AU703407B2 - Osmolyte regulator - Google Patents

Description

WO 96/23413 PCT/AU96/00045 1 OSMOLYTE REGULATOR The present invention relates generally to agriculture and in particular to methods of improving or controlling the growth of crops. More particularly, the present invention relates to methods of controlling stress and related conditions in plants during the growth of the plant, particularly in the early stages of growth and more particularly where the plant is pear, peach, cherries or the like. Even more particularly, the present invention relates to the use of an osmolyte regulator, particularly glycine betaine, to control stress in growing cherries so as to prevent the development of cracking in the skins and like areas of the cherries, thereby increasing the value of the cherry crop. The present invention finds particular application in administering glycine betaine to cherries by spraying at predetermined times to reduce or eliminate the number of cherry fruits which develop cracks in their skins.

Although the present invention will be described with particular reference to the use of glycine betaine as one example of the osmolyte regulator administered to cherry plants to control stress in the plants, particularly the tendency of the skins of the cherries to crack, it is to be noted that the scope of the present invention is not restricted to the described embodiment but rather the present invention is more extensive so as to include the use of other osmolyte regulators, to other ways of WO 96/23413 PCTAU96/00045 2 administering the regulators, and to other uses of the chemical compounds than as osmolyte regulators, and to the use of the regulators on crops other than those specifically described, particularly on pears, peaches and the like.

Cherries are a crop having considerable economic value. As cherries grow they are often subjected to stress in one or more forms. The cherries respond to stress by cracking their skins. Consequently, the development of stress within the cherries is easily and readily observable and demonstrable. The amount of cracking developed by the cherries is an indication of the amount of stress to which the cherries are being subjected. The main causes of skin cracking include too much or too little water, inadequate nutrition, low light intensity, low temperatures, and the like.

Cracks usually develop in cherries during the latter stages of ripening of the cherries and cracking is more severe under wetter conditions than under drier conditions. It is believed that cracking is caused by a build up of pressure within the fruit so that the skin is not strong enough to withstand the increase in pressure without splitting.

The development of cracks in the skin of ripe cherries reduces the economic worth of the crop since cherries with cracked skins cannot be sold at a premium price as high quality table fruit, but rather can only be used in lower quality applications, such as for example in making jams and the like, which lower quality applications do not produce the same economic returns to the growers as when the cherries are sold as high quality table fruit.

Additionally owing to the cracking of the skins secondary infection such as for example by moulds, brown rot, fungi and other pathogens can infect and develop within the cherries, so that a majority of cracked fruit is often not 3 suitable for any purpose and is simply dumped. Therefore, any reduction in the amount or extent of cracking in cherries will be beneficial to the grower as the fruit will be of a more uniform high quality and demand higher prices when sold, which increases the economic value of the crop and the monetary return to the grower.

The same applies to many other types of fruit, such as for example pears, peaches and the like.

Therefore, it is an aim of the present invention to provide a method of administering an osmolyte regulator to a plant, particularly to cherry plants for controlling the stress induced or developed in the growing plant so as to overcome S 15 or at least reduce the tendency of the skins of the plant to crack, particularly during ripening.

o oo S According to one aspect of the present invention there is o provided a method of controlling stress in a growing plant to reduce surface imperfections of the skin of the fruit of the plant, including administering an effective amount of osmolyte regulator to the plant by foliar application such that stress induced or developing in the plant can be "controlled to a satisfactory level in order to enhance the growth of the plant by eliminating or reducing the amount of surface imperfection, blemish or damage sustained to the skin of the fruit of the plant in order to provide a greater yield of the plant at a higher quality of product wherein the plant is selected from stone fruits, pome fruits and citrus fruits.

Typically, the plant to which the osmolyte regulator of the present invention can be administered includes stone and pome fruits, such as peaches, apricots, nectarines, pears, citrus fruit and the like, particularly including cherries.

Typically, the plant to which the osmolyte regulator of the present invention can be administered includes tropical H:\Sigrid\keep\patents\45314-96.doc 29/01/99 3afruits and nuts such as bananas, mangoes, macadamias and almonds. More typically, the cherries can be of any species and are preferably the Stellar variety.

