Methods and compositions comprising trichoderma hamatum
Patent Information
- Application Number
- PCT/NZ2026/050013
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-02-18
- Filing Date
- 2026-02-18
- Publication Date
- 2026-08-27
Smart Images
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Abstract
Description
[0001] Our Ref: 323254PCT
[0002] METHODS AND COMPOSITIONS COMPRISING TRICHODERMA HAMATUM
[0003] TECHNICAL FIELD
[0004] This invention relates to a novel Trichoderma hamatum strain Pal2T5 and compositions containing same. Methods for the biological control of increasing plant nitrogen use efficiency and reducing nitrogen wastage following urea application using Trichoderma hamatum strain Pal2T5 are also provided.
[0005] BACKGROUND ART
[0006] Trichoderma is a genus of fungi containing about 20 species. Over the last couple of decades, the efficacy of various Trichoderma species for biocontrol and plant growth promotion has been explored and discussed (see for example Stewart et al, Australian Plant Pathology Society, Adelaide, 2007).
[0007] However, despite extensive investigations, very few Trichoderma products are available for commercial use primarily because of the complex interactions that take place in the soil or on plants between Trichoderma and other microorganisms, the environment, and the plant or plant root.
[0008] Nonetheless, in 2009 a number of Trichoderma atroviride strains were identified in NZ587233 as highly effective as biocontrol agents and / or growth promotants either alone or in combination with one another. Specifically, these Trichoderma atroviride strains were:
[0009] • Trichoderma atroviride NMI No. V08 / 002387;
[0010] • Trichoderma atroviride NMI No. V08 / 002388;
[0011] • Trichoderma atroviride NMI No. V08 / 002389; and
[0012] • Trichoderma atroviride NMI No. V08 / 002390.
[0013] The inventors of this present invention have now identified a novel Trichoderma hamatum strain Pal2T5 which is also useful for one or more of: retaining plant available soil nitrogen, reducing nitrogen wastage, preventing nitrogen loss from soil, promoting plant growth and / or increasing plant tolerance of drought stress.James & Wells ref: 323254PCT
[0014] In addition, the inventors have discovered the new Trichoderma hamatum strain Pal2T5 is effective as an agent for increasing plant nitrogen use efficiency and reducing nitrogen wastage via ammonia volatilisation following urea application, either alone or in combination with other Trichoderma species.
[0015] It is an object of the present invention to provide a novel Trichoderma hamatum strain useful as an agent to increase plant nitrogen use efficiency and reducing nitrogen wastage following urea application to provide an alternative to application of urea with a chemical inhibitor e.g. such as NBPT.
[0016] Another object is to provide a composition comprising the novel Trichoderma hamatum strain of the invention, either alone or in combination with one or more other Trichoderma strains, such as those disclosed in NZ587233, to provide the public with a useful choice.
[0017] A still further object of the present invention is to provide an alternative to urease inhibitors such as, but not limited to N-(n-butyl) thiophosphoric triamide (NBPT) - for example such as that sold under the AGROTAIN trade mark or YaraVera AMIPLUS.
[0018] Another object is to provide increased plant tolerance to drought stress.
[0019] It is therefore an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.
[0020] All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.
[0021] Throughout this specification, the word "comprise", or variations thereof such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
[0022] Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.James & Wells ref: 323254PCT
[0023] DEFINITIONS
[0024] The term “effective amount” as used herein means an amount effective to increase nitrogen use efficiency and / or to reduce nitrogen wastage in a pasture or cropped soil.
[0025] The term “plant” as used herein encompasses not only whole plants but also extends to plant parts, cuttings and plant products. For example, roots, leaves, seeds, stems, callus tissue, and fruit.
[0026] The term “plant growth promoter” as used herein broadly refers to an agent which increases the total plant yield measured as dry weight mass in kilograms per plant, hectare or similar. The terms “promote plant growth”, plant growth promoter” and “growth promoter” should be similarly understood.
[0027] The term “yield” refers to the total plant yield measured as dry weight mass in kilograms per plant, per hectare or similar.
[0028] The term “strain” refers to a novel isolate of Trichoderma which has been identified selected and separated from other microbes found in situ including other strains of Trichoderma, as found in soil, the strain being capable of being grown in a culture.
[0029] The term “nitrogen use efficiency” refers to the ability of a plant to utilise soil available nitrogen.
[0030] The term “nitrogen wastage” refers to nitrogen in the soil being lost via nitrate leaching into waterways or escaping into the atmosphere as a nitrogen emission, such as N2O gas or ammonia volatilisation.
