WO2012048377A1

WO2012048377A1 - Control of weeds

Info

Publication number: WO2012048377A1
Application number: PCT/AU2011/001306
Authority: WO
Inventors: Victor Galea; Naomi Diplock
Original assignee: The University Of Queensland
Priority date: 2010-10-13
Filing date: 2011-10-13
Publication date: 2012-04-19

Abstract

The present invention discloses biocontrol methods for controlling weed 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

TITLE

CONTROL OF WEEDS

FIELD OF THE INVENTION 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

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 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 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 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 in some harsh environments, 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 areas. Thus, there is a need in the art for new methods and compositions for applying biocontrol agents to target weeds. 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 weed, the method including the steps of: a) introducing a biocontrol composition comprising one or more fungal strains into the weed and b) subjecting the weed to stress.

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 specimen, wherein the biocontrol composition comprises the isolated one or more fungal strains.

In one embodiment, introducing the biocontrol composition comprising one or more fungal strains into the weed comprises introducing the biocontrol composition into one or more openings formed in the weed and covering the one or more openings.

Suitably, according to the above embodiment, the one or more openings in the weed can be formed in the stem of the weed, in a branch of the weed, in the crown of the weed, in a fleshy part of the weed, or in any combination thereof.

In another embodiment, introducing the biocontrol composition comprising one or more fungal strains into the weed comprises introducing a device comprising the biocontrol composition into the weed.

Suitably, according to the above embodiment, the device can be a nail or a screw.

In some embodiments, the one or more fungal strains is an isolate from a diseased specimen of the weed or from another weed species.

In other embodiments, the biocontrol composition comprising one or more fungal strains further comprises a medium for supporting growth and/or viability of the one or more fungal strains.

In yet other embodiments, the biocontrol composition comprising one or more fungal strains further comprises a herbicide.

In further embodiments, 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 and/or a herbicide is encapsulated in a soluble container.

In some embodiments, subjecting the weed to stress comprises exposing the weed to a herbicide.

Suitably, according to the above embodiment, exposing the weed to a herbicide includes applying the herbicide to the stem, leaves and/or roots of the weed, or introducing the herbicide into an opening in the weed.

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 one embodiment, a herbicide is further located in the soluble container.

BRIEF DESCRIPTION OF THE FIGURE

Figure 1. Plot of disease rating mean values for Parkinsonia trees inoculated with six capsule fungal treatments in conjunction with three dose levels of glyphosate herbicide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to fungal biocontrol methods and compositions for controlling weed growth.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

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 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 philoxeroides (Alligator weed), Tamarix aphylla (Athel pine), Chrysanthemoides monilifera subsp. rotundata (Bitou bush), Chrysanthemoides monilifera subsp. monilifera (Boneseed), Rubus fruticosus L. agg. (Blackberry), Asparagus asparagoides (Bridal creeper), Ulex europaeus L. (Gorse), Lantana camara (Lantana), Prosopis spp. (Mesquite), Mimosa pigra (Mimosa), Parkinsonia aculeata (Parkinsonia), Parthenium hysterophorus L. (Parthenium), Annona glabra (Pond apple), Acacia nilotica subsp. lndica (Prickly acacia), Cryptostegia grandiflora (Rubber vine), and Salix spp. (Willows).

In one embodiment, the weed is Parkinsonia aculeata.

By "controlling growth of a weed" is meant interfering with the normal 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), inhibition of flowering and seed set, leaf loss, shoot tip damage, branch and/or shoot death, bare stems and/or branches, stem and/or branch splitting, and loss of bark.

In one embodiment, introducing a biocontrol composition into a weed comprises introducing the biocontrol composition into one or more openings formed in the weed and covering the one or more openings.

Formation of openings in a weed encompasses the preparation of a suitable repository in the weed for a biocontrol composition and or a herbicide. In some embodiments, multiple openings may be formed in the weed. Two, three, four, five, or more openings may be 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 both.

Openings may also be formed in the crown of a weed (e.g. , for a grassy weed) or in a fleshy part of a weed (e.g., leaf, stem or phyllode), for example, for a succulent weed (e.g., cacti). As will be understood by one of skill in the art, the preparation of a suitable repository in a weed for a biocontrol composition comprising one or more fungal strains includes an opening made in the soil in close proximity to the roots of the plant.

