EP0743821A1

EP0743821A1 - Trichodermas and metabolites as biological control agents

Info

Publication number: EP0743821A1
Application number: EP95907158A
Authority: EP
Inventors: Robert Anthony Hill; Horace G. Cutler; Stephen Robert Parker
Original assignee: Horticulture and Food Research Institute of New Zealand Ltd; US Department of Agriculture USDA
Current assignee: Horticulture and Food Research Institute of New Zealand Ltd; US Department of Agriculture USDA
Priority date: 1994-02-07
Filing date: 1995-02-03
Publication date: 1996-11-27
Also published as: WO1995020879A2; AU1547495A; JPH09508899A; WO1995020879A3; NZ250838A

Abstract

The present invention is directed to the use of (i) members of the family Trichoderma, and (ii) metabolites such as 6-pentyl-α-pyrone, delta-decanolactone, and massoialactone; as well as combinations thereof for controlling a range of afflictions of plants including, for example, botrytis, armillaria, silver leaf, and phytophthora. Various embodiments include methods for the prevention of a range of afflictions, as well as methods for the treatment and control of those afflictions. Methods of treating plant growth media are also discussed.

Description

TRICHODERMAS AND METABOLITES AS BIOLOGICAL CONTROL AGENTS

TECHNICAL FIELD

The present invention is directed to the use of biological control agents, and metabolites therefrom, for the control of fungal and microbial activity. The present invention will be directed primarily to the use of members of the genus Trichoderma, especially those producing as a metabolite at least one of 6-pentyl-α-pyrone (also known as 6-amyl-α-pyrone), delta-decanolactone, and massoialactone.

BACKGROUND ART

Synthetic fungicides are predominantly used for the control of fungi on crops. However many exhibit other toxic effects and could face future removal from the marketplace as controls and regulations governing agricultural chemicals tighten. In other instances, the public trend towards natural products may cause consumer resistance to the use of 'perceived' synthetic and non-natural substances.

Another problem of the art is the growing resistance of many targeted organisms substances to commonly used control agents. Accordingly there is a need for further alternatives to the currently used controlling agents, and ideally an alternative to existing control agents to which little resistance is exhibited by fungi and/or microbes.

ArmiUaria, a fungal pathogen of forest trees, was first identified 115 years ago, and is now recognised as a major problem in a variety of woody plant species world-wide. More than 500 different plant species are known to be susceptible to the organism. In undisturbed forests and native bush the organism rarely causes serious damage, but when trees are harvested, the rotting stumps and roots provide a rich source of nutrients so that the Armillaria may become destructively infective to any remaining shrubs and trees. Armillaria can be devastating to the forest industry, and billions of dollars are lost annually due to affected timber. The worst losses follow reforestation after clearing the natural tree cover. In New Zealand first time losses from less than 5 to over 90% for Pinus radiata, a major timber crop for local and export markets, have been attributed to Armillaria.

Another major crop affected by Armillaria is kiwifruit, in which the orchards are generally planted on cleared lands. Armillaria was listed as a new disease for kiwifruit in New Zealand in 1955. However, the first detailed account of Armillaria infection in kiwifruit was in a US Department of Agriculture orchard in California; and it described the decline and death of the vines from 1967-1971. Before 1980, the incidence of Armillaria in New Zealand kiwifruit was only occasional and it was considered to be a minor phytopathogen. Between 1980 and 1990 a dramatic increase occurred in the number of infected orchards, and the industry suffered as a consequence. Significant industry losses are expected if the disease continues to spread. Both kiwifruit and Pinus radiata are major export crops for New Zealand and treatment of these commodities with synthetic pesticides is unacceptable to many export markets, and the public consumer. Insofar as controlling Armillaria is concerned, Leach in 1936 reported some benefit from ring-barking forest trees; however, this is not advisable in New Zealand because willow trees treated this way appear to have high incidence of Armillaria, and willow is one of the shelter trees used in kiwifruit orchards.

Other plant pathogens are also commercially damaging. For instance Botrytis cinerea is well known for its effects on grapes and its responsibility for afflictions such as kiwifruit storage rot, as well as grain mould of grapes and strawberries, etc. Botrytis diseases are among the most common and most widely distributed diseases of vegetables, ornamentals, fruits and glasshouse crops throughout the world. In New Zealand, Botrytis cinerea attacks many economically important horticultural crops such as kiwifruit, bean and strawberry, and in particular is the causal agent of two important diseases - grey mould of tomato and bunch rot of grape. In the past, control of these diseases has relied extensively on the use of benzimidazole and dicarboximide groups of fungicides. however, the development of fungicide resistance has reduced the effectiveness of these chemicals and thus alternative control measures are required.

Phytophthora also effects commercially important crops and is responsible, among other things, for crown rot of apples. Again, while commercially available chemicals have been used to address problems associated with Phytophthora, there is a need for an improved substitute for currently available agents which are generally only partially effective against this family.

Silver-leaf is another plant disease for which currently available methods are only partially effective. The four general diseases (Botrytis, Armillaria, silver-leaf and Phytophthora) are characterised in that they all affect commercially important crops, and are difficult to control using commercially available agents, which are generally chemical based fungicides.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only. DISCLOSURE OF INVENTION

According to one aspect of the present invention there is provided a method for the control or prevention of at least one member of a group of targeted afflictions comprising botrytis, armiUaria, silver leaf, and phytophthora, said method comprising the administration of at least one member of an "active Trichoderma metabolite" group comprising 6-pentyl-α-pyrone, delta-decanolactone, and massoialactone.

According to a further aspect of the present invention there is provided a method for the control or prevention of at least one member of a group of targeted afflictions comprising botrytis, armiUaria, silver leaf, and phytophthora, said method comprising the administration of at least one active member of the Trichoderma family which produces at least one "active Trichoderma metabolite" of a group comprising 6-pentyl-α-pyrone, massoialactone, and delta-decanolactone.

