AU2020102683A4 - Recombinant broad-spectrum Metarhizium and its use for promoting plant root growth - Google Patents

Abstract

The present invention relates to a recombinant broad-spectrum Metarhizium and its use for promoting the growth of plant roots, belonging to the field of agricultural biotechnology. The recombinant broad-spectrum Metarhizium of the present invention expresses down-regulated monoamine oxidase or does not express monoamine oxidase, and can promote the growth of plant roots.

Description

Recombinant broad-spectrum Metarhizium and its use for promoting plant root growth

Technical Field

The present invention relates to a recombinant fungus and a preparation method and use thereof, in particular to a recombinant broad-spectrum Metarhizium capable of improving the production of tryptamine and its use for promoting plant root growth.

Background Art

The extensive use of chemical fertilizers in farmland can quickly provide the nutrients needed in the early stages of crops, but it will lead to soil acidification, uneven fertility and groundwater pollution. Microbial fertilizers can replace part of chemical fertilizers to solve the problem of soil degradation caused by long-term application of chemical fertilizers. Microbial fertilizers can be divided into five categories according to the types of microorganisms: (1) bacterial fertilizers (e.g., rhizobia fertilizer, nitrogen-fixing bacterial fertilizer, phosphate-solubilizing bacterial fertilizer, potassium-solubilizing bacterial fertilizer, photosynthetic bacterial fertilizer); (2) actinomycete fertilizers (e.g., antibiotic bacterial fertilizer); (3) fungal fertilizers (e.g., mycorrhizal fungal fertilizers: including ecto-mycorrhizal inoculants and endo-mycorrhizal inoculants); (4) algae fertilizers (e.g. nitrogen-fixing cyanobacteria fertilizer); (5) compound microbial fertilizers, which refer to a fertilizer consisting of two or more species of microorganisms combined in a certain proportion. Non-limiting examples of microbial isolates that can directly promote plant growth and/or yield include nitrogen-fixing bacteria: Rhizobium and Bradyrhizobium species, which can form nodules on legume roots through symbiotic nitrogen fixation; in the nodules, they can convert atmospheric N2 into ammonia; in contrast to atmospheric N2, ammonia can be used as a nitrogen source by plants. Other examples include Azospirillum species, which are independent nitrogen-fixing bacteria that can fertilize and increase the yield of cereal crops such as wheat, sorghum and corn. Despite the nitrogen-fixing ability of Azospirillum, the increase in yield caused by inoculation of Azospirillum is often still attributed to the increase in root development and therefore the increase in water and mineral uptake rates. In this regard, several rhizobacteria (e.g., Azotobacter spp.) have been reported to be capable of producing various plant hormones (e.g., auxin, cytokinin) and enzymes (e.g., pectinase). It has been confirmed that many of these plant hormones and enzymes are closely involved in the infection process of the bacterial-plant symbiosis combination that has a regulatory influence on the nodules formed by rhizobia.

Metarhizium fungi are widely used as biological insecticides. Compared with chemical insecticides, they have the advantages of environmental friendliness, strong stress resistance, large diffusion, and high selectivity. At present, more than 200 kinds of agricultural and forestry pests can be controlled by Metarhizium preparations. However, their use for promoting the growth of plant roots has not been reported in the field.

Contents of the Invention

The present inventors surprisingly discovered through research that a recombinant broad-spectrum Metarhizium with down-regulated expression of monoamine oxidase or without expression of monoamine oxidase can promote the growth of plant roots. Thus, the present invention is completed.

In one embodiment, the present invention provides a use of a recombinant broad-spectrum Metarhizium for promoting the growth of plant roots, wherein the recombinant broad-spectrum Metarhizium does not express or expresses down-regulated monoamine oxidase. The recombinant broad-spectrum Metarhizium is its strain per se, its progeny, a conidia produced by it, or a mycelium produced by it, or any combination thereof.

In one embodiment, the expression of monoamine oxidase in the recombinant broad-spectrum Metarhizium provided by the present invention is down-regulated by more than 50%. Preferably, the expression of monoamine oxidase in the recombinant broad-spectrum Metarhizium is down-regulated by more than 60%, 70%, 80%, 90% or 95%, and most preferably, the expression of monoamine oxidase in the recombinant broad-spectrum Metarhizium is down-regulated by 100%, that is, no monoamine oxidase is expressed. Compared to a wild-type broad-spectrum Metarhizium, the recombinant Metarhizium of the present invention has increased production of tryptamine in vivo.

