Application of citral in inhibition of Botrytis cinerea and preservation of picked peppers

Abstract

The invention relates to the technical field of hot pepper preservation, in particular to application of citral in inhibition of Botrytis cinerea and preservation of picked hot peppers. According to the method, citral fumigation can effectively delay the increase of the weight loss rate and the rotting rate of the chilies, and the rotting index of the chilies is obviously lower than that of a positive control 1-MCP group (Plt; the red value and the reddening index of the peppers in the citral fumigation group are lower than those of the control group, which indicates that the citral treatment can inhibit chlorophyll degradation and delay the color change of the peppers in the storage process. The citral treatment can effectively delay the red turning of the chilies and delay the red turning period of the chilies.

Description

Technical Field

[0001] The invention relates to the technical field of pepper preservation, and more specifically to application of citral in inhibiting Botrytis cinerea and preserving pepper after harvest. Background Art

[0002] Citral 10 H 16 O, 3,7-dimethyl-2,6-octadiene-1-aldehyde) is also called geranyl aldehyde or citronellal, a colorless or light yellow oily liquid with a strong lemon aroma. Natural citral is an acyclic monoterpenoid compound with cis-trans isomers, of which cis-citral is also called citral b or neral, and trans-citral is also called citral a or geranial. Natural citral is commonly found in aromatic plants such as Litsea cubeba (Lour.) Pers., Cymbopogoncitratus (DC.) Stapf, Verbena officinalis L., C. tortilis (J. Presl) A. Camus, and Cinnamomum camphora (L.) J. Presl. Existing technical research has found that the combined treatment of tomatoes with 1-MCP and 1×MFC citral can significantly reduce the incidence of tomatoes during storage; 40μg / mL citral immersion treatment can effectively reduce the weight loss rate and rot index of strawberries; citral treatment can significantly reduce the rot rate of kiwifruit and maintain the storage quality of kiwifruit; after kiwifruit is soaked in 0.8μL / mL citral, the weight loss rate and respiration rate of the fruit are reduced, delaying the post-harvest ripening time of kiwifruit.

[0003] Storage and preservation of peppers after harvest is one of the main methods to reduce losses and preserve value. However, peppers are susceptible to infection by pathogens, causing various diseases and rotting and deterioration after harvest. Therefore, it is necessary to take some measures to prevent peppers from rotting and deteriorating during storage and transportation. No prior art has studied the effects of citral on Botrytis cinerea and postharvest preservation of peppers. Summary of the invention

[0004] The invention aims to provide application of citral in inhibiting Botrytis cinerea and preserving pepper after harvest.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] Application of citral in inhibiting Botrytis cinerea.

[0007] Application of citral in postharvest preservation of peppers.

[0008] In a preferred embodiment, citral is applied by fumigation.

[0009] In a preferred embodiment, the concentration of citral is 0.15-0.30 mg / mL.

[0010] In a preferred embodiment, citral reduces the weight loss rate and decay rate of pepper.

[0011] In a preferred embodiment, citral reduces the red value and redness index of pepper.

[0012] In a preferred embodiment, citral inhibits the degradation of chlorophyll.

[0013] In a preferred embodiment, citral delays the increase of the Vc content of pepper.

[0014] For the first time, it was found that citral fumigation treatment has a good preservation effect on post-harvest peppers and significantly reduces the rot index of post-harvest peppers.

[0015] The beneficial effects of the present invention are:

[0016] In the present invention, citral can effectively inhibit the proliferation and growth of Botrytis cinerea.

[0017] Citral fumigation can effectively delay the increase of pepper weight loss rate and decay rate, and its decay index is significantly lower than that of the positive control 1-MCP group (P<0.05).

[0018] The red value and red turning index of peppers in the citral fumigation group were lower than those in the control group, indicating that citral treatment could inhibit chlorophyll degradation and delay the color change of peppers during storage.

[0019] The Vc content of peppers in the citral treatment group was significantly lower than that in the control group, which shows that citral treatment can effectively delay the redding of peppers and delay the peppers entering the redding period. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The colony morphology, spore morphology (40×) and inoculation symptoms (5d) of Botrytis cinerea;

[0021] Figure 2 It is agarose gel electrophoresis band;

[0022] Figure 3 Phylogenetic tree analysis of Botrytis cinerea;

[0023] Figure 4 The inhibitory effect of citral on Botrytis cinerea;

[0024] Figure 5 The effect of citral fumigation on pepper weight loss rate (A), decay index (B) and redness index (C);

[0025] Figure 6 The effect of citral fumigation on the appearance of peppers during storage;