Typically, the osmolyte regulator is an organic solute, an amino acid, a betaine, a sugar, a e \\melb~l\home$\Sigidkep\Ptents\4 6.doc 9/11/98 WO 96/23413 PCT/AU96/00045 4 polyol, or related compounds to the foregoing, and the like. More typically, the osmolyte regulator is an ammonio compound, such as a n-methyl substituted amino acid, proline, choline or a betaine, such as glycine betaine (oxyneurin) and other betaine member compounds and the sulphonio analogues of the betaines. Other betaines include proline betaine, 3 -alanine betaine, tryptophan betaine, histidine betaine, 2 -mercaptohistidine betaine, and the like. Even more typically, the osmolyte regulator is a nitrogenous compatible solute, such as stachydrine, trigonelline, homostachydrine (pipecolate betaine).

Other osmolytes or osmoregulators include glycine, methylene glycine, dimethyl glycine, glutamic acid, yaminobutyric acid, trimethylamine y-butyric acid, or the like.

Typically, the osmolyte regulator is administered alone or in combination with one or more other materials, such as adjuvants. Typically, the other materials include additives such as wetting agents, defoliants, growth regulators, pesticides, fungicides, vegetable and mineral oils, nutrients and the like. The other materials can be added separately or in combination to the osmolyte regulator.

Even more typically, the osmolyte regulator and other materials if present have a synergistically beneficial effect on the plant.

Typically, the amount of osmolyte, typically glycine betaine, administered to the growing plant is such so as to reduce or eliminate the tendency of the plant to develop surface irregularities, imperfections, blemishes, damage or the like, including cracks in the skin as they ripen, thus increasing the economic worth of the crop of cherries.

Typically, the stress induced or developed in the cherries includes environmental stresses such as, for example, WO96/23413 PCT/AU96/00045 5 stress related to temperature, water, salinity, light, nutritional stresses and the like. More typically, the stress induced results from too little or too much water, too low a temperature, too high a salt concentration, too low a light intensity, too low nitrogen concentration, and the like.

Typically, the glycine betaine is administered to the cherries in a concentration of up to about 20,000 ppm, preferably up to about 10,000 ppm, more preferably from 1000 to 10,000 ppm, most preferably 3,000 to 10,000 ppm.

The dosage rate of glycine betaine administered to the plants typically is in the range of up to 100 kg/100 L preferably up to 5 kg/100 L, more preferably up to kg/100 L, most preferably from about 0.1 to 1 kg/100 L of glycine betaine in aqueous solution.

Typically, the regulator either alone or in combination with the other materials is applied to the leaves and fruit of the cherry plants, more typically as an aqueous solution.

Typically, the osmolyte regulator can be applied to the crops at any time, preferably late in the life of the crops, typically shortly before the crops ripen, more typically just before the fruit is ready for harvesting.

More preferably the glycine betaine is applied at a time from when about 30% to 40% of the cherries are pink to red fruit to when about 70% to 80% of the cherries are pink to red fruit. More typically, the glycine betaine is applied to the cherries to the point of runoff. Dose rate is related to canopy size and thus the term "dose" is referred to as "to the point of runoff". This means the point when the leaves are covered by the solution and excess begins to run off.

It is to be noted that the osmolyte regulator can be WO 96/23413 PCTIAU96/00045 6 administered to the plants at any time until harvesting, including before, during or after flowering of the plants, development of fruit, ripening of the fruit and the like.

It is further to be noted that there can be a single administration of the osmolyte regulator or it may be administered in two, three, four or more separate applications.

Typically, the osmolyte regulator is applied to the plants by spraying, more typically spraying of aqueous solution.

However, the osmolyte regulator may be applied in any suitable manner.

Typically, the more advanced the fruit form of the plant when the osmolyte regulator is applied, the more the plant responds to the addition of the osmolyte regulator, and more control over stress induced in the plant is obtained.

More typically, the proportion of individual cherries having cracked skin decreased with an increase in fruit size when the glycine betaine is administered. Increasing the dose of glycine betaine significantly reduces the proportion of cracked fruit and the effectiveness of treatment with glycine betaine increased with the maturity of the fruit.

The present invention will now be described by way of example with reference to the following examples.