In one embodiment, the recombinant broad-spectrum Metarhizium in the present invention is a recombinant broad-spectrum Metarhizium robertsii or a recombinant broad-spectrum Metarhizium anisopliae. In a specific embodiment, the recombinant broad-spectrum Metarhizium anisopliae in the present invention is a recombinant broad-spectrum Metarhizium robertsii, its deposit number is CGMCC NO.14152, its classification name is Metarhizium robertsii, it was deposited on August 29, 2017 at the China General Microbiological Culture Collection Center (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing); see: Chinese invention patent CN107916232.

Another aspect of the present invention provides a microbial fertilizer, which comprises the recombinant broad-spectrum Metarhizium of the present invention and an agricultural carrier. The agricultural carrier can be soil or plant growth medium. Other usable agricultural carriers include water, fertilizers, vegetable oils, humectants, or combinations thereof. Alternatively, the agricultural carrier may be solid, such as diatomite, loam, silica, alginate, clay, bentonite, vermiculite, fruit peel, other plant and animal products or combinations thereof, including granules, pellets, or suspensions.

In one embodiment, through gene recombination, nucleotide sequence related with monoamine oxidase expression is knocked out or modified to down-regulate or avoid the expression of monoamine oxidase.

In a specific embodiment of the present invention, the recombinant broad-spectrum Metarhizium does not express monoamine oxidase by knocking out nucleotide sequence related with monoamine oxidase expression, which specifically comprises the following steps: respectively amplifying the upstream sequence and downstream sequence of monoamine oxidase nucleotide sequence in a wild-type broad-spectrum Metarhizium robertsii (MAA), seamlessly ligating the amplified upstream sequence and the downstream sequence, preferably, seamlessly ligating the amplified upstream sequence and the downstream sequence to Bar gene or Ben gene.

In the specific embodiment of the present invention, there is no limitation to the type of plasmid, as long as it contains the Bar gene (herbicide glufosinate resistance gene) and/or the Ben gene (benomyl resistance gene).

In a specific embodiment of the present invention, a PDHt-Bar plasmid containing the Bar gene is selected, and the method for preparing the recombinant broad-spectrum Metarhizium specificallycomprises:

designing primers, MAA_03753Fs and MAA_03753Rs, MAA_03753Fx and MAA_03753Rx, using the genomic DNA of MAA as template to amplify the upstream and downstream sequences respectively; after digesting the amplified upstream and downstream sequences, seamlessly ligating them to the upstream and downstream of Bar in the PDHt-Bar plasmid to form a recombinant plasmid; transferring the recombinant plasmid into MAA by an Agrobacterium tumefaciens-mediatedmethod.

In another aspect, this document provides a method for increasing the growth and/or yield of a plant. In some embodiments, this method comprises applying an effective amount of the recombinant broad-spectrum Metarhizium strain or a culture thereof according to the present invention to the plant or to surrounding environment of the plant. In some other embodiments, the method comprises allowing the recombinant broad-spectrum Metarhizium strain or a culture thereof according to the present invention to grow in growth medium or soil of the host plant, and subsequently or simultaneously allowing the host plant to grow in the growth medium or soil. In a preferred embodiment, the plant is a cruciferous plant. In a specific embodiment, the plant is selected from rape, Arabidopsis, potato, red, ginger, onion, garlic, white radish, carrot, leek, and soybean.

The present invention has the following advantages:

The recombinant broad-spectrum Metarhizium of the present invention can significantly increase the concentration of tryptamine in its spores and can promote the growth of plant roots. As a microbial fertilizer, the recombinant broad-spectrum Metarhizium of the present invention is harmless to the environment, has good biological safety, and is non-toxic to humans.

Brief Description of the Drawings

Figure 1 shows the schematic diagram of the structure of the recombinant plasmid in an example of the present invention;

Figure 2 shows the agarose gel electrophoresis diagram of the recombinant broad-spectrum Metarhizium robertsiiin an example of the present invention;

Figure 3 shows the content of tryptamine in hyphae of the recombinant broad-spectrum Metarhizium robertsii in an example of the present invention, and the content of tryptamine in migratory locusts after the infection by the recombinant broad-spectrum Metarhizium robertsii.