[0031] The term “plant tolerance” refers to a plant’s innate ability to survive, regrow and maintain fitness despite facing stress (eg. drought).
[0032] The term “drought stress” refers to environmental stress when plant water demands exceed soil supply, severely hindering plant growth, limiting photosynthesis and reducing yield.
[0033] SUMMARY OF THE INVENTION
[0034] According to a first aspect of the present invention there is provided a Trichoderma hamatum strain deposited in the National Measurement Institute laboratories (NMI), 1 / 153 Bertie Street, Port Melbourne, VIC 3207, Australia on 31stJanuary 2025 according to the Budapest Treaty forJames & Wells ref: 323254PCT
[0035] the purposes of patent procedure. The strain has been accorded accession number NMI No. V25 / 001998. This strain is also referred to as Pal2T5 throughout this specification.
[0036] According to a second aspect there is provided a use of the Trichoderma hamatum NMI No. V25 / 001998 substantially as described above to increase nitrogen use efficiency and / or to reduce nitrogen wastage in a pasture or cropped soil.
[0037] According to a third aspect there is provided a use of the Trichoderma hamatum NMI No. V25 / 001998 substantially as described above, wherein the use involves application to an area of land following urea application and / or urine deposition.
[0038] According to a fourth aspect there is provided a use of the Trichoderma hamatum NMI No. V25 / 001998 substantially as described above, wherein the nitrogen wastage includes ammonia volatilisation, nitrous oxide emissions, and / or nitrate leaching.
[0039] According to a fifth aspect there is provided a composition comprising in a reproductively viable form and amount: Trichoderma hamatum deposited at National Measurement Institute laboratories (NMI), 1 / 153 Bertie Street, Port Melbourne, VIC 3207, Australia and accorded accession number NMI No. V25 / 001998 and an agriculturally acceptable carrier.
[0040] According to a sixth aspect there is provided a composition substantially as described above, wherein the composition further comprises an equal mixture of Trichoderma atroviride NMI No. V08 / 002387, Trichoderma atroviride NMI No. V08 / 002388, Trichoderma atroviride NMI No. V08 / 002389, and Trichoderma atroviride NMI No. V08 / 002390 (collectively named PBI) in a reproductively viable form.
[0041] According to a seventh aspect there is provided a method for increasing nitrogen use and / or reducing nitrogen wastage on an area of land wherein the method comprising directly or indirectly applying a composition substantially as described above to said area of land.
[0042] According to an eighth aspect there is provided a method of increasing nitrogen use and / or reducing nitrogen wastage of an area of land, the method comprising delivering to said area of land Trichoderma hamatum NMI No. V25 / 001998 in a reproductively viable form and amount.
[0043] According to a ninth aspect there is provided a method of increasing plant tolerance of drought stress on an area of land, the method comprising delivery to said area of land Trichoderma hamatum NMI No. V25 / 001998 in a reproductively viable form and amount.James & Wells ref: 323254PCT
[0044] According to a 10th aspect there is provided a use of Trichoderma hamatum strain of according to the first aspect as described above to increase drought tolerance of a plant compared to a plant untreated with said Trichoderma hamatum.
[0045] According to a 11th aspect there is provided a use substantially as described above, wherein the plant is selected from the group consisting of: perennial ryegrass; white clover; plantain; and lucerne.
[0046] For example, one gram of seed may be coated with 10 million conidia (colony forming unit) and if the coated seeds are being applied by direct drilling, it is envisaged one may use 15 kg prills per hectare.
[0047] Other possible options for direct or indirect application to land and crops include coated fertiliser granules and foliar spraying. This list should not be seen as limiting as other forms of direct or indirect application may be used without departing from the scope of the present invention.
[0048] BRIEF DESCRIPTION OF THE DRAWINGS
[0049] 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:
[0050] Figure 1 Figures 1 A and 1 B show the effect of Trichoderma hamatum strain Pal2T5 on perennial ryegrass plant growth in the glasshouse following urea application; Figure 1C shows the effect of Trichoderma hamatum strain Pal2T5 and NBPT on dry weight of perennial ryegrass roots at 35 days after urea application; Figures 1 D and 1 E show the effect of Trichoderma hamatum strain Pal2T5 and NBPT on whole nitrogen content of perennial ryegrass shoots and roots at 10 and 35 days after urea application, respectively (1- Urea, 2- Urea + PBI, 3- Urea + Pal2T5 and 4- Urea + NBPT.
