AU2009201231B2

AU2009201231B2 - Control of weeds

Info

Publication number: AU2009201231B2
Application number: AU2009201231A
Authority: AU
Inventors: Naomi Diplock; Victor Galea
Original assignee: University of Queensland UQ
Current assignee: University of Queensland UQ
Priority date: 2009-03-30
Filing date: 2009-03-30
Publication date: 2015-05-14
Anticipated expiration: 2029-03-30
Also published as: AU2009201231A1

Abstract

OF THE INVENTION The present invention discloses biocontrol methods for controlling weed 5 growth, particularly fungal biocontrol methods for control and suppression of weed growth. The present invention also provides biocontrol compositions comprising one or more fungal strains for the control and suppression of weed growth.

Description

P/00/011 Regulation 3.2 AUSTRALIA Patents Act 1990 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title: "CONTROL OF WEEDS" The following statement is a full description of this invention, including the best method of performing it known to me/us: I TITLE CONTROL OF WEEDS FIELD OF THE INVENTION 5 THIS INVENTION relates to biocontrol methods and compositions for suppressing weed growth. More particularly, this invention relates to fungal biocontrol methods and compositions for suppression of weed growth. BACKGROUND TO THE INVENTION 10 Control of weeds is an important aspect of natural and managed ecosystems. Of particular concern are introduced and invasive weed species. Weeds degrade the environmental and economic value of land that they invade, and the spread of weeds threatens biodiversity, the health of river systems and wetland areas and the productivity of pastoral enterprises. Additionally, weeds can establish impenetrable 15 thickets on low stocking rate pastoral land, and create problems by harbouring feral animals. Methods of using chemical herbicide to control or eliminate weeds are known in the art. However, there are a number of drawbacks associated with the use of herbicides to control weeds. Herbicides are expensive and if improperly used there is 20 an increased risk that some weeds may develop herbicide resistance. In addition, there are also concerns about the short and long term safety of herbicides, both to consumers and the environment. A number of bacteria and fungi are natural pathogens of weeds and it has been suggested that bioherbicides, or weed killers made from biological agents rather 25 than chemical agents, may provide an alternative to chemical herbicides. However, biocontrol agents delivered onto target weeds using traditional dusting, spreading, broadcasting, and/or spraying methods provide poor weed control, due to problems with retention and/or survival of the biocontrol agent on the weeds. Additionally, these traditional application methods are generally not suitable for treatment of large 30 areas. Thus, there is a need in the art for new methods and compositions for applying biocontrol agents to target weeds.

2 SUMMARY OF THE INVENTION The present invention is directed to methods and compositions for controlling weed growth. In one aspect, the invention provides a method of controlling growth of a 5 weed, the method including the steps of: a) introducing a biocontrol composition comprising one or more fungal strains into one or more openings formed in the weed and b) covering the one or more openings in the weed. Optionally, the method includes the steps of: c) identifying a diseased specimen of the weed and d) isolating one or more fungal strains from the diseased 10 specimen, wherein the biocontrol composition comprises the isolated one or more fungal strains. In one embodiment, the one or more fungal strains is an isolate from a diseased specimen of the weed. In another embodiment, the biocontrol composition comprising one or more 15 fungal strains further comprises a medium for supporting growth and/or viability of the one or more fungal strains. In a further embodiment, the biocontrol composition comprising one or more fungal strains and a medium for supporting growth and/or viability of the one or more fungal strains is encapsulated in a soluble container. 20 Suitably, according to the aforementioned aspects, the one or more openings in a weed can be formed in the stem of the weed or a branch of the weed, or in both. In another aspect, the invention provides a biocontrol composition suitable for location in an opening of a weed, the biocontrol composition comprising a soluble container in which is located: a) one or more fungal strains, and b) a medium 25 for supporting growth and/or viability of the one or more fungal strains. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to fungal biocontrol methods and compositions for controlling weed growth. 30 Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the 3 inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. In one aspect, the invention provides a method of controlling growth of a weed, the method including the steps of: a) introducing a biocontrol composition 5 comprising one or more fungal strains into one or more openings formed in the weed and b) covering the one or more openings in the weed. In some embodiments, the method optionally includes the additional steps of: c) identifying a diseased specimen of the weed and d) isolating one or more fungal strains from the diseased specimen, wherein the biocontrol composition comprises 10 the isolated one or more fungal strains. As used herein, "weed" includes any undesired plant, for example, a plant growing out of place or in a place where a different kind of plant or no plant at all is desired. Non-limiting examples of weeds include, but are not limited to, Alternanthera 15 philoxeroides (Alligator weed), Tamarix aphylla (Athel pine), Chrysanthemoides monilifera subsp. rotundata (Bitou bush), Chrysanthemoides monilifera subsp. moniifera (Boneseed), Rubus fruticosus L. agg. (Blackberry), Asparagus asparagoides (Bridal creeper), Ulex europaeus L. (Gorse), Lantana camara (Lantana), Prosopis spp. (Mesquite), Mimosapigra (Mimosa), Parkinsona aculeata 20 (Parkinsonia), Parthenium hysterophorus L. (Parthenium), Annona glabra (Pond apple), Acacia nilotica subsp. Indica (Prickly acacia), Cryptostegia grandiflora (Rubber vine), and Salix spp. (Willows). In one embodiment, the weed is Parkinsona aculeata. By "controlling growth of a weed" is meant interfering with the normal 25 growth and development of a weed, including death of the weed. Interference with the normal growth and development of a weed, including death of the weed, can be assessed using methods well known to one of skill in the art. For example, weeds that are stressed exhibit symptoms including, but not limited to, changes in foliage colouration (e.g., from green to yellow and/or brown), leaf loss, shoot tip damage, 30 branch and/or shoot death, bare stems and/or branches, stem and/or branch splitting, and loss of bark.

