LU501276B1 - Culture Method and Application of Botrytis Cinerea with Herbicidal Effect - Google Patents

Abstract

The invention discloses a culture method and application of Botrytis cinerea with herbicidal effect, and relates to the technical field of microorganisms and weeding. In the present invention, the strain is Botrytis fungus HZ-011, for Botrytis fungus HZ-011 has herbicidal effect, enriching microbial species with herbicidal activity; according to the invention, the optimal conditions of fermentation culture medium of Botrytis fungus HZ-011 are determined through single factor optimization test, and the optimal proportion of carbon source, nitrogen source and inorganic salt is screened through central composite response surface. The optimized culture medium improves the spore yield of strain HZ-011, which is suitable for the development of related herbicide types and lays a foundation for biological control of weeds.

Description

DESCRIPTION HUS0T276 Culture Method and Application of Botrytis Cinerea with Herbicidal Effect

TECHNICAL FIELD The invention relates to the technical field of microorganisms and weeding, in particular to a culture method and application of Botrytis cinerea with herbicidal effect.

BACKGROUND Weed control is one of the main problems in agricultural ecosystem and an important link in agricultural production. At present, the control of weeds all over the world mostly depends on the use of chemical agents. The long-term and extensive use of chemical agents has also brought many ecological and environmental problems in farmland, such as the emergence of herbicide-resistant weed plants, soil pollution, water quality degradation and harm to non-target organisms.

With the emerging safety problems of agricultural products and the new requirements of modern agricultural development, microbial herbicides have been studied in major countries of the world for nearly 60 years, and many strains with biological control and weeding activity have been separated and screened from a large number of diseased weeds by researchers. At present, the selected microorganisms with herbicidal activity cover 40 genera, include FExserohilum, Bipolaris, Phytophthora, Fusarium, Alternaria, Colletotrichum, Cercospora, Curvularia, Sclerotinia, Ascochyta, Puccinia, Colleototrichum and Entyloma, about 80 kinds of active biological species are used to control more than 70 kinds of weeds. Microbial herbicides have become the research focus in the field of biological weed control, and the research results in this field are constantly emerging. With the continuous separation and research of weed pathogens, the research and development of microbial herbicides, especially fungal herbicides, has made breakthrough progress. However, there is no related literature about the herbicidal effect of Botrytis fungus HZ-011.

SUMMARY

The purpose of the present invention is to provide a culture method of Botrytis 001278 cinerea with herbicidal effect and its application, so as to enrich the microbial species with weeding activity, determine the optimal culture conditions of Botrytis cinerea fungus HZ-011, and provide basis for batch production of Botrytis cinerea fungus HZ-011.

To achieve the above objective, the present invention provides the following scheme: According to one of the technical schemes of the invention, a strain with herbicidal effect is a Botrytis cinerea fungus HZ-011.

According to the second technical scheme of the present invention, the cultivation method of the above-mentioned strain with herbicidal effect comprises the following steps: inoculating the strain into a liquid culture medium or a solid culture medium, and culturing at pH 6-8, temperature 20-30°C, and shaking table rotation speed 160-200 r/min for not less than 4 days.

Further, the strain is inoculated into a liquid culture medium or a solid culture medium, and cultured for 5 days under the conditions of pH 7, temperature 25°C and shaking table rotation speed 180 r/min.

Further, the components of the liquid culture medium include: sucrose 45-60 g/L, peptone 14-16 g/L, NaCl 0.1-0.3 g/L and K,HPO4 0.3-0.5 g/L.

Further, the components of the liquid culture medium include: sucrose 48.744 g/L, peptone 15.626 g/L, NaCl 0.214 g/L and K,HPO4 0.428 g/L.

Further, the liquid medium has a liquid content of 68-88%.

Further, the liquid medium has a liquid content of 72%.

Further, the components of the solid medium include: 30 g/L. sucrose, 10 g/L peptone, 0.3 g/L K2HPO4, 0.1 g/L NaCl and 20 g/L agar.

The third technical scheme of the present invention is the application of the above-mentioned strains with herbicidal effect in preparing herbicides.

