GB2239800A - Treatment or prevention of serpula lacrymans infection - Google Patents

Abstract

Trichoderma and Hyalodendron species, in particular Trichoderma harzianum, Trichoderma aureoviride and Trichoderma pseudokongingii, are effective in the treatment or prevention of Serpula lacrymans infections. Serpula lacrymans causes dry-rot in wood and is difficult and expensive to eradicate from wood by conventional means which involve removal of infected wood followed by sterilisation with fungicide. Such conventional treatments may even promote regrowth of Serpula lacrymans. An effective substance obtainable from Trichoderma or Hyalodendron, or the intact organisms, may be applied to treat or prevent Serpula lacrymans infection.

Description

TREATMENT OR PREVENTION OF SERPULA LACRYMANS INFECTION The present invention relates to a fungicidal agent.

More particularly, the present invention relates to a fungicidal agent in use against dry-rot caused by Serpula lacrymans.

The wood decay caused by the dry-rot fungus Serpula lacrymans (S.lacrymans) is found in cool temperate regions such as Britain and Northern Europe. Through the reduction of cellulose and hemicellulose in, for example groundfloor timber, it causes substantial damage to wood. In Switzerland, for example, up to one third of all remedial treatments of damages in houses were related to this fungus (Waelchli and Raschle, 1983). S.lacrymans differs substantially from other fungi.

It rarely exists outdoors. Long exposure to heat, e.g. warm summer, is sufficient to kill it. Specialised hyphae (strands) allow S.lacrymans to transport water from a wet place to a dry place so that it can spread to a considerable extent. These strands, together with the potential to grow in an alkaline pH range, allow it to grow even through brick and mortar. It is acknowledged in, for example, "Fungal Decomposition of Wood" (Rayner and Boddy, Wiley 1988, page 456) that it is "more difficult to treat and eradicate S.lacrymans than other fungi". Indeed at present the only treatment of dry rot is removal of the infected area.This involves the removal of all rotten wood, additional cutting away of timber of about 300-450 mm beyond the last indications of fungus sterilising the whole area with fungicide and replacement of the wood with timber treated with preservative. (Recognising Wood Rot and Insect Damage in Buildings - Bravey/Beny/Carey/Cooper BRE 1987, pages 16-19). This removal of wood is very wasteful and may cause considerable inconvenience. Moreover, S.lacrymans can respond to such treatment by a flush of new growth, and the moisture in the fungicide often seems to aid its regeneration.

Wood decay can also be caused by other fungi. In numerous publications particularly the brown rot fungus Lentinus lepideus (L.lepideus) has been studied which causes the decay of creosoted wood structures outdoors, such as fence posts, transmission poles and other atmospherically exposed wood. It has been suggested that mycoparasites such as Trichoderma spp. and Scytalidium sp.

could be used in the control of wood decay fungi such as L.lepideus. For example, Murmanis et al (Material und Organismen, 23 BD. 1988, page 271-279) describe hyphal interaction of Trichoderma harzianum Rifai and T.

polysporum with, L.lepideus which led to the total consumption of its cytoplasmic content and concomitantly to its death.

Highley and Ricard (Material und Organismen, 23/3, page 157-169) also described that Trichoderma harzianum could cause the death of the brown-rot fungus Lentinus lepideus, but showed no effect on white-rot fungi in wood.

In two articles, Bruce and King (Material und Organismen, 21/3, pages 165-179 and Material und Organismen, 21/1, pages 113) study the biological control of decay in creosote treated distribution poles caused by Lentinus lepideus. A combined innoculum of Trichoderma harzianum and Trichoderma polysporum was found to prevent or slow down an infection with L.lepideus in poles. In a review article (Commonwealth Institute of Biological Control, Biocontrol News and Information, Volume 2, No. 2, June 1981), Ricard proposes the use of a Trichoderma based mycofungicides for use against, for example, heart rot (Heterobasidion annosum).

