Application of porphyrin compound TMePPTos in prevention and treatment of citrus penicilliosis

Abstract

The invention discloses an application of a porphyrin compound TMePPTos in prevention and treatment of citrus penicilliosis. According to the application, the porphyrin compound is used for preventing and treating the citrus penicilliosis for the first time, and it is found that 5 ppm of the porphyrin compound TMePPTos can effectively inhibit growth of penicillium italicum and penicillium digitatum. The invention provides a new choice for preventing and treating citrus penicilliosis, provides a new green bactericide and a new green prevention and control idea for postharvest preservation of citrus and even other fruits and vegetables, and has important application value in the aspects of reducing the use of harmful chemical preservatives and green sustainable development of citrus storage and preservation.

Description

Technical Field

[0001] This invention belongs to the field of plant protection, specifically relating to the application of the porphyrin compound TMePPTos in the control of Penicillium citrus. Background Technology

[0002] Citrus is the largest fruit category in my country, with a national planting area of ​​nearly 50 million mu (approximately 3.3 million hectares) and a total output of about 60 million tons. Most citrus fruits mature after October. To extend their shelf life and facilitate off-peak sales, harvested citrus fruits need to be stored and preserved. The post-harvest rot rate of citrus is about 25%, causing significant economic losses for fruit farmers. Post-harvest fungal infection is a major factor affecting citrus spoilage and decay. More than 20 major post-harvest diseases of citrus have been reported, the most important being Penicillium mold (…). Penicillium spp.), stem rot ( Phomopsis citri), black rot ( Alternaria spp.), sour rot ( Geotrichum candidum ),anthrax( Colletotrichum spp.), including Penicillium italicum ( Penicillium italicum ) and Penicillium finger ( P. digitatum The most serious damage to citrus is caused by *Penicillium italicum* and *Penicillium citrinum* (hereinafter referred to as citrus *Penicillium*). Citrus *Penicillium* conidia are spread through the air to the surface of healthy fruit and infect the fruit through wounds caused by mechanical damage during harvesting, transportation, and storage. Due to the biological importance and persistence of the conidia, directly inactivating them inhibits the spread of decay and helps extend the shelf life of the fruit. Currently, in China, thiamethoxam, imazalil, sodium o-phenylphenolate, imazalil, and biguanide trioctylbenzene sulfonate are mainly used for pre-harvest spraying or post-harvest fruit dipping of citrus to inhibit the invasion of citrus pathogenic fungi, thereby achieving the purpose of preservation. However, these chemical preservatives all have varying degrees of toxicity, and are often concentrated, leave residues, and do not meet the current consumer demand for green and healthy fruits. Therefore, the development and application of compounds that have significant inhibitory effects on *Penicillium italicum* and *Penicillium digitatum*, and are harmless, degradable, and safe for citrus quality, has become the main development direction for post-harvest preservation of citrus (control of citrus *Penicillium*).

[0003] Photodynamic inactivation (PDI) based on photosensitizers (PS) is a novel non-thermal antibacterial technology. Due to its high efficiency in killing microorganisms, low risk of drug resistance, and the fact that most photosensitizers are safe for plants, humans, and the environment, photosensitizers are considered a highly promising green preservative. Photodynamic inactivation requires the simultaneous action of three non-toxic elements (light source, target microorganism, and photosensitizer) to generate reactive oxygen species (ROS). Porphyrin compounds absorb visible light to generate singlet oxygen (¹O2(¹Δg)), i.e., type II ROS, thus these compounds have the potential to be used as fungicides. Porphyrin compounds are non-toxic to humans, the environment, and plants, while also exhibiting high bactericidal activity, making them a promising candidate for controlling agricultural microorganisms. Summary of the Invention

[0004] The purpose of this invention is to solve the problems of existing technology: traditional citrus preservatives have disadvantages such as being toxic, easy to leave residues, and difficult to degrade, which seriously threaten human health and cannot meet the current consumers' pursuit of green and healthy fruits and the green and sustainable development direction of citrus fruit preservation.

[0005] This invention provides a novel use for the porphyrin compound TMePPTos, specifically its application in the control of Penicillium mold in citrus. Specifically, 5 ppm of TMePPTos has shown a strong inhibitory effect on the growth of *Penicillium italicum* QM and *Penicillium fingernail* 7-5, both of which cause Penicillium mold, and this inhibitory effect strengthens with increasing TMePPTos concentration.

