CN116445297A - Alternaria spinosa and application thereof in biological weeding - Google Patents

Abstract

The invention discloses Alternaria spinosa and application thereof in biological weeding, and belongs to the technical field of biological weeding. The Li Heiban Alternaria alternata GD-011 is preserved in China general microbiological culture Collection center (CGMCC) in the 04 th and 06 th year 2023, and the preservation number is CGMCC No.40558; the invention also discloses an application of the Li Heiban Alternaria alternata GD-011 in biological weeding or preparation of biological herbicide. The invention screens a Alternaria pyrifolia strain for biological weeding aiming at broadleaf weeds such as highland barley, wheat, rape, broad beans, pea fields, and the like, has the advantages of effective and safe control and prevention of most broadleaf weeds, low cost, no pollution, low residue and environmental friendliness.

Description

Alternaria spinosa and application thereof in biological weeding

Technical Field

The invention relates to the technical field of biological weeding, in particular to Alternaria spinosa and application thereof in biological weeding.

Background

Weeds cause great losses in agricultural production, and chemical herbicides are mainly applied to control weeds in agriculture at present. However, the application of chemical herbicides presents potentially serious problems of environmental pollution and threat to food safety. Biological herbicides provide an effective approach to the problem resolution. The biological herbicide is used for preventing and controlling weeds by utilizing natural pathogenic bacteria of the weeds, and has the advantages of high prevention and control efficiency, lasting prevention and control effect, no environmental problem and the like. The active substances in weed pathogenic microorganisms and fermentation liquor or metabolites thereof are used as lead compounds, and the development of microbial source herbicides is an important direction for the research of novel herbicides. The development of novel herbicides by novel organisms such as microorganisms has been urgent.

Over the last 20 years, some microbial herbicides have been successfully developed abroad, and certain economic benefits are achieved, bialaphos (biamphos) is the first metabolite of actinomycetes developed as a commercial herbicide. Bialaphos is a non-selective herbicide that kills monocotyledonous and dicotyledonous plants and has been widely marketed. However, the existing biological pesticides have weak capability and few new varieties, so that the pattern of single chemical herbicide production in the existing herbicide industry for a long time needs to be changed, and a new biological herbicide industry is formed, so that the screening of potential strains of more varieties is necessary.

Disclosure of Invention

The invention aims to provide Alternaria spinosa and application thereof in biological weeding, so as to solve the problems in the prior art, and the Li Heiban Alternaria spinosa GD-011 can effectively and safely control and prevent most broadleaf weeds, has low cost, no pollution and low residue, is environment-friendly, and can be used for biological weeding.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides Alternaria melanogaster (Alternaria gaisen) GD-011 which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for the year 2023, wherein the preservation address is North Chen Xili No. 1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.40558.

The invention also provides an application of the Alternaria spinosa GD-011 in biological weeding or preparation of biological herbicide.

The invention also provides a biological herbicide which comprises at least one of the strain, fermentation liquor or metabolite of Li Heiban Alternaria alternata GD-011.

The present invention also provides a method of biological weeding comprising the step of applying the strain, broth or metabolite of Li Heiban Alternaria GD-011 to a weed plant.

Further, the weed plant is a broadleaf weed.

Further, the broadleaf weeds include mallow, elsholtzia, polygonum aviculare, pig seedling, polygonum aviculare, quinoa, amaranthus retroflexus, wild oat and blue-green-grass-containing.

The invention discloses the following technical effects:

1) The invention utilizes the microbial resources to prevent and remove weeds, has the advantages of small influence on crops, less environmental negative effect, high safety and the like, accords with the development of sustainable agriculture, and has received great attention from countries around the world. The evaluation of microbial herbicides is theoretically based mainly on two criteria, namely effectiveness (efficacy) and specificity (safety). The weed biocontrol bacteria have the two characteristics of strong pathogenicity to target weeds and relative safety to crops.

2) The invention screens a Alternaria pyrifolia strain for biological weeding aiming at broadleaf weeds such as highland barley, wheat, rape, broad beans, peas, and the like in a field of peas. The invention can effectively and safely control and prevent most broadleaf weeds, has low cost, no pollution and low residue, and is environment-friendly.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is the pathogenicity of strain GD-011 on ex vivo leaves; A. quinoa (L); B. herba Moslae Mimosae Purpureae; C. polygonum aviculare (S. Amara) Maxim); D. abelmoschus manihot; E. amaranth with branches; F. pig seedling; G. wild oat; H. the method comprises the steps of (1) suppressing the blue vegetables; I. herba Polygoni Avicularis;

FIG. 2 is the pathogenicity of strain GD-011 against potted weeds; A. quinoa (L); B. herba Moslae Mimosae Purpureae; C. polygonum aviculare (S. Amara) Maxim); D. abelmoschus manihot; E. amaranth with branches; F. pig seedling; G. wild oat; H. the method comprises the steps of (1) suppressing the blue vegetables; I. herba Polygoni Avicularis;

