AU734994B2 - Pest resistance enhancement method - Google Patents

Description

WO 98/37763 PCT/AU98/00123 1 PEST RESISTANCE ENHANCEMENT METHOD The present invention relates generally to agriculture and in particular to methods of improving or controlling the growth of plants. More particularly, the present invention relates to methods of controlling pests in plants during the growth of the plants, particularly in the early stages of growth. Even more particularly, the present invention relates to methods of enhancing the pest resistance of plants, particularly cotton plants. Even more particularly, the present invention relates to the use of certain selected chemical compounds, such as for example glycine betaine, for enhancing or improving pest resistance already present in the plants, so as to increase the yield and quality of cotton. The present invention finds particular application in methods of administering glycine betaine and other related chemical compounds to transgenic cotton varieties having the Bt gene introduced into the plant.

Although the present invention will be described with particular reference to the use of glycine betaine as one example of the chemical compounds promoting enhanced pest resistance when administered to the growing cotton plants to further inhibit or prevent attack from pests, it is to be noted that the scope of the present invention is not restricted to the described embodiment, but rather, the scope of the present invention may be more extensive by including the use of other similar chemical compounds, other ways of administering the chemical compounds, and the use of the chemical compounds on plants other than cotton or on varieties of cotton other than those regarded as Bt varieties.

n- WO 98/37763 PCT/AU98/00123 2 Cotton is a crop having considerable economic value.

However, cotton is susceptible to attack from a variety of pests. As the cotton plants grow and develop, they are often subjected to attack from a variety of pests which reduces the economic value of the cotton plants. When cotton plants are under attack from pests, the plants shed flower buds (known as "squares"), flowers and bolls.

Therefore, it is possible to monitor the severity and type of pest attack on the cotton plants by observing what happens to the squares during the growth and development of the cotton plants, particularly the number of squares which are shed at certain times during the growth of the plants.

By ascertaining the number of damaged squares and bolls of individual plants subjected to a given treatment, it is possible to provide an indication of the effectiveness of that treatment.

Examples of pests which can attack cotton plants at various stages during the life of the plants are the lepidopteran insect pests, such as tobacco budworms, bollworms, cotton bollworms, pink bollworms, podworms, earworms, fruitworms and the like.

If the maturing cotton plants are additionally subjected to stress at the time of suffering pest attack, such as stress induced by lack of water, excess water, excessively cold conditions, or other environmental conditions or the like, the deterioration of the plants is increased, thereby further reducing the economic value of such plants. This is manifest by the shedding of even more squares.

In the past, various attempts have been made at combating pest attack, such as by using insecticides or other chemical spraying means. However, such methods have not been entirely satisfactory for one reason or another and increasing concern has been expressed over the overzealous and indiscriminate use of pesticides and insecticides to WO 98/37763 PCT/AU98/00123 3 reduce the incidence of and amount of damage caused by pest attack in plants, based primarily on health and environmental considerations. Additionally, over time certain pests have acquired immunity to many of the popular insecticides and pesticides that have been widely used.

Another way of combating pest attack has been to genetically alter the plant so that it has enhanced resistance to pest attack. Examples of plants that have increased pest resistance are the transgenic cotton varieties, particularly the various transgenic cotton varieties produced by the Monsanto Company of the USA, such as the so-called "Bt" transgenic cotton varieties.

The Bt transgenic cotton varieties have been altered via genetic engineering techniques and contain a synthetic gene derived from Bacillus thuringiensis a common bacterium that naturally produces a protein toxic to a narrow range of insects. The genes inserted into the cotton plants encode proteins that are specific to lepidopteran (caterpillar) larvae, including the two Helicoverpa species which are a major pest in the production of cotton using the traditional non-modified cotton varieties. The transgenic plants produce the proteins, which are toxic to the Helicoverpa species so that insect feeding on the plants is inhibited which subsequently leads to the death of the insect larvae eating the plant. This pest resistance is an inherent property of such cotton plants which have been modified. The Bt toxin is non-toxic to humans and animals and is also readily biodegradable. The target specificity of the Bt protein and its location within the tissues of the plant ensure that the protein is active only against the attacking insect pest species. Incorporation of the Bt gene directly into the cotton plant greatly enhances the efficacy of the Bt protein in insect control.

WO 98/37763 PCT/AU98/00123 -4- However, whilst the incorporation of the Bt gene into the transgenic cotton varieties has met with some success, it has been discovered that under some circumstances, including some environmental conditions, such as for example when the cotton plant having the Bt gene is subjected to one form or another of stress, the pest resistance provided by the modified form of the cotton is reduced to a level which is below that which is desirable or effective, i.e. the pest resistance of the Bt cotton varieties does not meet its expected performance in times of stress occurring in the plants. Therefore, whilst the Bt cotton varieties do exhibit increased pest resistance over non-modified varieties, the extent or degree of this pest resistance is not satisfactory in all circumstances and varies widely in different conditions. Therefore, there is a need to improve or enhance the pest resistance of the Bt modified cotton plants, particularly in circumstances where the plant is subjected to stress or stress-inducing conditions.

