Application of myrtenol

Abstract

The invention discloses application of myrtenol in preparation of a preparation for inhibiting telosporium roseum, physosphaeria cheyi, fusarium graminearum, dispora piricola, alternaria alternata, colletotrichum gloeosporioides and botryosphaeria dothidea and treating chestnut fruit rot.

Description

Technical Field

[0001] The invention belongs to the field of forest protection and relates to an application of myrtenol. Background Art

[0002] Chinese chestnut (Castanea mollissima) is a perennial deciduous woody plant of the genus Castanea and the family Fagaceae. The chestnut fruit is rich in various nutrients, such as sugar, starch, protein, etc., and is one of the important woody food crops in my country. Chinese chestnut is a native species of Castanea in the world. According to the latest data from the Food and Agriculture Organization of the United Nations (FAO) in 2024, my country has become the world's largest chestnut exporter. The chestnuts produced in my country are sweet and of good quality, and are deeply loved by consumers, with broad market prospects.

[0003] Chestnut fruit rot is one of the major challenges for the sustainable development of the chestnut industry. It can greatly affect chestnut production and quality. The prevention and control of chestnut fruit rot has a far-reaching impact on the development of the chestnut industry. Since 1979, fruit rot has occurred year-round in major chestnut producing areas such as Hebei and Henan, causing huge economic losses to chestnut farmers. Chestnut fruit rot is a complex infectious disease with diverse pathogens and complex symptoms. The incubation period of the disease is relatively long, and the peak of the disease occurs during the storage period. Brown-black spots appear on the diseased part of the fruit, and the taste becomes bitter, affecting the quality of the chestnut. Although certain results have been achieved through continuous research, there is still a lack of environmentally friendly and efficient methods for the prevention and control of the disease. The fruit disease is still serious, and the economic threshold for the prevention and control of chestnut fruit rot is low. Therefore, it is necessary to explore a simpler, more feasible, effective, and cost-controlled green and safe prevention and control technology.

[0004] (1R)-(-)-Myrtenol (C 10 H 16 O) is a volatile compound belonging to the monocyclic monoterpenoid terpenoids. Other names: (1R)-2-pinene-10-ol, (1R)-6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-methanol. CAS number: 19894-97-4. It is one of the essential oil components of many aromatic plants (including Rosemary, Pelargonium, Artemisia, Melissa and Rhodiola). Myrtenol has been reported to have antioxidant, antidiabetic, sedative and gastroprotective effects. It has not been reported on chestnut diseases. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the first aspect of the present invention provides the use of myrtenol in preparing a preparation for inhibiting fungi in or outside a plant body, which is used alone or in combination with other materials. The fungi are zero, any one, any two, any three, any four, any five or six species of Polytermes roseus, Aspergillus laurel, Fusarium graminearum, Diplosporium citri, Alternaria interspersed, Colletotrichum gloeosporioides and Botrytis cinerea.

[0006] In some embodiments, the myrtenol is (1R)-(-)-myrtenol.

[0007] In some embodiments, the other material is a pharmaceutical excipient.

[0008] In some embodiments, the myrtenol is the only active drug.

[0009] In some embodiments, the other material is a fungal inhibitor.

[0010] The second aspect of the present invention provides the use of myrtenol in preparing a preparation for use alone or in combination with other materials to treat, prevent, slow down or improve plant diseases caused by fungi by inhibiting the fungi, wherein the fungi are zero, any one, any two, any three, any four, any five or six species of Polythene rosobasidium, Aspergillus laurel, Fusarium graminearum, Diplosporium citri, Alternaria interspersed, Colletotrichum gloeosporioides and Botrytis cinerea.

[0011] In some embodiments, the other material is a pharmaceutical excipient.

[0012] In some embodiments, the myrtenol is the only active drug.

[0013] In some embodiments, the other material is a fungal inhibitor.

[0014] In some embodiments, the myrtenol is (1R)-(-)-myrtenol.

[0015] In some embodiments, the plant disease is chestnut fruit rot.

