阅读说明：本技术 一种测定柑橘木虱虫生真菌致病力的方法 (Method for determining pathogenicity of diaphorina citri endophytic fungi ) 是由 廖咏梅 黄桂香 黄元腾吉 朱文倩 黄世炎 白铭 于 2020-11-20 设计创作，主要内容包括：本发明公开了一种测定柑橘木虱虫生真菌致病力的方法,包含以下操作步骤：(1)虫生真菌孢子悬浮液的准备；(2)柑橘木虱的准备；(3)九里香的准备；(4)虫生真菌的致病力测定。本发明针对柑橘木虱喜爱九里香嫩梢的特点,通过透明塑料杯种植九里香幼苗,将接种虫生真菌后的柑橘木虱成虫倒入杯中,柑橘木虱可自行爬行到九里香嫩梢上取食,能较长时间维持柑橘木虱的活力；同时本发明所用的塑料杯体积小,便携、独立,能更加灵活地控制试验条件并观察试验结果,为实验室条件下研究柑橘木虱提供便利。(The invention discloses a method for determining pathogenicity of diaphorina citri endophytic fungi, which comprises the following operation steps: (1) preparing entomogenous fungi spore suspension; (2) preparing diaphorina citri; (3) preparing murraya paniculata; (4) and (3) determining the pathogenicity of the entomogenous fungi. According to the method, aiming at the characteristic that the murraya paniculata young tips are favored by the diaphorina citri, murraya paniculata seedlings are planted through the transparent plastic cup, the adult diaphorina citri inoculated with entomogenous fungi is poured into the cup, and the diaphorina citri can automatically climb to the murraya paniculata young tips to eat, so that the activity of the diaphorina citri can be maintained for a long time; meanwhile, the plastic cup used in the invention has small volume, is portable and independent, can more flexibly control the test conditions and observe the test results, and provides convenience for researching the diaphorina citri under the laboratory conditions.)

1. A method for determining the pathogenicity of diaphorina citri endophytic fungi is characterized by comprising the following operation steps:

(1) preparation of entomogenous fungal spore suspension: culturing a test fungus strain on a PDA culture medium flat plate, scraping hypha and conidia, transferring into a triangular flask, pouring a Tween-80 solution, fully shaking, filtering a bacterium solution, adding the Tween-80 solution for dilution according to test requirements, or centrifugally concentrating a stock solution to obtain a conidia suspension with a target concentration, and cooling for later use;

(2) preparing diaphorina citri: planting murraya paniculata in a greenhouse, propagating diaphorina citri, capturing adult diaphorina citri, and paralyzing the adult diaphorina citri at low temperature;

(3) preparing murraya paniculata: fixing murraya paniculata seedlings with 5-7 mature leaves in a cup with holes at the bottom of the cup by using a matrix, covering light-colored absorbent paper on the surface of the matrix and compacting the light-colored absorbent paper after fixing the murraya paniculata seedlings; placing the cup on which the murraya paniculata is planted in a culture dish tray, and placing clear water or an improved Hoagland nutrient solution into the culture dish tray to obtain a murraya paniculata planting cup;

(4) pathogenicity determination of entomogenous fungi: putting ice blocks into a cup, covering the cup opening to be 0.5cm below the cup opening, covering the cup opening with gauze, pressing the gauze downwards to form a tuck net shape, pouring the citrus psyllid imagoes paralyzed at the low temperature in the step (2) into the tuck net shape gauze, spraying conidium suspension of the entomogenous fungi to be used in the step (1) onto the surface of the citrus psyllid body until the surface of the citrus psyllid body is sufficiently moistened for inoculation, spraying a Tween-80 solution as a contrast, taking out the gauze, reversely buckling the gauze on the cup opening of the murraya paniculata planting cup obtained in the step (3), fixing the gauze, placing the murraya paniculata planting cup at room temperature, removing the gauze on the cup opening after the citrus psyllid imagoes completely leave the gauze and disperse the murraya paniculata seedlings, covering the cup opening with a culture dish, covering a jack on the culture dish, maintaining the relative humidity of air in the cup to be more than 95% in a covered state, placing the murraya paniculata cup with the citrus psyllid, and in the photoperiod of 12D to 12L and in the environment with the air relative humidity of 70-85%, moving away the culture dish cover after 48h of inoculation, wrapping the cup mouth of the Murraya koenigii planting cup with gauze, observing the death condition of the adult diaphorina citri every 24h, continuously observing and recording for 10D, laying the diaphorina citri on the side when the diaphorina citri falls down or the tentacle is still marked as death after stimulation, timely picking the dead diaphorina citri for moisturizing treatment, and observing whether inoculated entomogenous fungi are generated.