[0006] The first objective of this invention is to provide the application of the porphyrin compound TMePPTos in the control of Penicillium wilt in plants.

[0007] Preferably, the penicillosis is caused by *Penicillium italicum* and / or *Penicillium finger*.

[0008] Preferably, the Italian Penicillium is Italian Penicillium QM, and the finger Penicillium is finger Penicillium 7-5.

[0009] Preferably, the control of plant penicillium disease involves inhibiting the growth of *Penicillium italicum* and / or *Penicillium fingernail* and the germination of conidia.

[0010] Preferably, the plant is a citrus fruit.

[0011] The second objective of this invention is to provide a formulation for the prevention and control of Penicillium wilt in plants, containing the porphyrin compound TMePPTos as an active ingredient.

[0012] Preferably, the concentration of the porphyrin compound TMePPTos in the preparation for preventing and controlling plant blue mold is 5 ppm or more.

[0013] Preferably, the concentration of the porphyrin compound TMePPTos in the preparation for preventing and controlling plant blue mold is 5 ppm - 50 ppm.

[0014] The third object of the present invention is to provide a method for preventing and controlling citrus blue mold, comprising the following steps: applying the porphyrin compound TMePPTos or the said preparation to the surface of citrus fruits and then placing them under light conditions.

[0015] Advantages of the present invention: The present invention provides a new use of the porphyrin compound TMePPTos, specifically relating to the application of the porphyrin compound TMePPTos in inhibiting the growth of Penicillium digitatum. Using the porphyrin compound TMePPTos to prevent and control Penicillium digitatum has the characteristics of being more environmentally friendly and non-toxic. Low-concentration TMePPTos can effectively inhibit the growth of Penicillium italicum and Penicillium digitatum, providing a new option for preventing and controlling citrus blue mold, providing a very meaningful basis for the development of new drugs and compound reagents for citrus preservatives, and having great potential application value in reducing the use of harmful chemical preservatives and the green sustainable development of citrus storage and preservation. Description of the drawings

[0016] Figure 1 is the analysis of the antibacterial effects of different porphyrin compounds on Penicillium; among them, (a), (c) are the inhibitory effects of TPP, T4PP, TMePPTos, TMePPI, TSPPA, and TPPP (final concentration 50 ppm) on Penicillium digitatum P. digitatum strains under light (Light) and dark (Dark) treatment conditions respectively, (b), (d) are the inhibitory effects of TPP, T4PP, TMePPTos, TMePPI, TSPPA, and TPPP (final concentration 50 ppm) on Penicillium italicum P. italicum strain QM under light and dark treatment conditions, Control is the control group added with sterile water, and the pictures were taken at the 3rd day after inoculation; (c), (d) are the radial growth diameters of Penicillium mycelia measured after 3 days of treatment, and the error bars represent the standard deviations of three independent experiments (n = 9); analyzed by Student's t-test, the data groups marked with asterisks are significantly different from the control group, ns indicates no significant difference, ** indicates 0.001 < P < 0.01, *** indicates P < 0.001.

[0017] Figure 2Analysis of the inhibitory effects of TMePPTos, TMePPI, and TSPPA on conidia of Penicillium digitatum at different concentrations; (a) and (b) are the inhibitory effects of TMePPTos, TMePPI, and TSPPA (final concentration 5 ppm) on Penicillium digitatum P. digitatum strain 7-5 and Penicillium italicum P. italicum strain QM at different concentrations of conidia (1×10 3 cells / ml, 1×10 4 cells / ml, and 1×10 6 cells / ml). Control is the control group with sterile water added. The pictures were taken on the 3rd day after inoculation.

[0018] Figure 3 Comparison and analysis of the inhibitory effects of TMePPTos and prochloraz on Penicillium digitatum; specifically, the inhibitory effects of TMePPTos (final concentration 5 ppm) and prochloraz (1000-fold dilution) on Penicillium digitatum P. digitatum strain 7-5 and Penicillium italicum P. italicum strain QM under light (Light) and dark (Dark) treatment conditions. Control is the control group with sterile water added. Picture (a) was taken on the 3rd day after inoculation, and (b) is the radial growth diameter of Penicillium digitatum hyphae measured 3 days after treatment. Error bars represent the standard deviation of three independent experiments (n = 9); analyzed by Student’s t-test, data groups marked with asterisks are significantly different from the control group, ns indicates no significant difference, and *** indicates P < 0.001.

