees to evaluate the preventive effect of the Trichoderma- strain T-15603.1 against colonization by wood decay fungi (Table 1). Tree wounds were treated with the antagonist as described above and three weeks after treatment were inoculated with three basidiomycetes: I. hispidus, G. adspersum and P. squamosus. The determination of the infection rate of the latter fungi was based on inoculated wounds from which the respective fungus could be re-isolated (GADGIL & BAWDEN, 1982). The biological treatment efficacy [E] of T-15603.1 was measured according the formula of ABBOTT (1925):

(Ix[%] – IT [%]) * 100 E = Ix[%]

Ix = Infection rate without treatment and IT = Infection rate after application of T-15603.1.

Re-isolation and analysis

Establishment in the wood substrate and the persistence of T-15603.1 were monitored with standardized re-isolations after 2, 8, 12, 18, 24 and 30 months. Wood samples (20 × 10 × 5mm) were extracted from the centre and peripheral regions of the wound with a sterile chisel. In the laboratory, the surface of the samples were sterilized with hydrogen peroxide, divided into 3 subsamples and placed onto Petri dishes containing malt extract agar (MEA), TSM and T-MEA. After thirty months sections of the treated wounds were extracted, bisected radially and re-isolations were performed along a gradient and in wood to a depth of 1, 3 and 5cm. Parameters like

TABLE 1. Data on inoculated tree species and sites used in the present study.

Tree species Location Quantity Inoculation

Platanus x hispanica Münchh. Ludwigshafen 54 – Acer pseudoplatanus L. Ludwigshafen/Freiburg-Lehen 40 P1,P2,P3 Tilia platyphyllos Scop. Freiburg-Lehen 24 P1,P2,P3 Populus nigra L. Ludwigshafen / Freiburg-Lehen 16 P1,P2,P3 Quercus rubra L. Freiburg-Lehen 16 P1,P2,P3 Robinia pseudoacacia L. Ludwigshafen 9 –

P1 = Inonotus hispidus P2 = Ganoderma adspersum P3 = Polyporus squamosus 252 ARBORICULTURAL JOURNAL

A B

FIGURE 1. Application of T-15603.1 on fresh pruning wounds. A: Conidial suspensions 1 and 2 were sprayed onto the wound surface. B: The third type of spore suspension containing the carrier substance sodiumpolyacrylate was applied with a conventional brush.

wound occlusion and discoloration were recorded and measured according to LIESE et al. (1988) and METZLER (1997) (Figure 2 A&B). Additional data such as site, orientation, climate data, ratio of sap- and heartwood and wound dimensions were also recorded periodically. A statistical interpretation and characterization of the isolated fungal populations, i.e. diversity and succession, was made using diversity indices (MARIA & SRIDHAR, 2002). Shannon Index H’:

S ni H'   (* pipi )Log Pi  i1 N S = number of different species or groups; N = total numbers of individuals; ni = number of individuals classified as ith species or group; pi = proportion of total samples classified as ith species. The Shannon Equitability E is the ratio between the observed species diversity (H’) and the maximum species diversity (Hmax) calculated as:

H' H' E = + H max log (S)

E = equitability (range 0-1, max. value when all species are equally

abundant); H = observed species diversity; Hmax = species diversity under conditions of maximal evenness. FIELD EXPERIMENTS TO EVALUATE THE APPLICATION OF TRICHODERMA STRAIN 253

A B

Discolouration a h b S r b

1 3 5 Marrow

 bh )**( 100C%  4 100*  *r 2

FIGURE 2. A: Re-isolations of fungi were performed along a gradient (1, 3, 5cm) and from the sapwood [S]. The extracted wood samples were incubated on MEA, T-MEA and TSM. Discolouration was measured in the axial and radial direction (a, b). The wood structure (reaction- and barrier zone) were additionally analysed in histological studies B: Wound occlusion was expressed by measuring the wound wood coefficient [C%].

