NZ250838A

NZ250838A - Antifungal treatment of plants with massoialactone and/or 6-pentyl-<alpha>-pyrone and/or delta-decanolactone

Info

Publication number: NZ250838A
Application number: NZ250838A
Authority: NZ
Inventors: Robert Anthony Hill; Horrace G Cutler
Original assignee: Horticulture & Food Res Inst; Us Agriculture
Priority date: 1994-02-07
Filing date: 1994-02-07
Publication date: 1998-05-27
Also published as: EP0743821A1; JPH09508899A; WO1995020879A3; AU1547495A; WO1995020879A2

Description

<div class="application article clearfix" id="description"> New Zealand No. International No. TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 07.02.1994 Complete Specification Filed: 03.02.1995 Classification: (6) A01N63/02.04 Publication date: 27 May 1998 Journal No.: 1428 250838 p cxjGNATijm PCT/ 25oss/ 2fc>t4 l-i/ 01034^ new zealand patents act 1953 COMPLETE SPECIFICATION Title of Invention: Improvements in and relating to the use of 6-pentyl-alpha-pyrone and trichoderma as control agents Name, address and nationality of applicant(s) as in international application form: The Horticulture and Food Research Institute of New Zealand Limited, Institute of Ruakura Research Centre, East Street, Hamilton, New Zealand; The United States Department of Agriculture, Department of Richard B Russell Center, Athens, GA 30613, USA 250838 / 250851 26417] /270249 PATENTS FORM NO. 5 Fee No. 4: $260.00 PATENTS ACT 1953 COMPLETE SPECIFICATION After Provisional Nos.: 250838/250851/264171/270249 Dated: 7 Feb 94/8 Feb 94/4 Aug 94/21 Dec 94 respectively TRICHODERMAS AND METABOLITES AS BIOLOGICAL CONTROL AGENTS We ' The Horticulture and Food Research Institute of New Zealand Limited, a New Zealand company of State Highway 57 (no street number), Palmerston North, New Zealand and The United States Department of Agriculture, Richard B Russell Centre, Athens, GA 30613, United States of America hereby declare, the invention for which I/We pray that a patent may be granted to me/us, and the method by which it is to be performed to be particularly described in and by the following statement: 1 250838 Technical Field The present invention is directed to the control of fungal and microbial activity. The 5 present invention will be directed primarily to the use of the compound massoialactone, alone or together with other antifungal compounds or with members of the genus Trichoderma, especially those producing at least one of 6-pentyl-a-pyrone (also known as 6-amyl-a-pyrone), delta-decanolactone, and massoialactone. Background Art 10 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. 15 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 currendy used controlling agents, and ideally an alternative to existing control agents to which litde resistance is exhibited by fungi and/or microbes. Armillaria, a fungal pathogen of forest trees, was first identified 115 years ago, and is 20 now recognised as a major problem in a variety of woody plant species world-wide. More than 560 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. 25 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 30 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. 2 initllectual property office of nz 0 9 apr 1998 .Received 'f 5©&38/2 5 085 l/26 4 1 7 t/^7 0249 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. 5 Both kiwifiruit.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 10 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, 15 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. 20 however, tl*e 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 25 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 30 control using commercially available agents, which are generally chemical based fungicides. > ^'v. It is an object of the present invention to address the foregoing problems or at least to ^ T^Aprovide the public with a useful choice. ' JTT f CO ^/further aspects and advantages of the present invention will become apparent from the 35 . ry i Cj 4 35./ ensuing description which is given by way of example only. 3 DISCLOSURE OF INVENTION 250838 The invention has various aspects. These are defined In the appended claims, to which specific reference should be made. 5 Broadly, the invention provides the following: 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 massoialactone to an afflicted site 10 or to a site susceptible to such affliction. 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 15 Trichoderma family which produces massoialactone to an afflicted site or to a site susceptible to such affliction. a method substantially as described above which involves the administration of both: 20 - massoialactone, and at least one active member of the Trichoderma family which produces at least one of a group comprising 6-pentyl-a-pyrone, delta-decanolactone, and massoialactone. 