Compositions and methods for preventing and treating cold-night-bright-day stress damage in tropical and subtropical trees

Abstract

The present invention is related to the induction of stomatai opening in order to bypass the impaired stomatai opening in the cold-stress mango leaves, reduce leaf cold damage, and improve the overall physiological state of the plant. The composition of the present invention is useful for preventing or treating cold-night-bright-day stress damage in tropical and subtropical trees, especially mango trees. The said composition comprising at least one stomatai opening inducer substance, and a surfactant. The invention is also related to a method of preventing cold-night-bright-day stress damage in tropical and subtropical trees, wherein said method comprises steps of: (a) foliar spray or cotton swab treatment with the compositions disclosed herein; (b) wherein the foliar spray or cotton swab is applied on the following lighted-day just before the sunrise; and (c) inducing the stomatai opening.

Description

[0001] COMPOSITIONS AND METHODS FOR PREVENTING AND TREATING COLD-NIGHT-BRIGHT-DAY STRESS DAMAGE IN TROPICAL AND SUBTROPICAL TREES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the general area to crop protection of trees vulnerable to weather. More particularly, the invention relates to compositions and methods for preventing and treating cold-night-bright-day stress damage in tropical and subtropical trees, mainly mango trees.

[0004] BACKGROUND

[0005] Mango (Mangifera indica L.) is one of the world's most important fruit trees. The crop stands at about 55.8 million tons and is second only to banana among the tropical and subtropical fruits. Israel is one of the northernmost regions of the world where mangoes are grown. Today, it is planted on over 30,000 Dunam, mainly concentrated in the Kinneret Valley. The average Mango yield in Israel is about 70,000 tons a year, with about one-third of the crop allocated for export, and is expected to rise.

[0006] Much research has been done on climate change and its effects on various crops in agriculture. Many forecasts indicate an increase in the frequency and extremes of weather changes and, as a result, exacerbation of weather damage in agriculture. Past and future data show an increase in the frequency of weather extremes and cold events in the world in general and in the Mediterranean in particular. Tropical and subtropical plants are particularly sensitive to cold (Allen et al., 2000;) with mango being the most sensitive tree among Israel's subtropical fruit trees (citrus, avocado, lychee). Most of the mango plantations in Israel are located around the Sea of Galilee, and the Valley of Springs, which are susceptible to night-chilling events.

[0007] In temperate regions, such as the Mediterranean, night-chilling events are often characterized by a temperature drop at night followed by a high-irradiance sunny day (Nir et al., 1997; Allen etal., 2000), termed ‘cold night - bright day’. This type of cold damage is expressed in the chlorosis of the leaves. Under severe cold stress, the damage can reach burning and dehydration of the leaves and branches and deciduous foliage, which requires aggressive pruning and sometimes uproot the damaged trees and re -planting.

[0008] Various studies show that in “Cold-night-bright-day-stress”, the damages in tropical and subtropical trees are mainly due to sunlight the trees are exposed to on the day after the cold night, and not directly due to the drop in night temperature. Following cold night stress, the outer parts of the tree, which are exposed to the sunlight, develop a damaged phenotype more than the inner parts of the tree, and the severity of the damage is correlated with illumination intensity (Nir et al., 1997; Feng & Cao, 2005).

[0009] A "Cold-night-bright-day-stress” is characterized by a decrease in transpiration and carbon fixation (Allen et al., 2000). This phenomenon results in the deficiency of electron-acceptors in the photosynthetic system and in radiation damage in some of the components of the light reaction (photoinhibition). Next, due to the accumulation of excess photon energy, there is increased production of active oxygen molecules (ROS) that cause damage to all cell systems which then induce chlorosis and at high levels, can cause cell death and necrosis.

[0010] It is known that cold stress impairs various mechanisms in the plant; however, many studies indicate that the decrease in photosynthesis values is directly due to the delay in stomata opening on the day after the cold night. This delay was reported in Mango (Nir et al., 1997; Allen et al., 2000) as well as in Tomato (Martin et al., 1981), Olive (Bongi & Long, 1987), Coffee (Bauer et al., 1985), Vine (Flexas et al., 1999) and Peanut (Bell et al., 1994).

[0011] Stomatai conductance regulation is crucial for plant life and growth. Each stomate includes two specialized guard cells, which increase and decrease in size to control the magnitude of the gap between them, thereby controlling the rate of transpiration and the diffusion of CO2(Matthews et al., 2017) to optimize assimilation of atmospheric CO2for photosynthesis and on the same time controlling water loss leaf temperature (via transpiration). It has been shown that the impairment in stomatai opening on the day that follows the cold-night cause a chain of events that includes a dramatic decrease in gas exchange (z.e., transpiration and photosynthetic CO2assimilation), a lack of electron acceptors, accumulation of radiation, and active oxygen molecules (ROS), which eventually results in cell damage, leaf chlorosis, and in severe cases necrosis.

[0012] Climate change and weather extremes are severe problems in agriculture in general, particularly in subtropical fruit trees, which are sensitive to cold. Cold-night- bright-day stress causes severe damage to mango plantations (and other tropical and subtropical species, and sometimes up to the need for uprooting and re -planting. Several studies have been done in Israel and worldwide to solve this agricultural problem, but effective and applied treatment has not been found yet. Furthermore, no differences in cold tolerance were reported between the various mango varieties grown in Israel so far. Presently, cold damage is handled by reducing light intensity using a grid coverage or water-sprayers, which increases the temperature around the tree. Grids and sprinklers installation costs for the entire plantation are relatively high. In addition, mango growth under nets leads to reduced yields (Adato, 2002; Sahak.Y. et al., 2009), probably due to impairments in flower differentiation. The use of sprayers during cold weather demands a highwater supply for a prolonged time and the initiation of water sprinklers even before temperature drops (to prevent water freezing in pipes). The high installation costs, the lack of efficient agricultural water infrastructure, and the availability of water in some areas are huge obstacles.

[0013] In light of the damage that "cold-night-bright-day" stress causes in the mango industry and the lack of a practical and cost-effective solution, there is a need to develop an applied treatment to reduce mango cold-night-bright-day stress damage.

[0014] SUMMARY OF THE INVENTION

[0015] According to the present invention there are provided compositions useful for preventing or treating tropical and subtropical trees from cold-night-bright-day stress damage, especially mango trees.

[0016] According to further features in preferred embodiments of the invention described below, the compositions comprise at least one stomatai opening inducer substance, and a surfactant.

[0017] According to still further features in the described preferred embodiments, the at least stomatai opening inducer substances is selected from the group consisting at least one of: phytohormone cytokinin-like plant growth regulator, a leaf hydraulics modulator, a competitive inhibitor of ethylene, a guard cell H+ pump activator, and any combination thereof.

[0018] According to still further features in the described preferred embodiments, the phytohormone cytokinin-like plant growth regulator is an abscisic acid (ABA) inhibitor cytokinin. The abscisic acid (ABA) inhibitor cytokinin is N-(2-Chloro-4-pyridyl)-N'- phenylurea (CPPU).

[0019] According to still further features in the described preferred embodiments, the CPPU is in an amount from about 40 pM to about 240 pM. More preferred, the CPPU is in an amount from about 40 pM to about 120 pM. Even more preferred, the CPPU is in an amount of 40 pM. According to still further features in the described preferred embodiments, the leaf hydraulics modulator is Potassium nitrate (KN03).

[0020] According to still further features in the described preferred embodiments, the Potassium nitrate (KN03) is in an amount of about 100 mM.

[0021] According to still further features in the described preferred embodiments, the competitive inhibitor of ethylene is 1 -Methylcyclopropene (1-MCP).

[0022] According to still further features in the described preferred embodiments, the 1- Methylcyclopropene (1-MCP) is in a concentration of 3.8% (active ingredient), sprayed on leaves until dripping.

[0023] According to still further features in the described preferred embodiments, the guard cell H+pump activator is Fusicoccin.

[0024] According to still further features in the described preferred embodiments, the Fusicoccin is in an amount of 10 pM.

[0025] According to still further features in the described preferred embodiments, the surfactant is 0.025% Triton X-100 or from about 0.02% Tween®20 to about 0.025% Tween®20.

[0026] According to another embodiment, the invention is also related to a method of preventing cold-night-bright-day stress damage in tropical and subtropical trees.

[0027] According to still further features in the described preferred embodiments, the method is comprised by the steps of (a) foliar spray or cotton swab treatment with the compositions disclosed herein; (b) wherein the foliar spray or cotton swab is applied on the following lighted-day just before the sunrise; and (c) inducing the stomatai opening.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Various embodiments are herein described, by way of example only, with reference to the accompanying drawings, wherein:

[0030] FIG. 1A-1E depict the improvement, by Fusicoccin, on gas exchange and CO2assimilation rates in mango leaves after moderate cold-night-bright-day stress;

[0031] FIG. 2A-2F depict the improvement, by Fusicoccin, on gas exchange and chlorophyll fluorescence in mango leaves after severe cold-night-bright-day stress;

[0032] FIG. 3A-3E depict foliar treatment of Fusicoccin after an extreme night-chilling event results in a greater gas exchange and CO2assimilation rates and rescue from an impairment in mango leaf photosynthetic performances. FIG. 4 depicts foliar treatment of Fusicoccin after an extreme night-chilling event reduces the mango leaf photosynthetic damage and enhances its recovery.