However, despite numerous publications and years of research there appears to be no suggestion that a Trichoderma based agent, or any other agent derived from a microorganism could be used against dry rot caused by S.lacrymans. As described above, S.lacrymans is very different from other fungi and in contrast cannot merely be eradicated by drying the infected area. Since S.lacrymans is capable of translocation of water it is rarely possible to completely dry out all possible moisture sources which would be necessary for its eradication as expressly stated in "Fungal Decomposition of Wood" (Rayner & Boddy, supra, p.462). Usually, for complete eradication there is also the need for sterilisation of the masonry. It would therefore be desirable to provide a treatment for dry rot.

According to one aspect of the present invention there is provided a method for the treatment or prevention of S.lacrymans infection in a material, which comprises applying to said material, an organism effective to treat or prevent said infection, or an effective substance obtainable from said organism. Said organism is preferably a Trichoderma species, but Hyalodendron species has been found also to be effective in culture. The organism or effective substance may be applied to the material after it is infected with S. lacrymans but is preferably applied as a preventive measure to material which is substantially lacking in S. lacrymans infection.

According to another aspect of the present invention there is provided a fungicidal agent against S.lacrymans comprising a Trichoderma organism or an effective substance as obtainable therefrom. The agent may conveniently be in powder or liquid form, for application by dusting, or by brush, spray or impregnation. Preferably, the agent comprises Trichoderma harzianum and/or Trichoderma aureoviride and/or Trichoderma pseudokoningii, most preferably T.harzianum. As will be shown below, it is not necessary to use the live Trichoderma organism to be effective, and its effectiveness therefore presumably arises from a part of that organism or a metabolite therefrom. Thus, the invention includes the use of such an effective substance as obtainable from the organism.

In one embodiment of the invention susceptible material, to be used in for example a building, is pretreated with Trichoderma and incubated for a period of time as a preventive measure against dry rot before being used. The treated material can be used while still carrying the live Trichoderma, or after the period of incubation the Trichoderma can be killed.

In another embodiment, the effective metabolite or the like derived from Trichoderma is applied to the wood as a prophylactic measure. In another embodiment of the invention Trichoderma or its effective metabolite or the like is administered to an area already infected with S.lacrymans.

The invention will now be illustrated by way of the following examples: The Trichoderma species used in the present invention are all readily available from the Commonwealth Mycological Institute, Kew, UK and are in particular: T.harzianum IMI 206040 Picea excelsa, Sweden 1976 (deposited at the CMI under this number), but many other strains exist; T.aureoviride IMI 91968 Fagus sylvaticus, Scotland, 1962 T.longibrachiatum IMI 53608 Coffea spp. Tanzania, 1953 The Serpula species (isolates) used were S.lacrymans (Schumacher ex Fr) IMI 152233, S.lacrymans (Schumacher ex Fr) Gray FPRL 12C (obtained from the Building Research Establishment, Garston, Watford, UK).

Experiment 1 Fungal Interaction on 2% Agar/5% Malt extract Each of three Trichoderma species interacted with two Serpula isolates, with five replicates per interaction.

Therefore, 30 plates were prepared containing 2% purified agar/5% malt extract.

Recipe: for 250mls of 2% purified agar/5% malt extract Purified Agar 2.0g Malt Extract 12.5g Distilled water 250.Omls The plates were marked with a bisecting line 7cm in length. The fungi (target and control) were sub-cultured at opposite ends of this line. Initially, S.lacrymans 12C and S.lacrymans CMI 152233 (target) were sub-cultured onto the plates first to allow the fungi to establish a reasonably mature colony. The plates were incubated at 220C until the target colony had attained a radial size of 2025cm. Once this had been achieved the Trichoderma species (control) were then sub-cultured onto the plate (at the opposite end of line to the target fungus). The plates were then returned to the 22"C incubator.

The radii of both fungal colonies were measured once a day, the measurement being taken along the line mark.