[0026] Figure 7 The effect of citral fumigation on the color of pepper fruit; A: yellow value; B: red value; C: blue value;

[0027] Figure 8 Effects of citral fumigation on pepper respiration intensity (A), chlorophyll content (B), Vc content (C) and capsanthin (D). DETAILED DESCRIPTION

[0028] The present invention is not limited to the following specific embodiments. A person skilled in the art can implement the present invention by adopting other specific embodiments according to the content disclosed in the present invention, or any simple changes or modifications made by adopting the design structure and ideas of the present invention fall within the protection scope of the present invention. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The "Xiangyan 55" pepper used in the present invention was picked from a pepper plantation in Hunan Province. Citral (purity ≧97%) was purchased from Yongzhou Shan Spice Co., Ltd. Potato dextrose agar (PDA) medium and potato dextrose water (PDB) liquid medium were purchased from Beijing Spectroscopy Technology Co., Ltd. Soil temperature 80 was purchased from Shanghai Hushi Experimental Equipment Co., Ltd., and propidium iodide staining solution (PI), Calcofluor fluorescent brightener staining solution (CFW) and 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent dye were all purchased from Sigma (Shanghai). Omega HP fungal DNA extraction kit (item number D3195) was purchased from Beijing Noble Technology Co., Ltd. MDA, CAT, POD, SOD, and GPX kits were purchased from Beijing Solebow Technology Co., Ltd. Other chemical reagents were of analytical grade and commonly available commercial products. Trizol kit was used to extract total RNA (Life technologies, USA). Vazyme reverse transcription kit (HiScript III RTSuperMix for qRCR (+gDNA wiper) was used. The Botrytis cinerea (accession number: OQ592390) used was a known strain.

[0029] The experimental data were plotted using Prism 8 software, and one-way analysis of variance (Duncan test) was analyzed using SPSS25.0 software. The data were expressed in the form of "mean ± standard deviation" and annotated with "letter notation". P < 0.05 indicated a significant difference between the experimental samples. The fluorescence staining rate was calculated using Fiji software.

[0030] Example 1

[0031] 1. Isolation and purification of Botrytis cinerea

[0032] The tissue block separation method was used to wash peppers with disease symptoms with sterile water, and the edge tissue blocks (0.5 cm × 0.5 cm) of the diseased tissue were cut off with sterile scissors on a clean bench, soaked in 75% alcohol for 20 seconds, rinsed twice with sterile water, drained, placed in the center of a PDA culture dish, and cultured in a 28°C incubator for 72 hours. The three-point method was used to separate and purify the peppers into single colonies.

[0033] 2 Pathogenicity detection of Botrytis cinerea

[0034] Prepare spore suspension (1×10 5 The peppers were soaked in 75% ethanol for 30 seconds, rinsed with sterile water 4 times and dried. A sterile inoculation needle was used to make an inoculation hole (4 mm × 5 mm) on the surface of the pepper fruit, and 15 μL of 1 × 10 5 The spore suspension was treated with sterile water as a negative control. The growth of pathogenic fungi in pepper wounds was observed every other day.

[0035] The results are as follows Figure 1 As shown in Figure 1. Botrytis cinerea was isolated from the pathogenic junction of peppers with disease symptoms. The pathogenicity of Botrytis cinerea to peppers (cultured for 5 days) is shown in Figure 1. Figure 1 As shown, the symptoms of pepper disease are not obvious.

[0036] 3 Identification of pathogenic fungi in postharvest peppers

[0037] (1) Morphological identification

[0038] The purified strains were inoculated onto PDA plates and cultured at 28°C. The growth of the strains, including the color and texture of the colonies, was observed every day. The mycelium and spore morphology of the pathogenic fungi were observed using an optical microscope and photographed and recorded. According to the Fungal Identification Manual and related literature, preliminary identification of the observed fungi was performed.

[0039] The colony morphology and conidia morphology of Botrytis cinerea on PDA are shown in Table 1.

[0040] Table 1 Morphological identification results of Botrytis cinerea

[0041]

[0042] According to the Manual of Fungal Identification and Chinese Fungi, it was preliminarily identified as the genus Botrytis.

[0043] (2) Molecular Biological Identification

[0044] After the purified strain was activated, DNA was extracted and ITS1 / ITS4 (fungus) primers were used to amplify the sample DNA ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). PCR experiments were performed using TOYOBO's high-fidelity PCR polymerase product KOD OneTM PCR Master Mix (Cat. No. KMM-101).