[0016] The third aspect of the present invention provides a method for inhibiting plant fungi, wherein the plant fungi are zero, any one, any two, any three, any four, any five or six of Polythene rosenbergii, Aspergillus laurelii, Fusarium graminearum, Diplodia citri, Alternaria interspersed, Colletotrichum gloeosporioides and Botrytis cinerea, and the method is: placing the fungi in a space containing myrtenol, and the amount of myrtenol in each mL of the space is 0.1-1.0 μL (for example, a range between any one or any two of 0.1 μL, 0.2 μL, 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, 0.7 μL, 0.8 μL, 0.9 μL, 1.0 μL).

[0017] In some embodiments, the myrtenol is (1R)-(-)-myrtenol.

[0018] A fourth aspect of the present invention provides a method for treating, preventing, slowing down or ameliorating plant fruit rot, the method comprising: placing a plant fruit in a space containing myrtenol, wherein the amount of myrtenol in each mL of the space is 0.6-1.2 μL (for example, any one of 0.6 μL, 0.7 μL, 0.8 μL, 0.9 μL, 1.0 μL, 0.7 μL, 0.8 μL, 0.9 μL, 1.0 μL, 1.1 μL, 1.2 μL or a range between any two of them).

[0019] In some embodiments, the myrtenol is (1R)-(-)-myrtenol; and / or

[0020] The plant fruit rot is chestnut fruit rot.

[0021] In some embodiments, the pathogenic bacteria of fruit rot include zero, any one, any two, any three, any four, any five or six of Polythene rosobacter, Aspergillus laurelii, Fusarium graminearum, Diplodia citri, Alternaria interspersed, and Colletotrichum gloeosporioides, as well as Botrytis cinerea. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the effect of myrtenol on the growth of B. dothidea on the fifth day of growth at a higher concentration.

[0023] Figure 2 The effect of different concentrations of myrtenol on the growth of B. dothidea for 2-5 days.

[0024] Figure 3 This is the effect of myrtenol on the growth of B. dothide on the fifth day of growth under low concentration.

[0025] Figure 4This is a statistical chart of the antibacterial rate of low concentration myrtenol against B. dothidea.

[0026] Figure 5 This is the effect of adding myrtenol on the growth of Botrytis cinerea after one day of growth.

[0027] Figure 6 These are chestnut fruits treated with 0.2 μL / ml myrtle.

[0028] Figure 7 The control group was chestnut fruit. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0030] Materials and instruments not described in the present invention are conventional materials and instruments in the art, operation details not described in the present invention are conventional operations in the art, the software used in the present invention is operated by conventional methods with reference to the instructions of the software provider, and the kit used in the present invention is operated by conventional methods with reference to the kit instruction manual.

[0031] Example 1: Isolation and identification of chestnut fruit rot pathogen

[0032] (I) Isolation and identification of pathogenic fungi

[0033] For a batch of chestnuts with chestnut fruit rot (variety: Yanshan Zaofeng), chestnuts with fruit rot were selected and the strain was isolated by tissue separation method. Under the clean bench, the tissue at the junction of the diseased fruit, kernel, and healthy fruit was cut into small pieces of 0.5 cm 3 , soaked in 75% alcohol aqueous solution for 3 minutes, 2% NaClO aqueous solution for 5 minutes, 75% alcohol aqueous solution for 3 minutes, rinsed with sterile water 3 times, and the surface moisture was dried with sterile paper. The small pieces were placed in PDA plates and cultured in the dark at 25°C. Observe every day. Once some fungal hyphae are found growing on the tissue block, the top hyphae are picked and transferred to a new PDA plate for pure culture and expansion. The pure culture colonies obtained are used for pathogenicity verification of fungal strains and identification of strains and species.

[0034] (II) Verification of pathogenicity of isolated fungal strains infecting chestnuts

[0035] The chestnuts (variety: Yanshan Zaofeng) used for inoculation had healthy appearance and uniform size. They were soaked in 75% alcohol for 3 minutes, 2% NaClO for 5 minutes, and 75% alcohol for 3 minutes, rinsed with sterile water 3 times, and the surface moisture was dried with sterile paper for pathogenicity determination.

