USE OF (Z)-3-HEXENYL ESTERS AND METHOD FOR PROTECTING PLANTS AGAINST PESTS

Abstract

The present invention relates to the use of esters acetate, propionate and (Z)-3-hexenyl butyrate, or of a composition comprising these compounds, to protect plants, in particular agricultural crops, against pests, by stimulating the defense mechanisms of the plants themselves. The invention also relates to a method for protecting plants against pests by bringing said esters into contact with the plants.

Claims

1-7. (canceled)

8. A method for protecting plants against pests by stimulating the natural defense mechanisms of said plants, characterized in that it comprises contacting at least one compound of formula I, or of a composition comprising the same, with the plants: ##STR00002## where R=—CH.sub.3, —CH.sub.2—CH.sub.3 or —CH.sub.2—CH.sub.2—CH.sub.3.

9. The method according to claim 8, characterized in that the compound of formula I is (Z)-3-hexenyl propanoate.

10. The method according to claim 8, characterized in that the compound of formula I, or of a composition comprising same, is contacted with the plant by dispensers.

11. The method according to claim 10, characterized in that the dispenser is a passive emitter consisting of a container of the compound of formula I configured to allow the diffusion of said compound into the environment surrounding the plant.

12. The method according to claim 10, characterized in that the dispenser consists of a nebulizer that produces an aerosol.

13. The method according to claim 10, characterized in that the dispenser is a liquid or gel composition that contains the compound of formula I and is formulated to allow diffusion of said compound into the environment.

14. The method according to claim 10, characterized in that the dose of compound applied to the environment surrounding the plants by the dispenser is between 25 mg/ha/day and 25 g/ha/day.

15. The method according to claim 8, characterized in that the natural defense mechanisms stimulated in plants cause the repellency of pests selected from the list comprising: whiteflies Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood), Aleurothrixus floccosus (Maskell), Dialeurodes citri (Ashmead) and Paraleyrodes minei laccarino (Hemiptera: Aleyrodidae), the thrips Frankliniella occidentalis Pergande, Pezothrips kellyanus Bagnall, Chaetanaphothrips orchidii (Moulton) and Thrips tabaci Lindeman (Thysanoptera: Thripidae), red spiders Tetranychus urticae, Eutetranychus orientalis (Klein), Eutetranychus banksi (McGregor), T. urticae, Panonychus citri (McGregor) Koch and Tetranychus evansi Baker & Pritchard (Acari: Tetranychidae), lepidoptera Tuta absoluta (Meyrick), Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), Spodoptera exigua Hübner and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae); and psyllids Diaphorina citri Kuwayama (Hemiptera: Liviidae) and Trioza erytreae (Del Guercio) (Hemiptera: Psyllidae).

16. The method according to claim 8, characterized in that the natural defense mechanisms stimulated in plants cause the attraction of parasitoids selected from the list comprising species of the genera: Encarsia spp., Aphytis spp. Cales spp., Eretmocerus spp., Aphelinus spp., (Hymenoptera: Aphelinidae), Aphidius spp. Lysiphlebus spp. (Hymenoptera: Braconidae) Metaphycus spp., Anagyrus spp. (Hymenoptera: Encyrtidae), Tamarixia spp. Citrostichus spp., Cirrospilus spp., Diglyphus spp. (Hymenoptera: Eulophidae), Trissolcus spp., Telenomus spp. (Hymenoptera: Scelionidae) and Trichogramma spp. (Hymenoptera. Trichogrammatidae).