[0051] Figure 2 Figures 2A and 2B show the effect of Trichoderma hamatum strain Pal2T5 on perennial ryegrass plant dry matter production (kg / ha) from two harvests (1-No urea, 2- Urea only, 3- Urea + CaT1, 4- Urea + Po3T26, 5- Urea + PBI, 6- Urea + Pal2T5, 7- Urea + NBPT); Figure 2C shows the effect of Trichoderma hamatum strain Pal2T5 on cumulative pasture dry matter collected from six harvests over 15 months from non-irrigated pasture; Figure 2D shows the effect of Trichoderma hamatum strain Pal2T5 on cumulative pasture dry matter collected from fiveJames & Wells ref: 323254PCT
[0052] harvests over 15 months from irrigated pasture; Figure 2E shows the effect of Trichoderma hamatum strain Pal2T5 on plant total nitrogen content compared to the PBI combination; Figure 2F shows the effect of Trichoderma hamatum strain Pal2T5 on plant 15N content compared to the PBI combination from non-irrigated pasture; Figure 2G shows the effect of Trichoderma hamatum strainPal2T5 on the percentage of 15N taken up by the plants compared to the PBI combination; Figure 2H shows the effect of Trichoderma hamatum strain Pal2T5 on soil urease activity compared to the PBI combination (values labelled with the same letter do not differ significantly according to Fisher’s Unprotected Least Significance Difference (P<0.05)); Figure 2I and 2L show the effect of Trichoderma hamatum strain Pal2T5 on cumulative ammonia released from soil compared to the PBI combination; Figure 2J shows the effect of Trichoderma hamatum strain Pal2T5 on nitrous oxide emissions compared to the PBI combination (values labelled with the same letter do not differ significantly according to Fisher’s Unprotected Least Significance Difference (P<0.05)); Figure 2K shows the effect of Trichoderma hamatum strain Pal2T5 on soil urease activity compared to the PBI combination Figure 2M shows the effect of Trichoderma hamatum strain Pal2T5 on soil nitrate compared to the PBI combination; Figure 2N shows the effect of Trichoderma hamatum strain Pal2T5 on soil urease activity compared to the PBI combination (values labelled with the same letter do not differ significantly according to Fisher’s Unprotected Least Significance Difference (P<0.05)).
[0053] Figure 3 Figure 3A shows the effect of Pal2T5 on perennial ryegrass plant growth in the glasshouse under drought stress; Figure 3B shows the visual comparison of perennial ryegrass plants treated with Pal2T5 (left) compared with the control (right) after two weeks of moisture stress; Figure 3C shows the effect of Pal2T5 on leaf chlorophyll content of perennial ryegrass under drought stress.
[0054] Figure 4 Figure 4A shows the effect of Pal2T5 on white clover plant growth (shoot dry weight) under drought stress; Figure 4B shows the effect of Pal2T5 on leaf chlorophyll content of white clover under drought stress; Figure 4C shows the visual comparison of white clover plants treated with Pal2T5 compared with the control after two weeks of moisture stress; Figure 4D shows the relative water content of white clover leaves treated with Pal2T5 compared with the control after two weeks of moisture stress.
[0055] Figure 5 Figure 5A shows the effect of Pal2T5 on plant growth of plantain under drought stress; Figure 5B shows the effect of Pal2T5 on leaf chlorophyll content of plantainJames & Wells ref: 323254PCT
[0056] under drought stress; Figure 5C shows the visual comparison of plantain plants treated with bio-inoculants compared with the control after two weeks of moisture stress; Figure 5D shows the effect of Pal2T5 on relative water content of plantain leaves after two weeks of moisture stress.
[0057] Figure 6 Figure 6A shows the effect of Pal2T5 on plant growth of lucerne under drought stress; Figure 6B shows the effect of Pal2T5 on relative water content of lucerne leaves after two weeks of moisture stress; Figure 6C shows the visual comparison of Pal2T5 treated lucerne plant roots and nodules under drought stress.
[0058] Figure 7 Figure 7A shows the cumulative perennial ryegrass dry matter production measured at pre-drought, middle-drought and post-drought stress in different Trichoderma (PBI + Pal2T5) application methods; Figure 7B shows the SPAD value of perennial ryegrass leaves measured at mid-drought stress in different Trichoderma (PBI+ Pal2T5) application methods.