Surprisingly, it has been discovered that applying certain chemical compounds to the transgenic cotton varieties has resulted in increased pest resistance and accordingly greater yields of cotton. It has been found that the application of the chemical compounds to the plants not only works in normal conditions but also works, perhaps even more effectively, when the plants are stressed or subjected to stress-inducing conditions.

Therefore, it is an aim of the present invention to provide a method of administering selected chemical compounds, called pest resistance enhancers, such as for example glycine betaine, to a growing plant, such as for example a cotton plant, and more typically a Bt modified cotton plant, to enhance or improve the pest resistance performance of the plant in certain circumstances so as to reduce or at least alleviate one or more of the drawbacks i~-T c WO 98/37763 PCT/AU98/00123 5 of growing cotton, and in particular to increase the yield of cotton plants so treated.

It is to be noted that the use of such terms as "selected chemical compounds", "pest resistance enhancers" and the like is meant to refer to those chemical compounds which generally are effective in providing at least some small degree of synergy between the inherent pest resistance already possessed by the plants and the chemical compound to produce greater pest resistance in the plants.

Additionally, such terms are used interchangeably throughout the specification and may be simply abbreviated to the term "enhancers". By this it is understood that the term "enhancer" refers to chemical compounds that provide some increase in pest resistance over that which is already possessed by the plant.

According to one aspect of the present invention, there is provided a method of treating a plant to improve or enhance the pest resistance of the plant comprising administering an effective amount of a selected pest resistance enhancing chemical compound to the plant such that the pest resistance of the plant is improved to a level above that which is already possessed by the plant when not so treated, in order to enhance the growth of the plant to provide a greater yield thereby increasing the economic value of the plant.

Typically, the plant to which the chemical compound promoting enhanced pest resistance (the enhancer) of the present invention is administered is cotton. More typically, the cotton plants are transgenic cotton varieties, typically Bt modified cotton varieties developed by Monsanto Company of the USA. More typically, the cotton varieties are marketed under the registered trade mark INGARDTM or BOLLGARDTM, which varieties were developed to deliver a natural insecticidal protein isolated from WO 98/37763 PCT/AU98/00123 6- Bacillus thuringiensis or Bt for the control of tobacco budworms, cotton bollworms and pink bollworms and the like.

However, it is to be noted that the present invention can be used on any plants having inherent pest resistance whether occurring naturally or as a result of genetic engineering or the like.

Typically, the selected chemical compound promoting the enhanced pest resistance is an organic solute, an amino acid, a betaine, a sugar, a polyol, or related compounds to the foregoing, and the like. More typically, the pest resistance enhancing chemical compound is an ammonio compound, such as a N-methyl substituted amino acid, proline, choline or a betaine, such as glycine betaine (oxyneurine) and other betaine member compounds including the sulphonio analogues of the betaines. Other betaines include proline betaine, P-alanine betaine, tryptophan betaine, histidine betaine, 2-mercaptohistidine betaine, and the like. Even more typically, the pest resistance enhancer is a nitrogenous compatible solute, such as stachydrine, trigonelline, homostachydrine (pipecolate betaine).

It is to be noted that particularly preferred pest resistance enhancers are the betaines, particularly the glycine derivative. Betaine refers to fully N-methylated amino acids. Glycine betaine has three methyl groups attached to the nitrogen atom of the glycine molecule and is usually called betaine, glycino-betaine, trimethyl glycine or 1-carboxy-N,N,N-trimethylmethanaminium hydroxide, and has the following structural formula:

CH

3 N (CH 3

)(CH

3

)CH

2

COO-

Other pest resistance enhancers include glycine, methylene glycine, dimethyl glycine, glutamic acid, y-aminobutyric acid, trimethylamine y-butyric acid, or the like.

:r I WO 98/37763 PCT/AU98/00123 7 Typically, the enhancer is administered alone or in combination with one or more other materials. Typically, the other materials include additives such as wetting agents, other adjuvants, defoliants, growth regulators, pesticides, nutrients and the like. The other material can be added separately or in combination with the enhancer.

Typically, the pest resistance enhancer is preferably applied with a wetting agent, such as for example MONSOONTM (obtainable from Cyanamid), or is applied in combination with a wetting agent, mineral oil and/or vegetable oil.

Although many types of mineral oil or vegetable oils may be used, specific examples of the mineral oil include DC- TRONTM (a registered trade mark of AMPOL Australia Ltd) which is a narrow range boiling point mineral oil, whereas specific examples of the vegetable oil include rapeseed based vegetable oils such as SYNETROLTM.

