CN116210697B - Application of trans-cinnamic acid in improving apple tree rot resistance - Google Patents

Application of trans-cinnamic acid in improving apple tree rot resistance Download PDF

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CN116210697B
CN116210697B CN202310191157.6A CN202310191157A CN116210697B CN 116210697 B CN116210697 B CN 116210697B CN 202310191157 A CN202310191157 A CN 202310191157A CN 116210697 B CN116210697 B CN 116210697B
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cinnamic acid
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apple tree
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CN116210697A (en
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刘长海
李春容
敬媛媛
马锋旺
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Northwest A&F University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/10—Aromatic or araliphatic carboxylic acids, or thio analogues thereof; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
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    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

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Abstract

The invention belongs to the technical field of pesticides, and relates to application of trans-cinnamic acid in improving apple tree rot resistance. After t-CA is dissolved by ethanol with the volume fraction of 75%, the growth of pathogenic bacteria of apple tree rot can be effectively inhibited at the concentration of 200 ug/mL. When the apple tree canker mycelium growth is inhibited after t-CA treatment and the mycelium is deformed and the sclerotium does not develop during in vitro inoculation on a Potato Dextrose Agar (PDA) culture medium. After living inoculation on leaves and annual branches of the Malus plant Aronia, the areas of leaves and branch lesions treated by t-CA are obviously reduced. the t-CA has the characteristics of strong antibacterial effect, small environmental pollution, no residual toxicity and the like, effectively avoids the harm of chemical pesticides, can improve the resistance of apples to rot diseases, and is suitable for large-scale popularization.

Description

Application of trans-cinnamic acid in improving apple tree rot resistance
Technical Field
The invention belongs to the technical field of pesticides, and particularly relates to application of trans-cinnamic acid (t-CA) in inhibiting apple tree rot.
Background
Apples (Malus domestica borkh.) are valued by consumers for their nutritional quality, unique mouthfeel and good storage characteristics. China is the largest apple producing country in the world, and the area and the yield are the first in the world.
Apple tree rot (VALSA MAIL) is a fungal disease caused by black rot fungi, and is one of the most destructive diseases to apples. It widely exists on the tree body, and the death of branches and trunks is caused, and finally the death of the whole tree is caused, so that the yield of apples is reduced, and the traditional multipurpose chemical pesticide is used for prevention and control. Fungicides have been the primary disease prevention and control agent for the last decades. The use of the aloma-ter on apple trees in the early years has been banned by the chinese department of agriculture due to its high toxicity and high residues. Methionine acetic acid is recommended for the treatment of rot disease according to recent reports, but it has not been registered yet. Currently, apple tree rot is controlled primarily by spraying effective fungicides, manually removing diseased tissue, or pruning weak/dead branches. Although the chemical has good control effect, the residual toxicity of the medicine is large, and the medicine is harmful to human bodies, pollutes the environment, and destroys ecological balance after long-term use. Therefore, there is an urgent need to find an efficient, low-toxicity, environment-friendly bactericide to reduce the occurrence and development of apple tree rot. Biological fungicides are considered as important substitutes for classical agrochemicals and play an important role in plant disease control.
Trans-cinnamic acid (t-CA) is widely used as a raw material. The natural extracts thereof are usually present in free form or in the form of esters. It is also present in cinnamon oil, basil oil, balsam and lithocarpus leaves. Have long been used as raw materials for plant-derived pesticides, such as growth promoters, long-acting fungicides, and fruit and vegetable preservatives. In addition, it can obviously increase root system activity and strengthen plant resistance to diseases. Although t-CA has wide application, the inhibition effect on apple tree canker is not reported yet.
Disclosure of Invention
In order to study the influence of trans-cinnamic acid on apple tree rot, the invention provides the following technical scheme:
in a first aspect of the invention, the use of trans-cinnamic acid for increasing resistance to rot of apple trees is provided.
Preferably, the trans-cinnamic acid is used for preparing an inhibitor of black rot fungi.
Preferably, the inhibitor is a liquid formulation.
More preferably, the concentration of trans-cinnamic acid in the liquid formulation is 178 ug/mL-229 ug/mL.
Preferably, the trans-cinnamic acid is used for the preparation of a plant elicitor.
Preferably, the plant resistance inducer is used to increase resistance to apple tree rot.
Preferably, the trans-cinnamic acid is used for preparing an accelerator for the accumulation of salicylic acid and polyphenols in plants;
the trans-cinnamic acid is used for preparing an inhibitor of jasmonic acid accumulation in plants.