17. The method according to claim 8, characterized in that the plant is a horticultural plant or a citrus fruit.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0059] FIG. 1.: Transcriptional response of defense genes PR1 (a marker gene for the salicylic acid signaling pathway, SA) (FIG. 1A) and SI-PI-I and SI-PI-II (two markers for plant proteinase inhibitors) (FIGS. 1B and 1C, respectively) in tomato plants exposed to (Z)-3-hexenyl acetate (HA), (Z)-3-hexenyl butanoate (HB) and (Z)-3-hexenyl propanoate (HP) and control. Data are presented as the mean of eight independent analyses of transcript expressions relative to a housekeeping gene±standard error (n=8). Significant differences based on the ANOVA test are marked with different letters (P<0.05). Response (%±SE (Standard Error)) of female Encarsia formosa (E.f.), Tuta absoluta (T.a.), Tetranychus urticae (T.u.), Bemisia tabaci (B.t.) and Frankliniella occidentalis (F.o.) in a Y-tube olfactometer when exposed to the control (1:10,000 methanol:water) and to (Z)-3-hexenyl propanoate ((Z)-3-HP) (1:10,000 with the volatile to test). Asterisks indicate significant differences in the distribution of side arm choices (X2 tests; P<0.05) (FIG. 1D). Number (mean±standard error) of Tetranychus urticae females per tomato plant when comparing mite development in tomato plants exposed to (Z)-3-hexenyl propanoate [(Z)-3-HP)] compared to unexposed tomato plants (Control). Exposure to (Z)-3-HP was tested in two types of bioassays, (24 h) and (Per): in the first type, the plants were only exposed for 24 hours to the volatile before the release of T. urticae (24 h); In the other type of test, the plants were permanently exposed to the volatile throughout the experiment (Per). Bars with different letters are significantly different (GLMM, repeated measures α<0.05) (FIG. 1E). Number of eggs (mean±standard error) laid by 2 Tuta absoluta females for 72 hours in plants exposed to (Z)-3-hexenyl propanoate [(Z)-3-HP)] compared to unexposed tomato plants (Control). Bars with different letters are significantly different (ANOVA, Tukey α<0.05) (FIG. 1F). Mortality percentage (mean±standard error) of T. absoluta from egg to adult when developed in tomato plants exposed to (Z)-3-hexenyl propanoate [(Z)-3-HP)] compared to untreated tomato plants (Control). Bars with different letters are significantly different (ANOVA, Tukey α<0.05) (FIG. 1G).

[0060] FIG. 2. Transcriptional response of the PIN2 gene (a marker gene for the jasmonic acid (JA) signaling pathway in tomato plants exposed under semi-field conditions to (Z)-3-hexenyl propanoate [(Z)-3-HP] with different volatile emission levels: 5 mg/day/dispenser in the low-density (LD) polyethylene emitter and 0.01 mg/day/dispenser in the high-density (HD) polyethylene emitter. The control treatment consists of plants not exposed to the volatile. Significant differences based on the ANOVA test are marked with different letters (P<0.05).

[0061] FIG. 3: Transcriptional response of defense genes PR1 (a marker gene for the SA signaling pathway) (FIG. 3A) and PIN2 (a marker gene for the jasmonic acid signaling pathway) (FIG. 3B) in tomato plants exposed under field conditions to (Z)-3-hexenyl propanoate [(Z)-3-HP] released in a 4-mL low-density (LD) polyethylene polymeric dispenser vial (Kartell) a day before, 4 and 8 weeks after the placement of the dispenser. Significant differences between treatments for each date are marked with an asterisk (t-test; P<0.05). Number of Nesidiocoris tenuis per plant (mean±standard error) (FIG. 3C) and number of infested leaflets per plant (mean±standard error) (FIG. 3D) in the commercial greenhouse (GLMM, P<0.05). Release profile of the dispenser used fitted from the linear regression model (FIG. 3E).

[0062] FIG. 4: Transcriptional response of defense genes LOX2 (precursor of JA jasmonic acid) (FIG. 4A) and PAL and NPR1, two genes related to the salicylic acid pathway (FIGS. 4B and C, respectively) in Carrizo citrange plants exposed to (Z)-3-hexenyl propanoate and control. Data are presented as the mean of six independent analyses of transcript expressions relative to a housekeeping gene±standard error (n=6). Significant differences based on the t-test are marked with (*) (P<0.05). Response (%±SE) of Diaphorina citri y Tamarixia radiata females in a Y-tube olfactometer when exposed to control (1:10,000 methanol:water) and to (Z)-3-hexenyl propanoate ((Z)-3-HP) (1:10,000 with the volatile to test). Asterisks indicate significant differences in the distribution of side arm choices (X2 tests; P<0.05) (FIG. 4D).

[0063] FIG. 5: Number of receptive shoots (mean±SE) and laying (mean±SE) of Diaphorina citri in the seedlings of Murraya paniculata used as sentinel plants with and without a dispenser loaded with HP. Different letters on the bars show significant differences (t-test P<0.05).

EXAMPLES

[0064] Next, the invention will be illustrated using assays carried out by the inventors.

