Abstract:
A method for the biological control of pollen in plants, including the thinning of fruits, reducing seeds in citrus plants and reducing the incidence of shotberries in grapes, by applying an effective amount of activated or inactivated RNase or mixtures of these to the flowers of the plants.

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to a method of the biological control of pollen in plants using RNase. The present invention particularly relates to the thinning of a large variety of fruit plants, the reduction of seeds in citrus plants and the reduction of the incidence of shotberries in grapes using activated RNase, inactivated RNase or mixtures of these. 
     The purpose of thinning fruit, whether it is done manually, chemically or mechanically, is to reduce crop load early in the fruit development period to maximize return of bloom (for apple) and ensure attainment of commercially acceptable fruit size (for peach, nectarine, plum, apple and table grapes). Although apples and grapes are routinely thinned in many districts, satisfactory results have not always been consistent, because sensitivity to any specific thinning agent varies from clone to clone, and climatic conditions enhance or diminish the efficiency of the chemicals. In peach and nectarines hand thinning is the only practical method that is currently employed, simply because none of the many chemicals and cultural practices employed, give consistent results. Among the many methods are: Control of flower buds initiation by gibberellic ac id (M.Sc. Thesis, A. Breuer-Mizrahi (1991), The Hebrew University of Jerusalem), flower thinners (R. Gaash, S. Lavee, A. Golan, D. Brown, &#34;Alon Hanotea&#34;, (1969), pp. 319-325), fruitlet thinners (S. Morini, G. Vitagliano, C. Xiloyannis, I. Ant. Soc. Hort. Sci., 40, (1976), pp. 237-247, and G. E. Stembridge, C. G. Gambrell, Ibid., 96, (1971) pp. 7-9), urea, as well as cold water. 
     Two major problems are associated with these methods: 
     1. Thinning intensity was inconsistent and difficult to control. 
     2. The broad range of effect makes it impossible to determine a precise developmental stage (of the flower or the fruitlet) for optimal treatment 
     In grapes, gibberellic acid is routinely used for thinning Tompson cultivar. The results, however, require always hand thinning for adjustment. The same treatment in Perlette is not practiced for the last 15 years, since it was found that in this variety, the gibberellic acid causes irregular berry size (shortberries), therefore hand thinning is practiced in Perlette. 
     The series of events that occur between pollination and fertilization include processes of specific recognition of pollen grains and pollen tubes by the pistil tissues. The control of the pistil on pollen germination and pollen tube growth has been mainly studied in self-incompatible systems, in which arrest of pollen tube is the consequence of a contact between pollen and pistil of the same genotype. In self-compatible matings there is some evidence that the pollen tube is directed toward the ovule by trophic and chemotropic effect in the style. However, there is still a paucity of information about the female control of the male gametophyte. Most economically important apple ((Malus domestica) cultivars require cross-pollination for fruit set. Peach (Prunus persica) is known as self-compatible. In these species the growth of pollen tubes in the style has been thoroughly investigated by physiological and histochemical studies. It has been established that the kinetics of pollen tube growth is controlled by each part of the gynoecium, starting with the stigma and ending with the ovule. However, the nature of this control, or any stylar product in which it is accomplished by, have not been characterized in these works. 
     Recently a major stylar protein (S-protein) was isolated from the self-incompatible species Nicotiana alata. The gene that encodes for the protein was cloned and sequenced and was found to share homology with fungal RNase. It was established that this protein is indeed RNase, and was found to arrest pollen tube growth (B. A. McClure et al., Nature, 1989, pp. 955-957). However, the RNase was also found in stylar diffusates of self-compatible species such as Nicotiana tabacum (McClure et al., 1989) and more recently in peach (Roiz and Shoseyov, 1993, unpublished data) and citrus (Roiz, Shoseyov and Goren. 1993, unpublished data). It is speculated that the RNase penetrate the pollen tube, degrade ribosomal and mRNAs and thereby cause its abortion. 
     Closely related to the thinning of the fruits is the reduction of seeds in citrus plants, which is very desirable from a practical as well as commercial point of view. Until now there is no known reliable method for reducing the seeds in citrus plants. 
     Similarly, there is no known reliable method for reducing the incidence of shotberry in grapes. 
     RNase is a known enzyme obtainable from a large variety of sources. One main source is the fermentation of various fungi. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention there is provided a method for the biological control of pollen in plants, comprising applying an effective amount of activated or inactivated RNase or a mixture thereof. The present invention results in the thinning of a large variety of fruit plants, the reduction of seeds in citrus plants and, in the case of grapes, the reduction of the incidence of shotberries. 
     DETAILED DESCRIPTION OF THE INVENTION 
     RNase may be obtained from sources such as fungi, bacteria, plants, animal and vital. When obtained from fungal sources, the RNase may easily be obtained by the fermentation of various fungal sources. Examples are Aspergillus niger, Aspergillus claritus, Aspergillus oryzae and Rhisopus miveus, preferably Aspergillus niger and Aspergillus oryzae. A most preferred fungus is Aspergillus niger B1(CMICC Number 324,626). This strain was deposited with the Commonwealth Mycological Institute, Ferry Lane, Kew, Richmo rid-upon-Thames, Surrey TW9 3A5, United Kingdom, on 20 May, 1988. 
     The plants where the method of the biological control of pollen is chosen from the group consisting of fruit trees, ornamental trees, vegetables, field crops, plantations, ornamentals and the like. The trees where the method of thinning was found to be applicable are chosen from the group consisting of deciduous plants, tropical plants, sub-tropical plants, citrus plants, nut tree and grape vines. Examples of deciduous plants are stone fruit, pears, apples, quince, peaches, plums, nectarines, cherries and apricots. 
     Examples of ornamental trees are olive and Fica trees. 
     Examples of field crops are cereals, especially hybrids. 
     Examples of citrus plants where the RNase in use in the thinning and the reduction of seeds are orange, lemon, grapefruit, clementins, mandarin, citron, pomela and their hybrids. 
     The method of the prevention of fruit set of field crops of the present invention was found to be especially useful in preventing fruit set of ornaments whose fruits and flowers cause environmental problems. 
     The RNase is added to the pollen in the form of crude or purified extract of the RNase in the form of spray, powder and the like. A really novel way to apply the RNase is to powder bees and let them distribute the RNase powder during their feeding on the pollen in the flowers. 
     The RNase is applied at a rate of 1 to 10,000 units per hectare. 
     While the invention will now be described in connection with certain preferred embodiments in the following examples, it will be understood that it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents, as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of procedures, as well as of the principles and conceptual aspects of the invention. 
    