Preferably, the method comprises the steps of,
The trans-cinnamic acid is used for preparing an enhancer of phenylalanine ammonia lyase, beta-1, 3 glucanase and chitinase activity in plants;
The trans-cinnamic acid is used for preparing an accelerator of the total antioxidant capacity of plants;
The trans-cinnamic acid is used for preparing inhibitors of hydrogen peroxide and malondialdehyde in plants.
Preferably, the solution containing the trans-cinnamic acid is sprayed on the spot to prevent and treat apple tree rot.
Compared with the prior art, the invention has the following beneficial effects:
(1) The natural extract t-CA of the invention can be used as a natural substitute of a commercial bactericide, solves the problems of drug resistance and chemical residue of the existing bactericide, and prevents apple tree rot caused by black rot fungi from spreading worldwide, thus causing serious economic loss.
(2) The natural extract t-CA of the invention can promote the accumulation of SA (salicylic acid) and polyphenols (mainly phenolic acid and flavonoids) in the leaves and branches of the Aronia melanocarpa, and has important application value for inhibiting the occurrence of apple tree rot.
(3) The natural extract t-CA of the invention can enhance the activities and the total antioxidant capacity (ABTS) of Phenylalanine Ammonia Lyase (PAL), beta-1, 3 Glucanase (GLU) and Chitinase (CHT) of the leaf and branch of the Aronia melanocarpa, and reduce the accumulation of hydrogen peroxide (H 2O2) and Malondialdehyde (MDA), thereby improving the resistance of the Aronia melanocarpa to rot.
(4) The natural extract t-CA provided by the invention has low price, can reduce chemical residues and environmental pollution when being applied to production, can improve the quality of apples and economic benefit, and is a natural substitute of a commercial bactericide or a novel bactericide for controlling apple tree rot.
Drawings
FIG. 1 is a screen for effective inhibitory concentrations of t-CA of example 1 of the present invention; (a-e) a growth phenotype map of the rot fungi cultivated in ethanol culture medium with concentration of 50, 100, 150, 200 and 300ug/mL respectively; (f-j): growing phenotype images of the rot fungi respectively cultured in the t-CA culture mediums with the concentrations of 50, 100, 150, 200 and 300 ug/mL;
FIG. 2 shows the inhibition of rot on the leaf and shoot of Aronia melanocarpa by EC 50 of t-CA of example 2 of the present invention; (a) a phenotype map after inoculation of leaves with a rot pathogen; (b) statistics of disease diameter after inoculation of the leaves; (c) a phenotype chart after inoculating the branch with the rot pathogen; (d) counting the length of the disease spots of the branches;
FIG. 3 shows the SA and JA contents of the leaf and twig of Aronia melanocarpa when the rot pathogen was inoculated after the EC50 treatment of t-CA of example 3 of the present invention; (a) SA levels in catalpa bungei leaves; (b) JA levels in catalpa bungei leaves; (c) SA levels in catalpa bungei shoots; (d) JA levels in catalpa branches;
FIG. 4 shows the levels of phlorizin and catechin in the leaves and shoots of Aronia melanocarpa when treated with EC 50 of t-CA of example 4 of the present invention and inoculated with a rot pathogen; (a) levels of phlorizin in the leaves of the Aronia melanocarpa; (b) levels of catechins in the leaves of the Aronia melanocarpa; (c) levels of phlorizin in branches of Aronia melanocarpa; (d) levels of catechins in branches of Aronia melanocarpa;
FIG. 5 shows the levels of gallic acid and hyperin in the leaves and shoots of Aronia melanocarpa when the EC 50 of t-CA of example 4 of the present invention was treated and then inoculated with rot pathogen; (a) the level of gallic acid in the leaves of the Aronia melanocarpa; (b) hyperin levels in the leaves of the Aronia melanocarpa; (c) the level of gallic acid in the branches of the Aronia melanocarpa; (d) hyperin levels in branches of Aronia melanocarpa;
FIG. 6 shows the levels of gallic acid and chlorogenic acid in the leaves and shoots of Aronia melanocarpa when the EC 50 of t-CA of example 4 of the present invention was treated and then inoculated with rot pathogens; (a) level of chlorogenic acid in the leaves of Aronia melanocarpa; (b) levels of coumaric acid in the leaves of the Aronia melanocarpa; (c) the level of chlorogenic acid in the branches of the Aronia melanocarpa; (d) levels of coumaric acid in the branches of the Aronia melanocarpa;
FIG. 7 shows the levels of ferulic acid and rutin in the leaves and shoots of the Aronia melanocarpa when the rot pathogen was inoculated after EC 50 treatment of t-CA of example 4 of the present invention; (a) levels of ferulic acid in the leaves of Aronia melanocarpa; (b) levels of rutin in the leaves of Aronia melanocarpa; (c) levels of ferulic acid in branches of Aronia melanocarpa; (d) levels of rutin in the branches of Aronia melanocarpa;