Example 1: Defensive Activation Assays in Tomato Via Exposure Under Laboratory Conditions

[0065] Methodology

[0066] To demonstrate that the exposure of volatiles (Z)-3-hexenyl propanoate, (Z)-3-hexenyl acetate, and (Z)-3-hexenyl butanoate activates plant defenses, Solanum lycopersicum cv. Moneymaker tomato plants were used. Two weeks after seed germination, the seedlings were individually transplanted into pots (8×8×8 cm). The plants were kept at 25±2° C., with a constant relative humidity of 65%±5% and a 14:10 h (light:dark) photoperiod. Tomato plants that had not received any pesticide treatment at four weeks of age (about 20 cm tall) were used. All volatiles used were obtained from Sigma-Aldrich (St. Louis, Mo., USA). Each volatile was exposed by using a 2×2 cm piece of filter paper on which 10 μl of a solution containing the corresponding volatile at a concentration of 1:10,000 [(Z)-3-hexenyl ester:methanol] was impregnated as an emitting source of the corresponding volatile (Pérez-Hedo, M. et al. Biocontrol, 63: 203-213 (2018)).

[0067] To activate a plant, two pieces of filter paper impregnated with the volatile were placed at the bottom of a 30×30×30 cm plastic cage (BugDorm-1 Insect Tents; MegaView Science Co., Ltd, Taichung, Taiwan) into which the plant was introduced. A control treatment was carried out in which the two pieces of filter paper were impregnated with 10 μl of methanol. Eight repetitions per treatment were performed. The plants of the treatments were kept exposed to the volatile without disturbing for 24 hours in separate and isolated climatic chambers to avoid any interference from the volatiles and were kept at 25±2° C., 65±10% RH, and a 14:10 h (L:D) photoperiod. After 24 hours, the expression of 3 genes related to plant defensive activation was studied in both treatments: the precursor of the protein related to basic pathogenesis (PR-1), a marker gene for the SA signaling pathway, and two markers for plant proteinase inhibitors (SI-PI-I and SI-PI-II) were studied.

[0068] The methodology followed for extracting and quantifying gene expression was described by Pérez-Hedo et al. Journal of Pest Science 3: 543-554 (2015). The primers used were forward primer: 5′-CTCATATGAGACGTCGAGAAG-3′ (SEQ ID NO: 1) and reverse primer: 5′-GGAAACAAGAAGATGCAGTACTTAA-3′ (SEQ ID NO: 2) for the quantification of PR1, forward primer: 5′-TGAAACTCTCATGGCACGAA-3′ (SEQ ID NO: 3) and reverse primer: 5′-TTTTGACATATTGTGGCTGCTT-3′ (SEQ ID NO: 4) for SI-PI-I, and forward primer: 5′-GGCCAAATGCTTGCACCTTT-3′ (SEQ ID NO: 5) and reverse primer: 5′-CAACACGTGGTACATCCGGT-3′ (SEQ ID NO: 6) for the SI-PI-II gene. The nucleotides of the constitutive gene EF1 were forward primer: 5′-GATTGGTGGTATTGGAACTGTC-3′ (SEQ ID NO: 7) and reverse primer: 50-AGCTTCGTGGTGCATCTC-30 (SEQ ID NO: 8).

[0069] After verifying that exposure to the three volatiles activated genes related to plant defenses, (Z)-3-hexenyl propanoate was selected from among the three volatiles as a model for the (Z)-3-hexenyl esters.

[0070] It was studied whether the defensive activation achieved by exposure to (Z)-3-hexenyl propanoate could affect repellency and/or attraction in herbivores that attack tomatoes, the whitefly Bemisia tobaci (Gennadius) (Hemiptera: Aleyrodidae), the thrip Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), the red spider Tetranychus urticae Koch (Acari: Tetranychidae) and the tomato borer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), and in a natural enemy that has been used as a model organism in this crop, the parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae).

[0071] The last instar nymphs of B. tabaci and the pupae of E. formosa were provided by Koppert Biological Systems, S. L. (Águilas, Murcia, Spain). The recently emerged B. tabaci adults (less than 2 days old) were released into tomato plants inside 60×60×60 cm plastic cages (BugDorm-2) and placed in a climatic chamber at 25±2° C., 65±10% RH and a 14:10 h (L:D) photoperiod in IVIA. Five-day-old B. tabaci adults were used in all the experiments. In the case of E. formosa, the pupae were placed in a Petri dish 9 cm in diameter and allowed to emerge under ambient laboratory conditions (25±2° C.), with a small drop of honey as food. E. formosa females less than two days old were used in all the experiments. F. occidentalis adults came from a brood kept in captivity established in the Valencian Institute of Agrarian Research (Instituto Valenciano de Investigaciones Agrarias, IVIA) in 2010, originally from Campo de Cartagena (Murcia, Spain). The thrips brood was kept in bean pods (Phaseolus vulgaris L.; Fabales: Fabaceae) under the same conditions described above. All the F. occidentalis females used for experimentation were less than five days old. T. urticae adults were obtained from a colony established in IVIA in 2011, which initially came from clementines located in La Plana (Castelló, Spain). The mites were kept on tomato plants in a climatic chamber under the same conditions described above. T. absoluta females were obtained from colonies on tomatoes maintained in a greenhouse at IVIA at 25±4° C., 60±15% RH, and under a natural photoperiod. Newly emerged adult females less than 5 days old were used in all the assays.