Typically, the amount of pest resistance enhancer, typically glycine betaine, administered to the growing cotton plant is such so as to increase the retention of squares, fruiting forms or bolls in cotton thereby increasing the yield of the cotton plants.

Typically, the plant has increased resistance to attack from members of the two Helicoverpa species or lepidopteran species, such as for example tobacco budworm, bollworm, cotton bollworm, pink bollworm, podworm, earworm, fruitworm and the like.

Typically, the glycine betaine is administered to the cotton plants in a dose of from about 0.1 to 30.0 kg/ha, typically from.0.1 to 10.0 or 20.0 kg/ha, preferably from to 7.0 kg/ha with typical dosages being about 0.5, 2.5, 3.5 kg/ha and preferable dosages being between about to 4.0 kg/ha, more preferably between about 2.5 to kg/ha, and most preferably about 3.0 kg/ha. More :I i r WO 98/37763 PCT/AU98/00123 8 typically, the enhancer is applied externally or exogenously to the plants, such as for example to the leaves of the plant.

Typically, the enhancer is applied by any suitable means such as spraying, including ground spraying, aerial spraying or the like.

Typically, the stress-inducing conditions or stress is attributable to environmental stress, such as for example relating to temperature, water, salinity, light, nutritional stress, pest attack, and the like, including too little or too much water, too low or too high temperatures, too high salt concentration, and the like.

Typically, the enhancer with or without additives is applied in one, two, three, four, five, six or more sprayings either on the one day or on separate days, either regularly or irregularly spaced apart from each other.

Typically, the enhancer is applied to the plants at any time from planting to harvesting, more typically from about days before first flowering to about 30 days after FF, even more typically from about slightly before FF, say a few days, to slightly after FF, say about a few days. Most typically, the plants are sprayed at FF.

Typically, the improvement (reduction) in the amount of damaged squares of the cotton plants is from about 1% or 2% to about 200%, more typically up to about 150%, preferably 100% or the like.

Typically, the greater the amount of stress suffered by the plant at the time of administering the enhancer, the greater the improvement and the more the plant responds to the treatment.

I WO 98/37763 PCT/AU98/00123 9 The present invention will now be described by way of example with reference to the following examples which are meant to be illustrative of the present invention only and not limiting to its scope in any way.

Example 1 The object of this example is to determine the effect of foliar application of glycine betaine on square and boll retention on cotton which has been treated with a pest resistance enhancer in both normal irrigation regimes and water-reduced irrigation regimes.

The following trial was conducted with all small plot trials being applied at a concentration of 25 L/ha plus MONSOON wetting agent at a concentration of 3 ml/10 L.

Various dosages of glycine betaine were prepared and applied to growing cotton plants in differing combinations and at differing times. The preparations applied to the plants are described with reference to Table 1 as follows: UT W no glycine betaine applied to the plants, and the plants subjected to a first irrigation. The conditions existing at the time of this treatment were in effect stress-inducing since shortly after irrigation there was a cold snap which resulted in the cotton plants suffering the stress of low temperature. This is an example of normal environmental factors combining to produce stress-inducing conditions on the cotton plants.

UT W no glycine betaine applied to the plants, and no first irrigation. The conditions existing at the time of this treatment produced some stress due to the small amount of water applied to the cotton plants but far less stress than produced by the watering in the treatment above because of WO 98/37763 PCT/AU98/00123 10 the cold snap, so that accordingly this treatment related to less stressful conditions than the UT W treatment.

3 W 3 kg of glycine betaine were applied per hectare to the plants together with a first irrigation.

As mentioned above, the results of this treatment were illustrative of applying the enhancer in stress-inducing conditions, in this case due to low temperatures.

3 W 3 kg of glycine betaine were applied per hectare to the plants without a first irrigation. The results of this treatment were illustrative of applying the enhancer in less stressful conditions even though one irrigation was removed which produced a small amount of stress due to the slight lack of water.

The variety of cotton used in this example was provided by the Monsanto Company under the name INGARDTM which is a cotton variety having the Bt gene.

In Table 1 is shown the results of the trial including the column headed Damaged Squares" which is the amount of damaged squares resulting for the various treatments shown in the column entitled "Treatment". This value is indicative of the success or otherwise of the various treatments since the value of the percentage of damaged squares shows the proportion of fruiting forms which will not contribute to the yield of cotton. Clearly, the higher the value of the damaged squares of cotton, the less effective the corresponding treatment.

In treatment UT W under stress-inducing conditions there was 31.6% damaged squares when the cotton plants were not treated with the enhancer, whereas in treatment 3 W there iC 1 i I WO 98/37763 PCT/AU98/00123 11 was only 21.4% damage when the cotton plants were treated with a dosage of 3.0 kg/ha of glycine betaine. Clearly, there is a marked improvement in the number of damaged squares of the cotton plants when glycine betaine is applied in stress-inducing conditions since the number of damaged squares was reduced.