[0045] The reaction system is as follows:

[0046] Table 2 Reaction system

[0047]

[0048] After the reaction, the PCR product was verified by 1% agarose gel electrophoresis. The PCR amplification product was sequenced using an ABI 3730XL gene sequencer with bidirectional primers, and the expected band size was approximately 600 bp. After sequencing, the sequencing results were spliced ​​using DNAMAN software, and the sequences were aligned using the Blast system in the NCBI database. A phylogenetic tree was constructed using the neighbor-joining method using MEGA software, and Bootstrap support analysis was performed, with the number of repetitions set to 1000.

[0049] PCR amplification test Figure 2 The gene length obtained by the strain was 543bp.

[0050] The sequencing results of the PCR products were compared with the rDNA-ITS gene sequences of strains with high similarity in NCBI, and a phylogenetic tree was constructed using MEGA11 software ( Figure 3 ).Depend on Figure 3 It was found that the strain (Accession No.: OQ595254) was Botrytis cinerea.

[0051] 4. Determination of the inhibitory effect of citral on Botrytis cinerea by disc diffusion method

[0052] Scrape spores from the pathogenic fungus plate cultured at 28°C for 7 days with sterile saline and adjust the concentration to 1.0×10 5 / mL, prepare spore suspension, add 1mL spore suspension to 20mL melted PDA medium at about 50℃, mix well and pour 3mL into PDA medium (forming a double-layer agar medium), after the plate solidifies, place a 4mm sterile filter paper in the center of the plate, add 10μL citral on the sterile filter paper, set up a negative control group (sterile water) and a positive control group (0.20mg / mL triadimefon), culture in a 28℃ incubator, observe after 3 days, record the results, and each treatment group has 3 parallels.

[0053] The inhibitory effect of citral on Botrytis cinerea Figure 4 and as shown in Table 3. The results show that citral has a good inhibitory effect on Botrytis cinerea.

[0054] Table 3 Antibacterial diameter of citral on postharvest pepper pathogenic fungi

[0055]

[0056] Note: Different letters indicate significant differences, P<0.05

[0057] 5. Citral fumigation induces pepper to produce resistance to Botrytis cinerea

[0058] Lesion diameter and incidence

[0059] After washing the peppers with tap water, wipe the surface with 75% ethanol, rinse twice with sterile water, dry, and use a sterile needle to make two wounds of the same size on each pepper. Inoculate 10 μL of a 1×10 8 The suspension of Botrytis cinerea spores was placed in a fresh-keeping box (28×14×7 cm 3 ), 3g cotton was placed in each fresh-keeping box, citral was dripped into the cotton to make the citral concentration in the fresh-keeping box 0 (blank control group CK, injury inoculation, no citral), MIC i.e. 0.30mg / mL, and cultured in a constant temperature incubator at 28℃. The diameter of pepper lesions was measured by the cross method every 2 days. The disease was considered to occur when the lesion diameter was not less than 2mm, and the incidence rate was calculated as follows.

[0060] Incidence rate % = number of peppers with disease / total number of peppers inoculated × 100%

[0061] The results showed that the lesion diameter and incidence rate of peppers in different treatment groups were stored for 4 days, and peppers in all treatment groups began to get sick. On the 6th day of storage, the lesion diameters of CK and MIC citral groups were 29.69±1.32mm and 11.23±0.49mm, and the incidence rates were 99.00±1.83% and 35.00±4.56%, respectively. During the entire storage period, the lesion diameter and incidence rate of peppers in the MIC citral group were significantly lower than those in the CK group (P<0.05).

[0062] Example 2

[0063] Study on the effect of citral on postharvest preservation of peppers

[0064] After washing the peppers with tap water, wipe the surface with 75% ethanol solution, rinse twice with sterile water, dry them and put them in a fresh-keeping box (28×14×7cm 3 ), 2g of cotton was put into each fresh-keeping box, and citral was dripped into the cotton to make the citral concentrations in the fresh-keeping box be 0 (blank control group CK2, without citral), MIC, 2×MIC, i.e. 0, 0.30, 0.60mg / mL, respectively. The commercial general-purpose fruit and vegetable preservative 1-methylcyclopropene (1-MCP, 1mg / L) was used as the positive control group. The boxes were stored in a constant temperature box at 28°C and 85-90% humidity. The relevant indicators were measured every 2 days and recorded by taking photos.

[0065] (1) Determination of pepper weight loss rate

[0066] The weight loss rate of peppers was calculated by weighing method. Thirty peppers were taken from each group, and the initial weight of 30 peppers was weighed respectively. The weight of the corresponding peppers was weighed every 2 days. The weight loss rate of peppers was calculated by dividing the difference between the initial weight of peppers and the weight of peppers at different storage periods by the initial weight.