25 - 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 massoialactone, in either or both the plant or its root zone. 30 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: MRBVC1P22237.001 INTELfcUUAL HHOPERTY OFFICE OF N.Z. 0 9 apr 1998 received W - massoialactone, and at least one active member of the Trichoderma family which produces massoialactone. 5 - 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 massoialactone alone or in combination with another antifungal compound selected from 6-pentyl-a-pyrone and delta-decanolactone, or in combination with, at least one active member of the Trichoderma family 10 capable of producing one or more of 6-pentyl-a-pyrone, delta decanolactone and massoialactone. treated plant growth media comprising media capable of supporting the growth of a plant to which has been introduced either or both: 15 - massoialactone, and at least one active member of the Trichoderma family which produces massoialactone. The term "targeted affliction" shall preferably refer to a member of the group 20 comprising "Botrytis, Armillaria, silver-leaf, and Phytophthora". It should also be appreciated that, many of the compounds 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, 25 Sclerotium cepivorum, Macrophomina phaseoltna, Fusarium oxysporum, Verticillium albostrum, Chondrostereum purpureum, Scletottnia sclerotiorwn, Pythium ultimum and Corticum rolfsii may also be addressed. 30 SPECIFIC ASPECTS OF THE INVENTION A specific and highly important aspect of the invention is a fungicidal composition. This composition contains the compound massoialactone, (R)-5,6-dihydro-6-pentyl-2H-pyran-2-one. Massoialactone has the structure: CK O MRBVC.P22237.001 ^ | 'NT^CTW OFFICE] o 9 apr 1998 received Massoialactone occurs as a racemate and as an enantiomer. For the avoidance of doubt, both forms are intended to be covered by the term "massoialactone'' as used herein. 5 Massoialactone is commercially available as an extract of the bark of Zryptocaria massoia from Frutarom Ltd, Haifa, Israel. It can also be synthesized chemically, both as its racemate and enantiomer. 10 It has also been found by the applicants to be present as a minor component of the crude extract of Trichoderma viride. Prior to the applicants invention, there has been no report of massoialactone as having antifungal activity. It is this finding of the applicants that forms the basis of the J 5 specific, and claimed, aspects of this invention. 20 The compositions of the invention can be employed in antifungal applications containing massoialactone as the sole antifungal compound. The compositions can be formulated for application by any conventional means, including as sprays, dips, pastes or powders. 25 While the composition? can contain only massoialactone as the antifungal agent, compositions which include other antifungal compounds are contemplated. For exampie, the antifungal compounds 6-pentyl-2H-pyran-2-one and (RS)-tetrahydro-6-pentyl-2H-pyran-2-oae can be employed in combination with massoialactone. These latter compounds are commonly called 6-pentyl-a-pyrone and delta-decanolactone, respectively and have the structures: 0 30 (6-peniyl-a -pyrone) and MRB\CtPi2Z37.00t intellectual property office 0fn.z. 0 9 apr 1998 received 25<>038 (delta-decanolactone). Other related lactones include: (RS )-dihydro-5 -hexyl-2H-furan-2-one, 10 15 (RS)-dihydro-5-octyl-2H-furan-2-one, 20 (RS)-tetrahydro-6-heptyl-2H-pyran-2-one, 0_0 25 (RSMetrahydro-6-hexyl-2H-pyran-2-one, O 30 All of these compounds are available commercially from Aldrich Chemical Co, Inc., Milwaukee, Winsconsin, United States of America. MRRCIP22237.001 intellectual property ofrceI of nz 1 09 apr 1998 .Received 250838 Joth 6-pentyl-a-pyrone and delta-decanolactone are produced as metabolites of a number of Trichoderma. The compositions of the invention can therefore include Trichoderma organisms which produce active compounds. Brtf.f 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: is a graph - Botrytis trials first assessment - week one; Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figured is a graph - Botrvtis trials first assessment - week two; is a graph - Botrytis trials second assessment - week one; is a graph - Botrytis trials second assessment - week two; is a graph - Botrytis trials third assessment - week one; is a graph - Botrytis trials first assessment - week two; Figure 7 incidence of Botrvtis 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 MR8VCIP22237.001 INiaiECTUAL PROPERTY OFFICE OF N.Z. 0 9 apr 1998 ^received 25 0 S 5 8 DESCRIPTION I The invention is broadly as defined above, and has a focus upon massoialactone. More ^jenerally, investigative research by the applicants has established that certain Trichoderma metabolites are particularly effective against the aforesaid targeted afflictions. Trials have indicated the effectiveness of the active Trichoderma compounds 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. These results indicate that the metabolite compounds are suitable secondary antifungal compounds for use in conjunction with massoialactone. The preferred metabolite compounds comprise 6-pentyl-a-pyrone and delta-decanolactone. 