In the relatively stress-free conditions, the amount of damaged squares in treatment UT W was about 18.1% when not treated with an enhancer, whereas in treatment 3 W when treated with an enhancer there was only 15.6% damage.

Again, there is a reduction in the amount of damaged squares when the cotton plants are relatively stress-free.

In both the stress-inducing conditions, i.e. the treatments involving irrigation and the cold snap, and the less stressful conditions, i.e. without one irrigation, there was a marked reduction in the amount of damaged squares when the plants were treated with 3.0 kg/ha of glycine betaine in accordance with the present invention.

The results provided in Table 1 indicate the following: (Note: Sample 40 (4 x 10) terminals/treatment) 1 way anova/plant Total square count is quite high; normal range would be 7-8 No early season stress due to ample natural rain; water-reduced cotton, i.e. less stressed cotton, had irrigation removed at first flower The economic damage level for larvae number in cotton is usually 0.15 After irrigation there was a cold snap which resulted in stress-inducing conditions which may have affected soil/root temperature which could have a flow-on effect to the performance of the Bt gene.

WO 98/37763 PCT/AU9800123 12 Glycine betaine significantly (P 0.001) increased total square retention on both the fully irrigated, cold stressed and reduced irrigated, less stressed cotton plants. There is significantly less square damage in the reduced irrigated, less stressed cotton plants than in the fully irrigated, cold stressed cotton plants. Without being bound by this explanation, it is thought that this may be due to lower soil temperature following irrigation, combined with two days of cloud (six and seven days after application). The larval numbers in the cotton plants suggest this. Glycine betaine significantly reduced the square damage irrespective of water status.

Example 2 This trial was to determine the effect of dosages of kg/ha of glycine betaine enhancer upon fruit retention in irrigated cotton when water stress occurs at different times within the boll formation period in transgenic cotton varieties. The water stress was introduced by withholding the first, second and third irrigations. The stress levels were quantified by periodic measurement of volumetric soil water using a neutron probe.

The results obtained from a further trial are shown in Table 2 which results were determined on and the assessment made 46 days after first flower Treatment 1 corresponds to a single administration of glycine betaine enhancer applied at a dosage rate of kg/ha at FF with the first irrigation omitted, which resulted in 29% damaged squares.

Treatment 2 corresponds to Treatment 1 (above) except that no glycine betaine enhancer was administered which resulted in 47% damaged squares.

Treatment 3 corresponds to a single administration of rpl I WO 98/37763 PCT/AU98/00123 13 glycine betaine enhancer applied at a dosage rate of kg/ha at FF with the second and third irrigations omitted, which resulted in 27% damaged squares.

Treatment 4 corresponds to Treatment 3 (above) except that no glycine betaine enhancer was administered which resulted in 43% damaged squares.

Treatment 5 corresponds to a single administration of glycine betaine enhancer at a dosage rate of 3.0 kg/ha at FF with all three irrigations carried out normal irrigation) which resulted in 19% damaged squares.

Treatment 6 corresponds to Treatment 5 (above) except that no glycine betaine was applied which resulted in 38% damaged squares.

Treatment 7 corresponds to two separate administrations of glycine betaine each at a dosage rate of 1.5 kg/ha of glycine betaine, the first being applied at FF and the second at 14 days after FF with normal irrigation being carried out, which resulted in 41% damaged squares.

Treatment 8 corresponds to a single administration of glycine betaine at a dosage rate of 3.0 kg/ha at 14 days after FF with normal irrigation being carried out, which resulted in 39% damaged squares.

On comparing the results obtained in Treatment 5 which is the treatment of the present invention involving the administration of glycine betaine with the normal irrigation regime leading to unstressed conditions, there was 19% damaged squares as compared to the results of Treatment 6 involving no treatment, resulting in 38% damaged squares. Hence, there is a clear reduction in the number of damaged squares of some 50%. Thus, in relatively unstressed conditions there is a clear improvement when the WO 98/37763 WO 9837763PCT/AU98/OOI 23 14 cotton plants are sprayed with glycine betaine as compared to not being sprayed with glycine betaine since the number of damaged squares is considerably reduced.

Comparing the results obtained in Treatment 2 with Treatment 1 which relates to a more stress-inducing environment due to the lack of the first irrigation, there is a reduction from 47% damaged squares for the untreated cotton as compared to 29% damaged squares for the treated cotton.

Similarly, comparing the results of Treatment 4 with Treatment 3 which relates to a more stress-inducing condition than experienced for Treatments 1 and 2 due to reduced watering because of the omission of both of the second and third irrigations, the amount of damaged squares changed from 43% for the untreated plants to 27% for the treated plants.