[0036] The pathogen inoculum is the mycelium tissue at the edge of the pure culture colony of each fungal strain obtained in step (a) by culturing on PDA medium at 25°C for 5 days. A sterile cork puncher (5mm in diameter) is used to make a hole (about 2mm deep) in the above chestnut material to form a wounded chestnut material; at the same time, a sterile cork puncher with the same aperture is used to take an inoculum cake from the edge of the above pathogen colony. The cake is covered on the wound of the chestnut fruit, with the mycelium surface of the cake facing the chestnut kernel. To prevent contamination by other microorganisms, a sealing film is wrapped around the wound. 30-50 chestnuts are inoculated for each treatment. Afterwards, they are placed in a sterile glass culture dish (20cm in diameter) and cultured at 25°C. After 7 days, the disease situation is observed, the incidence rate and disease level are counted, and the disease index is calculated. At the same time, sterile PDA medium is used to organize the inoculation block, and the wounded and unwounded chestnut materials are inoculated respectively to set up wounded and unwounded controls.

[0037] The chestnut pathogen was re-isolated, cultured and identified, and Koch's postulates were fulfilled to determine that it was the pathogen of Yanshan Zaofeng seed rot, and a batch of chestnut fruit rot pathogenic fungi were obtained.

[0038] (III) Identification of chestnut pathogenic fungi

[0039] Based on the morphological characteristics of the cultured colonies, vegetative hyphae and reproductive spores (conidia) of the chestnut fruit rot pathogenic fungi obtained in step (ii) and available for observation, identification is mainly carried out based on molecular phylogenetic methods.

[0040] Pathogen DNA extraction and ITS sequence amplification: The modified CTAB method was used for DNA extraction. Primer pair 1 (ITS1F and ITS4R) and primer pair 2 (ITS5F and ITS4R) were used to amplify the DNA templates of each strain by PCR. 25μL PCR reaction system: 2×Taq PCR MasterMix (Cowin Biotech Co., Ltd., Beijing, China), 10.5μL; 1μL of primers and DNA template each; 25μL of ddH2O. The PCR reaction program was: 95℃ pre-denaturation for 5min, 95℃ denaturation for 1min, 58℃ annealing for 30s, 72℃ extension for 1min, 35 cycles, and finally 72℃ extension for 10min, and stored at 4℃. The PCR products were entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0041] ITS1F (SEQ ID NO.1): 5'-CTTGGTCATTTAGAGGAAGTAA-3'

[0042] ITS4R(SEQ ID NO.2):5'-TCCTCCGCTTATTGATATGC-3'

[0043] ITS5F (SEQ ID NO.3): 5'-GGAAGTAAAAGTCGTAACAAGG-3'

[0044] The sequencing sequences were bidirectionally spliced ​​by SeqMan software to obtain the final ITS sequence. Using the blast tool of NCBI (under the default parameters of the website), the ITS sequences were preliminarily assigned, and then the reference strains (including the model strains) were selected to construct the phylogenetic tree. The ML analysis method was used to combine the available morphological and tissue characteristics to strive to identify the pathogen to the species. The reference strains (including the model strains) used to construct the phylogenetic tree of the invention are shown in each phylogenetic tree. After the target strain and the reference strain sequence combination were aligned and aligned under the default parameters of the online MAFFT tool, the ML phylogenetic tree was constructed using MEGA6.06 software, and the Bootsrap value was set to 1000. Seven fungi that cause chestnut fruit rot in the inoculum family, whose species were identified by the ITS method, were selected for subsequent research. The species identification information is shown in Table 1.

[0045] Table 1. Identification of strain species

[0046]

[0047]

[0048] (IV) Preservation of chestnut pathogenic fungi

[0049] The above seven kinds of fungi were placed in a medical ultra-low temperature refrigerator (Haier) and stored at -70°C for future use.

[0050] Example 2: Test on the inhibitory effect of higher concentrations of myrtenol on Botrytis cinerea, the main pathogen of chestnut fruit rot

[0051] Preparation of MEA (1L): 20g malt extract powder, 15g agar, 1000mL water, 7.0pH.

[0052] In this example, the bacterial cakes of the Botrytis cinerea isolated in Example 1 were inoculated in the center of 6 0 mm diameter culture dishes respectively under sterile conditions (in a clean bench). The required volume of myrtenol was drawn with a pipette, the lid of the culture dish with the bacterial block was opened, the drug solution was dripped into the center of the lid, the dish was covered again, and the dish was sealed with a sealing film, and the dish was kept stable when sealing.

