A colorless bacillus with antifungal activity and its applications

Abstract

This invention provides an antifungal Achromobacterium and its application, wherein the Achromobacterium is Achromobacterium NPDY20Z, which was deposited at the China Center for Type Culture Collection on August 10, 2021, with accession number CCTCC NO: 20211002. This Achromobacterium strain has antagonistic and rapid killing effects on 17 common pathogenic fungi of crops, providing antagonistic microorganisms for the biological control of fungal diseases in crops, and has practical significance for the biological control of fungal diseases in crops.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a colorless bacillus with antifungal activity and its applications. Background Technology

[0002] Corn flat-hilum spirulina ( Bipolaris ), Tea-like spores ( Pestalotiopsis theae ), genus *Zea* (maize stem mold) Phoma sp. Fusarium oxysporum (Tomato Fusarium) Fusarium oxysporum Fusarium oxysporum, the bacterium that causes banana wilt ( Fusarium oxysporum f.sp.cubense ), *Pseudomonas chinensis* ( Pestalotiopsis palmarum ), Fusarium wilt ( Fusarium verticillium ), gray mold ( Botrytis cinerea ), rice sheath blight ( Rhizoctonia solani Corn stalk rot () Rhizoctonia solani Asparagus neck withering ( Crown rot ), Banana coccidioides ( Mycosphaere musicoia Soybean anthrax ( Colletotrichum chlorophyti ), Phytophthora capsici ( Phytophthora capsici ), soybean root rot ( Fusarium Oxysporum Fusarium graminearum ( ) Fusarium graminearum Rice blast fungus ( Magnaporthe oryzae Microorganisms and fungi, such as wheat, rice, and corn, are common pathogens of crops. If crops are infected with any one or more of these pathogens, and favorable climatic conditions occur, the pathogens can spread and cause widespread outbreaks, posing a significant threat to crop production.

[0003] Currently, chemical fungicides are the main pesticides used to control fungal diseases in crops. However, chemical pesticides are prone to resistance and can lead to pesticide residues and environmental pollution. Microbial pesticides, on the other hand, have the advantages of being easily degradable, pollution-free, and residue-free, aligning with the national goal of encouraging and guiding pesticide research towards green and environmentally friendly practices. They hold promise as a replacement for traditional chemical fungicides. Screening for microorganisms with antifungal activity is of practical significance for the biological control of fungal diseases in crops. Research on microbial pesticides focuses primarily on Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus polymyxa, while reports on the use of Achromobacterium as a microbial pesticide are relatively few.

[0004] Existing research on Achromobacterium mainly focuses on its degradation of harmful substances such as heavy metals, pesticide residues, and dyes in soil, and has not reported any characteristics of Achromobacterium antagonizing plant fungal diseases or killing fungal pathogens. Summary of the Invention

[0005] One of the objectives of this invention is to provide a colorless bacillus with antifungal activity, which is isolated from the root tissue of spider plant. It has antagonistic and killing effects on 17 common pathogenic fungi of crops (such as wheat, rice, corn, etc.), providing antagonistic microorganisms for the biological control of fungal diseases of crops, and has practical significance for the biological control of fungal diseases of crops.

[0006] A type of achromobacterium with antifungal activity, namely Achromobacterium NPDY20Z ( Achromobacter sp. NPDY20Z, deposited on August 10, 2021, at the China Center for Type Culture Collection in Wuhan, with accession number CCTCC NO: 20211002. This invention obtained a strain of Achromobacterium, NPDY20Z, through screening. Analysis using the streak plate confrontation method and soaking in barley grains carrying various fungi revealed that this strain exhibits inhibitory and bactericidal activity against up to 17 types of fungi, demonstrating broad-spectrum antifungal activity and possessing extremely high application research and economic value.

[0007] The nucleotide sequence of the 16S rDNA of the achromobacter NPDY20Z is shown in SEQ ID NO.1. The nucleotide sequence length of the 16S rDNA of the achromobacter NPDY20Z is 1469 bp, and the strain has been identified as Achromobacter.

[0008] The second objective of this invention is to provide a microbial preparation for preventing and controlling fungal diseases in crops. This microbial preparation contains the above-mentioned antifungal Achromobacterium NPDY20Z, or the fermentation product of the above-mentioned antifungal Achromobacterium NPDY20Z, or the extract of the effective active ingredient of the above-mentioned antifungal Achromobacterium NPDY20Z.

