A microbial composition for preventing and treating white mold disease of Morchella esculenta and its uses

Abstract

The present invention discloses a microbial composition for preventing and treating white mold disease of Morchella and its use. The microbial composition consists of three strains, namely Streptomyces aurantiogriseus C4-A-9, Agromyces iriomotensis 15-B-11, and Kocuria rosea 17-B-11. The three strains all have good inhibitory effects on Paecilomyces, and after the combination of the three strains, they have a synergistic antibacterial effect on Paecilomyces. The combined microbial composition has a good prevention and treatment effect on white mold disease of Morchella.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a microbial composition for preventing and controlling white mold of Morchella esculenta and its use. Background Art

[0002] Morchella spp. is a rare and precious edible (medicinal) mushroom with rich nutrition and has always been a precious mushroom in short supply in the international market. In 2008, Morchella esculenta achieved artificial domestication and cultivation. In recent years, with the expansion of the cultivation scale, the yield of Morchella esculenta has increased year by year. The price of Morchella esculenta is more affordable compared to the prices of wild Morchella esculenta and when it was just successfully cultivated. Coupled with the rich nutrition and delicious taste of Morchella esculenta itself, it has become an ordinary dish on the tables of more people. However, with the continuous expansion of the cultivation scale of Morchella esculenta, the development of the Morchella esculenta industry is affected and restricted by more and more factors. Among these factors, pests and diseases have become one of the important factors restricting its development. Among them, white mold is one of the main diseases in the production of Morchella esculenta. White mold gets its name from the symptoms that occur in the field. It shows symptoms of white fluff on the stipe or cap of the Morchella esculenta fruit body. This disease is caused by Paecilomyces penicillatus. The impact of white mold on the yield and quality of Morchella esculenta is becoming increasingly prominent and shows a continuous aggravating trend. This disease can occur throughout the entire growth period of the fruit body and even in the initial storage period, and is more serious when the temperature is relatively high. Therefore, once white mold occurs in the production of Morchella esculenta, it will be devastating. As long as it occurs, it will greatly reduce the economic income of growers, seriously hitting their planting confidence, and showing an increasingly serious development trend in the impact on the Morchella esculenta industry.

[0003] Currently, there are no effective and safe products and methods to inhibit the occurrence of white mold of Morchella esculenta. The main methods for preventing and controlling white mold are only to strengthen field management, such as reducing temperature, humidity, strengthening ventilation in the shed, reducing the relative humidity of the air in the shed, etc., to keep the air in the shed fresh and the mushroom bodies dry to reduce the incidence probability. However, white mold is a soil-borne disease, and Paecilomyces penicillatus can spread to Morchella esculenta in the next season through the soil. Therefore, in order to avoid white mold and other factors affecting the continuous cropping of Morchella esculenta, many growers adopt the method of changing the planting land every year to plant Morchella esculenta, and even a large number of growers plant far away from their hometowns in other places, bringing many ecological problems and social problems. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a strain for preventing and controlling white mold of Morchella esculenta and a microbial composition composed thereof for preventing and controlling white mold of Morchella esculenta.

[0005] The technical solution of the present invention is as follows:

[0006] Streptomyces aurantiogriseus C4-A-9 was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on June 2, 2023, with the deposit number CGMCC No. 27524.

[0007] Agromyces iriomotensis 15-B-11 was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on June 2, 2023, with the deposit number CGMCC No. 27525.

[0008] Kocuria rosea 17-B-11 was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on June 1, 2023, with the deposit number CGMCC No. 27526.

[0009] Use of the above-mentioned Streptomyces aurantiogriseus C4-A-9 or Agromyces iriomotensis 15-B-11 or Kocuria rosea 17-B-11 in inhibiting Paecilomyces penicillatus.

[0010] A microbial composition containing Streptomyces aurantiogriseus C4-A-9, Agromyces iriomotensis 15-B-11 and Kocuria rosea 17-B-11.

[0011] A biological organic fertilizer containing the above-mentioned microbial composition.

[0012] Use of the above-mentioned microbial composition or biological organic fertilizer in preventing and controlling the white mold disease of Morchella caused by Paecilomyces penicillatus.