[0019] Figure 4 Analysis of the inhibitory effects of different concentrations of TMePPTos on Penicillium digitatum; (a) and (b) are the inhibitory effect diagrams of TMePPTos with a concentration gradient of 5 ppm - 30 ppm on Penicillium digitatum P. digitatum strain 7-5 and Penicillium italicum P. italicum strain QM growth under light (Light) and dark (Dark) treatment conditions. The pictures were taken on the 3rd day after inoculation; (c) and (d) are the radial growth diameters of Penicillium digitatum and Penicillium italicum hyphae measured 3 days after treatment, respectively. Error bars represent the standard deviation of three independent experiments (n = 9); analyzed by Student’s t-test, data groups marked with asterisks are significantly different from the control group, ns indicates no significant difference, ** indicates 0.001 < P < 0.01, and *** indicates P < 0.001.

[0020] Figure 5This study analyzed the effects of TMePPTos on Penicillium citrinum infection of citrus fruits. The disease incidence of Penicillium citrinum inoculated fruits was observed after treatment with TMePPTos (dissolved in distilled water and diluted to 15 ppm). Each treatment involved 48 inoculated fruits, replicated three times. Control-D represents water + darkness treatment, TMePPTos-D represents TMePPTos aqueous solution + darkness treatment, Control-L represents water + light treatment, and TMePPTos-L represents TMePPTos aqueous solution + light treatment. The light treatment involved 12 hours of light followed by 5 days of darkness, while the darkness treatment involved continuous darkness. The experiment was conducted at 26℃ and 90% humidity. After 5 days of cultivation, representative diseased fruits were photographed and recorded. Detailed Implementation

[0021] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0022] The following reagent information is provided: TMePPTos (5, 10, 15, 20-Tetrakis (1-methyl-4-pyridinio) porphyrintetra (p-toluenesulfonate)), purchased from Bioberry, USA, CAS No. 36951-72-1.

[0023] TSPPA (5, 10, 15, 20-(tetra-4-p-phenylenesulfonic acid)porphyrin tetraammonium salt), purchased from Hebei Bailingwei Ultrafine Materials Co., Ltd., CAS No. 39174-47-5.

[0024] TMePPI (5, 10, 15, 20-(Tetra-N-methy-4-pyridyl) porphyrintetraiodide), purchased from Bioberry, USA, CAS No. 36674-90-5.

[0025] TPPP (5, 10, 15, 20-(Tetra-4-phosphonatophenyl) porphyrin), purchased from Bioberry, USA, CAS No. 143969-69-1.

[0026] TPP (5, 10, 15, 20-(Tetraphenyl) porphyrin), purchased from Bioberry, USA, CAS No. 917-23-7.

[0027] T4PP (5, 10, 15, 20-(Tetra-4-pyridyl) porphyrin), purchased from Bioberry, USA, CAS No. 16834-13-2.

[0028] Example 1 Six commercially available, easily synthesized, and cost-effective free porphyrins (free porphyrins refer to porphyrin compounds that are not bound to metal ions) were initially selected, including cationic porphyrins TMePPTos and TMePPI, anionic porphyrins TPPP and TSPPA, and neutral porphyrins T4PP and TPP. The cationic porphyrin TMePPTos was screened out as exhibiting significant inhibitory effects against both *Penicillium italicum* and *Penicillium fingernail*. This compound maintained a significant inhibitory effect even at low concentrations (5 ppm). The specific experimental procedures and results are as follows.

[0029] (1) Preliminary screening of the anti-Penicillium activity of different porphyrin compounds After melting the pre-prepared PDA solid culture medium, place it in a 60°C constant temperature water bath and keep it at a stable temperature. Then, add the different porphyrin compounds purchased above (diluted with distilled water to a final concentration of 50 ppm) and antibiotics (ampicillin and kanamycin diluted with distilled water to a final concentration of 100 μg / ml) to the melted PDA solid culture medium. Pour the mixture into a petri dish and let it solidify again. Add the same volume of sterile water to the control group. Mycelial cakes were obtained by punching holes along the edges of well-grown *Penicillium italicum* strain QM (its ITS nucleotide sequence is shown in SEQ ID No. 2) or *Penicillium digitatum* strain 7-5 (its ITS nucleotide sequence is shown in SEQ ID No. 1) colonies using a sterile punch with a diameter of 5 mm. The 5 mm mycelial cakes were then placed on the surface of PDA medium containing different porphyrin compounds and antibiotics. One portion was cultured in a 26°C constant temperature and light incubator, while the other portion was cultured in the same incubator in the dark. On day 3, the colony diameter was measured using the cross-cross method. The inhibitory effect of different porphyrin compounds on *Penicillium italicum* strain (QM) or *Penicillium digitatum* strain (7-5) was determined, and the antibacterial activity of different porphyrin compounds was preliminarily analyzed to screen for porphyrin compounds with excellent antibacterial effects.