[158] .

[0067] (2) Determination of pepper color

[0068] Thirty peppers were selected from each group, and the peel color was measured using a Lovibond colorimeter. The results were expressed as yellow, red, and blue values.

[0069] (3) Determination of pepper decay index

[0070] The pepper rot index is calculated according to formula (6-2).

[0071]

[0072] The decay levels in this test are assessed as follows:

[0073] Level 0: No decay;

[0074] Level 1: The rotten area accounts for 0-10% of the total area of ​​peppers;

[0075] Level 2: The rotten area accounts for 11-30% of the total pepper area;

[0076] Level 3: The rotten area accounts for 31-50% of the total pepper area;

[0077] Level 4: The rotten area accounts for 51-100% of the total area of ​​peppers.

[0078] (4) Determination of redness index

[0079] According to the reddening area on the surface of pepper fruit, it is divided into 4 levels: Level 0 (green fruit without lesions or rotten spots), Level 1 (reddening area <1 / 3), Level 2 (1 / 3≤reddening area<2 / 3), Level 3 (reddening area≥2 / 3). The calculation formula is shown in formula (6-3).

[0080]

[0081] (5) Determination of respiratory intensity

[0082] Determination by static method

[160] .

[0083] (6) Determination of chlorophyll content

[0084] Determination by spectrophotometry

[160] .

[0085] (7) Determination of vitamin C content

[0086] Determination by 2,6-dichlorophenol indophenol titration

[160] .

[0087] (8) Determination of capsanthin

[0088] Pepper (1g) was ground by adding 4mL acetone, and the homogenate was diluted to 50mL with acetone, extracted with ultrasonic wave at 42℃ and 60W for 30min, and the filtrate was collected by filtration. 2mL of the above filtrate was diluted to 25mL, and its absorbance was measured at a wavelength of 460nm, and the acetone was adjusted to zero. The content of capsanthin was expressed as color value and calculated according to formula (6-4).

[0089]

[0090] Where: E is the color value; A is the absorbance of the actual test sample; F is the dilution factor; m is the mass of the sample (ɡ).

[0091] In all treatment groups, the weight loss rate of peppers showed an increasing trend during the entire storage period ( Figure 5 A), the weight loss rate of peppers in CK2 group was always the highest. On the 6th and 8th days of storage, the weight loss rate of 1-MCP group was the lowest, while there was no significant difference between the citral treatment group and CK2 group (P>0.05).

[0092] On the 6th day of storage, the CK2 group and the 1-MCP group began to decay, while the citral treatment group began to decay on the 8th day of storage. The decay index of all treatment groups increased with the storage time. On the 10th day of storage, the decay index of the CK2 group was the highest, which was 30.63±2.65%, followed by 1-MCP (21.25±1.77%), MIC citral group (7.50±3.54%) and 2×MIC citral group (12.50±3.54%) ( Figure 5 B), among which, the decay index of MIC and 2×MIC citral groups was not only significantly lower than that of CK2 group (P<0.05), but also significantly lower than that of 1-MCP group (P<0.05).

[0093] The redness index of all treatment groups increased with the storage time. During the entire storage period, the redness index of the CK2 group was always the highest, and the 1-MCP group was always the lowest, followed by the MIC citral group. Citral treatment can inhibit the accumulation of capsanthin and delay the color change of the fruit to red ( Figure 5 C).

[0094] The appearance of all treatment groups was Figure 6 shown.

[0095] During the storage of pepper, the yellow value ( Figure 7 A) and red value ( Figure 7 B) rises, blue value ( Figure 7 C) first increased and then decreased. On the 8th day and beyond, the yellow value of the CK2 group was always lower than that of the other treatment groups, while the red value and blue value were always higher than those of the other treatment groups. On the 10th day, the yellow value and red value of the citral treatment group had no significant difference with the 1-MCP group (P>0.05), and the blue value of the 2×MIC citral group had no significant difference with the 1-MCP group (P>0.05) ( Figure 7 ), the changes in the redness index during storage are similar to the changes in the redness value.

[0096] The respiration rate of peppers increased, metabolism accelerated, nutrients and water were lost, and the quality of peppers decreased. During the entire storage period, the respiration rate of peppers in all treatment groups showed a trend of first decreasing and then increasing, but the overall trend was increasing ( Figure 8 A); On the 6th and 8th days of storage, the respiration intensity of peppers in the CK2 group was significantly higher than that in the other treatment groups (P<0.05), and on the 6th day of storage, the respiration intensity of peppers in the 2×MIC citral treatment group was the lowest; on the 8th day of storage, the respiration intensity of peppers in the MIC and 2×MIC citral groups had no significant difference from that in the 1-MCP group (P>0.05), indicating that citral treatment can effectively reduce the respiration intensity of peppers during storage.