According to another aspect of the present invention there is provided a method substantially as described above in which the administered substances include both:

- at least one member of an "active Trichoderma metabolite" group comprising delta- decanolactone, 6-pentyl-α-pyrone, and massoialactone, and

- at least one active member of the Trichoderma family which produces at least one "active Trichoderma metabolite" of a group comprising 6-pentyl-α-pyrone, delta- decanolactone, and massoialactone.

According to yet a further aspect of the present invention there is provided a method for conferring, to plants, resistance to at least one of a group of targeted afflictions comprising botrytis, armiUaria, silver leaf, and phytophthora, said method comprising the establishment of an active population of at least one member of the Trichoderma family which produces at least one of a group of Trichoderma metabolites comprising massoialactone, 6-pentyl-α-pyrone, and delta-decanolactone, in either or both the plant or its root zone.

According to an even further aspect of the present invention there is provided a method of treatment of plant growlh media to address at least one of a group of targeted afflictions comprising botrytis, armiUaria, silver leaf, and phytophthora, said method comprising the introduction into the growth media of either or both:

- at least one active member of the Trichoderma family which produces at least one "active Trichoderma metabolite" of a group comprising 6-pentyl-α-pyrone, delta- decanolactone, and massoialactone. According to yet a further aspect of the present invention there is provided a composition for the control of at least one member of a group of targeted afflictions comprising botrytis, armillaria, silver leaf, and phytophthora, said composition comprising at least one member of an "active Trichoderma metabolite" group comprising delta-decanolactone, 6-pentyl-α-pyrone, and massoialactone, in combination with at least one active member of the Trichoderma family capable of producing one or more "active Trichoderma metabolites".

According to yet a further aspect of the present invention there is provided treated plant growth media comprising media capable of supporting the growth of a plant to which has been introduced either or both:

According to yet a further aspect of the present invention there is provided a method of protecting seedlings against plant disorders, said method comprising the application to the roots of said seedlings by dusting, spray, immersion, or other means, either or both of: - at least one member of an "active Trichoderma metabolite" group comprising delta- decanolactone, 6-pentyl-α-pyrone, and massoialactone, and

The term "active Trichoderma metabolite" wherever used herein refers to a metabolite of members of the Trichoderma family which exhibits activity in controlling or adversely affecting the growth, establishment and/or existence of, a targeted affliction. More specifically the group comprises 6-pentyl-α-pyrone, delta-decanolactone, and massoialactone. The terms "targeted affliction" shall preferably refer to a member of the group comprising "Botrytis, Armillaria, silver-leaf, and Phytophthora". It should also be appreciated that, many of the metabolites described herein will exhibit useful activity against other disorders and fungal afflictions and therefore use of the present invention need not be limited to the targeted afflictions. For instance, problems of other crop pathogens such as Nectria galligena, Sclerotium rolfsii, Rhizoctonia solani, Sclerotium cepivorum, Macrophomina phaseolina, Fusarium oxysporum, Verticillium albostrum, Chondrostereum purpureum, Scletotinia sclerotiorum, Pythium ultimum and Corticum rolfsii may also be addressed.

Investigative research by the applicants have established that certain Trichoderma metabolites are particularly effective against the aforesaid "targeted afflictions". Trials have indicated the effectiveness of the "active Trichoderma metabolites" against at least Botrytis cinerea. Further trials, and parallels in the prior art, have indicated that these results may be extrapolated to the control of other members of the group of targeted afflictions as well as to the other plant disorders and fungal afflictions listed above, in many instances.

The preferred metabolite compounds comprise 6-pentyl-α-pyrone, delta-decanolactone and massoialactone. These may be effectively used by application of the substantially pure compound to plants and plant matter. This will depend upon the situation; in some instances injection of the composition into the sapwood may be preferred, whereas on kiwifruit picking wounds, spraying or dabbing a composition in the region of the picking wound would be preferred. Compositions according to the present invention may include one or more of the "active Trichoderma metabolites". These may also be derived from a number of sources - for instance 6-pentyl-α -pyrone can be isolated from various Trichoderma organisms, or the synthetically produced 6-amyl-α-pyrone may also be substituted. Similarly, the other "active Trichoderma metabolites" may be synthetically produced or extracted from natural sources - various biosynthesis and other techniques may also be reUed upon.

While the active metabolites may be relied upon for control, the metabolite producing organisms may also be used as part of a delivery system. Introduction of Trichoderma family members to plants and crops is another alternative, as is the combination of the metabolite producing members in conjunction with supplemental metabolite. However, it is envisaged that often the use of 'living' control compositions will be predominantly used for plants rather than harvested crops, though exceptions may exist.

Observations by the applicants during their work include that micro-organisms grown in vitro produce the sought metabolite within a fairly narrow window of time, maybe as little as 2 to 3 days, and is then seen no more. However, in the natural state, where nutrients are abundant, they often produce secondary metabolites in a continuum which appears to last some weeks or even longer. This is the case in certain biocontrol situations.

Accordingly, where control agents comprising metabolite producing Trichoderma are applied to plants or their growth media, one is more likely to counter the extended metabolite producing window described above. In some instances it is more beneficial to include nutrients with a control composition to assist the establishment of the Trichoderma members once applied. It may be preferable to introduce, or co-apply, the nutrient at the time of application of any control agent. Another observation is that the active organisms readily secrete the desired metabolites into their immediate surroundings with relative facility; and in addition, they may deliver the materials to critical active sites and tissue conduits. Compositions containing active metabolite producing organisms may therefore provide an efficient delivery system for many applications. An applied metabolite, while sufficient for many situations such as treating fruit picking wounds, may not always provide the period of activity that a 'living' composition may. The continuing and residual activity of the active micro-organism containing embodiments will therefore find use in many situations and may provide considerable advantage over commonly used techniques and substances.