Example 1 The object of this example is to determine the effect of foliar applications of glycine betaine on the tendency of cherries to crack during ripening. The variety of cherries used in the following trial is Stellar. The cherry trees were mature trees about 15 years old and planted on a 5.5 m by 6.0 m grid having a shape described as "delayed-modified central leader". The dimensions of the plants were 4.5 to m high by 4.5 to 5.0 m in diameter of from 70 to 110 WO 96/23413 PCT/AU96/00045 7 cubic metres with moderate foliage density.

Layout of the trial The layout of the trial included the following details: design: randomised complete block replications: 4 plot size: single tree buffers: nil Treatments Details of the treatments included the following: Code Active Dose active ingredient ppm A glycine betaine 1000 B glycine betaine 3000 C glycine betaine 6000 D glycine betaine 1000 E glycine betaine 3000 F glycine betaine 6000 G untreated control Application The equipment used to apply the treatments to the cherry plants included a hand-held spray gun fitted with a single nozzle on a 1 m long extension.

WO 96/23413 PCT7AU96/00045 8 Application treatments Inspection and Assessment Inspection of the fruit of the cherry plants under test for colour development was carried out at 3 and 8 days after the first treatment application, which was denoted to be day 0, just before the second treatment application, which.

occurred at day 10, and two days after the second application, in order to determine the appropriate time to start the assessments.

At 13 days after the first application and 3 days after the second application two samples of 200 fruit per tree were examined. The first sample consisted of the 10 fruit nearest the point 0.5 metres down from the tip of each of branches. The branches selected were as evenly spaced WO 96/23413 PCT/AU96/00045 9 around the tree as possible. The number of fruit that were more than 50% red were recorded as well as the number showing symptoms of phytotoxicity which is typically seen as a discoloured area at the drip point which is surrounded by a prominent band of darker coloured tissue.

The second sample consisted of 10 fruit each more than red on each of 20 branches per tree. The branches selected were as evenly spaced around the tree as possible. The number of cracked fruit from each sample was recorded.

To ensure that classification of colour was constant, each of the two assessors frequently referred to examples of each classification selected before the assessment began.

At 16 days after the first application the proportion of red fruit was estimated again, using the same sampling procedure as at 13 days.

At 20 days after the first application, 15 fruit on each of limbs per tree were examined and the numbers with cracks and symptoms of phytotoxicity were recorded. At this time virtually all fruit were red.

Results The results of the trial are summarised in Table 1.

Fruit colour Compared with the untreated control (treatment none of the treatments B, C, D, E, F) had any significant effect on the proportion of red fruit that had developed at either assessment. This suggests that it was most unlikely that the glycine betaine treatments affected the maturation of the cherries and accordingly the yield of cherries was not significantly altered by the administration of the glycine betaine. However, the glycine betaine may assist in some way which was not WO 96/23413 PCTAU96/00045 10 readily observable in the actual trial being conducted.

Cracking The frequency of cracking in this trial was moderate, and also the severity of cracking was very light with cracks ranging from 1 mm to 3 mm in length and penetrating to a depth of no more than 1 mm.

The application of glycine betaine at 6000 ppm (treatment c) to predominantly dark green fruit had no significant (p>0.05) effect on the proportion of fruit which developed cracks at the time of harvest. But later application (treatment f) of this same dose to predominantly light green fruit significantly (p<0.05) reduced the proportion of fruit which developed cracks by 65%. At both times of application, the lower doses of glycine betaine tested, 1000 and 3000 ppm respectively, had no significant effect on the proportion of fruit that was cracked at harvest.

However, the trends in the results suggest that doses lower than 6000 ppm may be effective, particularly if such doses are applied at later times closer to harvest than at the timing used in this trial. Also at both assessments of the effects of the later application the proportion of cracked fruit was highly correlated with the dose of glycine betaine, i.e. first assessment: r=0.6803, P<0.010, n=12; second assessment: r=0.7623, p<0.001, n=12.

Cracks usually develop in cherries during the latter stages of ripening and cracking is more severe under wetter conditions than under drier conditions. All assessments of cracking were made on the ripest (red) fruit and as a result the samples used for the first and second assessments were comprised of different fruit. This explains the significant difference in the proportion of cracked fruit between the first and second assessments viz.

an average of 19.1% cracked fruit at the first assessment and 15.3% at the second assessment (p=0.037, paired t- WO 96/23413 PCT/AU96/00045 11 test).

