CN111418416A - Method for preventing and controlling strawberry gray mold by directionally propagating trichoderma harzianum spore powder by using apis cerana - Google Patents
Method for preventing and controlling strawberry gray mold by directionally propagating trichoderma harzianum spore powder by using apis cerana Download PDFInfo
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- CN111418416A CN111418416A CN202010181057.1A CN202010181057A CN111418416A CN 111418416 A CN111418416 A CN 111418416A CN 202010181057 A CN202010181057 A CN 202010181057A CN 111418416 A CN111418416 A CN 111418416A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/05—Fruit crops, e.g. strawberries, tomatoes or cucumbers
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Abstract
The invention discloses a method for preventing and controlling strawberry gray mold by directionally transmitting trichoderma harzianum spore powder by using apis cerana, which comprises the following process steps: 1) testing the optimal powder loading amount of the Chinese bees carrying the trichoderma harzianum spore powder; 2) testing the optimal release time of the Chinese bees carrying the trichoderma harzianum spore powder; 3) determining the optimal humidity of the Chinese bees carrying the trichoderma harzianum spore powder; 4) selection of diluent type and determination of optimal dilution factor.
Description
Technical Field
The invention belongs to the field of agricultural production, and particularly relates to a method for preventing and controlling strawberry gray mold by directionally transmitting trichoderma harzianum spore powder by using apis cerana.
Background
Strawberry gray mold (gray mold) is prepared from Botrytis cinereaBotrytis cinereaOne of the most devastating diseases of strawberries caused by infestation, untreated strawberries can cause yield losses of up to 25% and are also the main cause of losses of strawberries during storage and transport after harvesting. Botrytis cinerea is a widely-existing vegetative filamentous fungus pathogen, and often infects strawberry young fruits by colonizing parts such as flowers decayed by strawberries. At present, fungicides such as carbendazim and the like are commonly used in production to control the gray mold of strawberries, but the fungicides are not only harmful to human bodies and harmful to environmental health, but also easily cause drug resistance of botrytis cinerea. Therefore, the safe control of the gray mold of the strawberry can be carried out by using biological control means represented by biocontrol fungi.
At present, biological control of strawberry gray mold by using biocontrol fungi such as trichoderma harzianum is reported, but the biocontrol fungi such as trichoderma harzianum is applied by adopting a manual spraying method in agricultural production, but the manual spraying not only increases labor cost, pollutes strawberry fruits and causes mechanical damage, but also needs enough application times to ensure the control effect. On the other hand, because the space of the strawberries in the greenhouse in winter is closed and lacks of natural insect vectors, the strawberries are usually pollinated by bees in order to ensure fruit drop. The body surface of the bee is generally hairy, and the shape of the bee is favorable for carrying biocontrol fungal spores during pollination. Therefore, the bee can be used for carrying the biocontrol fungus preparation, and spores of the biocontrol fungus preparation are accurately released on strawberry petals in the pollination process, so that bee pollination and prevention and control of strawberry gray mold are combined, and win-win and accurate prevention and control effects are achieved.
Chinese beeApis cerana ceranaIs a unique native bee species in China, and has the advantages of strong collection ability, cold resistance, being good at utilizing sporadic honey sources and the like. The greenhouse strawberry blossoming season begins in winter, and the low temperature enables Chinese bees to be very suitable for pollinating the greenhouse strawberries. Therefore, the invention aims to utilize Chinese bees to carry trichoderma harzianum spore powder and directionally spread the trichoderma harzianum spore powder to strawberry petals, thereby achieving the purpose of preventing and treating strawberry gray mold. On the basis of toxicological test of biocontrol spore on bee, the determination of optimal powder loading quantity, optimal release time, optimal humidity, optimal diluent type and optimal dilution multiple is obtainedActively groping and developing a method for preventing and controlling strawberry gray mold by directionally transmitting trichoderma harzianum spore powder by using Chinese bees.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention aims to design and provide a technical scheme for directionally transmitting trichoderma harzianum spore powder by using apis cerana so as to control strawberry gray mold. The specific technical scheme is as follows:
a method for preventing and controlling strawberry gray mold by directionally transmitting Trichoderma harzianum spore powder by Chinese honeybees is characterized by comprising the following process steps:
1) testing the optimal powder loading amount of the Chinese bees carrying the trichoderma harzianum spore powder;
2) testing the optimal release time of the Chinese bees carrying the trichoderma harzianum spore powder;
3) determining the optimal humidity of the Chinese bees carrying the trichoderma harzianum spore powder;
4) selection of diluent type and determination of optimal dilution factor.