The invention discloses the following technical effects: (1) The invention found that the Botrytis fungus HZ-011 has herbicidal effect, enriching the microbial species with herbicidal activity;

(2) The culture medium of the invention improves the spore yield of the strain 001278 HZ-011, is suitable for the development of related herbicide types, and lays a foundation for the biological control of weeds.

BRIEF DESCRIPTION OF THE FIGURES In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention, and for ordinary technicians in the field, other drawings can be obtained according to these drawings without paying creative efforts.

Fig. 1 is the micromorphology of Botrytis fungus HZ-011; Fig. 2 is the PCR amplification product of ITS sequence of Botrytis fungus HZ-011; Fig. 3 shows the phylogenetic tree of Botrytis fungus HZ-011 and Botrytis strains constructed by adjacent connection method.

Fig. 4 shows the pathogenicity of Botrytis fungus HZ-011 to weed leaves; Fig. 5 shows the pathogenicity of Botrytis fungus HZ-011 to potted weeds; Fig. 6 shows the safety of Botrytis fungus HZ-011 to crops; Fig. 7 shows the pathogenic process of Botrytis fungus HZ-011 to Chenopodium album.

DESCRIPTION OF THE INVENTION Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present invention.

It should be understood that the terms used in this invention are only for describing specific embodiments, and are not used to limit the invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Any stated value or intermediate value within the stated range and any other stated value or every smaller range between intermediate values within 001278 the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary technicians in the field of this invention. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail.

Without departing from the scope or spirit of the present invention, it is obvious to those skilled in the art that many modifications and changes can be made to the specific embodiments of the present invention. Other embodiments obtained from the description of the present invention will be obvious to the skilled person. The description and example of that present invention are exemplary only.

The words "comprising", "including", "having" and "containing" used in this paper are all open terms, that is, they mean including but not limited to.

The Botrytis fungus HZ-011 used in this invention is preserved in the China General Microbiological Culture Collection Center, CGMCC, with the preservation number CGMCC 23894.

In this invention, Excel 2010 and DPS 9.01 are used for statistical analysis in data processing, and Duncan's new repolarization method is used for difference significance analysis.

Example 1 Step 1, inoculate Botrytis fungus HZ-011 into PDA culture medium (potato 200 g/L, glucose 20 g/L, agar 18 g/L, natural pH), culture at 25°C for 5 days, obtain activated strains, and beat the activated strains into bacteria cakes (p=8 mm);

Step 2, prepare liquid culture medium: sucrose 48.744g/L, peptone 15.626 g/L, 001278 NaCl 0.214g/L and K,HPO4 0.428 g/L, and put 180 mL of the above liquid culture medium into a 250 mL triangular flask; Step 3, inoculate the bacterial cake prepared in step 1 into the liquid culture medium prepared in step 2 (one bacterial cake per 50 mL liquid culture medium), adjust the pH value to 7, and shake culture at 180 r/min and 25°C for 5 days.

Results: the OD value of Botrytis fungus HZ-011 reached 1.056.

Example 2 Step 1, the same as step 1 of Example 1; Step 2, prepare solid medium: sucrose 30 g/L, peptone 10 g/L, K,HPO4 0.3 g/L, NaCl 0.1 g/L, agar 20 g/L; Step 3, inoculate the bacterial cake prepared in step 1 into the center of the solid medium plate prepared in step 2, and culture in a constant temperature incubator at 25°C for 4 days.

Results: the colony diameter of Botrytis fungus HZ-011 reached 7.52+0.148 cm.

Test example 1 Identification of Botrytis fungus HZ-011 (1) Morphological identification Culture strain HZ-011 on PDA medium, observe its mycelium and spore morphology under optical microscope, inoculate the 8 mm-diameter bacterial cake in different medium plates (p= 90 mm) and culture at 25°C, observe the colony growth rate, colony morphology and color change regularly, preliminary carry out identification in combination with the Fungus Identification Manual.