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 may include one or more of the active Trichoderma metabolites These may also be derived from a number of sources - for instance 6-pentyl-a-pyrone can be isolated from various Trichoderma organisms, or the synthetically produced 6-amyl-a-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 relied upon. While the active corfjounds may be relifld upon for control, the confound 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 confound producing members in conjunction with supplemental metabolite. However, it is envisaged that often the use of 'living' control compositions will be predominandy 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 mclude nutrients with a control composition to assist the establishment of the In PROPERTY OFFii OF NZ members once applied. It may be preferable to introduce, or co-apply, the nutr i time of application of any control agent. ent at the 0 9 APR 1998 250838 ^knother observation is that the active organisms readily secrete the desired confounds 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 ccnpound producing organisms may therefore provide an efficient delivery system for 5 many applications. An applied confound, 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. 10 It should also be appreciated that in some cases a combination of compound and active oonpound producing organisms may be relied upon. A composition which immediately provides active cxxqpound 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 aoiqpound, for a period exceeding that for which an isolated 15 compound 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, 20 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 25 New Zealand to include isolates of Trichoderma hamatum, T. harzianum, T. viride, and other Trichoderma spp. - particularly those collected from Armi/iaria-infected orchards and forest sites in the Bay of Plenty. Some Trichoderma strains were growing on and consuming Armillaria mycelium and rhizomorphs. On transfer to the laboratory, in vitro tests confirmed the activity of the Trichoderma isolates against Armillaria; and as the result 30 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. 35 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 je used in an intellectual property office of n.z. compatibility of the Trichoderma isolates with each other so that they could 10 0 9 apr 1998 received 25 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 mycelium, yellowing of the Armillaria mycelium, overgrowth of the Armillaria by Trichoderma. and extensive r'hizomorph initiation of the Armillaria colony. Importantly, there were differences in the antagonistic response of the accessed Trichnderma isolates to Armillaria; 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 Armillaria; 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 Armillaria 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 nonvolatile 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. Research has established that various Trichoderma species produce a number of antibiotics. The most common of these is 6-pentyl-a-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-a-pyrone has shown, for example, that a 1:40 dilution of the metabolite applied at the rate of 15 p.1/4 mm disk inhibited the growth of Aspe producer of aflatoxins. 11 * 25083 oth T. lignorum and T, viride produce trichodermin (4B-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 5 to certain herbaceous plants. However, it has relatively low toxicity in mice (LD50 lg/kg orally) compared to its congeners, and at one time was considered by the pharmaceutical 4 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 10 ±delta-decanolactone. Preliminary trials by the applicants have indicated useful activity by massoialactone against targeted afflictions. As for 6-pentyl-a-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 15 other "active Trichoderma metabolites", will be analogous to the use of 6-pentyl-a-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. muni J-J 20 hi OAc Structure of trichodermin (4B-acetoxy-12-13-epoxy trichothec-9-ene) Observed Effects Of trichoderma and 6-pentyl-a-pyrone ox armillaria Disease in Pinus radiata 25 Crude extracts from Trichoderma containing 6-pentyl-a-pyrone and synthetic 6-pentyl-a-pyrone (hereafter referred to as 6-amyl-a-pyrone to distinguish between the 'natural' product arud 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 (il per disk with 6-amyl-a-pyrone, concomitantly the crude extract was active. 