[0059] BEST MODES FOR CARRYING OUT THE INVENTION
[0060] Example 1: Effect of Trichoderma hamatum strain Pal2T5 on Plant Growth in the Glasshouse Following Urea Application
[0061] 1.1 Materials and Methods
[0062] To measure plant response to nitrogen uptake following urea application and the effect of Trichoderma strains on this process, plastic containers (9 x 9 x 28.3 cm) were used. Soil from a field at Lincoln University was sieved and mixed with pumice (3:1 w / w) to fill the containers (711 g in each container). As the containers were clear, black tape was used to cover their lower half to prevent any likely effect of light on soil microbes. Two Trichoderma bio-inoculants (Trichoderma hamatum’. strain Pal2T5 and T. atroviride: PBI, which is an equal mixture of four strains, Trichoderma atroviride NMI No. V08 / 002387, Trichoderma atroviride NMI No.
[0063] V08 / 002388, Trichoderma atroviride NMI No. V08 / 002389, and Trichoderma atroviride NMI No. V08 / 002390), were cultured on 30 g sterilised wheat bran, peat and water (1:1:1 v / v / v) in tissue culture jars, incubated at 23°C for two weeks, following which 1.5 g culture was mixed into the top 200 g soil in a container. Perennial ryegrass seeds (Cv. One50; 15 seeds for each container) were placed in the container using forceps at 1 cm depth then covered with soil and left to grow in a glasshouse at Lincoln University (daily average temperature was 17.2°C). The containers were watered daily when needed and seedling emergence was recorded after 20 days. T o add nitrogen to the soil at a rate equivalent to 50 kg nitrogen / ha the area of the jar wasJames & Wells ref: 323254PCT
[0064] calculated, and the required amount (0.088 g / container) of urea solution (containing 10% 15N labelled urea) was applied to each container 59 days after sowing. NBPT was also applied at a rate equivalent to 1 Kg / Ha along with urea as one of the treatments to compare results with the Trichoderma treatments. The shoots were cut to the top of the container level at 58 days after sowing (one day prior to urea application) and again at 69 days after sowing and fresh and dry weights recorded. For dry weight the shoots were oven dried at 60°C for three days before weighing. The final harvest was at 94 days after sowing, when the entire plants were carefully removed from the containers washed to remove soil and oven dried at 60°C for three days before root and shoot dry matter was determined. The 15N content of the dry shoot and root material was measured using a stable isotope ratio mass spectrometer (IRMS) at the Faculty of Agriculture and Life Sciences, Lincoln University.
[0065] 1.2 Results
[0066] Following urea application, shoot dry weight at 10 days after urea application for both the Trichoderma treatments and NBPT was significantly greater than the urea only control (Fig.1 A, 1B). At 35 days after urea application root dry weight in plants treated with both the Trichoderma treatments and NBPT was also greater than that of the urea only control, and Pal2T5 root dry weight was significantly greater than that of NBPT (Fig. 1C). All the treatments increased the shoot and root nitrogen content over that of the urea only control (Fig. 1 D, 1 E). Roots from plants treated with Pal2T5 had a 62% higher nitrogen content than the control and also a significantly higher nitrogen content than roots of plants treated with NBPT (Fig. 1E).
[0067] Example 2:
[0068] 2.1 Materials and Methods
[0069] 2.1.1. Lincoln University H8 field trial
[0070] Following conventional cultivation and seed bed preparation, this field trial was sown on 24th April 2015. Trichoderma treatments were applied in a seed coating provided by Dr. Craig Bunt of Lincoln University. Perennial ryegrass seeds (Cv. One 50) were sown at 20 kg / ha using a precision seed drill. The treatments were Pal2T5 + urea, PBI + urea, two other Trichoderma strains (CaT1 and Po3T26) both + urea, no urea, urea only, and N-(n-butyl) thiophosphoric triamide (NBPT) (1 Kg / ha) + urea. Specifically, Po3T26 was isolated from soil in Westport and CaT1 from soil in Southbridge using Trichoderma selective medium. Seedling emergence was recorded 24 days after sowing. Urea (25 kg N / ha) was manually applied to each plot on 2ndJames & Wells ref: 323254PCT
[0071] November. Plants growth was assessed at 16 and 83 days after the urea application. Plants were cut to 5cm above ground level and fresh and dry weight recorded.