[0033] FIG. 5 depicts foliar treatment of Fusicoccin after an extreme night-chilling event protects from bright-day induced membrane damage in mango leaves.

[0034] FIG. 6 depicts foliar treatment of Fusicoccin after an extreme night-chilling event protects from bright-day induced chlorosis and necrosis in mango leaves.

[0035] FIG. 7A-7C depict the physiological reduction in mango leaf gas exchange was revealed by analyzing the CO2response curve (A / Ci) parameter after a moderate nightchilling event.

[0036] FIG. 8 depicts the increased production of ethylene in mango leaves following cold-night-bright-day stress;

[0037] FIG. 9A-9E depict the improvement, by 1-MCP, on gas exchange and CO2assimilation rates in mango leaves after moderate cold-night-bright-day stress;

[0038] FIG. 10A-10E depict the improvement, by foliar CPPU treatment, on gas exchange, net CO2assimilation rates, and chlorophyll-based fluorescence parameters in mango leaves after sever cold-night-bright-day stress;

[0039] FIG. 11A-11E depict the improvement, by foliar KNO3treatment, on gas exchange, net CO2assimilation rates, and chlorophyll-based fluorescence parameters in mango leaves after extreme cold-night-bright-day stress;

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention discloses that bypassing the stomatai impaired response of the cold-night-stressed mango leaves will increase the stomatai conductance during the lighted day, resolve the lack of electron acceptors, and prevent the accumulation of active oxygen molecules (ROS). The present invention discloses that removing this bottleneck using specific compound / s that induce stomatai opening can bypass this bottleneck, decreases the physiological damage of cold-night-bright-day stress, and improves the overall physiological state of the plant. The present invention is related to the induction of stomatai opening in order to bypass the impaired stomatai opening in the cold-stress mango leaves, reduce leaf cold damage, and improve the overall physiological state of the plant.

[0042] The principles and operation of preventing and treating cold-night-bright-day stress damage in tropical and subtropical trees according to the compositions and methods disclosed by the present invention may be better understood with reference to the drawings and the accompanying description.

[0043] Introduction to the Technology

[0044] It has been noted that Fusicoccin, 1-MCP, CPPU and Potassium Nitrate (KNO3), are potential compounds for targeting this problem.

[0045] Described below are components of the present invention.

[0046] 1. The guard cell H+ pump activator, Fusicoccin: One known substance is Fusicoccin, a toxin produced by a fungus, which induces stomatai opening by activating the H+ pump within the guard cells. To test whether the induction of stomatai to open following “cold-night-bright-day” will reduce the physiological damage, treatment with Fusicoccin was tested.

[0047] 2. The ethylene inhibitor 1-Methylcyclopropene (1-MCP). Cold stress causes an increase in ethylene levels (Guye et al., 1987; Michaeli et al., 2002) and ethylene has been shown to mediate stomatai conductance (Azoulay-Shemer et al., 2023) and to induce stomatai closure (Desikan et al., 2006). The applicants hypothesized that inhibition of ethylene reception, using the ethylene competitive inhibitor 1-MCP (Sisler & Serek, 2003), may result in the bypass of the night-chilled induced impairment of stomatai opening.

[0048] 3. The abscisic acid (ABA) inhibitor cytokinin N-(2-Chloro-4-pyridyl)-N'-phenyl urea (CPPU). The cytokinin CPPU is a phytohormone that has been shown to inhibit the central signaling molecule for stomatai closure ABA (Agurla et <2 / ., 2018; Bharath et al., 2021), by reducing NO within the guard cells (Xiao-Ping & Xi-Gui, 2006). Previous studies indicate increased concentrations of the phytohormone ABA following cold stress (Lang et al., 1994). Based on these data, the applicants hypothesized that treatment with the artificial cytokinin CPPU may result in stomatai opening of night-chilled mango tree leaves.

[0049] 4. The leaf hydraulics modulator Potassium Nitrate (KNO3). KNO3has previously been found to enhance transpiration and photosynthetic levels (Borowski & Michalek, 2010) via mechanisms that increase stomatai conductance (Elhindi, 2016) and leaf hydraulics (Gorska et al., 2008). To test whether KNO3can induce stomatai opening and improve the physiological state of night-chilled mango trees, the applicants tested its effect.

[0050] TERMS AND DEFINITIONS

[0051] The term “Composition” herein is defined as a composition useful for preventing or treating cold-night-bright-day stress damage in tropical and subtropical trees. The composition comprises at least one stomatai opening inducer substance, and a surfactant. The term “Stomatai opening inducer substance” herein is defined as substances selected from the group consisting at least one of: phytohormone cytokinin-like plant growth regulator, a leaf hydraulics modulator, a competitive inhibitor of ethylene, a guard cell H+pump activator, and any combination thereof.

[0052] The term “Phytohormone cytokinin-like plant growth regulator” herein is defined as abscisic acid (ABA) inhibitor cytokinin. More particularly, “abscisic acid (ABA) inhibitor cytokinin” is N-(2-Chloro-4-pyridyl)-N'-phenylurea (CPPU).

[0053] The term “Leaf hydraulics modulator” herein is defined as Potassium nitrate (KNO3).

[0054] The term “Competitive inhibitor of ethylene” herein is defined as 1- Methylcyclopropene (1-MCP).

[0055] The term “Guard cell H+pump activator” herein is defined as Fusicoccin.

[0056] The term “Surfactant” herein is defined as Triton X-100 and / or Tween®20.

[0057] The term “Method of preventing or treating cold-night-bright-day stress damage” herein is defined as a method comprising steps of (a) foliar spray or cotton swab treatment with a composition defined in the claimed invention; wherein (b) the foliar spray or cotton swab is applied on the following lighted-day just before the sunrise; and (c) inducing the stomatai opening.

[0058] The term “Tropical and subtropical trees” herein are preferably Mangifera indica L (mango trees).

[0059] The term “Cold-night-bright-day stress damage” herein is defined as mild or severe physiological damage.

[0060] DETAILED DESCRIPTION OF THE FIGURES Reference is now made to Figures 1A-11E describing compositions and methods for preventing and treating cold-night-bright-day stress damage in tropical and subtropical trees.

[0061] Fig. 1A-1E illustrate the improvement, by Fusicoccin, on gas exchange, CO2assimilation rates and photosynthetic performances in mango leaves after moderate cold-night-bright-day stress. Young mango trees were exposed to a moderate nightchilling event (Cold; 7°C for 6 h) or kept under ambient growth conditions (Control). On the next day, just before sunrise, the cold stressed trees were treated with either 10 pM Fusiccocin in 0.02% Tween-20 (Cold + FC) or just with 0.02% TWEEN-20 solution (Cold). Control trees were treated with 0.02% TWEEN-20 solution (Control). All trees were then moved back to the greenhouse under natural light (-1000 pmol m'2s'1). After -2 h, gas exchange and light-adapted chlorophyll fluorescence parameters were measured. Figure 1A: stomatai conductance (gs; mmol H2O m'2s'1), Figure IB: net CO2assimilation rates (A; pmol CO2m'2s'1), Figure 1C: intercellular CO2(Ci; pmol CO2mol air'1), Figure ID: actual efficiency of PSII (<PPSIP), and Figure IE maximal efficiency of PSII (Fv / Frri) 7 h after exposure to natural light followed by one h of dark. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s multiple comparison test. The bars shown are means ± SEM; n = 3 trees for each treatment, and five leaves were measured from each tree. When a statistically meaningful Effect Size was found, the Pvaiue is noted.

[0062] Fig. 2A-2F illustrate the positive protective effect of Fusicoccin, on gas exchange and chlorophyll fluorescence in mango leaves after severe cold-night-bright-day stress. The experiment was carried out as described in Fig. 1A-1E where young mango trees were tested under severe cold night stress (4°C for 12 h). Gas exchange and light-adapted chlorophyll fluorescence parameters were measured. Data shown is the average of stomatai conductance (gs; mmol H2O m'2s'1, Figure 2A), net CO2assimilation rates (A; pmol CO2m'2S'1, Figure 2B), intercellular CO2(Ci; pmol CO2mol air'1, Figure 2C), actual efficiency of PSII ((PPSII, Figure 2D), and maximal efficiency of PSII Fv / Frri) 7 h after exposure to natural light followed by one h of dark (Figure 2E). Statistical analysis was conducted using one-way ANOVA followed by Tukey’s multiple comparison test. The bars shown are means ± SEM; n = 5 trees for each treatment, and five leaves were measured from each tree. When a statistically meaningful Effect Size was found, the Pvaiue is noted. Pictures were taken 15 days after the treatment. The most common phenotypes of cold-night-bright-day induced necrosis and chlorosis are presented in Figure 2F, upper panel and lower panel, respectively.