2. The method of claim 1, wherein: the culture in the step (1) is carried out for 10 days in a constant temperature incubator at 27 ℃.

3. The method of claim 1, wherein: the mass concentration of the tween-80 solution in the step (1) and the step (4) is 0.1 percent.

4. The method of claim 1, wherein: and (3) placing the diaphorina citri adults in the step (2) at a temperature of 15 ℃ for low-temperature paralysis.

5. The method of claim 1, wherein: the substrate in the step (3) is perlite: the vermiculite is mixed evenly according to the volume ratio of 1:4, and the total volume is 120mL-130 mL.

6. The method of claim 1, wherein: drilling three small holes with the diameter of 0.3cm at the bottom of the plastic cup by using a red metal strip before filling the matrix in the step (3), and thus obtaining the cup with the hole at the bottom; the cup is a transparent polypropylene plastic cup with the caliber of 8cm and the height of 13.5 cm.

7. The method of claim 1, wherein: the light-colored absorbent paper in the step (3) is white absorbent paper.

Technical Field

The invention relates to the field of diaphorina citri research, in particular to a method for determining pathogenicity of diaphorina citri entomogenous fungi.

Background

Diaphorina citri (Diaphorina citri Kuwayama) belongs to the Psyllidae (Psyllidae) of the Hemiptera (Hemiptera) and has piercing-sucking mouthparts, mainly with rutaceae plants as hosts. The nymphs can damage the young shoots of the oranges, so that the young shoots are malnutritional and even withered, the nymphs can also excrete a large amount of honeydew to induce the sooty mould of the oranges, and adults can spread the citrus yellow dragon disease through flying diffusion. Citrus yellow shoot is caused by the candidate of the genus bastnaeus (libertacter), namely, the candidate of the genus libertus asiaticus (Candidatus Liberibacter asiaticus), and is an invasive disease, and the tree vigor of citrus plants infected with the yellow shoot is reduced, the yield is reduced, and even the plants die. At present, the citrus yellow shoot is mainly controlled by a three-board axe technology, namely planting disease-free seedlings, controlling citrus psyllids and cutting diseased trees. The main method for preventing and controlling the citrus psylla is to spray a chemical agent, but the long-term application of the chemical agent can easily cause the citrus psylla to generate drug resistance, so that the citrus psylla can rampant again, the ecological balance of an orchard can be damaged, the environment pollution can be caused by pesticide residue, and the like. Along with the implementation of the policies of 'double reduction' of the application amount of pesticides and fertilizers and 'green plant protection' in the ministry of agriculture of China, the biological control position of the diaphorina citri is becoming more and more obvious. Besides various animal natural enemies, various entomogenous fungi also have pathogenic effects on the diaphorina citri in nature.

Examples of entomogenous fungi which have been reported to infect psyllid citri include Paecilomyces variotii varioti, Paecilomyces fumososea, Isaria javanicus evansia, Beauveria bassiana, Metarhizium anisopliae, Hirsutella citrifolia, Erysipelothrix lecanii, Lecanicillium psilliotae, Lecanicillium acutiloba, etc. Entomogenous fungi separated from the diaphorina citri comprise body surface saprophytic bacteria, pathogenic bacteria, in-vivo symbiotic bacteria and the like, and in order to screen strains which are pathogenic to the diaphorina citri from the separated fungi, the separated fungi are required to be inoculated to the body surface of the diaphorina citri imagoes; the individual size of the diaphorina citri adults is small, so that the expression of the diaphorina citri after inoculation of entomogenous fungi is difficult to observe; meanwhile, to obtain accurate data, it is necessary to ensure that the control diaphorina citri, which is not sprayed with entomogenous fungi, remains viable, so that the diaphorina citri can remain viable only if the diaphorina citri is taken normally on a living host plant.

