Streptomyces sp. producing amylase for controlling konjac stem rot and application thereof

Abstract

This invention discloses an amylase-producing chromogenic Streptomyces diastatochromogenes resistant to konjac stem rot and its application. The chromogenic Streptomyces diastatochromogenes A-dyzsc04-2 was deposited on December 23, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20222046. The amylase-producing chromogenic Streptomyces A-dyzsc04-2 isolated in this invention exhibits a highly beneficial inhibitory effect against *Fusarium solani*, with its fermentation broth showing a 100% inhibition rate against *Fusarium solani*. Field trials have demonstrated that its fermentation broth has excellent control effects against konjac stem rot caused by *Fusarium solani*.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of amylase-producing Streptomyces resistant to konjac stem rot and its application. Background Technology

[0002] Guizhou boasts a warm climate with mild winters and cool summers, abundant rainfall, relatively little sunshine, and significant diurnal temperature variations. Its extensive mountainous forests provide ideal natural conditions for konjac production. Konjac (Amorphophallus konjac) is a perennial herbaceous plant belonging to the Araceae family and the Amorphophallus genus. It is a semi-shade, moisture-loving plant with wide adaptability, capable of being cultivated from tropical to temperate mountainous regions. The entire growth period is 100–200 days. During this period, it requires temperatures of 15–22℃. Root development begins at 12℃, sprouting begins at 15℃, and the tuber enlargement stage requires temperatures around 20℃.

[0003] The above conditions indicate good growth, but growth is inhibited above 35℃, and the above-ground parts naturally wither below 12℃; during dormancy, it can tolerate temperatures as low as -5℃, but the underground tubers will be damaged by frost below -8℃.

[0004] Konjac is one of Guizhou's famous, high-quality, and special agricultural crops. Also known as taro, it was anciently called "Yaoweng." Its corms contain abundant starch, protein, vitamins, glucomannan, glutamic acid, glycine, and 17 other essential amino acids, as well as various trace elements such as phosphorus, potassium, sodium, iron, and zinc. It plays an important role in regulating, balancing, and improving human metabolism, enhancing immunity, and providing health benefits. It has broad development prospects in food, medicine, health care, cosmetics, and industry. Guizhou Province is a superior production area for konjac cultivation, with a climate suitable for its growth. In recent years, the konjac industry has developed rapidly, with large-scale cultivation emerging in Bijie City, Weining County, Liupanshui City, Xingyi City, Dafang County, Nayong County, Anlong County, and other places. With the expansion of konjac cultivation in Xingyi, an integrated konjac industry encompassing cultivation, harvesting, and processing has been formed. However, the increasing severity of diseases such as soft rot, white mold, stem rot, leaf blight, and sunscald has led to a significant reduction in konjac production, causing severe economic losses to konjac growers.

[0005] Konjac stem rot, caused by Fusarium, is a significant soil-borne disease in konjac cultivation. Large-scale outbreaks can be devastating, increasing the risks for farmers. Although research and reports on control methods have been published and have shown some effectiveness, konjac stem rot remains a major challenge in actual konjac production, causing substantial losses to the industry. Currently, there is limited research on the isolation and identification of the pathogen causing konjac stem rot in Guizhou. Stem rot causes the konjac corms to rot, and since the corms are a crucial source of economic value for konjac, stem rot can lead to severe economic losses for farmers, even resulting in total crop failure. Summary of the Invention

[0006] The technical problem to be solved by this invention is: how to provide a strain of amylase-producing Streptomyces resistant to konjac stem rot and its application.

[0007] The technical solution of the present invention is as follows: a strain of amylase-producing Streptomyces diastatochromogenes A-dyzsc04-2 was deposited at the China Center for Type Culture Collection on December 23, 2022, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20222046.

[0008] Application of strain with accession number CCTCC NO: M 20222046 in the control of konjac stem rot caused by Fusarium solani.

[0009] A formulation containing the aforementioned amylase-producing Streptomyces diastatochromogenes A-dyzsc04-2 or its metabolites.

[0010] A method for preventing stem rot in konjac involves irrigating the roots around the petioles of konjac with the fermentation broth of the amylase-producing Streptomyces diastatochromogenes A-dyzsc04-2.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The amylase-producing Streptomyces diastatochromogenes A-dyzsc04-2 isolated in this invention exhibits a highly beneficial inhibitory effect against Fusarium solani, with its fermentation broth showing a 100% inhibition rate against the fungus. Field trials have demonstrated that its fermentation broth has excellent control effects against konjac stem rot caused by Fusarium solani. Attached Figure Description

[0013] Figure 1 Morphological observation of Streptomyces diastatochromogenes A-dyzsc04-2, a strain of amylase-producing chromogen.

[0014] Figure 2 Phylogenetic tree of amylase-producing Streptomyces diastatochromogenes A-dyzsc04-2;

[0015] Figure 3 The plate inhibition effect of four different species of actinomycetes on Fusarium is shown. Fusarium is in the middle, and the four surrounding species are actinomycetes. A is the front side of the plate, B is the back side of the plate, and C is the negative control.