[0063] Fig. 3A-3E illustrate the protective effect of Fusicoccin from extreme cold-night- bright-day by partially rescue from the physiological drop in mango leaf gas exchange and from a drop of leaf PSII photochemical efficiency. The experiment was carried out as described in Fig. 1 A-1E, where young mango trees were tested under under extreme cold night stress (0.5°C for 12 h). Gas exchange and light-adapted chlorophyll fluorescence parameters were measured. Data shown is the average of: stomatai conductance (gsmmol H2O m'2s'1Figure 3A), net CO2assimilation rates (A; pmol CO2m'2s'1Figure 3B), intercellular CO2(Ci; pmol CO2mol air'1, Figure 3C), actual efficiency of PSII (<J>PSII, Figure 3D), and non-photochemical quenching (NPQ, Figure 3E). Statistical analysis was conducted using one-way ANOVA followed by Tukey’s multiple comparison test. The bars shown are means ± SEM; n = 4 trees for each treatment, and five leaves were measured from each tree. When a statistically meaningful Effect Size was found, the Pvaiue is noted.

[0064] Fig. 4 illustrates the alleviating effect of Fusicoccin after an extreme night-chilling event from mango leaf photosynthetic damage and its positive effect on photosynthesis recovery. The experiment was carried out as described in Fig. 1A-1E where young mango trees were tested under extreme cold night stress (0.5°C for 12 h). Measurements of the maximal quantum efficiency (FV / FM) were conducted at: 1) “Coldnight” at the end of the night, before exposure to light, 2) ’’Cold-night + light” 8 h after exposure to natural light (dark adapted for 1 h) and 3) “1 week of recovery” after plants were exposed to the natural sunlight for 7 h (plus 1 hour of leaf dark adaptation). Statistical analysis was conducted using two-way ANOVA, with treatment type and time point (as repeated measurement) as factors. Statistical interaction was evident (P = 0.0024). The bars shown are means (± SEM); n = 4 trees for each treatment; in each tree, 5 leaves were measured. When a statistically meaningful Effect Size was found within the same time point, the Pvaiue is noted. Fig. 5 illustrates the alleviating effect of Fusicoccin after an extreme night-chilling event from bright-day induced membrane damage in mango leaves. The experiment was carried out as described in Fig. 1 A-1E where young mango trees were tested under extreme cold night stress (0.5°C for 12 h). After 8 h under natural light conditions, leaf discs were excised from Control, Cold, and Cold+FC treated plants and measured for electrolyte leakage. Statistical analysis was conducted using 1-way ANOVA followed by Tukey’s multiple comparison test. The bars shown are means ± SEM; n = 4 trees for each treatment, and five leaves were measured from each tree. When a statistically meaningful Effect Size was found, the Pvaiue is noted.

[0065] Fig. 6 illustrates the protecting effect of Fusicoccin-induced stomatai opening from extreme cold-night-bright-day from chlorosis and necrosis. The experiment was carried out as described in Fig. 1A-1E where young mango trees were tested under extreme cold night stress (0.5°C for 12 h). Chlorophyll concentration was measured at 02:00 pm on days 1, 2, 4, and 7. Data shown is the chlorophyll concentration means ± SEM (pmol Chi nr2); n = 4 trees for each treatment, 5 leaves were measured from each tree. Statistical analysis was conducted using two-way ANOVA, with 2 factors: Treatment and Time point (repeated measurement). No statistical interaction effect was found (P=0.3508). When a statistically meaningful Effect Size was found within the same time point, the Pvaiue is noted.

[0066] Fig. 7A-7C illustrate the physiological reduction in mango leaf gas exchange following moderate cold-night. The physiological reduction in mango leaf gas exchange was revealed by analyzing the CO2response curve (A / Cz) parameter after a moderate night-chilling event. Leaves of young mango trees were measured for their CO2response curves at three-time intervals, (Figure 7A) 08:00 AM, (Figure 7B) 10:00 AM, and (Figure 7C) 12:00 PM, before or after cold night stress. On the first day, leaf CO2response curve measurement were taken and then the analyzed plants were split into two treatment groups, I) cold-night stress, where plants were subjected to moderate nightchilling event (Cold; 7°C for 6 h) or II) Control, where plants were kept under ambient growth conditions. On the proceeding day, the same plants were measured again for their CO2response curve at the same time of the day previously measured (08:00 AM / 10:00 AM / 12:00 PM). Fig. 8 illustrate the increase in mango leaf ethylene production in response to a nightchilling event. Young mango trees were exposed to a ‘moderate’ night-chilling event (Cold; 7°C for 6h) or kept under ambient growth conditions (Control). On the next morning (before sunrise), fully expanded mature leaves were excised, moved into a glass container, and incubated in a growth control room, under 22°C, 12h / 12h light cycle (light 600 pmol m2s '), for 4.5h, 5.5h or 27h. Ethylene production was quantified in n=4 replicates per treatment (3 leaves in each replicate). ND - non-detectable level.

[0067] Fig 9A-9E. illustrate the protective effect of the ethylene inhibitor 1-MCP from moderate cold-night-bright-day by enhancing leaf gas exchange. Gas exchange and chlorophyll fluorescence-based parameters measurements show stomatai conductance (gs; mmol H2O m'2s’1, Figure 9A), net CO2assimilation rates (A; pmol CO2m’2s’1.Figure 9B), intercellular CO2(Cz; pmol CO2mol air’1Figure 9C), actual efficiency of PS II Figure 9D), and maximal efficiency of PSII (Fv / Fm) 7h after exposure to natural light followed by one h of dark (Figure 9E). Statistical analysis was conducted using one-way ANOVA, followed by Tukey's multiple comparison test. Bars represent means ± SEM, where each treatment comprised four trees, where five leaves were measured from each tree. The corresponding significant P-values (<0.05) are indicated above bars.

[0068] Fig 10. illustrates the protective effect of CPPU from a severe night-chilling event by enhancing leaf gas exchange, and complete protection of the light-harvesting and electron-transport systems Gas exchange and chlorophyll fluorescence-based parameters measurements show stomatai conductance (gsmmol H2O m’2s’1, Figure 10A), net CO2assimilation rates (A; pmol CO2m’2s’1, Figure 10B), intercellular CO2(Cz; pmol CO2mol air’1, Figure 10C), actual efficiency of PSII (PPSII. Figure 10D), and maximal efficiency of PSII (Fv / Fm) 7h after exposure to natural light followed by one h of dark (Figure 10E). Statistical analysis was conducted using oneway ANOVA, followed by Tukey's multiple comparison test. Bars represent means ± SEM, where each treatment comprised four trees, where five leaves were measured from each tree. The corresponding P-value is indicated above bars. Fig 11A-11E. illustrate the protective effect of KNO3from extreme night-chilling event, by increasing gas exchange and CO2assimilation rates and by the protective effect of mango leaf photosynthetic capabilities from declining. Gas exchange and chlorophyll fluorescence- based parameters measurements show stomatai conductance (gs; mmol H2O m'2s'1, Figure 11A), net CO2assimilation rates (A; pmol CO2m'2s'1, Figure 11B), (C) intercellular CO2(Cz; pmol CO2mol air'1), actual efficiency of PSII Figure 11D), and maximal efficiency of PSII (Fv / Fm) 7h after exposure to natural light followed by one h of dark (Figure 11E). Statistical analysis was conducted using one-way ANOVA, followed by Tukey's multiple comparison test. Bars represent means ± SEM, where each treatment comprised four trees, where five leaves were measured from each tree. The corresponding P-value is indicated above bars.

[0069] DETAILED DESCRIPTION OF THE INVENTION

[0070] The following description is provided, alongside all chapters of the present invention, so as to enable any person skilled in the art to make use of the invention and sets forth the best modes contemplated by the inventor of carrying out this invention.

[0071] EXAMPLES

[0072] To study and develop a cold-stress treatment for mango trees, the present invention combined basic and applicative study. The efficiency of different foliar spray treatments to prevent cold-night-bright-day stress damage in mango trees was tested by the present invention.

[0073] A great deal of research has been carried on stomatai movement (opening / closure), which identified different substances that increase stomatai conductance and, consequently, increase transpiration.