[0030] The results showed that ionic porphyrin compounds significantly inhibited the growth of *Penicillium citrinum*, the causal agent of citrus diseases. The antifungal effects of six free porphyrins against *Penicillium italicum* (QM) and *Penicillium fingering* (7-5) were as follows: Figure 1 As shown. Figure 1Images (a)-(b) show that cationic porphyrins (TMePPTos and TMePPI) exhibited strong inhibitory activity against *Penicillium italicum* strain QM and *Penicillium digitatum* strain 7-5 under continuous light conditions, completely inhibiting the growth of both strains. Although anionic porphyrins (TSPPA and TPPP) also significantly slowed the growth rate of both *Penicillium* strains, they did not completely inhibit their growth, especially against *Penicillium italicum* strain QM, and their effect was weaker than the cationic group. Neutral porphyrins (T4PP and TPP) did not cause significant changes in *Penicillium* growth under light conditions. It can also be seen that TMePPTos, TMePPI, and TSPPA showed significantly better inhibitory effects on *Penicillium digitatum* and *Penicillium italicum* under light conditions and under dark conditions. Under dark conditions, the growth levels of Penicillium citrinum were comparable to those of the control group, indicating that these free porphyrins lacked antibacterial activity or had weakened antibacterial activity under dark conditions. This also suggests that the anti-Penicillium activity of porphyrins depends on their photosensitivity.

[0031] (2) Analysis of the inhibitory effects of TMePPTos, TMePPI and TSPPA on conidia of different concentrations of Penicillium citrinum. Dissolve porphyrins TMePPTos, TMePPI, and TSPPA in distilled water. Dilute conidia of Penicillium italicum strain QM or Penicillium finger strain 7-5 to different concentrations: 1×10⁻⁶. 3 cells / ml, 1×10 4 cells / ml and 1×10 6 The concentration of conidia was increased to 1 / ml, and then different concentrations of conidia were transferred to PDA solid medium containing porphyrin compounds TMePPTos, TMePPI, or TSPPA (final concentration 5 ppm). The culture dishes were incubated at 26°C in darkness or light for 3 days, and the growth of *Penicillium citrus* was observed by photographing. Results are as follows: Figure 2 As shown, on PDA solid medium containing 5 ppm TMePPTos, the concentration was 1×10⁻⁶. 6 The germination of conidia of *Penicillium digitatum* was completely inhibited at concentrations of 1 × 10⁶ spores / ml, while that of *Penicillium italicum* was significantly inhibited; however, on culture dishes containing 5 ppm TMePPI or TSPPA, the germination was significantly inhibited at a concentration of 1 × 10⁶ spores / ml. 6 The growth and germination rate of *Penicillium citrinum* conidia at concentrations of 1 / ml did not differ significantly from the control group. Therefore, in vitro experiments showed that 5 ppm TMePPTos had the best inhibitory effect on *Penicillium citrinum* conidia.

[0032] (3) Comparative analysis of the antibacterial effects of TMePPTos and imazalil against Penicillium The porphyrin compound TMePPTos was dissolved in distilled water. *Penicillium italicum* strain QM or *Penicillium fingernail* strain 7-5, with a diameter of 5 mm, were transferred to PDA solid medium supplemented with 5 ppm TMePPTos or 1000 times dilution of imazalil. The culture dishes were incubated at 26°C in either dark or light conditions for 3 days, and photographs were taken afterward. Results are as follows: Figure 3 As shown, under dark conditions, the growth of both *Penicillium citrinum* strains was completely inhibited only on PDA medium supplemented with 1000 times diluted imazalil, while the growth of *Penicillium citrinum* strains on medium supplemented with 5 ppm TMePPTos showed no significant difference compared to the control group. However, under light conditions, the growth of both *Penicillium citrinum* strains was completely inhibited on PDA medium supplemented with 5 ppm TMePPTos or 1000 times diluted imazalil. Therefore, under light conditions, there was no significant difference in the effects of low concentrations (5 ppm) of TMePPTos and imazalil.