It should also be appreciated that in some cases a combination of metabolite and active metabolite producing .organisms may be relied upon. A composition which immediately provides active metabolite, perhaps in a relatively high level, to the plant or other substrate may be required in certain situations. However, there may also be a need for the continuing presence of a metabolite, for a period exceeding that for which an isolated metabolite would typically remain in place or available.

Many Trichoderma family members have been used successfully in field trials to control various crop pathogens. Examples include Nectria galligena in apples, Sclerotium rolfsii in tobacco, bean, iris; Rhizoctonia solani in radishes, strawberries, cucumbers, potatoes, and tomatoes; Sclerotium cepivorum in onions; Macrophomina phaseolina in maize, melons, beans, and other economically important crops; Fusarium oxysporum in tomatoes and Chrysanthemum and Verticillium albo-strum in tomatoes; Chondrostereum purpureum in pip fruit, stone fruits and other crops; and Botrytis cinerea in apples, kiwifruit.

ARMILLARIA CONTROL

Trials have indicated the more effective biological control agents for Armillaria in New Zealand to include isolates of Trichoderma hamatum, T. harzianum, T. viride, and other Trichoderma spp. - particularly those collected from Armillaria-infected orchards and forest sites in the Bay of Plenty. Some Trichoderma strains were growing on and consuming ArmiUaria mycelium and rhizomorphs. On transfer to the laboratory, in vitro tests confirmed the activity of the Trichoderma isolates against Armillaria; and as the result of many tests, superior strains were selected for field use, and different fermentation and formulation technology is presently underway.

In research the in vitro interactions between Trichoderma isolates and Armillaria novaezelandiae using dual plate techniques and visualisation with a light and scanning electron microscope, 11 potentially superior isolates of Trichoderma were evaluated. These included strains of T. hamatum, T. harzianum, and T. viride, and the evaluations covered two major points: the antagonistic potential against Armillaria and the compatibility of the Trichoderma isolates with each other so that they could be used in an inoculum blend. All the Trichoderma isolates antagonised Armillaria in dual culture; and the antagonism was manifest by the formation of brown residues on the surface of the Armillaria mycehum, yellowing of the Armillaria mycelium, overgrowth of the Armillaria by Trichoderma. and extensive rhizomorph initiation of the Armillaria colony. Importantly, there were differences in the antagonistic response of the accessed Trichoderma isolates to ArmiUaria; and in vitro cultures of T. harzianum were easily overgrown by T. hamatum and T. viride in paired assays.

In addition to the above observations, a temperature effect on the antagonism between Trichoderma and Armillaria was noted. The greatest antagonism was exhibited by T. hamatum and T. viride isolates between 20 and 25°C, while T. harzianum isolates were predominantly effective at 25°C. There was also a pH effect on the antagonism between Trichoderma and ArmiUaria; and this was greatest at a basic pH on malt extract agar, while on tap water agar acidic conditions were generally more favourable. Furthermore, the germination of Trichoderma spores on a low nutrient medium was enhanced under acidic conditions. There was competition for nutrients between Trichoderma and Armillaria in dual culture due to differences in the relative growth rates. Interactions between Trichoderma and ArmiUaria rhizomorphs indirectly indicated that hyperparasitism may be part of the control mechanism.

ACTIVE TRICHODERMA METABOLITES

Antibiotics were produced by some of the Trichoderma isolates in vitro in the New Zealand experiments, and antibiosis was detected using liquid culture and split plate techniques. However, the ability of the Trichoderma isolates to produce volatile and non-volatile antibiotics was found to differ within and between species. The culture filtrates of some of the isolates were also found to be inhibitory towards the growth of Armillaria.

Structure 1. Structure of 6-pentyl-α-pyrone (= 6-amyl-α-pyrone)

Research has established that various Trichoderma species produce a number of antibiotics. The most common of these is 6-pentyl-α-pyrone (Structure 1) which has potent antifungal activity. Its coconut/celery-like odour permeates the atmosphere on isolation, and can be easily detected in Trichoderma cultures by sniffing. In vitro assays with 6-pentyl-α-pyrone has shown, for example, that a 1:40 dilution of the metabolite applied at the rate of 15 μl/4 mm disk inhibited the growth of Aspergillus flavus, a producer of aflatoxins. Both T. lignorum and T. viride produce trichodermin (4β-acetoxy-12-13-epoxy trichothec-9-ene) - a natural product that has marked antibiotic effects against many fungi, including Candida albicans - but is relatively inactive against bacteria (Structure 2). Unfortunately, it also possesses plant growth regulatory properties and is selectively toxic to certain herbaceous plants. However, it has relatively low toxicity in mice (LD₅₀ 1g/kg orally) compared to its congeners, and at one time was considered by the pharmaceutical trade to be a candidate antibiotic. A variety of other Trichoderma metabolites with biological activity have subsequently been discovered and are discussed later (vide infra).

Further Trichoderma metabolites are massoialactone (also known as massoilactone), and ±delta-decanolactone. Preliminary trials by the applicants have indicated useful activity by massoialactone against targeted afflictions. As for 6-pentyl-α-pyrone, metabolites such as massoialactone may be introduced by the establishment of Trichoderma species i.e. by the establishment of the population of a massoialactone producing species of Trichoderma. It should be appreciated that the use of massoialactone, delta-decanolactone and many of the other "active Trichoderma metabolites", will be analogous to the use of 6-pentyl-α-pyrone producing Trichoderma species, for which many examples are given herein. It should also be appreciated that compositions containing a variety of "active Trichoderma metabolites" and/or active metabolite producing Trichoderma species, may all be prepared and used according to the present invention.