    
     EXAMPLE 1 
     Growth Conditions Of Aspergillus niger 
     Aspergillus niger was grown in medium that contained 1% wheat flour and 0.05% (NH 4 ) 2  SO 4  in distilled water. The solution was brought to pH 3.5 with HCl and then autoclayed. An inoculum of 10 6  spores of A. niger were suspended into 100 ml of the growth medium and the suspension was incubated at 30° C. and shaken at 150-200 rpm. 
     RNase activity in the growth medium reached its peak after 5-7 days of incubation. The medium was then passed through a millipore filter of 0.2 μm to remove any traces of hypha and spores. The medium was ultrafiltered to concentrate the enzyme. 
     EXAMPLE 2 
     RNase Assays 
     RNase activity in the growth medium of A. niger was determined by diffusion plate assay, using 0.1% torula yeast RNA (Sigma) in 1% agarose as a substrate. A drop of 10 μl was applied on the substrate in a 15 cm petri dish and then was incubated at 37° C. for one hour. The substrate was then stained with 0.02% toluidine blue in water, RNase activity was indicated as a white halo on a blue background. 
     A quantitative spectrophotometric assay of RNase was conducted as a modification of the procedure described by Brown &amp; Ho (1986). Each sample contained 100 μl of the examined enzyme mixed with and 4 μg torula yeast RNA and 1 ml of 20 mM buffer of choice. The sample was incubated in 37° for 30 minutes. The reaction was stopped by putting the tube into ice and adding 200 μl &#34;stop reagent&#34; (0.75% uranyl sulphate in 25% HClO 4 ). The solution was centrifuged in 12,000 rpm for 5 minutes. The supernatant was diluted to 1/20 in distilled water and OD in 260 nm was examined. The blank solution contained the same component and incubated by the same procedure, only the enzyme was added after the reaction was stopped. The results were given in units/ml, equivalent to standards of bovine pancreatic RNase. 
     EXAMPLE 3 
     Optimization of RNase Activity 
     The optimal pH for RNase activity in the growth medium was examined using 20 mM citrate buffer at the pH value oft 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. The above spectrophotometric procedure showed that the optimal pH for the fungal RNase activity was 3.5 (FIG. 1). 
     The effect of presence or lack of metal ions on RNase activity was also examined. Each of the following salts: MgCl 2 , FeCl 3  or CaCl 2  was supplemented into the reaction mixture to a final concentration of 10 mM. Additional samples contained the same concentration of DTT as a reductant and EDTA that has strong affinity to metal ions. RNase activity was examined by the above diffusion plate assay. No difference in the diameter of the white halo (1 cm) on the blue background was observed between the different treatments and the control. 
     EXAMPLE 4 
     The Effect of RNase on Pollen Germination and Pollen Tube Growth In Vitro 
     Pollen grains of peach cv. Texas were germinated in vitro in growth medium containing 15% (w/v) sucrose, 100 μg/ml H 3  BO 3 , 200 μg/ml MgSO 4  ·7H 2  O and 200 μg/ml Ca(NO 3 ) 2  ·4H 2  O (Galletta, 1983). In each 1.5 ml Eppendorf approximately 1000 pollen grains were suspended by the edge of a needle into 100 microliters of growth medium. RNase was suspended in the pollen growth medium to a final concentration of 0.05, 0.5, 5, 50 or 500 units/ml. The percentage of germination was examined after two hours of incubation at 25° C. in the dark and the length of the pollen-tubes was measured after incubations overnight at the same conditions. Table 1 shows that the RNase produced by A. niger has a clear inhibitory effect on both pollen germination (P&lt;0.02) and pollen tube growth (P&lt;0.01). For a comparison, the results obtained from the same conditions with 5 units/ml bovine pancreatic RNase are also demonstrated. 
     EXAMPLE 5 
     Field Experiments 
     The RNase produced by A. niger was examined in the field as a thinning agent of deciduous fruit trees. In peach (Prunus persica) and plum (Prunus salicina) the fungal RNase was applied exogenously directly on the stigmas, by spraying the flowers during anthesis. The flowers were exposed to open pollination and the fruit-set was examined four weeks after the blooming period. In trees of peach cv. Texas grown in an orchard in Kefar Vatburg different branches were treated as follows: 
     The flowers were left untreated (control) 
     The flowers sprayed by 10 mM citrate buffer pH 3.5 just once, when the percentage of open flowers was estimated as at least 50% (B-I) 
     The same buffer was used, only spraying procedure was repeated every 2-3days, starting from 10% open flowers till the branch ceased blooming (B-M). 
     The flowers were sprayed by RNase that was diluted by the above buffer to 1 or 100 units/ml. RNase was applied once (R1-I and R100-I, respectively) or repeatedly (R1-M and R100-M respectively) during anthesis. 
     Table 2 shows that RNase caused a significant reduction of about 40% (P&lt;0.01) of fruit-set. The repeated treatments showed better results than one treatment but interestingly, the low concentration was most effective in this case. 
     