[0074] Plant material and growth conditions

[0075] Young (~1- to 2-year-old) mango trees (Mangifera indica L.) of the 'Shelly' or 'Omer' cultivars, grafted onto clonal poly embryonic rootstock 13-1, were obtained from Zvieli Nursery (Moshava Kinneret, Israel). The trees were cultivated in 4-L pots with soil mixture (Klasmann-Deilmann) in a net-house / greenhouse at Newe Ya' ar Research Center, Israeli Ministry of Agriculture (GPS 32.70808, 3517937). During April to December, they were grown under a white net (50-mesh), receiving a maximum photosynthetic photon flux density (PPFD) of about 1400 pmol photons m2s '. In the winter (January to March), trees were shifted to a temperature-controlled greenhouse with a maximal PPFD of 900-1100 pmol photons m2s ', and temperatures ranging from 20 to 24°C during the day and 15 to 20°C at night. Trees were watered daily through an automated system and fertilized weekly with 7:3:7 NPK (Shefer, ICL). Irrigation was adjusted to twice daily in hot seasons and once daily in winter, at a rate of 1 L / h for 60 minutes each.

[0076] Materials tested (foliar application)

[0077] 1. Fusicoccin (FC) 10 pM + surfactant 0.02% Tween®20

[0078] 2. 1-MCP (“Harvista”, sprayed by Rimi .LTD Israel)

[0079] 3. CPPU 40-120 pM + surfactant 0.025% Triton X-100

[0080] 4. Potassium nitrate (KNO3) 100 mM + surfactant 0.025% Tween®20

[0081] Experimental design

[0082] A total of 16 young mango trees were moved, at dusk, from the green / net -house to a temperature-controlled dark room (18-22°C) and treated as follows: (1) "Control" plants were maintained under ambient temperatures (18-22°C) during the night. At the end of the night, leaves were applied with “mock” water-based solution (Tween®20 or Triton X-100 according to experiment, as a surfactant) (2) "Cold" treated plants were espoused to cold- night-stress (7°C or 4°C, as described in each experiment). At the end of the night, tree foliar were applied with “mock-solution” (Tween®20 or Triton X-100 in distilled water, DDW, according to experiment) (3) "Cold + treatment (FC, 1-MCP, CPPU or KNO3)" treated plants were subjected to the cold-night- stress (as described above). At the end of the night, leaves were applied with either 10 pM Fusicoccin (in 0.02% Tween®20), CPPU 40-120 pM (in 0.025% Triton X-100), 100 mM KNO3(in 0.025% Tween®20) or 1-MCP (“Harvista” sprayed by Rimi company). For each experiment, each treatment was comprised of four biological replications (n=4 trees).

[0083] Cold-night-stress

[0084] Trees were moved at dusk from the greenhouse or net-house to a temperature-controlled dark room (18-22°C) and randomly split into two treatments: (i) Control: trees were maintained during the night in the dark at ambient temperature (18-22°C); (ii) Cold: trees were exposed to a moderate (7°C), severe (4°C), or extreme (0.5°C) night-chilling event for different numbers of hours during the night (as described in each experiment). Exposure to night-chilling events was conducted in a temperature-controlled chamber (Inomak). The chamber was first set to room temperature (18-22°C), and the plants were moved into the chamber at dusk, after sunset. For the 6 h cold-night treatment (7°C, 4°C, or 0.5°C), the chamber started to cool at 11:00 PM, reaching the desired temperature at -11:15 PM. At 5:30 AM, the chamber stopped cooling, and the temperature gradually increased until it reached room temperature at 6:30 AM. Similarly, for the 12 h cold-night treatment, the temperature was gradually reduced starting at 7:00 PM and maintained at the desired low temperature till 7:00 AM (as described in each experiment). To prevent sudden temperature drops in the soil and root system, the applicantsconstructed a system in the cold chamber that maintains soil temperature between 13 and 15 °C. Two perspex containers (width 56 cm, length 61 cm, height 27.5 cm), wired with an electrical heating cable (Exo Terra), were installed in the temperature-controlled chamber. Mango tree pots were placed in the perspex containers and covered with a rockwool blanket (temperature insulator). A HOBO temperature-logger (Pendant MX2202, Onset, USA), which was installed in the pot, indicated that the soil temperature stayed between 13 and 15 °C during the night. At the end of the night (before exposure to direct sunlight), all trees were moved out of the chamber to the darkened room and acclimatized to room temperature together with the trees from the control treatment.

[0085] For post night-chilling-event treatment, two solutions were prepared: (i) Mock (0.02% Tween-20 surfactant in double distilled water (DDW)) and (ii) FC (0.02% Tween- 20 + 10 pM FC in DDW). For each experiment, the Mock and FC working solutions were freshly prepared in the morning. The 2mM Fusiccocin (Santa Cruz Biotechnology, catalog number SC-200754) stock solution, which was made in 100% ethanol and kept at -20°C, was diluted in 0.02% Tween-20 to the final concentration of lOmM of FC. At the end of the night, after 1 h of acclimation to room temperature in the dark, the trees from the Control (18-22°C) and Cold (7°C, 4°C or 0.5°C) treatments were randomly divided into two sub treatments: (i) Mock and (ii) FC . Using a cotton swab, the indicated solution was applied on both the adaxial and abaxial sides of healthy and fully expanded mature leaves ( -700 ul per each leaf). The solution was applied on more than 5 leaves of at least 4 trees, per each treatment. Trees were then placed outdoors, in the shade, for 30 min to acclimate under low light and so that the applied solution would evaporate from the leaf surface. Trees were then shifted to the net-house / greenhouse and exposed to a bright sunny day. More than 9 experiments were conducted under different cold-night temperatures (i.e., moderate (7°C), severe (4°C), or extreme (0.5°C) night-chilling event). For each min temperature, a representative dataset was generated to demonstrate the various physiological effects of the varied night-chill temperatures.

[0086] After acclimation to room temperature, control (18-22°C) and cold (7°C, 4°C or 0.5°C) exposed plants were randomly assigned into two treatments: A) “mock” waterbased solution (Tween®20 or Triton X-100 according to experiment, as a surfactant) or B) treatment: 10 pM Fusicoccin (in 0.02% Tween®20 ), 1-MCP (“Harvista” sprayed by Rimi company), CPPU 40-120 pM (in 0.025% Triton X-100) or 100 mM KNO3(in 0.025% Tween®20). Healthy and fully expanded mature leaves were applied with the above-mentioned different solutions (adaxial and abaxial sides of the leaves) using a cotton swab (Fusicoccin) or foliar spray (I-MCP / CPPU / KNO3Hervista). Plants were then kept in the shade for another half an hour to let the leaves dry and then shifted back to the net / greenhouse.

[0087] Gas exchange and chlorophyll-α fluorescence-based measurements

[0088] Gas-exchange and chlorophyll-fluorescence measurements were conducted with an LI- 6800 portable photosynthesis system (LICOR Biosciences, USA) equipped with a Multiphase Flash™ Fluorometer (measured leaf area = 2 cm2). All measurements were conducted on fully expanded mature leaves after at least 2 h of exposure to natural light. For gas-exchange measurements, reference CO2concentration was set to 400 ppm, and relative humidity and temperature were kept at ambient levels. Light, in the intensity of 1000 pmol m'2s'1PPFD, was provided by blue / red light-emitting diodes (10% blue and 90% red). The LL6800 chamber airflow was set to 500 pmol s'1with a boundary layer of ~3 mol m'2s'1. Measurements of gas exchange and light-adapted chlorophyll a fluorescence were recorded simultaneously. After leaf steady-state gas exchange was recorded for net CO2assimilation rate (A), stomatai conductance (g4), and internal CO2(Cz), the steady-state chlorophyll fluorescence signal (Fs) was logged. A saturating light pulse of 8000 pmol m'2s'1was used to determine the maximum light-adapted fluorescence (Fm'). The overall efficiency of the photosystem II (PSII) reaction center in the light, calculated as = (Fm-Fl)IFm'. where Fm is the maximum fluorescence signal (when all PSII centers are in the closed state) measured from dark-adapted material, Ft is the steady-state yield of fluorescence in the light, and Fm' is the maximum fluorescence signal (when all PSII centers are in the closed state), measured from light- adapted material. To determine the maximum PSII efficiency (F\ / Fm). by dividing the variable fluorescence (Fv) by the maximum fluorescence (Fni), chlorophyll-based fluorescence measurements were conducted in complete darkness, either predawn or 7 h after exposure to natural light (and 1 h of dark adaptation). For the evaluation of Fv / Fm at different time points, measurements were taken at each time point from the same leaves. For the calculation of non-photochemical quenching (NPQ), predawn Frn values were used as described in (Ruban, 2016).

[0089] Leaf chlorophyll concentration

[0090] Chlorophyll concentration (pmol Chi m'2) was estimated non-destructively using a chlorophyll meter (MC-100, Apogee Instruments, USA). Measurements were conducted on 4 leaves from 4 trees (n = 4) for each treatment; for each leaf, 2 measurements were collected from both sides of the main vein.