Conclusions The effectiveness of foliar applications of glycine betaine in reducing the proportion of cracked cherries increased with increasing dose and with the maturity of the fruit at the time of application of the glycine betaine. While only the highest dose at the latest time of application (6000 ppm, at 10 days before harvest) was significantly (p<0.05) effective in this trial, the trends in the results of other doses and other times of application suggested that lower doses are effective if applied at later times closer to the time of actual harvest.

The level of performance of the most effective treatment, which was a dosage of 6000 ppm at day 20, showing a reduction in the amount of cracked cherries, is more than sufficient for commercial utility. Table 2 shows the actual number of fruit which were assessed as being cracked for each of the treatments. Treatments 1 to 7 correspond to treatments A to G. The data relating to the actual number of fruit with cracks was used to calculate the results shown in Table 1.

WO 96/23413 PCT/AU96/00045 12 Table 1

TREATMENT

3od 6000o Dat0 41000 Day 0 2 3000 Day 0 6 6000 Day 0 7 Untreated Control

(UTC)

Mean Red .1 Fruit Day Day 13 16 16.5 51.4 17.1 58.3 19.3 577 14.9 57,9 14.2 55.7 15.6 53.9 16.5 57.0 Mean 0/ Fruit *1 with symptoms of phytotoxic~ty Day Dav 13 2 11.2 b 0.2 11.7 b 0.3 13.9 b 0.2 12.7 b 0.4 14.5 b 0.5 12.8 b 0.2 0.0Oa 0.0 Mean Red fruit with crack~s Vay Dav 13 2 .3 22.6 15.7 bcd 14.8 19.9 cd 21.9 12.2 abc 26.6 23.6 d 14.4 10.7 ab 8.6 5.9 a 21.1 17 0 bCd F prob.) UTC Out 0.797 j0.724 0.951 0.858 0.147 0.003 2 way ANOVA UTC In 0.865 0.965 <0,001 0.560 0.129 0.006 *1 All means de-transtormed~ from angles.

'2 Letters indicate statistical separation (p=0.05 Fishers protected LSD tesi *3 Statistical separation the same with control included or excluded.

A discussion of the results of Table 1 will now follow.

As can be seen from the results of Table 1, particularly from the results of the column headed "Mean red fruit with cracks", when treatment F (corresponding to code 6) is considered there is a reduction of from 21.1% of fruit with cracks from treatment 7 resulting from the controlled untreated treatment to 8.6% after 13 days. This means that.

WO 96/23413 WO 9623413PCT/AU96/00045 13 Table 2 Plot/ TRT. Appicn. Tree Row Code Time No. No.

1 1 2 1 1 1 22 2 1 1 13 1 2 1 26 2 2 1 .7 1 2 1 18 2 2 1 14 1 3 1 28 2 3 1 23 2 3 1 20 2 3 1 12 1 4 2 4 1 4 2 6 1 4 2 21 2 4 2 11 1 2 1i 1 2 24 2 2 8 1 2 16 2 6 2 I27 2 6 2 25 2 6 2 10 1 6 2 17 2 7 riia 3 1 rv 5 1 7 n/a 19 2 7 n/a 15 2 No. of Fr-uit with No. of Red sy tms of No of Cracked Fruit Fruit phytotoxicrty Day 13 Day 20 Day 13Dav 16 Day 1 2 Rep. per 200 per 300 per 200 per 0 e 0 e Q Red Fruit Fruitji Fruit Fruit Fruit ft I

I

125 102 87 97 156 107 ill 89 141 123 104 93 131 103 119 92 84 145 132 100 97 127 93 114 127 98 96 134 WO 96/23413 PCT/AU96/00045 14 within 3 days after application of the glycine betaine there was observed an improvement in the reduction of cracked fruit.

At 20 days after application, the percentage of cherries with cracks resulting from treatment 6 was 5.9% as compared to the untreated fruit which exhibited 17.0% of the fruit with cracks.

Thus, with larger dosages of treatments of glycine betaine such as at 6000 ppm at a later timing of application closer to harvest time, it can be seen that a greater proportion of the cherry crop was free from cracking and hence of greater economic value to the grower.

