CN114540213B - Actinomycetes with antibacterial activity and application thereof - Google Patents
Actinomycetes with antibacterial activity and application thereof Download PDFInfo
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- CN114540213B CN114540213B CN202111334350.8A CN202111334350A CN114540213B CN 114540213 B CN114540213 B CN 114540213B CN 202111334350 A CN202111334350 A CN 202111334350A CN 114540213 B CN114540213 B CN 114540213B
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/28—Streptomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of microorganisms, and particularly relates to actinomycetes with antibacterial activity and application thereof. The actinomycetes are Streptomyces sp.5-6, and the preservation number is GDMCC No:61984 has stable broad-spectrum antibacterial activity, has good antagonism on chestnut blight germ, banana large gray spot germ, banana leaf margin blight germ, banana fusarium wilt germ No. 4 physiological seed, cucumber fusarium wilt germ, pepper anthracnose germ, banana fusarium wilt germ No. 1 physiological seed, rubber corynespora leaf-drop germ, vegetable heart anthracnose germ, strawberry anthracnose germ, wheat scab germ and the like, provides a new path for green control of various plant diseases such as banana fusarium wilt and the like, and has wide development space and good development and application prospect.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to actinomycetes with antibacterial activity and application thereof.
Background
Bananas are musaceae musa plants, are mainly distributed in tropical and subtropical areas, and are one of the most important fruits in world yield and trade. When the banana industry is continuously developed and a certain effect is gradually obtained, the banana is affected by large-area outbreak of banana wilt, so that the traditional banana planting area is rapidly reduced, even phenomena of losing, barren and seed changing occur, serious economic loss is brought to banana farmers, and healthy development of the banana industry is restricted. Banana vascular wilt is a soil-borne disease caused by Fusarium oxysporum Guba specialization (Fusarium oxysporum f.sp.cube, foc) infection, and mainly occurs in banana producing areas such as Asia, australia, africa, philippines, tropical America and the like, and more serious banana vascular wilt occurs in Guangdong, guangxi, hainan, yunnan, taiwan and the like in China. The banana fusarium wilt bacteria have 4 physiological micro-seeds, wherein the 1 # and 4 # physiological micro-seeds have the greatest harm to banana industry in China: the No. 1 race mainly infects the banana and the variety of 'big honey ha', while the No. 4 race can infect almost all banana varieties, and has extremely strong pathogenicity. Banana vascular wilt has been a serious threat to the health development of the banana industry worldwide due to its high incidence, large destructive nature and rapid spread. Biological control is considered to be one of the most effective methods at present before chemical control is difficult to achieve and no better disease-resistant varieties are cultivated.
Actinomycetes are capable of producing abundant secondary metabolites against pathogenic microorganism growth, and also promote plant growth and maintain a balance of micro-ecology within plants by a range of different mechanisms (e.g., phytohormone synthesis, nitrogen fixation, phosphate solubilization, induction of defensive responses, reduction of abiotic stress by reduction of ethylene content, etc.), which have considerable agrobiotechnology potential. Although new species are continuously discovered, the types separated at present are still not 1% of the total quantity of actinomycete types in the nature, and actinomycete resources are still one of research hotspots. Streptomyces (Streptomyces) is an important microbial resource, and the metabolite thereof accounts for 80% of the natural metabolites of actinomycetes reported so far, is an important research object of actinomycetes in plants, has important application value in preventing and controlling agricultural diseases and insect pests, and provides a new path for green prevention and control of banana vascular wilt.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide actinomycetes which are novel streptomycetes species, have broad-spectrum antibacterial activity, have good antagonism on various pathogenic bacteria and provide references for green control of banana wilt.
In a first aspect, the present invention provides an actinomycete which is a novel species of Streptomyces, designated Streptomyces sp.5-6, deposited at the microorganism strain conservation center in Guangdong province on 10 months 27 of 2021 under the accession number GDMCC No:61984.
in a second aspect, the invention provides a fermentation broth of a strain of actinomycetes according to the first aspect of the invention, and/or a crude fermentation extract of actinomycetes.
Further, the preparation method of the fermentation liquid and the fermentation crude extract comprises the following steps: inoculating the actinomycete strain into a soybean meal fermentation medium for culturing to obtain strain fermentation liquor, adding pure ethanol for mixed extraction, filtering with filter paper to remove hypha, and rotationally evaporating the filtrate to obtain a fermentation crude extract.
Further, in the strain fermentation process, the strain is cultured on a constant temperature shaking table at the temperature of 28 ℃ and the ratio of the volume of the added pure ethanol to the volume of the strain fermentation liquid is 1:1.