Micro-morphological and molecular identification

Identification of the obtained isolates was undertaken both with traditional methods based on macro- and micro-morphological features and with RAPD-PCR as described by CASTLE et al. (1998). Morphological observations were made from cultures grown on 2% MEA at 25 (±1)°C under ambient laboratory conditions and diffuse daylight, relying on the microscopic characteristics of conidiophores. Identity was established using several diagnostic keys (RIFAI, 1969; BISSETT, 1984, 1991a,b,c, 1992; GAMS & BISSETT, 1998). For DNA isolation, fungal cultures were grown at room temperature on 2% MEA. Mycelia were harvested by filtration through a piece of filter paper and washed with distilled water. The samples were immediately frozen in liquid nitrogen and lyophilized. DNA extraction was carried out with a PhytoPure DNA extraction kit (Amersham Life Science, Buckinghamshire, UK) according to the manufacturer’s instructions. The DNA concentration was determined by measuring the absorbance at 260nm (Beckmann DU 7500i, Munich). RAPD characters were developed with primer 1 (5'-CACGGCGAGT-3') and primer 2 (5'-CTGTCCAGCA-3') (Carl Roth GmbH). The reaction volume of 50 μl contained 26.5 μl distilled water, 5μl Mg-reaction buffer, 5μl 25mM MgCl2, 5μl Primer 1 and 2, 1μl 10mM dNTP mix, 0.5μl Taq DNA polymerase, 2μl DNA. PCR amplification was performed in a Eppendorf Mastercycler 254 ARBORICULTURAL JOURNAL

Gradient (Eppendorf AG, Hamburg) programmed for 1 cycle of initial denaturation for 3 min. at 94°C, 7min at 74°C, followed by 39 cycles of 1 min. at 94°C (denaturation), 1 min. at 37°C (low stringency annealing), 2 min. at 72°C (elongation) and with a final extension step for 10 min. at 72°C. The DNA samples were separated for analysis by electrophoresis on 1 to 2% agarose gels (1× Tris-borate EDTA buffer). The fragments were visualized by staining with 0.5μg of ethidium bromide per ml and UV illumination.

Results

Monitoring results revealed successful re-isolation of the Trichoderma strain T-15603.1 from treated wounds thirty months after application (Figure 3). The establishment and colonization of T-15603.1 on the wound surface was highly dependent on the medium in which the conidia were suspended (Table 2). Table 3 shows contrast analysis of the different conidial suspensions tested. The highest mean re-isolation rate of 74.8% was recorded after application with conidial suspension 3 (P<0.05). Wounds were more weakly colonized after treatment with suspensions 1 (32.5%) and 2 (29.1%). Re-isolation rates from wounds treated with suspension 3 were significantly higher than from control wounds (P<0.001). After twelve months no differences between wound treatment with suspension 2 and control wounds were found. After thirty months no differences were observed between the re-isolation rates of suspension 1 and the controls. Correlation

1 2 3 4 5 6 7 8

FIGURE 3. RAPD-PCR: (1) = The applied strain T-15603.1 (reference). (2-6) = Trichoderma-isolates from the treated pruning wounds after 30 months. (7) = Trichoderma virens. (8) = Trichoderma fasciculatum. FIELD EXPERIMENTS TO EVALUATE THE APPLICATION OF TRICHODERMA STRAIN 255

TABLE 2. Re-isolation rate of T-15603.1 (%) from pruning wounds in relation to different conidial suspensions applied.

2 8 12 18 24 30 Mean months months months months months months

Control1 3.3 4.5 4.8 4.7 5 4.8 4.5 Suspension 1a 31.5 52.5 30 27 27.8 26.4 32.5 Suspension 2b 50 32.5 25 22.5 23 21.4 29.1 Suspension 3c 81 82.5 72.5 70 72 71 74.8 a = Conidial suspension (CFU 105 ml–1) without additives b = Conidial suspension (CFU 105 ml–1) with 0.1% urea; 0.2% glucose c = Conidial suspension (CFU 105 ml–1) with 0.1% urea; 0.2% glucose and 0.4% sodiumpolyacrylate

TABLE 3. Contrast analysis of the re-isolation rate of T-15603.1 from pruning wounds in relation to the different conidial suspensions applied.