The state of each interaction was noted daily. The plates were incubated until a final 'result' was obtained, e.g.

total coverage of the Serpula colony by the Trichoderma fungus.

The overall results are set out in Table A in the column headed "Agar".

Table A Table of Interaction Results Serpula lacrymans Interaction Result Trichoderma Agar Sawdust Agar T.harzianum Replacement of Replacement of target target T.aureoviride Replacement of Replacement of target target T.longibrachiatum Replacement of Replacement of antagonist antagonist Note: Both S.lacrymans isolates were affected in equal ways by the same Trichoderma species.

Antagonist - Trichoderma spp.

Target - S.lacrymans isolate The column "Sawdust Agar" relates to Experiment 2.

From the results, the three species of Trichoderma can be split into two main groups.

Group A: T.harzianum, T.aureoviride.

Group B: T.longibrachiatum.

The Group A Trichoderma species were the most efficient at covering the colonies of both S.lacrymans isolates and killing their mycelia. The time taken to reach the S.lacrymans cores ranged from 12-13 days. All of Group A initiated yellowing of the S.lacrymans mycelia a small distance behind the mycelial leading edge of the S.lacrymans colony, and there was a difference in the rates of coverage over the colonies of the two S.lacrymans isolates (the coverage of the S.lacrymans CMI 152233 colony being slower than the coverage of the S.lacrymans 12C colony).

The Group B Trichoderma was the least efficient at covering the S.lacrymans colonies but the mycelia that was covered was killed. Trichoderma longibrachiatum in Group B initiated yellowing of the S.lacrymans mycelia from the tip of the mycelial leading edge.

The Trichoderma species effective against both S.lacrymans isolates may kill the S.lacrymans mycelia by chemical release when mycelia from both colonies come into contact with each other. The other Trichoderma may release a chemical which has only a temporary effect on the S.lacrymans mycelia. Also, both S.lacrymans isolates may release chemicals. S.lacrymans 12C releases its chemical in response to the presence of the Trichodermas, even if no contact has yet been established. S.lacrymans CMI 152233 releases its chemical only when contact with the Trichoderma has been established.

Evidence that selected Trichoderma spp do indeed kill S.lacrymans rather than just stall its growth is provided by Experiment 3, where samples of S.lacrymans which had been covered by Trichoderma do not regrow in conditions where the Trichoderma growth is inhibited but S.lacrymans growth is possible.

These agar interaction experiments were repeated with a variety of other S.lacrymans strains and gave the results as shown above.

Experiment 2 Fungal Interaction on 5% Sawdust Agar Each of the original three Trichoderma species were interacted with the same two Serpula isolates, with five replicates per interaction. Therefore, 30 plates were prepared as described above but containing additionally 5% sawdust in 2% purified agar to more closely mimic the nutrient conditions for S.lacrymans when growing on wood.

The plates were firstly autoclaved, then, as in Experiment 1, the plates were marked with a bisecting line 7cm in length. The fungi (target and control) were subcultured at opposite ends of this line. Initially, S.lacrymans 12C and S.lacrymans CMI 152233 (target) were sub-cultured onto the plates first to allow the fungi to establish a reasonably mature colony. The plates were incubated at 22"C until the target colony had attained a radial size of 22-25mm. This, however, took so long to achieve that the agar began to dry out and shrink away from the plate edges. Therefore, the Trichoderma were subcultured in approximately the centre of the plate to initiate the interaction as quickly as possible. The plates were then returned to the 220C incubator.

The radii of the S.lacrymans isolates were measured until enough data was obtained to calculate growth rates.

After this it became too difficult to measure the radii as excessive condensation built up on the inside of the lid obscuring the colony edges. The plates were incubated until the final outcome of any particular interaction was obtained, e.g. total coverage of the Serpula colony by the Trichoderma fungus.

The overall results are set out in Table A in the column headed "Sawdust Agar". Despite major differences in the nutrient source available to the organisms in this experiment the overall results are similar to those reported in respect of Experiment 1.