Example 2 The object to this example was to determine the effect of dose and time of application on the effectiveness of foliar sprays of glycine betaine in preventing cracking in cherries. The variety of cherry is Stellar and the age of the trees is about 20 years with the trees planted on a 6 m by 6 m grid. The tress have modified central leader shape.

The dimensions of the plants were 5-6 m in height by 5-6 m in diameter, or from 80 to 130 cubic metres, with a moderate to high foliage density.

The trial was laid out as a randomised complete block experiment with 4 blocks, each containing one replicate of each treatment. The plot size was one tree.

WO 96/23413 PCT/AU96/00045 15 Code Material Dose Time of application A glycine betaine 3000 30% to 40% pink to B 6000 red fruit.

C 10000 Timing 1 (Tl) D glycine betaine 3000 70% to 80% pink to E 6000 red fruit.

F 10000 Timing 2 (T2) G untreated control MONSOON, an aqueous solution containing 300 g/L linear (C9 to Cll) alcohol ethylene oxide condensate, was added to all sprays at 30 mL/100 L.

The equipment used to apply the treatments to the cherry plants under test included a hand-held spray gun fitted with a single nozzle, on a 1 m long extension.

Timing and conditions of application of treatment Application No. 1 2 Treatments applied A,B,C D,E,F Day No. 0 8 Time 19.15 to 20.55 19.30 to 20.45 Nozzle designation 1/4 GG 3009 Nozzle manufacturer Spraying Systems Co Pressure at nozzle (kpa) 1700 Droplet size Medium spray Spray volume (1/ha) Trees sprayed to the point of runoff: 9 to 16 L/tree equivalent to 105 to 120 litres per 1000 cubic metres of plant volume.

Temperature (oC) 19 to 21 20 to 22 Relative humidity 80 to 85 65 to Wind speed (m/sec) Nil; dead calm Cloud cover 60 to 80% 50 to Spray drift Negligible due to wind, but where foliage of adjacent trees contacted each other, treatment of non-target tree was unavoidable.

WO 96/23413 PCT/AU96/00045 16 Application No. 1 2 Foliage condition Dry Spray drying time (hour) >1 Crop stage Refer assessments Crop condition Healthy, vigorous, no stress or pests evident. Very heavy fruit load.

Inspection and Assessments The circumference of all trees at 0.3 m above the soil (butt circumference) was measured when the trial was pegged out.

To estimate the proportion of pink/red fruit at application, 5 groups of 10 fruit were examined on each tree to be sprayed and on each untreated control tree.

This was repeated at the second application timing.

Fifty dark red (ripe) fruit were picked from the shoulder height circumference of the canopy of each tree. Fruit were not sampled from areas where the foliage contacted that of adjacent trees. After the samples were immersed for ca 20 hours in de-ionized water at 17 to 22 0 C, each fruit was examined and classified as either sound or cracked. On the last occasion, the volume of each sample of 50 fruit was measured.

The results of this example are provided in Table 3.

Cracking was artificially induced by immersing fruit in water because virtually none of the fruit on the tress had cracked due to the prevailing weather conditions at the time that the trials of this example were conducted.

When the data was analysed according to the original design, no statistically significant (p<0.05) responses were obtained (Table 3).

WO 96/23413 WO 96/34 13PCT/AU96/00045 17 Table 3 Initial analyses of results TREATMENT Mean Mean Mean Cracked Butt Pink/Red Fruit cherries Circumr1 Code fDose of Time (cm) T1 T2 As. 1 As. 2 I GB of (ppm) Appin j A 3000 Ti 85 34 81 1 37 1 37 B 6000 Ti 77 33 81 27 36 C 10000 TI 77 33 83 23 23 D 3000 T2 80 40 81 26 E 6000 T2 74 43 77 19 29 F 10000 T2 80 37 75 18 18 G Untreated 7642 75 25 36 L ~controlIIII 1-way ANOVA F f .3 f0.713 f 0.289 f 736 1.7 prob. 0.9 j0.664 0.934 0. 628 .1 *1Analyses calculated using original spatial blocking.

Further examination of the data showed that with fruit from untreated controls, the proportion of cracked fruit increased with an increase in fruit size (volume).

However, with fruit from glycine betaine treated trees, the proportion of cracked fruit decreased with an increase in fruit size (Table 4).