Murraya koenigii is a shrub or small arbor of Rutaceae, and is an important host of diaphorina citri. The citrus psylla is an ideal host for feeding citrus psylla in scientific research because of strong treetop-pulling capability and fresh and tender branches and leaves. If the isolated murraya paniculata branches are used for feeding the diaphorina citri, the murraya paniculata branches are gradually weakened in vitro, the palatability of the diaphorina citri is influenced, the diaphorina citri can die in advance due to lack of nutrition, and the accuracy of a test result is further influenced; if the murraya paniculata is potted in the insect cage to feed the diaphorina citri and then the entomogenous fungi are inoculated, the volume in the insect cage is too large, and the diaphorina citri is too small, so that the death condition of the diaphorina citri is difficult to observe and count.

Disclosure of Invention

Aiming at the technical problems, the invention provides a method for determining the pathogenicity of diaphorina citri endophytic fungi, and aims to obtain a method which is small in size, independent and portable, can more flexibly control test conditions and observe test results, and provides convenience for researching the pathogenicity of the diaphorina citri endophytic fungi under laboratory conditions.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a method for determining the pathogenicity of diaphorina citri endophytic fungi comprises the following operation steps:

(1) preparation of entomogenous fungal spore suspension: culturing a fungus strain to be tested on a PDA culture medium flat plate, scraping hypha and conidia, transferring the hypha and the conidia into a triangular flask containing a plurality of glass beads, pouring a Tween-80 solution, fully shaking, filtering the bacterial solution by using 4 layers of gauze, counting the number of the conidia in unit volume by using a blood counting plate, converting the conidia concentration of the bacterial solution, adding the Tween-80 solution for dilution according to test requirements, or centrifugally concentrating the stock solution at 4000 Xg to obtain a conidia suspension with a target concentration, and cooling the conidia suspension in a refrigerator at 4 ℃ for later use;

(2) preparing diaphorina citri: planting murraya paniculata in a greenhouse, breeding diaphorina citri, capturing diaphorina citri imagoes by using self-sealing bags, filling 30 heads of the diaphorina citri imagoes into each self-sealing bag, and placing the self-sealing bags filled with the diaphorina citri imagoes into a foam box at about 15 ℃ to cause low-temperature paralysis;

(3) preparing murraya paniculata: fixing 2 murraya jasminorage seedlings with 5-7 mature leaves in a cup with a hole at the bottom of the cup by using a substrate, covering light-color absorbent paper on the surface of the substrate after fixing the murraya jasminorage seedlings, compacting, and observing and counting the citrus psylla adults falling from the murraya jasminorage by using the color difference between the light-color absorbent paper and the brown body color of the citrus psylla adults so as to prevent the citrus psylla adults from falling into the gaps of the substrate and being difficult to be found; placing the cup on which the murraya jasminorage is planted in a disposable culture dish tray with the diameter of 9cm, placing clear water or an improved Hoagland nutrient solution into the disposable culture dish tray, infiltrating the clear water or the improved Hoagland nutrient solution into the matrix through holes in the bottom of the cup, and conducting the clear water or the improved Hoagland nutrient solution to the upper surface of the matrix through capillary action formed between the matrices to obtain the murraya jasminorage planting cup;