As in Experiment 1, T.harzianum, T.aureoviride, were more efficient at killing S.lacrymans isolates than T.longibrachiatum. In fact, T.longibrachiatum had no fatal effect on the isolates.

Experiment 3 Benomyl Agar Experiment Samples of S.lacrymans from interaction plates of Experiments 1 and 2 were placed on agar containing benomyl (1:100 dilution of 0.1% benomyl solution). This substrate inhibits the growth of Trichoderma but has no effect on S.lacrymans. Therefore, any viable S.lacrymans should colonise the benomyl agar plates.

There were two sets of results derived from this experiment: 1) Any S.lacrymans mycelia covered by the Trichoderma mycelia did not resume growth. Such covered areas in fact showed evidence of autolysis and S.lacrymans mycelia in these areas were reddish brown in colour.

2) Areas of the S.lacrymans colony not covered by the Trichoderma mycelia did resume growth.

These results are consistent with the proposal that the Trichoderma species do kill the S.lacrymans mycelia when they advance over and cover the S.lacrymans colony.

Experiment 4 Anti-fungal metabolites produced by Trichoderma spp All Trichoderma species used were grown in 3% Malt Extract Broth, at 250C with continuous rotation (150 r.p.m.). The broth was then filtered through a Whatman No.

1 filter and the filtrate spun for 10 minutes at 4000 r.p.m. The supernatant was then removed and stored at 20"C. When required for use, the supernatant was filtered through a 20pm filter which would sterilise the supernatant.

1. The broth supernatants of the following Trichodermas were used in this experiment: T. harzianum, T. aureoviride.

The supernatants were incorporated into a liquid agar solution which was then allowed to solidify. S.lacrymans 12C was then sub-cultured onto the solid agar and the increase in the colony diameter was measured daily.

The following results were obtained: T.harzianum reduced the S.lacrymans growth rate by 77%, T.aureoviride reduced the S.lacrymans growth rate by 48%.

2. Agar cores were removed from agar plates and the supernatant from the T.harzianum broth filtrate was placed into the holes. S.lacrymans was sub-cultured centrally onto the plate. Results show that S.lacrymans growth towards such holes was slower than growth towards controls which contained normal growth medium. This system therefore serves as a rapid microassay method for detecting the presence of anti-S.lacrymans metabolites.

The nature of the anti-fungal metabolites was further demonstrated in the following experiment. The surface of an agar plate was covered with a single layer of dialysis tubing and a T.harzianum core was sub-cultured onto the tubing. After covering 75% of the tubing, the tubing (and the fungus) was removed and the plate was exposed to W light to ensure that no viable Trichoderma was present.

S.lacrymans 12C was then sub-cultured centrally onto the plate. Results show that the S.lacrymans mycelia turned yellow and growth was inhibited for six days. After that time the S.lacrymans 12C did begin to grow but this growth was extremely slow.

Since dialysis tubing inhibits the movement of large molecules only relatively small ones (molecular weight < 1000) would have been able to penetrate the dialysis tubing and enter the agar layer. The experiment indicates that at least one component produced by Trichoderma spp.

which is effective against S.lacrymans must be relatively small, and hence a simple molecule.

Experiment 5 Interaction of Serpula lacrymans and Trichoderma spp on pine wood blocks One cm square wood blocks, were cut from a piece of pine sapwood. The blocks were then sanded down to remove any loose fragments of wood which may interfere with the results. The blocks were then dried in an oven set at 1020C for three hours and weighed to four decimal places (after cooling in a dessicator). The blocks were then sterilised by the use of gamma irradiation (exposed to 2 mega-rad for 12 hours).

Glass jars (500 ml) were prepared with 2% purified agar/5% malt extract in 2oomls of water. An air vent in the screwtop lid was closed with a foam bung. The jars were then autoclaved and allowed to cool. Once the agar had set the jars were inoculated. In the case of S.lacrymans 12C, this was two weeks before the blocks were due to be placed in the jars, and for Trichoderma this was four days beforehand. All jars were incubated at 220C.