In a third aspect the invention provides the use of an actinomyces and/or strain broth according to the first aspect of the invention for antagonising the physiological race No. 4 of banana vascular wilt (Fusarium oxysporum f.sp.cube race 4, foc 4), and/or the physiological race No. 1 of banana vascular wilt (Fusarium oxysporum f.sp.cube race1, foc 1), and/or the bacterial leaf spot of banana (Curvularia lunata), and/or the bacterial leaf spot of banana (Alternaria musae), and/or the bacterial leaf spot of banana (Colletotrchum musae), and/or the bacterial leaf anthracnose of vegetable heart (Colletotrichum higginsianum), and/or the bacterial anthracnose of capsicum (Colletotrichum acutatum), and/or the bacterial anthracnose of strawberry (Colletotrichum fragariae), and/or the bacterial chestnut blight (Cryphonectria parasitica), and/or the bacterial leaf spot of cucumber (Fusarium oxysporum) f.sp.cube, and/or the bacterial leaf blight of wheat (Fusahum graminearum), and/or the bacterial leaf spot of banana (Corynespora cassiicola).
A fourth aspect of the invention provides the use of an actinomycete and/or strain fermentation broth according to the first aspect of the invention for the preparation of a disease control agent for controlling a physiological race No. 4 of banana vascular wilt (Fusarium oxysporum f.sp. cube race 4, foc 4), and/or a physiological race No. 1 of banana vascular wilt (Fusarium oxysporum f.sp. cube race1, foc 1), and/or a bacterial leaf spot of banana (Curvularia lunata), and/or a bacterial leaf spot of banana (Alternaria musae), and/or a bacterial leaf spot of banana (Colletotrchum musae), and/or a bacterial leaf spot of vegetable heart (Colletotrichum higginsianum), and/or a bacterial anthracnose of capsicum (Colletotrichum acutatum), and/or a bacterial anthracnose of strawberry (Colletotrichum fragariae), and/or a bacterial chestnut blight (Cryphonectria parasitica), and/or a bacterial leaf spot of cucumber (Fusarium oxysporum) (Schcul.) F.sp. cube, and/or a bacterial leaf spot of wheat (Fusahum graminearum), and/or a bacterial leaf spot of banana (Corynespora cassiicola).
In a fifth aspect, the invention provides the use of the strain fermentation broth and/or crude fermentation extract of actinomyces as described in the second aspect of the invention for inhibiting banana fusarium wilt No. 4 physiological race.
The sixth aspect of the invention provides the application of the strain fermentation broth and/or the fermentation crude extract of actinomyces in the second aspect of the invention in inhibiting the physiological race pathogenic hypha of banana fusarium wilt No. 4.
In a seventh aspect, the present invention provides a biocontrol formulation comprising an actinomycete according to the first aspect of the invention, or a fermentation broth comprising a strain fermentation broth according to the second aspect of the invention, and/or a crude fermentation extract of actinomycete.
The actinomycetes disclosed by the invention are a new species of streptomyces, have stable broad-spectrum antibacterial activity, have good antagonism on banana fusarium wilt 4 physiological race, banana fusarium wilt 1 physiological race, banana large gray spot disease germ, banana leaf margin fusarium wilt germ, banana anthracnose germ, vegetable heart anthracnose germ, pepper anthracnose germ, strawberry anthracnose germ, chestnut blight germ, cucumber fusarium wilt germ and/or wheat scab germ, rubber corynespora leaf spot germ and the like, provide a new path for green control of various pathogenic bacteria such as banana fusarium wilt and the like, and have wide development space and good development and application prospects.
Drawings
FIG. 1 is a scanning electron microscope image of strains 5-6;
FIG. 2 is a genomic circle map of strains 5-6;
FIG. 3 is a phylogenetic tree of strains 5-6;
FIG. 4 shows the results of a broad-spectrum bacteriostatic activity assay for strains 5-6;
FIG. 5 shows the results of the antibacterial activity measurement of the crude fermentation extract of strain 5-6;
FIG. 6 shows the dissolution of hyphae by crude fermentation extracts of strains 5-6.
Detailed Description
The invention will be further described with reference to specific examples to provide a better understanding of the invention.
The invention provides actinomycetes which are a novel species of Streptomyces and are named as Streptomyces sp 5-6 (hereinafter referred to as strain 5-6), and are deposited in the microorganism strain deposit center of Guangdong province at 10 months and 27 days in 2021, wherein the deposit number is GDMCC No:61984 the deposit address is at Guangdong province microbiological institute of Guangzhou City, guangdong, first, china, no. 100, no. 59, 5. The actinomycetes of the invention are isolated from the root of the curculigo latifolia (Curculigo capitulata).