FIG. 8 shows the anthocyanin B 2 and anthocyanin B 1 levels in the leaves and shoots of the Aronia melanocarpa when the EC 50 of t-CA of example 4 of the present invention was treated and then inoculated with the rot pathogen; (a) The level of anthocyanin B 2 in the lamina of the liriodendron; (b) The level of anthocyanin B 1 in the lamina of the liriodendron; (c) The level of anthocyanin B 2 in the branches of the Aronia melanocarpa; (d) The level of anthocyanin B 1 in the branches of the Aronia melanocarpa;
FIG. 9 shows the GLU, CHT, and PAL levels of the lamina and twig of Sorbus pohuashanensis when re-inoculated with rot pathogen after treatment with EC 50 of t-CA of example 5 of the present invention; (a-c) glut, CHT, and PAL activity in the lamina of the Aronia melanocarpa, respectively; (d-f) GLU, CHT, and PAL activities in the branches of the Aronia melanocarpa, respectively;
FIG. 10 shows the levels of ABTS, H 2O2 and MDA in the lamina and twig of Aronia melanocarpa when the EC 50 of t-CA of example 6 of the present invention was treated and then inoculated with rot pathogen; (a-c) are ABTS, H 2O2 and MDA activities in the lamina of Aronia melanocarpa respectively; (d-f) ABTS, H 2O2 and MDA activities in the branches of Aronia melanocarpa, respectively.
Detailed Description
In order that those skilled in the art will better understand the technical scheme of the present invention, the present invention will be further described with reference to specific embodiments and drawings.
The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Rot caused by fungi v.mali is one of the most damaging apple diseases in apple major producing areas in our country and other eastern asia countries. The pathogen typically passes through the natural small Kong Qinru apple tree in the wound or bark and induces severe tissue maceration and necrosis. Trans-cinnamic acid (t-CA) is a phenylpropane compound with a chemical formula of C 9H8O2, a structural formula shown as a formula I, and naturally exists in a plurality of plants, has wide biological activities including antioxidant and antibacterial activities, and has great application potential in foods and cosmetics. Based on the above, the invention relates to the application of trans-cinnamic acid (t-CA) which is a natural extract in improving the rot resistance of apple trees.
Example 1
Effective antibacterial concentration
T-CA was weighed and dissolved with 75% ethanol to 50mg/mL as a mother liquor for ready use. It was diluted into PDA medium, 5 concentration gradients were set, 50, 100, 150, 200, 300ug/mL respectively, and the control was diluted to the same concentration with 75% ethanol by volume fraction. After the culture medium is solidified, it is sealed with a sealing film. Opening after three days, inoculating activated rot pathogen after ensuring no pollution, and culturing in a biochemical incubator at 25 ℃.
The results show (figure 1) that the antibacterial concentration range of cinnamic acid is 178 ug/mL-229 ug/mL, and the effective antibacterial concentration EC 50 is 200ug/mL. At this concentration, the growth of the rot pathogen is effectively inhibited.
Example 2
Inhibition effect of t-CA on rot diseases on leaf and branch of Aronia melanocarpa
(1) Blade and branch inoculation treatment
Subculturing the tissue-cultured catalpa bungei every 4 weeks. (Aronia melanocarpa seed subculture medium: 4.43g MS powder +30g sucrose +8g agar +0.3 mg/L6-BA +0.2mg/L IAA, pH was adjusted to 5.8-6.0 with NaOH, and solvent was water) the roots of the catalpa bungei were cultured on the rooting medium for 40 days, (Aronia melanocarpa seed rooting formulation: 2.22g MS powder +15g sucrose +8g agar +0.6 mg/L6-BA +0.6mg/LNAA, pH was adjusted to 5.8-6.0 with NaOH, and solvent was water). Then transferring into flowerpot (8X 8 cm) containing nutrient soil, vermiculite and perlite with mass ratio of 3:1:1, culturing in incubator for 30 days, transferring plant into larger plastic pot (30X 18 cm), filling with forest soil, sand and organic fertilizer (volume ratio of 5:1:1), storing in greenhouse, and inoculating leaf with consistent growth condition. The culture environment temperature of the tissue culture seedlings is 23 ℃, and the light period of 16 hours of illumination/8 hours of darkness is realized. When the plant grows to about 20cm, taking middle and upper blades with the same size for inoculation. The 1-year-old Aronia melanocarpa shoots with consistent growth vigor were collected from a national academy of sciences of northwest agriculture and forestry science and technology test station of China Yang Ling (34 DEG 20'N,108 DEG 24' E).