(2) Molecular level identification Extract genomic DNA according to the instructions of the provided fungal genomic DNA extraction kit, use ITS universal primers ITS 1: 5'-TCCGTaggtgaacctgegg-3' and ITS 4: 5'-TCCTCCTCCGCTTattgatgtge-3' to amplify the ITS region of strain 5.8S rDNA, detect the purity by 1% agarose gel electrophoresis, send the PCR amplified products to Shanghai Sangon Biotech Co., Ltd. for sequencing, analyse the 5.8 S rDNA sequence obtained by BLAST by homology sequence in GenBank's nucleic acid sequence library, select different similarity sequences, use MEGA 6.0 software for phylogenetic analysis, and use 001278 Neighbor-Joining (NJ) method to construct phylogenetic tree to determine the taxonomic status of strain HZ-011. The results are shown in Figures 1, 2 and 3.

Fig. 1 is the micromorphology diagram of strain HZ-011, in which A is the front of the colony, D is the back of the colony, and B, C, E and F are mycelium, conidiophore and conidia of HZ-011. As can be seen from Figure 1, the strain HZ-01 grows rapidly on PDA medium, the surface of the colony is white at first, and the aerial hyphae are underdeveloped, and after 5 days of growth, the center gradually turns dark yellow; at first, the back is white, then it gradually turns to orange after 3 days, and after 10 days of growth, it is accompanied by the growth of granular materials. Conidiophore is scattered and colorless, and the top of the cell is expanded into a ball with many small stalks. The conidia are oval, stick-shaped or oblong, and the small ridges are clustered into grape spikes. Conidia (14.5-25.5) umx(7.5-10.5) um; sclerotium is black, bright in surface, medium in thickness, irregular in shape, scattered in Petri dishes with many edges.

The DNA of strain HZ-011 is amplified by PCR (see Figure 2), and 500bp is selected for comparison after sequencing. The top 10 fungi with ITS gene sequence similarity greater than 95% with strain Hz-011 are selected for its phylogenetic tree analysis with MEGA 6.0 software. In this experiment, the neighbor connection method was used to construct the phylogenetic tree (see Figure 3; 0.005 in figure 3 indicates the genetic distance between two nucleosides; only>40% boot value is displayed at the branch); the results show that the strain HZ-011 is based on the same evolutionary branch as the first three closely related strains NR 111839.1 Botrytis caroliniana (100%), NR 164209.1 Botryotinia ficariarum (100%) and NR _159599.1 Botrytis byssoidea (99.78%), which proves that the strain HZ-011 is a new strain.

Test example 2 Pathogenicity of Botrytis fungus HZ-011 to weed leaves Collect the leaves of weeds with normal leaf color (Amaranthus blitum Linnaeus, Elsholtzia densa Benth., Malva verticillata var. crispa and Chenopodium album L.)

from the field, bring them indoors for surface washing, put them in a Petri dish (¢= 90 001278 mm) with 3-4 pieces of filter paper in each dish, and soak the filter paper with sterile water to provide a moist environment; punch the purified and cultured strain plate with a punch (¢=8 mm), and inoculate on each leaf, each of which is repeated for 3 times, with sterile PDA as control. Seven days later, the lesion area is measured, and the lesion area is 1/4xlengthxwidthx3.14.

The results are shown in Table 1 and Figure 4 (where A represents Amaranthus blitum Linnaeus, B represents FLlsholtzia densa Benth., C represents Malva verticillata var. crispa and D represents Chenopodium album L.).

Table 1 Area of lesion Weed name Pathogenic characteristics (cm’) Amaranthus 5471 The leaves produce brown disease spots, and a thick blitum Linnaeus white mold layer 1s attached to the whole leaves. Elsholtzia 3.567 Most of the leaves are all brown, and densa Benth. snowflake-shaped hyphae are attached to the leaves. The leaves are yellow, some leaves have waterlogged Malva verticillata

0.858 spots, and there are a few hyphae around the fungus var. crispa cake. Chenopodium 1.803 Yellow-brown irregular spots are produced, and a few From Table 1 and Figure 4, it can be seen that Botrytis fungus HZ-011 has good pathogenicity to weed leaves.