(4) pathogenicity determination of entomogenous fungi: putting crushed ice into a cup, covering the crushed ice to 0.5cm below the cup mouth, covering the cup mouth with a double-layer gauze, pressing the gauze downwards to form a pocket shape, pouring the citrus psylla adults paralyzed at the low temperature in the step (2) into the pocket-shaped gauze, spraying conidium suspension of the entomogenous fungi to be used in the step (1) onto the surface of the citrus psylla to fully moisten the surface of the citrus psylla, taking a sprayed Tween-80 solution as a control, quickly taking out the gauze, reversely buckling the gauze on the cup mouth of the murraya jasminorage planting cup obtained in the step (3), fixing the gauze by using a rubber band, standing at room temperature for 30min to restore the vitality of the citrus psylla adults, airing the redundant conidium suspension or the Tween-80 solution on the citrus psylla insects, and removing the gauze on the cup mouth after the citrus psylla adults completely leave the gauze and disperse into murraya jasminorage seedlings, covering a disposable culture dish with the diameter of 9cm on a cup mouth, inserting 1 round hole in the culture dish cover to keep gas exchange and maintain the relative air humidity in the cup to be more than 95% under the state with the cover, placing the murraya jasminorage planting cup with the adult citrus psylla in an environment with the temperature of 27 ℃, the photoperiod of 12D:12L and the relative air humidity of 70-85%, moving the culture dish cover away after inoculation for 48h, wrapping the cup mouth of the murraya jasminorage planting cup with two layers of gauze to ensure that the air in the cup circulates and the humidity is reduced, avoiding the normal movement of the citrus psylla due to high humidity, enabling the citrus psylla and the murraya jasminorage to normally survive, observing the death situation of the adult citrus psylla every 24h, continuously observing and recording for 10D, lying on the side with the citrus psylla falling or keeping the tentacle still after stimulation, and timely picking the dead citrus psylla to carry out moisture preservation treatment, observing whether the inoculated entomogenous fungi are produced on the body surface.

Preferably, the culture in step (1) is carried out in a 27 ℃ incubator for 10 d.

Preferably, the mass concentration of the tween-80 solution in the step (1) and the step (4) is 0.1 percent.

Preferably, the diaphorina citri adults in the step (2) are paralyzed at a low temperature of 15 ℃.

Preferably, the matrix in step (3) is perlite: the vermiculite is mixed evenly according to the volume ratio of 1:4, and the total volume is 120mL-130 mL.

Preferably, in the step (3), before filling the matrix, a red metal strip is used, three small holes with the diameter of 0.3cm are drilled at the bottom of the plastic cup for water seepage, and the cup with the hole at the bottom is obtained; the cup is a transparent polypropylene plastic cup with the caliber of 8cm and the height of 13.5 cm.

Preferably, the light-colored absorbent paper in the step (3) is white absorbent paper.

Compared with the prior art, the invention has the following beneficial effects:

according to the method, aiming at the characteristic that the murraya paniculata young tips are favored by the diaphorina citri, murraya paniculata seedlings are planted through the transparent plastic cup, the adult diaphorina citri inoculated with entomogenous fungi is poured into the cup, and the diaphorina citri can automatically climb to the murraya paniculata young tips to eat, so that the activity of the diaphorina citri can be maintained for a long time; meanwhile, the plastic cup used in the invention has small volume, is portable and independent, can more flexibly control the test conditions and observe the test results, and provides convenience for researching the diaphorina citri under the laboratory conditions.

Drawings

Fig. 1 is a picture of adult diaphorina citri after spray treatment raised by the method of example 1 of the present invention.

FIG. 2 is an observation of the method of example 1 of the present invention for feeding adult diaphorina citri in different treatment groups; wherein A is control group (CK) sprayed with 0.1% Tween-80 solution, and B is conidium suspension (concentration of 5 × 10) sprayed with Lecanicillium psalliotae LJLZJ strain⁷spores/mL), C is a conidia suspension (concentration 5X 10) of Aspergillus clavatus BQM strain, which is a spray entomogenous fungus⁷Spores per mL), D is conidium suspension (concentration of 5 × 10) of spraying entomogenous fungus purple spore⁷spores/mL).

FIG. 3 is a daily average death head number curve chart of adult diaphorina citri fed by the method of example 1 of the present invention in different treatment groups; wherein the curve BQM is Aspergillus clavatus BQM strain; the LJLZJ curve is Lecanicillium psalliotae LJLZJ strain; the DZZBM curve is a purple violet Purpureocillium lilacinum DZZBM strain; the CK curve is a control without spraying entomogenous fungi.

