Growth and Antagonism of Trichoderma spp . and Conifer Pathogen Heterobasidion annosum s . l . in Vitro at Different Temperatures

Variations in the radial growth rate of 24 isolates belonging to ten species of Trichoderma, three isolates of conifer pathogen Heterobasidion annosum s.s. and four isolates of H. parviporum were evaluated by incubation on a solid malt extract medium at a temperature of 4 ̊C, 15 ̊C and 21 ̊C. Trichoderma antagonism against Heterobasidion was investigated in dual culture in vitro. The slowest rate of growth was referable to all seven strains of Heterobasidion spp. All Heterobasidion spp. strains were overgrown by 63% of Trichoderma spp. strains after two weeks at 21 ̊C and by 33% of strains at 15 ̊C. 21% of Trichoderma strains did not grow and only four strains belonging to T. koningii, T. viride and T. viridescens demonstrated the ability to completely overgrow Heterobasidion spp. after two weeks incubation at 4 ̊C. According to the antagonistic efficiency, Trichoderma strains were divided into five groups with an Euclidean distance of 25. The groups contained isolates from different species. It was suggested that selected psychrotrophic fast growing T. viride, T. koningii and T. viridescens strains could be examined in different substrate conditions as suitable antagonist agents for the control of H. annosum and H. parviporum.


Introduction
Conifer root and butt rot disease caused by Heterobasidion annosum (Fr.)Bref.sensu lato is an economically important disease of coniferous trees in boreal and temperate forests [1].The fungus spreads through aerial basidiospores to stump surfaces and wounds, and by mycelia via root contacts from tree to tree [2,3].Heterobasidion spp.also has an anamorph stage preferably developing in laboratory conditions and forming conidia.Conidiospores also form on the stumps in moist weather and can survive in soil up to ten months [4].Heterobasidion spp.grow at a wide range of temperature with a minimum of 2˚C [5].
A biocontrol method to reduce H. annosum s.l.infection is to apply the wood degrading fungus Phlebiopsis gigantea in a spore suspension directly on the freshly cut stumps immediately after cutting [6].There are also a lot of other fungi examined for Heterobasidion biocontrol potential including Trichoderma spp.[7].
Trichoderma and its teleomorphic stage Hypocrea include cosmopolitan soil-borne species.At present, the International Subcommission on Trichoderma and Hypocrea Taxonomy lists 104 species (http://www.isth.info).Trichoderma are particularly prevalent in the humic layer of hardwood forests where they represent up to 3% of all fungal propagules [8].Some strains are commonly used to control different plant diseases.Possible mechanisms involved in disease control by Trichoderma include mycoparasitism, antibiosis, competition, and induction of plant defence responses [9].Trichoderma are also woodcolonising fungi which restrict the growth of H. annosum in vitro and under natural conditions [10,11].There are a number of other investigations of antagonistic action of Trichoderma against Heterobasidion [12][13][14][15][16].However, only one or a few strains per species of Trichoderma were involved [7].
In general, Trichoderma species are mesophylic.However, they possess different temperature minima, optima and maxima.Temperature also affects the metabolism features and antagonistic properties of Trichoderma spp.[17].Goldfarb et al. [18] ascertained variation of growth rate substantially within and among species.The investigations supported the opinion that Trichoderma isolates adapted to comparable or lower temperatures than that of plant pathogens provide superior disease control compared to Trichoderma isolates that are incapable of growth or activity at cold temperatures [19].
The aim of this work was to study the effect of temperature on antagonism of different Trichoderma isolates towards H. annosum s.l. with prospects of application in the biological control in boreal and temperate forests.

Fungal Strains
In total 24 Trichoderma spp.strains, as well as three H. annosum s.s.strains and four H. parviporum strains isolated primarily in Latvia were used (Table 1).For longterm storage, fungi were maintained frozen in liquid nitrogen in the Microbial Strain Collection of Latvia (MSCL) University of Latvia.

Molecular Identification of Species
A PCR was carried out with the DNA samples of 20 Trichoderma isolates.Genomic DNA of Trichoderma spp.isolates was extracted from mycelia using a Power-Soil™ DNA Isolation Kit (MO BIO Laboratories, USA).The rRNA gene region was amplified with primers

Growth Kinetics and Antagonism Assay
Paired cultures of Trichoderma and Heterobasidion spp.were grown from inocula placed 5 cm apart, on malt extract agar (MEA; Becton Dickinson, USA) at 4˚C, 15˚C and 21˚C.In controls, single cultures were grown in the centre of Petri dishes.Radii of fungal colonies have been recorded daily after 3 rd -28 th days.The rate of colony growth was calculated from measurements of radii after three days long incubation of fungi at 21˚C and 15˚C or after 10 days of incubation at 4˚C when the growth corresponded to the exponential growth phase.The results were expressed in mm•h -1 .The experiment was performed twice.The efficiency of Trichoderma in suppressing radial growth was calculated as follows: , where C is radial growth measurement of the pathogen in the control and T is radial growth of the pathogen in the presence of Trichoderma [22].