Structure 2. Structure of trichodermin (4β-acetoxy-12-13-epoxy

trichothec-9-ene)

OBSERVED EFFECTS OF TRICHODERMA AND 6-PENTYL-α -PYRONE ON ARMILLARIA DISEASE IN PINUS RADIATA

Crude extracts from Trichoderma containing 6-pentyl-α-pyrone and synthetic 6-pentyl-α-pyrone (hereafter referred to as 6-amyl-α-pyrone to distinguish between the 'natural' product and the 'synthetic copy' of the natural product) were evaluated with in vitro assays against Armillaria novae zelandiae. Potent anti-microbial activity was seen with as little as 4 μl per disk with 6-amyl-α-pyrone, concomitantly the crude extract was active. Other micro-organisms were also strongly inhibited; and these included Botrytis cinerea, Scletotinia sclerotiorum, Chondrostereum purpureum, Phytophthora fragariae, Pythium ultimum, and Corticium rolfsii, all important phytopathogens. These results led to field trials in their respective crops of importance.

As an initial step, because Trichoderma treatments appeared to be an efficient delivery system for 6-pentyl-α-pyrone to the necessary sites, selected Trichoderma spp. isolates were tested in laboratory assays with Pinus radiata tissue cultured plantlets. No pathogenicity or toxicity was seen, except in very aged cultures where nutrients were exhausted. Following this, private forest trials were initiated in summertime in New Zealand, and following treatment with Trichoderma. treated trees showed less mortaUty and were more vigorous compared to control treatments. Far fewer treated trees (5.9%) were infected and died from Armillaria compared with controls (22%) (P < 0.019). Treated trees were taller and had thicker trunks and wider canopy than untreated trees. Consequently, another 50ha of P. radiata have been treated with Trichoderma and various combinations of Trichoderma and 6-amyl-α-pyrone to determine effects on Armillaria and enhancement of vigour.

EFFECTS OF TRICHODERMA AND 6-PENTYL-α-PYRONE/6-AMYL-α-PYRONE ON ARMILLARIA DISEASE IN KIWIFRUIT

The stumps of shelter trees that had been cut down and were possible sources of Armillaria infection have been treated with Trichoderma formulations. Soil amendments have inhibited or prevented the spread of the organism within kiwifruit orchards, and in addition soil treatment in barrier trenches between infectious Armillaria sites and kiwi plantings have been very successful. Soil drenches, too, have been effective. Injections with formulations of Trichoderma directly into the trunks of kiwi vines have shown that infected plantings may recover; and pastes made up of Trichoderma applied directly to infected areas, where as much as four fifths of the vascular cambium has been destroyed, have completely healed the vines. As the vascular cambium grew, the vines regained their lost vigour and become productive. Root treatments with Trichoderma have reduced mortality in kiwifruit vine replants at diseases sites from approximately 50% of untreated plants to 5% of treated ones. Selected Trichoderma isolates have also been evaluated for antifungal use on stored kiwifruit, and Botrytis cinerea was totally inhibited. Other storage organisms, including Scletotinia sclerotiorum, treated with species of Trichoderma and Gliocladium were successfully controlled for the first time in kiwifruit.

Armillaria infected kiwifruit vines in the Bay of Plenty were injected in February with treatments ranging from 10 to 100 μl per vine of 6-amyl-α-pyrone; 10 to 50 μl per vine of 6-pentyl-α-pyrone (the natural product is more difficult to obtain in quantity relative to the synthetic material); and 300 μl of a crude extract, known to contain 6-pentyl-α-pyrone, from a high yielding isolate of T. hamatum. Other infected vines were injected with a mixed strain Trichoderma formulations with proven efficacy against Armillaria. All untreated Armillaria infected vines died within 6 months. Both 6-amyl-α-pyrone and 6-pentyl-α-pyrone treatments significantly increased the survival rate (to -50%) in infected vines. However, Trichoderma formulations were even more effective, and over 80% of the infected vines survived; while the crude extract was approximately as active as the 6-amyl-α-pyrone and 6-pentyl-α-pyrone.

OBSERVED EFFECTS OF TRICHODERMA ON THE CONTROL OF SILVER-LEAF DISEASE

Effective disease control using high 6-pentyl-α-pyrone producing strains of Trichoderma. especially T. hamatum, has been achieved in the North Island of New Zealand against silver-leaf disease (Chondrostereum), an organism that was controlled in vitro by Trichoderma isolates in laboratory assays. Injections with liquid formulations of Trichoderma gave rapid control of silver-leaf in Pyrus serotinia (nashi, Asian pear) with even severely affected trees recovering completely. Most treated trees remained disease free for two years following treatment. In addition, a pruning paste containing Trichoderma greatly reduced the spread of silver-leaf in infected nashi orchards.

POSTHARVEST TREATMENTS

Horticultural produce may be treated with fungicides immediately following harvest to increase shelf life. This is a critical stage because the treatment may be persistent; and depending on the nature of the fungicide, the implications as far as the consumer is concerned may be of enduring consequence. Some biocontrol alternatives to synthetic fungicides have been evaluated and the chemistry studied in some detail.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from the ensuing description which is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 is a graph - Botrytis trials first assessment - week one;

Figure 2 is a graph - Botrytis trials first assessment - week two;

Figure 3 is a graph - Botrytis trials second assessment - week one;

Figure 4 is a graph - Botrytis trials second assessment - week two;

Figure 5 is a graph - Botrytis trials third assessment - week one;

Figure 6 is a graph - Botrytis trials first assessment - week two; Figure 7 incidence of Botrytis storage rot on kiwifruit after three months in cool storage - trial one

Figure 8 incidence of Botrytis storage rot on kiwifruit after three months in cool storage - trial two Figure 9 table of the inhibition of various fungi by differing concentrations of various Trichoderma metabolites

BEST MODES FOR CARRYING OUT THE INVENTION

Trials directed to the control of Botrytis cinerea were conducted comparing the use of 6AAP (6-amyl-α-pyrone)) with other substances. The use of thyme oil, which has exhibited some effectiveness against certain fungi, was included in the trials. Also included was the commercial fungicide marketed under the name RONILAN®. The results of three trials, indicated as first, second and third assessments, were performed, and the results are summarised in tables 2 - 4 herein. Figures 1 through 8 are graphical representations of data accumulated during the trials.