Three days before inoculation, in order to activate the rot germs, the stored rot germs are inoculated onto a new PDA culture medium again, and are cultured on the PDA culture medium at 25 ℃ for 3 days.
Before inoculation, the fully developed apple leaves were surface sterilized with a 0.6% sodium hypochlorite solution by mass and sprayed with sterile water three times. The petioles were cut with scissors to about 0.5cm and wrapped with moist cotton, placed in a plastic tray, sprayed on the leaves with 200ug/mL cinnamic acid for 6 hours after the surface moisture was dried, sprayed on the leaves with 200ug/mL ethanol (solvent for cinnamic acid) for 6 hours as a control, and then agar blocks (5 mm each) were removed from the edges of growing colonies and placed on the backs of the leaves by needle punching. The shoots were cut into sections 20cm long and the ends of the small branches were sealed with wax. Washing with tap water, immersing in sodium hypochlorite with mass fraction of 0.6% for 6min, and washing with sterile water three times. After the surface water of the branches is dried, 200ug/mL of cinnamic acid is sprayed on the branches for 6 hours, 200ug/mL of ethanol (solvent for dissolving the cinnamic acid) is sprayed on the branches for 6 hours as a control, and each branch segment is perforated by a puncher. Immediately covering with ethylene film to maintain 25 deg.C humidity, placing into a dark incubator, culturing inoculated leaves for 3 days, and culturing inoculated shoots for 4 days. Leaf lesion size was measured by the crossover method and the lesion area was calculated from the diameter. The total length of the longitudinal lesions along the shoots is measured directly to determine the size of the lesions. Leaves at 3 days and bark at 2 days were collected, frozen in liquid nitrogen, and stored at-80 ℃.
The experimental results are shown in fig. 2, (a) is a phenotype chart after leaf inoculation, (c) is a phenotype chart after branch inoculation, (b) is a disease diameter statistic after leaf inoculation, and (d) is a disease length statistic of the branches. The diseased areas of the leaves and shoots treated with trans-cinnamic acid (t-CA) were significantly smaller than the diseased areas of the control (FIGS. 2a, c), and the sizes of the diseased areas of the leaves and shoots treated with t-CAEC 50 were counted to be 47.8% and 49.2% smaller than the control, respectively.
Example 3
The t-CA treatment increases the SA content of the Aronia melanocarpa and reduces the JA content.
Leaves at 3 days of treatment and bark at 2 days of treatment in example 2 were collected, frozen in liquid nitrogen, and stored at-80 ℃.
Cleaning a mortar, a hammer and a spoon, wiping, wrapping with tinfoil paper, baking in an oven at 180 ℃ for 3 hours, precooling with liquid nitrogen, grinding a sample into powder, weighing 0.1g in a 2mL siliconized centrifuge tube, adding precooled extracting solution at-20 ℃ (prepared extracting solution: methanol: isopropanol: acetic acid=20:79:1, volume ratio), vibrating and swirling for 5min, and processing overnight at-20 ℃. Then it was centrifuged at 12,000Xg for 10min in a centrifuge pre-chilled at 4℃and the supernatant was filtered through a 0.22um organic filter in a small palm flask and analyzed by HPLC-MS assay.
The experimental results are shown in fig. 3, (a) SA level in the lamina of the Aronia melanocarpa; (c) SA levels in branches of the Aronia melanocarpa; (b) JA levels in the leaf of the Aronia melanocarpa; (d) JA level in branches of Aronia melanocarpa. After t-CAEC 50 treatment, the SA content in leaves and shoots was increased by 47.4% and 18.9% respectively (figures 3a, c) while the JA content was decreased by 21.3% and 28.7% (figures 3b, d), antagonism was seen between SA and JA.
Example 4
At the time of V.mali infection, t-CA treatment increased polyphenol content in the leaves and bark of Aronia melanocarpa
Leaves at 3 days of treatment and bark at 2 days of treatment in example 2 were collected, frozen in liquid nitrogen, and stored at-80 ℃.