[0013] In the present invention, three microorganisms with antagonistic effects against the pathogen of the white mold disease of Morchella were isolated from the soil. Experiments showed that these bacteria had an inhibitory effect on the white mold pathogen but had no effect on the mycelial growth of Morchella. After combining the three strains, they had a synergistic antibacterial effect on Paecilomyces. The three microbial strains were combined and fermented and field experiments were carried out. Through three years of field experiments, it was found that the composition had a good control effect on the white mold disease of Morchella in the field.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The three strains of the present invention have good antagonistic effects against Paecilomyces, and have a synergistic effect of inhibiting Paecilomyces after combination. The microbial composition composed of them has a good control effect on the white mold disease of Morchella, and can reduce the incidence of the white mold disease of Morchella by 12.26%-27.82%.

[0016] Preservation information:

[0017] Streptomyces aurantiogriseus C4-A-9 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on June 2, 2023, with the deposit number CGMCC No. 27524, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing 100101, China.

[0018] Agromyces iriomotensis 15-B-11 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on June 2, 2023, with the deposit number CGMCC No. 27525, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing 100101, China.

[0019] Kocuria rosea 17-B-11 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on June 1, 2023, with the deposit number CGMCC No. 27526, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing 100101, China. Description of the drawings

[0020] Figure 1 Effect of three selected strains on the growth of Morchella

[0021] Figure 2 Symbiotic experiment of two of the three selected strains Detailed implementation manners

[0022] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from commercial channels unless otherwise specified.

[0023] Example 1 Screening and identification of antagonistic bacteria against white mold disease of Morchella

[0024] 1. Collection of soil samples

[0025] Collect different crop tillage soil samples in many places in Sichuan. During the process of soil sample collection, first remove the topsoil about 5 cm thick in the rhizosphere of the plants, and then collect about 1000 g of samples. Preserve them in an ice bag foam box and take them back to the laboratory to immediately isolate the microorganisms in them. If the samples cannot be immediately isolated, store them in a refrigerator at 4 °C and isolate the microorganisms in the samples in a timely manner.

[0026] 2. Isolate alternative microbial strains

[0027] 2.1 Culture medium

[0028] Beef extract peptone medium: 3.0 g of beef extract, 5.0 g of peptone, 5.0 g of NaCl, 20.0 g of agar, 1000 mL of distilled water, pH 6.8 - 7.2.

[0029] Gause's No. 1 medium: 1.0 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, 20.0 g of soluble starch, 20 g of agar, 1000 mL of distilled water, pH 7.2 - 7.4.

[0030] PDA medium: 200.0 g of potatoes, 20.0 g of glucose, 18.0 g of agar, 1000 mL of distilled water, pH natural.

[0031] LB medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, 20 g of agar, pH 7.4.

[0032] 2.2 Strain isolation

[0033] ⑴ Pour the plates

[0034] Put the medium sterilized at 121 °C for 30 min into the sterile operating table. When it cools to 50 °C - 60 °C, pour it into the petri dishes. 20 mL for each dish, and cool and solidify for standby.

[0035] ⑵ Serial dilution

[0036] Weigh 10.00 g of soil samples, add them to 100 mL of sterile water (in a 250 mL conical flask) with about 15 glass beads, and shake well at 180 r / min on a rotary shaker for 30 min to form the mother liquid bacterial suspension (10 -1 gradient). Transfer 1 mL of the liquid from the 10 -1 dilution to 9 mL of water as the 10 -2 dilution, and then transfer 1 mL of the liquid from the 10-2 dilution to 9 mL of water as the 10 -3 dilution, and so on. Each time, suck the suspension, pay attention to shaking the suspension evenly, and blow and suck it 3 times repeatedly before sampling.

[0037] (3) Coating separation

[0038] Absorb 100 μL of the dilution and transfer it onto the culture medium, then spread it evenly with a sterile glass rod. Make 4 consecutive gradients for the same soil sample, with 3 replicates for each gradient, and spread from low to high concentration. For bacterial culture, select 10 -4 、10 -5 、10 -6 、10 -7 . For actinomycetes and fungi, select concentrations of 10 -2 、10 -3 、10 -4 、10 -5 .

[0039] (4) Cultivation and observation

[0040] Place the spread plates of nutrient agar peptone medium, Gao's No. 1 medium, and PDA medium in an incubator at 30 °C for inverted cultivation. Take them out to observe the growth after 28 - 36 h of bacterial culture, 4 - 5 d of actinomycete culture, and 3 - 5 d of fungal culture.

[0041] 2.3 Isolation results

[0042] A total of 153 strains of various microorganisms were isolated from the rhizosphere soil of crops.