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 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 1 .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, 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 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 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 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 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 0° to about 85° 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.

In another embodiment, introducing a biocontrol composition into a weed comprises introducing a device comprising the biocontrol composition into the weed.

Such a "device" can take the form of a screw or nail (or other similar device as will be understood by one of skill in the art) containing a dose of the biocontrol composition (and, in some embodiments, a herbicide) which can be hammered or screwed directly into a weed. This process is carried out as a single step, thereby removing the need to form a hole in a weed and cover the hole after introducing the biocontrol composition.

The device can contain one or more holes, grooves, chambers, or the like in which the biocontrol composition is located, thus allowing it to colonise a weed after insertion into the weed. The device can be made of metal, plastic, wood, or a composite material, as will be understood by one of skill in the art. The inclusion of multiple holes, grooves, chambers, or the like in the device allows for the inclusion of more than one type of fungal inoculum ( . e. , multiple isolates) and/or the inclusion of a herbicide in the device. The device can also be used solely for introduction of the herbicide into a weed in the absence of a biocontrol composition.

The device can simply be driven or screwed into a target weed. For soft wooded species of weed, a nail-type device works well, and the device can be nailed by hand, using a hammer or a self-loading hand operating spear unit, or delivered by a mechanically operated purpose built apparatus in high-volume applications.

For hard wooded species of weed, a screw-type device may be desirable.

Screwing can be achieved through the use of existing technology, such as a drill (e.g. , a cordless drill) for low- volume applications, or a self-loading screw gun (e.g., as used in the building industry) for high-volume applications.

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 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 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 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 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 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 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 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 already adapted to, or originate from, the native habitat (i.e., are considered to be endemic organisms).

As used herein, "stress" includes any forcibly exerted influence, and includes, but is not limited to, physical stress (e.g. , removing one or more leaves from a weed; breaking one or more branches of a weed; girdling or ring barking a portion of a weed), chemical stress (e.g., application, either externally or as part of a biocontrol composition, of a herbicide, such as glyphosate [N-(phosphonomethyl) glycine], including the isopropylamine salt form of such herbicide, to a weed), burning, and animal grazing (i.e., herbivory).

A purpose of subjecting a weed to stress as described herein is to facilitate or enhance the ability of the one or more fungal strains in the biocontrol composition to suppress the growth of, or kill, the weed. That is, subjecting a weed to stress is not for the purpose of killing the weed. Rather, subjecting a weed to stress facilitates enhances colonisation of the weed by the one or more fungal strains in the biocontrol composition, as well facilitaing/enhancing the ability of the one or more fungal strains to spread to neighbouring weeds and seed banks. A preferred method of subjecting a weed to stress is by applying a sub-lethal dose of a herbicide as described herein.

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 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 before and after introduction into a weed.

As described herein, a biocontrol composition may still further comprise a herbicide, such as, for example, glyphosate (N-(phosphonomethyl) glycine), including the isopropylamine salt form of such herbicide.

As described herein, a biocontrol composition may be introduced into one or more openings formed in a weed or via a device containing the biocontrol composition. When the biocontrol composition is introduced into one or more openings formed in the weed, it is intended that the one or more openings formed in the weed are at least partially filled with the biocontrol composition. In some embodiments, the one or more openings formed in the weed are substantially filled with the biocontrol composition.

The biocontrol composition may be in any form, including, but not limited to, a liquid, a gel, a wet slurry, a sprayable formulation, 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 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.

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 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), 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^th edition).

In a further embodiment, a wet slurry or a sprayable formulation of the biocontrol composition can be infiltrated into holes, chambers or grooves of a device (e. g. , a nail or a screw) by processes such as dipping or spraying during manufacture. Alternatively, an encapsulated form of the biocontrol composition can be introduced into holes, chambers or grooves of the device. Other processes for introducing the biocontrol composition to the device are also possible, as will be understood by one of skill in the art. After drying, the device can be spray coated with an appropriate biodegradable sealant to maintain the integrity of the biocontrol composition and to provide protection for handling should it be required.

As described herein, subjecting a weed to stress comprises exposing the weed to a herbicide. By "exposing" is intended the application of the herbicide to the leaves and/or roots of the weed, as well as the application or delivery of the herbicide to an opening formed in the weed. In some embodiments, the herbicide is applied or delivered to an opening formed on the opposite side of the stem from the one or more openings into which a biocontrol composition is introduced.