Plastic mesh was cut to the size of the glass jars (so that it easily fitted into the jars) and autoclaved before insertion into the jars. Insertion was carried out in an isolation unit with sterile equipment, with the mesh being placed on top of the fungal colony. Once the mesh was inserted, the blocks were placed on top of the mesh. This was also carried out in an isolation unit, with everything being wiped with 70* alcohol beforehand. All the blocks were placed onto the mesh, any which landed on the agar were quickly lifted onto the mesh using sterile forceps.

Once the blocks were placed in the jars, they were incubated in a glass tank for an appropriate time period.

The tank was lined with moist paper towel and had an air inlet connected to an air pump, and an air outlet.

In this and the following experiments the jars containing S.lacrymans 12C and Trichoderma spp. were placed into different tanks, each with the same conditions. This was to prevent any detrimental effect on the S.lacrymans 12C caused by the release of volatiles by the Trichoderma EE After completion of the experiment, the blocks were removed from the jars, freeze-dried and weighed to determine weight loss.

Experiments were designed to investigate if preincubation of wood blocks with Trichoderma spp. (viz.

T.harzianum, T.aureoviride or T.longibrachiatum) could inhibit or reduce subsequent weight loss caused by infection of blocks with S.lacrymans 12C. Weighed, sterile, pine wood blocks (prepared as above) were exposed to Trichoderma spp. for 2, 4 or 6 weeks, then incubated with S.lacrymans 12C for 3 or 6 weeks and weight losses estimated at these latter times. Control blocks were also set up; these were either preincubated with Trichoderma and then harvested, or preincubated in the absence of Trichoderma then subsequently incubated with S.lacrymans 12C.

The results are shown in Table B: Table B first pre-inc. second inc. no. of mean organism time organism time samples weight (weeks) (weeks) loss (%) 1) Controls - 6 - - 4 -0.37 - - S.lacrymans 3 5 18.03 - - S.lacrymans 6 5 29.76 T.harzianum 2 - - 4 -1.58 T.harzianum 4 - - 4 0.16 T.harzianum 6 - - 4 -2.07 T.aureoviride 2 - - 4 -1.07 T.aureoviride 4 - - 4 0.28 T.aureoviride 6 - - 4 5.89 T.longi. 2 - - 4 -4.38 T.longi. 4 - - 4 -0.49 T.longi. 6 - - 4 -1.24 2) Results T.harzianum 2 S.lacrymans 3 5 -0.15 T.harzianum 4 S.lacrymans 3 5 0.00 T.harzianum 6 S.lacrymans 3 5 0.12 T.harzianum 2 S.lacrymans 6 5 -0.91 T.harzianum 4 S.lacrymans 6 5 -1.26 T.harzianum 6 S.lacrymans 6 5 -0.47 T.aureoviride 2 S.lacrymans 3 5 -0.84 T.aureoviride 4 S.lacrymans 3 5 -0.25 T.aureoviride 6 S.lacrymans 3 5 -1.73 T.aureoviride 2 S.lacrymans 6 5 -1.04 T.aureoviride 4 S.lacrymans 6 5 -0.81 T.aureoviride 6 S.lacrymans 6 5 5.12 T.longi. 2 S.lacrymans 3 5 -2.89 T.longi. 4 S.lacrymans 3 5 6.76 T.longi. 6 S.lacrymans 3 5 -1.37 T.longi. 2 S.lacrymans 6 5 -2.34 T.longi. 4 S.lacrymans 6 5 4.10 T.longi. 6 S.lacrymans 6 5 -0.98 Considering first the results of the controls, incubation with any one of the three Trichoderma spp. did not itself cause weight losses except in the case of the 6 week incubation with T.aureoviride. The explanation for this single anomalous result has not yet been determined though the raw data indicates that there was considerably more variation in replicates in this case than was normal.