                       TABLE 1______________________________________POLLEN GERMINATION AND POLLEN TUBE LENGTHIN DIFFERENT CONCENTRATIONS OF RNASE                PollenRNase Conc.     Number of  Germination Pollen tube(units/mililiter     Repetitions                %           length (mm)______________________________________0 (control)      3         77.22 ± 2.98     20                     0.57 ± 0.100.5        2         81.05 ± 8.95     10                     0.56 ± 0.145          2         50.63 ± 0.62     17                     0.33 ± 0.085.sup.a    5         42.89 ± 2.97     55                     0.31 ± 0.08______________________________________ a = pancreatic RNase. 
    
     
                       TABLE 2______________________________________FRUIT SET OF PEACH CV TEXAS IN KEFAR VARBURG             Total      Percentage   Number of number of  of fruitsTreatment   branches  flowers    per branch                                 P = 0.05______________________________________Control 7         166        98.45 ± 2.83                                 aB-I     8         188        88.91 ± 12.36                                 abB-M     9         186        84.08 ± 7.54                                 bR1-I    8         214        78.95 ± 15.32                                 bcR1-M    8         202        61.51 ± 20.97                                 dR100-I  7         160        66.85 ± 15.42                                 cdR100-M  7         217        75.32 ± 9.91                                 bcd______________________________________ 
    
     In nectarine cv. Fantasia in Rosh-Zurim the effect of the pH of the solvent and the effect of Triton x-100 as a surfactant were examined, All the branches were sprayed repeatedly, starting from 10% blooming. The treatment were designed as follows: 
     Untreated flowers as control. 
     Flowers that were sprayed by 100 units/ml of RNase that was dissolved in water or 10 mM citrate buffer pH 3.5 (RW and RB, respectively), without or with 0.025% Triton X-100 (RWT and RBT, respectively). Table 3 shows that RNase significantly reduced fruit set in nectarine trees. The use of a buffer in addition of Triton X-100 did not significantly alter the results. 
     In plum cv. Vikson branches were sprayed with 1, 10 or 100 units/ml of RNase, dissolved in water (R1, R10 and R100, respectively, supplemented with 0.025% Triton X-100 and applied once at 50% blooming (I) or three times, starting from 10% blooming (M), Table 4 shows that the combination of high concentration of RNase and repeated applications during anthesis were most effective in reducing fruit-set. 
     