WO 96/23413 PCT/AU96/00045 18 Table 4 Interaction between fruit size (volume) and treatment on the proportion of cracked fruit TREATMENT Linear regression of cracked fruit on volume of 50 Fruit (n=4) Code Dose Time Assessment 1 Assessment 2 Mean of both of GB of Assessments (ppm) Appln Slope Sig. Slope Sig. Slope Sig.

level level level A 3000 T1 -0.259 0.051 -0.436 0.023 -0.347 0.030 B 6000 T1 -0.264 0.049 -0.187 <0.001 -0.226 0.007 C 10000 T1 -0.281 0.131 -0.290 0.007 -0.289 0.027 D 3000 T2 -0.455 0.004 -0.390 0.004 -0.423 0.004 E 6000 T2 -0.136 0.130 -0.348 0.007 -0.242 0.027 F 10000 T2 -0.038 0.285 -0.124 0.118 -0.081 0.135 G 0 n/a 0.107 0.018 0.177 0.015 0.142 0.016 Since the responses to fruit size were opposed as indicated by the different directions of the slopes shown in Table 4, the glycine betaine data and the untreated control data were analysed separately. Additionally, the glycine betaine data was re-blocked on the basis of fruit size to allow for the response to this variable. This analysis showed (Table 5) that increasing the dose of glycine betaine applied to the fruit significantly reduced the proportion of cracked fruit and that the effectiveness of glycine betaine treatments increased with the maturity of the fruit, which is to say that the more mature the fruit when the glycine betaine is applied, the more effective is the treatment. Since the results of the two assessments were highly correlated (R sqrd=0.7307, p<0.001), the analyses were also calculated for pooled data.

WO 96/23413 PCT/AU96/00045 19 Table Analyses of data blocked on the basis of fruit size (volume) TREATMENT Mean cracked fruit (detransformed from angles) Code Dose of Time of Assessment Number GB (ppm) Appln 1 2 1 2 A 3000 T1 37 b *1 37 c 37 c B 6000 T1 27 ab 36 c 32 bc C 10000 Tl 23 a 23 ab 23 ab D 3000 T2 26 ab 30 bc 28 bc E 6000 T2 19 a 29 abc 24 ab F 10000 T2 18 a 18 a 18 a F (prob.) 2-way ANOVA 0.050 0.026 0.018 G *2 Untreated Control 25 36 31 Fruit Size-Block means: GB treatments only Size 1 smallest 38 b 48 c 43 c Size 2 31 b 34 c 33 c Size 3 18 a 22 b 20 b Size 4 largest 11 a 14 a 14 a F (prob.) 2-way ANOVA <0.001 <0.001 <0.001 *1 *2 Letters indicate statistical separation (p=0.05), Fishers protected LSD test Untreated control data not included in the analyses The relationship between the mean proportion of cracked fruit (Pt %),fruit size expressed as the volume of 50 fruit V mL), the dose/concentration of glycine betaine (D ppm) and time of application, where T1=l and T2=2) was well described by the- following equation.

WO 96/23413 PCT/AU96/00045 20 Pt=-36.658+756.512/V 0.00131 D 12.156 T. n=24.

Rsqr=0.7413.

Standard errors of 38.451, 110.35, 0.00054 and 3.137, respectively. Slopes significant at <0.001, 0.023 and <0.001, respectively.

For the untreated control the relationship between the mean proportion of cracked fruit (Px and fruit size expressed as the volume of 50 fruit (V mL) was: Px= -20.403+0.1425 V. n=4. Rsqr=0.9224.

Standard errors of 0.748 and 0.0292, respectively. Slope significant at 0.016. Solutions of the equations are plotted in Figure 1, with V expressed in terms of mean diameter per cherry, assuming they are spherical.

Figure 1 100 0- Zero 10000 ppm T1 6000 ppm @T1 3000 ppm T1 10000 ppm T2 S- 6000 ppm T2 3000 ppm T2 -2- CC~_-T~rl 21 22 23 24 25 26 Mean diameter of cherry (mm).

27 28 29 Figure 1. Effect of fruit size (diameter) and the dose and timing of Glycine Betaine on the proportion of cracked cherries.