[0043] 3. Screening of antagonistic microorganisms

[0044] 3.1 Pathogen of Morchella

[0045] The pathogen of Morchella, Paecilomyces penicillatus, was previously isolated from diseased fruiting bodies by the Sichuan Edible Fungi Research Institute. Inoculate Paecilomyces penicillatus, the pathogen of Morchella, into a PDA plate and culture it in an incubator at 25 °C for standby.

[0046] 3.2 Screening of antagonistic microorganisms by the plate confrontation method

[0047] Simultaneously inoculate the isolated strains and Paecilomyces penicillatus, the pathogen of Morchella, into a petri dish of LB medium and culture it upright at 28 °C, then observe the inhibition of the pathogen. A total of 153 strains of various microorganisms were isolated from the rhizosphere soil of crops. According to the plate confrontation method, screen the strains with antagonistic effects against the Morchella white mold pathogen. 33 strains were initially screened to have antagonistic effects, and then the plate confrontation method was repeated to screen for antagonistic bacteria. 3 strains with better effects were obtained, namely C4 - A - 9, 15 - B - 11, and 17 - B - 11.

[0048] 3.3 Effects of strains on the growth of Morchella

[0049] Sixth Sister Morchella is the Morchella variety with the largest cultivation area in China, and it is used as a representative strain to study whether the antagonistic bacteria have an impact on the growth of Morchella. The strain is inoculated in the center of the petri dish, and the antagonistic strains are inoculated at about 0.8 cm on both sides in a straight line. Observe the effect of the antagonistic strains on the mycelial growth of Sixth Sister Morchella, with the direct inoculation of Morchella strain without the antagonistic bacteria as the control (CK). The results of the effects of the screened strains on the mycelial growth of Sixth Sister Morchella are as Figure 1 shown. The results show that the antagonistic strains C4-A-9, 15-B-11 and 17-B-11 against white mold pathogen have no effect on the growth of Sixth Sister Morchella, preliminarily indicating that these strains can be applied to the prevention and control of white mold in Morchella cultivation.

[0050] 3.4 Compound of strains

[0051] (1) Symbiotic situation between two strains

[0052] The three screened antagonistic strains are streaked pairwise on the petri dish to observe whether there is inhibition or promotion of growth between the strains and judge the symbiotic ability of each strain. The growth situation after pairwise streaking of the three strains is as Figure 2 shown. It can be seen from the growth situation of the strains in Figure 2 that there is no mutual inhibition of growth among the strains, nor is there an obvious mutual promotion of growth, proving that the three strains can coexist.

[0053] (2) Construction of antagonistic bacterial system

[0054] LB culture medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, pH 7.4.

[0055] The antagonistic strains are not inoculated (CK), single strain, pairwise combination, and all three strains are inoculated, with a total of 8 combinations. One loop of the strains in each combination is picked from the test tube and inoculated into the same triangular flask containing sterile LB culture medium, and fermented and cultured at 30 °C and 180 r / min for 30 h; the white mold pathogen is inoculated into the triangular flask containing sterile LB culture medium and fermented and cultured at 25 °C and 180 r / min for 72 h. Pour 20 mL of LB medium accurately into a 9 cm petri dish. After solidification, 100 μL of the cultured pathogen culture solution is inoculated onto the medium and spread evenly with a sterile glass rod. Punch holes in the center of the petri dish with a 6 mm sterile puncher, and inoculate 30 μL of the culture solution of each antagonistic bacteria combination into the holes, with 3 replicates for each combination. After culturing upright at 30 °C for 30 h, measure the antagonistic zone of the antagonistic bacteria combination inhibiting the growth of the pathogen and judge the situation of each combination inhibiting the pathogen. As shown in Table 1, the results show that the inhibitory effect of the combination 123 on the growth of the pathogen after fermentation is extremely significantly better than other combinations, proving that the three strains have a synergistic effect, so this combination is determined as the optimal combination.

[0056] Table 1 Compatibility effects of antagonistic bacterial strains

[0057]

[0058] Note: The above data are the mean ± standard deviation of 3 replicates. A, B, and C indicate differences at the 1% level, and the same applies hereinafter. 1. C4-A-9; 2. 15-B-11; 3. and 17-B-11.