As described herein, the openings formed in a weed may be covered after a biocontrol composition (and, in some embodiments, a herbicide) has been introduced into the openings in the weed. In a preferred embodiment, the openings in a weed are sealed after the biocontrol composition herbicide has been introduced into the openings in the weed. As used herein, "sealed" includes an air-tight covering to the openings in the weed, such that the introduced biocontrol composition/herbicide is not disturbed and does not become desiccated. Sealing also reduces the possibility of 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 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".

In some embodiments, the medium comprises potato dextrose agar and/or millet seed.

In other embodiments, a herbicide is further located in the soluble container. An exemplary herbicide is glyphosate (N-(phosphonomethyl) glycine), including the isopropylamine salt form of such herbicide.

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.

So that the invention may be readily understood and put into practical effect, the following non-limiting Examples are provided.

EXAMPLES

Example 1

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% NaOCl for three minutes, followed by a rinse in sterile water for three minutes and a final rinse in sterile water for a duration of one minute. The segments were then placed on ½ potato dextrose agar (PDA) plates using sterile forceps, placed 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 ½ PDA plates incubated at 25°C until grown, and finally placed under UV light for 24 hours to promote sporulation.

A culture bank containing over 200 endemic isolates taken from naturally occurring field affected Parkinsonia plants has been generated. Within the collection, six key genera (Fusarium, Botryosphaeria, Lasiodiploidia, Phoma, Macrophomina, and Fusicoccum) have been identified.

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 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 ½ PDA to inoculate the millet seed. Inoculated millet seed was incubated at 25°C in a darkened incubator to encourage fungal colonization.

Control treatments consisted of autoclaved millet seed without inoculum and did not contain any additives.

Example 3

Production of Inoculum Capsules

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 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 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 mechanised by a machine operated procedure.

Example 4

Stem Inoculation of Parkinsonia aculeata With Fungal Isolates

Materials and Methods

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*, Selleys™ Roof and Gutter Silicone Sealant (translucent) and Selleys™ No More Gaps Multipurpose Gap Filler (white) to give the 15 treatments (Table Π). 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.

Planting

Parkinsonia seeds were provided by John Mc enzie, 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 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. Inoculation

A hole was bored into the plant stem to a depth of 3-4 mm, positioned at 50 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 Selleys™ No More Gaps Multipurpose Gap Filler (white) or Selleys™ Roof and Gutter Silicone 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

To investigate if the Selleys™ Roof and Gutter Silicone Sealant (translucent), and Selleys™ 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 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.

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 days following inoculation. Condition status rating was based on a scale of 1 to 3, where 1 = 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 experiment trial.

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 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.

Results

Antifungal Test Results

All fungal isolates tested grew over the sealants of Selleys™ Roof and Gutter Silicone Sealant (translucent) and Selleys™ No More Gaps Multipurpose Gap Filler (white). Sporulation was slowest in isolates NT027 and NT094. Growth of NT027 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 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 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 weeks following inoculation.

All replicates within the control treatment appeared healthy. On the other 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 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™ No More Gaps Multipurpose Gap Filler (white) and Selleys™ 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 Selleys™ No More Gaps Multipurpose Gap Filler (white) and Selleys™ 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.

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 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 treatments and sealants, and fungal treatments. Treatments NT039 No more gaps™, NT094 Parafilm, NT094 Silicone, QLD003(1) No more gaps™, and QLD003(1) Silicone, were significantly different to the control for both mean height and stem diameter.

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 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 Selleys™ No More Gaps Multipurpose Gap Filler (white) and Selleys™ Roof and Gutter Silicone Sealant (translucent) were more capable of initiating a death response.

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 = 0.001 respectively. Most importantly, a significant interaction can be seen between fungal isolates and sealant.

NT094 was incapable of initiating a significant disease response, while 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 Selleys™ No More Gaps Multipurpose Gap Filler (white) generated a slightly larger lesion length when compared to Selleys™ Roof and Gutter Silicone Sealant (translucent). However, it was not significantly different. For NT039 and QLD003, Parafilm^® was significantly less effective than either Selleys™ No More Gaps Multipurpose Gap Filler (white) or Selleys™ 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 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 length when compared to NT039.