[0053] The following five myrtoline alcohol concentration gradients were set up (myrtoline alcohol volatilizes and fills the volume inside the dish, and the concentration refers to the ratio of the original myrtoline alcohol volume used to the volume between the culture medium and the upper cover in the culture dish): 0.2μL / mL (add 10μL myrtoline alcohol), 0.4μL / mL (add 20μL myrtoline alcohol), 0.6μL / mL (add 30μL myrtoline alcohol), 0.8μL / mL (add 40μL myrtoline alcohol), 1.0μL / mL (add 50μL myrtoline alcohol). The headspace volume of a 90mm diameter culture dish is 50mL. Each concentration gradient was repeated 3 times. The control group was a culture dish without drug addition. The culture condition of the culture dish was 28℃. For the effect of myrtoline alcohol on the growth of B. dothidea on the fifth day of growth at a higher concentration, see Figure 1 , where the upper left, upper middle, upper right, lower left, and lower middle are respectively the cases where the concentration of myrtenol is 0.2μL / ml, 0.4μL / ml, 0.6μL / ml, 0.8μL / ml, and 1μL / ml, and the lower right is the control. It can be seen that the aforementioned concentrations of myrtenol can effectively inhibit Botrytis cinerea.

[0054] The inhibition rates of five concentrations of myrtenol on the growth of Botrytis cinerea were calculated. The results are shown in Table 2.

[0055] The calculation formula of inhibition rate = [(control pathogen colony diameter - pathogen colony diameter treated with liquid fumigation) / control pathogen colony diameter] × 100%.

[0056] The colony diameter was determined by the cross-sectional assay.

[0057] Table 2. Effects of high concentrations of myrtenol on Botrytis cinerea on the fifth day of culture

[0058] Concentration (μL / mL) 0.2 0.4 0.6 0.8 1 Inhibition rate (%) 100±0.00 100±0.00 100±0.00 100±0.00 100±0.00

[0059] Within the tested concentration, myrtenol can completely inhibit the growth of Botrytis cinerea. When the observation period was extended to 30 days, Botrytis cinerea still did not grow.

[0060] Figure 2 The figure shows the effect of different concentrations of myrtenol and control on the colony diameter of B. dothidea from 2 to 5 days. It can be seen that from 2 to 5 days, the colony of the control group (CK) gradually grew larger with the number of days, while the colony of the bacteria treated with different concentrations of myrtenol did not grow.

[0061] Example 3: Test on the inhibitory effect of low concentration myrtenol on Botrytis cinerea, the main pathogen of chestnut fruit rot

[0062] In this embodiment, the concentration lower than the lethal concentration of 0.2 μl / mL is referred to as low concentration. The concentration of myrtenol is respectively changed to 0 μL / ml (no myrtenol is added), 0.02 μL / ml (1 μL myrtenol is added), 0.05 μL / ml (2.5 μL myrtenol is added), 0.075 μL / ml (3.75 μL myrtenol is added), 0.1 μL / ml (5 μL myrtenol is added), 0.15 μL / ml (7.5 μL myrtenol is added), and other methods and steps are the same as those in Example 2.

[0063] The effect of myrtenol on the growth of B. dothidea on the fifth day of growth at low concentrations can be found in Figure 3 Among them, the concentrations in the first row from left to right are 0 (CK), 0.02 μL / ml, and 0.05 μL / ml, and the concentrations in the second row from left to right are 0.075 μL / ml, 0.1 μL / ml, and 0.15 μL / ml.

[0064] The inhibition rates of five concentrations of myrtenol on Botrytis cinerea were calculated, and the results are shown in Table 3.

[0065] Table 3. Effects of low concentration myrtenol on Botrytis cinerea on the fifth day of culture

[0066] Concentration (μL / mL) 0.02 0.05 0.075 0.1 0.15 Inhibition rate (%) 13.29±1.23 40.32±0.91 51.41±1.58 73.81±2.01 92.36±0.92

[0067] For inhibition rate statistics, see Figure 4 .