FIG. 3 is the safety of strain GD-011 to crops; a, broad beans; b: pea(s); c, rape; highland barley; e, wheat;

FIG. 4 is a morphological feature of strain GD-011; A. d: morphology of Strain GD-011 on PDA plates: b: conidiophores; c and E-F: conidium;

FIG. 5 is a phylogenetic tree of GD-011 constructed based on rDNA-ITS, EF-1a and Alt a1 gene sequences in combination;

FIG. 6 is a characteristic of a scanning electron microscope for observing invasion of strain GD-011 into leaf tissue of Elsholtzia ciliata; a: not inoculated; B-C: inoculating for 1-2d; D-E: inoculating for 3-4d; F-G: inoculating for 5-6d; h: inoculating for 7d; wherein s: air holes; h: hypha; ih: invasive hyphae; td: tissue destruction.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

Example 1

1 materials and methods

1.1 test strains

Alternaria pyriformis GD-011 is separated from alfalfa roots with natural disease, and classified and named Alternaria agarase; the preservation address of the China general microbiological culture Collection center is No. 1 and No. 3 of North Chenxi Lu of the Korean area of Beijing city, and the preservation number is CGMCC No.40558.

1.2 weed and crop tested

Common broadleaf weeds in Qinghai province: quinoa, elsholtzia, mallow, polygonum aviculare, amaranthus retroflexus, wild oat, herba aristolochiae, polygonum aviculare and pig seedling; main cultivated crops in Qinghai province: highland barley, wheat, rape, broad beans and peas.

1.3 test design and method

(1) Pathogenicity of biocontrol bacteria on weed blades

Collecting normal leaf-colored weeds (quinoa, elsholtzia, mallow, polygonum aviculare, amaranthus retroflexus, wild oat, blue-grass-containing, polygonum aviculare and pig seedling) leaves from a test field, taking the leaves back into a room to perform surface flushing, then sterilizing the leaves for 30s by using 75% alcohol, flushing for 3 times by using sterile water, naturally airing the leaves, placing the leaves in a culture dish (d=9 cm) filled with sterile filter paper, and soaking 3-4 leaves in the culture dish by using the sterile water to provide a moist environment. Hypha blocks (d=8mm) were inoculated onto the front of the leaf from the edge of the colony cultivated for 7d, and 3 replicates were set per treatment with the inoculated sterile PDA medium blocks as a control, and placed in an illumination incubator at (25±1) °c for 12h illumination and 12h darkness (12 l:12 d) for cultivation. After 7d the lesion area was measured, lesion area = 1/4 x length x width x 3.14.

(2) Pathogenicity of biocontrol bacteria to potted weeds and safety of crops

(1) Pathogenicity of potted weeds

The mycelium pellet was inoculated into PDB medium (250 mL/flask), and 5 pellets per flask were cultured with shaking at 25℃and 180r/min for 120 hours. The fermentation liquor is filtered by four layers of sterilizing gauze to obtain fermentation filtrate, and then is continuously inoculated to potted weed plants in the 4-7 leaf period of normal growth for 3 days by a spray inoculation method, wherein the inoculum size is 25 mL/basin. After 24 hours of moisture-preserving culture of inoculated weed plants by using a plastic bag, placing the weed plants in a 25-30 ℃ L// D=12h// 12h artificial climatic chamber, repeating the treatment for 3 times, taking the plants inoculated with sterile PDB culture solution as a control, observing the disease condition of inoculated weeds after 7 days, and calculating the disease rate and fresh weight control effect.

(2) Safety of crops

5 Qinghai main cultivated plants of broad bean V.faba, pea P.satium, highland barley H.vulgare, wheat T.aestinum. And rape B.napus are respectively planted in flowerpots with d=12 cm and are placed in indoor cultivation. After the strain fermentation broth is diluted, the strain fermentation broth is inoculated on crop plants in the 3-6 leaf stage according to the weed pathogenicity method. Each treatment was repeated 3 times and the disease condition was investigated after 7d with crops inoculated with sterile PDB medium as a control. The safety evaluation criteria of the crops are: NS means that the plant is asymptomatic (no lesions, normal plant growth); LS indicates a slight effect (scattered plaque on leaves, growth slightly controlled); MS represents moderate disease (1/5-1/4 of leaf area has disease spots and growth is inhibited); SS stands for severe disease (massive death of plants, severe control of growth and development).

(3) Identification of biocontrol bacteria

(1) Morphological identification

The strain was placed on a PDA plate and cultured alternately in an incubator at 25℃for L// D=12h// 12h light and dark, and the colony growth rate, colony appearance and color change were observed, and the hypha and spore morphology were observed under an optical microscope. Preliminary identification is carried out by combining with fungus identification handbook.