[0009] The third objective of this invention is to provide the application of the aforementioned antifungal-active achromobacterium in the control of fungal diseases in crops. The achromobacterium isolated by this invention has good environmental safety, is harmless to humans and animals, and can be used as a green fungicide. Furthermore, the achromobacterium exhibits a certain degree of inhibitory, antagonistic, and bactericidal effects against 17 common fungal pathogens affecting crops, including *Helicobacter maculatus*, *Helicobacter pylori*, *Helicobacter spp.*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium wilt*, *Fusarium graminearum*, *Botrytis cinerea*, *Rice sheath blight*, *Corn sheath blight*, *Asparagus neck blight*, *Helicobacter pylori*, *Anthracnose*, *Phytophthora capsici*, *Root rot*, *Fusarium graminearum*, and *Bacillus oryzae*. Therefore, the strains have broad-spectrum antifungal and bactericidal effects against fungal pathogens affecting crops and have potential application prospects.

[0010] Preferably, the fungal diseases of crops include fungal diseases caused by any one or a combination of several of the following pathogens: *Helicobacter maculatus*, *Pseudomonas chamae*, *Pseudomonas maculatus*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Pseudomonas rubrum*, *Fusarium wilt*, *Gyromitra esculenta*, *Rice sheath blight*, *Corn sheath blight*, *Asparagus neck blight*, *Pseudomonas spp.*, *Anthracnose*, *Phytophthora capsici*, *Soybean root rot*, *Fusarium graminearum*, and *Rhizoctonia solani*.

[0011] Preferably, the fungal disease of the crop is Fusarium head blight or rice blast. Fusarium head blight is one of the most common fungal diseases of barley, while rice blast is one of the most common fungal diseases of rice.

[0012] Preferably, the antifungal *Achromobacterium* is used to prepare a microbial preparation for controlling fungal diseases in crops, and this preparation is applied to the roots of the crops during the heading stage. Further, the microbial preparation for controlling fungal diseases in crops is a bacterial suspension obtained by inoculating *Achromobacterium* in a brown sugar and soybean powder culture medium, or an extract of the effective active ingredients of *Achromobacterium*. The formula for the brown sugar and soybean powder culture medium can be: 23.3 g / L brown sugar. -1 4.9 g·L⁻¹ CaCO₃ -1 11.1 g·L soybean flour -1 KNO3 8g·L -1 KH2PO4 0.5g·L -1 K2HPO4 1.2 g·L -1 MgSO4·7H2O 5 g·L -1 NaCl 3 g·L -1 .

[0013] Similarly, the microbial preparation for controlling fungal diseases in crops is sprayed onto the ears of the crops once each at the initial flowering stage, full bloom stage, and initial heading stage. The microbial preparation for controlling fungal diseases in crops is obtained by inoculating the *Achromobacterium* in a brown sugar and soybean powder culture medium to obtain a bacterial suspension, and then adding adjuvants to the bacterial suspension; or it is obtained by adding adjuvants to an extract of the effective active ingredients of the *Achromobacterium*. It has good dispersibility, solubility, and penetration, facilitating spraying. The adjuvant formulation can be: wetting agent D425 (alkyl sulfonic acid condensate) 4.8%, dispersant PVA (polyvinyl alcohol) 7.2%, protective agent FWA (fluorescent whitening agent) 0.1%, and stabilizer CMC-Na (sodium carboxymethyl cellulose) 2.0%.

[0014] With repeated subculturing of bacterial strains, the superior genetic traits inherent in the strain may be preserved, or they may mutate. Mutations can be positive (spontaneous mutation) or negative, with negative mutations, such as the deterioration of the strain's productive traits or the loss of certain genetic markers, being termed strain degeneration. To ensure that the preserved strain still possesses good antibacterial effects, this invention employs a streak plate confrontation culture method for strain preservation. Therefore, a fourth objective of this invention is to provide a method for preserving the aforementioned Achromobacterium with antifungal activity, employing a streak plate confrontation method for pre-preservation screening before implementing existing strain preservation processes. The screening process before preservation includes the following steps: First, barley grains carrying the pathogens of the crop fungal disease are inoculated into the center of a fungal culture medium. Then, a square or circular inoculation line is drawn with the antifungal-active Achromobacterium tumefaciens as the center of the barley grain. The presence of an inhibition zone between the inoculation line and the pathogen colony is observed. The wider the inhibition zone, the better the antifungal effect, indicating that the antifungal activity of the Achromobacterium tumefaciens has not degenerated. Conversely, the narrower the inhibition zone, the worse the antifungal effect, indicating that the antifungal activity of the Achromobacterium tumefaciens has degenerated. Finally, the Achromobacterium tumefaciens with undegenerated antifungal activity is selected for preservation. Attached Figure Description