[0033] (4) Analysis of the antibacterial effect of different concentrations of TMePPTos on Penicillium citrinum. To further clarify the inhibitory effect of TMePPTos on Penicillium citrinum, experiments were conducted at different concentration gradients (diluted with distilled water to 5 ppm-30 ppm) to explore its effect on Penicillium digitatum (…). P. digitatum strain 7-5 and Penicillium italicum ( P. italicum The inhibitory effect of strain QM. For example... Figure 4 As shown, TMePPTos exhibited significant fungicidal effects against *Penicillium citrinum* under light conditions: compared to the control group, a concentration of 5 ppm significantly slowed mycelial growth, and the fungal inhibition rate gradually increased with increasing TMePPTos concentration (up to 30 ppm); conversely, under dark conditions, the porphyrin compound TMePPTos showed almost no antibacterial activity. Figure 4 (c)-(d) in the middle.

[0034] (5) Analysis of the effect of TMePPTos on the pathogenicity of Penicillium citrinum on citrus fruits To clarify the practical application value of porphyrin compound TMePPTos as a fungicide during the storage period of citrus, it is necessary to determine its disease resistance effect on citrus fruits.

[0035] Based on preliminary assessments, TMePPTos is effective against Penicillium finger-shaped mold on citrus fruits. P. digitatum To investigate the inhibitory effect of 7-5, four treatment groups were set up: water + spore suspension (dark or light), 15 ppm TMePPTos + spore suspension (dark or light).

[0036] Select citrus fruits (Ehime 28) that are uniform in size and color, undamaged, and disease-free. Wash them with water and ethanol and air-dry them. Then, spray the surface of the citrus fruits with a 15 ppm TMePPTos aqueous solution and air-dry them in the dark for 1 hour. Use a 1 mL sterile syringe to prick 3-5 small holes on the surface of each fruit, and spray 1×10⁻⁶ ppm of a solution containing the target strain (Penicillium fingerlingae strain 7-5) into the holes. 6 A spore suspension of 1 spore / mL was prepared. Inoculated fruits were placed in covered, humidified trays to maintain humidity during incubation. Incubation conditions were set at 26°C, 90% humidity, and a 12-hour light / 12-hour dark photocycle (light treatment: initial 12 hours of light followed by darkness for 5 days; darkness treatment: continuous darkness for 5 days). The severity of disease on the fruits was photographed on day 5. A control group was treated with an equal volume of distilled water injected into the wells; 48 fruits were treated per cycle, repeated three times.

[0037] Figure 5 Data showed that when spores and TMePPTos were co-incubated under light or dark conditions, no obvious finger-shaped Penicillium conidia were produced on the surface of inoculated citrus, while the control group showed obvious conidia, indicating that TMePPTos can significantly inhibit the germination of its conidia. However, under dark conditions, the 15 ppm TMePPTos + spore suspension treatment group produced obvious water-soaked lesions, while under light conditions, the area of ​​water-soaked lesions was almost invisible to the naked eye.

Claims

1. Use of a porphyrin compound TMePPTos in the prevention and treatment of plant penicillium diseases.

2. Use according to claim 1, characterized in that, The penicillium disease is caused by Penicillium italicum and / or Penicillium digitatum.

3. Use according to claim 2, characterized in that, The Penicillium italicum is Penicillium italicum QM, and the Penicillium digitatum is Penicillium digitatum 7-5.

4. Use according to claim 2, characterized in that, The prevention and treatment of plant penicillium diseases is to inhibit the growth of Penicillium italicum and / or Penicillium digitatum and the germination of conidia.

5. The use according to claim 1, characterized in that, The plant is citrus.

6. An agent for controlling penicillium diseases of plants, characterized by comprising a compound represented by the formula (I) or a salt thereof as an active ingredient. The porphyrin compound TMePPTos is used as an active ingredient.

7. The formulation of claim 6, wherein, The concentration of the porphyrin compound TMePPTos in the preparation for preventing and treating plant penicillium diseases is 5 ppm or more.

8. The formulation of claim 7, characterized in that, The concentration of the porphyrin compound TMePPTos in the preparation for preventing and treating plant penicillium diseases is 5 ppm to 50 ppm.

9. A method of controlling penicillium decay of citrus fruit, characterized in that, The method comprises the following steps: applying the porphyrin compound TMePPTos or the preparation of claim 6 to the surface of citrus fruit and then placing it under light conditions.

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