[0072] To assess the preference of F. occidentalis, B. tabaci, T. urticae, T. absoluta and E. formosa for tomato plants that were previously exposed for 24 h to (Z)-3-HP relative to intact plants, a Y-shaped olfactometer was used. The plants were exposed to both volatiles as described above using the pieces of filter paper. The olfactometer (Analytical Research Systems, Gainesville, Fla.) consisted of a 2.4 cm in diameter Y-shaped glass tube with a 13.5 cm long base and two arms, each 5.75 cm long. Both side arms were connected via high-density polyethylene (HDPE) tubing to two identical glass jars (5-liter volume), each of which was connected to an air pump that produced a unidirectional humidified airflow at 150 ml/min (Pérez-Hedo et al. Journal of Pest Science 3: 543-554 (2015)).

[0073] For each species, a female was individually introduced into the inlet tube of the olfactometer and observed until she had walked at least 3 cm down one of the arms of choice or until 15 minutes had elapsed. A total of 40 valid replicates were recorded for each species for each pair of odor sources. Each individual was tested only once. Females that did not choose a side arm within 15 minutes were recorded as “no response” and excluded from data analysis. After recording five responses, the Y-tube was rinsed with soapy water, cleaned with acetone, and allowed to dry for 5 minutes. The odor sources were later switched between the left and right side arms to minimize any spatial effect on choice. The two types of plants (intact and induced) were used only once to assess the response of 10 females and were then replaced with new plants. The Y-tube experiment was performed under the following environmental conditions: 23±2° C. and 60±10% RH.

[0074] It was also studied whether the defensive activation induced by exposure to (Z)-3-hexenyl propanoate could influence the development of two highly important phytophages in tomato cultivation, the red spider, T. urticae, and the lepidoptera T. absoluta. The individuals used in these experiments came from the captive breeding of these species that are kept in the IVIA described above. The exposure time of the plant to the volatile was tested on both phytophages in two different ways, one where the plant was only exposed to the volatile for 24 hours and then the herbivore to be assayed was immediately released, and the other where the volatile was permanently exposed throughout the experiment.

[0075] For this purpose, two climatic chambers were used under the same environmental conditions [25±2° C., 65±5% HR and 14:10 h (L:D) photoperiod], wherein, to avoid interference between the volatiles, one was assigned to the (Z)-3-hexenyl propanoate treatments and one to the control treatment. In the climatic chamber in which the exposure to (Z)-3-hexenyl propanoate was assayed, there were placed 12 plastic cages (60 cm×60 cm×60 cm) (BugDorm-2. MegaView Science Co., Ltd., Taichung, Taiwan), six per treatment tested (24 h of exposure and permanent exposure), while six cages were placed in the climatic chamber where the control treatment was tested. The cages were equally distributed at a distance of one and a half meters between one another. Each cage represented a replica. In each cage, two pieces of impregnated filter paper were placed at the bottom as explained above, both for the treatment with (Z)-3-hexenyl propanoate and for the control. In the treatment where the volatiles were exposed throughout the experiment, the pieces of impregnated filter paper were replaced every two days.

[0076] Eight tomato plants (Lycopersicon esculentum cv. Moneymaker) were introduced in each cage. In the experiment with T. urticae, to prevent the movement of mites from one plant to another, the plants were isolated individually without touching one another or the walls of the cage. Furthermore, the plants were placed on a small brick inside a plastic tray filled with water, and all the pots were painted with a glue band. The plants were artificially infested with T. urticae from the laboratory population mentioned above. Twenty T. urticae females were released per plant, evenly distributed on all the leaves with the help of a fine brush. Naked eye sampling was carried out seven, 14, and 21 days after the release of T. urticae, where the total number of T. urticae females in each plant was counted.

[0077] Two consecutive experiments were performed to assess the effect of exposure to both volatiles on T. absoluta. The first experiment studied the effect on the laying of T absoluta. The selected eggs laid in the first experiment were later used in the second experiment to study the mortality of immature T. absoluta specimens developed in plants exposed to (Z)-3-hexenyl propanoate. The same three treatments described above for T. urticae were also assayed for T. absoluta. The laying of T. absoluta was assessed in 8 tomato plants (cv. Moneymaker) per treatment. Each of the plants was isolated inside a plastic cage (60×60×60 cm) (BugDorm-2) and kept in a climatic chamber at 25±2° C., 65±5% HR, 14:10 (L:D) h photoperiod following the same assay distribution described for T. urticae. Inside each cage (replica), 2 adult pairs were released from T. absoluta (males and females) and left undisturbed for 72 hours. After this time, T. absoluta adults were removed, and the number of eggs was counted.