[0091] Membrane damage evaluated by electrolyte-leakage assay

[0092] Leaf discs (1 cm diameter) were cut using a leaf puncher from fully developed leaves and rinsed with deionized water. To determine electrolyte leakage, electric conductivity (EC) was measured using a conductivity meter (CON 11 / 110, Oakton Instruments, USA). Samples were first immersed in 10 mL deionized water in a sealed 15-mL Falcon tube, incubated at room temperature on a shaker for 24 h, and then EC was measured (ECI). Next, the same samples were incubated at 100°C for 1 h, cooled to room temperature, and EC was measured again (EC2). Electrolyte leakage (EL%) was calculated as (EC1 / EC2) x 100 (Lutts et al., 1996).

[0093] A / Ci curves to study stomatai and biochemical photosynthesis limitations

[0094] Simultaneous measurement of leaf gas-exchange and modulated chlorophyll-fluorescence responses to light and CO2concentration enables to determine a wide range of key biochemical and biophysical limitations on photosynthesis. Leaf gas-exchange photosynthetic measurement can determine leaf CO2uptake (A) and the intercellular CO2concentration (Cz). Analysis of the ratio between them, using A / Ci curves, can quantitatively separate biochemical and stomatai limitations to photosynthesis and provide detailed information on the different photosynthetic components, including the maximal rate of carboxylation VCMax, electron transport rate for regeneration of ribulose 1,5-bisphosphate (RuBP) (J), and triose phosphate-utilization rate (TPU) (Long & Bernacchi, 2003). To measure the leaf A / Ci ratio before and after the cold-night-bright-day stress, leaf A / Ci curves at different hours during the day were obtained. To calculate the A / Ci baseline levels of non- stressed leaves, 2 young mango trees were moved from the net-house to a temperature- controlled darkroom (18-22°C). The following day, A / Ci response curves were obtained for 3 mature leaves per tree (see details below) using two LI-6800 portable photosynthesis systems equipped with the Multiphase Flash Fluorometer. At the end of the measurement, the plants were returned to the net-house for the rest of the day. At dusk on the second day, the same trees were moved back to the darkroom and randomly split into two treatments: (i) Control (18°C-22°C) and (ii) Cold (exposure to a moderate night-chilling event, 7°C for 12 h). On the morning of day 3, the same 3 leaves were measured again, in the same order as on the first day (before treatments), to determine their A / Ci response curves. To prevent differential light exposure, the leaves were kept in the dark by covering them with aluminum foil until the measurements were performed. By using this protocol, the applicantseliminated the effect of different cumulative light exposures and focused on the physiological effect produced by time of day. To test the impact of time on leaf photosynthetic performance, the first leaf from each tree was measured at 08:00 AM, the second leaf at 10:00 AM, and the third leaf at 12:00 PM. The experiment was performed in five independent time blocks within 2 weeks. Each replication was measured over 2 consecutive days, generating a repeated-measures design for the analysis. CO2response curves with the Dynamic Assimilation™ Technique (DAT) was recently developed by Saathoff and Welles (2021). Experiments were performed as recommended in the Li-Cor LTD user manual (https: / / www.licor.com / env / support / LI- 6800 / videos / dynamic-assimilation-technique.html). This technique represents an advanced and improved Rapid A / Ci Response technique, that features better accuracy of derived FcVB parameters and shorter replication time. In this study, the detailed rapid A / Ci curves were obtained after induction of photosynthesis for 1 h with linear PPFD ramping from 0 to 2000 pmol m'2s'1. Chamber conditions at the end of the light adaptation were as follows: CO2level - 415 pmol mol’1, PPFD - 2000 pmol nr2s’1, leaf temperature - 29°C, and relative humidity - 60%. Chamber CO2concentration was then reduced to 50 pmol mol'1and then increased at a linear rate (200 pmol mol'1min'1) to 1800 pmol mol'1min'1. Data were recorded every 5 s for -100 data points per curve (each curve took 9 min to complete). Data from A / Ci curves were fitted with the FvCB model at actual leaf temperature (Farquhar et al., 1980) using the msuRACiFit r script (https: / / github.com / poales / msuRACiFit).

[0095] Statistical Analysis The number of trees (biological replications) and of leaves measured within each replication was at least 3, as reported in the relevant figure legends. Results were analyzed using one-way ANOVA or two-way repeated-measures ANOVA followed by Tukey's or Sidak's multiple comparison tests. Data were checked for normality of the residuals and equal variance using the Shapiro-Wilk and Brown-Forsythe tests, respectively. In the few cases for which unequal variance was detected, Welch's ANOVA was used, followed by Dunnett's T3 pairwise comparison test. Results are reported as mean ± standard error of the mean (SEM) and P-values (adjusted for multiple comparisons) fromTukey, Sidak, or Dunnett's pairwise comparisons. When present, statistical interactions from ANOVA are reported as P-values. A / Ci curve data (Table 1) are presented as mean ± (SEM), mean difference, with 95% confidence intervals, and P-values for mean difference (PES). Analysis was conducted using GraphPad Prism version 9.5 (GraphPad Software, San Diego, CA, USA, www.graphpad.com).

[0096] Fusicoccin application results

[0097] 1. Moderate cold-night stress la. Foliar treatment with FC after a moderate night-chilling event results in full recovery of leaf stomatai conductance and photosynthetic performance.

[0098] To test whether FC can bypass the cold-induced impairment in stomatai opening of mango tree leaves and whether this treatment can reduce leaf physiological damage, young mango trees were exposed, during the night, to either ambient or moderate chilling (7°C for 6 h) conditions, followed by application of FC in the morning. After -2 h of natural light (-1000 pmol m'2s'1), leaves were measured for their gas-exchange and chlorophyll-fluorescence parameters; 5 h later (total of 7 h exposure to light), the same leaves were dark-adapted for 1 h and Fv / Fm was measured. The moderate night-chilling event induced an over 50% decrease in stomatai conductance (g4) the following morning compared to control plants (Fig. 1A; Cold 26 ± 4 vs. Control 53 ± 8.0 mmol H2O m'2s'1, PES= 0.142). FC treatment following the night chilling resulted in stomatai conductance that was similar to the control plants (Fig. 1 A; Cold+FC 48 ± 13 mmol H2O m'2s'1, PEScoid+Fc - control = 0.986). Furthermore, untreated mango plant leaves showed a reduction in the rate of net CO2assimilation (A) after exposure to the moderate night-chilling event compared to the control plants (Fig. IB; Cold 3.3 ± 0.4 vs. Control 5.9 ± 0.4 pmol CO2m'2s'1, PES= 0.028). The effect size of the cold-night stress reached a reduction of -50%, which was in agreement with the detected decrease in stomatai conductance. When leaves of cold-night stressed trees were treated with FC, net CO2assimilation levels were unaffected and remained stable and similar to those detected in the control trees (Fig. IB; Cold+FC 5.7 ± 0.3 vs. Control 5.9 ± 0.4 pmol CO2m'2s'1, PES= 0.992). A comparison of leaf intercellular CO2(Cz) levels showed no statistically significant differences among treatments (Fig. 1C; Control 186 ± 32, Cold 175 ± 40, and Cold+FC 173 ± 29 pmol CO2mol air1). In addition, chlorophyll fluorescence parameters (<PPSII and Fv / Fni) showed no statistically significant differences among treatments (PPSir. 0.11 ± 0.01, 0.11 ± 0.02 and 0.15 ± 0.02; Fv / Fnv. 0.79 ± 0.01, 0.79 ± 0.01 and 0.79 ± 0.01, for Control, Cold and Cold+FC, respectively (Fig. ID and Fig. IE, respectively).