[0016] Figure 4 The growth of Fusarium on a plate containing the fermentation broth of Streptomyces diastatochromogenes A-dyzsc04-2 containing amylase is shown. A is the front side of the plate, B is the back side of the plate; C is the front side of the negative control plate, and D is the back side of the negative control plate. Detailed Implementation

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0018] Example 1

[0019] Isolation, identification, and preservation of bacterial strains:

[0020] (I) Isolation of strains:

[0021] Actinomycetes were isolated from soil samples using the soil dilution spread method. 5g of soil sample was poured into a 45mL sterile conical flask containing water and incubated at 28℃ and 150r / min on a shaker for 30min to obtain a concentration of 10. -1 The sample suspension. Take 10 under aseptic conditions. -1 The sample suspension was prepared with a concentration of 10. -3 and 10 -4 Dilute the solution by 100 μL and spread it onto Gao's No. 1 medium agar plates (containing 0.05 g / L potassium dichromate), seal, and incubate in the dark at 28 °C. After two subculturings of the colony morphology, which is similar to that of actinomycetes, pure cultures can be obtained. The pure cultures are numbered and stored in sterile water at 4 °C and -20 °C, respectively, using sterile water and 25% glycerol.

[0022] (II) Screening of strains:

[0023] (1) Initial screening by confrontation culture: Fusarium solani mycelial cakes (D=6mm) of the konjac stem rot pathogen were punched with a sterile punch and inoculated into the center of a PDA plate. Isolated actinomycete mycelial cakes (D=6mm) were inoculated in four directions at a distance of 25mm from F. solani. Single inoculation of pathogen was used as a control. Three biological replicates were performed for each treatment. After incubation in the dark at 28℃ for 7 days, the colony radius of the target bacteria growing towards the actinomycetes was measured, and the inhibition rate of the actinomycetes against the pathogen was calculated.

[0024] Inhibition rate (%) = (Coronavirus diameter of control group - Coronavirus diameter of treatment group) / Coronavirus diameter of control group × 100%; Target bacterial mycelial discs (D = 6 mm) were punched using a sterile punch and inoculated into the center of a PDA plate. Actinomycete mycelial discs (D = 6 mm) were inoculated in four directions at a distance of 25 mm from the target bacteria. Single inoculation of pathogenic bacteria served as a control. Three biological replicates were performed for each treatment. After incubation in the dark at 28°C for 7 days, the colony radius of the target bacteria growing towards the actinomycetes was measured, and the inhibition rate of actinomycetes against the pathogenic bacteria was calculated.

[0025] The results showed that the strain A-dyzsc04-2 had an inhibition rate of 86.55% against *Fusarium solani*, the pathogen causing stem rot of konjac. Figure 3 )

[0026] (2) Mycelial growth rate method for secondary screening: The strain A-dyzsc04-2 with high inhibition rate in the initial screening was added to LB liquid medium at a ratio of mycelial cake (D=6mm) number:V medium = 1:10mL. The fermentation broth was obtained by shaking and culturing at 28℃ and 180r / min for 7 days. After filtration, the fermentation broth was centrifuged at 10000r / min for 10min. 10mL of supernatant was taken and filtered through a 0.22μm filter membrane to obtain sterile fermentation filtrate. The filtrate was poured into an Erlenmeyer flask containing 90mL PDA, mixed thoroughly, and then poured into a plate. The mycelial cake of the target bacteria was inoculated in the center of the plate. Single inoculation of the target bacteria was used as a control. Three biological replicates were performed for each treatment. After culturing at 25℃ for 7 days, the colony radius was measured by the cross-cross method, and the inhibition rate of the antagonistic actinomycete fermentation broth was calculated.

[0027] Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%

[0028] The results showed that the fermentation broth of A-dyzsc04-2 inhibited the growth of *Fusarium solani*, the pathogen causing stem rot of konjac, by 100.00%. Figure 4 )

[0029] (II) Identification of strain A-dyzsc04-2:

[0030] (1) Morphological characteristics: strain A-dyzsc 04-2 grew well on ISP2 solid medium. After 7-14 days of culture, the aerial mycelium was initially white and later turned grayish-white. The mycelium in the substrate had no distinguishable color, no water-soluble pigment, and produced an earthy smell. The spore chains were mainly flexible (Rectiflexibiles type), but also open ring, hook, or broad spiral (Retinaculiapertu type). After the spore chains broke, conidia were formed. The conidia were elliptical and irregular, and no sporangia were observed.