Treatment method of conidial Time [months] suspensions

2 8 12 18 24 30

Suspension 1a vs. Control 0.038 0.001 0.042 0.045 0.048 0.056 Suspension 2b vs. Control 0.001 0.046 0.052 0.058 0.078 0.167 Suspension 3c vs. Control 0.001 0.001 0.000 0.000 0.000 0.000 Suspension 2b vs. Suspension 1a 0.055 0.061 0.089 0.092 0.104 0.096 Suspension 3c vs. Suspension 1a 0.000 0.019 0.001 0.001 0.001 0.001 Suspension 3c vs. Suspension 2b 0.014 0.000 0.001 0.001 0.000 0.000 a = Conidial suspension (CFU 105 ml–1) without additives b = Conidial suspension (CFU 105 ml–1) with 0.1% urea; 0.2% glucose c = Conidial suspension (CFU 105 ml–1) with 0.1% urea; 0.2% glucose and 0.4% sodiumpolyacrylate F-test of significance P<0.05, P value is denoted, not significant atP ≥ 0.05 analysis according to Spearman’s rho showed a positive relationship between precipitation and re-isolation rate (=0.829, P<0.05) and a negative correlation between wound dimension and re-isolation rate (=0.714, P<0.05). In comparison to re-isolations from sapwood wounds (69.7%), an increase in wound size and proportion of heartwood resulted in a lower re-isolation rate (30.3%). No significant correlation between re-isolation rate and temperature was detected (=0.371, P<0.05), nor between re- isolation rate and tree species (=0.276, P<0.05). Thus the applied conidial suspensions, the moisture content of the wood and the ratio of sap- and heartwood significantly (P<0.05) influenced the re-isolation rate of T-15603.1 (Figure 4). T-15603.1 was re-isolated from the sapwood (69.7%) more often than from the heartwood (30.4%). After two months the re-isolation rate of T-15603.1 was similar from the sap- and 256 ARBORICULTURAL JOURNAL

heartwood, but after eight months a significant difference between sap- and heartwood was detectable (P<0.05). This trend persisted and after eighteen months the differences between re-isolation rates from sap-and heartwood were highly significant (P<0.001). Isolations from different wood depths showed that the highest re-isolation rate was obtained from the surface of the wounds (46.1%). Re-isolation of T-15603.1 decreased significantly (P<0.05) in deeper wood regions and at a depth of 5cm T-15603.1 was not detectable (Table 4). Analysis of wood discolouration resulting from pruning wounds showed that the maximum amount of wood discolouration was detected in Populus nigra (20.4cm2), followed by Tilia platyphyllos (16.74 cm2) and Acer pseudoplatanus (11.8cm2). The smallest amount of wood discolouration was observed in Quercus rubra (9.5cm2). In addition a positive correlation (=0.721) was observed for wood discolouration and wound dimension. Moreover the treatment method had a considerable effect on the expansion

TABLE 4. Re-isolation rate of T-15603.1 in different wood depths (%).

Suspension 1 Suspension 2 Suspension 3 Mean

1 cm 30a 36a 71.4b 46.1 3 cm 3.5a 2.3a 7b 13.5 5 cm 0a 0a 0a 0

Letters denote significant differences after contrasts analysis P <0.05

Sapwood Heartwood

** * ** 24** 19.2 * * 29.9 26.5 30.4 37.6 44.9

76 80.8 70.1 73.5 69.7 62.4 55.1 Ratio of sap- and heartwood

2 months 8 months 12 months 18 months 24 months 30 months Mean Time [m]

FIGURE 4. Re-isolation rate of T-15603.1 from the sap- and heartwood (%) of wounds. Asterixes indicate significant differences *=significant (P<0.05); **=highly significant (P<0.001). FIELD EXPERIMENTS TO EVALUATE THE APPLICATION OF TRICHODERMA STRAIN 257 of wood discolouration. Development of discoloured wood was more extensive from wounds inoculated with the wood decay fungi and without pre-treatment than wounds treated with T-15603.1 (Figure 5). Thus pre- treatment significantly (P<0.05) reduced development of dysfunctional wood. No differences in wood discolouration were recorded for the different wound decay fungi (P<0.05). After thirty months the woundwood coefficient (wound occlusion) was measured and analysed. A mean woundwood coefficient of 71.3% was determined (ccoef. 71.3% of the wound was occluded). Figure 6 provides statistical analysis of the woundwood development of different tree species.