[0015] Figure 1 The present invention demonstrates the antagonistic and inhibitory effect of Achromobacterium NPDY20Z on Fusarium graminearum, the causal agent of wheat scab.

[0016] Figure 2 The microscopic morphology of the colorless bacillus NPDY20Z of the present invention;

[0017] Figure 3 The colony morphology of the colorless bacillus NPDY20Z of the present invention;

[0018] Figure 4 This is an electrophoresis image of the 16S rDNA amplified fragment of Achromobacterium NPDY20Z according to the present invention. Lane marker is DNA marker, and lane 1 is 16S rDNA of Achromobacterium NPDY20Z. The molecular weights of the bands in the DNA marker lane from top to bottom are as follows: 3000 bp, 2000 bp, 1500 bp, 1200 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp.

[0019] Figure 5 The antagonistic effect of the achromobacterium NPDY20Z of this invention against nine fungi is described. Figure 5Petri dishes numbered 1-9 represent the experimental groups. Number 1: Antagonistic effect of strain NPDY20Z against *Helicobacter pylori* of maize; Number 2: Antagonistic effect of strain NPDY20Z against *Polytrichum chamaegi*; Number 3: Antagonistic effect of strain NPDY20Z against *Fusarium oxysporum* of tomato; Number 4: Antagonistic effect of strain NPDY20Z against *Polytrichum chamaegi* of wax apple; Number 5: Antagonistic effect of strain NPDY20Z against *Fusarium wilt* of Verticillium wilt; Number 6: Antagonistic effect of strain NPDY20Z against *Rhizoctonia solani* of rice; Number 7: Antagonistic effect of strain NPDY20Z against *Anthracnose* of soybean; Number 8: Antagonistic effect of strain NPDY20Z against *Phytophthora capsici* of pepper; Number 9: Antagonistic effect of strain NPDY20Z against *Bacillus oryzae* of rice.

[0020] Figure 6 The growth rate of the colorless bacillus NPDY20Z of the present invention in three culture media: NB, brown sugar bean powder culture medium, and PDB.

[0021] Figure 7 This is a schematic diagram of the streaked structure of the colorless bacillus NPDY20Z before preservation, according to the present invention. Detailed Implementation

[0022] The embodiments of the present invention are now described in detail: Example 1

[0023] (I) Isolation and purification of antagonistic strains

[0024] Clean the soil from the roots of the spider plant, soak the roots in 75% alcohol for 15 seconds, disinfect them in 0.1% mercuric chloride solution for 1 minute, rinse three times with sterile water, and grind with 10ml of sterile water. Dilute the supernatant with sterile water to a final concentration of 10. -2 10 -3 10 -4 Spray the mixture onto a fungal culture medium (PDA medium) inoculated with Fusarium graminearum, the fungus causing Fusarium head blight, and incubate at 28°C for 2 days. Select single colonies of antagonistic bacteria that can show inhibition zones on the fungal culture medium (i.e., single colonies that have antagonistic effects against Fusarium graminearum) for further purification.

[0025] The formula for the PDA culture medium is: 180-250g potato, 15-25g glucose, 15-20g agar, and 1000mL water.

[0026] (II) Screening of antagonistic strains

[0027] Antagonistic strains were screened using the streak plate confrontation method. Specifically: First, purified single colonies of the antagonistic bacteria and the test fungal strain (Fusarium graminearum) were activated on PDA medium. Next, a mycelial block of the test fungal strain was picked and inoculated into the center of a 9cm PDA medium. Then, a square (or circular) frame was drawn on the PDA medium at a distance of 3.5cm from the center of the mycelial block of the test fungal strain, serving as the experimental group. Simultaneously, a PDA plate containing only a mycelial block of the test fungal strain in the center, but without streaks around the activated single colony, served as the control group. Finally, all the petri dishes were sealed and incubated at 28℃ for 5-7 days, and growth was observed. When the mycelia of the test fungal strain in the control group completely covered the entire petri dish, the antibacterial effect of the experimental group was observed. In the experimental group, the larger the diameter of the inhibition zone centered on the activated single colony, the better the antibacterial effect of the activated single colony.

