Penicillium kluyvei with effect of promoting plant growth and application of penicillium kluyvei

Abstract

The invention discloses Penicillium kirscheri with a plant growth promoting effect and application thereof, and relates to the technical field of microorganisms, the Penicillium kirscheri has been preserved in China General Microbiological Culture Collection Center on January 22, 2024, and the preservation number is CGMCC No.41049. The Penicillium kirscheri has been preserved in China General Microbiological Culture Collection Center on January 22, 2024. The penicillium kluyverovii provided by the invention has the effect of remarkably promoting plant growth, especially promoting plant root growth, can be applied to preparation of biological agents in agricultural production, and can generate various agricultural benefits.

Description

TECHNICAL FIELD

[0001] The present application provides a paecilomyces fumosaegenus with plant growth promoting effect and its application, and relates to the field of microbial technology. BACKGROUND

[0002] Plant growth promoting microorganisms (PGPR) include a group of microorganisms that colonize in soil or plant roots, which have beneficial effects on plant growth, uptake of mineral nutrients and defense against harmful pathogens. PGPR can enhance seed germination, promote seedling growth, stimulate root development and improve the ability of plants to absorb and utilize mineral nutrients and water. In addition, they show antagonistic activity against various bacterial, fungal, nematode and even some viral diseases. PGPR uses various mechanisms to promote plant growth, including the secretion of hormones (such as auxins, gibberellins, cytokinins), three-dimensional production of various enzymes and phosphate solubility. For example, Aryabhattai Bacillus produces IAA, ABA, JA, GA and cytokinin hormones in culture. In addition, phosphate-solubilizing bacteria help root growth and rhizosphere acidification by hydrolytic enzymes and organic acids, promoting crop growth. In addition to promoting growth, PGPR can produce antibiotics to prevent pathogen invasion. They can produce various antibiotics, including DAPG, aniline and pyrrol, induce resistance to nematodes in plants and enhance plant defense mechanisms. In addition, PGPR can secrete various enzymes, such as dehydrogenase, phosphatase and urease, promote organic matter degradation and enhance plant salt tolerance. Many PGPR have cellulose-degrading ability, which is classified in the carbohydrate-active enzymes database, which is essential for catalyzing carbohydrate decomposition and biosynthesis. Bilaiae, a filamentous fungus isolated from the soil of Alberta, Canada, is a fungus that enhances phosphate availability by acid secretion. This fungus colonizes the roots, increases root length and density, thereby improving the absorption of nutrients and water for the sustained growth of crops, thereby increasing the growth of agricultural crops. In addition, it has been proven that Bilaiae can increase the yield of agricultural crops. Therefore, it is a research and development trend to find strains with more excellent agricultural potential in fungi. SUMMARY

[0003] Based on the above background, the present application provides a paecilomyces fumosaegenus with plant growth promoting effect and its application. The specific technical solutions are as follows:

[0004] The present application provides a paecilomyces lilacinus with plant growth promoting effect, the strain name is paecilomyces lilacinus 23301-20, the preservation number is CGMCC No.41049, the preservation time is January 22, 2024, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No.3, Beichen West Road, Chaoyang District, Beijing City;The paecilomyces lilacinus culture medium comprises a PDA culture medium;The colony character of the paecilomyces lilacinus is initially white, then becomes dark green, the conidial phialide is branched multiple times to form a typical broom structure, and the conidium is nearly spherical.

[0005] The present application also provides the application of the above-mentioned paecilomyces lilacinus with plant growth promoting effect in preparing biological agents with plant growth promoting effect.

[0006] Preferably, the application comprises the application in preparing liquid or solid biological agents.

[0007] Preferably, the biological agent comprises a biological agent with plant growth promoting effect and a biological agent with other agricultural benefits.

[0008] Preferably, the other agricultural benefits comprise one or more of promoting plant root growth, improving plant stress resistance and improving soil properties.