Cleaning a mortar, a hammer and a spoon, wiping, packaging with tinfoil paper, baking in an oven at 180 ℃ for 3 hours, precooling with liquid nitrogen, grinding a sample into powder, weighing 0.1g, adding 1mL of an extracting solution (prepared extracting solution: methanol: water: formic acid=25:24:1) into a 2mL siliconizing centrifuge tube, performing ultrasonic treatment (25 ℃ at 40Hz, 100W) for 20min, and shaking 150 r.min -1 at 25 ℃ for 20min.1000 Xg was centrifuged for 15min and the supernatant was filtered through a 0.22um organic filter in a small palm flask and analyzed by HPLC-MS.
The experimental results are shown in fig. 4 to 8. After apple tree rot germs are inoculated, the polyphenol content in the leaves and twigs of the Aronia melanocarpa treated by t-CA acid EC 50 is obviously higher than that of a control, in particular to gallic acid and p-coumaric acid in phenolic acid. Leaves and shoots increased 65% and 78.6% gallic acid (fig. 5a, c) and 66.1% and 38.7% p-coumaric acid (fig. 6b, d), respectively. In addition, ferulic acid was also increased by 7.9% and 25.1% in leaves and shoots, respectively (fig. 7a, c), while chlorogenic acid was increased by 14.3 and 1.3%, respectively (fig. 6a, c). Among flavonoids, under the treatment of cinnamic acid EC 50, phlorizin in leaves and branches is increased by 1.4% and 47.4% (fig. 4a and c), hyperin is increased by 30.8% and 70% (fig. 5B and d), procyanidine B 2 is increased by 17% and 56.3% (fig. 8a and c), and catechin (fig. 4B and d), rutin (fig. 7B and d) and procyanidine B 1 (fig. 8B and d) also show increasing trends to different degrees.
Example 5
Determination of enzyme Activity after t-CA treatment at V.mali infection
Leaves at 3 days of treatment and bark at 2 days of treatment in example 2 were collected, frozen in liquid nitrogen, and stored at-80 ℃.
The mortar, hammer and spoon were cleaned and wiped dry, then covered with tinfoil paper, baked in an oven at 180℃for 3 hours, pre-cooled with liquid nitrogen, the sample was ground to powder, 0.1g was weighed into a 2mL siliconized centrifuge tube, and the assay was performed as a kit (Suzhou Ming Biotechnology Co., ltd.).
(1) T-CA treatment increased the activity of the PAL, GLU and CHT of the Sorbus pohuashanensis following v.mali infection.
Experimental results referring to fig. 9, after inoculation with apple tree canker, GLU content in leaves and shoots after treatment with t-CAEC 50 was increased by 24.8% and 39.2% respectively (fig. 9a, d), CHT content was increased by 18.8% and 8.5% respectively (fig. 9b, e), and PAL content was increased by 6.1% and 8.6% respectively (fig. 9c, f). the content of PAL, GLU and CHT in the cotyledon and branch of Aronia melanocarpa can be increased after t-CA treatment, and the resistance of Aronia melanocarpa to rot can be improved.
(2) T-CA treatment increases ABTS activity and reduces accumulation of H 2O2 and MDA
The ABTS content in leaves and shoots treated with t-CA EC 50 was 24.1% and 21.7% higher than the control (fig. 10a, d), respectively, whereas the H 2O2 and MDA contents were the opposite, the H 2O2 content in leaves and shoots treated with EC 50 was reduced by 34.6% and 21.75% respectively (fig. 10b, e), and the MDA content was reduced by 22.9% and 33.3% respectively (fig. 10c, f). The result shows that the invasion of the leaf blade of the Aronia melanocarpa after t-CA treatment to the rot is smaller than that of the control, and the leaf blade of the Aronia melanocarpa shows good disease resistance.
It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (1)

1. The application of trans-cinnamic acid in improving apple tree rot resistance is characterized in that the trans-cinnamic acid is used for preparing an accelerator for accumulating salicylic acid and polyphenols; or (b)
The trans-cinnamic acid is used for preparing an inhibitor of jasmonic acid accumulation; or (b)
The trans-cinnamic acid is used for preparing enhancers of phenylalanine ammonia lyase, beta-1, 3 glucanase and chitinase activity; or (b)
The trans-cinnamic acid is used for preparing an accelerator with total antioxidant capacity; or (b)
The trans-cinnamic acid is used for preparing inhibitors of hydrogen peroxide and malondialdehyde.
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