4 The formation of one or more openings in a weed encompasses the preparation of a suitable repository in the weed for the biocontrol composition comprising the one or more fungal strains. In some embodiments, multiple openings may be formed in the weed. Two, three, four, five, or more openings may be 5 provided in a weed. The number of openings provided in a weed may depend on the circumference of the stem (trunk) of the weed or a branch (limb) of the weed. For example, a weed with a narrow stem may require three or fewer openings (e.g., two or one), while a weed with a larger stem may require more openings. Multiple openings may be positioned around a circumference of a weed stem or branch, or 10 both. Openings may have various cross-sectional shapes, such as substantially rectangular, substantially square, substantially oval, or irregular. An opening may have a substantially circular cross-sectional shape. An opening may be substantially in the centre of the stem or branch of a weed. Openings in a weed may have a 15 diameter or cross-sectional width of approximately 4.0 mm to approximately 20.0 mm, such as approximately 5.0 mm, 7.0 mm, 10.0 mm, 12.0 mm, 15.0 mm, 17.0 mm, and 19.0 mm. In one embodiment, openings may be formed in a weed by drilling into the weed. Drilling openings in a weed may include using a drill, such as a power drill, 20 tree borer, and/or manually operable drill. In another embodiment, openings may be formed in a weed by wounding the weed, for example, using an awl, a nail, a blade, or the like. In yet another embodiment, openings may be formed in a weed by driving, vibrating, rotating, and/or impacting a device (e.g., a punch, awl, nail, screw, blade, or the like) into the weed. After insertion, the device used to form the opening 25 may be removed. Openings may be formed at any height in the stem or branch of a weed to be treated. In one embodiment, openings may be formed in the weed at a height less than or equal to 100 cm above soil level, such as 90 cm, 80 cm, 70 cm, 60 cm, 50 cm, 40 cm, 30 cm, 20 cm, 10 cm, and 5 cm. In another embodiment, two or more rings 30 of openings may be formed in a weed. The openings in a first ring may be offset from the openings in a second ring so that one or more of the first ring openings are 5 not positioned vertically in line with the openings of the second ring. Openings formed in a weed may extend in the weed to a depth from approximately 10.0 mm to approximately 100.0 mm, such as approximately 15.0 mm, 20.0 mm, 25.0 mm, 30.0 mm, 35.0 mm, 40.0 mm, 45.0 mm, 50.0 mm, 55.0 5 mm, 60.0 mm, 65.0 mm, 70.0 mm, 75.0 mm, 80.0 mm, 85.0 mm, 90.0 mm, and 95.0 mm. The openings may extend into a weed at any angle, such as from about 00 to about 850 relative to an axis parallel to the weed stem or branch. Multiple opening in the same weed may extend in the weed at substantially the same angle, or the openings may be formed at different angles. 10 As used herein, a "biocontrol composition" includes one or more fungal strains that controls or suppresses the growth of, or kills, a weed. By "fungal strain" is meant a biologically active fungal species, or a biologically active fragment or component obtained (but not isolated) or isolated from a fungal species. By "fragment" or "component" of a fungal isolate is meant a 15 fragment of the mycelium, or one or more spores, pycnidia, conidia, chlamydospores or other propagules, or a combination thereof, obtained from the fungi. Fungal strains may be obtained from commercial sources well known to one of skill in the art, or, alternatively, isolated from a diseased specimen of a weed of interest. For example, samples from a diseased specimen of a weed of interest, such 20 as necrotic lesions on leaf and stem tissues, can be used to isolate fungal strains as described herein or using standard methods well known in the art. Diseased specimens may include weeds affected by the condition known as "dieback". Dieback symptoms include, but are not limited to, decline (little or no current season growth), staining of the vascular tissue (xylem and phloem), pith or 25 cambial layers, foliage yellowing, stem and branch lesions (often covered by black, superficial fungal growth), excessive exudation of resin, internal necrosis of stems, branches and/or roots, and death of peripheral foliar elements (leaves, branch tips and the like) progressing inwards to the central crown of the plant and possible eventual death of the weed. Dieback may also have the potential to be transferred naturally 30 to adjacent weeds through physical contact between the root systems of affected plants with those not yet affected by dieback, or through natural movement of the 6 fungal biological control agent through the soil via mycelia, conidia, chlamydospores or other propagules. By "isolated" is intended separated or purified from other biological components in a mixed sample (such as a fungal extract from a diseased weed). For 5 example, an "isolated" fungal strain is a fungal strain that has been separated from the other components of a sample in which the fungal strain was present (such as diseased weed or a sample taken from a diseased weed). An "isolate", such as a fungal isolate, is a component that has been isolated. It is preferred that the fungal strains used in the methods described herein 10 selectively control weed growth, and do not have any substantial effect on a plant for which growth is desired, for example a non-weed plant, such as an agriculturally important plant, or a residential plant. For this reason, fungal strains used in the methods described herein are preferably obtained from populations of weeds already growing in the native environment to select for biological control agents which have 15 already adapted to, or originate from, the native habitat (i.e., are considered to be endemic organisms). As described herein, a biocontrol composition may further comprise a medium for supporting growth and/or viability, both before and after introduction of the composition into a weed, of the one or more fungal strains. Examples of suitable 20 media include, but are not limited to, agar, supplemented agar, vermiculite, clay, starches, potato dextrose broth, whole grain (e.g., cereal grains, such as rice, wheat, corn, oats, or barley) or grain fragments, whole seeds (e.g., grass seeds, such as millet) or seed fragments, and legumes (e.g., lentil or chickpea), or any combination or variant thereof, provided that the medium allows the fungal strain to remain viable 25 before and after introduction into a weed. As described herein, the biocontrol compositions are introduced into one or more openings formed in a weed. By "introduced" is intended the application or delivery of the biocontrol composition such that the one or more openings formed in a weed are at least partially filled with the biocontrol composition. In some 30 embodiments, the one or more openings formed in the weed are substantially filled with the biocontrol composition.