[0068] It can be seen that within the test concentration range, myrtenol can inhibit the growth of Botrytis cinerea. It can be considered that the minimum inhibitory concentration of myrtenol on Botrytis cinerea is 0.02 μL / mL, and as the concentration increases, the antibacterial effect gradually increases.

[0069] Example 4: Test of the inhibitory effect of myrtenol on Botrytis cinerea grown one day earlier

[0070] In this embodiment, myrtenol was added to the bacterial cake inoculated with Botrytis cinerea one day later, and the concentration of myrtenol was changed to 0 μL / ml (no myrtenol was added), 0.2 μL / ml (10 μL myrtenol was added), 0.4 μL / ml (20 μL myrtenol was added), 0.6 μL / ml (30 μL myrtenol was added), 0.8 μL / ml (40 μL myrtenol was added), and 1 μL / ml (50 μL myrtenol was added), and other methods and steps were the same as those in Example 2.

[0071] The effect of adding myrtenol on the growth of Botrytis cinerea after one day of growth can be found in Figure 5Among them, the concentrations in the first row from left to right are 0 (CK), 0.2 μL / ml, and 0.4 μL / ml culture results, and the concentrations in the second row from left to right are 0.6 μL / ml, 0.8 μL / ml, and 1 μL / ml culture results.

[0072] The inhibition rates of five concentrations of myrtenol on Botrytis cinerea were calculated, and the results are shown in Table 4.

[0073] Table 4. Effect of high concentration of myrtenol on the fifth day of culture on Botrytis cinerea inoculated one day earlier

[0074] Concentration (μL / mL) 0.2 0.4 0.6 0.8 1 Inhibition rate (%) 93.33±0.84 100±0.00 100±0.00 100±0.00 100±0.00

[0075] It can be seen that high concentrations of myrtenol have a strong inhibitory and killing effect on the growing Botrytis cinerea. On the fifth day of culture, within the test concentration, only 0.2 μL / mL of myrtenol failed to completely inhibit the growth of Botrytis cinerea, with an inhibition rate of 93.33% ± 0.84%. In other high-concentration groups, the grown Botrytis cinerea hyphae became lighter in color and had sparse hyphae. Continuing to observe, the Botrytis cinerea treated with 0.2 μL / mL of myrtenol grew all over the plate on day 21, which was much lower than the control group (6 days). The hyphae in other groups did not grow until the 30th day.

[0076] Example 5: Inhibitory effect of myrtenol on other chestnut fruit rot pathogens

[0077] In this embodiment, the bacterial cakes of the seven chestnut fruit rot pathogens obtained in Example 1 were inoculated in the center of a 90 mm MEA culture medium. Myrtenol was dripped on the dish cover. The dripping concentration gradient was 0 μL / ml, 0.02 μL / ml, 0.05 μL / ml, 0.075 μL / ml, 0.1 μL / ml, 0.15 μL / ml, 0.2 μL / ml, 0.4 μL / ml, 0.6 μL / ml, 0.8 μL / ml, and 1.0 μL / mL. 11 concentration gradients were used for the first test. The culture method and conditions were the same as in Example 1. After 14 days of culture, the dish cover to which myrtenol was dripped was removed, replaced with a new sterile dish cover (without the agent), sealed with a sealing film, and continued to observe for 1 month. Within one month, if the colony did not grow or did not grow into a colony, it means that the hyphae of the original colony has been killed and has no ability to recover growth. An inhibition rate of 100% is a prerequisite for killing. The lowest myrtenol concentration titer at which the hyphae were killed was taken as the minimum lethal concentration for the corresponding fungus.

[0078] Two concentrations close to each other were selected above and below the minimum lethal concentration for further testing. If the lower concentration was not completely lethal and the higher concentration was completely lethal, the minimum lethal concentration was finally determined to be accurate. The minimum lethal concentration of myrtenol against seven chestnut pathogens is shown in Table 5.