Test example 3 Pathogenicity of Botrytis fungus HZ-011 to weeds and safety of crops (1) Pathogenicity of weeds Transplant Amaranthus blitum Linnaeus, FElsholtzia densa Benth., Malva verticillata var. crispa and Chenopodium album L. in the experimental field of Qinghai Academy of Agricultural and Forestry Sciences in China at 4-5 leaf stages, and culturing at room temperature (24+1°C) for one week in a flowerpot (p=15 cm). After the strains are cultured in PDA medium plate for 7 days, the bacterial pieces (¢=

8 mm) are taken from the edge of the colony, and inoculated into 250 mL/bottle of 001278 PDB culture solution, with 5 pieces in each bottle, and cultured for 7 days at 25°C and 180 r-min’!. The fermentation broth is filtered with sterilized gauze and diluted to a spore concentration of 1.0x10° CFU/mL-1.0x10" CFU/mL. The spore solution is poured into a 500 mL watering can sterilized with 75% alcohol, and 2 mL Tween 20 is added and sprayed onto the transplanted healthy weed plants at an inoculation amount of 25 mL per pot. The plants inoculated with PDB medium are used as the blank control. Repeat 3 times per processing setting. The inoculated weed plants are placed at room temperature, kept at a relative humidity of 60% with JBX-3.5 centrifugal industrial humidifier, and cultured alternately in light and dark for 12 hours.

Observe the incidence of inoculated weeds, investigate the incidence degree of weeds and count the distribution of disease grades 7 days later, and calculate the incidence and disease index according to the following formula. The severity of the disease is graded as follows. Grade 0: leaves without any disease spots; Grade 1: there are scattered spots on leaves; Grade 2: 1/3-2/3 leaves rot to death; Grade 3: more than 2/3 of the leaves are rotten to death; Grade 4: all the leaves are rotten to death.

Number of diseased leaves Incidencerate = — ————————————— Total number of investigated leaves Incidence rate = Humber of diseased i eaves x Number of corvesy onding gr ades Total number of Investigated leaves x Maximum number of levels Results are as shown in Figure 5 (pathogenicity of Hz-011 to potted weeds; in which a is Amaranthus blitum Linnaeus, b is Elsholtzia densa Benth., c is Malva verticillata var. crispa and d is Chenopodium album L.); it can be seen from fig. 5 that after weeds are inoculated with strain HZ-011 1d, Chenopodium album leaves show disease spots and curled leaf edges, and the disease index is 6.25%. The leaves of Amaranthus blitum Linnaeus and the leaf margin dry up and wilt, and the disease index is 3.12%. The leaves of Malva verticillata var. crispa gradually turn yellow from the root to the top, and the disease index is 1.31%. Elsholtzia densa Benth. has sporadic small spots, no obvious wilting phenomenon, and the disease index is 0%. HUS01270 After 4 days, 1/2 leaves of Chenopodium album wilt and dry, and the incidence rate reaches 51%. Nearly 2/3 of the leaves of Amaranthus blitum Linnaeus are green and brown, and some of them are dead, with the disease index of 31.25%. 25% of Malva verticillata var. crispa leaves turn yellow, and the disease index is 13.1%. 35% of the leaves of Elsholtzia densa Benth.are dry with brown spots, and the disease index is

17.50%. After 7 days, the leaves of Chenopodium album and Amaranthus blitum Linnaeus all die, and the disease index is 100%. The incidence of Malva verticillata var. crispa is only 40%, and the symptom is that half of the whole leaf is yellow, and the disease index is 29.6%. 75% of the leaves of Elsholtzia densa Benth. die, and the disease index is 56.25%.

(2) Safety of crops Wheat, rape, broad bean, pea and highland barley are planted in pots with ¢=15 cm respectively, and cultured indoors.

The preparation and inoculation of fermentation broth are the same as the steps in (1) in pathogenicity of weeds as follows: 7 days after inoculation, the incidence of the tested crops is observed. The incidence is recorded according to the following standards: NS means asymptomatic (no disease spots, normal plant growth); LS indicates slight reaction (scattered spots on leaves, slightly inhibited growth); MS indicates moderate susceptibility (1/5-1/4 of the leaf area has disease spots and growth is inhibited); SS indicates severe disease (more than 1/4 of the leaf area has disease spots and the growth is severely inhibited).