30 Other micro-organisms were also strongly inhibited; and these included office 12 (followed by page 17k) 0 9 apr 1998 received 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 5 system for 6-pentyl-a-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 10 mortality 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-a-pyrone to determine 15 effects on Armillaria and enhancement of vigour. EFFECTS OF TRICHODERMA AND 6-PENTYL-a-PYRONE/6-AMYL-a-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 20 have inhibited or prevented the spread of the organism within kiwifruit orchards, and in addition soil treatment in barrier trenchcs 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 25 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 30 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 |il per vine of 6-amyl-a-pyrone; 10 to 50 pi per vine of 35 6-pentyl-a-pyrone (the natural product is more difficult to obtain in quantity relative to the synthetic material); and 300 fxl of a crude extract, known to contain 6-pentvl-a-pyrone. from a high yielding isolate of T. hamatum. Other infected vines were in 12A (followed by page 12B) flyjWWV OFFICE 0 9 apr 1998 .received ZSOgr 8 mixed strain Trichoderma formulations with proven efficacy against Armillaria. All untreated Armillaria infected vines died within 6 months. Both 6-amyl-a-pyrone and 6-pentyl-a-pyrone treatments significantly increased the survival rate (to ~50%) in infected vines. However, Trichoderma formulations were even more effective, and over 80% of 5 the infected vines survived; while the crude extract was approximately as active as the 6-amyl-c. -pyrone and 6-pentyl-a-pyrone. Observed Effects Of trichoderma on the control of Silver-Leaf Disease Effective disease control using high 6-pentyl-a-pyrone producing strains of Trichoderma. 10 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 15 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 20 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. 12B (followed by page 12C intellectual property office of n.z. 0 9 apr 1998 received 250838 DETAILED DESCRIPTION Trials directed to the control of Botrytis cinerea were conducted comparing the use of 6AAP (6-amyl-a-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-a-pyrone (6PAP) and its synthetic equivalent 6-amyl-a-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-a-pyrone in Trichoderma samples. This experimental procedure is outlined below and includes details of the typical 6-pentyl-a-pyrone contents of various Trichoderma samples. As can be appreciated, strains exhibiting higher levels of 6-pentyl-a-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-a-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 haruanum US2/NZ Trichoderma koningii US3/NZ Trichoderma hamatum HPP1 Trichoderma harizarutm D Trichoderma viride TV Trichoderma hamatum TBHPP7 Trichoderma hamatum GT4 Trichoderma hamatum KEK 12C (followed by page 13) intellectual property office of nz 0 9 apr 1998 received 250838/25085? 264171 / 270249 EXPERIMENTAL Extraction method tests The method PAP-1 may be summarised as follows: 1. Take a subsample of solid, typically lOg. 5 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. 10 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 15 An initial screen of the 11 samples was done by extracting lOg 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 20 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 25 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 30 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, 13 250838/250851 264)71 /270249 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 5 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 10 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 15 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 farther 6ml of cyclohexane was passed through the column and the eluate collected as Fl. 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 acetale-methanol. All fractions 20 were evaporated and the residues weighed before re-dissolving in 2ml of cyclohexane for analysis. RESULTS Extraction solvent test Initial test 25 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 30 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. 14 S,38 7 250851 264171/270249 Table 1: Analysis of 11 Trichoderma culture samples for 6PAP after extraction with two solvent systems. Sample 6PAP (mg/kg) with extraction solvent methanol-water (50 + 17) acetontrile-methanol-water (80:5:15) OG3 7.5 7.3 Hend <5 n.d. US3/NZ <5 n.d. D <5 n.d. - TBHPP7 69.0 61.4 KEK <5 n.d. NZ164/US 193.1 142.5 US2/NZ <5 n.d. HPP1 86.9 49.1 TV <5 2.6 GT4 <5 n.d. control <5 n.d. n.d. is not detected at a limit of about lmg/kg 15 Partition solvent tests Results with ethyl acctate 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, 20 especially when applied to cultures of T. koningii such as NZ164/QS. 