Example 5

Stem Inoculation of Parkinsonia aculeata With

Fungal Isolates and Glyphosate

Materials and Methods

Experimental Design

A total of eighteen (6 x 3) treatments (including control) with five replicates were used. The trial followed the design outlined below.

Major Treatment Set - (6)

Replications: (4) each rep unit = 8 trees. Four replicates per treatment.

Overall Trial Structure: 6 x 3 x 32 = 576 trees.

Plants The trial was conducted in a naturally occurring infestation of Parkinsonia trees at "Stradbroke", a cattle grazing property located south of Duchess in North Western Queensland with permission from the station owner and support from the station manager. The trial was established on 12 April 2011.

Inoculation

Trees were treated in groups of eight (one replicate group) and separated from other groups by a distance of at least five metres. The first tree in each replicate group was identified by attaching a labelled tree tag at chest height and the circumference of its stem measured and recorded. A GPS reading was taken at this tree position. All eight trees per replicate were sprayed with red paint at the base to further identify them.

Where only a single capsule was required, a single hole (10 mm diameter) was drilled into the tree approximately 200 mm above ground level, a capsule inserted into the hole, and the hole sealed with silicone sealant (domestic roof and gutter type silicone). Where more than one capsule was required, multiple holes were drilled in close proximity, inoculated singularly and sealed.

Where herbicide co-treatment was performed, a hole was drilled at a 45 degree angle on the opposite side of the tree to the capsule insertion point and a 1 ml volume of the herbicide , solution applied using a herbicide syringe pump. This herbicide hole was left un-sealed.

Disease Evaluation

Trees were assessed for tree health on the day of trial establishment using a five level assessment score based on the following categories.

Rating score Description

0 Tree dead - 100% of stem material dead

1 Tree severely stressed 1-30% of stem material green

2 Tree moderately stressed 31-50% of stem material green 3 Tree slightly stressed 51-70% of stem material green

4 Tree healthy - 71-100% of stem material green

This same rating score was applied at subsequent evaluation of the trial.

Statistical Analysis

The first data set was collected on 10 October 2011, 26 weeks after trial establishment. The plants were assessed using the rating system previously described. Data is shown in Table ΙΠ and Figure 1.

Disease Progress

Field observations and data collected 26 weeks after trial establishment indicated that disease symptoms were becoming apparent in some treatments compared to the control plots. Individual trees in some treatments rated health scores as low as 2, 1 or 0. However, treatment means indicated that overall health scores were still high at this early stage of the trial.

Comparison Between Treatments

For all trees not treated with herbicide (Nil Herbicide) the decline in health when inoculated with any of the five fungal treatments was significant when compared to the control trees. Trees inoculated with all three isolates as separate capsules showed the greatest decline in health over this period.

For those trees treated with the lowest dose of herbicide (1/4 strength) the tree health ratings were somewhat variable, but were generally higher (but not significantly so) for all fungal treated trees when compared to the control containing no fungi.

For those trees treated with the highest dose of herbicide (1/2 strength) tree health ratings for fungal treated trees were either comparable with or lower than the control treatment containing no fungi. Trees inoculated with NT039 alone, NT039 + QLD003 + NT094 as separate capsules in the same tree or a blend of all three isolates as a single capsule showed reduced (but not significantly) tree health when compared to the control trees.

Overall Results

The preliminary results indicate that there is a clear relationship between the health status of Parkinsonia trees and the use of fungal capsules as inoculants. Furthermore, this data shows that co-treatment of Parkinsonia plants with fungal agents and sub-lethal doses of herbicides can lead to an enhanced level of tree health decline as a result of accelerated fungal dieback. A clearer picture will be available at the 12-month and 18-month assessment points.

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 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.

Table I. Treatments used in stem inoculation

Treatment / Isolate Identification Relative Aggressiveness

QLD003(1) Fusicoccum spp. High

NT027 Fwarium spp. Medium

NT094 Botryosphaeria spp. High

NT039 Lasiodiplodia spp. High

Control Autoclaved millet Nil.

