CN114045223B - Pediospora pseudodiscus strain P6 from yellow pear, breeding method and application - Google Patents
Pediospora pseudodiscus strain P6 from yellow pear, breeding method and application Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
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Abstract
The invention discloses a Mucor pseudodiscus strain Pestalotiopsis arceuthobii P from yellow pear, which is deposited in the Guangdong province microorganism strain collection center with the deposit number of: GDMCC No:61995. the invention also discloses a breeding method of the mistletoe, which comprises the following steps: spraying fermentation liquor of the strain P6 on mulberry branches with the parasitic loranthus seeds for culture. The invention also discloses application of endophytic fungi in laccase production, lignin degradation and cellulose degradation, parasitic loranthus seed germination, parasitic loranthus cultivation, plant prevention and control and scientific research. The invention also discloses a primer pair amplification sequence adopted in the construction of the phylogenetic tree by the endophytic strain. The invention not only provides application guidance and theoretical basis for the breeding of the mistletoe, but also provides a new research thought for other mistletoe plant invasion host plant mechanism.
Description
Technical Field
The invention belongs to the field of parasitic loranthus breeding, and relates to a trichoderma pseudodisc strain P6 derived from yellow pear, a breeding method and application.
Background
The herba Taxilli is dry leaves, stems and branches of herba Taxilli Taxillus chinensis (DC.) Danser of Loranthaceae, is a traditional common bulk Chinese medicinal material, has effects of dispelling pathogenic wind and dampness, nourishing liver and kidney, strengthening tendons and bones, an Taiyuan, etc., and is a collection variety of Chinese pharmacopoeia (2020 edition) (national pharmacopoeia Committee, 2020). Loranthus mulberry mistletoe is also a very characteristic road-land mistletoe medicinal material in Guangxi province, and as early as Guangxi general Zhi Ling, guangxi Tong Zhi, in the past, it is recorded that Loranthus mulberry mistletoe and Loranthus mulberry mistletoe wine are the main features of Guangxi province (Zhu Lunhuan, 2002), and the Wuzhou mistletoe tea developed by taking Loranthus mulberry mistletoe as a raw material is listed as a traditional Chinese food health-care tea as early as 1992, and exported to the near 30 countries in southeast Asia (Li Yonghua, etc., 2006). Along with the increasing market demand, the mistletoe has a very important role in Guangxi and even national Chinese herbal medicine markets. Because the wild resources of the Chinese taxillus herb are less and less, the development of artificial cultivation is imperative, and the improvement of the parasitic rate and the solution breeding mechanism of the Chinese taxillus herb seed is the key point and the difficulty of the artificial breeding and cultivation of the Chinese taxillus herb.
Disclosure of Invention
It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below.
It is still another object of the present invention to provide a Pelargonium pseudodisc strain Pestalotiopsis arceuthobii P of Solanum annuum.
The invention also aims to provide a breeding method of the mistletoe.
It is another object of the present invention to provide the use of the Mucor strain Pestalotiopsis arceuthobii P6 in laccase production, lignin degradation, cellulose degradation and parasitic loranthus seed germination.
Another object of the invention is to provide the use of the Mucor pseudodisci strain Pestalotiopsis arceuthobii P in the breeding and cultivation of Loranthus parasiticus, plant control and scientific research.
It is a further object of the present invention to provide primer pair amplification sequences for use in the construction of phylogenetic tree of endophytic strains.
For this purpose, the technical scheme provided by the invention is as follows:
a trichoderma strain Pestalotiopsis arceuthobii P6 derived from yellow pear, said trichoderma strain Pestalotiopsis arceuthobii P6 being deposited with the cantonese collection of microorganism strains under accession number: GDMCC No:61995, the preservation time is: 2021, 10 and 18, the deposit unit addresses are: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.
The breeding method of the mistletoe comprises the following steps:
1) Inoculating the trichoderma pseudodisc Pestalotiopsis arceuthobii P strain according to claim 1 into a culture medium for culturing;
2) Harvesting the fermentation broth of the strain P6 grown in step 1);
3) And (3) planting a plurality of mistletoe seeds on each mulberry branch, and spraying the fermentation liquor obtained in the step (2) on the mulberry branches every day in the culture, wherein the dosage of the fermentation liquor is 1-10 ml/one strain at a time.
Preferably, in the breeding method of the mistletoe, in step 1), the culture medium adopts PDA liquid culture medium.
Preferably, in the breeding method of the mistletoe, in step 1), the culturing is: strain P6 was cultured with shaking at a constant temperature of about 28℃for about 3 days.
Preferably, in the breeding method of the mistletoe, in the step 2), the culture solution obtained by culturing in the step 1) is filtered by sterile gauze and filtered by filter paper, and then centrifuged, and the supernatant is taken to obtain the fermentation liquor.
Preferably, in the breeding method of the mistletoe, in the step 3), the dosage of the fermentation liquid is 5 ml/one time/one strain.
Preferably, in the method for breeding mistletoe, in step 3), the culturing lasts from about 30 days to the germination of mistletoe seeds.
The application of the Mucor pseudodiscus strain Pestalotiopsis arceuthobii P in laccase production, lignin degradation, cellulose degradation and parasitic loranthus seed germination.
The application of the Mucor pseudo-discus strain Pestalotiopsis arceuthobii P in parasitic loranthus breeding and cultivation, plant prevention and control and scientific research.
Primer pair amplification sequences adopted in the construction of phylogenetic tree of the endophyte, wherein the primer pair amplification sequences comprise: primer pairs shown as SEQ ID NO. 1 and SEQ ID NO. 2, and primer pairs shown as SEQ ID NO. 3 and SEQ ID NO. 4.
The invention at least comprises the following beneficial effects:
the invention explores the action mechanism of the parasitic loranthus endophytic fungi in the parasitic loranthus invading host plant cell wall degradation, identifies and obtains better lignocellulose degradation strain, can produce lignin peroxidase, manganese peroxidase, laccase and cellulase, and lays a theoretical foundation for the action mechanism of the parasitic loranthus invading host plant cell wall degradation. Meanwhile, the endophytic fungus Mucor pseudodiscus strain Pestalotiopsis arceuthobii P6 is applied to parasitic loranthus breeding, so that the seed germination rate of the parasitic loranthus is improved. The invention not only provides application guidance and theoretical basis for the breeding of the mistletoe, but also provides a new research thought for other mistletoe plant invasion host plant mechanism.
Reference to the literature
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Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a graph of the growth of different strains on a chromogenic medium in an example of the present invention (7 d).
FIG. 2 is a graph of the growth of different strains on Congo red medium in an example of the invention (7 d).
FIG. 3 is a graph showing the enzyme activity of laccase produced by different strains at different times in the embodiment of the invention.
FIG. 4 is a graph showing the activity of manganese peroxidase produced by different strains at different times in the example of the present invention.
FIG. 5 is a graph showing the activity of lignin peroxidase produced by different strains at different times in the examples of the present invention.
FIG. 6 is a graph showing the enzyme activities of different strains for producing cellulase at different times in the embodiment of the invention.
FIG. 7 is a phylogenetic tree constructed based on the combination of ITS rDNA and tubulin beta-tublin sequences in an embodiment of the present invention.
FIG. 8 is a photograph showing germination of Mucor pulmonale strain Pestalotiopsis arceuthobii P after acting on Taxus chinensis seeds in the examples of the present invention.
FIG. 9 is a photograph showing the growth of Mucor strain Pestalotiopsis arceuthobii P6 after acting on Loranthus seed in the examples of the invention.
Detailed Description
The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not exclude the presence or addition of one or more other elements or combinations thereof.
The applicant observes that the parasitic loranthus mainly ingests fruits of the parasitic loranthus through some birds with pulp in a natural state, seeds after the pulp is digested are discharged and attached to branches of a host plant, then a sucker formed by germination of the parasitic loranthus seeds is found on the surface of the host plant to penetrate through the epidermis of the host plant and extend to form a physiological bridge with the host plant, and finally successful breeding of the parasitic loranthus is realized, and the surface of the host plant is intact when the parasitic loranthus seeds which fail to successfully parasitize are peeled off from the host plant. Then, it might be critical to develop parasitic plant penetration through the epidermis of the host plant and formation of a physiological bridge.
The mechanism by which the suction of the parasitic plant penetrates the parasitic plant has also attracted considerable attention because of the barrier that the suction of the parasitic plant forms a physiological bridge "with the host plant must penetrate the epidermal tissue of the host plant (Nagar et al,1984;Ranjan et al,2014;Johnsen et al,2015;Olsen et al,2016). There have been studies to find that the penetration of the aspirator does not significantly damage the host plant cells, such as found in the case of the Leptoradix (Striga hermonthica) aspirator penetrating the endothelial layer of the host plant, the aspirator does not cause damage to the endothelial layer cells (Neumann et al,1999;Naseer et al,2012;Perez-de-Luque, 2013). And during this process, enzymes associated with cell wall degradation have been found in large numbers, such as in most Orobanchaceae (Orobanchaceae) parasitic plants (Ben-Hod et al,1993; perez-de-Luque, 2013), as has been found in the aspirator puncture sites of Orobanches cumana and Phelipanche aegyptiaca parasitic plants as a pectin methyl esterase believed to degrade pectin (Losner-golden et al, 1998); there are other cell wall modifying enzymes, expandases, such as the cell wall modifying endoglucanases in the penetration phase of dodder infection, which peak, in contrast, when inhibited, reduce the chance of successful aspirator penetration (Olsen and Krause, 2017). The effect of fungi in degrading the cell walls of a host plant in the process of invading the host plant has been reported more, and can provide a reference for the research of the effect of parasitic endophytic fungi in the process of parasitic hosts. Studies on lignin and cellulose, the major components in plant cell walls, have also been reported, but have focused mainly on several modes of wood rot fungi, such as Phanerochaete chrysosporium Phanerochaete chrysosporium Burds and Ceriporiopsis entomophaea Ceriporiopsis subvermispora (Pil t) Gilb. & Ryvarden, and the brown rot fungus Postina planta (Fr.) M.J.Larsen & Lombard et al (Martinez et al,2004,2009; fernandez-Fueyo et al 2012). White rot fungi are considered to be the most effective and dominant microorganism for degrading lignin, and form a unique set of degradation systems in the long-term biological evolution process, laccase, manganese peroxidase and lignin peroxidase together form a lignin degradation enzyme system of white rot fungi (Xu Congfeng and the like, 2019) and can degrade all components of plant cell walls, including lignin, cellulose and hemicellulose (Hakala et al,2005;Kirk and Jeffries,1996). The invention aims to separate endophytic fungi from the mistletoe of different hosts, the obtained endophytic fungi screen strains producing color development rings and transparent rings on a selection medium and a cellulose solid medium which take guaiacol as indicators, researches and reports that the strains producing color development rings and transparent rings have the capability of degrading lignin and cellulose (Anderet al,1977 and Xiong Yi, 2019, xu Anmin and the like, 2020), the strains producing color development rings and transparent rings are subjected to qualitative measurement of laccase, lignin peroxidase, manganese peroxidase and cellulase enzyme activities, the high-yield strains of the mistletoe endophytic fungi lignocellulose degrading enzyme are screened, classification positions of the strains are established according to morphological characteristics and molecular biology, and the action mechanism of the mistletoe endophytic fungi degrading in the cell wall of the mistletoe invasion host plant is explored, so that application guidance and theoretical basis are provided for the breeding of the mistletoe, and a new research idea is provided for the mechanism of other mistletoe plants invading the host plant.
The invention provides a Pediophora strain Pestalotiopsis arceuthobii P derived from yellow pear, wherein the Pediophora strain Pestalotiopsis arceuthobii P is preserved in the microorganism strain collection of Guangdong province, and the preservation number is: GDMCC No:61995, the preservation time is: 2021, 10 and 18, the deposit unit addresses are: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.
The invention also provides a breeding method of the mistletoe, which comprises the following steps:
1) Inoculating the trichoderma pseudodisc Pestalotiopsis arceuthobii P strain according to claim 1 into a culture medium for culturing;
2) Harvesting the fermentation broth of the strain P6 grown in step 1);
3) And (3) planting a plurality of mistletoe seeds on each mulberry branch, and spraying the fermentation liquor obtained in the step (2) on the mulberry branches every day in the culture, wherein the dosage of the fermentation liquor is 1-10 ml/one strain at a time.
In one embodiment of the present invention, preferably, in step 1), the culture medium is PDA liquid culture medium.
In one embodiment of the present invention, preferably, in step 1), the culturing is: strain P6 was cultured with shaking at a constant temperature of about 28℃for about 3 days.
In one embodiment of the present invention, preferably, in step 2), the culture solution obtained by culturing in step 1) is filtered by sterile gauze and filtered by filter paper, and then centrifuged, and the supernatant is collected to obtain a fermentation broth.
In one embodiment of the present invention, preferably, in step 3), the amount of the fermentation broth is 5 ml/one at a time/one strain.
In one embodiment of the present invention, preferably, in step 3), the culturing is continued for about 30 days to germination of the mistletoe seeds.
The invention also provides application of the Mucor pseudo-disci strain Pestalotiopsis arceuthobii P in laccase production, lignin degradation, cellulose degradation and parasitic loranthus seed germination.
The invention also provides application of the Mucor pseudo-disci strain Pestalotiopsis arceuthobii P in parasitic loranthus breeding and cultivation, plant prevention and control and scientific research.
The invention also provides a primer pair amplification sequence adopted in the construction of the phylogenetic tree of the endophytic strain, wherein the primer pair amplification sequence comprises the following components: primer pairs shown as SEQ ID NO. 1 and SEQ ID NO. 2, and primer pairs shown as SEQ ID NO. 3 and SEQ ID NO. 4.
For a better understanding of the technical solution of the present invention, the following examples are now provided for illustration:
1 materials and methods
1.1 materials
1.1.1 sample collection of Loranthus mulberry mistletoe
1 month in 2019, the parasitic stems and branches of different hosts including mulberry, plum, yellow tree, yellow pear, bauhinia, persimmon and longan are collected at the plant base of Loranthus mulberry in Cen Funing village. 3-5 branches are collected from the same plant.
Raw wheat bran was purchased from the company limited by the modern agricultural development group of the chinese crane. Pretreatment of Wheat Bran (WBDF) reference Tao Yanjuan method (Tao Yanjuan, 2008) and slight modification. Pulverizing testa Tritici, sieving with 40 mesh sieve, adding 10 times volume of distilled water, mixing with colloid mill, and pulverizing for 25min. The liquid impurities were filtered off with a 150 mesh sieve, and the solid fraction was dried in an oven at 60 ℃ for 24h and then crushed. Adding 10 times volume of distilled water, heating at 95deg.C for 30min, adjusting pH to 5.6 with HCL, adding 1.5% (w/w) high temperature resistant alpha-amylase, stirring at 95deg.C, reacting for 30min, and detecting whether the reaction is complete with iodine solution. Cooling to 50 ℃, regulating the pH to 9.0 by NaOH, adding 3% (w/w) alkaline protease, and stirring for reaction for 2h. The supernatant was discarded, washed by filtration through a 150 mesh screen in clear water until the wash was clear, and the remaining solid material was dried in an oven at 60 ℃ for 24 hours. Pulverizing the wheat bran obtained by drying in a micro pulverizer, sieving with a 100 mesh sieve, and oven drying at 50deg.C overnight for use.
1.1.2 Medium
PDA culture medium, potato 200g/L, glucose 20g/L, agar 20g/L, natural pH, 1×10 5 Pa sterilization for 30min.
Seed liquid culture medium comprising 20g/L glucose, 2g/L yeast extract, and KH 2 PO 4 3g/L，MgSO 4 ·7H 2 O 1.5g/L，V B1 0.5g/L, natural pH 1X 10 5 Pa sterilization for 30min.
Guaiacol solid medium: 200g/L potato, 20g/L glucose and agarFat 20g/L, KH 2 PO 4 3g/L，MgSO 4 ·7H 2 O 1.5g/L，V B1 0.02g/L, guaiacol 1g/L, pH naturally 1X 10 5 Pa sterilization for 30min.
Liquid fermentation basic culture medium, wheat bran 30g/L, KH 2 PO 4 3g/L，MgSO 4 ·7H 2 O 1.5g/L，(NH 4 ) 2 SO 4 1.4g，CaCl 2 0.3g，FeSO 4 ·7H 2 O 5mg/L，MnSO 4 ·H 2 O 1.6mg/L，VB 1 0.02g/L, natural pH, and packaging into 250mL triangular bottles with 100mL of 1×10 each 5 Pa sterilization for 30min.
The cellulose solid medium was congo red staining medium, see reference (Kim et al, 2018), for details as follows:
cellulose congo red solid medium: sodium carboxymethylcellulose 5g/L, yeast extract 0.5g/L, peptone 0.5g/L, beef extract 0.3g/L, KH 2 PO 4 3g/L，K 2 HPO 4 5g/L，(NH 4 ) 2 SO 4 2g/L，MgSO 4 0.4g/L，CaCl 2 0.1g/L of microelement solution 1mL, agar powder 20g/L, and Congo red powder 0.5g were dissolved in 50mL of sterile water.
1.2 method
1.2.1 isolation and purification of endophytic fungi
Selecting healthy and disease-free mistletoe branches of different hosts, and cutting the tissues into 5cm fragments; sequentially sterilizing with 75% ethanol and 0.1% mercuric chloride for 2.5min, and washing with sterile water for 3 times; it was cut into tissue pieces of about 5mm size with sterile forceps and a scalpel, 5 pieces per plate, 3 plates per sample, cultured at 28℃until mycelia grow out at the edges of the tissue pieces, transferred to PDA plates for culture, purified and preserved.
1.2.2 preliminary screening of lignocellulose degrading enzyme Strain
Primary screening of lignin degrading enzyme strain: plates (Sun Jianghui, 2012) were made by adding 0.1% of the guaiacol to PDA medium, and the purified 6mm diameter pieces were picked on a super clean bench and incubated for 10d at 28℃in 3 replicates per strain. The size of colony circle and color circle diameters, and the color change of the colonies in the assay plate were observed and counted.
Primary screening of cellulose degrading enzyme strains: after preparing a cellulose solid medium by a transparent circle method, tissue blocks (diameter: 6 mm) of the purified strain were inoculated on the medium, each group was repeated 3 times, incubated at 28℃for 10 days, stained with 0.1% Congo red for 15 minutes, and then decolorized with 1M NaCl for 15 minutes, and the diameter of a transparent circle of a colony of a unit was observed and recorded, and the diameter of the transparent circle of a colony of a unit = the diameter of the transparent circle-the diameter of the colony. The larger diameter of the transparent circle indicates a higher enzyme-producing activity (Kim et al, 2018).
1.2.3 re-screening of lignocellulose degrading enzyme Strain
The lignocellulose degrading enzyme strain with the color-developing ring and the transparent ring is re-screened according to the method of 1.2.2.
1.2.3 preparation of enzyme solution
The lignocellulose degradation strains with the color development rings and the transparent rings are selected for culturing for 5-7d, respectively and quantitatively inoculated, 10mL of the uniformly shaken seed liquid is absorbed and added into 250mL triangular flasks filled with 100mL of liquid fermentation culture medium, each strain is inoculated with 2 flasks, each flask is connected with 5 strain blocks with the diameter of 6mm, and the strains are cultured for 11d in a constant temperature shaking table at 26 ℃ and 140 r/min. Sampling from day 4, sampling 1 time every 1d, filtering the fermentation liquor with 4 layers of gauze, centrifuging the filtrate at 3 000r/min for 15min, and collecting the supernatant as crude enzyme solution.
1.2.4 enzyme Activity assay
Measurement of laccase enzyme Activity ABTS was used as substrate (Buswell et al 1995), defined as the amount of enzyme required to catalyze the oxidation of 1. Mu. Mol of substrate ABTS per minute at 30℃as a laccase enzyme activity unit, oxidation of substrate ABTS was determined by measuring absorbance of the reaction solution at 420nm, and molar extinction coefficient was 36000Lmol -1 ·cm -1 The method comprises the steps of carrying out a first treatment on the surface of the Measurement of lignin peroxidase Activity A method of producing veratraldehyde using veratrole as an oxidation substrate at 30℃was used (Tien et al, 1988), and the enzyme activity per unit was defined as a change in absorbance per minute per milliliter of the reaction solution of 0.1 unit; manganese peroxidase activity was measured by phenol red (Orth et al, 1993), oneThe enzyme activity per unit was defined as an increase in absorbance per minute per milliliter of the reaction solution at 610nm of 0.1 units, U.mL -1 And (3) representing.
The cellulase activity is determined by using a Cellulase (CL) activity detection kit (Beijing Soy Bao technology Co., ltd.) and adopting a 3.5-dinitrosalicylic acid (DNS) method, under the action of the cellulase, reducing sugar is produced by cellulose degradation, and the enzyme activity is determined by measuring the content of the reducing sugar. The kit 10mg anhydrous glucose standard (weight loss on drying < 0.2%) was taken out, and 1mL of ultrapure water was added to prepare 10 mg.mL -1 Glucose solution, and then diluted in gradient to 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0 mg.mL -1 . At 540nm, 0 mg.mL -1 Zeroing, reading the absorbance value of each concentration gradient, and establishing a standard curve by taking the concentration (X) as an abscissa axis and the absorbance A (Y) as an ordinate axis. 1mL of fermentation broth is weighed, and the thallus is broken by ultrasonic ice bath. And (5) centrifuging at a temperature of between 8000g and 4 ℃ for 10min, taking supernatant as crude enzyme liquid, filling the crude enzyme liquid into a 2mL centrifuge tube, and placing the crude enzyme liquid on ice for detection. The reaction substrate and 50. Mu.L of the sample form 350. Mu.L of a reaction system, the reaction is stopped by boiling to obtain a saccharification liquid, 50. Mu.L of the saccharification liquid is taken, 150. Mu.L of DNS reagent is added for uniform mixing, 1050. Mu.L of double distilled water is added for measuring absorbance at 540nm under an ultraviolet spectrophotometer (Xie Changjiao, ke Lixia, etc., 2015).
1.2.5 identification of endophytic fungi
The method of reference in the square records colony morphology (square in the square in 1998), the method of reference Wei Jingchao (Wei Jingchao, 1979) and the International Classification website (http:// www.indexfungorum.org) for preliminary identification of colony morphology. Referring to the method of Kumar et al (Kumar et al, 2016), molecular biology identification was performed using the orthotopic ligation of MEGA 7.0 to construct ITS rDNA (ITS 15 '-TCCGTAGGTGAACCTGCGG-3' SEQ ID NO:1 and ITS 45 '-TCCTCCGCTTATTGATATGC-3' SEQ ID NO: 2) in combination with tubulin beta-tublin (βt-2a 5'-AACATGCGTGAGATTGTAAGT-3' SEQ ID NO:3 and βt-2b5'-ACCCTCAGTGTAGTGACCCTTGGC-3' SEQ ID NO: 4) phylogenetic tree (White et al,1990;Weir et al,2012); using MightyAmp DNA Polymerase ver.3 (1.25U/50 μl) kit (Takara Bio inc., japan, cat.no. r076 a), picking colonies of the cultured endophytic fungi as templates for direct PCR reaction, detecting the target bands by gel imaging, and delivering the PCR products of the target bands to the large gene (guangzhou) stock company for sequencing; sequencing results were BLAST aligned with sequences in GenBank of NCBI. The method of Neighbor-Joining of MEGA 6.0 software was used to construct ITS rDNA phylogenetic tree.
1.2.6 statistical analysis of data
Data statistics are expressed by x+ -s, s is the mean, s is the standard deviation, each group of data is analyzed by single-factor ANOVA using SPSS19.0 software, variance homogeneity is checked, and P <0.05 is statistically significant.
2 results and analysis
2.1 isolation of endophytic fungi
Endophytic fungus strain 147 is obtained by co-separating seven host mistletoe of mulberry, plum, wampee tree, yellow pear, bauhinia, persimmon, longan and the like, wherein mulberry 20, plum 20, wampee tree 9, yellow pear 27, bauhinia 22, persimmon 23 and longan 26. And selecting strains of different types, namely 72 strains in total, and performing lignin degrading enzyme and cellulase activity flat-plate detection.
2.2 Lignin degrading enzyme Activity Flat plate detection
Microorganisms producing color circle on the selective culture medium with guaiacol as an indicator have the ability to degrade lignin, and hyphae grown on the culture medium by laccase-producing strains can produce obvious reddish brown color. Culturing 72 strains of 11d on plates of guaiacol selection medium, and observing and recording the color change of each plate every other day, so that 11 strains of strains without colony rings and color development rings are found; 17 strains are arranged in the colony ring diameter and the color development ring diameter ratio of less than 1; the ratio of the colony circle diameter to the color development circle diameter is more than 1, and 26 strains exist; 7 strains with color development circles and without colony circles; there were 11 strains with colony circles and without color circle. According to the study reported by the literature report of Eriksson et al (Eriksson et al 2012), the ratio of the diameters of colony rings and chromogenic rings can be used as a basis for judging whether the bacterium can selectively degrade lignin, and if the ratio is smaller than 1, the bacterium can selectively degrade lignin. In summary, after the guaiacol method-based primary screening and secondary screening, 5 strains which can produce large and obvious color circle were screened out of 24 strains (the ratio of the diameter of the color circle to the diameter of the color circle is less than 1 and the strains with color circle and without color circle) together for the subsequent enzyme activity test (Table 1). As can be seen from Table 1, the 5 strains increased over time, and the colony circle and the color development circle increased, and neither the colony circle nor the color development circle increased up to 11 days. FIG. 1 shows the growth of these 5 strains on PDA plates for 7 days.
TABLE 1 variation of colony circle and color circle diameters of different strains with time
Table 1 Diameter changes of colony circle and chromogenic circle of different strains with time\cm
2.3 Flat plate detection of cellulose degrading enzyme Activity
Culturing 72 strains 11d on cellulose solid culture medium plates, and observing and recording the color change of each plate every other day, wherein 14 strains are found out from strains with colony rings and without transparent rings; the ratio of the colony circle diameter to the transparent circle diameter is less than 1, and 58 strains exist; strains with a ratio of colony free circle diameter to transparent circle diameter greater than 1. The same strain as lignin degrading enzyme was selected from the strains having a ratio of colony circle diameter to transparent circle diameter of less than 1 for subsequent enzyme activity test (Table 2). As seen from Table 2, these 5 strains were gradually increased with time, and were not changed until 9d later, maintaining the original range. FIG. 2 shows the growth of these 5 strains on cellulose solid medium at 7 d.
TABLE 2 variation of colony circle and color circle diameters of different strains with time
Table 2 Diameter changes of colony circle and chromogenic circle of different strains with time\cm
2.4 Lignin degrading enzyme System enzyme Activity analysis
2.4.1 laccase enzyme Activity assay
As can be seen from FIG. 3, the peak time of laccase production by these 5 strains was 7d, and decreased from 9 d. The laccase production capability of 5 strains is obvious (P is less than 0.05). Wherein, the laccase production capability of the P6 is strongest, the enzyme activity is 117.66U/mL, the enzyme activities are 51.82U/mL, 32.11U/mL and 21.85U/mL respectively, the laccase production capability of the strain 4 is weakest, and the enzyme activity is 9.76U/mL. The sequence of the enzyme activities of the 5 endophytic fungi is that P6 is more than N6 is more than 15 is more than 31 is more than 4.
2.4.2 manganese peroxidase Activity assay
As seen in FIG. 4, the highest value of the manganese peroxidase produced by strain No. 4 was 7d, the enzyme activity was 11.61U/mL, followed by strains No. N6 and No. 31, the highest enzyme activities were 8.47U/mL and 5.58U/mL, respectively. The 15 and P6 strains had the highest enzyme productivity at 11d, and the enzyme activities were 8.59U/mL and 6.79U/mL, respectively. The maximum enzyme activity of manganese peroxidase of the 5 endophytic fungi is respectively 4 & gt 15 & gtN 6 & gtP 6 & gt31, and the difference is obvious (P is less than 0.05).
2.4.3 Lignin oxide enzyme Activity assay
As shown in FIG. 5, the lignin peroxidase activity curves of endophytic fungi strains N6, 4 and 31 are all raised with time until reaching the enzyme activity peak, and then start to decline, the highest lignin peroxidase production capacity of the 3 strains is 7d, and the enzyme activities are 6.64, 3.0 and 2.9U/mL respectively. The lignin peroxidase activities of the P6 strain and the P15 strain are respectively increased, then decreased, and increased again when the enzyme activity reaches 11d, and the highest enzyme activities are respectively 4.21U/mL and 3.4U/mL. The order of the lignin peroxidase activity of the 5 endophytic fungi is N6 & gtP 6 & gt15 & gt4 & gt31, and the difference between the value of the 5 endophytic fungi strain with the lowest enzyme activity and the value of the 5 endophytic fungi strain with the highest enzyme activity is remarkable (P & lt 0.05)
2.4 analysis of cellulose degrading enzyme Activity
As seen from FIG. 6, the cellulase activity curves of 4 strains P6, N6, 31, 4 and the like were all increased with time until the highest peak of the enzyme activity was reached, then the decrease was started, the peak was increased at 7d in addition to the sudden decrease of strain 15 at 5d, and the cellulase activities were 1.66, 1.61, 0.67, 0.61 and 0.59U/mL, respectively, when the highest cellulase production time of these 5 strains was 7 d.
2.5 identification of strains
The ITS rDNA sequence and tubulin beta-tublin sequence are determined and combined to construct a phylogenetic tree, and the results (figure 7) show that strains 4, 15, 31, N6 and P6 are respectively gathered on the same branch with Colletotrichum acutatum (CBS MH 860607), nigrospora sphaerica (CBS MH 854878), exserohilum rostratum (Solanum nigrum CBS MH 865917), diaporthe phaseolorum (CBS KC343174\KC343175\KC 343176) and Pestalotiopsis arceuthobii (CBS MH 858656), and the similarity is 99-100%. Thus, these 5 strains were identified as C.acutatum, N.sphaerica, E.rossmatum, D.phaseolorum, P.arceuthobii, respectively. The 5 bacterial sequences were submitted to the GenBank database with accession numbers \ (ITS and beta-tublin) MZ2823601/MZ964759, MZ2823600/MZ934421, MZ2823597/MZ934418, MZ2823599/MZ934420, MZ2823598/MZ934419, respectively.
Simultaneously performing biological preservation on a trichoderma pseudodisc strain Pestalotiopsis arceuthobii P from yellow pear, wherein the trichoderma pseudodisc strain Pestalotiopsis arceuthobii P is preserved in the Guangdong province microorganism strain preservation center, and the preservation number is as follows: GDMCC No:61995, the preservation time is: 2021, 10 and 18, the deposit unit addresses are: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.
3 specific operation steps and method for applying endophytic fungi to breeding of mistletoe
Endophytic fungi (endophytic fungi) are fungi that are an important component of the plant microbial community that do not cause significant disease symptoms to plant tissue during some or all cycles within healthy plant tissue. Endophytic fungi have abundant biodiversity, can grow asymptomatically in different healthy tissues such as stems, leaves, roots and the like of plants, naturally exist in temperate and tropical rain forests, and are distributed with about 30 ten thousand land host plants. Each plant has one or more endophytic fungi. It is estimated that there are over 100 tens of thousands of endophytic fungi species in nature. The relationship of endophytes to host plants is generally a reciprocal symbiotic relationship, on the one hand, where the endophytes need to grow and where the nutrients come from the host, and on the other hand, the endophytes play an important role in the growth and development of the host and in the systematic evolution. Research shows that the existence of endophytic fungi can enhance the immunity of a host, promote the germination capacity of seeds and resist diseases, and the endophytic fungi are important microbial resources in the technical field of plant prevention and control.
The mistletoe is a semi-parasitic plant, the hosts have diversity, the mistletoe seeds are truly typical recalcitrant seeds, the dehydration and low temperature sensitivity are strong, the propagation of the mistletoe is severely restricted, and the mistletoe is a technical bottleneck for standardized cultivation.
The fermentation liquid of the prepared strain P6 is used for breeding the parasitic loranthus seeds parasitized on the mulberry by a spraying mode so as to improve the parasitizing rate of the parasitic loranthus seeds. The method comprises the following specific steps:
(1) Strain P6 was activated on PDA medium, incubated at 28℃for 5-7d with 60% humidity.
(2) Under aseptic conditions, 5 tissue blocks with the diameter of 7 mm are taken from each strain, placed into (300 ml triangular flask) liquid PDA culture medium without agar, vibrated at constant temperature of 28 ℃ for 3 days at 180r/min, filtered by aseptic gauze, filtered by filter paper, centrifuged at 10000r/min for 15 minutes, and the supernatant is taken.
(3) The supernatant of the fermentation liquor is taken 5 ml/one time/one strain every day, one strain of mulberry branch is inoculated with 20 seeds, each treatment is repeated 5 times, 100 seeds are processed in total, the indoor temperature is kept for 30 days at 25 ℃, and the control is that clear water is sprayed.
(4) As shown in fig. 8 and 9, the calculated seed germination rate was found to be 50% higher than the control, and 100 seeds sprayed with the fermentation broth were able to germinate 50 seeds at maximum, whereas the control was 25 seeds only.
(5) Observing the growth vigor of seed germination, wherein the seed germination of the sprayed fermentation liquid is 3-5d earlier than that of the control.
Discussion 4
The wheat bran is selected as a substrate of the lignocellulose biomass to ferment, and enzyme activities of laccase, manganese peroxidase, lignin peroxidase, cellulase and the like are measured at different times, so that the production is foundThe highest enzyme activity time of 5 strains of laccase and cellulase is 7d, wherein the highest enzyme activity of laccase and cellulase are P6 strains, the enzyme activities are 117.66 and 1.66U/mL respectively, the morphology and molecular biology identification prove that the fungus is P.arceutophilus, the fungus can produce relatively high laccase and cellulase, the study of effectively degrading forest litter and the like is reported (Hao Jiejie and 2006), and the fermentation liquor extract of the fungus P.arceutophili is obviously shallower than that of the other 4 strains because of laccase can be decolorized and degraded with fuel (Thurston, 1994; wong et al, 1999); the highest 117.66U/mL laccase activity produced by P.arceuthobii is a relatively high value in the crude enzyme activity data reported so far, and its production is not dependent on the addition of some enzymes such as soil temperature 80, ferulic acid, cu 2+ Or an inducer such as dimethylaniline, and Cao Yongjia et al (Cao Yongjia et al 2021) reported that soil temperature 80 and Cu were added 2+ The laccase enzyme activity of the inducer was indeed much higher within a week, but the average laccase enzyme activity of 2 other white rot fungi (Mao Shuankong fungi hirsuta and fomes fomentarius Fomes fomentarius) besides white rot fungi Ganoderma lucidum Ganoderma applanatum did not reach this peak, indicating that p.
Manganese peroxidase activities of the 4 strains N6, 4, 15 and 31 were found to be not the highest at 7d, but at 11d were found to be the second highest at 15 and P6, respectively, at 8.59U/mL and 6.79U/mL, respectively, and lignin peroxidase activities were found to be 3.4 and 4.21U/mL, respectively, consistent with the reported lignin enzymes and cellulases of some white rot fungi (Ruttimannet al, 1992;Raghukumar et al,1999; arora et al, 2000), the second peak was found probably due to the fact that the mycelia autolyzed so that the corresponding intracellular enzymes originally bound to the cell membrane were released (Hao Jiejie et al, 2006). N6, 4, 15, 31 and other 4 strains are respectively identified as D.phaseolorum of the genus Deuterina, C.acutatum of the genus Celastrus, N.sphaerica of the genus Celastrus and E.rossmatum of the genus Celastrus by morphological and molecular biology, which are the first reports that 4 fungi produce lignocellulose degrading enzymes by taking wheat gluten fiber biomass as a substrate, and enzyme activities (Hao Jiejie, etc.) of 6 fungi (Alternaria, penicillium sp., cephalosporium sp., tricherdermasp., pestalotiopssp. And Aspergillus) of degrading Marathon to produce laccase, lignin peroxidase, manganese peroxidase, cellulase and the like are also studied by the former (2006).
Lignin and cellulose degradation is not solely by 1 enzyme action, but rather the result of the combined action of several enzymes, lignin degrading enzymes are mainly completed by lignin peroxidase, manganese peroxidase and laccase, namely 3 enzymes (Chi Yujie and Hong Wei, 2007), and the size of a color development circle is positively correlated with the activity of laccase but not linearly (Du Haiping, etc., 2006); the cellulose degrading enzymes are mainly endoglucanases, exoglucanases and beta-glucosidase, and the synergistic action of these 3 enzymes is required for complete hydrolysis of cellulose to monosaccharides (Wilson 2009;Linton 2020;Siqueira et al.2020). According to the invention, 5 good lignocellulose degradation strains are screened and identified, lignin peroxidase, manganese peroxidase, laccase and cellulase are produced in different degrees, and the fact that the enzymes produced by the lignin peroxidase, the manganese peroxidase, the laccase and the cellulase can break through epidermal cells of a host is indicated, so that the enzymes can be successfully parasitic in the host, and a theoretical foundation is laid for an action mechanism of parasitic loranthus invading a plant cell wall of the host for degradation. The test still needs to measure the content of the enzyme activities and the degradation rate of lignocellulose-producing degrading enzyme by taking the mulberry bark serving as a parasitic host as a lignocellulose biomass substrate, and considers the optimization of different solid culture media, the extension of the fermentation period and the addition of soil temperature 80, ferulic acid and Cu 2+ Or induction factors such as dimethylaniline and the like, and whether the lignocellulose degradation enzyme activity of the strain can be improved is judged, which is to analyze the action mechanism of parasitic endophytic fungi on the degradation of host cell walls, so that theoretical basis and application guidance are provided for the breeding of the parasitic.
The number of modules and the scale of processing described herein are intended to simplify the description of the present invention. Modifications and variations to the formate-based single cell protein strains of the present invention and their use will be apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
SEQUENCE LISTING
<110> Guangxi Zhuang nationality medicinal plant garden
<120> Pelargonium sp.sp.6 derived from Pyrus annua, breeding method and use
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Claims (8)
1. Pediophora strain derived from yellow pearPestalotiopsis arceuthobii P6, the Pediopsis strainPestalotiopsis arceuthobii P6 was deposited at the Guangdong province microorganism strain collection under the accession number: GDMCC No:61995, the preservation time is: 2021, 10 and 18, the deposit unit addresses are: building 5, guangzhou City first middle road No. 100, university, no. 59, guangdong province scientific microbiological institute.
2. The breeding method of the mistletoe is characterized by comprising the following steps:
1) Taking a strain of Pelaralopecia as defined in claim 1Pestalotiopsis arceuthobiiP6 is inoculated in a culture medium for culture;
2) Harvesting the fermentation broth of the strain P6 grown in step 1);
3) And (3) planting a plurality of mistletoe seeds on each mulberry branch, and spraying the fermentation liquor obtained in the step (2) on the mulberry branches every day in the culture, wherein the dosage of the fermentation liquor is 1-10 ml/one strain at a time.
3. The method for breeding mistletoe according to claim 2 wherein in step 1), said medium is PDA liquid medium.
4. The method for breeding mistletoe according to claim 2, wherein in step 1), said culturing is: shake culturing at 28deg.C for 3 days.
5. The method for breeding mistletoe according to claim 2 wherein in step 2), the culture solution obtained by culturing in step 1) is filtered by sterile gauze and filtered by filter paper, and then centrifuged, and the supernatant is collected to obtain a fermentation broth.
6. The method for breeding mistletoe according to claim 2, wherein in step 3), the amount of said fermentation liquid is 5 ml/one at a time/one strain.
7. The method of breeding mistletoe according to claim 2, wherein in step 3), said culturing is continued for 30 days to germination of mistletoe seeds.
8. A strain of Pelaralopecia as claimed in claim 1Pestalotiopsis arceuthobii Application of P6 in laccase production, lignin and cellulose degradation and improvement of parasitic loranthus seed germination.
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| CN105263965A (en) * | 2013-03-15 | 2016-01-20 | 斯波根生物技术公司 | Fusion proteins and methods for stimulating plant growth, protecting plants, and immobilizing bacillus spores on plants |
| CN106434371A (en) * | 2016-09-29 | 2017-02-22 | 贵州医科大学 | Neopestalotiopsis zerumbet and method of using same to identify Alpinia zerumbet leaf diseases |
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