Conidiospore Germination
Conidia from H. annosum 1020 were obtained from fully sporulated agar cultures.Droplets (100 l) of spore suspension (about 10 7 conidia•ml -1 ) were transferred to ster-ile cellophane strips and placed onto a surface of Trichoderma spp.growing in the MEA medium for three days.Germinated spores were counted after one, two and three days of incubation at 21˚C using a microscope.Germination of 100 randomly selected conidia in each droplet was evaluated with a microscope at ×600 magnification.Enumeration of germinated conidia was performed in five replicates (on five cellophane strips).A conidium was considered germinated, if the length of the germ tube was not shorter than the diameter of the conidium.The experiment was repeated three times.An average of measurements from 15 replicates was calculated.

Statistical Analysis
The significance of the difference in values was determined through ANOVA analysis at a significance level of 0.05.The efficiency of different Trichoderma strains in suppressing of Heterobasidion spp. was analysed using hierarchial cluster analysis with complete linkage method and Euclidean distance matrix.

Identity of Trichoderma Species
According to molecular identification, six Trichoderma strains proved to be T. asperellum and five belonged to T. viride, two strains belonged to T. koningii and two belonged to T. viridescens but five further species (T.citrinoviride, T. hamatum, T. harzianum, T. longibrachiatum and T. rossicum) were represented by single isolates (Table 1).

Growth and Antagonism between
Trichoderma was referable to all seven strains of two Heterobasidion species.Only some strains formed a sterile zone between antagonistic colonies.For example, it was in the case of T. asperellum 1011 and H. parviporum 981 (Figure 2).We also observed changes in the Trichoderma pigmentation from green to yellow near of the pathogen while the  strains after two weeks at 21˚C and by 33% of strains at 15˚C (Table 3).The restriction of Heterobasidion was especially difficult and slow at 4˚C. 21% of Trichoderma strains did not grow and only four strains (T.koningii 585, T. viride 595 and 969, and T. viridescens 472) clustered in the group (Figure 4) what demonstrated the ability to completely overgrow Heterobasidion spp.also after two weeks incubation at 4˚C. T. asperellum 309 was not able to grow at 4 ˚C and allowed to extend H. parviporum 981 (Figure 3(c)) with minimal restriction.

Trichoderma Impact on the Germination of Heterobasidion Species
All  4), especially on the first day.3).  1) and the obtained means differed significantly (P < 0.05) between species only at 4˚C (Table 2).The significant influence of the temperature (P < 0.05) was estimated on the growth rate of different Trichoderma strains and species allowing their division into three, four and five groups respectively at a temperature of 21˚C, 15˚C and 4˚C (Table 2).Already Danielson and Davey [23] recognized that some Trichoderma species are typical for cool geographic regions (T.viride, T. polysporum) and others are typical for warm climatic regions (T.koningii, T. hamatum, T. harzianum, T. pseudokoningii, T. saturnisporum).Today, T. polysporum is identified as one of the psychrotrophic Trichoderma species present in temperate [18] and arctic environments [24].Our fungal strains were isolated from the temperate region mainly from Latvia (Table 1).In addition, our single T. polysporum strain (origin-Sweden) showed a high rate of growth (0.055 mm•h -1 ) at 4˚C.However, three other species, T. viride, T. koningii and T. viridescens, demonstrated a growth rate of 0.073 -0.080 mm•h -1 and overcame T. polysporum (Table 2).
Jaklitsch et al. [25] estimated that T. viride and T. viridescens is characterised by north-and south-temperate distribution, relatively slow growth and temperature optimum of about 25˚C.They showed very little variation in growth rate among their many isolates that corresponds to the results of our study.Lieckfeldt et al. [26] found that T. viride has an optimum temperature of 22.5˚C but T. asperellum has an optimum of 30˚C.We did not attempt to estimate optimum temperature, however, all six of our investigated T. viride strains showed significantly faster growth (P < 0.05) than all six of T. asperellum strains at all of the used temperature regimes.