[0059] 3.5 Strain identification

[0060] Physiological and biochemical identification and sequence analysis results showed that the partial 16S rRNA gene sequence of strain 17-B-11 is shown in SEQ ID No.2, with a length of 1433bp. Combining the physiological and biochemical reaction results of the strain and phylogenetic analysis, this bacterium is Kocuria rosea; the partial 16S rRNA gene sequence of strain 15-B-11 is shown in SEQ ID No.3, with a length of 1434bp. Combining the physiological and biochemical reaction results of the strain and phylogenetic analysis, this bacterium is Agromyces iriomotensis; the partial 16S rRNA gene sequence of strain C4-A-9 is shown in SEQ ID No.1, with a length of 1430bp. Combining the physiological and biochemical reaction results of the strain and phylogenetic analysis, this bacterium is Streptomyces aurantiogriseus.

[0061] Table 2 Physiological and biochemical test results of three strains

[0062]

[0063]

[0064] 3.6 Preservation of strains

[0065] On June 2, 2023, Streptomyces aurantiogriseus C4-A-9 was deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No.27524, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0066] On June 2, 2023, Agromyces iriomotensis 15-B-11 was deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No.27525, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0067] On June 1, 2023, Kocuria rosea 17-B-11 was deposited in the General Microbiology Center of the China Microbial Culture Collection Center, with the deposit number CGMCC No. 27526 and the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0068] Example 2 Field Experiment

[0069] 1. Preparation of biological organic fertilizer

[0070] The complex bacterial system composed of three strains, namely strain 17-B-11, 15-B-11, and C4-A-9, was co-fermented in liquid, and then subjected to matrix-binding solid biological amplification → raw material composting: 40% livestock and poultry manure, 60% straw. After adding the complex bacterial system and mixing, it was naturally composted for more than 1 month → primary crushing and sieving (removing impurities) → secondary fermentation → re-crushing and sieving (removing impurities) → adding auxiliary materials such as inorganic nutrients, and continuing to compost for more than 1 week → obtaining biological organic fertilizer.

[0071] 2. Field test

[0072] (1) Experimental design:

[0073] Test sites: Jianyang, Xindu

[0074] Test time: Conducted at both locations for 3 consecutive years from 2019 to 2021

[0075] Treatment: 3 treatments, 4 replicates

[0076] ①: Blank

[0077] ②: Matrix (equal amount of matrix obtained by natural composting without adding bacteria in the preparation of biological organic fertilizer)

[0078] ③: Organic fertilizer (biological organic fertilizer obtained from the preparation of 1. biological organic fertilizer)

[0079] Fertilization rate: 0.75 kg / m 2

[0080] (2) Test results

[0081] The statistical results of the 3-year field test are shown in Table 2. From the table, we can see that at the two test sites in three years, the effect of the organic fertilizer treatment on the incidence of white mold showed a highly significant reduction compared with the blank control and the matrix control, which could reduce the incidence of Morchella esculenta by 12.26% - 27.82%, and the prevention and control effect was better. It was proved that the organic fertilizer prepared from the screened complex bacterial system had a good control effect on Morchella esculenta in the field production of Morchella esculenta and had the potential for large-scale production for the prevention and control of white mold disease of Morchella esculenta.

[0082] Statistical Table of Incidence Rates for Each Treatment

[0083]

[0084]

[0085] Note: The data of mean ± standard deviation are the mean ± standard deviation of 4 replicates. A, B, and C indicate differences at the 1% level, and a, b, and c indicate differences at the 5% level.

Claims

1. Agromyces iriomotensis 15-B-11 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 2, 2023, with the deposit number CGMCC No. 27525.

2. Kocuria rosea 17-B-11 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 1, 2023, with the deposit number CGMCC No. 27526.

3. Use of Streptomyces aurantiogriseus C4-A-9 or Agromyces iriomotensis 15-B-11 as claimed in claim 1 or Kocuria rosea 17-B-11 as claimed in claim 2 in inhibiting Paecilomyces penicillatus; said Streptomyces aurantiogriseus C4-A-9 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 2, 2023, with the deposit number CGMCC No. 27524.

4. A microbial composition, characterized in that, 4. Comprising Streptomyces aurantiogriseus C4-A-9, Agromyces iriomotensis 15-B-11 as claimed in claim 1 and Kocuria rosea 17-B-11 as claimed in claim 2; said Streptomyces aurantiogriseus C4-A-9 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 2, 2023, with the deposit number CGMCC No. 27524.

5. A biological organic fertilizer, characterized in that, 5. Comprising the microbial composition as claimed in claim 4.

6. Use of the microbial composition as claimed in claim 4 or the bio-organic fertilizer as claimed in claim 5 in controlling white mold disease of Morchella caused by Paecilomyces penicillatus.

Images