[0009] Beneficial effects:

[0010] The present application provides a paecilomyces lilacinus strain with significant plant growth promoting effect. The strain is particularly good at promoting the development and extension of plant roots, thereby improving the absorption efficiency of water and nutrients by plants, and further promoting the growth and development of the whole plant. This characteristic makes the paecilomyces lilacinus have wide application prospects in agricultural production. By applying the strain in the preparation of biological agents, the yield and quality of crops can be effectively improved, the dependence on chemical fertilizers can be reduced, and the efficiency of agricultural production can be improved. The application of the paecilomyces lilacinus strain not only promotes the healthy growth of crops, but also improves the soil microbial community and promotes the sustainable development of agriculture. BRIEF DESCRIPTION OF DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiment or prior art description will be briefly introduced below.

[0012] Figure 1 For the whole genome analysis and phylogenetic tree of paecilomyces lilacinus;The genome analysis is from inside to outside: the first circle: GCskew;The second circle: G+C content;The third circle: Cazy;The fourth circle: DFVF The fifth circle: COG to which each CDS on the minus strand belongs;The sixth circle: COG to which each CDS on the plus strand belongs;The seventh circle: scale;

[0013] Figure 2 Figure 1. Metabolic pathway diagram for promoting growth, alkaloids, and hormone-related pathways.

[0014] Figure 3 Figure 2. Photographs of the roots of some plants obtained in experimental group 1.

[0015] Figure 4 Figure 3. Photographs of the roots of some plants obtained in experimental group 2.

[0016] Figure 5 Figure 4. Photographs of the potted plants obtained in experimental group 1.

[0017] Figure 6 Figure 5. Photographs of the potted plants obtained in experimental group 2.

[0018] Figure 7 Figure 6. Photographs of some plants obtained in experimental group 1.

[0019] Figure 8 Figure 7. Photographs of some plants obtained in experimental group 2. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, and all other embodiments obtained by a person of ordinary skill in the art without any creative work on the basis of the embodiments of the present application shall fall within the scope of the present application.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art. In addition, any method and material similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are only used for demonstration, but cannot limit the content of the present application.

[0022] In the following examples, the experimental methods are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0023] The culture media involved below include:

[0024] (1) PDA solid culture medium: 200 g of potatoes, 20 g of glucose, 20 g of agar, 1 L of distilled water, natural pH, 121 °C high-pressure sterilization for 20 min.

[0025] (2) PDB liquid fermentation medium: 200 g of potatoes, 20 g of glucose, 1 L of distilled water, natural pH, 121 °C high-pressure sterilization for 20 min.

[0026] Example 1 Isolation and identification of Penicillium camembertii strain:

[0027] The strain was isolated and purified by gradient dilution plate coating method. 5 g of soil sample was weighed and added to 45 mL of sterile water to prepare a soil suspension with a concentration of 10-1. The suspension was incubated at 30°C with shaking at 180 r / min for 15 min. After shaking, the suspension was allowed to stand, and 5 mL of the soil suspension was added to 45 mL of sterile water to prepare a soil suspension with a concentration of 10-2. The dilution process was repeated to obtain a series of soil suspensions with concentrations of 10-1-10-7. 100 μL of the 10-4-10-7 soil suspensions was inoculated into solid PDA medium, and the bacterial solution was spread with a sterilized coating rod. The plates were placed in a 30°C incubator and incubated for 3 days. The appropriate concentration plates were selected, and the different types of single colonies formed were transferred to new PDA medium and incubated in a 30°C incubator. The culture was transferred for 3 times to obtain the purified single strain.

[0028] Strain morphological characteristics observation: The purified strain was inoculated into solid PDA medium using the three-zone streak method to obtain single colonies. The colony morphology was observed, and the mycelium and spores were picked under a light microscope to observe the spore and mycelium morphology.

[0029] Strain morphological characteristics: The initial colony was white, and then turned into dark green. The mycelium and spores were observed under a light microscope (400x lens) to observe the typical broom-like structure of the conidial antheridia, which continuously produced conidia at the top. The conidia were nearly spherical.