[0028] Using the above-mentioned streak plate confrontation method for screening antagonistic strains, the applicant screened an antagonistic strain with good activity against Fusarium graminearum. The antagonistic inhibitory effect of the antagonistic strain on Fusarium graminearum was further verified by the plating method, and the colony state is as follows: Figure 1 As shown.

[0029] The antagonistic strains exhibited moist, smooth, and transparent colonies on PDA solid medium, with raised, irregular edges and a gelatinous appearance. After 10 days of culture in petri dishes, the color gradually deepened to a pale yellow, with strong viscosity. Microscopic observation revealed rod-shaped cells measuring (1.0–2.5) μm × (1.0–1.5) μm (e.g., ...). Figure 2 (As shown).

[0030] The colony morphology of the antagonistic strains, such as Figure 3 As shown.

[0031] And based on the electrophoresis image of the 16S rDNA amplified fragment of the antagonistic strain (e.g.) Figure 4 As shown in the figure, the nucleotide sequence length of the 16S rDNA of the antagonistic strain is 1469 bp, and this was confirmed by DNA sequencing. The homology of the antagonistic strain was also determined to be... Achromobacter (Achromobacterium). The DNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd. DNA sequence analysis was conducted using the Blast tool available at www.ncbi.nkm.nlh.gov. Therefore, the applicant names the obtained antagonistic strain as: Achromobacterium NPDY20Z.

[0032] The antifungal bacillus NPDY20Z ( Achromobacter sp.The NPDY20Z was deposited on August 10, 2021, at the China Center for Type Culture Collection in Wuhan, with accession number CCTCC NO: 20211002.

[0033] (III) Antibacterial effect of Achromobacterium acnes NPDY20Z

[0034] The antifungal effect of Achromobacterium ningpoensis NPDY20Z against various fungi was observed using the streak plate confrontation method. The antagonistic activity of this strain against 17 fungi (all common pathogens of fungal diseases in crops) was determined. Table 1 shows the antifungal effect against these 17 fungi. Figure 5 The study investigated the antagonistic effects of *Achromobacterium acnes* NPDY20Z against nine of the 17 fungi listed. The results showed that *Achromobacterium acnes* NPDY20Z exhibited varying degrees of antagonism against all 17 fungi (also known as "pathogenic fungi"), demonstrating broad-spectrum antifungal properties.

[0035] Table 1. Antibacterial effect of Achromobacterium acnes NPDY20Z

[0036]

[0037] Note: The data in Table 1 are the averages of three replicates for each treatment. The same letter after the data indicates that the multiple comparisons were not significant at the 0.05 level.

[0038] The sources of the 17 fungi (“pathogenic fungi”) mentioned in Table 1 are as follows:

[0039] The rice blast fungus and *Fusarium graminearum* were isolated by our research group; the *Helicobacter cuspidata*, *Pseudomonas aeruginosa*, *Stemum cuspidata*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Pseudomonas aeruginosa*, *Fusarium wilt*, and *Botrytis cinerea* fungi were provided by the College of Plant Protection, Fujian Agricultural and Forestry University; the rice sheath blight was provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences; the corn sheath blight, asparagus neck blight, *Pseudomonas aeruginosa*, and soybean anthracnose were provided by the College of Plant Protection, Fujian Agricultural and Forestry University; and the *Phytophthora capsici* and soybean root rot were provided by the Sichuan Mianyang Agricultural Research Institute.

[0040] (iv) Killing effect of Achromobacterium acnes NPDY20Z on the 17 fungi.