1. Materials and methods
1.1 test strains
The measurement of the antibacterial spectrum is performed by banana fusarium wilt 4 physiological race (Fusarium oxysporum f.sp.cube race 4, foc 4), banana fusarium wilt 1 physiological race (Fusarium oxysporum f.sp.cube race1, foc 1), banana large gray leaf spot (Curvularia lunata), banana leaf margin blight (Alternaria musae), banana anthracnose (Colletotrchum musae), vegetable heart anthracnose (Colletotrichum higginsianum), pepper anthracnose (Colletotrichum acutatum), strawberry anthracnose (Colletotrichum fragariae), chestnut blight (Cryphonectria parasitica), cucumber fusarium wilt (Fusarium oxysporum (Schl.) F.sp.cube, wheat scab (Fusahum graminearum), and rubber corynespora leaf spot (Corynespora cassiicola). Is preserved in Tropical biotechnology institute of Tropical agricultural academy of sciences of China.
1.2 test Medium
The culture medium for identifying the physiological biochemistry, the culture characteristics and the like of actinomycetes is referred to actinomycetes phylogenetic-principle, method and practice. PDA culture medium is used for antagonism test, and soybean meal fermentation culture medium is used for fermentation culture. The specific composition is as follows.
Table 1 formulation of the Medium
1.3 Strain 5-6 culture characteristics and physiological Biochemical identification
Identification of antagonistic actinomycetes the identification of physiological and biochemical characteristics, culture characteristics and the like of the strain is carried out by referring to actinomycete phylogenetic principles, methods and practices.
1.4 scanning electron microscope observations
Sample: strains 5 to 6 were inoculated onto ISP2 medium and cultured in reverse at 28℃for about 7 to 10 days. Cube patches containing actinomycete colonies were excised.
Front fixing: square pellets containing actinomycete colonies were immersed in glutaraldehyde at a volume fraction of 2.5% and allowed to stand at 4 ℃ overnight.
Rinsing: the immobilized samples were rinsed with 0.1mol/L Phosphate Buffered Saline (PBS) for 15min each, and repeated 3 times.
Dehydrating: sequentially dehydrating the rinsed sample with ethanol with volume fraction of 30%,50%,70%,80%,90% and 95% for 15min each time; eluting with 100% ethanol for 2 times each for 20min.
Intermediate liquid replacement: with V (ethanol): the mixed solution of V (isoamyl acetate) =1:1 is treated for 30min for preliminary replacement, and then the sample is treated for 2h with the volume fraction of 100% isoamyl acetate to finish the replacement of the intermediate solution.
The treated sample was placed in a sterile super clean bench for natural air drying, then gold plating was performed, and the microscopic structures of the mycelia and spores of actinomycetes were observed by a scanning electron microscope (Zhang Wenjuan, 2013).
1.5 extraction of Strain 5-6 Total genome
Strains 5-6 were activated on ISP2 plates, incubated at 28℃for 3d, single colonies were picked and inoculated into a triangular flask (250 mL) containing 100mL of ISP2 liquid medium, and after 5d incubation at 28℃at 150rpm, the cells were collected and subjected to genome sequencing.
1.5.1 sequencing and Assembly of the 5-6 genome of Strain
Strains 5-6 were subjected to genome sequencing and splicing using a second generation sequencer (Shanghai Meiji Biotechnology Co., ltd.). Genome sequencing is carried out on an Illumina Hiseq multiplied by 10 platform, a small fragment genome library is constructed by adopting a Paired-End technology, a fragment with an insert of 400bp is constructed for a DNA sample with qualified quality, and the genome is sequenced through the Illumina Hiseq multiplied by 10 platform. And (3) splicing a plurality of Kmer parameters of the optimized sequence after the second generation of sequencing by using short sequence assembly software SOAPdenovo2 (http:// soap. Genes. Org. Cn /), obtaining an optimal contigs assembly result, then comparing reads to contigs, and carrying out partial assembly and optimization on the assembly result according to the relationship between the paired-end and overlap of reads to form scaffoldes.