In all of the above situations from the most stressed to the less stressed conditions, there was a marked improvement when spraying the cotton plants with glycine betaine.

The comparison of Treatment 7 with Treatment 5 suggests that splitting the administration of the enhancer into two separate applications is probably not as effective as a single treatment of the same amount of enhancer since the split administration results in 41% damaged squares as compared to 19% for the single treatment.

Similarly, a comparison of Treatment 8 with Treatment shows that the late administration of the enhancer at 14 days after FF, resulting in 39% damaged squares, does not produce the same improvement as does a single administration in the same conditions at FF which results in 19% damaged squares.

WO 98/37763 PCT/AU98/00123 15 Irrespective of the irrigation regime, all applications of kg/ha at FF significantly increased the number of undamaged squares and the hence potential yield of the cotton plants so treated (Table These applications also significantly increased the height of the plants as shown by the results in the column entitled "Mean Crop Height The delayed application (14 days after FF) had no significant effect on the number of undamaged squares or the height of the crop (Table While splitting the application had no significant effect on height it significantly reduced the number of undamaged squares.

Based on undamaged squares, the response to glycine betaine appeared to increase with the level of water stress, i.e.

the order of increasing response (relative to the control) was Treatment 5, Treatment 1 and Treatment 3 (Table 3).

Indeed, based on total squares, this trend was significant.

There is no doubt that increasing the supply of water and glycine betaine applied at 3.0 kg/ha significantly increased the potential yield of cotton (Table 4).

The attractiveness of cotton to Helicoverpa spp moths increases with the number of squares it is carrying. As a result, in the cotton not treated by glycine betaine the number of Helicoverpa spp larvae increased significantly as the number of squares increased. Hence to determine the effect of glycine betaine on Bt toxin production as measured by the control of Helicoverpa spp, it was necessary to account for the increased numbers of squares on glycine betaine treated cotton.

The relationship between the number of squares and larvae on untreated cotton (Figure 1) was used to calculate the WO 98/37763 PCT/AU98/00123 16 number of larvae in glycine betaine treated cotton expected on the basis of the number of squares it was carrying. In cotton treated at either FF or 14 days later with glycine betaine at 3.0 kg/ha the actual number of larvae was significantly less than the expected number (Table However, in the split application the difference was not statistically significant.

The relationship between the number of squares and larvae on cotton treated at first flower is also plotted in Figure 1. The slopes of the two lines are significantly different, p<0.02.

Thus, there is evidence that glycine betaine increases the effectiveness of Bt cotton against Helicoverpa spp.

However, without being bound by the following, presumably it does this by reducing the effect of stress on protein and hence Bt toxin production.

Example 3 The objective of this example was to determine the effect of glycine betaine treatment on subsequent infestations of Helicoverpa spp. The cultivars used in the trial of this example were: Siokra L-22 (Okra leaf ex CSD), a conventional cotton species; and Two transgenic (Bt) varieties, Siokra L-23i (Okra leaf ex CSD) and NuCotton 37 (broad leaf ex DeltaPine).

The trial of these three cotton cultivars was laid out as a split block experiment with respect to 3 irrigation regimes. Each regime was applied to a randomised complete block experiment comprising 3 cultivars of cotton, with one plot of each in each of 4 (sub)blocks. The plot size was metres of bed (two rows of plants) and one half of each WO 98/37763 PCT/AU98/00123 17 plot was treated with glycine betaine.

There were 6 treatments: 2 levels of glycine betaine application, being 0 and 3 kg/ha, by 3 cultivars of cotton NuCotton 37, Siokra L-22 and Siokra L-23i.

A compressed gas-operated sprayer fitted with a flat boom carrying 3 nozzles at 30 cm spacings, designed to spray a single row, with the central nozzle above the row, was used to spray the cotton plants of this trial with the glycine betaine. The nozzles were about 25 cm above the top of the crop and were aimed vertically down. Glycine betaine at kg/ha plus Agral 600 surfactant at 25 ml/100L was sprayed onto the cotton plants being treated. All plants and the plants with flowers in the central 25 m of each row of each plot were counted. Crop height was measured at each of 5 pre-determined locations in each row of each plot, viz. 6, 10, 14, 18 and 22 m from the tail ditch end in the eastern row and 8, 12, 16, 20 and 24 m from the tail ditch end in the western row. The plants tipped by mirids in 25 consecutive plants in about the middle of each row of each plot were counted.

The crop was inspected on several occasions for Helicoverpa infestation, such as at 72, 82 and 98 days after application of the glycine betaine to the various cotton plants, respectively. Boll assessment occurred at 106 to 110 days after application of the glycine betaine.