[0041] Seventeen fungi, including *Fusarium graminearum*, were cultured in bottles containing autoclaved barley grains. After the mycelia of each fungus had covered the barley grains, each fungus was separately inoculated with either a 36-hour fermentation broth containing NPDY20Z and a sterile fermentation broth containing only brown sugar and soybean flour (CK). The mixture was shaken and soaked for 6 hours. After soaking, 20 barley grains were placed on PDA medium to observe mycelial growth. The experiment was repeated three times. The results of soaking in the sterile fermentation broth were compared to calculate the killing effect of NPDY20Z on various fungi. The results are as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] After soaking barley grains infected with 17 fungi for 6 hours, mycelia germinated on PDA medium, with a germination rate of 100%. After soaking the 17 fungi in brown sugar bean flour fermentation broth containing NPDY20Z for 6 hours, except for Fusarium oxysporum tomatoense, 80% of the fungi were still able to germinate mycelia, while the other fungi could not germinate mycelia. That is, the killing rate of Fusarium oxysporum tomatoense by brown sugar bean flour fermentation broth containing NPDY20Z for 6 hours was only 20% (but it could kill them all after 48 hours), while the killing rate of the other 16 fungi reached 100% within 6 hours (see Table 2 below).

[0042] Table 2. Fungal killing effect of Achromobacterium acnes NPDY20Z

[0043]

[0044] Note: The data in Table 2 are the averages of three replicates for each treatment. The same letter after the data indicates that the multiple comparisons were not significant at the 0.05 level.

[0045] This experiment shows that Achromobacterium tumefaciens NPDY20Z has a short-term killing effect on a variety of fungi, rather than simply an inhibitory effect. The discovery of this functional effect is very important for the development and utilization of Achromobacterium tumefaciens. If Achromobacterium tumefaciens NPDY20Z is soaked in soil with serious soil-borne diseases, its broad-spectrum sterilization effect can kill most harmful fungi within 6 hours. In particular, it can achieve a unified killing effect on some soil-borne diseases caused by multiple fungal infections. Therefore, the application potential of this bacterium is huge.

[0046] (V) Field trials of Achromobacterium NPDY20Z

[0047] Experimental Site Overview: The experiment was conducted from autumn 2020 to spring 2021 at the experimental base of the Nanping Agricultural Science Research Institute in Fujian Province. Fusarium head blight occurs naturally and stably at this base annually, making it a national regional trial site for identifying natural resistance to Fusarium head blight in winter wheat, thus eliminating the need for inoculation with the pathogen. Winter wheat (Yangfumai 4, a highly susceptible Fusarium head blight variety provided by the Yangzhou Lixiahe Agricultural Science Research Institute) was sown on November 16th, began heading on February 11th, and began flowering on February 14th. The incidence of Fusarium head blight was investigated on April 15th.

[0048] Field control efficacy of Achromobacterium tumefaciens NPDY20Z against wheat scab: The field control efficacy of Achromobacterium tumefaciens NPDY20Z was evaluated by root drenching and spraying fermentation broth onto the ears, and compared with the chemical fungicide tebuconazole and carbendazim. A total of 4 treatments with 3 replicates were set up, and the experimental protocol is as follows:

[0049] The fermentation broth of Achromobacterium tumefaciens NPDY20Z was used for root irrigation once during the wheat heading stage; the fermentation broth of Achromobacterium tumefaciens NPDY20Z was: a bacterial suspension obtained by inoculating Achromobacterium tumefaciens NPDY20Z into brown sugar bean flour culture medium (or other common bacterial culture medium) (also called "brown sugar bean flour fermentation broth").

[0050] The fermentation broth (containing adjuvants) of Achromobacterium Ⅱ-NPDY20Z was sprayed on the ears of wheat once each at the initial heading stage, initial flowering stage, and full flowering stage. The fermentation broth was obtained by inoculating Achromobacterium Ⅱ-NPDY20Z into a brown sugar bean flour culture medium (or other common bacterial culture medium) to obtain a bacterial suspension, and then adding adjuvants to the bacterial suspension.

[0051] Treatment III: Spray the panicle once at the beginning of flowering and once at the full bloom stage with 60% tebuconazole carbendazim water-dispersible granules (Hebei Guanlong Agricultural Chemical Co., Ltd.).

[0052] Treatment IV - the water control (CK) was sprayed once at the beginning of flowering and once at the peak of flowering.

[0053] Each treatment has 3 replicates. There are a total of 12 cells, each cell is 20m. 2 The effective active ingredient content of the antagonistic bacteria fermentation broth sprayed on the panicle was 15 trillion CFU / hm². 2 The effective active ingredient content in soil irrigation is 300 trillion CFU / hm². 2 .