7 The biocontrol composition may be in any form, including, but not limited to, a liquid, a gel or a solid (e.g., a granular form). In one embodiment, the biocontrol composition is in a granular or pelletised form, for example, pesta. By "pesta" is meant a granular product made from a cereal grain flour and one or more fungal 5 strains. The process of producing pesta encapsulates the one or more fungal strains in pasta-like products (Connick et al., J. Nematol. 25:198-203, 1993). Fungal strains formulated in such media exhibit extended shelf and field-life, which characteristics are desired in a product which may be stored prior to use or shipped over long distances prior to being used for weed control in the field. 10 In one embodiment, the biocontrol composition may be maintained as loose particles (e.g., colonised grain that has been dried and separated) which can be introduced to a weed by various means, such as through one or more openings formed in the weed, or by placement on the soil around the weed's roots or by rolling into a cylinder or a similar package made of paper, such as rice paper, cigarette paper 15 or other materials with similar capabilities. In another embodiment, the biocontrol composition is encapsulated in a soluble container. Soluble containers for the packaging of products are well known to those of skill in the art, and include, for example, capsules made of a soluble material, such as gelatine, alginate, water-soluble polysaccharides (e.g., pullulan), 20 cellulose derivatives (e.g., hydroxypropylmethyl cellulose), polyethylene glycol (PEG), and mixtures thereof. Additional soluble materials and methods of preparing soluble containers (e.g., capsules) are known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy Pharmaceutical Sciences, Lippincott Williams and Wilkins (A. R. Gennaro editor, 20* edition). 25 As described herein, the one or more openings formed in a weed are covered after a biocontrol composition has been introduced into the one or more openings in the weed. In a preferred embodiment, the one or more openings in a weed are sealed after the biocontrol composition has been introduced into the one or more openings in the weed. As used herein, "sealed" includes an air-tight covering to the one or 30 more openings in the weed, such that the introduced biocontrol composition is not disturbed and does not become desiccated. Sealing also reduces the possibility of 8 secondary (contaminant) microorganisms or insects gaining access to the inoculation site and interfering with the development of disease. Non-limiting examples of products that can be used for forming an air-tight seal include, but are not limited to, elastomeric sealants comprising silicone, latex 5 and/or acrylic, such as those used for caulking and sealing in the building industry. In one embodiment, the weed to be controlled is free of visible disease prior to treatment with the biocontrol composition. By "free of visible disease" is meant that the weed is healthy and does not appear to be stressed, and lacks outward signs of disease. A weed that is free of visible disease may also be termed "disease-free". 10 In another aspect, the invention provides a biocontrol composition suitable for location in an opening of a weed, the biocontrol composition comprising a soluble container in which is located: a) one or more fungal strains, and b) a medium for supporting growth and/or viability of the one or more fungal strains. In some embodiments, the medium comprises potato dextrose agar and/or 15 millet seed. In other embodiments, the soluble container is a gelatine capsule, an alginate capsule, a water-soluble polysaccharide capsule, a cellulose-derived capsule, or a polyethylene glycol-containing capsule. 20 So that the invention may be readily understood and put into practical effect, the following non-limiting Examples are provided. EXAMPLES Example 1 25 Fungal Isolate Acquisition Fungal strain isolation involves the collection of stems or other weed parts expressing symptoms of dieback disease from naturally occurring infected sites. Stems were cut into segments approximately 1 cm in length and surface sterilized with 4% NaOCI for three minutes, followed by a rinse in sterile water for three 30 minutes and a final rinse in sterile water for a duration of one minute. The segments were then placed on % potato dextrose agar (PDA) plates using sterile forceps, placed 9 in an incubator at 25*C and daily observation was conducted. Once growth of fungal hyphae from stem segments was detected, sub cultures were made onto new PDA plates incubated at 25*C until grown, and finally placed under UV light for 24 hours to promote sporulation. 5 A culture bank containing over 200 endemic isolates taken from naturally occurring field affected Parkinsonia plants has been generated. Within the collection, five key genera (Fusarium, Botryosphaeria, Lasiodiploidia, Phoma, and Fusicoccum) have been identified. 