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Evaluation of an antagonistic Trichoderma strain for reducing the rate of wood decomposition by the white rot fungus Phellinus noxius

ARTICLE in BIOLOGICAL CONTROL · MAY 2012 Impact Factor: 1.64 · DOI: 10.1016/j.biocontrol.2012.01.016

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Available from: Mark Schubert Retrieved on: 30 March 2016 Biological Control 61 (2012) 160–168

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Biological Control

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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

highlights 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.

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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 8 February 2012 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). 162 F.W.M.R. Schwarze et al. / Biological Control 61 (2012) 160–168

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 fungi were used as an indication of its activity (Naár and Kecskes, Difference) test (P<0.05) was additionally performed. The statisti- 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 F.W.M.R. Schwarze et al. / Biological Control 61 (2012) 160–168 163

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). 164 F.W.M.R. Schwarze et al. / Biological Control 61 (2012) 160–168

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. F.W.M.R. Schwarze et al. / Biological Control 61 (2012) 160–168 165

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 166 F.W.M.R. Schwarze et al. / Biological Control 61 (2012) 160–168 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 indi- cates 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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