Further, in the step 1), the maximum value of the powder carrying is reached after the honeybees are contacted with the spore powder for about 3min, and the maximum value is about 5.35 × 107CFU/bee。
Further, in the step 2), the powder carrying amount of the bees reaches the minimum value about 5min, namely the release amount of the spore powder of trichoderma harzianum carried by the bees reaches the maximum value, and is 1.55 × 107CFU/bee。
Further, in the step 3), when the humidity of the trichoderma harzianum spore powder reaches about 60%, the quantity of spore powder carried by the bees is obviously reduced, namely 60% is the optimal humidity.
Further, in the step 4), after the flour is mixed with the trichoderma harzianum spore powder, the number of the spore powder carried by the bees is the maximum, and the powder carrying amount reaches 2.81 × 107CFU/bee, and the flour and the spore powder are mixed according to the mass ratio of 1:1, so that the flour is used as the optimal diluent for carrying the powder.
Further, the method also comprises a greenhouse strawberry oriented prevention and control strawberry gray mold verification step, which specifically comprises the following steps:
in the field test of strawberries in a greenhouse, bees can spread spore powder carried by the bees on strawberry petals while visiting the flowers, and the number of spores on the strawberry flowers visited by the bees carrying the spore powder is 3.25 × 105To 2.85 × 106Between CFU/flower, the average number of spores was 1.31 × 106CFU/flower。
The invention aims to utilize Chinese bees to carry trichoderma harzianum spore powder and directionally spread the trichoderma harzianum spore powder to strawberry petals, thereby achieving the purpose of preventing and treating strawberry gray mold. On the basis of toxicological tests of the biocontrol spores on honeybees, the optimal powder loading amount, the optimal release time, the optimal humidity, the optimal diluent type, the optimal dilution multiple and other aspects are obtained for active research, directional strawberry gray mold prevention and control verification is carried out in greenhouse strawberry fields, and a set of method for preventing and controlling strawberry gray mold by directionally propagating trichoderma harzianum spore powder by using Chinese bees is developed. The practical application of the technology in agricultural production can greatly simplify the labor intensity of spraying in artificial fields, expand the biological control means of fungal preparations, change the traditional plant disease control from ' treatment-oriented to ' prevention-oriented ', and provide a typical case for implementing precise agriculture.
Drawings
FIG. 1 is a graph showing the amount of Trichoderma harzianum spore powder carried by Apis cerana as a function of contact time;
FIG. 2 is a graph showing the amount of Trichoderma harzianum spore powder carried by Apis cerana as a function of release time;
FIG. 3 is a graph showing the variation of the amount of Trichoderma harzianum spore powder carried by Apis cerana Fabricius with humidity;
FIG. 4 is a graph of the effect of different types of diluents on the amount of spore powder carried by Chinese bees;
FIG. 5 is a graph showing the effect of different dilution ratios on the amount of powder carried by Apis cerana and the amount released;
FIG. 6 is a comparison graph of the amount of bee carried powder at different humidity before and after dilution of the flour;
FIG. 7 shows the result of bee pollination paired pollen carrying amount test of strawberries in a greenhouse.
Detailed Description
The present invention is further illustrated by the following examples.
Example 1: determining the maximum powder carrying amount of bees and the influence of the contact time of the bees and the spore powder on the powder carrying amount
In order to investigate the influence of different contact time of bees and spore powder on the number of spores carried by the bees, the flour-carrying bees with good health activity are grabbed and enter a large test tube paved with a layer of trichoderma harzianum spore powder, the bees are grabbed at 6 time points of 30s, 1min, 2min, 3min, 4min, 5min and the like respectively, the spore powder of the bees on the bodies is eluted by PBS buffer solution with the pH of 5m L =7.4, counting is carried out by a blood counting plate under an optical microscope, and each group is repeated for 5 times, and the shortest contact time of the bees and the spore powder is the shortest time when the number of the spore powder carried by the bees reaches the maximum value, namely the maximum powder carrying amount is taken as the optimal contact time of the bees and the spore powder.
As shown in FIG. 1, the bee can carry a certain amount of spore powder after contacting with the biocontrol fungal spore powder for a certain time, and the powder carrying amount of the bee shows an S-shaped ascending mode along with the approximate increase of the time, and reaches the maximum value of carrying, namely the maximum carrying amount, about 5.35 × 10 after about 3-4 min7CFU/bee. In combination with the fact that the actual time of the bees passing through the distributor is short, the bees are contacted with the spore powder for 3min to serve as the shortest time required by the bees to achieve the maximum powder carrying amount, and the time interval is uniformly adopted in subsequent experiments to carry out experiments.