10 Example 2 Inoculum Production and Storage Millet seed was rinsed twice with distilled water and placed into a beaker filled with distilled water and soaked for 24 hours at room temperature. Excess water was poured off and the millet seed was placed into 500 ml Erlenmeyer flasks, capped 15 with cotton wool and aluminium foil, and autoclaved twice for 25 minutes, 24 hours apart. Following cooling, approximately 5 ml of sterile water was added to each flask together with portions of fungal cultures previously grown on '/2 PDA to inoculate the millet seed. Inoculated millet seed was incubated at 25*C in a darkened incubator to encourage fungal colonization. 20 Control treatments consisted of autoclaved millet seed without inoculum and did not contain any additives. Example 3 Production of Inoculum Capsules 25 Inoculum (colonized millet seed as described herein) is moist at the end of the first phase of production. For ease of handling, and improved storage and transport qualities, the inoculum was dried, for example, by spreading the inoculum decanted from the incubation flasks onto flat sterile metal or plastic trays. The trays were placed onto the bench of a Biohazard Hood, where a filtered, sterile air flow allows 30 the inoculum to dry out (generally in 12 to 24 hours). Dried inoculum was then stored in sterile glass jars with Silica gel (desiccant) at the base to maintain a low moisture 10 level. Dried inoculum was crumbled into individual grains, and used to fill gelatine capsules of the type used for the formulation of medical or food supplement (e.g., vitamin) capsules. This was done by way of a capsule filling tray, but may also be 5 mechanised by a machine operated procedure. Example 4 Stem Inoculation of Parkinsonia aculeata With Fungal Isolates Materials and Methods 10 Experimental Design A total of 15 treatments (including control) with 3 replicates were used. Four fungal isolates plus a control were used (Table I), in combination with three sealants: Parafilm*, SelleysTM Roof and Gutter Silicone Sealant (translucent) and SelleysTM No More Gaps Multipurpose Gap Filler (white) to give the 15 treatments (Table II). 15 Control treatments consisted of un-inoculated autoclaved millet seed. Plants were randomly selected for inoculation with no bias reference to overall height. Plants were randomly placed and moved around the glasshouse bench every week to ensure randomisation. 20 Planting Parkinsonia seeds were provided by John McKenzie, Department of Primary Industries and Fisheries, Charters Towers, Queensland, Australia. Three days following germination, Parkinsonia seedlings were placed in standard potting media acquired from the University of Queensland Gatton Nursery. Each seedling was 25 planted in a 1.4 L pot and a total of 45 pots were used in the experiment. Plants were grown in a glasshouse. Drip irrigation was supplied twice a day for 3 minutes, with intervals increasing to 5 minutes after 18 weeks of growth. Fertiliser was added 6 weeks after inoculation. 30 Inoculation A hole was bored into the plant stem to a depth of 3-4 mm, positioned at 50 11 mm above the soil level, using a 4.5 mm diameter drill bit. Three colonised millet seeds from each fungal isolate were placed into the stem of each plant. The inoculated wound was then covered over with Parafilm*, or sealed with SelleysTM No More Gaps Multipurpose Gap Filler (white) or Selleys T M Roof and Gutter Silicone 5 Sealant (translucent). Implements used to handle inoculum were subjected to alcohol sterilization to prevent cross contamination throughout the inoculation process. Testing Sealants for Antifungal Activity 10 To investigate if the SelleysTM Roof and Gutter Silicone Sealant (translucent), and SelleysTM No More Gaps Multipurpose Gap Filler (white), exhibited anti-fungal properties, a PDA culture plate test was conducted. One half PDA contained within 90 mm Petri dishes was used. Using a sterilised 5 mm diameter cork borer, two holes were bored at opposite ends of the Petri dish, measured 10 mm from the 15 circumference of the dish. One inoculated millet seed was placed in the centre of the plate, while each hole was filled with either of the two sealants. The inhibition of fungal growth around the filled holes would indicate the presence of anti-fungal properties in these compounds. Four fungal isolates (Table I) with three replicates were used. 20 Disease Evaluation To evaluate the effect of the fungal isolate in combination with the various sealants on disease progression in Parkinsonia plants, stem lesion length was measured 9 days after inoculation and a condition status rating was conducted 30 25 days following inoculation. Condition status rating was based on a scale of 1 to 3, where I = healthy, 2 = stressed and 3 = permanently wilted. Measurements were recorded on a weekly basis and tabulated for a period of 8 weeks. Stem diameter and overall height (mm) was conducted at the end of the 30 experiment trial.