WO 96/23413 PCT/AU96/00045 21 The proportion of cherries cracked after 20 hours immersion in water decreased significantly as the dose of glycine betaine was increased and as the maturity of the cherries at application increased. But the cracking of fruit from glycine betaine treated trees decreased with increase in fruit size whilst the cracking of fruit from untreated trees increased with fruit size.

Assuming that the immersion technique predicts what will happen to fruit on the trees when conditions are favourable for cracking, treatments of glycine betaine may either decrease or increase the proportion of cracked fruit. This depends on the size of the cherries. In this trial, glycine betaine applied at 10,000 ppm at about 80% pink/red fruit, decreased cracking in fruit greater than 24 mm in diameter and increased cracking in fruit smaller than this critical diameter. It also showed that cracking decreased as the dose of glycine betaine increased and as the maturity of the fruit at application increased.

Glycine betaine appears preferably suited for use in light crops with large fruit where fruit are most likely to crack.

Example 3 The objectives of this example are: 1. To determine the effect of foliar applications of glycine betaine on the maturity, size and finish of deciduous fruit, and 2. To determine the effect of foliar applications of glycine betaine on the tolerance of deciduous fruit trees to damage caused by two-spotted mite (Tetranychus urticae).

Number of trials and crops On trial in pears cv Packham's Triumph and one in a cultivar of canning peach that matures about mid-season eg.

WO 96/23413 PCT/AU96/00045 22 Taylor Queen.

Treatments: (both crops) A) glycine betaine at 1 kg/100 L applied at early fruit development B) glycine betaine at 1 kg/100 L applied at early fruit development and ca 4 weeks later C) glycine betaine at 0.5 kg/100 L applied at early fruit development and ca 4 weeks later D) glycine betaine at 0.25 kg/100 L applied at ca 4 week intervals commencing at early fruit development E) untreated control Adjuvant MONSOON (wetting agent) to be added to all sprays at the rate of 1-2 mL/100 L.

Application All treatments applied to dry foliage to the point of runoff, 4 times at ca 4 week intervals.

Maturity size and finish Pome fruit examination of 250 fruit per tree/plot about 1 week before harvest. Count number of fruit above the approximate control median size (using standard size gauges). Score each fruit for colour on a 2 or more point scale for colour. Score each fruit on the basis of surface area russeted using an appropriate scale. (Both colour and russet scales to be determined after an initial examination of the fruit.) Score foliage colour using an appropriate scale.

Peaches Stone hardness to be determined on 25 fruit per tree at standard time for the particular variety. One week before harvest examine 250 fruit per tree and assess as for pome fruit.

WO 96/23413 PCT/AU96/00045 23 Note, with both pome fruit and peaches the fruit assessments will be conducted on 1 control per block.

Tolerance of Mites The timing of the mite assessment will depend upon the development of the infestation. The mite infestation in each tree/plot will be scored at each application, but it is likely that one or two further inspections/scores may be required to determine the appropriate time.

The mite assessment will only be conducted if the Inspections/Scores indicate there is a response. It will entail counting motile T. urticae and Phytoselid mites (predators) on 24 leaves per tree; 3 inner and 3 outer at shoulder height from each quadrant. The proportion of bronzed foliage in each quadrant will then be estimated.

If glycine betaine increases the tolerance of the trees to mites then for a given infestation level, the damage in the untreated will be higher than in the treated. Twelve untreated controls are required to quantify the relationship between infestation level and damage.

From the results of this trial, it has been demonstrated that peaches have shown a significant increase in water shoot growth, which is important for growth rate and establishment of peach orchards, and that pears are growing more rapidly and are bigger in size than control. Thus, it is clear that the administration of glycine betaine to pears and peaches is beneficial for both of these crops.

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 herein disclosed.

WO 96/23413 PCTIAU96/00045 24 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.