The trials involved the mechanical application of droplets of 6-pentyl-α-pyrone (6PAP) and its synthetic equivalent 6-amyl-α-pyrone (6AAP) applied at various rates to the picking wound of kiwifruit.

Experiments have also yielded an extraction test which is suitable for determining the amount of 6-pentyl-α-pyrone in Trichoderma samples. This experimental procedure is outlined below and includes details of the typical 6-pentyl-α-pyrone contents of various Trichoderma samples. As can be appreciated, strains exhibiting higher levels of 6-pentyl-α-pyrone production will be preferred in compositions and methods according to the present invention. With reference to table 1 herein, strains exhibiting 7.5mg/kg of 6PAP for methanol-water extractions solvent will generally be most suitable. Strains exhibiting much higher levels, typically 25mg/kg or higher will typically be preferred in most embodiments of the present invention. However, also to be taken account of, is the period over which a particular strain will produce 6-pentyl-α-pyrone should also be taken into account.

For the purposes of the experimental procedures, the following codes are used:

Trichoderma hamatum OG3 Trichoderma koningii NZ164/US

Trichoderma hamatum Hend Trichoderma harizanum US2/NZ

Trichoderma koningii US3/NZ Trichoderma hamatum HPP1

Trichoderma harizanum D Trichoderma viride TV

Trichoderma hamatum TBHPP7 Trichoderma hamatum GT4

Trichoderma hamatum KEK EXPERIMENTAL

Extraction method tests

The method PAP-1 may be summarised as follows:

1. Take a subsample of solid, typically 10g.

2. Blend sample with a mixture of water plus methanol.

3. Filter.

4. Partition an aliquot of extract into cyclohexane after diluting with buffered saline solution.

5. Centrifuge to give phase separation.

6. Filter the supernatant organic layer through anhydrous sodium sulphate and collect.

7. Analyse by GC/FIC.

Aspects of this procedure were tested.

Extraction solvent tests

An initial screen of the 11 samples was done by extracting 10g subsamples of each sample with two different solvent systems, namely; water (17ml) plus methanol (50ml) as for PAP-1, and 80:5:15 acetonitrile-methanol- water (50ml). After blending, each mixture was filtered under vacuum.

It was noted that while the acetonitrile-methanol-water extractant gave mixtures which clarified rapidly on standing and filtered rapidly, the water-methanol extract mixtures did not readily clarify, were slow to filter (typically 20-30 min) and yielded cloudy filtrates. The use of a filter aid (celite) allowed these water-methanol extracts to be filtered in 1-2 min.

Partition solvent tests

Three alternative partition solvents, namely; cyclohexane, ethyl acetate and toluene, were tested using the sample water-methanol extracts.

Extraction solvent v/s partition diluent test

Some preliminary tests with water as the partition diluent instead of buffered saline solution showed that phase separation was often difficult or unobtainable with the acetonitrile-methanol-water extracts. The two T. koningii extracts produced gels which were not broken by centrifugation

A detailed investigation into the affect of extraction solvents and saline concentration on the method and 6PAP analytical results was carried out using subsamples of the T. koningii culture NZ164/US. The solvents tested were different mixtures of methanol, water and acetonitrile, and the saline concentrations used were 10%, 5% and 2.5%. Each test was done in duplicate.

Reproducibility test with new extractant solvent and method

During the course of these studies a new extractant solvent, 85:15 methanol-water, was developed to replace the mixture recommended in PAP-1. The extract mixture was no longer filtered, but was allowed to clarify while standing. The partition step was as described in PAP-1, and after separation of the phases, the cyclohexane layer was dried and analysed as in PAP-1.

The reproducibility of this new method was tested using samples TBHPP7 and NZ164/US. Five replicate subsamples of each were extracted, and each extract solution worked-up and analysed in duplicate.

Fractionation of extract on silica

A 20ml sample from extraction of NZ164/US with 85:15 methanol-water was partitioned into cyclohexane according to the method (see 4.1). The total cyclohexane fraction was collected, evaporated and weighed, than re-dissolved in cyclohexane (4ml) and applied to a column of dry silica (Dabisil) (2g in a 8mm I.D. column). A further 6ml of cyclohexane was passed through the column and the eluate collected as F1. Six further fractions (F2-F7) were collected by eluting the column with 50ml, of each 10:90, 20:80, 50:50, 75:25, ethyl acetate-cyclohexane, ethyl acetate, and 90:10 ethyl acetate-methanol. All fractions were evaporated and the residues weighed before re-dissolving in 2ml of cyclohexane for analysis.

RESULTS

Extraction solvent test

Initial test

Results of the comparison of two extraction solvent systems is shown in Table 1. The estimated values for 6PAP concentrations in the samples were similar for the two systems, although higher values were obtained using the methanol-water extraction system described by Klaffenbach (PAP-1).

It was noted that while the acetonitrile-based solvent system gave extracts which clarified rapidly and did not need filtering, there were problems at the partition step. For some samples an emulsion formed which was difficult to break even with centrifugation, and this may have contributed to low estimations for 6PAP. The methanol-based system, on the other hand was slow to clarify and difficult to filter without a filter aid.

Partition solvent tests

Results with ethyl acetate and toluene were similar to those with cyclohexane. Cyclohexane remained the solvent of choice.