The highest woundwood coefficient was measured for Populus nigra (ccoef. 79.6%), followed by Tilia platyphyllos (ccoef. 72.7%), Acer pseudoplatanus (ccoef. 69.2%) and Quercus rubra (ccoef. 63.9%). By contrast no significant differences were observed between the tree species Tilia platyphyllos, Acer pseudoplatanus and Quercus rubra; the coefficients for Populus nigra and Acer pseudoplatanus, and for Quercus rubra, were significantly different (P<0.05). The effect of treatment with T-15603.1 on the inoculated wood decay fungi and wound occlusion is apparent from Figure 7. The woundwood coefficients were divided into 3 categories (1=ccoef. <30%, 2=ccoef. 31–60%, 3=ccoef. >60%). Wounds treated with T-15603.1 and untreated controls could be classified in the highest category 3 (>60% wound closure), whereas wounds inoculated with wood decay fungi could be classified in category

Populus Quercus Acer Tilia

T-15603.1 b

P. squamosus b

G. adspersum b

I. hispidus 0 10 20 30 40 50 60 70 80 90 Extension of discolouration [cm2]

FIGURE 5. Extent of wood discolouration developing from pruning wounds. Letters denote significant differences after contrasts analysis P<0.05 258 ARBORICULTURAL JOURNAL

FIGURE 6. Measurement of the woundwood coefficient. Letters denote significant differences (P<0.05) of wound occlusion for tree species. (1) <30% (2) 31–60% (3) >60%

T-15603.1 10 [19.2%] 15 [28.8%] 27 [51.9%] Wood decay fungi 15 [26.3%] 26 [45.61%] 22 [38.6%] Control 4 [18.2%] 5 [22.7%] 13 [59.1%]

FIGURE 7. Wound occlusion rate (woundwood coefficient) for individual treatments was classified in 3 woundwood coefficient categories (1=ccoef.<30%, 2=ccoef. 31- 60%, 3=ccoef. >60%).

2 (31–60% wound occlusion). The results indicated that the wood decay fungi negatively influenced wound occlusion, whereas neither a positive nor negative effect on woundwood formation was recorded for T-15603.1. In addition to tree species and wound treatment, wound size influenced wound occlusion. In Figure 8 the influence of the wound size on the woundwood coefficient is illustrated. According to Spearman’s rho analysis a negative correlation (=0.6879) was recorded for woundwood coefficient and wound size (P<0.05). Thus with increasing wound size the woundwood coefficient decreased. FIELD EXPERIMENTS TO EVALUATE THE APPLICATION OF TRICHODERMA STRAIN 259

100

30 R2 = 0.4733 20 Woundwood coefficient [%] 10

0 0 2 4 6 8 10 12 14 16 Wound size [r]

FIGURE 8. Correlation analysis showed a significant relationship between wound size and woundwood coefficient (P<0.05).

In addition to bacteria a range of fungal genera and species were isolated from the untreated wounds (Table 5). The application of T-15603.1 reduced the microbial diversity of colonized wounds (Table 6). Thus the mean indices of the untreated wounds (control) H'=1.547 and E=0.846 were higher than the indices of the treated wounds (H’=0.829 and E=0.414). Wounds treated with conidial suspension 3 showed the lowest diversity indices (suspension 1 H'=0.811 E=0.468; suspension 2 H'=1.024 E=0.534; suspension 3 H'=0.651 E=0.242). Only 25 genera of fungi but no basidiomycetes were isolated from the treated wound which is equivalent to a reduction in microbial diversity of 28.6%. The evaluation of the artificial inoculation tests showed a high biocontrol efficacy of T-15603.1 against I. hispidus, G. adspersum and P. squamosus on pruning wounds (Figure 9). Control wounds that were inoculated with wood decay fungi but not treated with Trichoderma, showed a mean infection rate of 78.7%. G. adspersum caused the highest infection rate (81%) followed by P. squamosus (79%) and I. hispidus (76%). All fungi showed similar statistical performance (P<0.05). Analysis of variance showed that the treatment of pruning wounds with conidial suspensions of T-15603.1 reduced colonization significantly (P<0.001) from 78.7% to 13.9%; which corresponds to a mean wound treatment efficiency of 82.3%. T-15603.1 showed the highest efficiency (93.7%; P<0.001) against I. hispidus. A similar reduction in infection rate (91.1%; P<0.001) was also determined for G. adspersum, whereas efficacy against P. squamosus (62.4%; P<0.05) 260 ARBORICULTURAL JOURNAL sp. Myxomycetes Physarum Basidiomycetes Flammulina velutipes Stereum hirsutum Trametes versicolor Zygomycetes Absidia Mortierella Rhizopus sp. sp. sp. sp. sp. sp. sp. Ascomycetes Aleuria Apiognomonia Ascocoryne Chaetomium sp. Daldinia Erysipha Hypocrea Nectria sp. Peziza sp. Verticillium sp. Xylaria sp. sp. sp. sp. sp. sp. Pestalotia Penicillium Phialophora Phoma sp. Nodulisporium Stemphylium sp. sp.