1.5.2 genome component analysis
After obtaining the genome data, the genome is subjected to structural analysis using related software. The coding sequence (CDS) in the genome was predicted using Glimmer (Version 3.02) software (http:// ccb.jhu.edu/software/Glimmer/index. Shtml). Tandem repeats in the DNA sequence were searched using Tandem Repeats Finder software. The nucleotide sequence information, anticodon information and secondary structure information of tRNA in each sample genome can be obtained by predicting tRNA contained in the genome using tRNAscan-SE v2.0 software (http:// tRNA. Ucsc. Edu/software /). The rRNA contained in the genome was predicted by using Barrnap software (https:// github. Com/tseemann/Barrnap) to obtain the kind, position and sequence information of all rRNAs in each sample genome.
1.5.3 phylogenetic tree construction
After homology alignment of the 16SrDNA sequences in the EzBioCloud database, phylogenetic trees were constructed in a contiguous manner using MEGA software. The genome of the strain with the highest homology was downloaded in NCBI database, and the Average Nucleotide Identity (ANI) of the whole genome among the strains was calculated using the alignment tool ANI Calculator of EzBioCloud database, strain 5-6.
1.6 broad-spectrum antibacterial Activity assay
The broad-spectrum antibacterial activity of the strain 5-6 is measured by adopting a flat plate counter culture method. Taking bacterial cakes of pathogenic bacteria with consistent growth vigor by using a puncher with the diameter of 5mm, inoculating bacterial cakes of 4 bacterial strains 5-6 at the position which is 2.5cm away from bacterial colonies of the pathogenic bacteria in the middle of each PDA flat plate, and placing the bacterial cakes in an incubator with the temperature of 28 ℃ for inverted culture of 5-7 d so as to only inoculate the pathogenic bacteria as a control. The diameter of the colony of pathogenic fungi was measured by the crisscross method, and the inhibition rate against pathogenic fungi was calculated according to the following formula.
Colony diameter (mm) =average value of colony diameter measurement-5.0
1.7 bacterial strain 5-6 fermentation crude extract antibacterial Activity
Inoculating the strain 5-6 into a soybean meal fermentation medium, and culturing for 7d on a constant temperature shaking table at 28 ℃ and 200 r/min. Adding pure ethanol at a ratio of 1:1 (v: v) to mix, extracting active crude extract, filtering hypha with filter paper, rotary evaporating to obtain fermented crude extract, and preparing into fermented crude extract solution with concentration of 500 μg/mL with sterilized water for use.
The antibacterial activity of the crude fermentation extract of strain 5-6 on Foc was determined by the filter paper method. The filter paper sheet is completely soaked in the fermented crude extract solution, taken out and air-dried. At a distance of 2.5cm from the cake of the test pathogenic bacteria, 4 filter paper sheets containing 500. Mu.g/mL of the crude fermentation extract of strain 5-6 were attached, respectively, to inoculate only the test pathogenic bacteria as a control, and each treatment was repeated three times and cultured upside down at 28℃for 7d. The diameter of the colony of pathogenic fungi was measured by the crisscross method, and the inhibition rate against pathogenic fungi was calculated according to the following formula.
Colony diameter (mm) =average value of colony diameter measurement-5.0
1.8 dissolution of crude fermentation extracts from strains 5-6 on slide-attached pathogenic hyphae
The influence of the crude extract on the morphology of Foc germ hyphae was observed by a dissolution method of the attached pathogenic hyphae of the slide. The PDA plate culture medium is connected with Foc germ cake, a sterile cover glass is inserted around the germ cake, and the temperature is constant at 28 ℃ for culture until Foc hyphae grow on the cover glass. The coverslip was carefully removed with forceps, the fungus silk attachment surface was placed on the slide, 50. Mu.L of 500. Mu.g/mL of the crude fermentation extract solution of strain 5-6 was added dropwise, the treated slide was placed in a sterile petri dish, and the mycelia of the crude fermentation extract solution without strain 5-6 was used as a control, and hydrolysis was carried out at 28℃for 48 hours, and each treatment was repeated 3 times. The morphological changes of Foc germ hyphae were observed using an optical microscope.
2. Results and analysis
2.1 identification of Strain 5-6
2.1.1 Strain 5-6 culture characteristics and physiological Biochemical assays
Strains 5-6 grew best on ISP2, ISP3 and ISP4 media, forming abundant endophytes, but grew poorly on ISP5 (Table 2). The strain has protease activity, can liquefy gelatin, and is positive for nitrate reduction and urease test through physiological and biochemical measurement; can utilize sucrose, glucose, soluble starch, asparagine, tyrosine, valine and other carbon nitrogen sources to grow well; can grow in the pH range of 4.0-8.0, and the optimal growth pH value is 7.0; the optimum growth salt concentration was less than or equal to 5% (table 3).