[0072] 2.1.2. Lincoln University Kowhai Farm field trials (irrigated and rainfed plots)
[0073] These two field trials were established to investigate the effects of Trichoderma strains on urea-N losses and pasture DM production Seeds of perennial ryegrass (Cv. One50, 15 kg / ha) and white clover (cv.Aran, 4 kg / ha) were coated with either PBI or PalT25 Trichoderma strains and sown into a conventionally prepared seed bed on 3 / 05 / 2019. There were eight replicates of each treatment arranged within eight blocks in a randomised complete block design for both the rainfed and irrigated trials. The treatments in each trial were: Urea only, Urea + PBI, Urea + Pal2T5. The plots at Kowhai farm were cut during the year to produce a cumulative set of dry matter data to determine the effects of the treatments in both irrigated and rainfed conditions. For both trials, plants were cut to measure DM six times during the year on 24 / 9 / 2019, 31 / 10 / 2019, 7 / 1 / 2020, 11 / 2 / 2020, 1 / 5 / 2020 and 24 / 12 / 2020. The dry matter collected from the non-irrigated section over 15 months is an indicative of drought stress tolerance increase by Pal2T5 as the field was not irrigated during this time.
[0074] A further experiment using 15N labelled urea was conducted on the Kowhai farm rainfed trial area in late January 2020. Plants were first cut to 5 cm above the ground level following which chambers were installed to collect ammonia and nitrous oxide from the soil. A 47 cm diameter chamber was used for nitrous oxide collection (Di and Cameron 2022) and a 35 cm diameter chamber was used for ammonia collection) (Han et al., 2014, Pan et al., 2022). One large and one small chamber were installed in each plot (Fig. 1-13). Urea fertiliser (70 kg urea-N containing 10% 15N) was applied to each plot before applying 25mm water to irrigate the plots. Ammonia released from the soil after urea application was trapped in a plate containing 50 ml 0.05N sulphuric acid which had been placed in the small chamber. Nitrous oxide released from the soil into the large chambers was collected every 24h for 10 days. The ammonia trapped in acid samples was analysed using flow injection analysis (using a FOSS FIAstar 5000 twin channel analyser) and a gas chromatograph (Model 8610C, SRI Instruments, California, USA) configured with two Haysep-D™ packed columns and an Electron Capture Detector (ECD) was used to quantify nitrous oxide samples. Each large container had a hole on the lid with a rubber bung fitted in which a needle (with a tap) could pass through to be used to collect nitrous oxide in small, vacuumed bottles. The collection started with putting the lid on the chamber to collect the gas present at time zero and then leaving the lid on before collecting the gas again at time 40 minutes. The difference between the gas amount released at time 40 minutes and time zero was the amount of the gas which had been produced over the 40 minutes time period. The gas collected was analysed and compared among the different treatments. Soil samples (20 gramsJames & Wells ref: 323254PCT
[0075] from each plot) were collected using a soil sampling core, for analysis. Soil urease activity was measured according to Gianfreda et al. (1994). Briefly, one g of soil was mixed with 4 ml Tris buffer 20mM pH 7 and mixed. Then one ml urea solution (6%) was added to the mixture and incubated for one hour at 37°C. For the blank sample, one ml distilled water was used instead of urea solution. After one hour of incubation, 10 ml KCL 2N was added to each tube on ice and the samples were centrifuged at 8500 RPM for 10 minutes. The supernatant was separated and 500pL was mixed with one ml distilled water in Eppendorf tubes before adding 500pL Nessler’s reagent to each tube. Absorbance of each sample was read at 450 nm using a Genesys 10 UV scanning spectrophotometer, Thermo Scientific, Helios gamma. The blank sample was used to remove the background yellow colour which appeared from the background ammonium in the soil. An increase in absorbance was considered as urease activity (Gianfreda et al., 1994). To measure soil nitrate content, 10 g dry soil was mixed with 40 ml KCI 2N and was shaken for 2 hours. The mixture was then centrifuged at 8500 RPM for 10 minutes and the supernatant was filtered and analysed using a SmartChem® analyser (Blakemore et al., 1987).
[0076] 2.1.3. Lincoln H11 field trial
[0077] On 14 / 02 / 2022 a prill formulation of PBI and PalT25 was direct drilled at 15kg / ha into the existing pasture plots at this site. On 12 / 04 / 2022, plants were cut to 5 cm above the ground level. Urea (70 kg N / ha) was then applied to each plot and soil samples were analysed for urease activity as described in 2.1.2.
[0078] 2.2 Results
[0079] 2.2.1. Lincoln University H8 field trial
[0080] By 16 days after urea application plants treated with urea only had produced significantly more shoot dry matter than the plants which had not received urea (Treatments 1 and 2, Fig.2A). Two treatments, Pal2T5 and NBPT had produced significantly more shoot dry matter than the urea only control but CaT1, Po3T26 and PBI did not (Fig. 2A).