[0031] (2) Molecular developmental analysis: Actinomycete DNA was extracted according to the method of Ezup column-based bacterial genomic DNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.), and the 16S rDNA gene was amplified; PCR amplification system (25μL): 12.5μL GoldenStar Tb Super PCR Mix (1.1x) (Beijing Qingke Biotechnology Co., Ltd.), 2μL DNA template, 1μL each of 10μmol / L primers, and ddH2O to make up to 25μL. Primers and all PCR reaction procedures and conditions are shown in Table 1. PCR products were sequenced by Beijing Qingke Biotechnology Co., Ltd. The sequenced sequences were subjected to homology searches using NCBI's BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and Ezbiocloud, sequence alignment was performed using MAFFT, sequence cutting was performed using Bioedit software, and sequence assembly was performed using MEGA X software. Maximum-likelihood phylogenetic trees were constructed using the 16S rDNA gene and 16S rDNA-gyrB gene sequences, respectively. The phylogenetic tree nodes only showed values ​​with a bootstrap value greater than or equal to 50%, and the superscript "T" indicated the type strain.

[0032] Table 1 Primers and PCR Procedure

[0033]

[0034] The amplified 16S rDNA and gyrB gene fragments of strain A-dyzsc04-2 were 514 bp (SEQ ID No. 1) and 686 bp (SEQ ID No. 2), and 923 bp (SEQ ID No. 3) and 951 bp (SEQ ID No. 4), respectively. A phylogenetic tree of the spliced ​​16S rDNA and gyrB gene sequences was constructed using known model strains of the genus *Streptomyces*, which exhibited amylase production. The results showed that the 16S rDNA-gyrB gene sequence of A-dyzsc04-2 belonged to the same clade as *S. diastatochromogenes* on the phylogenetic tree, with a sequence similarity of 88%. Based on morphological characteristics, A-dyzsc04-2 was confirmed to belong to *S. diastatochromogenes*.

[0035] (III) Preservation of strain A-dyzsc04-2:

[0036] The amylase-producing chromogenic Streptomyces (S. diastatochromogenes) A-dyzsc04-2 was deposited on December 23, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20222046.

[0037] Example 3

[0038] This example demonstrates an experiment using the amylase-producing Streptomyces diastatochromogenes A-dyzsc04-2 to control stem rot caused by Fusarium solani.

[0039] Preparation of fermentation broth for *Streptomyces diastatochromogenes* A-dyzsc04-2: The mycelium of strain *Streptomyces diastatochromogenes* A-dyzsc04-2 was added to LB liquid medium at a ratio of mycelium cake (D=6mm) number:V medium = 1:10mL. The mixture was cultured in a shaker at 28℃ and 180r / min for 7 days. The fermentation broth was obtained by filtration through 4 layers of gauze.

[0040] The experiment was conducted at the experimental base in Hewang Village, Tianlong Town, Pingba District, Anshun City, Guizhou Province. Each treatment had 3 replicates, and the plot area was approximately 52 m². 2 The fermentation broth of *Streptomyces diastatochromogenes* A-dyzsc04-2, an amylase-producing strain, was randomly arranged and applied to the roots around the petioles of konjac leaves in a 30 mL drenching manner. Spraying with water served as a control. The application was repeated once at 10 days and 20 days, for a total of 3 applications. After 30 days, the incidence and disease index of konjac stem rot were investigated.

[0041] Field survey grading standards:

[0042]

[0043] The results are shown in the table below. Under conventional control (blank control), the incidence of konjac stem rot was 32.67%, and the disease index was 28.27. After applying fermentation broth of *Streptomyces diastatochromogenes* A-dyzsc04-2, the incidence of konjac stem rot was 16.33%, and the disease index was 10.6. The control efficacy of *Streptomyces diastatochromogenes* A-dyzsc04-2 against konjac stem rot was 62.50%.

[0044] Table 1. Control effect of fermentation broth of *Streptomyces diastatochromogenes* A-dyzsc04-2 on konjac stem rot.

[0045] deal with Incidence rate Disease index Prevention Blank control 32.67％ 28.27 - A-dyzsc04-2 16.33％ 10.6 62.50％

Claims

1. A strain of amylase-producing Streptomyces ( Streptomyces diastatochromogenes A-dyzsc04-2 was deposited on December 23, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20222046.

2. The Streptomyces di azotrophicus of claim 1, wherein the Streptomyces di azotrophicus is a strain of Streptomyces di azotrophicus NRRL B-50591. Streptomyces diastatochromogenes ) A-dyzsc04-2 for use in the control of yam stem rot caused by Fusarium solani f. sp. radicis-lycoperci. Fusarium solani ) A-dyzsc04-2 for use in the control of yam stem rot caused by Fusarium solani f. sp. radicis-lycoperci.

3. A formulation comprising the amylase-producing Streptomyces of claim 1 ( Streptomyces diastatochromogenes )A-dyzsc04-2.

4. A method for preventing and controlling konjac stem rot, characterized in that, The amylase-producing Streptomyces as described in claim 1 ( Streptomyces diastatochromogenes Apply A-dyzsc04-2 fermentation liquid to the roots around the petioles of konjac leaves.

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