Figure 4 shows the promotion results of the recombinant broad-spectrum Metarhizium robertsiito the growth of Arabidopsisroot system in the embodiment of the present invention.

Specific Models for Carrying Out the Invention

The technical solutions in the examples of the present invention are clearly and completely described as follows. Obviously, the described examples are only a part of the examples of the present invention, rather than all of the examples. Based on the examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The present invention will be described in detail below in conjunction with specific examples, which are used to understand but not limit the present invention.

Example 1: Preparation of recombinant broad-spectrum Metarhizium robertsii

In this example, knocking out the monoamine oxidase gene (denoted as MAA_03753) in broad-spectrum Metarhizium robertsii (denoted as MAA) was taken as an example for illustration, in which the gene bank accession number of MAA_03753 was NW_011942171.1, monoamine oxidase [EC:1.4.3.4], and the specific sequence was shown in SEQ ID NO:1. The present example did not limit the broad-spectrum Metarhizium robertsii, but could also include other broad-spectrum Metarhizium species with monoamine oxidase, such as Metarhizium anisopliae. In the present example, the type of plasmid was not limited, as long as it contained Bar gene and/or Ben gene. For example, it could be pDHt-Bar plasmid or pDHt-Ben plasmid. The pDHt-Bar plasmid was used as an example for illustration.

1. Knocking out plasmid construction of MAA_03753

Designing primers, MAA_03753Fs and MAA_03753Rs, MAA_03753Fx and MAA_03753Rx, using wild-type MAA genomic DNA as template to amplify its upstream and downstream sequences, respectively. SmaIand Spel restriction sites were added to the end of the product. The sequences of specific primers were as follows:

MAA_03753Fs (as shown in SEQ ID NO: 2):

ATTCCTGCAGCCCGGGATGGCGACAACCCAAATC

MAA_03753Rs (as shown in SEQ ID NO: 3):

CGACGGATCCCCCGGGGTGTCAACCCTCGTTCTATT

MAA_03753Fx (as shown in SEQ ID NO: 4):

GATCTGATGA3ACTAGTGTTTCGGAACATTCACTTTG

MAA_03753Rx (as shown in SEQ ID NO: 5):

CCGCTCTAGAACTAGTCGGGCAAGATTCCGTTCGT

The MAA_03753Fs and MAA_03753Rs primer pairs were used to amplify the upstream sequence of monoamine oxidase, and a 880bp fragment (denoted as MAO-S) was amplified. After single digestion with SmaI, MAO-S was seamlessly ligated to the upstream of Bar in the PDHt-Bar plasmid (as shown in Figure 1).

The MAA_03753Fx and MAA_03753Rx primer pairs were used to amplify the downstream sequence of monoamine oxidase, and a 646bp fragment (denoted as MAO-X) was amplified. After single digestion with Spel, MAO-X was seamlessly ligated to the downstream of Bar in the PDHt-Bar plasmid (provided by Shanghai Institute of Plant Physiology and Biochemistry, specifically referring to: Yixiong Chen, Zhibing Duan, Peilin Chen, Yanfang Shang & Chengshu Wang, The Bax inhibitor MrBI-1 regulates heat tolerance, apoptotic-like cell death, and virulence in Metarhizium robertsii, Scientific Reports 5, Article number: 10625 (2015); and Wei Huang, Yanfang Shang, Peilin Chen, Kai Cen and Chengshu Wang, Basic Leucine Zipper (bZIP) The downstream of Bar in the Domain Transcription Factor MBZl Regulates Cell Wall Integrity, Spore Adherence, and Virulence in Metarhizium robertsii*, Journal of Biological Chemistry 290(13): 8218-8231.) (as shown in Figure 1). In this way, the 580bp fragment in the middle of MAA-03753 was replaced by the Bar sequence with a length of 938bp.

The PCR reaction mixture was: 2.5pL of 1OxEx Taq Buffer polymerase buffer, 2pL of 2.5mM dNTP, lpL of each of 10pM upstream and downstream primers, lpL of template, 0.25pL of Takara Ex Taq DNA polymerase, added with ultrapure water to the total volume of 25pL;

PCR reaction conditions: 95°C pre-denaturation for 5min, 94°C for 30sec, 54°C for 30sec, 72°C for 1min (35 cycles); finally, 72°C extension for 10min. After the PCR reaction product was electrophoresed on an agarose gel with a mass fraction of 1.0%, the product was recovered with a gel recovery kit.