TABLE 2 culture characteristics of strains 5-6 on 6 media
TABLE 3 physiological and biochemical characteristics of strains 5-6
Note that: in the results of carbon source and Nitrogen source' ++ + + + is shown the growth is good, the quality of the product is good, "++" indicates growth in general, "+" indicates growth possible, and "-" indicates no growth. In the physiological and biochemical test results, "+" indicates positive and "-" indicates negative.
2.1.2 scanning Electron microscope observations
The morphology of hyphae and spores of strains 5-6 on ISP2 medium was observed by scanning electron microscopy, as shown in FIG. 1. The strain 5-6 has developed hyphae, the spore chain is in a compact spiral shape, and the surface is provided with wrinkles.
2.1.4 Strain 5-6 genome sequencing and analysis
The Streptomyces 5-4 genome circle map is shown in FIG. 2, and comprises 11,162,522 p sequences, 69 tRNA's and 2 rRNA's, and the average G+C% content is 71.395%. The genome predicts 10018 CDS genes, the total length is 9,508,390 bp, accounting for 90% of the total length of the genome. 2234 tandem repeats, 296,932bp in total length, account for 3.12% of the genome's full length.
2.1.3 Strain 5-6 phylogenetic Tree construction and genome average nucleotide identity
The sequences of the strains were aligned for homology in the ezbiocoud database to construct a phylogenetic tree (fig. 3). The relativity of the strain 5-6 with the strain Streptomyces rapamycinicus and Streptomyces iranensis is recent, the similarity of the 16S rDNA sequence is 99%, and the strain 5-6 presents typical streptomycete morphological characteristics in combination with the aspects of culture characteristics, physiological and biochemical characteristics and the like, so that the strain 5-6 is attributed to Streptomyces.
Strains 5-6 showed a genomic comparison of the most homologous strains Streptomyces rapamycinicus (Lei Palian mould) and Streptomyces iranensis (Streptomyces lividans) with ANI values of 93.96% and 94.09%, respectively (Table 4 and Table 5). According to the fact that the ANI value of the whole genome is more than 96% and is used as the basis for judging the same strain, the strains 5-6 are novel strains, and the novel strains are named as follows: streptomyces sp.5-6.
Table 4 genomic comparison of strains 5-6 with closely related model strains
TABLE 5 Average Nucleotide Identity (ANI) alignment results
2.2 broad-Spectrum antibacterial Activity assay
The bacterial strains 5-6 have in-dish antibacterial activity on 12 pathogenic fungi, and the antibacterial rate is above 64.31% (figure 4). The bacterial strain 5-6 has the best antibacterial activity on the epidemic disease bacteria of the wheat, the antibacterial rate reaches 92.55 +/-0.49%, the antibacterial activity on Foc bacteria is better, the antibacterial rate reaches 73.18 +/-1.61%, but the antibacterial effect on the wheat scab bacteria is slightly poor, and the antibacterial rate is only 64.31 +/-0.36%.
2.3 bacterial strain 5-6 antibacterial Activity of crude fermentation extract
The fermented crude extract of the strain 5-6 has an inhibition effect on Foc bacteria, and the antibacterial activity is 54.84+/-1.61% (shown in figure 5), which shows that the fermented crude extract of the strain 5-6 also has a good inhibition effect on Foc bacteria.
2.4 dissolution of crude fermentation extract of Strain 5-6 on slide-attached pathogenic hyphae
The dissolution of the attached pathogenic hypha of the glass slide is shown in figure 6, and after the pathogenic hypha is hydrolyzed for 48 hours by the crude extract fermented by the bacterial strain 5-6, the pathogenic hypha is obviously broken, dissolved and the like, thereby inhibiting the growth of pathogenic bacteria.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (5)
1. An actinomycete which is Streptomyces sp.5-6, deposited under the accession number GDMCC No:61984; is preserved in the microorganism strain collection center of Guangdong province.
2. Use of actinomycetes according to claim 1 for antagonizing physiological race 4 of banana vascular wilt (Fusarium oxysporum f.sp.document 4, foc 4).
3. Use of actinomycetes according to claim 1 for antagonizing physiological race1 of banana vascular wilt (Fusarium oxysporum f.sp.document 1, foc).
4. The use of actinomycetes according to claim 1 for preparing a biocontrol agent for controlling diseases caused by banana fusarium wilt 4 physiological race (Fusarium oxysporum f.sp.cube race 4, foc 4).
5. A biocontrol formulation comprising the actinomycete of claim 1.
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