Fungal Antagonism at Different Temperatures
The interaction between Trichoderma and Heterobasidion spp. was highly dependent on temperature.It is recognized that different Trichoderma spp.strains have different thermal requirements for biocontrol [27,28].Köhl and Schlösser [29] established the potential of some Trichoderma isolates to decay sclerotia of Botrytis cinerea at low temperatures, even at 5˚C.In the present investigation we found out that all 24 of the investigated Trichoderma strains showed antagonistic activity against Heterobasidion.The number of Trichoderma strains overgrowing pathogens decreased with a decrease in temperature from 21˚C to 15˚C and especially to 4˚C (Table 3).According to the antagonistic efficiency, Tricho- Copyright © 2012 SciRes.AiM derma strains were divided into five groups with an Euclidean distance of 25 (Figure 4).The groups contained isolates from different species.We did not find any Trichoderma strain that demonstrated a significant difference in antagonism against several strains of Heterobasidion.This is in contrary to the investigation of Bell et al. [30], which showed that a single isolate of the antagonist can be highly effective against one isolate (in particular Rhizoctonia solani) but may have only minimal effects on other isolates of the same species.Napierala-Filipiak and Werner [31], who studied interactions between 42 mycorrhizal fungi and six strains of Heterobasidion spp., ascertained that although antagonism is dependent on growth rates of fungi, the fungi do not display antagonistic properties to all strains of the pathogen.
Obtained results show that Trichoderma strains inhibited germination of Heterobasidion especially in the beginning (Table 4).A direct influence of hyphae and macromolecular compounds on the conidia was excluded by plating cellophane strips onto the surface of the colonies.Therefore, we can assume that soluble low molecular and/or volatile substances produced this impact.
Possible mechanisms of antagonism among our isolates of Trichoderma and Heterobasidion such as mycoparasitism and antibiosis will be studied in our further experiments.The experiments will also include investigation of the ability of Trichoderma to replace Heterobasidion in different substrate conditions including wood media.
It was suggested that selected psychrotrophic fast growing T. viride, T. koningii and T. viridescens strains could be examined in different substrate conditions as suitable antagonist agents for the control of H. annosum and H. parviporum.

Figure 2 .Figure 3 .
Figure 2. Dual growth of T. asperellum 1011 (on the left) and H. parviporum 981 (on the right) after one week (a), two weeks (b) and four weeks (c) incubation at 15˚C.
eight of the randomly chosen Trichoderma spp.strains (T.koningii 1025, T. polysporum 1024, T. viride 585, 945, 946, 969 and 1026, and T. viridescens 472) caused inhibition of H. annosum 1020 conidia germination after one day of incubation (data not shown).After two days, the inhibition effect was retained by only four T. viride strains and T. polysporum 1024.Both T. viride 585 and T. viride 969 demonstrated the ability to significantly (P < 0.05) slowing down the conidiospore germination of pathogen (Table Trichoderma sp.584 100.0 ± 0.0 81.9 ± 8.5 7.0 ± 6.3 Data are presented as means of efficiency according to three Heterobasidion strains (H.521, H. 980 and H. 981)  SD.The efficiency of Trichoderma was calculated as follows:   100 C T C   , where C is radial growth measurement of the pathogen in the control and T is radial growth of the pathogen in the resence of Trichoderma.p Copyright © 2012 SciRes.AiM

Figure 4 .
Figure 4. Dendrogram, based on the Euclidean distance, of the Trichoderma strains according to efficiency in suppressing linear growth of Heterobasidion spp. in paired cultures at a temperature of 21˚C, 15˚C and 4˚C (Table3).

Table 1 . Trichoderma and Heterobasidion spp. isolates used in this study and corresponding homologue sequences in the NCBI data base.
[21]and ITS4[21].The reactions in Eppendorf Mastercycler Personal were carried out in 25 μl volume.The mixture contained 0.2 μl Hot Start Taq DNA Polymerase, 2.5 μl 10X Hot Start PCR Buffer, 2.5 μl dNTP Mix, 2 mM each, 2 μl 25 mM MgCl 2 (all reagents from Fermentas, Lithuania), 0.5 μl of each 25 μM primer (OPERON Biotechnologies), 15.43 μl sterile distilled water and 1 μl of DNA template.The PCR conditions were as follows: the initial denaturation step of 4 min at 95˚C, 40 s of denaturation at 95˚C, 40 s of annealing at 52˚C, 1 min of primer extension at 72˚C (30 cycles) and final extension 10 min at 72˚C.PCR products (20 -25 µl) were purified using 0.5 μl Exonuclease I (20 u•μl -1 ) and 2.0 µL Shrimp Alkaline Phosphatase (1 u•μl -1 ) (both from Fermentas, Lithuania).Samples were further processed in a PCR machine 30 min at 37˚C, 15 min at a : Ac cording to molecular identification presented in this paper.ITS1F 85˚C.Both strands of the amplification products were sequenced by using an ABI Prism Big Dye terminator cycle sequencing ready reaction kit (version 3.1, Applied Biosystems, Foster City, USA) and primers ITS1F and ITS4.Sequencing of strains 309 -845 and 1011 -1012 was done in CBS, Utrecht, the Netherlands.Sequencing of strains 867 -969 was done in the Biomedical Study and Research Center at the University of Latvia.Obtained sequences were analyzed using Staden Package 1.6.0.release.The resulting consensus sequences were used in the BLASTN homology search against The National Center for Biotechnology Information (NCBI) nucleotide database (http://www.ncbi.nlm.nih.gov).