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. 25 b) The use of acetonitrile at levels of 50% and higher caused problems with ti\e 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 30 obtained increased with lower saline strength. 15 250838 / 250851 264171 /270249 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. 5 Table 2: Analysis of 6PAP in NZ164/US culture using different extraction 10 15 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. 20 Table 3: Reproducibility test. 6PAP (mg/kg) TBHPP7 NZ164/US Extracl No a b a b I 61 73 212 204 2 74 74 203 214 3 89 86 202 198 4 94 89 208 202 5 81 86 206 204 solvent-partition diluent combinations. Extraction solvent Replicate 6PAP (mg/k l) with saline concentrations methanol-water-acetonitrile 10% saline 5% saline 2.5% saline j 65:25:10 A 190 235 250 B 195 225 243 50:25:25 A 183 175 215 j B 205 225 225 25:25:50 A 190 255 260 B 203 250 250 85:15:0 A 215 228 230 B 258 258 250 0:15:85 A 23 58 193 B 25 68 223 16 2508381250851 264171 1270249 range 61-94 198-214 mean 80.7 205.3 % vari2ncc 12.4 2.4 Silica fractionation A 20ml aliquot of extract from NZ164AJS (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 i 7.5mg. After silica 5 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 10 Extraction and workshop a) The sample (lOg) 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 15 (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/FDD. e) When analysing for 6PAP along use programme A below; when screening for 20 other peaks use programme B. GC conditions Column: 25 m HP-5,0.2 mm iA, 0.33 fim film Detector: FID at 280°C Injector: 280°C, split 1:20, l|il injection 25 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. 17 250838 / 25085 264171 / 27Q24 Table 4 Initial Accessment Summary 14.6.93 1993 Botrytis Trials Treatments Day ! 1. Ronilan 75 ng Al 2. Ronilan 9 p.g AI 3. 4. 5. 6 AAP 100% 6. 6 AAP diluted to 25% with H2O 7. Thyme 100% 8. 9. Control - untreated Day 2 10. Ronilan 75 p.g AI 11. Ronilan 9 ng AI 12. 13. 14. 6 AAP 100% 15. 6 AAP diluted to 25% with H2O 16. Thyme 100% 17. 18. Control - untreated Day 3 19. Ronilan 75 ng AI 20. Ronilan 9 jig AI 21. 22. 23. 6 AAP 100% 24. 6 AAP diluted to 25% with H?0 25. Thyme 100% 26. 27. Control - untreated Percenta; Trial 4 • 6 May Reo 1 12 28 0 0 12 60 0 0 0 0 0 20 0 4 0 0 0 Rep 2 12 X5 0 0 8 48 0 4 0 0 0 0 4 0 0 0 Ren 3 12 24 0 0 12 52 12 0 0 0 0 12 0 0 0 4 i 16 e Botrytis Duplicate Trial 11-1} May Rep 4 12 8 0 0 0 20 8 44 0 0 0 48 0 4 0 0 0 Reo S 28 32 0 0 0 24 12 20 0 0 0 44 12 12 0 0 0 Rep 6 16 20 0 0 0 20 40 28 0 0 0 52 16 8 0 0 0 8 i 24 16 44 18 250838 / 25085T 264171 /270249 Table 5 2nd Accessmenl Summary 14.7.93 1993 Botrytis Trials (Cumulative Results) Treatments Day 1 1. Ronilan 75 jig AI 2. Ronilan 9 jig AI 3. 4. 5. 6 AAP 100% 6. 6 AAP diluted to 25% with H2O 7. Thyme 100% 8. 9. Control - untreated Percen Trial 4 ■ 6 May fUg \ 12 28 0 4 16 64 EsslI 12 23 0 0 8 60 tage Botrytis Duplicate Trial 11-13 May BgP 3. 28 24 0 0 12 52 Rep A 12 . 8 0 4 0 Emi 28 60 0 0 0 32 36 Ren 6 20 24 0 0 0 32 Day 10. 11. 12. 13. 14. 15. 16. 17. 18. Ronilan 75 p.g AI Ronilan 9 |ig AI 6 AAP 100% 6 AAP diluted to 25% with H2O Thyme 100% Control - untreated 0 0 0 0 0 24 0 4 0 0 0 12 4 0 0 0 16 16 56 0 0 0 56 12 40 0 0 0 68" Day 3 19. 20. 21. 22. 23. 24. 25. 26. 27. Ronilan 75 ng AI Ronilan 9 |ig AI 6 AAP 100% 6 AAP diluted to 25% with H2O Thyme 100% Control - untreated 4 4 0 0 0 0 4 0 0 0 20 12 12 0 0 0 8 8 0 0 0 16 12 0 0 0 48 32 0 0 0 60 20 24 28 24 0 0 56 19 Table 6 250838 / 25085» 264)7) /270249 Final Assessment Summary 18.8.93 1993 Botrytis Trials (Cumulative Results) Treatments Day 1 I. Ronilan 75figAl 2.. Ronilan 9 jxg AI 3. 4. 5. 6 AAP 100% 6. 6 AAP diluted to 25% with H2O 7. Thyme 100% 8. 9. Control - untreated Percentag Trial 4 • 6 May RioJL 12 28 0 4 16 64 Reo 1 16 23 0 0 8 60 e Botrytis Duplicate Trial U-J3 May Rep 3 28 28 0 0 24 52 Rcd-4 12 8 0 4 4 32 Rep. 5 28 60 0 0 4 36 Reo 6 20 24 0 0 4 32 Day 2 10. 11. 12. 13. 14. 15. 16. 17. 18. Ronilan 75 jig AI Ronilan 9 jig AI 6 AAP 100% 6 AAP diluted to 25% with H2O Thyme 100% Control - untreated 0 0 0 0 0 24 0 4 0 0 0 12 4 0 0 0 16 16 60 0 0 0 56 12 40 0 0 0 68 48 32 0 0 0 60 Day 3 19. 20. 21. 22. 23. 24. 25. 26. 27. Ronilan 75 p.g AI Ronilan 9 p.g AI 6 AAP 100% 6 AAP diluted to 25% with H2O Thyme 100% Control - untreated 4 4 0 0 0 0' 4 0 0 4 12 12 0 0 0 20 8 12 8 0 0 0 2 H 16 12 0 0 0 24 20 24 0 0 4 20 250838/25085? 264171 /270249 Table 7 Final Assessment Summary 18.8.93 1993 Botrytis Trials (Cumulative Results') Treatments Day 1 1. Ronilan 75|ig AI . 2. Ronilan 9 jig AI 3. 4. 5. 6 AAP 100% 6. 6 AAP diluted to 25% with H2O 7. Thyme 100% 8. 