Test of alternative extraction solvents and partition diluents

The initial tests (3.1.1) had shown practical problems with both solvent systems tested, especially when applied to cultures of T. koningii such as NZ164/US. A further series of tests were conducted to establish a better extraction/partition system. The results are shown in Table 2.

Points to note are:

a) Clarification rate of extracts was 5>>4>3>2> 1.

b) The use of acetonitrile at levels of 50% and higher caused problems with the saline partition, often giving three phases instead of two. For 50% acetonitrile this occurred with the 10% saline, but with 84% acetonitrile it occurred with the 10 and 5% saline solutions.

c) When the extraction solvent contained acetonitrile, the 6PAP concentration obtained increased with lower saline strength. d) The 85: 15 methanol-water extractant gave the most consistent set of 6PAP values.

As a result of these tests, the extraction solvent of 85: 15 methanol-water was used for the reproducibility test.

Reproducibility of analytical method for 6PAP

Five replicate subsamples of each of two samples were extracted with 85: 15 methanol-water and analysed for 6PAP concentration in duplicate. The results are presented in Table 3, and show that the method gave good reproducibility.

range 61-94 198-214 mean 80.7 205.3

% variance 12.4 2.4

Silica fractionation

A 20ml aliquot of extract from NZ164/US (Table 3, extract 3) was used for this experiment. Based on the results in 3.1.4, this should yield 0.8mg of 6PAP. The total weight of extract residue partitioned into cyclohexane was 17.5mg. After silica fractionation the total weight recovered was 13.8mg, which was found mainly in F2 (39%), F3 (29%), F4 (7%) and F7 (14%). The total 6PAP recovered was 0.54mg, which was found in F2 (87%) and F3 (13%). All other GC/FID peaks observed in the original extract were also eluted in F2 and F3.

RECOMMENDED METHOD FOR 6PAP ANALYSIS

Extraction and workshop

a) The sample (10g) is blended with 85: 15 methanol-water (50ml) at high speed for 2 min, and the mixture decanted into a boiling tube.

b) The sample is allowed to stand for lhr or until there is a clear supernatant.

c) An aliquot (4ml) of extract solution is added to 10% buffered saline solution

(10ml), then cyclohexane (2ml) added, and the mixture shaken and allowed to settle,

d) A portion of the cyclohexane layer is filtered through anhydrous sodium sulphate and then analysed by GC/FID.

e) When analysing for 6PAP along use programme A below; when screening for other peaks use programme B.

GC conditions

Column: 25 m HP-5, 0.2 mm i.d., 0.33 μm film

Detector: FID at 280°C

Injector: 280°C, split 1:20, 1μl injection

Programme: A: 160°C for 5 min, then to 230°C at 30°C/min, hold 10 min, reset.

Retention time for 6PAP is 3-4 min.

B: 120°C for 12 min, then to 230°C at 30°C/min, hold 10 min, reset.

Retention time for 6PAP is 11-12 min.

The present invention can be put into practice in many ways. In some embodiments the invention may be used as a curative measure to address plants already infected by the targeted afflictions. In other cases, the invention may find a preventative role, acting to prevent the targeted afflictions establishing themselves in various plants and produce. The role in which the invention is to be used will have some bearing, in most instances, on the particular embodiment which may be relied upon by the user.

For instance, in curative type roles, compositions containing "active Trichoderma metabolites" such as 6-pentyl-α-pyrone, delta-decanolactone and massoialactone may be sufficient in their own right to address targeted afflictions present in plants. However, in most cases such preparations are relatively short lived in their effect (e.g. they may be washed from foliage), unless means is provided to maintain the presence of sufficient levels of the active metabolite in the required portion of the plant. The use of non-aqueous constituents in compositions, as well as many other known means of retarding the dilution or removal of active constituents, may be relied upon.

As another option, a composition may seek to establish a population of a "active Trichoderma metabolite" producing organism though in a curative role. Supplementing such compositions with added active metabolite (regardless of its source) is yet a further option. This ensures that active components are immediately available to the plants. A relatively high initial concentration of 6-pentyl-α-pyrone and/or other metabolites may be more effective in reducing the level of the organisms responsible for the plant affliction to manageable levels, which can thereafter be controlled by the establishment of a population of "active Trichoderma metabolite" producing organisms.

Where the present invention is used in a preventative role, the higher initial levels of active metabolites such as 6-pentyl-α-pyrone may not be required and thus the establishment of a population of active metabolite producing organisms may be sufficient to provide long term and lasting control of targeted afflictions. As can be appreciated, according to the needs of the user, the use of "active Trichoderma metabolite" producing organisms, and combinations of the two may be relied upon.

In some embodiments of the present invention, compositions based on the foregoing description may be introduced directly into plant tissue, and in woody plants this is typically the sapwood. For seedlings or where direct introduction into plant tissue is not practical, introduction into the root zone may be satisfactory. In many instances the establishment of an effective "active Trichoderma metabolite" producing population in the root zone of a plant may be readily established. This may be accomplished, by way of example only, by the introduction of suitable organisms into soil or growing media, the treatment of potting and seedling mixes, the coating of seeds, and the roots of seedlings in treated compositions etc. Many other methods, including the application of various compositions to external surfaces of the plant may be relied upon. Some various examples follow:

Example 1

A composition including a live population of a Trichoderma species producing an "active Trichoderma metabolite" is introduced into the sapwood, or equivalent, of a plant. Typically this is by injection though introduction into an incision is another of many possible techniques. The quantity and nature of the introduction should be such that growing population of the Trichoderma species is established within the plant.

Compositions may comprise more than one Trichoderma species and the various Trichoderma species need not produce the same metabolites nor the same metabolites in the same proportions.