(2) Molecular identification and construction of phylogenetic tree

The CATB method is adopted to extract the genome DNA of the strain. PCR amplification was performed with the universal primers ITS1 and ITS4, EF1-728F and EF1-986R, alt-for and Alt-rev (Table 1), respectively, which were all synthesized by Shanghai Biotechnology Co., ltd. The PCR reaction systems were all (25. Mu.L): forward and reverse primers (10. Mu. Mol/L) each 0.5. Mu.L, DNA template 0.5. Mu.L, 10 XPCR buffer 2.5. Mu.L, taq enzyme 0.2. Mu.L, dd H ₂ And (3) supplementing O. The PCR cycle was set as follows: pre-denaturation at 94℃for 5min, denaturation at 94℃for 45s, annealing at 55℃for 45s, extension at 72℃for 1min,30 cycles, extension at 72℃for 10min, and incubation at 4 ℃; after amplification, electrophoresis detection is carried out, a SanPrep column DNAJ gel recovery kit (SK 8131, shanghai) is used for recovering and purifying products, the products are sent to Shanghai to carry out bidirectional sequencing, the nucleotide sequence obtained by sequencing is subjected to BLAST comparison analysis with sequences in GenBank, clustal X1.8 and MEGAX 7.0 are used for carrying out comparison analysis on the sequences, an adjacent-joining method (NJ) is used for carrying out clustering analysis on multiple sequences,constructing a polygenic sequence phylogenetic tree. And testing by using a bootstrap method (1000 times to analyze the relationship between the strain and different strains of the same genus).

Primer information used in Table 1

1.4 scanning electron microscope to observe the infection process of the strain to the leaves of Elsholtzia spatholobus Dunn

Filter paper was laid in sterilized petri dishes (Φ=90 mm) and leaves of elsholtzia pachyrhizi were placed, the filter paper was soaked with sterile water to provide a moist environment, a punch-out pad (Φ=8 mm) was placed in the center of the leaves, and a sterile PDA pad was used as a control, each treatment and control was repeated 3 times. The test was carried out at 25℃to 28℃and samples were taken daily after inoculation to observe the invasive process of the mycelium for 7 days.

After inoculation of bacterial strain mycelium blocks 1 to 7d, 3-6 leaf segments (0.5-1.0 cm) were prepared with fresh razor blades per inoculated leaf and fixed in 2.5% (v/v) glutaraldehyde in 0.1M phosphate buffer (pH 7.2) under vacuum for 2 hours at room temperature and in the same fixation buffer at 4 ℃. Leaf samples were washed three times with 0.1M phosphate buffer (pH 7.2) and dehydrated through a graded ethanol series (70%, 80%, 90% and 100%) for 30 minutes in each gradient. At the critical point with liquid CO ₂ The sample was dried. The immobilization material was coated with a layer of 10nm gold/palladium and observed using a scanning electron microscope.

1.5 statistical analysis

Statistical analysis of the experimental data was performed using Excel and SPSS 25.0 and differential significance tests were performed using one-way analysis of variance (ANOVA) and Least Significant Difference (LSD) mean comparison.

2 analysis of results

2.1 identification of the pathogenicity of Alternaria pyriformis GD-011 against weeds, the safety of crops and the Strain

2.1.1 pathogenicity of Alternaria pyriformis GD-011 against weed leaves

As shown in figure 1, after the GD-011 bacterial cake is inoculated for 7 days, the in-vitro leaves are damaged, the yellow and green phenomena of the leaves of the quinoa, the polygonum aviculare and the mallow appear, and hypha on the back surface of the inoculated part can be seen to penetrate and grow; the elsholtzia californica, the pig seedling and the amaranthus retroflexus have grey hyphae, and have wilting symptoms in the later period; the wild oat, the herba aristolochiae and the polygonum aviculare have the phenomena of fading green and yellowing and blackening after being inoculated for 7 days. The GD-011 mycelium blocks show that the pathogenicity of the mycelium blocks to different weeds in vitro is as follows: abutilum > herba Moslae, herba Polygoni Avicularis > pig seedling, herba Polygoni Avicularis > herba Polygoni Capitati > herba Chenopodii, herba Amaranthi Tricoloris > herba Avenae Fatuae, and herba Hedyotidis Diffusae.