[0078] To study the mortality of T. absoluta in the plants exposed to the 3 treatments described above, 6 T. absoluta eggs per plant were equally distributed on all the leaves with the help of a fine brush. The eggs used in each treatment came from the corresponding treatment of the first experiment. Mortality of T. absoluta was assessed in 8 tomato plants (cv. Moneymaker) per treatment. Each of the plants was isolated inside a plastic cage (60×60×60 cm) (BugDorm-2 insect tents) and kept in a climatic chamber at 25±2° C., 65±5% HR, 14:10 (L:D) h photoperiod following the same treatment distribution described above. The plants were left intact until T. absoluta adults emerged. As soon as the adults began to emerge, they were counted and removed daily from the cages.

[0079] Result

[0080] Exposure to the three volatiles (Z)-3-hexenyl propanoate, (Z)-3-hexenyl acetate, and (Z)-3-hexenyl butanoate of intact tomato plants significantly overexpressed the three markers studied compared to the control, finding no differences between them (FIG. 1 A,B,C). PR-1: F.sub.1-19=14.09; P<0.0001 SPI-1: F.sub.1-19=21.91; P<0.0001 and SPI-2: F.sub.1-19=16.28; P<0.0001.

[0081] The activated plants exposed 24 hours to (Z)-3-hexenyl propanoate turned out to be repellent against highly important pests in tomato, such as the lepidoptera T. absoluta (.sup.2=9.80; P=0.0017), the white fly B. tabaci (.sup.2=12.80; P=0.0003) and the thrips F. occidentalis (.sup.2=5.00; P=0.0253) (FIG. 1D). Furthermore, plants exposed to this volatile were more attractive to the parasitoid E. formosa (.sup.2=5.00; P=0.0253). The red spider T. urticae was the only species that showed no preference (.sup.2=1.80; P=0.1797) (FIG. 1D).

[0082] In the case of the red spider, 21 days after release, a reduction in the number of mites per plant of 50.3±6.3% and 83.9±5.0% with respect to the control was obtained in the treatments where the plant was kept exposed for 24 h and throughout the entire experiment to (Z)-3-hexenyl propanoate, respectively (F.sub.4.85=4.437; P=0.003) (FIG. 1E). In the case of T. absoluta, in the treatment of permanent exposure to (Z)-3-HP, a reduction in laying of 67.2±15.0% was obtained when compared to the control (F.sub.2.23=3.746; P=0.0406) (FIG. 1F). Furthermore, mortality from T. absoluta egg to adult in plants permanently exposed to (Z)-3-HP was around 80%, which was significantly higher than the control treatment (F.sub.2.23=6,944; P=0.0048) (FIG. 1G), the laying (F.sub.2.23=1.147; P=0.3367) or in mortality (F.sub.2.23=0.5881; P=0.5643) of T. absoluta.

Example 2: Semi-Field Release Assays with Different Application Doses of (Z)-3-Hexenyl Propionate from Polymer-Type Passive Dispensers

[0083] Methodology

[0084] Two different types of polymeric emitters were used to test the activation level that different emission levels of (Z)-3-hexenyl propionate can provide: 4-mL low-density (LD) polyethylene vial and 4-mL high-density (HD) polyethylene vial. The highest emission level (5 mg/day) was achieved through previous laboratory studies with LD dispensers loaded with 20 mg of the pure substance. The lowest emission level (0.01 mg/day) was provided by HD vials loaded with a 1:100 mixture of (Z)-3-hexenyl propionate and paraffinic oil (0.02:2; g:g). The emission kinetics of the emitters used during the experiments was controlled by studying weight loss (gravimetric method). Additional dispensers were placed in the same environmental conditions, measuring their weight weekly with a precision scale (0.0001 g). The difference in weight recorded in each period will be the emitted quantity of the substance for each type of dispenser. To obtain the mean emission level of each dispenser, the recorded weights (y) are related to the aging time (x) using a multiple regression analysis.