[0097] With the extension of storage time, the chlorophyll content of peppers in all treatment groups continued to decrease, and the chlorophyll content of peppers in CK2 group was always lower than that in other treatment groups ( Figure 8 B). After storage for 6 days or more, the chlorophyll content of peppers in other treatment groups was significantly higher than that in CK2 group (P<0.05). On the 10th day of storage, the chlorophyll content of peppers in 2×MIC citral group was the highest, 0.1098 mg / L, 2.54 times that of CK2 group, followed by 1-MCP group, 0.0830 mg / L, 1.92 times that of CK2 group, then MIC citral group, 0.0684 mg / L, 1.58 times that of CK2 group, and finally CK2 group, 0.0432 mg / L. It can be seen that citral treatment can effectively delay the degradation of chlorophyll in peppers during storage.

[0098] During the entire storage period, the Vc content of peppers in all treatment groups showed a trend of increasing first and then decreasing. On the 4th day of storage, the Vc content in peppers reached a peak, and the CK2 group had the highest content. On the 8th and 10th days of storage, the Vc content in CK2 peppers was significantly higher than that in other treatment groups (P<0.05) ( Figure 8 C).

[0099] As peppers ripened, the capsanthin content in the CK2 group and the citral treatment group gradually increased. However, in the late storage period (8th to 10th day), the increase in capsanthin content in the citral group was lower than that in the CK2 group (P<0.05). On the 10th day of storage, the capsanthin content in the 2×MIC and MIC citral groups was only 65.74% and 22.85% of that in the CK2 group, respectively. Figure 8 D), it can be seen that citral treatment can effectively delay the conversion of capsanthin during pepper storage.

[0100] In the present invention, citral fumigation can effectively delay the increase of pepper weight loss rate and decay rate. After storage for 10 days, the weight loss rates of peppers in MIC and 2×MIC citral groups were 11.77% and 14.07%, respectively, which were 2.33% and 16.76% higher than those in CK2 group, respectively; the decay indexes of peppers in MIC and 2×MIC citral groups were 7.5% and 12.5%, respectively, which were 75.51% and 59.19% lower than those in CK2 group, and 64.71% and 41.18% lower than those in 1-MCP group, respectively. It can be seen that the decay indexes of peppers treated with MIC and 2×MIC citral fumigation were significantly lower than those of the positive control 1-MCP group when stored at 28°C for 10 days (P<0.05).

[0101] The red value and redness index of peppers in the citral fumigation group were lower than those in the CK2 group (P<0.05), and the redness became slower. The color change may be caused by chlorophyll degradation. The chlorophyll content of all treatment groups decreased during storage, while the chlorophyll content of peppers in the citral treatment group decreased more slowly than that in the CK2 group, indicating that citral treatment can inhibit chlorophyll degradation and delay the color change of peppers during storage.

[0102] The Vc content of peppers in all treatment groups showed a gradual upward trend during storage. On the 4th day of storage, the Vc content in the CK2 group reached the highest value, which may be due to the accumulation of Vc in the peppers in the CK2 group as they entered the red-turning stage. On the 8th and 10th days of storage, the Vc content of peppers in the citral-treated group was significantly lower than that in the CK2 group (P<0.05). This trend was consistent with the trend of capsanthin, indicating that citral treatment can effectively delay the red-turning of peppers and delay the peppers from entering the red-turning stage.

[0103] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Citral inhibits Botrytis cinerea Botrytis cinerea Applications on.

2. The use according to claim 1, characterized in that: Citral is applied by fumigation.

3. The use according to claim 1, characterized in that: The concentration of citral is 0.15-0.30 mg / mL.

4. Application of citral in post-harvest preservation of peppers.

5. The use according to claim 2, characterized in that: Citral is applied by fumigation.

6. The use according to claim 2, characterized in that: The concentration of citral is 0.15-0.30 mg / mL.

7. The use according to claim 2, characterized in that: Citral reduces the weight loss rate and decay rate of peppers.

8. The use according to claim 2, characterized in that: Citral reduces the red value and redness index of peppers.

9. The use according to claim 2, characterized in that: Citral inhibits the degradation of chlorophyll.

10. The use according to claim 2, characterized in that: Citral delays the increase of vitamin C content in pepper.

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