Table II. Fungal and sealant treatments for stem inoculation

Table III. Disease rating for Parkinsonia aculeata inoculated with fungal isolates and glyphosate herbicide

Nil Herbicide ¼ strength herbicide ½ strength herbicide

Mean Standard Mean Standard Mean Standard rating error rating error rating error

Control 4.00 0.00 3.44 0.15 3.41 0.12

NT039 3.91 0.07 3.59 0.12 3.28 0.13

QLD003 3.66 0.10 3.52 0.13 3.45 0.11

NT094 3.71 0.09 3.66 0.11 3.47 0.09 NT039 + 3.54 0.12 3.54 0.12 3.22 0.17 QLD003

+ NT094

Cocktail 3.83 0.10 3.75 0.09 3.28 0.14

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 said weed; and

b) subjecting said weed to stress.

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 strains.

3. The method of claim 1, wherein introducing said biocontrol composition comprising one or more fungal strains into said weed comprises introducing said biocontrol composition into one or more openings formed in said weed and covering said one or more openings.

4. The method of claim 3, wherein said one or more openings in said weed are in the stem of said weed.

5. The method of claim 3, wherein said one or more openings in said weed are in a branch of said weed.

6. The method of any one of claims 3-5, wherein said one or more openings in said weed are formed with a drill.

7. The method of claim 6, wherein said one or more openings in said weed have a diameter of approximately 4.0 mm to approximately 20.0 mm.

8. The method of claim 6, wherein said one or more openings in said weed have a depth of approximately 10.0 mm to approximately 100.0 mm.

9. The method of any one of claims 3-8, wherein covering said one or more openings in said weed comprises sealing said one or more openings in said weed.

10. The method of claim 9, wherein sealing said one or more openings in said weed comprises applying an elastomeric sealant to said one or more openings in said weed.

11. The method of claim 1, wherein introducing said biocontrol composition comprising one or more fungal strains into said weed comprises introducing a device comprising said biocontrol composition into said weed.

12. The method of claim 11 , wherein said device is a nail.

13. The method of claim 11 , wherein said device is a screw.

14. The method of any one of claims 11-13, wherein said device comprises a hole, a grove or a chamber in which said biocontrol composition is located.

15. The method of any one of the preceding claims, 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., Lantana camara, Prosopis spp., Mimosa pigra, Parkinsonia aculeata, Parthenium hysterophorus L., Annona glabra, Acacia nilotica subsp. Indica, Cryptostegia grandiflora, and Salix spp.

16. The method of claim 15, wherein said weed is Parkinsonia aculeata.

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

18. 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.

19. The method of any one of the preceding claims, wherein said 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.

20. The method of claim 19, wherein said medium comprises potato dextrose agar and/or millet seed.

21. The method of any one of the preceding claims, wherein said biocontrol composition comprising one or more fungal strains further comprises a herbicide.

22. The method of claim 21 , wherein said herbicide is glyphosate.

23. The method of any one of the preceding claims, wherein said biocontrol composition is encapsulated in a soluble container.

24. The method of claim 23, 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.

25. The method of any one of the preceding claims, wherein subjecting said weed to stress comprises subjecting said weed to physical stress or chemical stress.

26. The method of claim 25, wherein subjecting said weed to chemical stress comprises exposing said weed to a herbicide.

27. The method of claim 26, wherein exposing said weed to a herbicide comprises applying said herbicide to the leaves and/or the roots of said weed.

28. The method of claim 26, wherein exposing said weed to a herbicide comprises introducing said herbicide into an opening formed in said weed.

29. The method of claim 28, wherein said biocontrol composition comprising one or more fungal strains and said herbicide are introduced into openings formed on opposite sides of the stem of said weed.

30. The method of any one of claims 26-29, wherein said herbicide is glyphosate.

31. The method of any one of the preceding claims, wherein said biocontrol composition comprising one or more fungal strains is introduced prior to subjecting said weed to stress.

32. The method of any one of claims 1 -30, wherein said biocontrol composition comprising one or more fungal strains is introduced after subjecting said weed to stress.

33. The method of any one of claims 1 -30, wherein said biocontrol composition comprising one or more fungal strains is introduced simultaneously with subjecting said weed to stress.

34. 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.

35. The biocontrol composition of claim 34, wherein said medium comprises potato dextrose agar and/or millet seed.

36. The biocontrol composition of claim 34, wherein a herbicide is further located in said soluble container.

37. The biocontrol composition of claim 36, wherein said herbicide is glyphosate.

38. The biocontrol composition of any one of claims 34-37, 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.