12 Statistical Analysis Data were analysed using the general linear model option in Minitab release 15 to test the effect of the fungal isolate. Least square means were compared using a least significant difference procedure. Analysis was carried out on the plant growth 5 per day, maximum lesion size, overall delta (change in) height and diameter of stems. As results obtained from NT027 and control reported a constant zero for lesion length, it was not involved in the analysis for lesion. Only the 3 active isolates were analysed. 10 Results Antifungal Test Results All fungal isolates tested grew over the sealants of SelleysTM Roof and Gutter Silicone Sealant (translucent) and SelleysTM No More Gaps Multipurpose Gap Filler (white). Sporulation was slowest in isolates NT027 and NT094. Growth of NT027 15 and NT094 was observed twenty eight days after plating. Disease Progress The progress of lesion length was documented on a fortnightly basis after inoculation to the conclusion of the experiment. Comparisons were made between 20 treatments 2 weeks and 8 weeks after inoculation. No lesions were recorded on the control treatment and NT027. Pigmentation around the region of wounding present in controls was noted. Lesion length continued to grow on fungal treatments NT094, NT039 and QLD003, before reaching a constant at week 4 after inoculation. The lesions of QLD003 appeared to 25 have a girdling effect. Comparison Between Treatments The comparison between treatments was documented on a fortnightly basis after inoculation to the conclusion of the experiment, at 4 weeks, 6 weeks and 8 30 weeks following inoculation. All replicates within the control treatment appeared healthy. On the other 13 hand, an interaction between fungal isolates and sealants was observed. Plants inoculated with fungal isolate NT027 displayed no significant disease expression but was marginally shorter in height when compared to controls. Replicates within NT027 all appeared healthy. Plants inoculated with NT094 were 5 shorter overall when compared against NT027, with no distinct changes occurring among sealant treatments. It should be noted that a death occurring in the silicone replicate of NT094 was caused by mealy bug infestation. Disease expression was apparent and achieved almost similar results on isolate NT039 sealed with Selleys T M No More Gaps Multipurpose Gap Filler (white) 10 and SelleysTM Roof and Gutter Silicone Sealant (translucent). These two sealant treatments produced significantly shorter plants when compared to the replicates sealed with Parafilm*. All replicates sealed with Parafilm* appeared healthy. Significant results were yielded from fungal isolate QLD003. Distinctive overall height reductions were noticed on replicates sealed with SelleysTM No More Gaps 15 Multipurpose Gap Filler (white) and Selleys T M Roof and Gutter Silicone Sealant (translucent). Parafilm replicates appeared fairly healthy with the presence of death occurring in only one replicate. Overall, all fungal isolate treated plants were relatively shorter than the controls. 20 Overall Results Average stem diameter and overall mean delta (change in) plant height was greatest in the controls. Some evidence of differential response to treatments was noticed. A similar pattern of interaction between the sealants and fungal treatments 25 can be seen in stem diameter and delta height. The ranking of suitability of sealants was variable between the isolates. A significant difference was noticed in the interaction between fungal TM treatments and sealants, and fungal treatments. Treatments NT039 No more gaps TM NT094 Parafilm, NT094 Silicone, QLD003(l) No more gaps , and QLD003(1) 30 Silicone, were significantly different to the control for both mean height and stem diameter.