sp. sp.

sp. sp. sp. sp. Deuteromycetes sp. Fungal Fungal genera and species isolated from untreated pruning wounds. 5. ABLE Acremonium Acremonium

Alternaria T Mortierella Aureobasidium Curvularia sp. Epicoccum sp. Exophiala Fusarium Gliocladium Monocillium sp. sp. Paecilomyces sp. Cladosporium FIELD EXPERIMENTS TO EVALUATE THE APPLICATION OF TRICHODERMA STRAIN 261

TABLE 6. Influence of T-15603.1 application on microbial diversity

Diversity indices Suspension 1 Suspension 2 Suspension 3 Control

H' 0.811 1.024 0.651 1.547 E 0.468 0.534 0.242 0.846

100 control T-15603.1

81 79 78.7 80 76

40 29.7* Infection rate [%] 20 13.9** ** 4.8 ** 7.2

0 I. hispidus G. adspersum P. squamosus Mean

FIGURE 9. Reduction of the infection rate of wood decay fungi on pruning wounds after treatment with T-15603.1. Asterices indicate significant differences (F-test) for untreated controls and treated wounds (* = P<0.05; ** = P<0.001).

was more limited. In Figure 10 the influence of T-15603.1 on infection rates of treated and untreated (control) wounds are illustrated.

Discussion

The field screening trials were undertaken to evaluate the potential ofthe selected T. atroviride isolate 15603.1 for biological control of wood decay fungi under natural conditions. Optimal establishment of the antagonist in the wood substrate prior to colonization by a wound pathogen appears to be a good strategy for a successful wound protection treatment. Establishment is highly dependent on the adhesion and the viability of conidia of the biocontrol agent and this is more difficult in the natural environment than in conditions found in the laboratory. One way to improve the performance of biocontrol agents is by formulating conidial suspensions that enhance the adhesion and viability. In this study a range of different conidial suspensions of T-15603.1 was formulated and tested, which significantly 262 ARBORICULTURAL JOURNAL