The results of the trial are provided in Table 6 which are the results of the assessment of damage to the bolls as a result of Helicoverpa infestation damage. The proportion of bolls damaged by Helicoverpa spp was significantly r- WO 98/37763 PCT/AU98/00123 18 greater in Siokra L-22 which is a conventional cotton species than in either of the transgenic cultivars, NuCotton 37 and Siokra L-23i. Also, the proportion of bolls damaged was significantly lower in transgenic cotton treated with glycine betaine than in untreated transgenic cotton. However, there was no indication of a similar response in Siokra L-22 (Table Thus, there is strong evidence that treatment with glycine betaine increased the effectiveness of transgenic cotton against Helicoverpa spp.

Further examination of the data showed that the proportion of damaged bolls in each treated cultivar and in untreated Siokra L-22 and Siokra L-23i, was independent of the total number of bolls. However, in untreated NuCotton 37 the two variables were significantly positively correlated. It followed that the difference between the effectiveness of treated and untreated NuCotton 37 against Helicoverpa spp may have been due to differences between the number of bolls, rather than glycine betaine enhancing the insecticidal effectiveness of NuCotton 37. This possibility was tested by comparing the expected proportion of damaged bolls in untreated NuCotton 37, on the basis of boll numbers, with the observed proportion. The relationship between the proportion of damaged bolls,

P

and boll numbers, BN, in untreated NuCotton 37 used to calculate the expected proportion of damaged bolls

P)

was: P 1.877 0.0302N, Rsqr 0.349.

slope significant at p 0.043.

The difference between the mean expected proportion of damaged bolls, 12.4%, and the observed proportion, 9.8%, was significant at p 0.009 (paired t test, n 12).

Thus, most of the difference between the proportion of damaged bolls on treated and untreated NuCotton 37 was due WO 98/37763 PCT/AU98/00123 19 to glycine betaine increasing the effectiveness of NuCotton 37 against Helicoverpa spp.

As can be readily seen from the results of Table 6 when NuCotton 37 was treated with glycine betaine the reduction was a 51% reduction in the amount of damaged bolls caused by Helicoverpa spp as compared to only 26% reduction when glycine betaine was not used. Similarly, with the Siokra L-23i species of cotton, when the cotton plants were treated with glycine betaine there was a 52% reduction in damage to the bolls from Helicoverpa spp infestation as compared to only 26% reduction when the plants were not treated with the glycine betaine.

From the results of this trial as provided in Tables 6 and 7, it is clearly demonstrated that the application of glycine betaine significantly increased the effectiveness of transgenic (Bt) cotton, NuCotton 37 and Siokra L-23i, against Helicoverpa spp. The glycine betaine had no effect on conventional cotton, Siokra L-22, in this respect.

Untreated and treated transgenic cotton reduced the proportion of damaged bolls at harvest by about 25% and respectively, compared with conventional cotton.

The foregoing results demonstrate that in all conditions from relatively unstressed to stress-inducing, the administration of an enhancer such as glycine betaine to cotton plants increases the pest resistance of the Bt transgenic cotton varieties with the greater improvement being demonstrated when the cotton is exposed to more stress-inducing conditions, so that when the Bt transgenic cotton varieties are placed under more stress and would be expected to perform unfavourably, the administration of doses of glycine betaine produces a marked improvement in the pest resistance of this variety of plant, and thereby its performance to provide a greater yield of cotton.

WO 98/37763 PCT/AU98/00123 20 The described arrangement has been advanced by explanation and many modifications may be made without departing from the spirit and scope of the invention which includes every novel feature and novel combination of features hereindisclosed.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope.

i-i Table 1 Treatment Damaged Average Total Average Damaged Total Larvae Squares Squares Squares UT W 1.675 b 10.77 a 31.6 b 0.35 b UT W 0.85 a 1.26 11.15 ab 10.96 a 18.1 a 0.15 ab 3 W 1.2 ab 11.75 bc 21.4 ab 0.225 ab 3 W 0.75 a 0.97 12.1 c 11.92 b 15.6 a 0.075 a LSD 0.68 0.69 0.489 10.81 0.206 P 0.035 0.25 0.001 0.000 0.023 0.059 ij Table 2 TREATMENT Mean number per 10 terminals *1 Mean Mean Code Dose (kg/ha) Irrigation total damaged undgd total damaged crop of Betaine Regime squares squares squares larvae squares height Glycine I (cm) FF FF+14 ist 2nd 3rd 1 3 0 0 1 1 48b 13a 31 bc 1.0 ab 29 a 92 b 2 0 0 0 1 1 33 a 15 ab 17a 1.4 ab 47 a 75 a 3 3 0 1 0 0 91 de 24 bcd 67 ef 0.8 a 27 a 107 d 4 0 0 1 0 0 45 b 19 abc 26 ab 2.1 a-d 43 a 94 b 3 0 1 1 1 109 e 21 a-d 87f 4.2 cd 19a 113 e 6 0 0 1 1 1 86d 32d 52 de 5.1 d 38a 106cd 7 1.5 1.5 1 1 1 66 c 27 cd 39 bcd 1.7 abc 41 a 103 c 8 0 3 1 1 1 76 cd 29 cd 46 cde 2.9 bcd 39 a 104 cd 1-way ANOVA, F 25.57 3.226 11.91 3.046 2.203 85.35 F prob. <0.001 0.015 <0.001 0.019 0.071 <0.001 1 Numbers of squares, proportions and heights are transformed from square roots, larvae from logs. Letters indicate statistical separation P 0.05 Fisher's protected LSD test.