9. Control - untreated Percentage Botrytis Trial 4 - 6 May Duplicate Trial 11-13 May Rep.l 12 28 0 4 16 64 Rep 2 16 23 0 0 8 60 Reo 3 28 28 0 0 24 52 Ren 4 12 • 8 0 4 4 32 Rep 5 28 60 0 0 4 36 Rej-6 20 24 0 0 4 32 Day 2 10. Ronilan 75 jig AI 11. Ronilan 9 fig AI 12. 13. 14. 6 AAP 100% 15. 6 AAP diluted to 25% with H2O 16. Thyme 100% 17. 18. Control - untreated 0 0 0 0 0 24 0 4 0 0 0 12 4 0 0 0 16 16 60 0 0 0 56 12 40 0 0 0 68 48 32 0 0 0 60 Day 3 19. Ronilan 75 (ig AI 20. Ronilan 9 |ig AI 21. 22. 23. 6 AAP 100% 24. 6 AAP diluted to 25% with H2O 25. Thyme 100% 26. 27. Control - untreated 4 4 0 0 0 0' 4 0 0 4 20 12 12 0 0 0 12 8 0 0 0 28 16 12 0 0 0 2d 20 24 0 0 4 56 21 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 plant pathogens. In other cases, the invention may find a preventative role, acting to prevent the targeted plant pathogens establishing themselves in various plants and 5 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, fungicidal compositions may be sufficient in their own right to address targeted plant pathogens present in plants. 10 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 compound in the required portion of the plant. Hie 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. 15 As another option, a composition may seek to establish a population of a . Trichoderma compound 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-a-pyrone and/or other metabolites may be 20 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-a-pyrone may not be required and thus the establishment of a 25 population of active metabolite producing organisms may be sufficient to provide long term and lasting control of targeted plant pathogens. 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 30 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 35 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 22 initllectijal property office of N.Z. 0 9 APR 1998 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: 5 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 hiiruducticn 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. 10 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 15 plant, may be included. To the composition of example 1, is included at least one "active Trichoderma metabolite". Particular metabolites of interest include 6-pentyl-a-pyrone, massoialactone, and delta-decanolactone. 20 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 25 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. 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 30 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 1 Example 2 23 2508 38/25085l/26 4 17.i/7 0 24 9 Example 5 I \ 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 5 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 10 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, 15 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 carricd over with it. 20 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 fiirtlier enhance the effects. Applying an active metabolite containing composition may also be useful. 25 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 nonaqueous or hydrophobic components may also be relied upon to resist washing of the 30 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 '>s=!=jy^-v. as use of a paste which dries or sets to provide a cap or cover for the wound) are rf oj considerations which may be addressed in the various compositions. « ^ /' Jill . >-* f ^i1 Co ; § vW X - ?/ j 24 / 25 0 8 38 Example 7 25085 l/26417 V 27 0 2 4 9 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, 5 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 10 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 15 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 20 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. 25 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 30 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-a-pyrone. 25 :/ 250838 / 250851 264171 /270249 Table 8 IriaLL Number fruit/treatment* 100 Volume treatment/application: 4 mg Application method: hand pipetted Harvest Dale: 16/5/94 (1) Hrs after inoculation treatment applied Treatment % botrytis rots @ 9 weeks after treated 4 1 - AANB 33 2 - CaCU(0.12%) 44 3 - CaCh (1/10) 41 4 - Beta-ionone 54 • 5 - Ronilan 75|X AI 9 6 - 6AAP 100% 0 7 - 6AAP 50% 0 8 - 6AAP 25% 2 9 - 6AAP 10% 15 10 • Control untreated 42 51 11 - AANB 60 12 - CaClj (0.12%) 60 13 - CaClj(l/l0) 63 14 - Beta-ionone 18 15 - Ronilan 15\i AI 18 16-6AAP 100% 0 17 - 6AAP 50% 0 18 - 6AAP 25% 0 19 - 6 AAP 10% 1 20 - Control untreated 74 26 250838 / 250851 264171 /270249 Table 9 Harvest Dale: 23/5/94 (2) Hrs after inoculation treatment applied Treatment % botrytis rots @ 8 weeks after treated 4 1-AANB 27 2-CaCla(0.12%) 29 3-CaCU(l/10) 16 4 - Beta-ionone 32 5 - Ronilan 75p.g AI 1 6 - 6 AAP 100% 0 7 - 6AAP 50% 0 8 - 6AAP 25% o 9 - 6AAP 10% 3 10 - Control untreated 36 51 11 - AANB 38 12-CaC!i (0.12%) 35 13-CaCUO/lO) 23 14 - Beta-ionone 2 • 15 - Ronilan 75jig AI 4 16 - 6AAP 100% 0 17 - 6AAP 50% 0 18 - 6AAP 25% 0 19 - 6AAP 10% 0 20 - Control untreated 30 27 Table 10 Harvest Date: 30/5/94 (3) Hrs after inoculation treatment applied Treatment % botrytis rots @ 7 weeks after treated 4 1 -CAH 40 2-CAL 49 3-BNB 34 4 - delta decanolactone 30 5 - Ronilan 75jJtg AI 5 6 - 6AAP 100% 0 7 - massoilactone 0 8-6AAP259& 0 9 - 6AAP 10% 0 10 - Control untreated' 47 51 11-CAH 61 12-CAL 47 13 - BNB 54 14 - delta decanolactone 2 15 - Ronilan 75|ig AI 55 16 - 6AAP 100% 0 . 