                       TABLE 3______________________________________FRUIT SET OF NECTARINE CV. FANTASIA IN ROSHZURIM             Total      Percentage   Number of number of  of fruitsTreatment   branches  flowers    per branch                                 P = 0.05______________________________________Control 5         100        63.60 ± 13.64                                 aRW      5         81         41.80 ± 8.70                                 bRWT     5         89         43.75 ± 7.74                                 bRB      5         97         45.0 ± 9.56                                 bRBT     5         85          41.0 ± 14.16                                 b______________________________________ R = Spraying with RNase (extract from Aspergillus niger) W = Water as Solvent B = Buffer as a solvent T = Triton × 100 
    
     
                       TABLE 4______________________________________FRUIT SET OF PLUM CV VIKSON IN HELETZ.sup.a    Concentration               Percent of Fruit Set In          of RNase     Experiment                               ExperimentNo.  Treatment Per ml       A.sup.b B.sup.c______________________________________1    Control                6.2  ab   13.0  a2    RNase      1           9.0  a    9.7   ab3    RNase      10          5.7  ab   7.8   ab4    RNase     100          3.4  b    5.5   b______________________________________ .sup.a = At a certainty of P = 0.05 .sup.b Experiment A = Three sprayings starting at 10% flowering .sup.c Experiment B = Single spraying at 50% flowering. 
    
     EXAMPLE 6 
     Effect on Shotberries in Grape-Vines 
     The vineyard tested did not receive any sort of spraying of gibberellin to increase the size of the grapes. The vine was sprayed by hand during flowering on 6/5/93. On 11/7/93 the bunches were weighed and the number of shot berries counted. The results are shown in Table 5. 
     EXAMPLE 7 
     RNase samples from the fermentation of Aspergillus niger niger were loaded on 15% polyacrylamide gel electrophoresis (SDS PAGE). The proteins were resolved and after the run the proteins were renatured in situ by removing the SDS using an isopropanol buffer. After the run the gel was divided into two lanes. One lane was subjected to RNase zymogram detection (overlayed on 0.1% yeast RNAS and 1.5% agarose for several hours) and then stained with 0.1% toluidine blue. RNase bands were visualized as evident by a white halo around an active band at approximately 30 KDa. The corresponding band from the parallel lane was dissected and inserted into peach pollen germinating tubes. The percent of germination and pollen tube growth were recorded after 24 hours. The results (pollen tube length) are shown in Table 6. 
     
                       TABLE 5______________________________________THE EFFECT OF RNASE ON SHOTBERRIES IN GRAPES.sup.a          Con.     No. Shotberries                             No. ShotberriesNo.  Treatment RNase    Per Bunch Per 100 g Grapes______________________________________1    control   --       77.1 a    45.1 a2    RNase     1 u/ml   84.5 a    46.9 a3    RNase     10 u/ml  45.5 b      33.8 ab4    RNase     100 u/ml 38.9 b    18.6 b______________________________________ .sup.a = At a certainty of p = 0.05 
    
     
                       TABLE 6______________________________________POLLEN TUBE LENGTH OF RNASE SAMPLESISOLATED FROM THE FERMENTATION OF A. NIGERUntreated     Gel Without Proteins                        Bands at 30 Da______________________________________Average   0.48 a    0.664 b        0.33 cStandard   0.19      0.38           0.24STE.sup.a   0.03      0.06           0.03______________________________________ .sup.a = At p = 0.05 
    
     EXAMPLE 8 
     The effect on the germination and the pollen tube length of peach pollen with boiled RNase (originating from Aspergillus niger) was studied. The results shown in Tables 7 and 8 show a marked effect even after boiling the RNase. 
     
                       TABLE 7______________________________________PEACH POLLEN GERMINATION IN BOILED RNASE.sup.aPercent Germination          Rnase Concentration (unit/ml______________________________________45.0 a         0  40.5 ab      0.0930.4 b         0.90              9______________________________________ .sup.a = From Aspergillus niger 
    
     
                       TABLE 8______________________________________PEACH POLLEN TUBE LENGTH IN BOILED RNASE.sup.aPollen Tube Length          Rnase Concentrations(mm)           (units/ml)______________________________________0.55 a         00.48 b         0.090.30 c         0.9______________________________________ .sup.a = from Aspergillus niger.