Fresh effect (96) = Control fresh weight — Treated fresh weight 7 Control fresh weight The results are shown in Figure 6 (where a is broad bean, b is pea, c is rape, d is highland barley and e is wheat). As can be seen from Figure 6, after inoculating the strain HZ-011 to crops, highland barley, wheat, rape, broad bean and pea are not pathogenic. Compared with the control, the growth and plant height of crops are not affected at all, showing no response (NS), indicating that the strain HZ-011 is not pathogenic to crops and relatively safe. The fresh weight and dry weight of highland barley, wheat, rape, broad bean and pea had no significant difference compared with 001278 the control, indicating that strain HZ-011 had no effect on crops.

Test example 4 Electron microscopic observation on pathogenic process of Botrytis fungus HZ-011 to Chenopodium album Put filter paper and weed leaves in sterilized Petri dish (p=90 mm), soak the filter paper with sterile water to provide moist environment, punch the fungus cake (p=8 mm) in the center of the leaves, and take sterile PDA fungus cake as control, with 3 replicates for each treatment and control. The experiment is carried out at 25°C -28°C. After inoculation, samples are taken every day to observe the invasion process of mycelium for 7 days.

The results are shown in Figure 7, which shows that the tissue surface of control Chenopodium album is intact (Figure A). After inoculation of Chenopodium album strains for 1 day, hyphae penetrated through the stomata, and the tissues are not damaged (Figure B). Two days later, many hyphae are distributed around the stomata of Chenopodium album tissue, and a single spore landed on the tissue surface (Figure C). Three days later, as hyphae and spores parasitize the tissue surface, hyphae and spores can grow by using the nutrients in the tissue, forming a bacterial net parasitizing on the tissue surface (Figure D). After 4-5 days, the tissue surface was damaged, the parasitic hyphae absorbed nutrients, and the plants showed obvious diseases (Figures E and F). After 6 days, the tissue is completely wrapped by mycelium, and the mycelium grows vigorously (Figure G). After 7 days, the spore stalks flourish scattered spores, forming a dense spore pile, which accumulated on the surface of the damaged tissue (Figure H).

The above-mentioned embodiments only describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims (9)

CLAIMS LU501276

1. A strain with herbicidal effect, characterized in that the strain is a fungus of the genus Botrytis HZ-011.

2. Method for cultivating a strain with herbicidal effect according to claim 1, characterized by comprising the following steps: inoculating the strain into a liquid culture medium or a solid culture medium, and culturing at pH 6-8, temperature 20-30°C, and shaking table rotation speed 160-200 r/min for not less than 4 days.

3. The method for cultivating a strain with herbicidal effect according to claim 2, characterized in that the strain is inoculated into a liquid culture medium or a solid culture medium, and cultured for 5 days under the conditions of pH 7, temperature 25°C and shaking table rotation speed 180 r/min.

4. The method for cultivating a strain with herbicidal effect according to claim 2, characterized in that the components of the liquid culture medium include: sucrose 45-60 g/L, peptone 14-16 g/L, NaCl 0.1-0.3 g/L and K,HPO4 0.3-0.5 g/L.

5. The method for cultivating a strain with herbicidal effect according to claim 4, characterized in that the components of the liquid culture medium include: sucrose

48.744 g/L, peptone 15.626 g/L, NaCl 0.214 g/L. and K,HPO4 0.428 g/L.

6. The method for cultivating a strain with herbicidal effect according to claim 2, characterized in that the liquid medium has a liquid content of 68-88%.

7. The method for cultivating a strain with herbicidal effect according to claim 6, characterized in that the liquid medium has a liquid content of 72%.

8. The method for cultivating a strain with herbicidal effect according to claim 2, characterized in that the components of the solid medium include: 30 g/L sucrose, 10 g/L peptone, 0.3 g/L. K,HPO4, 0.1 g/L NaCl and 20 g/L agar.

9. Application of a strain with herbicidal effect according to claim 1 in preparing herbicides.