CN110616160B

CN110616160B - Hansenula anomala SY1805 and application thereof in preparation of mulberry stem borer attractant

Info

Publication number: CN110616160B
Application number: CN201910879386.0A
Authority: CN
Inventors: 贺福平; 吴建华
Original assignee: Beijing Engeland Environmental Technology Co ltd
Current assignee: Beijing Engeland Environmental Technology Co ltd
Priority date: 2019-09-18
Filing date: 2019-09-18
Publication date: 2020-11-13
Anticipated expiration: 2039-09-18
Also published as: CN110616160A

Abstract

The invention belongs to the field of fruit tree pest attractants, particularly relates to abnormal hansenula polymorpha SY1805 and application thereof in preparation of a mulberry stem borer attractant, and particularly discloses abnormal hansenula polymorpha (Hansenula anomala) SY1805 with a preservation number of CCTCC NO: and M2019719. The wort fermentation liquor of the strain has an attraction effect on the mulberry borers, and the mixed fermentation liquor added with the mulberry juice has a better attraction effect. The invention also provides a multocida attractant which is prepared by mixing ethanol, acetic acid, white granulated sugar, high fructose corn syrup, anhydrous calcium sulfate and hansenula anomala fermentation broth and can be used for green prevention and control of multocida.

Description

Hansenula anomala SY1805 and application thereof in preparation of mulberry stem borer attractant

Technical Field

The invention belongs to the field of fruit tree pest attractants, and particularly relates to Hansenula anomala SY1805 and application thereof in preparation of a mulberry stem borer attractant.

Background

The mulberry is an important perennial economic crop in China, the cultivation range is wide, and the northeast is south of Harbin; northwest to Xinjiang, Qinghai; south to two guang, east to taiwan; the distribution is from west to Sichuan and Yunnan; and most plants are cultivated in various places in the middle and lower reaches of the Yangtze river. The mulberry has wide application, and the mulberries, the mulberry branches, the mulberry leaves and the white mulberry root-bark all contain special medicinal ingredients, so that the mulberry has better medicinal effect. The mulberry leaves are the only feed source of silkworms, and the quality and the yield of the mulberry leaves are decisive factors influencing the development of the sericulture and are also main restriction factors of the development of silk industry in China.

The mulberry borer is one of the important pests of the mulberry, and the larva thereof invades mulberry leaves in summer and autumn, so that the larva is most harmful to the mulberry leaves in late autumn, the yield of the mulberry leaves is seriously reduced, the quality of the mulberry leaves is deteriorated, and the silkworm industry production is further influenced. The mulberry borer is threshed and studded into rolled leaves or folded leaves by larvae in summer and autumn, the larvae hide the chewing mesophyll, residual veins and upper epidermis to form transparent gray brown films, and then the transparent gray brown films are broken to form holes, which are called as 'skylight opening'. The excrement pollutes the leaves and affects the quality of the mulberry leaves. The insect can cause the mulberry leaf withered and yellow in 9-10 months, and affects silkworm feeding in autumn or causes silkworm diseases.

The prevention and control of the multocida is mainly based on pesticide prevention and control at present, and has the advantages of convenient use, low cost, quick response and the like. But a large number of beneficial natural enemy insects are killed while pests are killed, and after the pesticide is used for many times, the mulberry borers generate drug resistance, so that the mulberry borers are easy to outbreak into disasters, in addition, the ecological environment of the mulberry field is also polluted by the pesticide, and the safety of the leaf for silkworm breeding is influenced. Therefore, the growth characteristics of the mulberry trees, the silkworm breeding layout and the driving characteristics of the mulberry stem borers are combined, the non-chemical pesticide control technologies such as trapping and killing by a solar insecticidal lamp, sticking plates, attractants, radiation sterility, releasing natural enemy insects and the like are adopted to control the mulberry stem borers, and the method has great significance for improving the control effect of the mulberry stem borers, protecting ecological balance and reducing environmental pollution.

The food attractant developed based on the preference of pests to food sources or volatiles thereof is an important green pest-proof product. With the continuous and deep understanding of information substances in the insect food-borne volatile matters, a food attractant (also called as a plant-borne attractant) which is a natural and artificially-synthesized volatile matter component is combined to develop a green pest prevention and control product for various female and male pests, and the food attractant plays an important role in the prevention and control of various major pests such as fruit flies, noctuids, beetles, thrips and the like at present and becomes an important component in a comprehensive prevention and control technical system for the pests. Meanwhile, the food attractant has the characteristics of wide source, easy material acquisition, low cost and the like, and is easy to popularize and apply. Therefore, the attractant with food-luring effect on the mulworms can be developed, and the purpose of green prevention and control is achieved.