[0054] The results showed that, in the two application methods of Achromobacterium NPDY20Z (Treatment I and Treatment II), the disease control efficacy of the ear fermentation broth and root irrigation treatment was 31.88% and 32.17%, respectively. Regarding the prevention of diseased ears, the two treatments mainly slowed the spread of Fusarium head blight in the ear to a certain extent. Soil irrigation (Treatment I) and ear fermentation broth (Treatment II) of Achromobacterium NPDY20Z both reduced the incidence of Fusarium head blight to some extent compared to the control (Treatment IV), but their effects were still somewhat different from those of the chemical fungicide tebuconazole and carbendazim.

[0055]

[0056] Note: The data in Table 3 are the averages of three replicates for each treatment. The same letter after the data indicates that the multiple comparisons were not significant at the 0.05 level.

[0057] The formula for the brown sugar and soybean powder culture medium is existing technology, and its formula is: 23.3 g·L brown sugar. -1 4.9 g·L⁻¹ CaCO₃ -1 11.1 g·L soybean flour -1 KNO3 8 g·L -1 KH2PO4 0.5g·L -1 K2HPO4 1.2 g·L -1 MgSO4·7H2O 5 g·L -1 NaCl 3 g·L -1 .

[0058] Meanwhile, adding adjuvants to microbial preparations for controlling fungal diseases in crops is an existing technology. These adjuvants typically include dispersants, spreaders, adsorbents, and UV stabilizers, which can be selected and combined as needed. The formulation of the adjuvant in this invention is as follows: wetting agent D425 (alkyl sulfonic acid condensate) 4.8%, dispersant PVA (polyvinyl alcohol) 7.2%, protective agent FWA (fluorescent whitening agent) 0.1%, and stabilizer CMC-Na (sodium carboxymethyl cellulose) 2.0%.

[0059] (vi) Screening of culture media

[0060] The applicant inoculated *Achromobacterium tumefaciens* NPDY20Z into three culture media: NB, fermentation broth (i.e., brown sugar and bean flour culture medium), and PDB. The growth of *Achromobacterium tumefaciens* NPDY20Z was observed every 8 hours. The results showed that *Achromobacterium tumefaciens* NPDY20Z grew fastest and had the highest growth rate in the brown sugar and bean flour culture medium, while it grew slowest in the PDB culture medium, with a maximum growth rate not exceeding 0.3 billion colonies per milliliter. In the fermentation broth (i.e., brown sugar and bean flour culture medium), the maximum growth rate of 200 million colonies per milliliter was reached after 32 hours, after which the number of colonies rapidly decreased, reaching 0.1 billion colonies per milliliter by 56 hours. In the NB culture medium, the maximum growth rate of 1.7 billion colonies per milliliter was reached after 48 hours, and then rapidly decreased (e.g., ...). Figure 6 (As shown). Therefore, the Achromobacterium NPDY20Z is preferably cultured using brown sugar and bean flour broth.

[0061] (vii) Determination of the temperature sensitivity of Achromobacterium NPDY20Z

[0062] The results showed that Achromobacterium affine NPDY20Z survived after being treated with a temperature gradient of 50℃ to 100℃ in a water bath, and still had an antagonistic effect on Fusarium graminearum, but was not sensitive to high temperature.

[0063] Table 4. Sensitivity of antagonistic achromobacterium NPDY20Z to temperature

[0064]

[0065] Note: "+" indicates that the strain is alive and still has an antagonistic effect on Fusarium graminearum, and "-" indicates that the strain is dead.

[0066] Based on the excellent and broad-spectrum antibacterial and bactericidal properties of Achromobacterium NPDY20Z described in this invention, this invention also provides a microbial preparation for preventing and controlling fungal diseases in crops, the microbial preparation comprising the aforementioned Achromobacterium NPDY20Z.