[0085] Once the mentioned emission levels have been established, 5 and 0.01 mg/day/dispenser, tomato plants were exposed to the different emission levels through an experiment under semi-field conditions in a glass greenhouse. For that purpose, there were three 24 m.sup.2 cabins for the different treatments: (CONTROL) without volatile emitters, (HD) with 1 0.01 mg/day emitter, and (LD) with 1 5 mg/day emitter. The relative humidity was 65%±10%, and the photoperiod was natural (approximately 14:10 h L:D) in all three cubicles. Environmental conditions were monitored and recorded using a Mithra clima data logger (ver. 1.01.03, Priva nutricontrol Ibérica S.L). In each of the three cubicles, 30 tomato plants were transplanted into individual 20-liter polyethylene pots filled with a mixture of sand and peat (1:2 w:w, respectively). The pots were distributed in each cabin in four rows of five plants each (2 plants/m.sup.2). The typical cultivation techniques of greenhouse tomato cultivation in Spain were followed: staking to a guide for each plant, weekly pruning of secondary shoots, application of a standard nutrient solution for tomato using an automated irrigation system with an irrigation frequency adjusted to environmental conditions and an irrigation time of 15 min. The dispensers were placed 50 cm above the apex of the tomato plants and adapted in height according to the growth of the plants.

[0086] The dispensers were kept inside the corresponding cabins throughout the assay and samples were taken from the plants of each cabin 4 weeks after the implementation of treatment. From these samples, the transcriptional response of the defensive gene PIN2 (a marker gene for the jasmonic acid, JA, signaling pathway). Five repetitions per treatment were performed. The methodology followed for the extraction and quantification of gene expression was described by Pérez-Hedo et al. Journal of Pest Science 3: 543-554 (2015). The sequences of the primer used for the quantification of the PIN2 gene were forward primer: 5′-GAAAATCGTTAATTTATCCCAC-3′ (SEQ ID NO: 9) and reverse primer: 5′-ACATACAAACTTTCCATCTTTA-3′ (SEQ ID NO: 10), while for the constitutive gene EF1 the sequences were forward primer: 5′-GATTGGTGGTATTGGAACTGTC-3′ (SEQ ID NO: 7) and reverse primer: 50-AGCTTCGTGGTGCATCTC-30 (SEQ ID NO: 8).

[0087] Results

[0088] Expression studies showed that the lowest level of (Z)-3-hexenyl propionate emission (0.01 mg/day) did not affect exposed plants. However, a 5 mg/day emission significantly activated the jasmonic acid metabolic pathway (FIG. 2).

Example 3. Defensive Activation Assays in Tomato Via Exposure Under Field Conditions

[0089] Methodology

[0090] To know the behavior of the dispensers loaded with (Z)-3-hexenyl propionate under real cultivation conditions, four commercial greenhouses for tomato cv Raf located in Xilxes (Castellón province), with a history of problems with T. absoluta contamination at the end of winter, were selected. An experimental design of randomized blocks was carried out where each greenhouse was considered a block with two treatments per block and 4 repetitions per treatment. In these tomato greenhouses, the zoophytophagous predator Nesidiocoris tenuis Reuter (Hemiptera: Miridae) has been used, being released from the seedbed at the end of August and keeping phytophagous pest populations under control after being transplanted in the greenhouse at the end of August until the arrival of winter. However, the populations of this predator decline sharply under the environmental conditions that occur in winter. At the end of winter, their populations begin to recover slowly so that populations of T. absoluta recover more quickly. It is common to resort to selective insecticides during the spring to keep T. absoluta at bay. Therefore, these greenhouses were considered an ideal setting in which to confirm the effect on T. absoluta found under laboratory conditions.

[0091] Cultivation began on 4 Sep. 2018 with the transplant with a 0.4×1 m planting frame, which resulted in 1.2 plants per square meter (25,450 plants in 21,200 m.sup.2). Common cultivation techniques in the area were followed: the main stem was brought to two arms, secondary shoots and senescent leaves were pruned weekly, and a standard tomato nutrient solution was applied weekly through an automated drip irrigation system. The transplanted plants were inoculated in the seedbed with the predator N. tenuis. A dose of 1 N. tenuis per plant in the nursery was released, and E. kuehniella eggs were used as alternative prey (Urbaneja-Bernat et al. Journal of Applied Entomology 139: 61-167 (2015)). From the sowing date to the day the dispensers were hung, none of the greenhouses received any chemical treatment, and pest control resided only in the predator N. tenuis.

[0092] As mentioned earlier, 2 zones were selected in each of the greenhouses in which, on 22 Feb. 2019, a dispenser (4-ml low-density (LD) polyethylene vial) loaded with 4 ml of (Z)-3-hexenyl was hung every 20 m.sup.2 in one of them, and the other with the same surface area was used as a control treatment. In the four zones with volatile, a total of 260 dispensers were placed in a total surface area of 5,200 m.sup.2 (equivalent to a density of 500 dispensers/ha). As described in Example 2, the dispensers' emission kinetics was studied using the gravimetric method.