The invention patent applications 201811440146.2 and 201811440132.0 disclose two mulberry stem borer attractant formulas, wherein the main attractants are camphor, star anise, sugar and other substances, and are compounded with other plant-derived bacteriostatic and insecticidal components to achieve the effect of trapping and killing the mulberry stem borers. However, these attractants are conventional materials, have poor specificity, and are relatively complicated in extraction and preparation processes. In the preliminary report of experiments on the biological food attractant Ku Chi Ling for preventing and controlling mulberry borers, Ku Chi Ling mainly uses sugar, aromatic alcohol, caryophyllene and the like to lure the mulberry borers, has trapping and killing effects on male and female adults, but has relatively single component.

Disclosure of Invention

The invention aims to provide a Hansenula anomala SY1805 strain, wherein the Hansenula anomala SY1805 strain is the Hansenula anomala SY1805 strain, and the preservation number is CCTCC NO: and M2019719.

Another objective of the invention is to provide application of a Hansenula anomala SY1805 strain.

The last purpose of the invention is to provide a multocida attractant which comprises fermentation liquor of SY1805 of Hansenula polymorpha, high fructose corn syrup, ethanol, acetic acid and white granulated sugar. The attractant has good attracting effect on the mulberry stem borers, and has good application prospect in the prevention and control of the mulberry stem borers in mulberry fields.

In order to achieve the purpose, the invention adopts the following technical measures:

the applicant obtains a strain of hansenula anomala by separating and screening rhizosphere soil of a certain mulberry field in Ningnan county of Liangshan mountain, Sichuan province, the mixed fermentation liquor of wheat juice and mulberry juice of the yeast has an attraction effect on the mulberry borers, the strain is delivered to a China center for type culture collection for collection in 2019, 9, 12 days, and is classified and named: hansenula anomala SY1805, accession number: CCTCC NO: m2019719, address: wuhan university in Wuhan, China.

The colony formed by the Hansenula anomala propagating and growing on the PDA culture medium is round, milky white, smooth in surface, viscous and flat in edge; microscopic examination shows that the strain is spherical or ellipsoidal, has size of 3.0-4.2 (x 3.0-5.0) μm, produces 1-4 ascospores, and cannot form pseudohypha; glucose, galactose, maltose and sucrose can be fermented, and lactose, xylose and arabinose can not be fermented; assimilable are raffinose, galactose, maltose, sucrose, soluble starch, and assimilable are lactose, xylose, arabinose, citric acid, arabinose, and trehalose; nitrate and ammonium salts can be assimilated; can be grown on a vitamin-free medium; cannot grow at 37 ℃; resistant to 14% ethanol.

Application of Hansenula anomala SY1805 comprises preparing mulberry borer attractant from the yeast or fermentation liquid of the yeast;

in the above application, preferably, the fermentation liquid is prepared by fermenting a mixed liquid of wort and mulberry juice with Hansenula anomala SY 1805;

in the above application, preferably, the fermentation liquid is prepared by fermenting a mixed liquid of 5-10 parts by volume of wort and 0.1-1 part by volume of mulberry juice with 0.1-0.3 part by volume of fermentation liquid of Hansenula anomala SY 1805;

in the above application, preferably, when the attractant is prepared, besides the fermentation liquor, the attractant also comprises ethanol, acetic acid, white granulated sugar and high fructose corn syrup;

in the above application, preferably, the main effective components of the attractant are as follows: 5-10 parts of ethanol, 2-5 parts of acetic acid, 10-20 parts of white granulated sugar, 1-10 parts of high fructose corn syrup, 20-40 parts of anhydrous calcium sulfate and 50-100 parts of hansenula anomala fermentation broth, wherein the above parts are in parts by weight;

in the above applications, most preferably, the main effective components of the attractant are as follows: 7.5 parts of ethanol, 2.5 parts of acetic acid, 15 parts of white granulated sugar, 5 parts of high fructose corn syrup, 20 parts of anhydrous calcium sulfate and 100 parts of hansenula anomala fermentation broth, wherein the above parts are all parts by weight.