[0067] This invention also protects the application of the aforementioned *Achromobacterium* NPDY20Z in the control of fungal diseases in crops. The *Achromobacterium* NPDY20Z isolated by this invention has good environmental safety, is harmless to humans and animals, and can be used as a green fungicide. Furthermore, the *Achromobacterium* exhibits a certain degree of inhibitory and antagonistic effects against 17 common fungal pathogens affecting crops, including *Helicobacter maculatus*, *Pseudomonas aeruginosa*, *Pseudomonas spp.*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Pseudomonas aeruginosa*, *Fusarium wilt*, *Fusarium wilt*, *Botrytis cinerea*, *Rice sheath blight*, *Corn sheath blight*, *Asparagus neck blight*, *Cyclocarya paliurus*, *Anthracnose*, *Phytophthora capsici*, *Soybean root rot*, *Fusarium graminearum*, and *Bacillus oryzae*. Therefore, the strain has broad-spectrum antibacterial and fungicidal effects against fungal pathogens affecting crops and has potential application prospects. Preferably, the crop fungal disease is a fungal disease caused by any one or a combination of several of the following pathogens: *Helicobacter maculatus*, *Pseudomonas aeruginosa*, *Pseudomonas maculatus*, *Fusarium oxysporum* (tomato), *Fusarium oxysporum* (banana wilt), *Pseudomonas aeruginosa* (wax apple), *Fusarium wilt* (Verticillium wilt), *Botrytis cinerea*, *Rice sheath blight*, *Corn sheath blight*, *Asparagus neck blight*, *Pseudomonas aeruginosa* (banana), *Anthracnose*, *Phytophthora capsici*, *Soybean root rot*, *Fusarium graminearum*, and *Rhizoctonia solani*. Preferably, the crop fungal disease is Fusarium head blight or rice blast. Fusarium head blight is one of the most common fungal diseases of barley, while rice blast is one of the most common fungal diseases of rice.

[0068] Preferably, the above-mentioned Achromobacterium NPDY20Z is used to prepare microbial preparations for the prevention and control of fungal diseases in crops.

[0069] To ensure that the preserved strain retains good antifungal activity, this invention employs a streak plate confrontation culture method for strain preservation. Therefore, this invention also protects the preservation method for the aforementioned *Achromobacterium* NPDY20Z, employing a streak plate confrontation method for pre-preservation screening before performing existing strain preservation processes. The pre-preservation screening includes the following steps: first, inoculating barley grains 10 carrying the pathogen of the crop fungal disease into the center of the fungal culture medium 20; then, using the *Achromobacterium* with antifungal activity, drawing a square or circular inoculation line 30 centered on the barley grain (e.g., ...). Figure 7 However, the shape of the inoculation line 30 is not limited to the square shape shown in the attached figure; it can also be a circle or other common shapes. The inoculation line is observed to determine whether an inhibition zone appears between it and the pathogen colony of the fungal disease of the crop. The wider the inhibition zone, the better the antifungal effect, indicating that the antifungal activity of the Achromobacterium with antifungal activity has not degenerated. Conversely, the narrower the inhibition zone, the worse the antifungal effect, indicating that the antifungal activity of the Achromobacterium with antifungal activity has degenerated. Finally, the Achromobacterium with antifungal activity that has not degenerated is selected for preservation.