B RZ

C D E

D D

F G

RZ D

H I Figure 10. A: Completely occluded wound of Populus sp. B: Occluded poplar wound pre -treated with Trichoderma sp. (longitudinal section). C: Longitudinal section of pruning wound of Tilia sp. FIGUREDiscoloured 10. A: Completely wood showed a occluded bleached a ppearancewound of [B]. Populus RZ= r eaction sp. B zone.: Occluded D: Pre-treated poplar wound wound pre-treatedsurface of withQuercus Trichoderma sp. show ing no signs sp. of (longitudinal degradation. E: section). Visible degradation C: Longitudinal o f wound surface section (Tilia sp.) inoculated with G. adspersum without pre -treatment. F + G: Zone lines indicat e of pruningsuccessful wound colonization of ofTilia wounds sp. by DiscolouredP. squamosus . H: wood Wood showed of Tilia wounds a bleached inoculated appearance with I. [B]. hispidus RZ= reaction was strongly zone. degraded D: a Pre-treated fter 3 years [ D]. wound RZ=reaction surface zone. of I: UntreatedQuercus wound sp. (control) showing no signswith of fruitdegradation. bodies of Stereum E: Visible hirsutum degradation(Willd.) Pers. of wound surface (Tilia sp.) inoculated with G. adspersum without pre-treatment. F + G: Zone lines indicate successful colonization of wounds by P. squamosus. H: Wood of Tilia wounds inoculated with I. hispidus was strongly degraded after 3 years [D]. RZ=reaction zone. I: Untreated wound (control) with fruit bodies of Stereum hirsutum (Willd.) Pers. FIELD EXPERIMENTS TO EVALUATE THE APPLICATION OF TRICHODERMA STRAIN 263 influenced the establishment (P<0.001) of the antagonist in pruning wounds. The concentration of all suspensions was adjusted to 105 CFU/ml. HJELJORD et al. (2001) observed that higher concentrations of conidia resulted in a reduction in conidial germination. Conidial suspension 2 was amended with urea and glucose. HJELJORD et al. (2001) showed that the addition of nutrients (C and N sources) resulted in an increase in germination rate and conidia viability and demonstrated the existence of a significant relationship between an increase in germination rate, viability and biocontrol efficacy of Trichoderma spp. However the authors also mentioned that conidia that are enriched in nutrients showed an increased water requirement resulting in a reduced germination rate due to a reduction in water potential. A comparison with climate data showed a significant correlation between re-isolation rate and precipitation on different sites. This may explain why no significant effect (P<0.05) could be observed between the amended suspension 2 and the non-amended suspension 1. The viability of the enriched conidia was possibly limited due to an increased water requirement and due to desiccation of the wood substrate i.e. the establishment, as indicated by re-isolation rate of T-15603.1, was hampered. Furthermore, the effect of the additives was not limited to the Trichoderma isolate but may also have promoted microflora competition in the wood substrate as indicated bythe higher diversity indices from wounds treated with conidial suspension 2 than from wounds treated with suspension 1. In previous studies SIMON & SIVASITHAMPARAM (1988) observed the inhibition of Trichoderma spp. by different bacteria in dual culture tests. NAÁR & KECSKÉS (1998) demonstrated that in several in vitro tests the bacteria Clavibacter michiganese and Pseudomonas syringae caused a significant inhibition of Trichoderma spp. by the production and excretion of antibiotic substances. LUTZ et al. (2003) observed a negative influence of the chitinase gene expression and therefore a reduction of antagonistic activity by the fungi Fusarium culmorum and F. graminearum. Carriers of inocula are inert substances in the sense that they do not have a disease control capacity; however they can profoundly affect time of germination as well as viability of conidia (FRAVEL et al., 1998). In the present study, the conidia of T-15603.1 enriched by additives in a humidity-storing gel formulation provided a constant moisture reservoir and thus increased conidial viability and effectiveness, as indicated by the significantly (P<0.001) higher re-isolation rates from wounds treated with the conidial suspension 3. In addition, conidial adhesion to the wood substrate was improved by the use of the gel as carrier substance (BATTA, 2004; JAYARAJ et al., 2006). Both increased viability and adhesion enhanced the establishment of T-15603.1 and thus improved efficiency as indicated by the lowest diversity indices recorded from wounds inoculated with the conidial suspension 3. In particular the low E index 0.242 (high ratio of 264 ARBORICULTURAL JOURNAL