Compared with the prior art, the invention has the following characteristics:

1) the invention provides a Hansenula anomala SY1805 for the first time, and the fermented mixed solution of the wheat juice and the mulberry juice has a strong attraction effect on the mulberry borers and can be used for green biological control of the mulberry borers.

2) The mulberry borer attractant provided by the invention is simple to prepare, non-toxic and harmless, and safe and convenient to use.

3) The invention takes the mixed fermentation liquor of the aroma-producing yeast as the main food attractant, and is supplemented with syrup and other auxiliary materials, and various mulberry-derived substances and rich volatile components contained in the mixed fermentation liquor have strong attractant to mulberry borer, long-lasting effect, simple preparation process, low cost and good application prospect.

Drawings

FIG. 1 is a colony morphology of a SY1805 plate of Hansenula anomala.

FIG. 2 is a morphological diagram of SY1805 of Hansenula anomala as a microscopic examination.

FIG. 3 is a view showing a Y-type olfactometer.

FIG. 4 is a gas chromatogram of a fermentation broth of SY1805, Hansenula anomala.

Detailed Description

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.

Example 1:

acquisition of Hansenula anomala SY 1805:

screening of yeast strains:

1. preparation of a culture medium:

YPD medium, peptone 20 g; 10g of yeast extract powder; 20g of glucose; 1000mL of distilled water; the pH value is 6.5.

PDA culture medium: 200g of potatoes; 20g of glucose; 1000ml of distilled water; the pH is natural.

Solid medium 1.5% agar was added based on liquid medium and all media were sterilized at 115 ℃ for 20 min.

2. Primary screening: taking rhizosphere soil and putrefactive leaf samples in a certain mulberry field in Ning-nan county of Liangshan, Sichuan province, taking 10g of the samples, adding the samples into a triangular flask containing 100mL of sterile normal saline, and oscillating for 30min at 200r/min at 30 ℃; the bacterial suspension was serially diluted with sterile physiological saline, and the appropriate dilution was selected, applied to YPD plates (supplemented with ampicillin), and cultured at 30 ℃ for 2-3d to observe colony morphology. Colonies of the suspected yeast were selected and streaked out using YPD plates and cultured at 30 ℃ for 48 hours to obtain 15 strains of yeast.

3. Re-screening: the 15 strains obtained from the preliminary screening were rescreened with wort. 50ml of wort is filled into a triangular flask of 250ml, sterilized at 115 ℃ for 20min, cooled, inoculated with a ring strain from a primary sieve plate, and fermented at 30 ℃ for 100r/min for 72h to obtain wort fermentation liquor.

The preparation method of the wort comprises the following steps: crushing 30g of barley malt, adding the crushed barley malt into 100g of warm water at 60 ℃, carrying out water bath at 60 ℃ for 1 hour, and stirring once at 10 min; then heating to 80 ℃ and maintaining for 30min, and stirring once in 10 min; and finally, filtering the wort by gauze to obtain the required wort, wherein the sugar degree is about 15 degrees Brix.

The repellence of the mulberry borer to different wort fermentation liquids is detected by a Y-type olfaction tester (figure 3, is a commercial product), so that a yeast with stronger attraction to the mulberry borer by the wort fermentation liquid is selected, the attraction rate to the mulberry borer is about 65 percent, and the attraction rates of female and male are similar.

The procedure of measuring the attraction rate by the Y-type olfactometer is as follows:

dripping 0.1ml of wort fermentation liquor of different strains on a filter paper strip, and plugging a short arm of a Y-type olfaction tester as an odor source; the other arm is dripped with the same amount of sterile culture solution as a blank odor control; the test insects are adults which are eclosion for about 2 days, the adults are placed in the long arms one by one in a male-female mode, the selection of the test insects is observed and recorded after 15min, and the experiment is carried out in a dark environment, so that the influence of the selective tropism of the test insects caused by the difference of the internal illumination of the Y-shaped tube is avoided. The experimental environment temperature was 25-27 deg.C, 50 replicates for each scent male and female.

After 5 adults per experiment, the Y-shaped tube was cleaned with absolute ethanol and dried, and the positions of the two short arms were changed to eliminate systematic errors.