Table 3 TREATMENT Mean increase relative to the nearest relevant control *1 Code Dose (kg/ha) Irrigation Total Damaged Undamaged Crop Height of Betaine Regime Squares Squares Squares (cm Glycine FFJFF+14 Ist 2nd 3 r d No.1I No.jI No.I No. 1 3 0 0 1 1 14 d 48 c -1la 13a 15 bc 95b 16 d 22 d 3 3 0 1 0 0 45 f 108 d 5 a 34 a 41d 167 b 13 cd 14 cd 3 0 1 1 1 23 e 28 bc -11a -28 a 34cd 84 b 7 bc 6 bc 7 1.5 1.5 1 1 1 -20 a -23 a -6 a -10 a -14 a -20a -3 a -3 a 8 0 3 1 1 1 -10 b -11a -3 a -2 a -7 a -7 a -3 a -3 a No Betaine Glycine Control Oc 0Qab Oa Oa 0Qab Oa 0Oab 0Oab 1 -way ANOVA, F 8.1 160 .49 0.609 11.22 6.908 13.81 14.51 F prob. <0.001 <0.001 0.474 0.694 <0.00 1 0.001 1<0.00 1 I<0.001 *1 Letters indicate statistical separation p =0.05 Fisher's protected LSD test.

Table 4 TREATMENT Mean number per 10 terminals *.1 Code Dose of Irrigation Mean Mean Crop Betaine Regime Itotal damaged undmgd total Damaged Height Glycine sqaesquares squares larvae Squares (cm (kg/ha) .1 1st 2nd 1,2 3,0 0o 1 1 40 a 14 a 24 a 1.2 a 37 a 83.3 a 3,4 3,0 1 0 0 66 b 22 ab 44 b 1.3 a 34 a 100.4 b 5,61 3,0 j1 1 1 97 c 26 b 68 c 4.6 b 2810.c 2-way ANOVA F prob. <0.001 0.019 <0.00 1 0.001 0.400 <0.001 1,3,5 3 Al 8b 19a 59b 1.6 a 25 a 103.7 b 2,4,6 0Al 53a 22 a 30a 2.6 a 43 b 91.5 a 2-way ANOVA F prob. <0.001. 0.441 <0.001 0.100 0.006 <0.001 *1 Numbers of squares, proportions and heights detransformed from square roots, larvae from logs. Letters indicate statistical separation p 0.05 Fisher's protected LSD test.

i-( Table TREATMENT Mean Number of Significance Larvae per 10 terminals level of the Code Dose (kg/ha) Irrigation difference of Betaine Regime Actual Expected (paired Glycine *1 ttest) FF FF+14 1st 2nd 3rd 1 3 0 0 1 1 1.0 2.6 0.0071 3 3 0 1 0 0 1.0 6.2 0.0064 3 0 1 1 1 4.3 7.6 0.0040 7 1.5 1.5 1 1 1 2.5 4.1 0.2368 8 0 3 1 1 1 3.0 5.0 0.0336 *1 According to the relationship between the numbers of squares and larvae in untreated cotton.

WO 98/37763 PCT/AU98/00123 26 Table 6 The effect of glycine betaine, on the damage caused by Helicoverpa spp in conventional and transgenic (Bt) cotton TREATMENT Mean number of Mean Mean Cultivar Dose damaged boils damaged boils reduction of per 10 plants *2 of glycine damaged betaine

I

(kg/ha) jjboils NuCotton37 0 64. 3bc 64 .3 b 14. 8b 14.8 b 26 NuCotton37 3 35.0Oa 35.0Oa 9. 8a 9. 8a 51 *1 Siokra 0 7 0. 7c 20 .4 c L-22 63 .9 b 19.9 C 0 Siokra 3 57.1lb 19.4 c L-22 Siokra 0 54.5 b 54. 5b 14.7 b 14.7 b 26 L-23i Siokra 3 28.2 a 28 .2 a 9 .5 a 9.5 a 52 L-23i [ANOVA F prob. <0.001 <0.001 <0.001 <0.001 n/a *1 Letters indicate statistical separation (p=0.05 Fisher's protected LSD test.

*2 Within 2 weeks of harvest.