Detailed Description

The following detailed description is to be read in connection with the accompanying drawings, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials, reagents and strains used in the examples were all commercially available unless otherwise specified. The nutrient solution in the examples is a modified Hoagland nutrient, which is commercially available.

Obtainment of Murraya koenigii seedlings used in example 1: collecting mature Murraya koenigii seeds, culturing seedlings in a seedling culture plate by taking a nutrient medium as a carrier, covering the seeds with the nutrient medium with the thickness of 1-2 cm, keeping the medium wet before breaking the soil of the seeds, and breaking the soil after 8-10 days of sowing at the temperature of 25-30 ℃. Transplanting after the seedlings grow to 7-9 cm in height and 5-7 leaves with 1-2 tender compound leaf stalks.

Example 1

A method for determining pathogenicity of diaphorina citri endophytic fungi comprises the following specific operation steps:

(1) preparation of entomogenous fungal spore suspension: culturing strains of 3 entomogenous fungi (Lecanicillium psalliotae, Purpureocillium lilacinum and Aspergillus clavatus) to be tested on a PDA culture medium flat plate in a constant temperature incubator at 27 ℃ for 10 days, scraping hyphae and conidia, transferring the hyphae and the conidia into a triangular flask containing a plurality of glass beads, pouring 100mL of Tween-80 solution with the mass concentration of 0.1%, fully shaking, filtering the bacterial liquid by 4 layers of gauze, counting the number of the conidia in unit volume by using a blood counting plate, converting the conidia concentration of the bacterial liquid, adding a proper amount of Tween-80 solution with the mass concentration of 0.1% according to test requirements, diluting to obtain the conidia concentration of 5 multiplied by 10⁷Cooling the conidium suspension at 4 ℃ in a refrigerator for later use;

(2) preparing diaphorina citri: planting murraya paniculata in a greenhouse, breeding diaphorina citri, capturing diaphorina citri adults by using self-sealing bags, filling 30 heads of diaphorina citri adults into each self-sealing bag, and placing the self-sealing bags filled with the diaphorina citri adults into a foam box filled with ice blocks and having the temperature of about 15 ℃ to paralyze the bags at low temperature;

(3) preparing murraya paniculata: fixing the 2 murraya jasminorage seedlings with 5-7 mature leaves in a transparent polypropylene plastic cup with the caliber of 8cm and the height of 13.5cm by using a matrix (formed by uniformly mixing perlite and vermiculite in a volume ratio of 1: 4), drilling three small holes with the diameter of 0.3cm at the bottom of the plastic cup for water seepage by using a red burning metal strip before filling the matrix, covering white absorbent paper on the surface of the matrix after fixing the murraya jasminorage seedlings and compacting the white absorbent paper, observing and counting the citrus psylla adults falling from the murraya jasminorage by using the color difference of the brown body colors of the white absorbent paper and the citrus psylla adults, and preventing the citrus psylla adults from falling into gaps of the matrix and being difficult to find; placing the plastic cup on which the murraya paniculata is planted in a disposable culture dish tray with the diameter of 9cm, placing clear water or an improved version of hoagland nutrient solution into the disposable culture dish tray (the nutrient solution is added into the culture dish tray at the bottom of the murraya paniculata planting cup, and can provide nutrients and water for the murraya paniculata through small holes at the bottom of the cup and capillary action of a matrix, so that the murraya paniculata can be ensured to grow healthily in the plastic cup, and the problem that the diaphorina paniculata is watered by opening the cup mouth or the nutrient solution causes escape of adult diaphorina citri is avoided), infiltrating the nutrient solution into the matrix through the small holes at the bottom of the transparent planting cup, and conducting the nutrient solution to the upper surface of the matrix through;