Enzyme digestion system: 5pL of 1Ox cutsmart buffer, lpg of plasmid DNA, lpL of endonuclease (NEB), supplemented with ddH20to 50pL.

Seamless ligation: Clone Express@IIOne Step Cloning Kit (Vazyme)

4 L of 5x buffer, 2L of Exnasell, vector in an amount of 0.02x vector base number ng, insert fragment in an amount of 0.04x insert fragment base number ng, supplemented with H20 to 20[L, incubated at 37°C for 30min and then transformed.

2. Construction of engineering strains:

The amplified upstream sequence and downstream sequence were inserted into the vector PDHt-Bar, respectively, and the successfully constructed knockout vector was confirmed after sequencing verification (as shown in Figure 1). Afterwards, the knockout plasmid was transformed into MAA by Agrobacterium tumefaciens-mediated transformation.

Construction of fungal genetic transformation system by Agrobacterium tumefaciens-mediated transformation (ATMT): the obtained vector was transformed into

Agrobacterium AGL-1, positive Agrobacterium AGL-1 transformed strain was selected after PCR identification, YEB medium (containing 50mg/mL Carb and 50mg/mL Kan) was used for expanded culture. After bacteria were collected, they were resuspended with an appropriate amount of IM liquid medium to OD66 of 0.15, and cultured at 28°C in the dark until the bacterial solution concentration OD660 was 0.5-0.8.

At the same time, conidia suspension of wild-type broad-spectrum Metarhizium robertsii (denoted as MAA) was prepared. The wild-type MAA was inoculated on PDA plate and cultured. After culturing for 14 days, an appropriate amount of wild-type broad-spectrum Metarhizium robertsii MAA conidia was scraped from the PDA plate into 1 mL of sterile water containing 0.05% Tween-20, subjected to vortex shaking, filtrated with glass wool to remove hyphae, and the filtrate was collected. After centrifugation at 12000 rpm for 3 minutes, sterile water with Tween-20 was used for washing twice; after resuspension, counting was carried out with a hemocytometer, and the wild-type MAA spore suspension was adjusted to contain approximately 1.Ox106 conidia per mL of suspension for later use.

The above-mentioned AGL-1 bacterial solution cultured in IM medium and the wild-type broad-spectrum Metarhizium robertsii MAA conidia suspension each in amount of 100 L were mixed and uniformly coated on IM medium plate. After co-cultivation for 48 hours, the co-culture was washed with sterile water, and cultivated for 7-10 days in the dark in M-100 medium containing cepholothin and glufosinate until the resistant colonies appeared; after single spores were separated, the resistant fungal tissues were reserved. The genome of the resistant fungal tissues was extracted and the transformant was verified with specific primer PCR.

3. Verification of fungal genome

The genome of the transformant was verified using the TranGen Plant Tissue PCR Kit (AD301).

The above-mentioned resistant fungal tissue was picked, added with 40 L of PD1 Buffer, vortex mixed well or blown and beated with pipette. After incubation in metal bath at 95°C for 10 minutes (the equipment was preheated in advance), 40 L of PD2 Buffer was added and mixed well, and this could be directly used as a template for PCR verification. After sequencing verification, it was confirmed to be the fungal tissue with successfully transformed knockout plasmid. The fungal tissue into which the knockout plasmid was successfully transformed was inoculated on PDA medium and cultivated until conidia grew. The spores were inoculated on SDB medium, cultured at 28°C, 180rpm in the dark for 3 days; after the hyphae was collected by suction filtration, the mycelium was ground with liquid nitrogen, then Trizol was added to extract the RNA. The extracted RNA was reverse transcribed into cDNA template, and subject to PCR. In this example, the expression of Tublin was used as a reference, and the specific primers used were as follows:

MAA_03753-ORF-F: CAAGCTGGGCTACTACTCA (as shown in SEQ ID NO: 6);

MAA_03753-ORF-R: AAGCATCAATAACCTCCCTC (as shown in SEQ ID NO: 7);

Tublin-F: GATCTTGAACCTGGCACCAT (as shown in SEQ ID NO: 8);

Tublin-R: CCATGAAGAAGTGCAGACGA (as shown in SEQ ID NO: 9)

The PRC system was as follows:

Tissue Extract 1.2pL

Forward Primer (10PM) 0.4pL

Reverse Primer (10PM) 0.4pL

2xTansDirect PCR SuperMix 10pL

ddH20 8pL

Total amount 20pL

The obtained PCR products were electrophoresed on a 1% agarose gel, and the experimental results were shown in Figure 2. In Figure 2, the first lane was the marker, the second lane was the expression of tublin in the wild-type MAA, the third lane was the expression of tublin in the MAA_03753 with knockout plasmid, the fourth lane was the expression of monoamine oxidase in the wild-type MAA_03753, and the fifth lane was the expression of monoamine oxidase in the MAA_03753 with knockout plasmid. It could be seen from Figure 2 that the MAA_03753 with knockout plasmid did not express monoamine oxidase, indicating that the gene sequence of monoamine oxidase had been knocked out. The above-mentioned resistant fungus was the recombinant broad-spectrum Metarhizium robertsii (denoted as MAA-KO, abbreviated as KO).

Example 2: Determination of tryptamine content in the recombinant broad-spectrum Metarhizium robertsii

The wild-type MAA, the recombinant MAA-KO selected in Example 1, and the wild-type obligate locust Metarhizium (denoted as MAC) were cultured on PDA plates. After culturing for 6 days in a dark incubator at 28°C, the hyphae were collected, and the medium was washed off with ddH20 twice, and then freeze-dried at -20°C. 1mg of dried hyphae was weighed, lysed with

100pL of 0.1M perchloric acid, ground, centrifuged at 5200g, 4°C for 30min, then the supernatant was taken, neutralized with Na2CO3 to leave the pH value at about 6, the

supernatant was taken and filtrated with 0.22pm microporous membrane for later use. HPLC was used to detect the tryptamine content in the supernatant.

HPLC detection:

Agilent 1100, G1315A fluorescence detector (FLD), the chromatographic column was a C18 column;

Mobile phase A: [0.05M acetic acid solution/tetrahydrofuran (96/4)]: methanol (V:V) was 60:40. Mobile phase B was methanol.

Sample introduction procedure:

A (in%): 75.00 (0min), 75.00 (8min), 66.67 (12min), 50.00 (25min), 0 (30min), 66.67 (35min), 75.00 (40min);

B (in%): 25.00 (0min), 25.00 (8min), 33.33 (12min), 50.00 (25min), 100 (30min), 33.33 (35min), 25.00 (40min).

Sample preparation:

0.4N boric acid buffer (pH 10.2) was prepared;

1 mg of the derivatization reagent o-phthalaldehyde (OPA) was dissolved in 100 pL of methanol, 900 pL of 0.4N boric acid buffer was added after complete dissolution, and then 10 pL of 3-mercaptopropionic acid (3-MPA) was added to prepare a mixed solution H .

The mixed solution H was mixed uniformly with the wild-type MAA, recombinant MAA-KO and MAC supernatants respectively and injected. The injection volume was 0.5 pL. The experimental results were shown in Figure 3A.

It could be seen from Figure 3A that the tryptamine concentration in the wild-type MAA was 34.47 ng/mg, the tryptamine concentration in the MAC was 85.07 ng/mg, and the tryptamine concentration in the recombinant MAA-KO was 84.53 ng/mg, there was no significant difference in tryptamine concentration between the recombinant MAA-KO and the MAC (both a), but there were significant differences between the recombinant MAA-KO and the wild-type MAA (a and b, respectively), indicating that the monoamine oxidase gene in the recombinant MAA-KO screened in Example 1 had been knocked out, so that it could significantly increase the concentration of tryptamine.

The spores of the wild-type MAA, the recombinant MAA-KO selected in Example 1 and the wild-type obligate locust Metarhizium (denoted as MAC) were used to infect migratory locusts respectively. Four days later, the hemolymph fluids of the migratory locusts were taken and the tryptamine concentrations thereof were measured, which were denoted as MAA-4d, MAC-4d and KO-4d, respectively. The hemolymph fluid of uninfected normal migratory locust was used as a control, and denoted as CK. The experimental results were shown in Figure 3B.