[0099] 2. Severe cold-nigh stress

[0100] 2a. Foliar treatment with FC after a severe night-chilling event offers partial protection with respect to leaf CO2assimilation (A) and stomatai function (gA, and complete protection of the light-harvesting and electron-transport systems ((PPSII and Fv / FmY To evaluate the efficiency of FC protection from leaf physiological damage following a stronger night-chilling event, a similar experiment with a severe night-chilling event of 4°C for 12 h was conducted. Severe cold-night stress resulted in a large decrease in stomatai conductance to -40% of the control (Cold 20 + 4 vs. Control 50 + 8 mmol H2O m'2s'1, PES= 0.008, Fig. 2A). Following the severe night-chilling event, FC-treated leaves showed higher (although not statistically significant) gsthan the untreated trees exposed to the cold night (Cold 20 + 4 vs. Cold+FC 36 + 4 mmol H2O m'2s'1, PES= 0.165, Fig. 2A). Nevertheless, their gswas somewhat (although not statistically significant) lower than in control non-stressed plant leaves (Control 50 + 8 mmol H2O m'2s'1, PEScoia+Fc- controi = 0.223, Fig. 2A). A significant reduction in leaf CO2assimilation, by -50%, was observed following the severe night- chilling event (Cold 2.59 + 0.23 vs. Control 5.1 ± 0.35 pmol CO2m'2s'1, PES= 0.006, Fig. 2B ;). However, when FC treatment was applied after the cold night, leaf CO2assimilation rate remained intact and was not substantially different from the control (Cold+FC 4.23 + 0.60 pmol CO2m'2s'1, PEScoid+Fc - control = 0.394, Fig. 2B). Similar Ci levels were identified in leaves of both control and cold-stressed mango tree (Cold 203 + 28 vs. Control 207 ± 17 pmol CO2mol air'1, PES= 0.991, Fig. 2C) and although not statistically significant, Ci levels in leaves of FC- treated cold-stressed plants were markedly lower (Cold+FC 146 + 10 pmol CO2mol air'1, PESCold+FC - Control = 0.153, Fig. 2C). Chlorophyll a fluorescence measurements of plant leaves from the different treatments revealed that after a severe night-chilling event and exposure to a bright sunny day, both the effective quantum yield of PSII and the maximum quantum yield of PSII reaction centers (Fv / Fm measured after 1 h of recovery in the dark) were considerably reduced compared to non-stressed control leaves. The cold-night stress resulted in a large decrease (by -54%) in leaf ( vs. Control 0.008, Fig. 2D). Interestingly, here it was found that using FC after the night-chilling event protected mango leaves from the subsequent alterations in the efficiency of linear flow through PSII (Cold+FC 0.090 ± 0.010, PEScoid+Fc - control - 0.467, Fig. 2D). Leaf Fv / Fm measurements, conducted following exposure of the tree leaves to sunlight for 7 h (including 1 h of dark adaptation), revealed no noticeable difference between the Control and Cold±FC-treated plants, both showing higher values compared to the cold- stressed plants (Control 0.699 ± 0.024, Cold 0.571 ± 0.002, and Cold±FC 0.731 ± 0.039, PEScold - control = 0.019, PEScold - coid+Fc = 0.005, PEScoid+Fc - control = 0.69, Fig. 2E).

[0101] To evaluate foliar damage following severe cold-night-bright-day stress and evaluate the effect of Fusicoccin treatment, leaf phenotype of the “Control”, “Cold” and “Cold+FC” treated mango trees were recorded every day for a week (Fig. 2F). “Cold” treated plants developed wilted, chlorotic, and necrotic leaves, in particular in the younger leaves that were exposed to light. This phenotype developed 7 days after the cold-night-stress and was the most pronounced 15 days post the cold-night (Figure 2F, upper panel). Remarkably, Fusicoccin treatment of cold-stressed plants (Cold+FC) showed pra protective effect and minor symptoms (Figure 2F, lower panel).

[0102] 3. Extreme cold-nigh stress

[0103] 3a. Foliar application of FC has a mild positive effect on leaf physiology after an extreme night-chilling event.

[0104] It has been well-documented that physiological damage following a night-chilling event is typically time- and temperature-dependent, meaning that the damage increases as the temperature decreases and as the exposure time to the low -temperature increases (Nir et al., 1997; Ying et al., 2002). To further explore the protective effect of FC from the physiological outcome imposed by radical cold night-bright day stress, we repeated the experiment described in figure 1 under an extreme night-chilling event of 0.5°C for 12 h. This event induced full stomatal closure. FC treatment resulted in a smaller (yet not statistically significant) reduction in stomatal conductance (Control 37 + 8.0, Cold 1.1 ± 0.4, Cold+FC 11.1 ± 2.2 mmol FFO m'2s'1, PEScoia - controi = 0.001, PEScoia - coid+Fc = 0.345, PEScoid+Fc - control = 0.009, Fig. 3 A). In correlation with gs, a complete reduction in A was observed in leaves following the night-chilling event, reaching negative values (Cold -0.7 ± 0.1 pmol m'2s'1, Fig. 3B). However, treatment with FC induced minimal stomatal opening, and increased A levels to positive values (Cold+FC 1.2 ± 0.4 pmol m'2s'1, Fig. 3B). Both treatments, Cold and Cold+FC, differed from the control (Control 5.0 ± 0.6 pmol m'2s'1, PEScold - control < 0.0001, PEScoid+Fc- control = 0.001, PEScoid - coid+FC = 0.06, Fig. 3B).

[0105] Unlike moderate and severe night chilling (Fig. 1C and 2C), extreme night-chilling stress resulted in a massive increase in Ci compared to the control non-stressed plants (Cold 1074 + 274 vs. Control 151 + 9 pmol CO2mol air'1, PES= 0.067, Fig. 3C). On the other hand, Ci of Cold+FC leaves was found to be lower than that of the untreated nigh-chill- induced plants, which further supports the positive effect of FC on the CO2assimilation process after the night-chilling event (Cold+FC 410 + 75 pmol CO2mol air'1, PEScoid+Fc - Control = 0.968, Fig. 3C).

[0106] Chlorophyll a fluorescenceparameters measured following the extreme night-chilling event revealed a reduction of -40% in <PPSII in cold-stressed plants (Cold 0.110 + 0.015 vs. Control 0.182 + 0.008, PES= 0.002, Fig. 3D). FC treatment of the cold-stressed plants could partially preserve the effective quantum yield of PSII, which was reduced by only -13% compared to control plants (Cold+FC 0.157 + 0.007, PEScoid+Fc - control = 0.258, PEScold - coid+Fc = 0.021, Fig. 3D). Since cold stress might negatively affect the plant’s photoprotection mechanism(s), nonphotochemical quenching of excessive excitation energy (NPQ) was calculated. Data showed slightly higher NPQ (yet not statistically significant) in leaves of both FC-treated / untreated cold-stressed plants (Control 3.1 + 0.2, Cold 3.3 + 0.4, Cold+FC 3.7 + 0.2, Fig. 3E).

[0107] 3b. Foliar application of FC after an extreme night-chilling event protects from chronic photoinhibition.

[0108] The Fv / Fm ratio is a chlorophyll fluorescence-based parameter representing the maximum potential quantum efficiency of PSII (Maxwell & Johnson, 2000). This parameter is a sensitive measurement tool that has been used as an indicator of environmental and temperature stress (Schulze & Caldwell, 1995). To assess the effect of FC foliar application on leaf photosynthetic performance after an extreme nightchilling event (0.5°C for 12 h), and whether FC also has an effect on its recovery', leaf Fv / Fm measurements were recorded from the Control, Cold, and Cold+FC treatments (as described in section 3.3) at three different time points: (i) pre-dawn: after the cold stress and before any exposure to light: (ii) natural light 7 h: after the cold stress and exposure to 7 h of light (followed by 1 h of dark adaptation); and (iii) 1 week recovery: pre-dawn measurement conducted 1 week after the cold stress on trees that were grown under control conditions.

[0109] At the end of the night, Fv / Fm ratios of the cold-stressed plants (i.e., Cold and Cold+FC treatments) were similar to that in the control plants (Control 0.837 ± 0.007, Cold 0.826 + 0.014, Cold+FC 0.852 + 0.001, Fig. 4), suggesting that the cold stress by itself did not affect the potential light capture and electron transport. We then measured Fv / Fm ratios after plants were exposed to natural sunlight for 7 h (plus 1 h of leaf dark adaptation). There was some reduction in the control non-stressed plants compared to pre-dawn values (Control 0.737 + 0.001). In contrast, Fv / Fm ratio dramatically declined in the cold- stressed plant leaves, by -27%, compared to the control. Fv / Fm ratio in the FC-treated cold-stressed plants was only reduced by -16% of the control plants (Cold 0.524 + 0.027, Cold+FC 0.617 + 0.021, PESCold - Control — 0.0063, PESCold - Cold+FC — 0.0791, PESCold+FC- controi = 0.0171, Fig. 4), further supporting the positive protective effect of FC against extreme night-chilling damage. To assess the physiological status of the leaves after 1 week of recovery, trees were maintained under ambient growth conditions in the net- house, and the leaves that were previously measured were analyzed again for their predawn Fv / Fm. ratios. Data showed that foliar application of FC results in full recovery, similar to the control plant leaves (Cold+FC 0.833 ± 0.022 vs. Control 0.835 ± 0.011, PES- 0.994, Fig. 4). In comparison, untreated cold-stressed plants showed some sustained photoinhibition, with lower Fv / Fm ratios, and did not fully recover after 1 week (Cold 0.786 + 0.033, PEScoid-controi = 0.436).

[0110] 3c. Foliar application of FC after an extreme night-chilling event minimizes cellular damage and improves membrane stability in the leaves.

[0111] Cold stress has been found to disturb membrane integrity (Campos et al., 2003). Electrolyte leakage (EL%) is an indicator of membrane stability, where increasing values of EL% indicate a decline in membrane stability and cellular damage. To test whether foliar application of FC after an extreme night-chilling event can protect from cellular damage, a similar experiment as in section 3.3 was conducted. After mango trees were exposed to night-chilling events and then to natural light conditions for 7 h, leaf discs were excised from the three different treatments (i.e., Control, Cold, and Cold+FC) and measured for electrolyte leakage. Results showed an -37% increase in electrolyte leakage from mango leaf tissue following an extreme night-chilling event (Cold) compared to the control leaves (Cold 21.21 ± 1.08 vs. Control 16.07 ± 0.30, PES= 0.002, Fig. 5). However, the EL% of FC-treated cold-stressed mango trees was similar to that of the control plants (Cold+FC 17.78 ± 0.66, PESCol+ddFC - control = 0.287, PEScoia - coid+Fc = 0.025. Fig. 5). These findings suggest that foliar application of FC on mango tree leaves after a night chill can increase leaf tolerance to cold night-bright day stress and prevent membrane damage.