14 For analysis purposes, overall plant condition status ratings 1 represented healthy plants, while ratings 2 and 3 were combined to categorise plants as dead, as plants with rating 2 were limited. NT094 had an effect on influencing stem diameter and growth rate as 5 compared to NT039, but had a higher percentage of healthy plants in comparison to NT039. Parafilm had no significant effect on the death of plants, while SelleysTM No More Gaps Multipurpose Gap Filler (white) and Selleys T M Roof and Gutter Silicone Sealant (translucent) were more capable of initiating a death response. 10 A reasonable evidence of interaction can be seen. A significant effect of fungal isolate alone and sealant alone can be seen, when P < 0.001 and P = 0.00 1 respectively. Most importantly, a significant interaction can be seen between fungal isolates and sealant. NT094 was incapable of initiating a significant disease response, while 15 NT039 and QLD003 have the ability to generate a reasonable infection, with the latter growing slightly faster. It can be said that the effect of sealant was obvious on two of the isolates: QLD003 and NT039. Documentation had shown that the use of SelleysTM No More Gaps Multipurpose Gap Filler (white) generated a slightly larger lesion length when compared to SelleysTM Roof and Gutter Silicone Sealant 20 (translucent). However, it was not significantly different. For NT039 and QLD003, Parafilm* was significantly less effective than either SelleysTM No More Gaps Multipurpose Gap Filler (white) or SelleysTM Roof and Gutter Silicone Sealant (translucent). NT094 had no significant response on the interaction between lesion growth and sealants. Based on the evaluation of chi-square test, a significant 25 association between both analysis of isolates and plant death (p = 0.016), and sealant and death (p = 0.025), when p = 0.05. However, final data assessed showed that NT094 had a significant effect on average stem diameter and low plant growth. On an overall analysis, QLD003 generated a greater growth or development of lesion per day, but was marginally lower in the expression of maximum lesion 30 length when compared to NT039.