17 • massoifacionc I 18 - 6AAP 25% 0 19 - 6AAP 10% 0 20 - Control untreated 78 28 250838/250851 264171 /270249 Tables 11 Number froit/utauncne 12S< Volume treatment/application: 4 mg Application method: automated droplet Harvest Dale: 10/5/94 (4) Treatment % botrflis rou @ 10 weeks after ucaled 1 ■ 6 AAP 100% 0 2-6AAP 10% 0.88 3 • Control untreated 1.42 Harvest Date: 24/5/9* (5) Treatment % botrytis rou <3> 8 weeks after vested 1 - 6 AAP 100*. 0 2 • 6 AAP 10% 0 3 ■ Control untreated 1.16 Number fruit/treatment: 10S0 Volume treatment/application: 4 mg Application method: automated droplet Harvest Dale: 10/3/94 (6) Treatment % botrytis rots <§> 10 weeks after treated Single Layers Trays Europacks 1 • 6 AAP 100* 0.19 0 2 • Control untreated 12.4 6.57 Harvest Date: 24/5/94 (7) Treatment % botrytis rots @ g weeks after treated Single Layers Trays Europacks 1 • 6 AAP 100% 0 0.29 2 - Control unseated 4.58 0.86 Treatment application method: automated droplet Assessment time after treatment: 7 • 9 weeks Treatment No. fruit treated Botrytis rots No. % 6AAP (100%) • contact wound 6010 3 0.05 ■ outside wound 698 2 0.29 6AAP (10%) ■ contact wound 2409 10 0.42 ■ outside wound 99 1 1.01 COHTtLOL. 3t>4- S-4-J 29 /' 2508 38 25 08 5 1/26 4 17.1 /27 0 249 As can be appreciated from the data, 6-pentyi-a-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 5 observation from the trial was that 6-pentyl-a-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. Tb:s compound appears to be more selective in those plant afflictions against which it is 10 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-a-15 pyrone, while Treatment 2 was a mixture of 6-pentyl-a-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 20 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 fungi prefixed by fk arc all fungi associated with sapstain, and comprise: 25 fkl50 fk36 Ceratocystis sp. fk64 Ceratocystis sp. Further trials were performed using a variety of "active Trichoderma metabolites", as well 30 as other substance, for the control of Botrytis cinerea. These results are listed in Tables ^ i Ss 12 onwards and exhibits the actual activity of 6-pentyl-a-pyrone (6AAP) and. Under tin conditions, delta-decanolactone exhibited high activity, and at worst an activity fk 304 [parable to other prior art treatments. 30 Table 12 250838/250851 264171 /270249 GAAP Botrvtis Trial 1 - Treatment after 4 hours harvest Treatment % botrytis rots % botrytis rots No. @ 9 w&eks after treated @ 18 weeks 1 1. AANB 33 33 2. CaCI2 (0.12%) 44 44 3. CaCI2 (1/10) 41 41 4. Beta-ionone 54 87 5. Ronilan 75u Ai 9 1 0 6. 6AAP 100% 0 0 7. 6AAP 50% 0 0 8. 6AAP 25% 2 2 9. 6AAP 10% 15 20 10. Control untreated 42 42 2 1. AANB 27 27 2. CaCI2 (0.12%) 29 29 3. CaCI2 (1/10) 16 17 4. Beta- ionone 32 45 5. Ronilan 75ug AI 1 2 6. 6AAP 100% 0 0 7. 6AAP 50% 0 0 8. 6AAP 25% 0 0 9. 6AAP 10% 3 6 10. Control untreated 36 40 3 i < o 40 43 2. CAL 49 50 3. BNB 34 40 4. delta decanolactone 30 41 5. Ronilan 75ug AI 5 5 6. 6AAP 100% 0 0 7. massoilactone 0 0 8. 6AAP 25% 0 0 9. 6AAP 10% 0 1 10. Control untreated 47 48 31 Tables 13-14 250838 /25085] 26417]J270249 GAAP Botrvtis Trial 2 - Automated droplet (rialuraUy_iugJai&ted) Harvest Treatment % botrytis rots % botrytis rots No. @ lOweeks alter treated @ 19 weeks 1 1. 6AAP 100% 0 0 2. 6AAP 10% 0.08 1.12 3. Control untreated 7.42 7.74 Harvest Treatment % botrytis rots % botiytis tots No. @ 8 weeks after treated @ 17 weeks 2 1. 6AAP 100% 0 0.08 2. 6AAP 10% 0 0.64 Control untreated 1.36 3.51 6AAP Botrvt's Trial 3 - Automated droplet (naturally inoculated) Harvest Treatment % botrytis rots % botrytis rots No. @10 weeks after treated @ 19 weeks Single Layer Europacks Single Layer Europacks Trays Trays 1 1. 6AAP 100% 0.19 0 0.38 0.1 2. Control untreated 12.4 6.57 15.00 7.33 Harvest Treatment % botrytis rots % botrytis rots No. @ 10 weeks after treated @ 17 weeks Single Layer Europacks Single Layer Europacks Trays Trays 2 1. 6AAP 100% 0 0.29 0.67 0.57 2. Control untreated 4.38 0.86 7.90 3.43 32 25Cc-o8 / 25085 T 26417] /270249 Table 15 6AAP Botrytis Trial 1 - Treatment after 51 Jbaprs Harvest Treatment % botrytis rots % botrytis rots No. @ 9 weeks after treated @ 18 weeks 1 1. AANB 60 60 2. CaCI2 (0.12%) 60 60 3. CaCI2 (1/10) 63 63 4. Beta-ionone 1 0 41 5. Ronilan 75u AI \ 8 18 6. 