[0081] For the next harvest (67 days later), none of the treatments had produced more shoot dry matter than the urea only control (Fig. 2B). As the urea had been applied 83 days earlier this result indicates that an increase in shoot dry matter requires the presence of urea, and that the increase at 16 days after urea application is because of an increase in plant nitrogen uptake.James & Wells ref: 323254PCT
[0082] 2.2.2. Lincoln University Kowhai Farm trials
[0083] Over 15 months the cumulative dry matter of plants treated with either PBI or Pal2T5 was significantly greater than the urea only control (3,311 kg / ha more dry matter in plots treated with Pal2T5 = 15%) in the rainfed trial (Fig. 2C) and a similar result occurred in the irrigated block with a 3574 kg / ha increase (Fig. 2D). A comparison between dry matter from the rainfed trial inoculated with Pal2T5 (which produced 25,609 kg / ha) and the urea only control in the irrigated trial (which produced 24376 kg / ha) indicates that Pal2T5 increased dry matter production under drought to the level of production in irrigated conditions.
[0084] In the rainfed trial total N content (including 14N and 15N) of plants treated with both Trichoderma strains was greater than the urea only control, with a 24% increase in N uptake (Fig, 2E).
[0085] Plants in the presence of the Trichoderma strains also had a higher 15N content (36%) compared with the urea only control (Fig. 2F), indicating that the plants were able to increase N uptake in non-irrigated conditions. Plants in the presence of the bio-inoculants took up 33% of the urea applied to the soil while plants in the absence of the Trichoderma strains were only able to take up 24% of the nitrogen applied (Fig. 2G). No data can be provided for the amount of applied nitrogen that may have been immobilised in the soil by soil inhabiting microbes including Trichoderma.
[0086] In the rainfed trial the soil from plants treated with both PalT25 and PBI had a significantly lower urease activity than that of the urea only control (Fig. 2H), and both cumulative ammonia released over the 12 days following urea application (Fig. 2I) and nitrous oxide emissions (Fig.
[0087] 2J) were significantly lower than that of the urea only control. Results for soil urease activity (Fig. 2 K) and cumulative ammonia release (Fig. 2L) in the irrigated trial were similar to those for the rainfed trial, and both Trichoderma treatments reduced soil nitrate content (Fig. 2M).
[0088] 2.2.3. Lincoln H11 field trial
[0089] Soil urease activity was significantly reduced in plots treated with NBPT (by 44%), Pal2T5 (by 29%) and PBI (by 18%) compared with the urea only control (Fig. 2N) indicating that NBPT and the Trichoderma strains had reduced the activity / population of urease producing soil microbes at this site.James & Wells ref: 323254PCT
[0090] Example 3: Effect of PBI and Pal2T5 on perennial ryegrass plant drought stress tolerance in the glasshouse
[0091] 3.1. Materials and Methods
[0092] To assess plant response to drought stress and the effect of Trichoderma strains on this process, plants were grown in 2.5L plastic pots. Soil from a field at Lincoln University was sieved and mixed with pumice (3:1 w / w) to fill the pots (2243 g / pot ). Trichoderma treatments were applied in a seed coating provided by Dr. Craig Bunt of Lincoln University. Perennial ryegrass seeds (Cv. Base AR1) were sown (20 seeds in each pot). Treatments were no Trichoderma (control), PBI and Pal2T5, with eight replicates of each treatment. Plants were drought stressed on four different occasions. The pots were initially watered several times during one day and left to reach soil field capacity before they were weighed. The plants in the moisture-stressed pots were then left until plant wilting was first observed and the pots were weighed again before adding water equivalent to 4% of the soil weight to each pot, at a level sufficient to keep plants alive (Umar et al., 2021). The stress was given for two weeks. Plant chlorophyll content was measured using a SPAD meter ((502 plus, Konica Minolta Inc., Japan). Leaves of plants were cut and fresh weight (FW) measured. They were then dried at 65°C for two days and the dry weight was recorded.
[0093] 3.2. Results -perennial ryegrass
[0094] After two weeks of drought stress both Pal2T5 and PBI treated perennial ryegrass plants had significantly greater shoot dry weight than the untreated control (Fig 3A). Plants treated with Pal2T5 are more erect (less wilted) than those of the control (Fig 3B). In drought stressed perennial ryegrass plants, those treated with Pal2T5 and PBI had significantly greater leaf chlorophyll content (measured as SPAD value) than control plants (Fig 3C).