Identification of the yeast strains:

the screened strains are subjected to morphological observation, biochemical characteristic test, growth characteristic test and DNA identification according to a fungal identification manual, and morphological characteristics are as follows: the colony formed by the propagation and growth on the PDA culture medium is round, milky white, smooth in surface, sticky and flat in edge; microscopic thallus is spherical or ellipsoidal, has size of 3.0-4.2 (x 3.0-5.0) μm, produces 1-4 ascospores, and cannot form pseudohypha. The biochemical characteristics are as follows: glucose, galactose, maltose and sucrose can be fermented, and lactose, xylose and arabinose can not be fermented; assimilable are raffinose, galactose, maltose, sucrose, soluble starch, and assimilable are lactose, xylose, arabinose, citric acid, arabinose, and trehalose; nitrate and ammonium salts can be assimilated; can be grown on a vitamin-free medium; cannot grow at 37 ℃; resistant to 14% ethanol.

Extracting a yeast genome, carrying out 26SrDNA D1/D2 region amplification sequence, then sequencing and comparing the sequences on GeneBank, wherein the similarity with the Hansenula anomala is the highest and reaches 99 percent, and the strain can be determined to be the Hansenula anomala.

The strain is sent to China center for type culture Collection in 2019, 9, 12 and is classified and named: hansenula anomala SY1805, accession number: CCTCC NO: m2019719, address: wuhan university in Wuhan, China.

Example 2:

analysis of the components of the fermented wort and mulberry juice of the Hansenula anomala SY 1805:

wort having a sugar degree of 15 ° Brix was prepared as described in example 1, and 20% (V/V) by volume of the wort was added to fresh mulberry juice (squeezed directly by a juicer and filtered without adding water), and 5% by volume of the wort was inoculated to fermentation broth of SY1805 (YPD liquid medium fermentation culture, OD 1805) of Hansenula polymorpha₆₀₀5.0), then fermenting for 72 hours at the temperature of 30 ℃ and the speed of 100r/min to obtain fermentation liquor.

The other group is directly fermented with wort without adding mulberry juice, and neither group of liquid needs sterilization. The mutagenicity of the two fermentation broths to mulberry borer was tested with the Y-type olfactometer described in example 1.

The result shows that the attraction rate of the fermentation liquor containing the mulberry juice to the mulberry borers is up to 75 percent and is higher than 67 percent of the fermentation liquor not containing the mulberry juice. The result shows that the wort fermentation liquor added with the mulberry juice has stronger luring property on the mulberry borers.

Example 3:

analysis of volatile components of a mixed fermentation liquid of a Hansenula anomala SY1805 wort and mulberry juice:

the odor of the two fermentation broths of example 2 was identified by gas chromatography-mass spectrometry (GC-MS) in combination. The gas chromatography was an Agilent 6890A, the mass spectrum was 5975C, DB-5 column (30 m.times.0.25 mm. times.0.25 μm). The temperature rising procedure is as follows: maintaining the furnace temperature at 40 deg.C for 1min, then heating to 120 deg.C at 5 deg.C/min, and maintaining for 2 min; then the temperature is raised to 250 ℃ at the speed of 10 ℃/min and maintained for 2 min. Mass transfer line temperature 250 ℃, ion source temperature 230 ℃, ion flux 70e V; the scanning range is 50-300 m/z.

Table 1 shows: 50 volatile compounds are detected from 2 fermentation liquid volatile components, wherein 21 esters, 4 alcohols, 4 acids, 2 ketones, 7 aldehydes, 2 phenols and 10 other compounds are detected.

The fermentation liquor mixed by the wort and the mulberry juice detects 36 aroma components comprising 18 esters, 5 aldehydes, 2 alcohols, 2 ketones, 2 olefins and 7 other types, and the relative contents are 29.667%, 6.435%, 5.064%, 0.826%, 0.714% and 26.190%, respectively. The aroma components with high relative content comprise ethyl benzoate (8.152%), ethyl caproate (6.126%), isoamyl alcohol (4.832%), phenylacetaldehyde (4.789%), ethyl caprylate (4.189%), ethyl phenylpropionate (3.360%) and the like.