Claims (21)

1. 2. A method of controlling stress in a growing plant oi. to reduce surface imperfections of the skin of the fruit of ego• the plant, including administering an effective amount of oo* osmolyte regulator to the plant by foliar application such 20 that stress induced or developing in the plant can be controlled to a satisfactory level in order to enhance the growth of the plant by eliminating or reducing the amount of surface imperfection, blemish or damage sustained to the skin of the fruit of the plant in order to provide a greater yield of the plant at a higher quality of product wherein the plant is selected from tropical fruits and nuts.
2. 3. A method according to claim 1 in which the plant is a cherry plant of the Stellar variety.
3. 4. A method according to claim 1 in which the plant is a pear plant. A method according to claim 1 in which the stone fruit is chosen from apricots, peaches, cherries or nectarines. H:\Sigrid\keep\patents\45314-96-doc 29/01/99 26
4. 6. A method according to claim 2 in which the tropical fruits and nuts include bananas, mangoes, macadamias or almonds.
5. 7. A method according to any one of claims 1 to 6 in which the osmolyte regulator is ammonio compound such as n- methyl substituted amino acid, proline, choline or a betaine such as glycine betaine (oxyneurin), or a sulphonic analogue of the betaines.
6. 8. A method according to claim 7 in which the betaines include proline betaine, 9-alanine betaine, tryptophan betaine, histidine betaine, or 2- mercaptohistidine betaine.
7. 9. A method according to any preceding claim in which the osmolyte regulator is a nitrogenous compatible :solute, such as stachydrine, trigonelline or homostachydrine (pipecolate betaine).
8. 10. A method according to claim 7 in which the osmolyte regulator is glycine, methylene glycine, dimethyl So glycine, glutamic acid, y-aminobutyric acid, trimethylamine or y-butyric acid.
9. 11. A method according to any preceding claim in which the osmolyte regulator is administered alone or in combination with one or more other materials.
10. 12. A method according to claim 11 in which the other material includes additives such as wetting agents, defoliants, growth regulators, pesticides, fungicides, vegetable and mineral oils, or nutrients.
11. 13. A method according to any preceding claim in which the stress induced or developed in the plants such as I cherries includes environmental stresses, such as stress \\melbOl\home$\Sigrid\kep\ptents\453 1 4. 96 .doc 9/11/98 27 related to temperature, water, salinity, light, or nutritional stresses such as stress induced by too little or too much water, too low a temperature, too high a salt concentration, too low a light intensity level, too low nitrogen concentration or the like.
12. 14. A method according to any preceding claim in which the osmolyte regulator is administered to the plants including cherries in a concentration of up to 20,000 ppm. A method according to claim 14 in which the osmolyte regulator is administered in a concentration of up to 10,000 ppm. 15 16. A method according to claim 15 in which the osmolyte regulator is administered in a concentration of from 1,000 to 10,000 ppm. S17. A method according to claim 16 in which the osmolyte regulator is administered in a concentration of from 3,000 to 10,000 ppm.
13. 18. A method according to any preceding claim in Swhich the dosage rate of osmolyte regulator administered to 25 the plant is in the range of up to 10 kg/100 L.
14. 19. A method according to claim 18 in which the dosage rate of osmolyte regulator administered to the plant is in the range of up to 5 kg/100 L. A method according to claim 19 in which the dosage rate of osmolyte regulator administered to the plant is in the range of up to 2.5 kg/100 L.
15. 21. A method according to claim 20 in which the dosage rate of osmolyte regulator administered to the plant is in the range of from 0.1 to 1 kg/100 L. \\melbO\homeS\Sigrid\keep\patents\4531496.doc 9/11/98 28
16. 22. A method according to any preceding claim in which the osmolyte regulator is applied to the leaves and fruit of the plant.
17. 23. A method according to any preceding claim in which the osmolyte regulator is applied to the plants at any time.
18. 24. A method according to claim 23 in which the osmolyte regulator is applied to the plants late in the life of the plants. A method according to claim 23 in which the osmolyte regulator is applied to the plants shortly before 15 the fruit of the plants ripens.
19. 26. A method according to claim 23 in which the S;o osmolyte regulator is applied to the plants just before the fruit is ready for harvesting.
20. 27. A method according to any preceding claim in which the osmolyte regulator is applied to the plants by spraying. 25 28. A method according to claim 27 in which the S osmolyte regulator is applied to the plants by spraying an aqueous solution of the osmolyte regulator.
21. 29. A method of controlling stress in a growing plant as hereinbefore described with reference to any one of the foregoing examples. Dated this 9 th day of November, 1998 AB TALL (HOLDINGS) PTY LTD By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent Attorneys of Australia \\melb01\home$\Sigrid\keep\patents\45314-96.doc 9/11/98