[0093] Twenty plants randomly chosen by repetition were sampled weekly for 11 weeks, beginning 30 Jan. 2019. First, the number of leaflets infested by T. absoluta per plant was counted. Then the number of N. tenuis (adults and nymphs) throughout the apical third of each plant (leaves, flowers and shoots) was counted. Following the methodology described above, 6 samples were taken from the apical part of each plant per repetition, and these samples were immediately introduced into liquid nitrogen to quantify the expression of the PR1 and PIN2 genes (described in Example 1 and Example 2). The expression of these genes was quantified one day before, 4, and 8 weeks after hanging the dispensers in the greenhouses.

[0094] Result

[0095] At the start of the assay, the expression of both marker genes for defense pathways was the same, but at 30 and 60 days, the expression of both genes was significantly higher in the plants exposed to the volatile compared to the plants in the control zone (FIG. 3 A, B) (Table 1).

TABLE-US-00001 TABLE 1 Probability values for pairwise comparison of the transcriptional response of the defense genes PR1 (a marker gene for the SA signaling pathway) and PIN2 (a marker gene for the JA signaling pathway) in tomato plants exposed to (Z)-3-hexenyl propanoate [(Z)-3-HP] released in a 4-mL low-density (LD) polyethylene polymeric dispenser vial (Kartell) and in control tomato plants 24 hours before and 4 and 8 weeks after the establishment of dispensers in tomato greenhouses. Bold values correspond to statistically significant values. t-Test (P < 0.05). PR1 PIN2 Control vs(Z)-3-HP t.sub.14 P t.sub.14 P 1 day before 0.2464 0.8089 0.4412 0.6658 4 weeks 2.379 0.0321 3.037 0.0089 8 weeks 3.490 0.0040 2.263 0.0400

[0096] The population of the plant bug N. tenuis remained the same in both treatments (F.sub.1.86=2.112; P=0.150) (FIG. 3C), but not so the infestation of T. absoluta, which was significantly lower (about 58%) in the treatment with volatile (F.sub.1.86=11.375; P<0.0001) (FIG. 3D). The volatile (Z)-3-hexenyl propanoate was emitted in a substantially consistent manner over the study period at a mean rate of 12.2 mg/day, according to the slope of the fitted model (FIG. 3E), which represents an approximate application dose of 6 g/ha/day.

Example 4. Defensive Activation Assays in Citrus Fruits Via Exposure Under Laboratory Conditions

[0097] To demonstrate that exposure to the volatile (Z)-3-hexenyl propanoate activates defenses in citrus plants, seedlings of the Carrizo citrange rootstock (Citrus sinensis Osb.×Poncirus trifoliata L. Raf.). Two weeks after seed germination, the seedlings were individually transplanted into pots (8×8×8 cm). The plants were kept in a greenhouse with a plastic cover at approximate climatic conditions of 25±5° C., a relative humidity of 65%±5%, and a natural light photoperiod, about 14:10 h (light:dark). Carrizo citrange plants that had not received any pesticide treatment at 8 weeks of age (approximately 20 cm tall) were used. The volatile (Z)-3-hexenyl propanoate was obtained from Sigma-Aldrich (St. Louis, Mo., USA). The volatile was exposed by using a 2×2 cm piece of filter paper on which 10 μl of a solution containing the corresponding volatile at a concentration of 1:10,000 [(Z)-3-hexenyl propanoate:methanol] was impregnated as an emitting source of the corresponding volatile (Pérez-Hedo, M. et al. Biocontrol, 63: 203-213 (2018)).

[0098] To activate a plant, two pieces of filter paper impregnated with the volatile were placed at the bottom of a 30×30×30 cm plastic cage (BugDorm-1 Insect Tents; MegaView Science Co., Ltd, Taichung, Taiwan) into which the plant was introduced. A control treatment was carried out in which the two pieces of filter paper were impregnated with 10,000 μl of methanol. Six repetitions per treatment were performed. The plants of both treatments were exposed to the volatile without disturbing for 24 hours in separate and isolated climatic chambers to avoid any interference from the volatile and were kept at 25±2° C., 65±10% RH, and a 14:10 h (L:D) photoperiod. After 24 hours, the expression of 3 genes related to plant defensive activation was studied in both treatments: a precursor of JA jasmonic acid (LOX2) and two related to the downstream salicylic acid pathway (PAL) and a precursor (NPR1) were studied.