[0095] Example 4: Glasshouse Clover experiment
[0096] 4.1 Materials and Methods
[0097] This experiment was conducted as described in 3.1 except ten white clover seeds (cv. Mainstay) were sown in each pot. Leaves of plants were cut and fresh weight (FW) measured. To measure turgidity, the leaves were suspended in deionized water and kept in a refrigerator overnight. The leaves were then taken out and the surface moisture removed using tissue paper before they were weighed for turgid weight (TW). Then the leaves were dried at 65°C for two days and the dry weight was recorded. The relative waterJames & Wells ref: 323254PCT
[0098] content (RWC) was calculated using the following formula (Gonzalez and Gonzalez-Vi lar, 2001).
[0099] RWC (%) = (FW-DW)Z(TW-DW) x 100
[0100] 4.2. Results - white clover
[0101] After two weeks of drought stress plants treated with Pal2T5 and PBI had significantly greater shoot dry weight than the control (Fig 4A). Drought stressed plants treated with Pal2T5 and PBI had significantly higher leaf chlorophyll content (measured as SPAD value) than control plants (Fig 4B). As can be seen in Fig 4C (left set of two pots) the Pal2T5 treated plants are more erect and less wilted than those of the control. The right set of two pots illustrates the greater witling severity in the control plants (right) than the Pal2T5 treated plants (left). In drought stressed white clover plants leaf relative water content is greater than the control for both Pal2T5 and PBI treatments (Fig 4D).
[0102] Example 5: Glasshouse Plantain experiment
[0103] 5.1. Materials and Methods
[0104] The treatments for the plantain experiment (10 seeds in each pot, cv. Tonic) were the same as for ryegrass and white clover. The plants were assessed for plant growth, chlorophyl content and leaf relative content after moisture stress as previously described.
[0105] 5.2. Results - plantain
[0106] Drought stressed plantain plants treated with Pal2T5 and PBI had significantly greater shoot dry weight than control plants (Fig 5A). Pal2T5 and PBI treated plants had a significantly greater leaf chlorophyll content (SPAD value) than the control (Fig 5B). After two weeks of drought stress plants treated with PBI (left) and Pal2T5 (right) were more erect (less wilting) than control plants (Fig 5C). Plantain leaf relative water content after two weeks of drought stress was significantly greater than the control for Pal2T5 and PBI treated plants (Fig 5D).
[0107] Example 6: Glasshouse Lucerne experiment
[0108] 6.1. Materials and Methods
[0109] Lucerne seeds (10 seeds in each pot, cv. Stamina GT5) were inoculated with Nodule N (New Edge Microbials Ltd, NSW, Australia) before sowing. The treatments were the same as forJames & Wells ref: 323254PCT
[0110] perennial ryegrass, white clover and plantain. Leaf relative water content, root nodule number, and plant dry matter were recorded.
[0111] 6.2. Results - Lucerne
[0112] After two weeks of drought stress lucerne shoot dry weight was significantly greater than the control for Pal2T5 and PBI (Fig 6A). Lucerne relative leaf water content was significantly greater for Pal2T5 and PBI than the control after two weeks of moisture stress (Fig 6B). After two weeks of drought lucerne plants treated with Pal2T5 had a greater root mass (Figure 6C, left) and more nodules / plant (26%) (Fig 6C, right) than the control.
[0113] Example 7:
[0114] Field Trial Kowhai Farm, Lincoln University.
[0115] 7.1 Materials and Methods
[0116] This trial was established at Kowhai Farm in a non-irrigated block to investigate the effects of a mixture of PBI and Pal2T5 (named Panch ) on perennial ryegrass drought tolerance. Perennial ryegrass (cv. LPH1702) seeds were sown into a prepared seed bed at 20kg / ha on 9 / 10 / 2023. The Panch was applied using two different methods: (i) Panch prills applied to the soil surface; and (ii) prills direct drilled into soil on 4 / 10 / 2024, one year after the plots were established. Treatments are referred to as: control, Panch surface application, Panch direct drilling. The plots were covered using rain-out shelters during January and February 2025 and dry matter cuts taken at the end of December 2023 (before), the end of January 2024 (middle) and early February (after) the drought period. Harvested plant material was dried in an oven at 65C for two days then weighed to obtain dry weight. Leaf chlorophyl content was measured at the middle of the drought stress using SPAD meter as previously described.