Example 2: influence of maximum release amount and release time of spores on bee pollen carrying amount
After the bees are fully contacted with the spore powder for 3min, the bees are transferred into a clean test tube, the bees freely fly in an empty large test tube, the bees are clamped out for microscopic examination by eluting with 5m L PBS solution, the number of spores carried by the bees after the bees are released for different time is calculated, and the steps are repeated for 5 times at each time point.
As shown in the result of FIG. 2, the carrying capacity of Trichoderma harzianum is gradually reduced with the increase of the release time, but the Trichoderma harzianum is not completely released, i.e. biocontrol fungus spore powder remains on bees. At 5miAbout n, the minimum value of the bee powder carrying amount is reached, namely the maximum value of the release amount of the bee powder carrying trichoderma harzianum spore is 1.55 × 107CFU/bee.
Example 3: influence of humidity on bee powder carrying amount
Adjusting the temperature of an incubator to 25 ℃, adjusting the humidity to 40% + -10%, 60% + -10% and 80% + -10% in sequence, spreading the trichoderma harzianum spore powder in a culture dish with the diameter of 5cm, respectively opening the culture dish and putting the culture dish into a constant-temperature incubator overnight, spreading the spore powder at the bottom of a large test tube after the spore powder fully absorbs the moisture and reaches the required humidity, allowing bees to contact the spore powder in the large test tube for 3min, clamping the bee powder out, performing elution microscopic examination by using 5m L PBS solution, calculating the number of spores carried by the bees, and repeating the steps for 5 times in each group.
The result is shown in fig. 3, when the humidity of the trichoderma harzianum spore powder reaches 60% ± 10%, the quantity of the spore powder carried by bees is remarkably reduced, and in combination with the actual high humidity condition of the strawberry greenhouse, the fresh spore powder preparation is recommended to be replaced in time in the process of actually applying the spore powder.
Example 4: selection of diluent type and testing and determination of optimal dilution factor
4.1 selection and determination of optimal diluent type
As the spore powder of the trichoderma harzianum is tiny, the bee carried spore powder is difficult to be fully released, the flying of the bee and the safety of a honeycomb can be influenced, the waste of raw materials in the using process can be caused, and the principle of high efficiency and environmental protection is not met. Therefore, the invention adopts the diluent to dilute the spore powder and selects three powders of flour, starch and granulated sugar powder as candidate diluents.
Uniformly mixing the three diluents with the trichoderma harzianum spore powder according to the mass ratio of 1:1, spreading the mixture at the bottom of a large test tube, allowing bees to contact the large test tube for 3min, carrying out elution microscopic examination by using 5m L PBS solution, calculating the number of spores carried by the bees, repeating each diluent for three times, and selecting the diluent with the largest number of spores carried by the bees as the optimal diluent type.
As shown in FIG. 4, among the three alternative diluents, flour and HatzAfter the trichoderma spore powder is mixed, the number of spore powder carried by bees is maximum and reaches 2.81 × 107CFU/bee. We therefore chose flour as the optimal diluent type for the experiments to follow.
4.2 determination of optimal dilution multiple of Trichoderma harzianum spore powder
Setting a series of dilution ratios, namely flour: trichoderma harzianum spore powder =8:1, 4:1, 2:1, 1:2 and 1:4, and the mixture mixed in proportion is laid on the bottom of a large test tube. The experimental procedure of example 4.1 was repeated to calculate the maximum powder carrying capacity of bees at each concentration. And taking fresh bees and fully contacting the fresh bees with the spore powder for 3min, repeating the experimental steps in the example 2, and calculating the maximum release amount which can be achieved by the bees carrying the mixture of the flour and the spore powder at each concentration. The concentration gradient with the largest release amount is taken as the optimal dilution factor.
As shown in figure 5, the mixture obtained by mixing the flour and the spore powder according to the mass ratio of 1:1 enables the bees to carry the maximum number of spores within a certain time, and the number of spores reaches 2.81 × 107CFU/be and at this concentration, the number of spores released by bees in a certain time also reaches a maximum of 1.65 × 107CFU/bee. The fact that the bees can not only carry the maximum amount of spores at the concentration, but also release the spores to the maximum extent meets the purpose that the bees are used for directionally carrying the spore powder to strawberry petals, which is expected by experiments, is indicated, and therefore the ratio of the spore powder to the spore powder is determined as the ratio of flour: spore powder =1:1 ratio was diluted to the optimal dilution factor.