[0030] After the strain was purified, DNA was extracted using the improved CATB method. Then, the fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGC-3') and ITS4 (5'-TCCTCCGCTTGAT ATGC-3') were used for PCR amplification. The PCR amplification system was 25 μL: template 1.5 μL, 2x PCR Taq Master Mix 12.5 μL, forward and reverse primers 1 μL each, and sterile double distilled water 9 μL. The amplification conditions were: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 20 s, 72°C extension for 90 s, 30 cycles; and 72°C extension for 10 min. Then, the PCR product was verified by 1% agarose gel, and the strain with a band was sent to Beijing Aovisen Biotechnology Co., Ltd. for sequencing. The obtained gene sequence was submitted to the NCBI database and compared with known sequences. MEGAX software was used to construct a phylogenetic tree using the Neighbourjioning method. Through strain morphology observation and molecular biology identification, the strain was determined to be Penicillium camembertii of the Basidiomycota phylum, Ascomycota class, Penicillales order, Penicillaceae family, and Penicillium genus.

[0031] The preservation condition of the Penicillium chrysogenum is as follows: the well-grown strain on the solid PDA culture medium plate is selected, the spores of the strain are eluted with sterile water, the spore suspension and 30% glycerol are moved into 1 mL strain preservation tube at a ratio of 1:1, and are stored in a -80℃ ultra-low refrigerator.

[0032] Example 2 Whole genome sequencing of Penicillium chrysogenum strain

[0033] Genomic DNA extraction: the conidiospores of Penicillium chrysogenum are inoculated into PDA liquid medium, and are cultured in a constant temperature incubator at 180 r / min and 28℃ for 48 h, then are filtered to collect mycelium, are washed with sterile water for 3 times, are rapidly frozen with liquid nitrogen and are ground, and the genomic DNA is extracted by using the Ezup column type fungal DNA extraction kit of Shanghai Biosciences Co., Ltd.

[0034] Protein coding gene prediction: three software, Augustus (v3.03), Glimmer HMM (v3.0.1) and Gene Mark-ES (v4.35), are used to predict the genome sequence of the strain from scratch; Exonerate software (v2.2.0) and protein sequences of close species are used for homologous prediction. The results of the prediction from scratch and the homologous prediction are integrated by using EVidenceModeler software to generate the prediction results of Penicillium chrysogenum.

[0035] Table 1 Whole genome results of Penicillium chrysogenum

[0036]

[0037] Table 2 Sequencing results

[0038]

[0039] From the sequencing results (Table 2), it can be seen that the total number of bases of the Penicillium chrysogenum genome is 4,392,696,002 bp, the total number of original reads is 29090702, 28895022 high-quality reads are obtained after filtering, the GC content is 46.33%, the genome size is 2,941,706 bp, the minimum sequence length is 55 bp, the maximum sequence length is 287510 bp, and the N50 is 1559217 bp.

[0040] The genome of P. chrysogenum has 11123 protein-coding genes, with a total gene sequence length of 17,387,264 bp, an average length of 1563.1 bp per gene, a total number of 31880 exons, an average number of 2.8 exons per gene, a total length of 15992151 bp, a proportion of 42.32% of the total length of the genome, an average length of 501.6 bp of the exons, an average length of 67.2 bp of the introns, a total length of 15992151 bp of the CDS, a proportion of 42.32% of the CDS in the genome, and an average length of 1437.7 bp of the CDS.

[0041] Example 3 Whole genome analysis and functional annotation of P. chrysogenum

[0042] Through gene annotation and KOG prediction classification of the egg-NOG database on the P. chrysogenum strain, a total of 10253 genes were annotated, which were divided into 24 categories. In addition to the unknown function category, the categories with a larger number of genes involved were carbohydrate transport and metabolism (G), with 898 genes, accounting for 8.07%; biosynthesis, transport, and catabolism of secondary metabolites (Q), with 704 genes, accounting for 6.33%; post-translational modification, protein turnover, chaperones (O), with 533 genes, accounting for 4.79%; amino acid transport and metabolism (E), with 432 genes, accounting for 3.88%; energy production and conversion (C), with 428 genes, accounting for 3.85%; and lipid transport and metabolism (I), with 413 genes, accounting for 3.71%.