As a variation, non-Trichoderma species which are capable of producing "active Trichoderma metabolites", and which do not show any pathogenic tendencies towards the plant, may be included.

Example 2

To the composition of example 1, is included at least one "active Trichoderma metabolite". Particular metabolites of interest include 6-pentyl-α-pyrone, massoialactone, and delta-decanolactone.

Example 3

A composition comprising one or more "active Trichoderma metabolites" is used for this example. Typically such a composition will differ from the composition of example 2 in that there is substantially little, or no, living Trichoderma material in the composition. Generally these compositions are used primarily in a curative or controlling role rather than a preventative or long term control role. Methods of use may be as described for examples 1 and 2 or the other examples herein.

Example 4

The compositions of examples 1, 2 or 3 are applied to the foliage and/or reproductive material of a plant. In this case reproductive material will often include the fruit or seed bearing portions. In an artificial environment or where there is human intervention, reproductive material shall also include cuttings, and various portions used for propagation. Typically application is by spray, dipping, dusting or some other coating process. Example 5

This method generally uses the compositions of examples 1 and 2 though the composition of example 3 may be used where long term or continuing effects are not desirable or necessary. According to this method compositions are introduced into the immediate interactive environment of the targeted plants, which generally means the soil and root zone.

Methods of application include drenching of plant growth media, which will be suitable for established plants. Mulches and fertiliser compositions containing the compositions of the present invention may also be relied upon to introduce the active metabolites and/or Trichoderma species into the plants' environment.

The preparation of potting mixes and other growth media which have been fumigated and/or inoculated with the various compositions of the present invention are other means to introduce. the preferred agents into the plants' environment. It is also envisaged that where the population of Trichoderma species is established in the root zone of seedlings, the seedlings when transplanted will carry along sufficient living organisms to establish a new population in the new site.

For instance, for Pinus radiata seedlings, their propagation in growth media containing a Trichoderma population would be relied upon. When the seedlings were transplanted to their final growth site, a living Trichoderma population would be carried over with it. Depositing some of the seedlings' growth media when planting seedlings in their new site would assist the establishment of the new Trichoderma population. The further application of a Trichoderma containing composition immediately prior to re-planting could be used to further enhance the effects. Applying an active metabolite containing composition may also be useful.

Example 6

The composition substantially as described in examples 1 through 3 is used to treat wounds on plants. Typically such compositions will be fluid or paste-like so that they may be applied to wounds such as caused through pruning. Incorporation of non-aqueous or hydrophobic components may also be relied upon to resist washing of the active metabolites and/or living organisms from the region of application.

Compositions for wound application may also provide a suitable growth media for establishing a population of a Trichoderma species. Nutrients and a suitable support (such as use of a paste which dries or sets to provide a cap or cover for the wound) are considerations which may be addressed in the various compositions. Example 7

A method for protecting seedlings against plant disorders comprising the introduction to the roots of the seedlings, either or both "active Trichoderma metabolites" and active metabolite producing members of the Trichoderma family. In this instance, cuttings, seedlings, etc. may be dipped in a liquid composition containing a metabolites and/or metabolite producing members. Alternatively, various compositions may be dusted or sprayed onto the roots or appropriate portions of the seedlings or cuttings etc.

Example 8

According to another embodiment, a substantially solid pellet may be prepared which is able to slowly decay in the environment in which it is to be used. Various slow decay compositions and techniques are known and recorded in the art - these may be relied upon.

The pellets will typically contain either or both "active Trichoderma metabolites", and active metabolite producing Trichoderma organisms. Nutrients, for the plants, and/or the Trichoderma species, may be included in the pellets. Other substances, such as pesticides, fungicides, plant hormones, etc. may also be included in a pellet. It is noted that these other substances may also be included in various other embodiments of the present invention.

Example 9

A composition comprising an "active Trichoderma metabolite" may be applied to harvested produce, typically in the region of the picking wound. While 'living' Trichoderma populations may be relied upon, these are not generally necessary -metabolites will generally remain on the produce (depending on its handling) sufficiently long to offer adequate protection.

Trials were performed by the applicant to determine the relative effectiveness of various "active Trichoderma metabolites" in addressing botrytis cinerea. The trials involved the application of 4 mg of each trial substance to the picking wound of kiwifruit. The results are summarised in the tables 8-11.

Other substances used in the trials included Ronilan™, a proprietary fungicide whose use is widespread for this type of application. Beta-ionone and calcium chloride were also included in the trials. AANB, CAH, CAL and BNB are various experimental compounds extracted from kiwifruit. 6AAP represents 6-pentyl-α-pyrone.

As can be appreciated from the data, 6-pentyl-α-pyrone is extremely effective against Botrytis cinerea. It is also noted that the technique of hand pipetting the selected substance onto the picking wound was not always accurate and in some cases the selected substance was delivered to a site adjacent to the picking wound rather than on it. An observation from the trial was that 6-pentyl-α-pyrone delivered next to the picking wound still provided relatively effective control against Botrytis cinerea in those cases.

Massoilactone, an "active Trichoderma metabolite" was also very effective though delta-decanolactone (another "active Trichoderma metabolite") was less effective. This compound appears to be more selective in those plant afflictions against which it is effective, though it still provided good results against Botrytis cinerea and in some cases was more effective than the commercially used fungicide, RONILAN™.

Example 10

Figure 9 is a table of data from petri dish trials of the effectiveness of various Trichoderma metabolites against a variety of fungi. In these trials, Treatment 1 represented 6-pentyl-α-pyrone, while Treatment 2 was a mixture of 6-pentyl-α-pyrone, massoialactone, and delta-decanolactone. Treatment 3 comprised delta-decanolactone while treatment 4 comprised.

The experimental procedure placed a portion of the treatment in the centre of the Petri dish while the figures in mm on the table represent the distance of closest approach of fungus induced to grow on the Petri dish.