15 Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can 5 be made in the particular embodiments exemplified without departing from the scope of the present invention. All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

16 Table I Treatments used in stem inoculation Treatment / Isolate Identification Relative Aggressiveness QLD003(l) Fusicoccum spp. High NT027 Fusarium spp. Medium NT094 Botryosphaeria spp. High NT039 Lasiodiplodia spp. High Control Autoclaved millet Nil. 5 Table II Fungal and sealant treatments for stem inoculation Fungal Isolates QLD003(l) NT027 NT094 NT039 CONTROL Parafilm Parafilm Parafilm Parafilm Parafilm Silicone Silicone Silicone Silicone Silicone No More Gaps No More Gaps No More Gaps No More Gaps No More Gaps

Claims

1. A method of controlling growth of a weed, said method including the steps of: a) introducing a biocontrol composition comprising one or more fungal strains into one or more openings formed in said weed; and 5 b) covering said one or more openings in said weed.

2. The method of claim 1, further including the steps of: c) identifying a diseased specimen of said weed; and d) isolating one or more fungal strains from said diseased specimen, wherein said biocontrol composition comprises said isolated one or more fungal 10 strains.

3. The method of claim 1 or claim 2, wherein said weed is selected from the group consisting of: Alternanthera philoxeroides, Tamarix aphylla, Chrysanthemoides monilifera subsp. rotundata, Chrysanthemoides monilifera subsp. monilifera, Rubus fruticosus L. agg., Asparagus asparagoides, Ulex europaeus L., 15 Lantana camara, Prosopis spp., Mimosa pigra, Parkinsona aculeata, Parthenium hysterophorus L., Annona glabra, Acacia nilotica subsp. Indica, Cryptostegia grandiflora, and Salix spp.

4. The method of claim 1 or claim 2, wherein said weed is Parkinsona aculeata.

5. The method of any one of the preceding claims, wherein said weed is free of 20 visible disease.

6. The method of any one of the preceding claims, wherein said one or more openings in said weed are in the stem of said weed.

7. The method of any one of claims 1-5, wherein said one or more openings in said weed are in a branch of said weed. 25

8. The method according to any one of the preceding claims, wherein said one or more openings in said weed are formed with a drill.

9. The method of claim 8, wherein said one or more openings in said weed have a diameter of approximately 4.0 mm to approximately 20.0 mm.

10. The method of claim 8, wherein said one or more openings in said weed have 30 a depth of approximately 10.0 mm to approximately 100.0 mm.

11. The method of any one of the preceding claims, wherein said one or more fungal strains are isolates from a diseased specimen of said weed.

12. The method according to any one of the preceding claims, wherein said 2242082v] 18 biocontrol composition comprising one or more fungal strains further comprises a medium for supporting growth and/or viability of said one or more fungal strains.

13. The method of claim 12, wherein said medium comprises potato dextrose agar and/or millet seed. 5

14. The method of claim 12 or claim 13, wherein said biocontrol composition is encapsulated in a soluble container.

15. The method of claim 14, wherein said soluble container is a gelatine capsule, an alginate capsule, a water-soluble polysaccharide capsule, a cellulose-derived capsule, or a polyethylene glycol-containing capsule. 10

16. The method according to any one of the preceding claims, wherein covering said one or more openings in said weed comprises sealing said one or more openings in said weed.

17. The method of claim 16, wherein sealing said one or more openings in said weed comprises applying an elastomeric sealant to said one or more openings in said 15 weed.

18. A biocontrol composition when used for location in an opening of a weed wherein the opening of said weed is covered, the biocontrol composition comprising a soluble container in which is located: a) one or more fungal strains, and 20 b) a medium for supporting growth and/or viability of the one or more fungal strains.

19. The biocontrol composition of claim 18, wherein said medium comprises potato dextrose agar and/or millet seed.

20. The biocontrol composition of claim 19, wherein said soluble container is a 25 gelatine capsule, an alginate capsule, a water-soluble polysaccharide capsule, a cellulose-derived capsule, or a polyethylene glycol-containing capsule.

21. The method of any one of claims 1-17, or the biocontrol composition of any one of claims 18-20, wherein the one or more fungal strains are of a genera selected from the group consisting of: Fusarium, Botryosphaeria, Lasiodiploidia, Phoma, and 30 Fusicoccum.

22. The method or biocontrol composition of claim 21, wherein the one or more fungal strains are of a species selected from the group consisting of: Fusicoccum, Botryosphaeria, and Lasiodiplodia spp. 2242082vl