[0099] The methodology followed for the extraction and quantification of gene expression was described by Pérez-Hedo et al. Journal of Pest Science 3: 543-554 (2015). The primers used were forward primer: 5′-GAACCATATTGCCACTTTCG-3′ (SEQ ID NO: 11) and reverse primer: 5′-CGTCATCAATGACTTGACCA-3′ (SEQ ID NO: 12) for the quantification of LOX2, forward primer: 5′-CACATTCTTGGTAGCGCTTTG-3′ (SEQ ID NO: 13) and reverse primer: 5′-AGCTACTTGGCTGACAGTATTC-3′ (SEQ ID NO: 14) for PAL and forward primer: 5′-TACCTCCACCTCTCTCATTCTT-3′ (SEQ ID NO: 15) and reverse primer: 5′-GTGCGAGAGAAGGTTAGCTATG-3′ (SEQ ID NO: 16) for the NPR5 gene. The nucleotides of the constitutive gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were forward primer: 5′-GGAAGGTCAAGATCGGAATCAA-3′ (SEQ ID NO: 17) and reverse primer: 5′-CGTCCCTCTGCAAGATGACTCT-3′ (SEQ ID NO: 18).

[0100] To assess the preference of D. citri females and the parasitoid T. radiata to Carrizo citrange plants that were previously exposed for 24 h to (Z)-3-HP in relation to intact plants, a Y-shaped olfactometer was used in the same way as that described in Example 1, and the plants were exposed to both volatiles as described above with the use of pieces of filter paper. A total of 40 valid replicates were recorded for each species for each pair of odor sources. Each individual was tested only once. Females that did not choose a side arm within 15 minutes were recorded as “no response” and excluded from data analysis. After recording five responses, the Y-tube was rinsed with soapy water, cleaned with acetone, and allowed to dry for 5 minutes. The odor sources were later switched between the left and right side arms to minimize any spatial effect on choice. The two types of plants (intact and induced) were used only once to assess the response of 10 females and were then replaced with new plants. The Y-tube experiment was performed under the following environmental conditions: 25±1° C. and 60±10% RH.

[0101] The D. citri adults were obtained from a colony established at SWFREC (University of Florida), initially originating from citrus fruits located in their experimental plots. The parasitoids were collected in these experimental fields. Newly emerged adult females less than 5 days old were used in all the assays.

[0102] Result

[0103] Exposure to intact Carrizo citrange plants' volatile (Z)-3-hexenyl propanoate significantly overexpressed the three markers studied (FIGS. 4A, B and C). Activated plants exposed 24 hours to (Z)-3-hexenyl propanoate turned out to be highly attractive to the parasitoid Tamarixia radiata (.sup.2=6.914; P=0.0043) and showed themselves to be indifferent to Diaphorina citri (.sup.2=1.429; P=0.1160) (FIG. 4D).

Example 5. Response to Defensive Activation in Citrus Fruits Under Field Conditions

[0104] Methodology

[0105] Efficacy of the use of dispensers loaded with HP in the field.

[0106] A Valencia orange field with a high presence of receptive shoots and a population of D. citri adults located in the experimental fields of SWFREC (University of Florida) was selected. The field had an approximate area of 0.3 ha. Sentinel plants that were hung inside the tree canopy were used. The sentinel plants were seedlings of Murraya paniculata with shoots receptive to the laying of D. citri. 7 seedlings with a dispenser (polymeric dispenser that was loaded with 1.5 ml of (Z)-3-hexenyl propanoate) and 7 seedlings without a dispenser were used. Each seedling was distributed in the field following a random block design (using a row of trees as a block), and one seedling with a dispenser and another one without was placed in each block. The seedlings were located at a minimum distance of 50 meters from each other to avoid any edge effect.

[0107] After 4 days of placing the seedlings, these were collected from the field and taken to the laboratory, where the number of eggs per shoot in each of the seedlings was counted under a binocular loupe.

[0108] Result

[0109] No differences were found between the number of receptive shoots between both treatments. As previously observed in the laboratory and in the greenhouse, the placement of the volatile dispensers managed to reduce D. citri by more than 70% (FIG. 5).

[0110] Although specific experiments carried out with tomato and citrus plants have been described, the person skilled in the art will understand that the compound described in the present invention ((Z)-3-hexenyl propanoate) will be equally useful for its use in the protection of other types of plants by stimulating or inducing defense mechanisms. Likewise, although experiments in which tomato plants have been subjected to certain pests have been described, the person skilled in the art will understand that the compound described in the present invention will be equally useful for use in the protection of plants against other pests.