S~ WO 98/37763 WO 9837763PCT/AU98/00123 -27 Table 7- The relationship between the total number of boils and the proportion of boils damaged by Helicoverpa spp Cultivar Glycine Correlation Significance (kg/ha) Co-efficient~ Level nIf 12 NuCotton 37 0 0.5908 0.043 NuCotton 37 3 0.3303 0.294 Siokra L-22' 0 -0.0915 0.777 Siokra L-22 3 0.4026 0.194 Siokra L-23i 0 0.4368 0.156 Siokra L-23i 3 0.0820 0.800 *1 Linear: number of boils vs damaged boils.

Claims (18)

1. A method of treating a cotton plant to improve or enhance the pest resistance of the cotton plant, including applying an amount of a selected pest resistance enhancing chemical compound being a betaine to the cotton plant to enhance the pest resistance of the cotton plant, wherein the amount of chemical compound applied is such that the pest resistance of the cotton plant is improved to a level greater than that possessed by the cotton plant when not so treated, so that the growth of the cotton plant is improved to provide greater yield, thereby increasing the economic value of the cotton plant. 15 2. A method according to claim 1, in which the cotton plant is a transgenic variety of cotton such as a Bt modified cotton.

3. A method according to any one of the preceding 20 claims, in which the betaine is glycine betaine (oxyneurine) or other betaine member compound such as following structural formula: CH 3 -N (CHA)(CH 3 )CH 2 COO-

5. A method according to any one of the preceding claims, in which the chemical compound acting as the pest resistance enhancer is administered alone or in combination with one or more additives. aa combination with one or more additives. H:\PClarke\Keep\Retypes\60815-98 AB TALL claims.doc 27/04/01 71, 29

6. A method according to claim 5 in which the additive includes wetting agents, adjuvants, defoliants, growth regulators, pesticides or nutrients.

7. A method according to claim 6, in which the additive is a wetting agent or a mineral oil or a vegetable oil.

8. A method according to any one of the preceding claims, in which the plant has increased resistance to attack from members of the two Helicoverpa species or lepidopteran species, such as tobacco budworm, bollworm, cotton bollworm, pink bollworm, podworm, earworm or 15 fruitworm. e*

9. A method according to any one of the preceding claims, in which the pest resistance enhancing chemical compound is administered to the cotton plants in a dose of 20 from 0.1 to 30 kg/ha. A method according to claim 9, in which the pest resistance enhancing chemical compound is administered to the cotton plants in a dose of from 0.1 to 20.0 kg/ha.

11. A method according to claim 10, in which the dosage is from 0.1 to 10.0 kg/ha.

12. A method according to claim 11, in which the dosage is from 0.5 to 7.0 kg/ha.

13. A method according to any one of claims 9 to 12, in which the dosage of pest resistance enhancing chemical H:\PClarke\Keep\Retypes\60815-98 AB TALL claims.doc 27/04/01 30 compound is from 0.5 to 4.0 kg/ha.

14. A method according to claim 13, in which the dosage is from about 2.5 to 3.5 kg/ha. A method according to any one of the preceding claims, in which the pest resistance enhancing chemical compound is applied externally or exogenously to the plant.

16. A method according to claim 15, in which the chemical compound is applied to the leaves of the plant.

17. A method according to any one of the preceding 15 claims, in which the pest resistance enhancing chemical compound is sprayed onto the plant either alone or in combination with an additive.

18. A method according to claim 17, in which the 20 leaves of the plant are sprayed.

19. A method according to any one of the preceding claims, in which the pest resistance enhancing chemical compound is applied when the plant is subjected to stress 25 or to stress-inducing conditions. A method according to claim 19, in which the stress or stress-inducing conditions includes environmental stress, stress relating to temperature, water, salinity, light, nutritional stress or pest attack.

21. A method according to any one of the preceding claims in which the pest resistance enhancing chemical H:\PClarke\Keep\Retyps\60815-98 AB TALL claims.doc 27/04/01 .i i li.i-;; i. l 31 compound, with or without additives, is applied to the plant in a single application or in two or more applications.

22. A method according to any one of the preceding claims, in which the pest resistance enhancing chemical compound is applied to the plant at any time from planting to harvesting.

23. A method according to claim 22, in which the pest resistance enhancing chemical compound is applied in the period beginning from 20 days before first flowering to days after first flowering. 15 24. A method according to claim 22 or 23, in which the first application of the chemical compound to the plant is at first flowering of the plant. A method of treating a plant to improve or 20 enhance pest resistance of the plant, substantially as hereinbefore described with reference to any one of the foregoing examples. o Dated this 27th day of April 2001 S* 25 AB TALL (HOLDINGS) PTY LTD By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\PClarke\Keep\Retypes\60815-98 AB TALL claims.doc 27/04/01