[0112] 3d. FC induction of stomatai opening can rescue mango leaves from chlorosis following an extreme night-chilling event.

[0113] Chlorophyll concentration after an extreme night-chilling event (as described in section 3.3) was determined 1, 2, 4, and 7 days after the chilling event. The chlorophyll concentration of the cold-stressed plants, measured after 7 h of light, was lower than that of the controls. Interestingly, leaves of the Cold+FC-treated plants showed only a negligable reduction (Cold 469 ± 13, Control 528 ± 9, Cold+FC 517 + 21 μmol m'2, PESCold - Control = 0.0263, PESCold - Cold+FC - 0.222, PESCold+FC - Control = 0.877, Fig. 6). On day 2, the difference (ES) between the cold-treated and control leaves significantly increased (Fig. 6; Cold 437 vs. Control 530 μmol m'2, PES= 0.007), suggesting that chlorophyll catabolism was still higher than its anabolism. On the other hand, although FC -treated cold-stressed plants (Cold+FC) showed some reduction in chlorophyll level on day 2 (compared to that on day 1), only a small, non-significant difference was found when compared to the control (Cold+FC 490 μmol m'2, PEScoid+Fc - control = 0.239, Fig. 6). Similarly, analysis on day 4 revealed a further decrease in chlorophyll concentration in the cold-stressed plant leaves. On the other hand, leaves of FC-treated cold-stressed plants (Cold+FC) maintained the same chlorophyll concentration as on day 2, which was not statistically different from the control plants (Cold 414, Control 513, Cold+FC 488 μmol m'2, PESCold - Control — 0.038, PESCold - Cold+FC — 0.107, PESCold+FC - Control — 0.663, Fig. 6). After 7 days of recovery, the average leaf chlorophyll concentration increased in all treatments and reached similar levels (Cold 490 + 9, Control 541 + 26, Cold+FC 525 + 30 μmol m’2, PESCold - Control — 0.284, ?ES Cold - Cold+FC — 0.552, PESCold+FC - Control — 0.923,

[0114] Fig. 6), showing recovery from the night-chilling event.

[0115] 3e. Photosynthetic leaf performance of night-chilled mango trees is affected by diurnal rhythm.

[0116] To further study the mechanism involved in the inhibition of gas exchange and the subsequent leaf damage induced by cold-night stress, we tracked leaf photosynthetic performance, measuring A / Ci curves at different times after the night-chilling event. We measured 3 leaves from 2 different trees sequentially at different times (leaf 1 at 8:00 AM, leaf 2 at 10:00 AM, and leaf 3 at 12:00 PM) as a control on the first day, before exposure to chilling. During the following night, one plant was exposed to a moderate night-chilling event (7°C for 12 h), and the other was maintained under ambient temperature (Control). The same leaves that were measured on the previous day (nonstressed, steady-state levels) were measured again in the same sequence and at the same times after 1 h of photosynthetic induction (ramping of PPFD from 0 to 2000 pmol m’2s’J). As no variations were found in control non-stressed trees between the first and second day of measurements, Fig. 7A-7C present only the before-cold and after-cold A / Ci curves. The representative data show that the night-chilling event did not affect the rate of leaf photosynthetic CO2assimilation as a function of CO2concentration inside the leaf in the early morning (8:00 AM, Fig. 7A). Two hours later (10:00 AM), the night chilling substantially decreased the A / Ci relationships (Fig. 7B), whereas after an additional 2 h (12:00 PM), partial recovery was observed (Fig. 7C). To quantify the effect of a moderate night-chilling event on photosynthesis, the FvCB model parameters were fitted to the whole dataset, and average values (n = 5) are summarized in Table 1. Solving the model for the control non-stressed plants revealed that all three parameters — VcMax, J, and TPU — were very similar on the two consecutive measurement days, in all three leaves (measured at 08:00 AM, 10:00 AM, and 12:00 PM). In contrast, mango plants exposed to a moderate night-chilling event showed an interesting response. The effect of night chilling on leaf photosynthetic performance depended on the time during the diurnal cycle at which it was assessed. When cold-stressed leaves were measured at 08:00 AM, J decreased considerably (79.2 ± 3.1 before vs. 66.9 ± 5.3 pmol m’2s’1after the night chilling, PES= 0.0052), whereas VcMax and TPU were slightly lower VcMax 50.8 ± 8.7 before vs. 38.1 ± 4.4 pmol m'2s_ |after night chilling, PES= 0.3930; TPU 5.6 ± 0.1 before vs. 4.6 ± 0.6 μmol m’2s'1after night chilling, PES= 0.1983). On the other hand, when cold-stressed leaves were measured at 10:00 AM, all three parameters were substantially reduced, with VcMax decreasing from 50.4 ± 5.6 to 34.5 ± 4.3 μmol m’2s'1(PES= 0.0205), / from 73.4 ± 6.2 to 52.6 ± 5.1 μmol m'2s_ |(PES= 0.0382) and TPU from 5.0 ± 0.6 to 3.9 ± 0.3 μmol m’2s'1(PES= 0.0593) before and after the night-chilling event, respectively. When cold-stressed leaves were measured at 12:00 PM, recovery was observed in all three parameters with no statistically significant differences compared to their previous levels (VcMax was 52.6 ± 4.2 vs. 42.0 ± 3.9 μmol m’2s’1, PES= 0.3248; J was 82.9 ± 7.7 vs. 71.6 + 2.1 μmol m’2s-1, PES= 0.2304, and TPU was 5.6 + 0.5 vs. 4.9 + 0.2 μmol m’2s-1, PES= 0.5144, before and after the night-chilling event, respectively).

[0117] Table 1

[0118] The achieved results provide solid evidence that the severe leaf damage in mango trees (i.e., wilting, chlorosis, and necrosis) results from the impaired stomatai opening following cold-night-bright-day stress. Furthermore, the present invention showed that cold-night-bright-day physiological damage could be rescued by increasing stomatai conductance on the following lighted-day, using Fusicoccin, in mango trees.

[0119] Research and development of applicative cold-night-bright-day foliar treatment Based on the results, induction of stomatal opening in mango leaves following cold-night- bright-day can rescue mango trees from severe physiological damage (i.e., wilting, chlorosis, and necrosis). In addition to Fusicoccin, the present invention also screened for alternative effective compound / s. The said compounds were approved for agricultural use in the plantation (for other applications) and induce stomatal opening. The said compounds are involved in stomatal opening via distinct signaling pathways. A) 1-MCP, a competitive inhibitor of ethylene, which is involved in stomatal conductance regulation and plant stress responses. B) N-(2-Chloro-4-pyridyl)-N'-phenylurea, CPPU, is a highly active cytokinin-like plant growth regulator, which inhibits ABA-induced-stomatal- closure signal within the guard cells. C) Potassium Nitrate (KNO3) induces an increase in transpiration and photosynthesis via modulation of leaf hydraulics.

[0120] Mango leaves produce ethylene in response to a night-chilling event.

[0121] To test whether ethylene biosynthesis is induced in mango leaves in response to night chilling, ethylene levels were quantified using gas chromatography from leaves of young mango trees that were exposed to a moderate night-chilling event (Cold; 7°C for 6h) or kept under ambient growth conditions (Control). While there were no detectable levels of ethylene produced in un-stressed control plant leaves, significant levels of ethylene were observed in leaves of trees that were subjected to the moderate night-chilling event and then exposed to light (Fig. 8).

[0122] The ethylene inhibitor 1-MCP enhances leaf gas exchange and protects the photosynthetic machinery of mango trees challenged by night chill.