[0070] Of course, the microbial preparation for controlling fungal diseases of crops made using *Achromobacterium affine* NPDY20Z of the present invention can also be a fermentation product of *Achromobacterium affine* NPDY20Z, or an extract of the effective active ingredients of *Achromobacterium affine* NPDY20Z, etc. The fermentation broth of *Achromobacterium affine* NPDY20Z of the present invention can also be: an extract of the effective active ingredients of *Achromobacterium affine* NPDY20Z, etc. The fermentation broth of *Achromobacterium affine* NPDY20Z of the present invention (containing adjuvants) can also be obtained by adding adjuvants to the extract of the effective active ingredients of *Achromobacterium affine* NPDY20Z, etc. sequence list <110> Nanping Agricultural Science Research Institute, Fujian Province <120> A colorless bacillus with antifungal activity and its applications <130> DS-P21216 <141> 2021-09-07 <160> 1 <170> SIPOSequenceListing 1.0 <210> 2 <211> 1469 <212> DNA <213> Achromobacter sp. NPDY20Z <400> 2 ctggctcaga ttgaacgcta gcgggatgcc ttacacatgc aagtcgaacg gcagcacgga 60 cttcggtctg gtggcgagtg gcgaacgggt gagtaatgta tcggaacgtg cccagtagcg 120 ggggataact acgcgaaagc gtagctaata ccgcatacgc cctacggggg aaagcagggg 180 atcgcaagac cttgcactat tggagcggcc gatatcggat tagctagttg gtggggtaac 240 ggctcaccaa ggcgacgatc cgtagctggt ttgagaggac gaccagccac actgggactg 300 agacacggcc cagactccta cgggaggcag cagtggggaa ttttggacaa tgggggaaac 360 cctgatccag ccatcccgcg tgtgcgatga aggccttcgg gttgtaaagc acttttggca 420 ggaaagaaac gtcatgggct aataccccgt gaaactgacg gtacctgcag aataagcacc 480 ggctaactac gtgccagcag ccgcggtaat acgtagggtg caagcgttaa tcggaattac 540 tgggcgtaaa gcgtgcgcag gcggttcgga aagaaagatg tgaaatccca gagcttaact 600 ttggaactgc atttttaact accgggctag agtgtgtcag agggaggtgg aattccgcgt 660 gtagcagtga aatgcgtaga tatgcggagg aacaccgatg gcgaaggcag cctcctggga 720 taacactgac gctcatgcac gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780 ccacgcccta aacgatgtca actagctgtt ggggccttcg ggccttggta gcgcagctaa 840 cgcgtgaagt tgaccgcctg gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg 900 gggacccgca caagcggtgg atgatgtgga ttaattcgat gcaacgcgaa aaaccttacc 960 tacccttgac atgtctggaa tgccgaagag atttggcagt gctcgcaaga gaaccggaac 1020 acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080 gagcgcaacc cttgtcatta gttgctacga aagggcactc taatgagact gccggtgaca 1140 aaccggagga aggtggggat gacgtcaagt cctcatggcc cttatgggta gggcttcaca 1200 cgtcatacaa tggtcgggac agagggtcgc caacccgcga gggggagcca atcccagaaa 1260 cccgatcgta gtccggatcg cagtctgcaa ctcgactgcg tgaagtcgga atcgctagta 1320 atcgcggatc agcatgtcgc ggtgaatacg ttcccgggtc ttgtacacac cgcccgtcac 1380 accatgggag tgggttttac cagaagtagt tagcctaacc gcaagggggg cgattaccac 1440 ggtaggattc atgactgggg tgaagtcgt 1469

Claims

1. A colorless bacillus with antifungal activity, namely, colorless bacillus NPDY20Z ( Achromobacter sp.NPDY20Z was deposited on August 10, 2021, at the China Center for Type Culture Collection in Wuhan, with accession number CCTCC NO: 20211002.

2. The colorless bacillus with antifungal activity according to claim 1, characterized in that: The nucleotide sequence of the 16S rDNA of the achromobacterium is shown in SEQ ID NO.

1.

3. A microbial preparation for controlling fungal diseases in crops, characterized in that: The microbial preparation comprises the antifungal bacillus NPDY20Z as described in claim 1.

4. The application of the antifungal Bacillus of claim 1 in the control of fungal diseases in crops, characterized in that: The aforementioned fungal diseases of crops include fungal diseases caused by any one or a combination of several of the following pathogens: *Helicobacter maculatus*, *Pseudomonas chamae*, *Pseudomonas zebrina*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Pseudomonas rubra*, *Fusarium wilt*, *Botrytis cinerea*, *Rice sheath blight*, *Corn sheath blight*, *Asparagus neck blight*, *Bacillus cereus*, *Anthracnose*, *Phytophthora capsici*, *Soybean root rot*, *Fusarium graminearum*, and *Rhizoctonia solani*.

5. The application of the antifungal Bacillus achromis as described in claim 4 in the control of fungal diseases in crops, characterized in that: A microbial preparation for controlling fungal diseases in crops is prepared using the aforementioned Achromobacterium with antifungal activity, and the microbial preparation for controlling fungal diseases in crops is used for root irrigation treatment of the crops during the heading stage.

6. The application of the antifungal Bacillus achromis according to claim 4 in the control of fungal diseases in crops, characterized in that: A microbial preparation for controlling fungal diseases in crops is prepared using the aforementioned Achromobacterium with antifungal activity. The microbial preparation for controlling fungal diseases in crops is then sprayed onto the ears of the crops once each at the initial flowering stage, full bloom stage, and initial heading stage.

7. The application of the antifungal Bacillus achromis as described in claim 6 in the control of fungal diseases in crops, characterized in that: The microbial preparation for preventing and controlling fungal diseases in crops is obtained by inoculating the Achromobacterium into a brown sugar and bean powder culture medium to obtain a bacterial suspension, and then adding adjuvants to the bacterial suspension.

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