Overall the results confirm that Trichoderma spp. do not cause weight loss of pine blocks.

The wood blocks incubated only with S.lacrymans showed a degree of weight loss in the range expected, viz. 18.03* at 3 weeks, 29.76* at 6 weeks. All blocks preincubated with any of the three Trichoderma spp. showed considerable protection, though weight loss, albeit reduced, was found in some blocks preincubated with T.longibrachiatum or T.aureoviride. T.harzianum therefore produced the most effective inhibition of weight loss. The weight losses associated with T.aureoviride were variable. In particular in the 6 week preincubation group individual blocks showed either substantial weight loss or no weight loss indicating perhaps an 'all or nothing' type protection.

It is possible that the results are due to two modes of action employed by the Trichoderma harzianum and aureoviride strain. The first is that T.harzianum is parasitising the S.lacrymans 12C. T.harzianum mycelia have to get through the mycelia on the surface and reach the mycelia inside the block which is producing the decay quickly enough to stop any serious decay.

The second possibility is that T.aureoviride is acting by producing a volatile substance, only some of which have been identified, e.g. 6-pentyl-pyrone.

Overall the experiment confirms that Trichoderma spp., and in particular T.harzianum, can protect wood samples from S.lacrymans decay for a period of up to 6 weeks, i.e.

the duration of this experiment.

Experiment 6 Experiment 6 was designed to see if the protective effect of Trichoderma spp. on wood requires viable organism or if the metabolites of Trichoderma spp., present after destruction of the organism, could have a similar protective effect.

For Experiment 6 the preincubation protocol used in Experiment 5 was repeated. However, after the appropriate period (2, 4 or 6 weeks), blocks were gamma irradiated to render the Trichoderma non-viable and then exposed to S.lacrymans. Again weight loss was determined at 3 to 6 weeks and control blocks were incubated only with S.lacrymans (i.e. no preincubation with Trichoderma).

This experiment was undertaken at the same time as Experiment 5 and therefore the Trichoderma control blocks were not repeated.

The results are shown in Table C.

Table C first inc. second inc. no. of mean organism time organism time samples weight (weeks) (weeks) loss(%) T.harzianum 2 S.lacrymans 3 5 -2.06 T.harzianum 4 S.lacrymans 3 5 -3.28 T.harzianum 6 S.lacrymans 3 5 -2.45 T.harzianum 2 S.lacrymans 6 5 -3.02 T.harzianum 4 S.lacrymans 6 5 -0.10 T.harzianum 6 S.lacrymans 6 5 0.01 T.aureoviride 2 S.lacrymans 3 5 -2.99 T.aureoviride 4 S.lacrymans 3 5 -4.50 T.aureoviride 6 S.lacrymans 3 5 10.97 T.aureoviride 2 S.lacrymans 2 6 -1.14 T.aureoviride 4 S.lacrymans 6 5 -5.31 T.aureoviride 6 S.lacrymans 6 5 11.64 T.longi. 2 S.lacrymans 3 5 3.56 T.longi. 4 S.lacrymans 3 5 5.14 T.longi. 6 S.lacrymans 3 5 -1.09 T.longi. 2 S.lacrymans 6 5 13.69 T.longi. 4 S.lacrymans 6 5 -2.90 T.longi. 6 S.lacrymans 6 5 3.08 - - S.lacrymans 3 5 9.36 (2 blocks undecayed) - - S.lacrymans 6 5 27.06 Blocks preincubated in the absence of Trichoderma, gamma irradiated and then incubated with S.lacrymans 12C for 3 weeks showed a 9.36% weight loss (this is an underestimate since 2 blocks were completely undecayed), a 6 week incubation gave a mean weight loss of 27.06%. Again preincubation of blocks with T.harzianum protected the blocks from subsequent S.lacrymans 12C induced weight loss, particularly after 4 or 6 weeks preincubation, similar results were found with T.aureoviride except for the 6 week preincubation period, the results with T.longibrachiatum were variable.