It could be seen from Figure 3B that the concentration of tryptamine in the control group CK was 32.11 pg/ul, the concentration of tryptamine in the wild-type MAA was 152.67 pg/ul, the concentration of tryptamine in the MAC was 266.89 pg/ul, and the concentration of tryptamine in the recombinant MAA-KO was 247.02 pg/ul. The tryptamine concentration in the recombinant MAA-KO showed no significant difference with that in the MAC (both a), while showed significant difference with that in the wild-type MAA (a and b, respectively), and that in the control group (a and c, respectively),too. It indicated that infecting migratory locusts with the recombinant MAA-KO selected in Example 1 from which the monoamine oxidase gene was knocked out could significantly increase the tryptamine content in the migratory locusts, reaching the same level as the obligate bacteria MAC, which was significantly higher than the content after infection with the wild-type MAA and the tryptamine content of the control group.

Example 3: Promotion of plant root growth with recombinant broad-spectrum Metarhizium robertsii

The wild-type MAA and the recombinant MAA-KO prepared in the above example were respectively inoculated on PDA medium and cultured, the spores of the MAA and the MAA-KO were scraped respectively, added with appropriate amounts of peanut oil respectively to suspend the spores, vortex shaken, filtrated with glass wool, then the spores were collected, and resuspended in peanut oil. The counting was carried out using a cell counting chamber under a microscope; after resuspending and counting for multiple times, the final concentration was 1x106 spores/ml.

Arabidopsis thaliana was purified at 4 °C for 4 days and then placed on a medium (0.5xMSP21), in which the MSP21 medium did not contain nitrogen, then it was placed into a climate chamber, the temperature was kept at 21°C, the day-night ratio was 16 hours: 8 hours, the culture plate was placed at 30°C. When the plant had grown to two leaves, 2uL of 5ug/ p L

locust protein extract was added at 2cm below. For the control group, 2 p L of L-15 medium was

mixed therein, while for the treatment groups, 2 p L of 1x106 spores/mL fungal solutions of the

wild-type MAA and the recombinant MAA-KO were mixed therein. After cultivating for 14 days at 30°C, the fresh weight of plant, the length of main root and the number of lateral roots were measured.

The results show:

After the co-cultivation of the MAA and Arabidopsis thaliana, the plant root was longer than that of the plant without fungus co-cultivation; after the co-cultivation of the MAA-KO and the plant, the length of the root was greater than that of the plant co-cultured with the MAA and that of the plant without co-cultivation with fungi (Figure 4). After co-cultivation of plants and the MAA-KO, the whole plant weight and the number of lateral roots were significantly higher than that of the plant cultured without fungi or the plant co-cultured with MAA fungi. The results showed that the MAA-KO could significantly promote plant growth.

The above descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement and so on made within the spirit and principle of the present invention shall fall into the protection scope of the present invention.

Claims (5)

What is claimed is:

1. A microbial fertilizer composition comprising a recombinant broad-spectrum Metarhizium and/or a progeny, conidia, or mycelium of said recombinant broad-spectrum Metarhizium, wherein the recombinant broad-spectrum Metarhizium expresses down-regulated monoamine oxidase or does not express monoamine oxidase, wherein the recombinant broad-spectrum Metarhizium and/or a progeny, conidia, or mycelium thereof is formulated with one or more agricultural carriers.

2. A method for promoting growth of plant roots, comprising applying to the roots of a plant a recombinant broad-spectrum Metarhizium, and/or a progeny, conidia, or mycelium of said recombinant broad-spectrum Metarhizium, wherein the recombinant broad-spectrum Metarhizium expresses down-regulated monoamine oxidase or does not express monoamine oxidase.

3. A method of producing a microbial fertilizer composition comprising combining (i) a recombinant broad-spectrum Metarhizium and/or a progeny, conidia, or mycelium thereof, and (ii) one or more agricultural carriers.

4. The microbial fertilizer composition of claim 1 or the method of claim 2 or 3, wherein: the recombinant broad-spectrum Metarhizium is recombinant broad-spectrum Metarhizium robertsii or recombinant broad-spectrum Metarhizium anisopliae;or the recombinant broad-spectrum Metarhizium is recombinant broad-spectrum Metarhizium robertsiihaving the deposit number CGMCC NO.14152.

5. The microbial fertilizer composition of claim 1or 4 or the method of any one of claims 2 to 4, wherein the recombinant broad-spectrum Metarhizium promotes growth of a plant selected from the group consisting of rape, Arabidopsis, potato, red, ginger, onion, garlic, white radish, carrot, leek, and soybean.