[0123] To test whether ethylene involves in the nigh-chilled induced impaired stomatal opening and whether this impairment can be bypassed, young mango trees were subjected to either 1) "Cold": moderate night-chilling event (7°C for 6h) OR 2) "Control": non-stressed ambient temperatures (18-22°C). On the next day, just before sunrise, cold-stressed trees were sprayed with 1-MCP ("Hervista" from Rimi LTD.). Fully developed leaves were analyzed for gas exchange -2 hours after trees were exposed to natural sunlight. The results revealed a dramatic decrease, of -47%, in leaf stomatal conductance in night-chill stressed trees when compared to non-stressed trees (gs25 + 5 vs. 47 + 5 mmol m-2s-1, Fig. 9A). As the results that were found when Fusicoccin treatment was tested (Fig. 1A- 1E), cold-night-stressed mango trees treated with 1-MCP exhibited stomatal conductance values that were indistinguishable from non-stressed control trees (Fig. 9A). Furthermore, leaves of untreated cold-stressed mango trees showed a reduction by 42% in net CO2assimilation rates compared to the control non-stressed trees (A; Cold vs. Control: 4 ± 0.5 vs. 7 ± 0.3 pmol m2s ', Fig. 9B). Conversely, net CO2assimilation rates of 1-MCP treated cold-stressed mango trees were unaffected and remained similar to those detected in the control non-stressed trees (Fig. 9B). The calculated leaf internal CO2did not show any statistically significant differences between the treatments (Fig. 9C). In addition, the overall efficiency of the photosystem II reaction center in the light, showed no statistically significant differences among treatments (Fig. 9D). Notably, 1-MCP treatment of cold- stressed trees significantly reduced the photoinhibitory effect, characterized by a decline in Fv / Fm, as observed in the night-chill-stressed trees (Fig. 9E).

[0124] Foliar spray of CPPU protects mango tree leaves from night-chilling physiological damage

[0125] To test whether foliar treatment of mango trees with the cytokinin CPPU can induce stomatai opening following night-chill and alleviate from physiological damage, young mango trees were exposed to severe night-chilling events (4 °C for 12 h) or were kept under ambient conditions. The subsequent morning, cold-stressed trees received mock or CPPU treatments (80 pM or 240 pM) with 0.025% Triton X-100 as a surfactant. A severe night-chilling event led to a pronounced decline in stomatai opening (gs, 8 ± 1 mmol m2s ' compared to 66 ± 2 mmol m2s ' in Control, Fig. 10A), and to a significant reduction in net CO2assimilation rates (-0.31 ± 0.04 pmol m2s ' compared to 6 ± 0.28 pmol m2s ' in Control, Fig. 10B). Treating the cold-stressed trees with CPPU (Both 80 pM and 240 pM) led to a significant increased stomatai conductance (19 ± 2 and 17 ± 1 mmol m2s ', respectively, Fig. 10A) and improved net CO2assimilation rates (0.37 ± 0.12 and 0.90 ± 0.17 pmol m2s ', respectively, Fig. 10B) compared to cold-stressed trees, yet still considerably lower than the control. Leaf internal CO2levels (Cz) were significantly lower in non-stressed “Control” trees (231 ± 5 pmol mol-1, Fig. 10C) than in cold-stressed trees (472 ± 18 pmol mol1). Treatment with 80 pM CPPU resulted in reduction of Ci to 356 ± 8 and to a greater extent following treatment with 240 pM CPPU, reaching 298 ± 20 pmol m2s ' (Fig. 10C). Analyses of and Fv / Fm showed the highest levels in non-stressed control mango leaves 0.17 ± 0.01; Fv / Fm'. 0.73 ± 0.02, Fig. 10D & 10E), while the lowest levels were observed in cold-stressed trees 0.06 ± 0.01; Fv / Fm: 0.42 ± 0.01, Fig. 10D & 10E). Foliar application of 80 pM or 240 pM CPPU on cold-stressed trees exhibited a positive protective effect, enhancing (0.08 ± 0.00 and 0.11 ± 0.00 respectively, Fig. 10D) and Fv / Fm'. (0.70 ± 0.02 and 0.69 ± 0.02 respectively, Fig. 10E). Overall, the effect of the higher concentration of CPPU yielded a better protective effect.

[0126] Foliar spray with KNO3enhances gas exchange and protects leaf photosynthetic apparatus following chilling events KNO3effects on plant gas exchange and chlorophyll fluorescence parameters were analyzed in young 'Omer' mango, another widespread Israeli cultivar. Mango trees were exposed to extreme night chilling (1°C for 12h). Our results show that extreme chilling night stress impairs stomatai opening the following bright day (gs: Cold 16 ± 3 mmol m2s ' vs. Control 54 ± 5 mmol m2s ', Fig. 11 A), leading to substantial reductions in net CO2assimilation rates (A: Cold 2.9 ± 0.02 pmol m2s ' vs. Control 6.3 ± 0.2, Fig. 11B). KNO3treatment on cold-stressed mango trees demonstrated a positive impact on gas exchange (gs) and carbon fixation (A), notably surpassing cold-stressed trees effects (gs: Cold+ KNO335 ± 4 mmol m2s1, Fig. 11A), (A: Cold+KNO34 ± 0.3 pmol m2s1, Fig. 11B). Intracellular CO2levels (Cz) significantly rose due to cold-night stress. KNO3induced stomatai opening, reducing Ci levels in Cold+KNCh treated trees (147 ± 14 pmol mol1). However, these levels weren't significantly lower from the control and cold- stressed trees (Cz: Control 179 ± 11 vs. Cold 188 ± 50 pmol mol-1, Fig. 11C).

[0127] For assessing KNO3's foliar spray treatment effectiveness against physiological damage caused by night chilling, leaf photosynthetic performance (Fv / Fni) was examined through chlorophyll α-based fluorescence PAM analysis. Night chilling led to a notable decline in Fv / Fm ratio compared to unstressed "Control" trees (Cold 0.47 ± 0.03 vs. Control 0.60 ± 0.03). Conversely, the foliar application of KNO3on cold-stressed trees significantly improved the Fv / Fm ratio (Cold+ KNO30.54 ± 0.03, Fig. 11E). Furthermore, a similar enhancement in photosynthetic efficiency was observed in KNCE-treated trees was highest in Control trees, followed by Cold+ KNO3and Cold-stressed trees Control 0.12 ± 0.01, Cold+ KNO30.12 ± 0.00, Cold 0.09 ± 0.01, Fig. 11D)

[0128] CONCLUSION

[0129] The present invention focused on mango, which is the most sensitive tree to cold- night-bright-day stress from the tropical and subtropical trees grown in Israel. The present invention achieved data that confirmed that induction of stomatai opening can bypass the impaired stomatai opening in the cold-stress mango leaves, reduce leaf cold damage, and improve the overall physiological state of the plant. In addition, the present invention brought supportive data proving the use of 3 different materials that induce stomatai opening in mango leaves and can be used in the plantation by foliage spray, following the cold-night stress: (1) The abscisic acid (ABA) inhibitor cytokinin (CPPU); (2) Potassium Nitrate (KNO3) and (3) The ethylene inhibitor 1-MCP.

[0130] While the invention has been described with respect to a limited number of embodiments, it will be appreciated that many variations, modifications and other applications of the invention may be made. Therefore, the claimed invention as recited in the claims that follow is not limited to the embodiments described herein.

Claims

CLAIMS1. A composition useful for preventing or treating cold-night-bright-day stress damage in tropical and subtropical trees, said composition comprising at least one stomatai opening inducer substance, and a surfactant.

2. The composition of claim 1, wherein said at least stomatai opening inducer substance is selected from the group consisting at least one of: phytohormone cytokinin-like plant growth regulator, a leaf hydraulics modulator, a competitive inhibitor of ethylene, a guard cell H+pump activator, and any combination thereof.

3. The composition of claims 1 or 2, wherein said phytohormone cytokinin-like plant growth regulator is an abscisic acid (ABA) inhibitor cytokinin.

4. The composition of claim 3, wherein said abscisic acid (ABA) inhibitor cytokinin is N-(2-Chloro-4-pyridyl)-N'-phenylurea (CPPU).

5. The composition of claim 4, wherein said CPPU is in an amount from about 80 pM to about 240 pM.

6. The composition of claims 1 or 2, wherein said leaf hydraulics modulator is Potassium nitrate (KNO3).

7. The composition of claim 6, wherein said Potassium nitrate (KNO3) is in an amount of about 100 mM.

8. The composition of claims 1 or 2, wherein said competitive inhibitor of ethylene is 1 - Methylcyclopropene (1-MCP).

9. The composition of claim 10, wherein said 1 -Methylcyclopropene (1-MCP) is in an amount of approximately 3.8% active ingredient.

10. The composition of claims 1 or 2, wherein said guard cell H+pump activator is Fusicoccin.

11. The composition of claim 12, wherein said Fusicoccin is in an amount of 10 pM.

12. The composition of any of claims 1 to 13, wherein said surfactant is 0.025% Triton X-100 or from about 0.02% Tween®20 to about 0.025% Tween®20.

13. A method of preventing or treating cold-night-bright-day stress damage in tropical and subtropical trees, wherein said method comprises steps of: a. applying foliar spray or cotton swab treatment with a composition defined in any of claims 1 to 14; wherein b. the foliar spray or cotton swab is applied on the following lighted-day just before the sunrise; and c. inducing the stomatai opening.

14. The method of claim 13, wherein said cold-night-bright-day stress damage is moderate, sever or extreme physiological damage.

15. The method of claim 13, wherein the said tropical and subtropical trees are Mangifera indica L (mango trees).

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