[0117] 7.2 Results:
[0118] Leaf chlorophyll content of direct drilled Panch was significantly greater than that of the control at the middle point of the drought (Figure 7B). A similar response was obtained from the cumulative (before+ middle+ after drought) dry matter produced over the two months (Figure 7A) with the direct drilled Panch producing 750 kg / ha more dry matter than the control.James & Wells ref: 323254PCT
[0119] The invention detailed herein may provide one or more advantages over the prior art Trichoderma strains and / or combination of such strains or at least offer the public a useful choice.
[0120] The invention as detailed above may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.
[0121] 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.James & Wells ref: 323254PCT
[0122] REFERENCES
[0123] Blakemore, L.C., Searle, P.L., Daly, B.K. 1987. Methods for chemical analysis of soils. NZ Soil Bureau Scientific Report, 80: 27-29.
[0124] Gianfreda, L, Sannini, F., Ortega, N. Nannipieri, P. 1994. Activity of free and immobilized urease in soil: effects of pesticides. Soil Biology and Biochemistry, 26: 777-784.
[0125] Gonzalez L., Gonzalez-Vilar, M. 2001. Determination of Relative Water Content. In: Reigosa Roger M.J. (eds) Handbook of Plant Ecophysiology Techniques. Springer, Dordrecht.
[0126]
[0127] Han, K., Yang, Y., Zhou, C., Shangguan, Y., Zhang, L, Li, N., Wang, L. 2014. Management of furrow irrigation and nitrogen application on summer maize. Soil Fertility & Crop Nutrition, 10: 1402-1410.
[0128] Pan, S.Y., He, K.H., Lin, K.T., Fan, C., Chang, C.T. 2022. Addressing nitrogenous gases from croplands toward low-emission agriculture. Climate and Atmospheric Science, 5:43.
[0129] Stewart et al. "How Much of Biocontrol is Enough"; In: Biological Control: a Global Perspective; eds C. Vincent, M.S. Goettel and G. Lazarovits. 2007, pages 185-196.
[0130] Umar, A., Hampton, J., Kandula, D., Rolston, M., & Chng, S. (2021). The impacts of take-all, drought and their interaction on Bromus wildenowii seed yield and the alleviation of these stresses by Trichoderma atroviride. Biocontrol Science and Technology, 31 (9), 976-989. https;.tfdoi.Qfg / lQ;.1080^)9583
[0131]
Claims
James & Wells ref: 323254PCTWHAT WE CLAIM IS:
1. A Trichoderma hamatum strain deposited at the National Measurement Institute, Australia on 31stJanuary 2025 and accorded accession number NMI No. V25 / 001998.
2. Use of the Trichoderma hamatum strain of claim 1 to increase nitrogen use efficiency and / or to reduce nitrogen wastage of a cropped soil.
3. Use of the Trichoderma hamatum strain according to claim 2, wherein the use follows urea application and / or urine deposition.
4. Use of the Trichoderma hamatum strain according to claim 2 or 3, wherein the nitrogen wastage includes ammonia volatilisation, nitrous oxide emissions, and / or nitrate leaching.
5. A composition comprising in a reproductively viable form and amount: Trichoderma hamatum deposited at the National Measurement Institute and accorded accession number NMI No. V25 / 001998 and an agriculturally acceptable carrier.
6. A composition according to claim 5 which further comprises an equal mixture of Trichoderma atroviride NMI No. V08 / 002387, Trichoderma atroviride NMI No. V08 / 002388, Trichoderma atroviride NMI No. V08 / 002389, and Trichoderma atroviride NMI No.V08 / 002390 in a reproductively viable form and amount.
7. A method for increasing nitrogen use and / or reducing nitrogen wastage of the method comprising directly or indirectly applying to said area of land a composition according to claim 5 or 6.
8. A method of increasing nitrogen use and / or reducing nitrogen wastage of an area of land, the method comprising delivering to said area of land a Trichoderma hamatum strain according to claim 1 in a reproductively viable form and amount.
9. A method of increasing plant tolerance of drought stress on an area of land, the method comprising delivery to said area of land Trichoderma hamatum NMI No. V25 / 001 in a reproductively viable form and amount.
10. Use of Trichoderma hamatum strain of claim 1 to increase drought tolerance of a plant compared to a plant untreated with said Trichoderma hamatum.James & Wells ref: 323254PCT11. The use of claim 10 wherein the plant is selected from the group consisting of: perennialryegrass; white clover; plantain; and lucerne.