35 kinds of aroma components including 17 kinds of esters, 5 kinds of aldehydes, 2 kinds of alcohols, 2 kinds of ketones, 2 kinds of olefins, 2 kinds of acids and other 5 kinds are detected in the wort fermentation liquor, and the relative contents are respectively ester 31.526%, aldehyde 9.462%, alcohol 3.978%, ketone 0.6535%, olefin 0.773%, acid 0.585% and other 8.136%. The components with higher relative contents comprise phenylacetaldehyde (4.832%), ethyl benzoate (8.436%), ethyl laurate (8.095%), isoamyl alcohol (7.865%), ethyl 3-phenylpropionate (3.454%), benzaldehyde (3.326%), ethyl caprylate (2.310%), ethyl caproate (2.167%), ethyl caprate (2.039%) and the like.

TABLE 1 analysis of the volatile constituents of two fermentation broths

Example 4:

two mulberry borer attractants and application effects of the mulberry field:

experimental groups: 7.5 parts of ethanol, 2.5 parts of acetic acid, 15 parts of white granulated sugar, 5 parts of high fructose corn syrup, 20 parts of anhydrous calcium sulfate and 100 parts of fermentation liquor of SY1805 which is Hansenula polymorpha, wherein the above parts are all parts by weight.

Control group: 7.5 parts of ethanol, 2.5 parts of acetic acid, 15 parts of white granulated sugar, 5 parts of high fructose corn syrup, 20 parts of anhydrous calcium sulfate and 100 parts of sterile distilled water.

The hansenula anomala fermentation broth in the experimental group was: mixing 5 volume parts of wort and 1 volume part of mulberry juice, inoculating 0.25 volume part of YPD fermentation liquor of SY1805 of Hansenula polymorpha, and fermenting at 30 ℃ for 72 hours.

The two attractants are prepared into paste according to the formula, the paste is placed into a purchased trap, 5g of 10% wettable powder of methomyl and 2-3 times of tap water are added and uniformly mixed, the trap is placed in a 10-mu mulberry field in Ningnan county of Liangshan, Sichuan province, and 5 attractants are arranged in each group. And (4) counting the number of the mulberry borers in the trapper for 1 time every 3d, recording results, supplementing tap water, changing the attractant once a week, and carrying out an experimental period of one month.

The experimental results are shown in table 2, the number of the mulberry borers trapped by the experimental group is obviously higher than that of the control group, and the continuous effect is better. Along with the experiment, the number of the mulberry borers in the mulberry field is gradually reduced, and the number of the mulberry borers caught by each trap is also gradually reduced. In general, the attractant prepared by the experimental group formula has better trapping and killing effect on the field mulberry stem borers and has popularization and application prospects.

Table 2 two attractants luring and killing of mulworms in mulberry field (trap/trap)

Claims

1. An isolated strain of Hansenula anomala, wherein the Hansenula anomala is Hansenula anomala (Hansenula anomala)) (Hansenula anomala ) SY1805, preservation number is CCTCC NO: and M2019719.

2. The use of Hansenula anomala or its fermentation broth as claimed in claim 1 for preparing an attractant for mulberry borer.

3. The use of claim 2, wherein the fermentation broth is obtained from Hansenula anomala (Hansenula polymorpha) ((III))Hansenula anomala) SY1805 is prepared by fermenting the mixture of succus Hordei Germinatus and Mori fructus juice.

4. The use of claim 2, wherein the fermentation broth is prepared from 0.1-0.3 parts by volume of Hansenula anomala (Hansenula polymorpha)) (Hansenula anomala) SY1805 fermentation liquid, which is prepared by fermenting a mixture of 5-10 parts by volume of wort and 0.1-1 part by volume of mulberry juice.

5. The use of claim 2, wherein the attractant comprises Hansenula anomala (Hansenula polymorpha)Hansenula anomala) SY1805 fermenting liquid, ethanol, acetic acid, white sugar and high fructose syrup.

6. The use of claim 5, the attractant comprising: 5-10 parts of ethanol, 2-5 parts of acetic acid, 10-20 parts of white granulated sugar, 1-10 parts of high fructose corn syrup, 20-40 parts of anhydrous calcium sulfate and 50-100 parts of hansenula anomala fermentation broth, wherein the above parts are in parts by weight.

7. The use of claim 6, wherein the attractant comprises: 7.5 parts of ethanol, 2.5 parts of acetic acid, 15 parts of white granulated sugar, 5 parts of high fructose corn syrup, 20 parts of anhydrous calcium sulfate and 100 parts of hansenula anomala fermentation broth, wherein the above parts are all parts by weight.