the antagonist on total number of individuals or groups) demonstrates that T-15603.1 was the dominant species in the wood substrate. The isolation rate of naturally occurring Trichoderma spp. from the untreated control wounds was consistently very low (4.5%). There is no explanation for this at present other than the speculation that the presence of Trichoderma spp. on urban sites may be limited due to the absence of organic matter, which is essential for growth, survival and sporulation of the fungus. More detailed studies are needed to confirm this hypothesis. The highest diversity indices were detected from control wounds. In particular the low E index (0.846) is indicative of the fact that on untreated wounds a natural succession of microorganisms occurred in the absence of T-15603.1. Not only the type of conidial suspensions and abiotic factors, especially wood moisture, influenced the establishment of the biocontrol agent, but also the wood substrate appeared to be a decisive factor. The re-isolation rate was significantly (P<0.05) reduced in the heartwood. SHIGO & HILLS (1973) investigated the specific characteristics of the heartwood from several tree species and they described heartwood as a substrate with low wood moisture content and with fungistatic properties, which hampers colonization by microorganisms. Thus, the conidial viability and consequently the biocontrol efficacy of T-15603.1 is reduced on large wounds with a high ratio of heart- to sapwood. Successful infection and colonization of untreated wounds by wood decay fungi depends on the ability to overcome host barriers in the wood and to circumvent and/or degrade phenolic compounds (SCHWARZE et al., 1999; SCHWARZE & FERNER, 2003). Inonotus hispidus and Polyporus squamosus are both classified as wound parasites and are able to infect and colonize small wounds (MCCRACKEN & TOOLE, 1974; SCHWARZE et al., 1999). The ability of G. adspersum to degrade polyphenolic deposits in reaction zones was demonstrated by SCHWARZE & FERNER (2003) and explains the high infection rate (78.7%) of the untreated wounds after thirty months. Pre- treatment of the wounds with T-15603.1 resulted in a strong preventive effect against colonisation by wood decay fungi, particularly I. hispidus and G. adspersum (Figure 9). This observation is in good agreement with the results previously obtained in laboratory tests that showed a high susceptibility of both basidiomycetes to antagonism by T-15603.1 (SCHUBERT et al., 2008). A lower effect (62.4%) was measured for P. squamosus, which is also in good agreement with results obtained in the in vitro studies. The active mechanisms of antagonism in the wood substrate are determined by rapid growth, mycoparasitism and antibiosis. A further passive effect of T-15603.1 could be related in maintaining the tree’s defence boundaries. SMITH et al. (1981) postulated that T. harzianum can tolerate high levels of phenols produced by the host in response to colonization without the need to FIELD EXPERIMENTS TO EVALUATE THE APPLICATION OF TRICHODERMA STRAIN 265 modify compounds. Other fungi, such as Phialophora melinii (Nannf.) Cont. which is also tolerant to fungistatic substances, may reduce the efficiency of defence boundaries by metabolizing large amounts of polyphenols (SMITH et al., 1981). Inhibition and exclusion of such fungi may maintain high concentrations of polyphenols thus preventing colonization by decay fungi which are sensitive to fungistatic substances. Whether the treatment with T- 15603.1 helped maintaining tree defence boundaries could not definitely be demonstrated, but the present work strongly indicates that the inoculation of T-15603.1 not only inhibits colonization by wood decay fungi but also of many other micoorganisms as demonstrated by the diversity indices. Results obtained from in vitro studies are helpful to eliminate non- performing isolates from the screening tests but are not representative, as in vitro assays do not completely mimic all ecological and endemic factors. Factors such as wood moisture content and competition with the indigenous microflora are impossible to reproduce in the laboratory. For this reason field studies are essential to test the efficacy of selected biocontrol agent under natural conditions. Results of field studies demonstrate that the Trichoderma strain T-15603.1 can be successful when applied as a biological wound treatment method against I. hispidus and G. adspersum on urban sites. But there is still a need to optimize the formulation of conidia i.e. to improve the establishment and therefore the biocontrol efficacy, particularly on pruning wounds with a high heart- to sapwood ratio. In addition it is possible that isolates exist that are more effective and persistent than T-15603.1. Therefore further screening trials of competitive strains against a range of common wood decay fungi should be undertaken. The application of T-15603.1 on tree wounds can not completely inhibit colonization by all wood decay fungi. In addition genotype and virulence of wood decay fungi may vary from those used in this study. A strategy to enhance the effect of a biological wound treatment could be based on the use of a mixture of biocontrol strains, which may provide a greater protection under different environmental conditions than the application of individual biocontrol strains (MEYER & ROBERTS, 2002).

Acknowledgments

We would like to express our appreciation to Mr. Dieter Keck (BASF AG Ludwigshafen) and Mr. Christoph Marx (city of Strasbourg) not only for their assistance, but also for their continuing interest in the progress of the field experiments. We would also like to thank Mr. Robert Dietrich and Mr. Karl Merz (Institute for Forest Botany, University of Freiburg, Germany) for technical support. Finally the financial support from the Ev. Studienwerk Villigst e.V. is gratefully acknowledged. 266 ARBORICULTURAL JOURNAL

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