[0043] The carbohydrate active enzyme genes in the gene sequence of the P. chrysogenum strain were predicted, including 341 glycoside hydrolase genes (GH), 123 carbohydrate esterase genes (CE), 104 glycosyltransferase genes (GT), 90 auxiliary activity enzyme genes (AA), 10 carbohydrate-binding module genes (CBM), and 6 polysaccharide lyase genes.

[0044] After KEGG Pathway annotation of P. chrysogenum, it was found that P. chrysogenum could correspond to 3996 genes of metabolic pathways, which were enriched in 52 pathways. The metabolic pathways with a larger number of genes involved were the following 7: genetic information processing metabolic pathway, with 2393 genes; signal and cellular processes pathway, with 649 genes; metabolism pathway, with 557 genes; signal transduction pathway, with 550 genes; carbohydrate metabolism pathway, with 523 genes; amino acid metabolism pathway, with 440 genes; and transport and metabolism pathway, with 384 genes.

[0045] By classifying and comparing the genomic information of *Penicillium clarithrum* strains, the results show that the genes in *Penicillium clarithrum* related to plant growth mainly involve phosphorus-solubilizing genes, siderophore-producing genes, hormones such as IAA production, and alkaloid synthesis genes. There are 44 phosphorus-solubilizing genes in *Penicillium clarithrum*, including GPI and pfkA. These genes mainly participate in regulating the glycolysis (EMP) pathway, the tricarboxylic acid cycle (TCA) pathway, the pentose phosphate pathway, and the oxidative phosphorylation pathway. Figure 3 ); 13 are related to hormones such as IAA, and 11 are related to alkaloids. Figure 4 ).

[0046] Example 4: Analysis of metabolites of Penicillium clarithii:

[0047] Experimental method: Activated Penicillium clarkii was inoculated into PDA liquid medium and cultured at 28℃ and 180 rpm for 3 days to obtain the strain seed culture. The seed culture was then inoculated into fresh PDA liquid medium at a 1% inoculation rate and cultured at 28℃ and 180 rpm for 10 days to obtain the strain fermentation broth. The fermentation broth was centrifuged at 4℃ and 3000 rpm for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane to obtain sterile filtrate, which was then flash-frozen in liquid nitrogen and temporarily stored at -80℃. The filtrate was then sent to Shanghai Ling'en Biotechnology Co., Ltd. for non-targeted metabolomics analysis of the strain fermentation broth.

[0048] Experimental Results: Under the screening conditions of (FC≥1.2||FC≤1 / 1.2)&OPLS-DA_VIP≥1&P-value≤0.05, a total of 841 differential metabolites were screened from the metabolites of *Penicillium baicalensis* and *Penicillium kurineumoniae*. These mainly consisted of alkaloids and their derivatives, benzene ring compounds, hydrocarbons, lipids and lipid molecules, nucleosides, mucopeptides and analogues, organic acids and stimulants, organic nitrogen compounds, organic carbon compounds, organic heterocyclic compounds, phenylpropionamides, and polyketides. Regarding plant growth promotion, the secretion of *Penicillium kurineumoniae*-related metabolites mainly consisted of IAAs and alkaloids (see Table 3).

[0049] Table 3. Genes and metabolites related to plant growth

[0050]

[0051]

[0052] Example 5: Mechanism of Penicillium's effect on promoting root growth in maize Zhengdan 958 (hydroponic experiment)

[0053] Experimental device: flowerpot, sterilized substrate soil 1 kg; test sample: penicillium crustosum; test crop variety: corn Zhengdan 958; culture conditions: room temperature, natural light. Test treatment: experimental group 1 normal culture conditions, no addition of penicillium crustosum, addition of equal amount of purified water, experimental group 2 addition of penicillium crustosum (spore concentration: 10 7 individuals / ml, PDA solid culture medium culture, then diluted with purified water to about the specified concentration), added around the seeds, method reference to irrigation, amount 0.8 L / m 2 ; after 21 days of culture, plant data were observed and measured, the specification drawings show part of the root system, potting and plant photos of part of the plants of each experimental group, 50 plants were guaranteed to survive in each experimental group, 50 plants were taken from each experimental group for plant part measurement and root scanning, plant parameters were obtained, the obtained parameters were processed to obtain the average value, the parameter processing method was:

[0054] The obtained data take the root system length Length (cm) in the root scanning data as an example:

[0055] 1. Data analysis:

[0056] The average value and standard deviation SD of all root system length values of 50 plants in the experimental group or comparative experimental group were calculated.

[0057] 2. Remove extreme values:

[0058] According to the range of standard deviation, remove extreme values that deviate from the average value, remove data that is lower than the average value minus 2 times the standard deviation and data that is higher than the average value plus 2 times the standard deviation. Reduce the influence of extreme values on the average value.

[0059] 3. Repeat steps 1 and 2 for the remaining root system length data points until no new abnormal values are found, the obtained root system length data is valid data, and the average value of all valid data is taken.

[0060] Other data are processed in this way, and the results are shown in Table 4.

[0061] Table 4 Experimental plant data

[0062] Parameter Experiment group 1 Experiment group 2 Change rate % Plant fresh weight (g) 1.41 2.12 50.35 (aboveground part) plant dry weight (g) 0.24 0.33 37.50 Root fresh weight (g) 0.73 1.42 94.52 Root dry weight (g) 0.15 0.21 40.00 Aboveground biomass (g) 0.56 0.67 19.64 Plant height (cm) 24.13 41.28 71.07 Root length (cm) 101.0582 181.4835 79.58 Root surface area (cm 2 )]]> 40.6739 55.9837 37.64 Average diameter (mm) 1.0032 0.8961 -10.68 Root volume (cm 3 ) 1.314 1.496 13.85

[0063] Test results:

[0064] 1. The influence of penicillium crustosum on corn plant growth:

[0065] After inoculation of penicillium crustosum, the plant growth can be promoted; the plant fresh weight and plant height are increased by 50.35% and 71.07%, respectively.

[0066] 2. The influence of penicillium crustosum on corn root system:

[0067] After inoculating paecilomyces fumosafractus, the growth of the root system of the plant can be promoted; the fresh weight of the root, the dry weight of the root, the length of the root system, the surface area of the root system and the volume of the root system are respectively increased by 94.52%, 40.00%, 79.58%, 37.64% and 13.85%.

[0068] The above results verify the application effect of the paecilomyces fumosafractus in promoting the growth of plants through metabolite analysis and plant growth experiments, the paecilomyces fumosafractus has the effect of significantly promoting the growth of plants, especially the growth of the root system of plants, and can be applied to the preparation of biological agents in agricultural production, and various agricultural benefits can be generated.

[0069] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

[0070] The above describes the present application and its embodiments, which is not limited, and the embodiments shown in the drawings are only one of the embodiments of the present application, and the actual application is not limited thereto. In summary, if a person skilled in the art is inspired, without departing from the purpose of the present application, without creative design, similar ways and embodiments of the technical solution should belong to the protection scope of the present application.

Claims

1. A type of Penicillium keloidea that promotes plant growth, characterized in that: The strain is named *Penicillium clarithroptus* 23301-20, with accession number CGMCC No. 41049, accession date January 22, 2024, and deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The culture medium for *Penicillium clarithroptus* includes PDA medium. The colony characteristics of *Penicillium clarithroptus* are: initially white, later turning dark green; conidiophores branch multiple times to form a typical broom-like structure; conidia are continuously produced at the top; and the conidia are nearly spherical.

2. The use of Penicillium keloidea as described in claim 1, which has the effect of promoting plant growth, in the preparation of biological agents with the effect of promoting plant growth.

3. The application according to claim 2, characterized in that: The applications include those used in the preparation of liquid or solid biological agents.

4. The application according to claim 2, characterized in that: The biological agents include those that promote plant growth and those that have other agricultural benefits.

5. The application according to claim 4, characterized in that: The other agricultural benefits include one or more of the following: promoting plant root growth, improving plant stress resistance, and improving soil properties.

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