[0079] Table 5. Minimum lethal concentration of myrtenol against seven chestnut pathogens (μL / ml)

[0080]

[0081] Example 6. Inhibitory effect of myrtenol on the chestnut fruit rot pathogen Botrytis cinerea

[0082] Select chestnuts of similar size and without worm eyes and disease symptoms on the surface. Wash the chestnuts with tap water, and remove the floating fruits in the process. Then disinfect the surface: soak in 75% ethanol aqueous solution for 3 minutes, then soak in 2% sodium hypochlorite aqueous solution for 5 minutes, soak in 75% ethanol aqueous solution for 3 minutes again to elute the sodium hypochlorite, and finally wash with sterile water 3 times. Use a hole puncher to punch a hole in the center of the waist of the chestnut and remove the inner seed coat, inoculate with grape seat cavity fungus cake, one chestnut inoculates a piece of cake, the size of the diameter is 0.5cm, the thickness is 0.1-0.2cm, the side with bacteria faces inward, and the hole is sealed with sealing film. Place it in a 20cm diameter glass culture dish, add 10μL / ml of myrtenol to the sterilized 1.5m centrifuge tube cap, and place it in the 50mL space of the culture dish (0.2μL / ml of myrtenol). Place wet cotton in the culture dish to keep it moist, put 30 chestnuts in each dish, and repeat 3 times for each treatment. Incubate in a light incubator at 25°C and 90% humidity for 20 days.

[0083] The control group did not use any drugs, and other operations were the same as those of the chestnuts treated with myrtenol.

[0084] Myrtenol-treated chestnut Figure 6 , the chestnut of the control group see Figure 7 See Table 6 for chestnut morbidity statistics.

[0085] Table 6. Disease status of chestnut after myrtenol treatment

[0086]

[0087]

[0088] It can be seen that after treatment with 0.2 μL / ml myrtenol, the incidence of chestnut fruit decreased by 79.1%, and the severity of the disease was also alleviated, indicating that myrtenol can not only inhibit the growth of fruit rot pathogens on the plate, but also protect chestnut fruit from infection.

[0089] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. Use of myrtenol in preparing a preparation for inhibiting fungi in plants or outside plants, which is used alone or in combination with other materials. The fungi are zero, any one, any two, any three, any four, any five or six of Polythene rosenbergii, Aspergillus laurel, Fusarium graminearum, Diplosporium citri, Alternaria interspersed, Colletotrichum gloeosporioides and Botrytis cinerea.

2. The use according to claim 1, characterized in that The myrtenol is (1R)-(-)-myrtenol.

3. Use of myrtenol in preparing a preparation for treating, preventing, slowing down or ameliorating plant diseases caused by fungi by inhibiting the fungi alone or in combination with other materials. The fungi are zero, any one, any two, any three, any four, any five or six of Polythene rosobasidium, Aspergillus laurel, Fusarium graminearum, Diplosporium citri, Alternaria interspersed, Colletotrichum gloeosporioides and Botrytis cinerea.

4. The use according to claim 3, characterized in that The myrtenol is (1R)-(-)-myrtenol.

5. The use according to claims 3 and 4, characterized in that The plant disease is chestnut fruit rot.

6. A method for inhibiting plant fungi, wherein the plant fungi are zero, any one, any two, any three, any four, any five or six of Polythene rosobasidium, Aspergillus lauraceus, Fusarium graminearum, Diplodia citri, Alternaria interspersed, Colletotrichum gloeosporioides and Botrytis cinerea, and the method is: placing the fungi in a space containing myrtenol, and the amount of myrtenol in each mL of the space is 0.1-1.0 μL.

7. The method according to claim 6, characterized in that The myrtenol is (1R)-(-)-myrtenol.

8. A method for treating, preventing, slowing down or improving plant fruit rot, the method comprising: placing plant fruit in a space containing myrtenol, wherein the amount of myrtenol in each mL of the space is 0.6-1.2 μL.

9. The method according to claim 8, characterized in that The myrtenol is (1R)-(-)-myrtenol; and / or The plant fruit rot is chestnut fruit rot.

10. The method according to claim 9, characterized in that The pathogenic bacteria of the fruit rot include zero, any one, any two, any three, any four, any five or six species of Polythecium roseum, Aspergillus laurelii, Fusarium graminearum, Diplodia citri, Alternaria interspersed, Colletotrichum gloeosporioides and Botrytis cinerea.

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