Since it is not clear whether Trichoderma kills S.lacrymans in wood (although this might be implied from the experiments involving agar) or whether it expresses some other mechanism of holding S.lacrvmans in check, isolation experiments have been conducted using the benomyl agar system discussed under Experiment 3. These were designed to determine if S.lacrymans can be isolated from infected blocks which have been decayed with Serpula for 2 weeks then incubated with T.harzianum or T.aureoviride for 2 to 4 weeks. If S.lacrymans could be isolated from such blocks then it would indicate that the organism was not being killed by the Trichoderma. Control blocks were also tested, these blocks having merely been incubated with Serpula.

The results showed that S.lacrymans cannot be isolated from the blocks incubated with Trichoderma but can from the control blocks. Such a result is consistent with the S.lacrymans being killed by Trichoderma. However, even if the S.lacrymans is not killed the result suggests that the organism is unable to move on to a fresh nutrient source and is being held in check by the Trichoderma.

We have also found by experiment that application of Trichoderma to wood already infected with S.lacrymans, and which shows significant weight loss, can result in further weight loss in the wood even though the S.lacrymans is checked or killed by the Trichoderma. The probable explanation is that metabolites of wood components partially digested by S.lacrymans have become readily accessible to digestive enzymes of Trichoderma.

Growth of Serpula lacrymans in experiments where filtrate from Hyalodendron cultures were mixed with normal growth medium in a ration of 50:50 resulted in complete inhibition of growth as compared to controls cultured on 100% normal growth medium.

Results with Trichoderma pseudokoningii indicated that when both this organism and S.lacrymans were grown on the same agar culture that the T.pseudokoningii rapidly killed and overgrew the S.lacrymans. The result obtained was similar to that found for both T.harzianum and T.aureoviride.

Similar results were found with a second isolate of S.lacrymans which was also effectively killed by T.pseudokonongii.

Claims (15)

1. A method for the treatment or prevention of S.

lacrymans infection in a material, the method comprising applying to said material an organism effective to treat or prevent said infection, or an effective substance obtainable from said organism.

2. A method according to claim 1 wherein said organism or effective substance is applied to material which is substantially not infected with S. lacrymans.

3. A method according to claim 1 or claim 2 wherein the organism is a Trichoderma species.

4. A method according to claim 3 wherein the organism is Trichoderma harzianum and/or Trichoderma aureoviride and/or Trichoderma pseudokoningii.

5. A method according to claim 1 or claim 2 wherein the organism is a Hyalodendron species.

6. A method according to any one of the preceding claims wherein said organism or effective substance is applied in powder or liquid form, by dusting, brushing, spraying or impregnation.

7. A method according to any one of the preceding claims wherein the material to which said organism or effective substance is applied is wood.

8. A fungicidal agent against S. lacrymans comprising a Trichoderma and/or a Hyalodendron organism or an effective substance as obtainable therefrom.

9. A fungicidal agent according to claim 8 comprising Trichoderma harzianum or an effective substance as obtainable therefrom and/or Trichoderma aureoviride or an effective substance as obtainable therefrom and/or Trichoderma pseudokoningii or an effective substance as obtained therefrom.

10. A fungicidal agent according to claim 8 or claim 9 in powder or liquid form.

11. The use of a Trichoderma species in the manufacture of a fungicidal agent against S.lacrymans.

12. The use of a Hyalodendron species in the manufacture of a fungicidal agent against S.lacrymans.

13. A method for the treatment or prevention of S.lacrymans infection in a material substantially as herein described and exemplified.

14. A fungicidal agent against S. lacrymans substantially as herein described and exemplified.

15. Use of a Trichoderma or Hyalodendron species in the manufacture of a fungicidal agent against S. lacrymans, substantially as herein described and exemplified.