4.3 Effect of humidity after dilution on the amount of spores carried by bees
After the flour and the trichoderma harzianum spore powder are mixed according to the mass ratio of 1:1, the experimental step 2.4.3 is repeated to explore the influence of the humidity of the diluted flour on the number of trichoderma harzianum spores carried by bees.
The results are shown in fig. 6, before and after mixing and diluting the trichoderma harzianum spore powder in the mass ratio of flour and the like, the number of trichoderma harzianum spores carried by bees shows a descending trend along with the increase of humidity, but the trend before the reduction of the fungus carrying amount of the bees after dilution is gentler than that before dilution. Therefore, the significant influence of high humidity conditions on the bee bacteria carrying capacity can be weakened by diluting the trichoderma harzianum spore powder with the flour, so that the spore powder is more suitable for practical application in greenhouse facilities.
Example 5: test for testing effectiveness of directional propagation trichoderma harzianum spore powder of bees in field test of strawberry greenhouse
The method comprises the steps of assembling a biocontrol fungal spore distributor filled with a mixture of flour and trichoderma harzianum spore powder in a mass ratio of 1:1 at a honeycomb outlet, enabling bees to carry the spore powder when the bees exit from a nest, observing strawberry flowers visited by the bees carrying the spore powder, carefully shearing off petals from a base, and performing microscopic examination by using an elution of 5m L PBS solution to calculate the number of spores on the strawberry flowers visited by the bees.
As shown in FIG. 7, in the field test of strawberry greenhouse in this experiment, the number of spores on strawberry flowers visited by bees carrying spore powder was 3.25 × 105To 2.85 × 106Between CFU/bee, the average number of spores is 1.31 × 106CFU/flower. Therefore, the bees can smoothly carry the spore powder through the distributor, and the carried spore powder can be scattered on strawberry petals while flowers are visited, so that the effect of directionally preventing and treating strawberry gray mold is achieved.
Claims (6)
1. A method for preventing and controlling strawberry gray mold by directionally transmitting Trichoderma harzianum spore powder by Chinese honeybees is characterized by comprising the following process steps:
1) testing the optimal powder loading amount of the Chinese bees carrying the trichoderma harzianum spore powder;
2) testing the optimal release time of the Chinese bees carrying the trichoderma harzianum spore powder;
3) determining the optimal humidity of the Chinese bees carrying the trichoderma harzianum spore powder;
4) selection of diluent type and determination of optimal dilution factor.
2. The method of claim 1, wherein the directional propagation of trichoderma harzianum spore powder by apis cerana is used for prevention and controlThe strawberry gray mold method is characterized in that in the step 1), the maximum value of the powder carrying is reached after bees are contacted with spore powder for about 3min, and the maximum value is about 5.35 × 107CFU/bee。
3. The method for preventing and controlling strawberry gray mold by directionally transmitting trichoderma harzianum spore powder by using apis cerana as claimed in claim 1, wherein in the step 2), the powder carrying amount of the bees reaches the minimum value about 5min, namely the release amount of the trichoderma harzianum spore powder carried by the bees reaches the maximum value, namely 1.55 × 107CFU/bee。
4. The method for preventing and controlling strawberry gray mold by directionally transmitting trichoderma harzianum spore powder by using apis cerana as claimed in claim 1, wherein in the step 3), when the humidity of the trichoderma harzianum spore powder reaches about 60%, the quantity of spore powder carried by bees is remarkably reduced, namely 60% is the optimal humidity.
5. The method for preventing and controlling strawberry gray mold by directionally transmitting trichoderma harzianum spore powder by using apis cerana as claimed in claim 1, wherein in the step 4), the number of spore powder carried by the apis cerana is the maximum after the flour is mixed with the trichoderma harzianum spore powder, and the powder carrying amount reaches 2.81 × 107CFU/bee, and the flour and the spore powder are mixed according to the mass ratio of 1:1, so that the flour is used as the optimal diluent for carrying the powder.
6. The method for preventing and controlling strawberry gray mold by directionally transmitting trichoderma harzianum spore powder by using apis cerana as claimed in claim 1, further comprising a step of verifying directional prevention and control of strawberry gray mold of strawberries in a greenhouse, which comprises the following specific steps:
in the field test of strawberries in a greenhouse, bees can spread spore powder carried by the bees on strawberry petals while visiting the flowers, and the number of spores on the strawberry flowers visited by the bees carrying the spore powder is 3.25 × 105To 2.85 × 106Between CFU/flower, the average number of spores was 1.31 × 106CFU/flower。
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Application publication date: 20200717 |