(4) pathogenicity determination of entomogenous fungi: placing crushed ice into a plastic cup with the caliber of 8cm and the depth of 13.5cm, covering the crushed ice to 0.5cm below the cup mouth, covering the cup mouth with double-layer gauze, pressing the gauze downwards to form a tuck net shape, pouring the low-temperature paralyzed adult citrus psylla in a foam box filled with ice and at the temperature of about 15 ℃ into the tuck net-shaped gauze (the citrus psylla is subjected to low-temperature paralysis in the foam box filled with ice and at the temperature of about 15 ℃, inoculating entomogenous fungi on the ice, the temperature change of the adult citrus psylla has a gradual process, the citrus psylla is easy to restore vitality), spraying a handheld spray bottle on the conidia suspension of the entomogenous fungi to be used in the step (1) to the body surface of the citrus psylla to be fully moistened and inoculated, spraying a Tween-80 solution with the mass concentration of 0.1% as a control, quickly taking out the gauze and reversely covering the cup mouth of the nine-mile cup obtained in the step (3) after the spraying treatment is finished, fixing gauze with rubber band, standing at room temperature for 30min to restore activity of diaphorina citri imago, air drying excessive conidium suspension or Tween-80 solution on diaphorina citri, removing gauze on the cup mouth after diaphorina citri imago completely leaves gauze and disperses onto Murraya koenigii seedling, covering the cup mouth with disposable culture dish with diameter of 9cm, inserting 1 round hole in the center of the culture dish cover to keep gas exchange and maintain relative air humidity in the cup at more than 95% under the state of cover, placing Murraya koenigii planting cup containing diaphorina citri imago in a constant temperature illumination incubator with temperature of 27 deg.C, photoperiod 12D:12L and relative air humidity of 70% -85% (as shown in figure 1), removing culture dish cover after inoculation for 48h, changing to the cup mouth of Murraya koenigii planting cup with two layers of gauze to circulate air in the cup and reduce humidity, the normal activity of the diaphorina citri is prevented from being influenced by high humidity, the diaphorina citri and the murraya paniculata can normally survive, the death condition of the adult diaphorina citri is observed every 24 hours, the diaphorina citri is continuously observed and recorded for 10 days, the death condition is recorded by the fact that the diaphorina citri falls off and lies on the side or the tentacle is still after stimulation (as shown in figure 2), the dead diaphorina citri is timely picked for moisture preservation, and whether inoculated entomogenous fungi are generated on the body surface or not is observed. The results are shown in Table 1, and the daily mortality of diaphorina citri after inoculation with entomogenous fungi is shown in FIG. 3. One treatment was repeated three times, and one was repeated for 30 citrus psyllids.

TABLE 1 determination of the virulence of the three Pediculus citri parasites on Pediculus citri

Calculating the difference significance of different treatments under the significance level P of 0.05 by using a new double-polarization difference method, wherein the difference of the same treatment with letters is not significant, and the difference of the different treatments with letters is significant.

2. The calculated formula for correcting mortality is:

corrected mortality (%) - (treatment mortality-control mortality)/(1-control mortality) ] × 100

As can be seen from the table 1, the virulence of different kinds of entomogenous fungi to the diaphorina citri can be effectively measured, after 10 days of treatment, the diaphorina citri of a control group which is not sprayed with the entomogenous fungi still maintains high survival rate, the average death rate is only 4.44 percent, and the requirement that the death rate of the control group in a conventional virulence test is not higher than 20 percent is met; the pathogenicity difference of different entomogenous fungi is obvious, wherein the pathogenicity of Lecanicillium psallonoid of Ericerus lecanii is strong, the average death rate of 4d after inoculation reaches 100%, the pathogenicity of Purpureocillium lilacinum is medium, the average death rate of 10d after inoculation is 44.33%, the pathogenicity of Aspergillus clavatus is zero, and the average death rate of 10d after inoculation is only 2.22%.

The transparent plastic cup used in the method has small volume, is portable and independent, and can easily control test conditions such as temperature, illumination and the like; the white absorbent paper is laid on the fixed substrate, so that the diaphorina citri can be prevented from entering the substrate, and meanwhile, the color difference can be formed between the diaphorina citri and the transparent cup body, and the diaphorina citri in the cup can be observed conveniently; the small holes are drilled at the bottom of the cup, clear water or nutrient solution is added into the tray of the culture dish, and the aims of avoiding tearing off gauze, adding water and fertilizer and avoiding the diaphorina citri from escaping can be realized by utilizing the capillary action of the holes between the matrixes.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.