2.1.2 pathogenicity of Alternaria pyriformis GD-011 against potted weeds

After the GD-011 fermentation product is sprayed for 7 days, the morbidity and fresh weight prevention effect of the okra reach 94.83%, 90.81%, and the herba chenopodii, the elsholtzia pachyrhizi, the polygonum aviculare and the cleaver show leaf curl, and the lower leaf wilting and yellowing are 75.5%, 80.87%, 77.13% and 68.5% respectively; the malva leaves lose green, necrotize and fall off after 7d; the leaves of the wild oat are yellow, 2/3 of the leaves die, and the morbidity is 82.13 percent respectively; the leaves of Amaranthus retroflexus and herba Polygoni Avicularis have sporadic spots, which are marked by black and dead stems and leaves, the morbidity is 56.2% and 42.07%, and the disease index shows that the strain fermentation filtrate is most sensitive to the malva, the cleavers and the wild oat. After 7d, the investigation found that the inoculated weeds had no resuscitative phenomenon, and the disease was continuously aggravated until the whole pot of weeds died (fig. 2 and table 2).

TABLE 2 pathogenicity of strain GD-011 fermentation filtrate against different weeds

2.1.3 safety of Alternaria pyrifolia GD-011 against crop plants

The GD-011 strain fermentation liquor has no pathogenicity on peas, broad beans and wheat, no matter the growth vigor and the plant height of crops are not affected by the comparison with that of blank control plants, and healthy growth is shown as no reaction (NS); rape is slightly pathogenic, black spots appear on 5% of leaf edges, plants wilt and show slight reaction (LS); the highland barley has slight pathogenicity, a small part of leaf veins have scattered spots, the leaves turn yellow, and the whole shows slight reaction (LS) (figure 3).

2.1.4 morphological identification of Alternaria melanogaster GD-011

The GD-011 bacterial colony is white in the early stage on a PDA plate, gradually develops into an olive or dark green velvet shape in the later stage, and has neat edges. Aerial hyphae are dense. The mycelium was colorless with a septum. Conidium is brown or black brown, elliptic, oval, rod-shaped or inverted pear-shaped, 2-5 transverse septa and 0-3 longitudinal septa are arranged, the separation part is slightly overflowed, and the beak is cylindrical or conical. Based on the culture characteristics and morphological characteristics of the strain, the pathogenic bacteria were initially identified as Alternaria sp (FIG. 4).

2.1.5 molecular biological identification of Alternaria pyriformis GD-011

PCR amplification is carried out on rDNA-ITS, EF-1 alpha and Alt a1 gene sequences of the strain GD-011, so that 3 gene fragments with the lengths of 533bp, 264bp and 477bp are obtained. According to the strain sequence, ulocladium consortiale is taken as an outer group to construct a phylogenetic tree (figure 5) of the strain, GD-011 and Alternaria gaisen are found to be gathered together on the phylogenetic tree, the support rate is 99%, and Alternaria pyriformica and other species can be accurately separated through the 3 gene sequences, so that the phylogenetic tree has good conservation as a whole. According to phylogenetic analysis of 3 gene sequences of GD-011, the strain GD-011 is identified as Alternaria pyriformis Alternaria gaisen.

2.1.6 observation of pathogenic Process of Elsholtzia ciliata by Strain GD-011

FIG. 6 shows that GD-011 strain affects the ultrastructure of elsholtzia ciliata after infecting its leaves. The uninoculated healthy elsholtzia ciliata leaf cells were normal in structure and organized in order (fig. 6 a). After 1-2d inoculation, the stomata had hyphae penetrated and a few hyphae were produced around (B and C in FIG. 6). After 3-4D inoculation, a large number of hyphae attached to the tissue surface and the leaf tissue was broken (D and E in FIG. 6). After 5-6d, bacterial nets are formed along with hyphae and parasitic on the surface of the tissue, the hyphae parasitic tissue absorbs nutrients, the plant tissue is diseased (F and G in figure 6), obvious damage phenomenon appears on the surface of the tissue along with infection of a large number of hyphae, after 7d, the hyphae grow vigorously and spread over the surface of the tissue, the metabolism of the leaf tissue of the elsholtzia spatulosa is disturbed, and the infected cells die gradually (H in figure 6).

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (6)

1. Alternaria alternata (Alternaria aisen) GD-011 is characterized in that it has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the day 04 and 06 of 2023, and has a deposit address of Hospital No. 3 of North Chen West Lu of the Korean region of Beijing city and a deposit number of CGMCC No.40558.

2. Use of alternaria caliae GD-011 as claimed in claim 1 for the biological control or for the preparation of biological herbicides.

3. A bioherbicide comprising at least one of the strain, fermentation broth, or metabolite of Li Heiban alternaria alternata GD-011 of claim 1.

4. A method of biological weeding comprising the step of applying the strain, broth or metabolite of Li Heiban alternaria GD-011 to a weed plant.

5. The method of claim 4, wherein the weed plant is a broadleaf weed.

6. The method of claim 5, wherein the broadleaf weeds comprise mallow, elsholtzia pachyrhizi, polygonum aviculare, pig seedling, polygonum aviculare, quinoa, amaranthus retroflexus, wild oat and blue-grass-suppressing.