The results indicate that the various Trichoderma metabolites are effective against a range of different fungi and also indicate that delta-decanolactone is more effective against some fungi than others though still remains active against all the fungi included in the trials.

In the trials, the fiingi prefixed by fk are all fungi associated with sapstain, and comprise: fk150

fk36 Ceratocystis sp.

fk64 Ceratocystis sp.

fk 304

Further trials were performed using a variety of "active Trichoderma metabolites", as well as other substance, for the control of Botrytis cinerea. These results are listed in Tables 12 onwards and exhibits the actual activity of 6-pentyl-α-pyrone (6 AAP) and. Under certain conditions, delta-decanolactone exhibited high activity, and at worst an activity comparable to other prior art treatments.

Claims

THE CLAIMS DEFINING THE INVENTION ARE:

1. A method for the control or prevention of at least one member of a group of targeted afflictions comprising botrytis, armillaria, silver leaf, and phytophthora, said method comprising the administration of at least one member of an "active Trichoderma metabolite" group comprising 6-pentyl-α-pyrone, delta-decanolactone, and massoialactone.

2. A method for the control or prevention of at least one member of a group of targeted afflictions comprising botrytis, armillaria, silver leaf, and phytophthora, said method comprising the administration of at least one active member of the Trichoderma family which produces at least one "active Trichoderma metabolite" of a group comprising massoialactone, 6-pentyl-α-pyrone, and delta-decanolactone.

3. A method as claimed in claim 2 in which a said active Trichoderma family member comprises Trichoderma hamatum. Trichoderma harzianum. Trichoderma lignorum. Trichoderma koningii. or Trichoderma viride.

4. A method as claimed in claim 1 or claim 2 in which the administered substances are applied to exposed surfaces of a plant.

5. A method as claimed in claim 1 or claim 2 in which the administered substances are applied to growth media in the root zone of a plant.

6. A method as claimed in claim 1 or claim 2 in which the administered substances are injected into the sapwood, or equivalent thereof, of a plant.

7. A method as claimed in claim 1 or claim 2 in which the administered substances are applied to the picking wounds of harvested fruit.

8. A method as claimed in claim 1 or claim 2 in which the administered substances include both:

- at least one member of an "active Trichoderma metabolite" group comprising deltadecanolactone, 6-ρentyl-α-pyrone, and massoialactone, and

- at least one active member of the Trichoderma family which produces at least one "active Trichoderma metabolite" of a group comprising 6-pentyl-α-pyrone, deltadecanolactone, and massoialactone.

9. A method as claimed in claim 1 or claim 2 in which the group of targeted afflictions also includes:

Nectria galligena, Sclerotium rolfsii, Rhizoctonia solani, Sclerotium cepivorum, Macrophomina phaseolina, Fusarium oxysporum, Verticillium albostrum, Chondrostereum purpureum, Scletotinia sclerotiorum, Pythium ultimum and Corticum rolfsii.

10. A method as claimed in claim 1 in which there is administered at least two of the "active Trichoderma metabolites".

11. A method for conferring, to plants, resistance to at least one of a group of targeted afflictions comprising botrytis, armillaria, silver leaf, and phytophthora, said method comprising the establishment of an active population of at least one member of the Trichoderma family which produces at least one of a group of Trichoderma metabolites comprising 6-pentyl-α-pyrone, delta-decanolactone, and massoialactone, in either or both the plant or its root zone.

12. A method as claimed in claim 11 in which the group of targeted afflictions also includes:

13. A method as claimed in claim 11 or claim 12 in which the population is established by the introduction of active Trichoderma organisms into the sapwood, or equivalent thereof, of the plant.

13. A method as claimed in claim 11 or claim 12 in which active Trichoderma family members are introduced into the growth media of the plant.

14. A method of treatment of plant growth media to address at least one of a group of targeted afflictions comprising botrytis, armillaria, silver leaf, and phytophthora, said method comprising the introduction into the growth media of either or both:

15. A method for the protection of seeds and seedlings against botrytis, armillaria, silver leaf, or phytophthora comprising their planting in treated growth media as claimed in claim 14.

16. A composition for the control or prevention of at least one member of a group of targeted afflictions comprising botrytis, armillaria, silver leaf, and phytophthora, said composition comprising at least one member of an "active Trichoderma metabolite" group comprising delta-decanolactone, 6-pentyl-α-pyrone, and massoialactone, which has been extracted from one or more members of the Trichoderma family.

17. A composition for the control of at least one member of a group of targeted afflictions comprising botrytis, armillaria, silver leaf, and phytophthora, said composition comprising at least one member of an "active Trichoderma metabolite" group comprising delta-decanolactone, 6-pentyl-α-pyrone, and massoialactone, in combination with at least one active member of the Trichoderma family capable of producing one or more "active Trichoderma metabolites".

18. A composition for the control of biological disorders of plants comprising an agronomically acceptable carrier, at least one active member of the Trichoderma family capable of producing one or more "active Trichoderma metabolites", and optionally at least one member of an "active Trichoderma metabolite" group comprising delta- decanolactone, 6-pentyl-α-pyrone, and massoialactone.

19. A composition as claimed in any one of claims 16 through 18 which is in a fluid form suitable for spray application.

20. A composition as claimed in any one of claims 16 through 18 which is in a paste like form.

21. A composition as claimed in claim 20 which includes a barrier layer forming component.

22. A composition as claimed in any one of claims 16 through 18 which is in a substantially dry and powdered form for dusting.

23. Treated plant growth media comprising media capable of supporting the growth of a plant to which has been introduced either or both:

24. A method of protecting seedlings against plant disorders, said method comprising the application to the roots of said seedlings by dusting, spray, immersion, or other means, either or both of:

5. Seeds coated with either or both: