CN115305206B - Endophyte for rapid fragrance formation of atraumatic photinia fraseri seedlings, preparation method and application thereof - Google Patents
Endophyte for rapid fragrance formation of atraumatic photinia fraseri seedlings, preparation method and application thereof Download PDFInfo
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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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Abstract
The preservation number of the endophyte for rapidly forming fragrance of the non-traumatic photinia fraseri seedling provided by the invention is as follows: CGMCC No.40065; the classification was named as pseudococoa trispora (Lasiodoplotione seudotobaromomae). The bacteria are a method for inducing rapid aroma formation under non-traumatic culture conditions and endophytes. The method overcomes the damage of traditional wound method to Phoebe ziqi. Meanwhile, based on unified strains, the quality of agilawood is comparable with that of phoenix, the meat is red brown, the fragrance is sweet, the teeth are stuck, the tongue is tingling, the primary fragrance is elegant and has flower fragrance, the fragrance is warm, thick and mellow, the penetrating power is strong, and the tail fragrance has frankincense taste; the quality is very stable.
Description
Technical Field
The invention relates to fungi and application thereof, in particular to endophyte for quickly forming fragrance of atraumatic photinia fraseri seedlings, a preparation method and application thereof.
Background
The phoenix is a finely divided category of phoenix. This grease of phoenix is red brown and looks purple, thus is named. The interior of the fiber is hollowed out, and the fiber is carbonized, so the fiber is named. The interior of the fiber is hollowed, the fiber is carbonized, the leather is thicker, the appearance is reddish brown, the outer layer is cut, and the oil in the inner layer is purple black. Most of them are small, and if they have a large volume, they are difficult to obtain. The phoenix is divided into two types, and one type of phoenix is gray purple in pulp and takes the shape of a sheet; the other meat is red-purple and takes the shape of twisted silk. The gray purple plate is very rare and can be called the absolute product in purple. The primary fragrance is elegant and has flower fragrance, five or six minutes later, the primary fragrance is changed into strong sweet and cool original fragrance which is impressive, and the tail fragrance has almond milk fragrance. The purple twisted silk phoenix has the advantages that the primary fragrance is also elegant and has flower fragrance, the fragrance is warm, thick and mellow, the fragrance is almond fragrance, the fragrance is cool, and the tail fragrance is obvious in frankincense. Such top-level notes are comparable to other classifications among the Qilan agilawood, such as Bai Jinan and Qilan, and can be said to be one of the top-level agilawood materials.
The prior research reports show that endophytes of the agilawood mainly comprise endophytes distributed in penicillium (Penicillium sp.), fusarium (Fusarium sp.), cocois (Botryodilopida sp.), fuscoporia aureobasidioides (Melanotus flavvolifers) and Trichoderma (Trichoderma sp.), wherein representative bacteria mainly comprise Fusarium longibrachiatum (Lasiodiplodia pseudotheobrome), cocois (Botryosphaeria rhodina), penicillium disease pathogenic bacteria (Penicillium italicum), fusarium solani (Fusarium solani), penicillium maculatum (Penkillium naeleagrinum), trichoderma pseudokoningii (Trichodernaa koningiopsis) and Trichoderma atroviride (Trichoderma atroviride).
Wherein the dominant heart wood bacteria at the initial stage of aroma formation are Brevibacterium paracoccus and Mycoplasma pneumophilum. The dominant bacteria of the core material in the middle period of aroma formation are as follows: rhizobium tropicalis, mycoplasma pulmonis, pseudomonas aeruginosa Pseudomonas aeruginosa, methoxybacteria Methylobacterium radiotolerans, boletus Labrtyporocalalis and Sphingomonas sp.CC-MHH0546. The dominant bacteria of the heartwood in the later stage of aroma formation are as follows: rhizobium tropicalis Rhizobium tropici, mycoplasma pulmonary Mycoplasma pulmonis polyaromatic monoas sphingamonaspolyaromacivora and methoxybacteria Methylobacterium radiotolerans.
Agilawood substance formation is reported to be closely related to microorganisms such as fungi, bacteria, and the like. The open wound of the xylem of the tree is invaded by certain kinds of fungi, bacteria, which regulate enzyme activity and stimulate signals by promoting the metabolism of xylem parenchyma cells and active cells, thereby inducing tree defensive responses and producing heartwood. The folk shannon also promotes the production and accumulation of agilawood substances by coating sugar or other sweeteners on the tree body, attracting ants, bees and other insect eating agilawood trunks, making the trunks porous, increasing the contact area with air. However, the method has a limited application range and is difficult to popularize and apply on a large scale, so that the method has not been reported in the literature.
Disclosure of Invention
The invention aims to provide a plant defense reaction of a photinia seedling caused by artificially introducing endophytes and an exciton (eibitor) of the endophytes; thereby forming fragrance. A method for realizing aroma formation of the phoenix tree seedlings under non-invasive conditions and endophytes thereof are provided.
The non-traumatic Photinia seedling provided by the invention is an endophyte (classified and named as "Russian cacao Mao" with a preservation number of CGMCC No.40065, a preservation time of 2022, 1 month and 21 days, a preservation unit of China general microbiological culture Collection center).
The preparation method of the endophyte for rapidly forming fragrance of the atraumatic phoenix seedling comprises the following steps:
1) Preparing Qilan agilawood powder;
2) Ultrasonic extracting the agilawood powder, and filtering the extracting solution to obtain endophyte mixed solution;
3) Placing the endophyte liquid mixture on a solid culture medium for culture, and selecting bacteria to obtain endophyte seed liquid;
4) Fermenting the endophyte seed liquid in a liquid fermentation tank, adding a liquid culture medium into the fermentation tank in a fed-batch mode, controlling the phosphorus concentration in the fermentation tank to be 0.03-0.06g/L in the first 5 days, and culturing for 2 days after 5 days, controlling the phosphorus source to be less than 0.01g/L to obtain endophyte fermentation liquid;
5) And diluting and centrifuging the endophyte fermentation liquid to obtain the endophyte liquid for quickly forming fragrance of the atraumatic phoenix seedling.
The preparation method of the sina agilawood powder in the step 1) comprises the following steps: planing lignum Aquilariae Resinatum produced by QINAO with flaker into slices smaller than 1 mm, sterilizing in 60mg/L ozone for 2 hr, pulverizing in aseptic environment with universal pulverizer, sieving with 0.5mm sieve, freezing in liquid nitrogen freezing grinder for 10 min, and pulverizing for 4 min to obtain pretreated QINAO heavy powder.
The formula of the solid culture medium is as follows: 40% of bran, 30% of bean cake powder, 15% of phoenix tree bark, 15% of corn niblet, 5g of sucrose, 15g of peptone and 15g of NH as additional components and inoculum size (/ kg of matrix) 4 NO 3 3.00 g、KH 2 PO 4 3 g、CaSO 4 3g、MgSO 4 1g, 10mg of vitamin PP and 500g of distilled water.
The liquid is beef peptone 8-10g, yeast powder 1.5-3g, glucose 4-6g, K 2 HPO 4 0.2-0.3g, formic acid 0.1-0.2g, caSO 4 0.5g，FeSO 4 0.05g,MgSO 4 0.05g, naCl 0.05g, sterile water 1L.
Endophytes for rapidly forming fragrance of atraumatic phoenix seedling induce rapid fragrance under the atraumatic condition of phoenix seedling; the method comprises the following steps: the photinia serrulata endophyte injection is injected into the photinia serrulata seedlings of more than one year through a special photinia serrulata endophyte injection device, and the whole plant can be fragrant after two years.
The beneficial technical effects of the invention are as follows: the invention provides a method for inducing rapid aroma formation under a non-traumatic culture condition and endophytes. The method overcomes the damage of traditional wound method to Phoebe ziqi. Meanwhile, based on unified strains, the quality of agilawood is comparable with that of phoenix, the meat is red brown, the fragrance is sweet, the teeth are stuck, the tongue is tingling, the primary fragrance is elegant and has flower fragrance, the fragrance is warm, thick and mellow, the penetrating power is strong, and the tail fragrance has frankincense taste; the quality is very stable.
Drawings
Fig. 1 shows the case of the phoenix fine branch forming fragrance for 2 months;
FIG. 2 shows the aroma formation after three months of Phoebe cheilis inoculation;
FIG. 3 shows the aroma formation after one year of Phoebe cheilis inoculation;
FIG. 4 shows the case of one year of aroma formation of the Phoebe fraxinus thin branch;
fig. 5 is a three-year finished product of the phoenix incense.
Detailed Description
EXAMPLE 1 isolation of endophytes
Planing lignum Aquilariae Resinatum produced by QINAO into slices smaller than 1 mm by a flaker, sterilizing in 60mg/L ozone for 2 hr, pulverizing in a sterile environment with a universal pulverizer, sieving with 0.5mm sieve, freezing in a liquid nitrogen freezing grinder for 10 min, and pulverizing for 4 min to obtain pretreated QINAO lignum Aquilariae Resinatum powder;
pouring the inula sinica powder into sterile water according to the weight ratio of 1:10, ultrasonically extracting for 1 hour at normal temperature by using a 40KHZ ultrasonic cleaner, centrifuging for 5 minutes at 3000 rpm, and taking supernatant to pass through a 0.22 microporous filter membrane.
Placing the microporous filtering membrane into 50 milliliters of sterile water, and performing ultrasonic treatment for 10 minutes at normal temperature by using a 40KHZ ultrasonic cleaner to obtain a clarified endophyte mixed solution;
inoculating the endophyte mixed solution on a solid microorganism culture medium for culture activation; the solid microorganism culture medium comprises 40% of bran, 30% of bean cake powder, 15% of photinia fraseri, 15% of corn nibbles, 5g of sucrose, 15g of peptone, NH4NO33.00 g of KH2PO43 g, 43g of CaSO, 41g of MgSO, 10mg of vitamin PP, 500g of distilled water and 18m of inoculum size, wherein the added components and the inoculum size (/ kg of substrate) are 5g of sucrose, and the endophyte seed liquid is obtained; the seed liquid of the endophyte is identified as lasiodulodiapeuticeobimane; see in particular the accessory: a China general microbiological culture Collection center (China general microbiological culture Collection center) microbiological culture collection report; named as 'Chengxiang purple chess No. 1'.
EXAMPLE 2 investigation of endophyte Activity
1. Preparation of endophyte injection
1g of endophyte seed liquid of Chengxiang purple chess No. 1 in the embodiment 1 and a liquid culture medium are added into a fermentation tank for culture to obtain high-density endophytes; the liquid culture medium comprises the following components: 8-10g of beef peptone, 1.5-3g of yeast powder, 4-6g of glucose, K2HPO40.2-0.3g of formic acid, 0.1-0.2g,CaSO4 o.5g,FeSO40.05g,MgSO4,0.05g,NaCl 0.05g g of sterile water and 1L of liquid culture medium are added into a fermentation tank in a fed-batch mode, the phosphorus concentration in the fermentation tank is controlled to be 0.03-0.06g/L in the first 5 days, and the phosphorus source is controlled to be less than 0.01g/L after 5 days and then 2 days of culture is performed to obtain endophyte fermentation liquor; diluting the cultured endophyte fermentation broth by 100 times, and centrifuging for 5 minutes at 3000 rpm to obtain the Photinia endophyte injection.
Application of endophyte 2-Qihuang injection
20 seedlings of Violet or more than one year old which are not infected with Penicillium, fusarium, cocois, monilinia flavescens, trichoderma and the like are selected. Injecting the endophyte injection into the photinia fraseri seedling. And constantly observe the process of fragrance, along with the time, the secondary xylem cells of the photinia fraseri grow thicken and lignify appears, the content of chromone substances in the xylem is obviously increased, along with the infection of new cells by mycelium, hyphae in old cells are decomposed completely at the same time, thereby promoting the 'defending reaction' of the photinia fraseri grow, and because the nutrient conveying channels of ducts and ray cells in the xylem of the photinia fraseri grow are blocked due to the wound, the nutrition obtained by the hyphae is reduced, the activity becomes low, the speed of infecting the new cells is slowed down, fragrance forming phenomenon can be observed from the second month, and the fragrance forming phenomenon is shown in figures 1 and 2. The aroma of the phoenix is increased in one year, and the fragrance is particularly shown in figures 3 and 4.
After two years, the 20 plants are all fragrant, and 17 of the 20 plants are fragrant; the agilawood quality is comparable with that of the phoebe nanensis, the meat quality is red brown, the fragrance is sweet, the teeth are stuck, the tongue is tingled, the primary fragrance is elegant and has flower fragrance, the fragrance is warm, thick and mellow, the penetrating power is strong, and the tail fragrance has frankincense taste; is completely consistent with the 'Chengxiang purple chess No. 1'; as shown in particular in fig. 5.
Claims (1)
1. An endophyte for enabling atraumatic phoenix seedling to quickly produce fragrance, which is characterized in that: the preservation number of the endophyte is: CGMCC No.40065; the classification name is pseudo-cocoa haircolor two spores @ Lasiodiplodia pseudotheobromae） 。
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