6AAP 100% 0 0 7. 6AAP 50% 0 0 8. 6AAP 25% 0 0 9. 6AAP 10% 1 1 10. Control untreated 74 74 2 1. AANB 38 S8 2. CaCI2 (0.12%) 35 36 3. CaCI2 (1/10) 2 3 26 4. Beta- ionone 2 10 5. Ronilan 75ug AI 4 6 6. 6AAP 100% 0 0 7. 6AAP 50% 0 0 8. 6AAP 25% 0 1 9. 6AAP 10% 0 0 10. Control untreated 30 30 3 1. CAH 61 64 2. CAL 47 48 3. BNB 54 56 4. delta decanolactone 2 8 5. Ronilan 75ug AI 55 57 6. 6AAP 100% 0 0 7. massoilactone 1 2 8. 6AAP 25% 0 0 9. 6AAP 10% 0 1 10. Control untreated 78 79 33 250838/250851 264171 /270249 Tables 16 6AAP Trial 4 ■ Automated droplet (naturally inoculated) Treatment No. fruit Botrytis rots @ 7-9 weeks treated No. % 6AAP (100%)-contact wound 6010 3 0.05 -outside wound 698 2 0.29 6AAP (10%)-contact wound 2409 1 0 0.42 -outside wound 99 1 1.01 Control 6708 364 5.43 Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims. 3.4 WHAT WE CLAIM IS: i A fungicidal composition comprising massoialactone as an active antifungal compound 2. A composition according to claim 1 which is in a fluid form suitable for spray application. 10 3. A composition according to claim 1 which is in the form of a paste. 4. A composition according to claim 1 which is in the form of a powder. 5. A composition according to claim 1 including at least one additional antifungal 15 compound. 6. A composition according to claim 5 wherein said at least one additional antifungal compound is selected from 6-pentyl-a-pyrone and delta-decanolactone. 20 7. A composition according to claim 6 wherein said additional antifungal compound is 6-pentyl-o-pyrone. 8. A composition according to claim 1 which includes a Trichoderma isolate which produces a metabolite selected from 6-pentyl-a-pyrone and delta-decanolactone. 9. A method of preventing or at least inhibiting growth of a fungus which comprises the step of applying massoialactone to said fungus or to the locus thereof. 10. A method according to claim 9 which Involves applying a composition as claimed in 11. A method according to claim 9 which involves applying a composition as claimed in claim 5. 35 12. A method according to claim 9 which Involves applying a composition as claimed in 5 together with an agronomically acceptable carrier therefor. 25 30 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> claim 1. claim 8. MRBVCU>22237.001 35 initllectual property OFFICE OF N.Z 0 9 apr 1993 received i 13. A method according to claim 9 wherein the fungus to be Inhibited is Botrytis cinerea, Armillaria, Phytophthora, Nectria galllgena, Sclerotium rolfsii, Rhlzoctonla solanl. Sclerottum ceplvorum, Macrophomlna phaseollna, Fusarlum oxysporum, Vertlcllllum albostrum, Chondrostereum purpureum, Scletotlnla sclerotiorum, Pythlum ultlmum and Cortlcum rolfsii. 14. A method of treating a biological surface susceptible to unwanted fungal growth to prevent or inhibit fungal growth which comprises the step of applying massoialactone to said surface. 10 15. A method according to claim 14 which Involves applying a composition as claimed in claim 1. 16. A method according to claim 14 which involves applying a composition according to 15 claim 5. 17. A method according to claim 14 which involves applying a composition as claimed in claim 8. 20 18. A method according to claim 14 wherein said surface is the external surface of a fruit or vegetable. 19. A method according to claim 14 wherein said surface is a timber surface. 25 20. A method according to claim 14 wherein said surface is an external surface of a plant or plant seed. 21. A method according.to claim 14 wherein the fungus is Botrytis cinerea, Armillaria, Phytophthora, Nectria galllgena, Sclerotium rolfsii, Rhlzoctonla solanl, Sclerotium 30 ceplvorum, Macrophomlna phaseollna, Fusarlum oxysporum, Vertlcllllum albostrum, Chondrostereum purpureum, Scletotlnla sclerotiorum, Pythlum ultlmum and Cortlcum rolfsii. 22. Treated plant growth media comprising media capable of supporting the growth of a 35 plant and an amount of massoialactone effective to prevent or at least Inhibit fungal growth. MRB\CIP22237.001 36 > in1ellectual property offjce of n.z. 0 9 apr 1998 received 250838 ^^23. Treated plant growth media comprising media capable of supporting the growth of a plant and a composition as claimed In claim 1. 24. Treated plant growth media comprising media capable of supporting the growth of a 5 plant and a composition as claimed in claim 5. 25. Treated plant growth media comprising media capable of supporting the growth of a \ plant and a composition as claimed in claim 8. 10 26. Plant seeds coated with a fungicidally-effective amount of massoialactone. RUSSELL MeVEMH WEST WALKER per ATTORNEYS FOR TVE APPLICANT END OF CLAIMS MRBVCIP22237.001 37 intellectual property office of n.z. 0 9 apr 1998 received </div>