CN110982872B - Inoculation auxiliary agent for promoting wheat root rot infection - Google Patents
Inoculation auxiliary agent for promoting wheat root rot infection Download PDFInfo
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Abstract
The invention provides an inoculating auxiliary agent for promoting wheat root rot infection, which comprises the following raw materials in parts by weight: wheat leaf powder 1-50 wt%; cellulase 0.01-0.2 wt%; lignin enzyme 0.01-0.1 wt%; microcrystalline wax 1-5 wt%; 0.05-0.5 wt% of surfactant; 0.25-1 wt% of lubricant; 1-5% wt% of adhesive; the filling agent is added to 100 percent. The inoculation auxiliary agent provided by the invention is beneficial to the infection incidence rate of pathogenic bacteria of the wheat root rot, water is not needed when the inoculation auxiliary agent is used, the yield reduction of wheat caused by other non-disease factors is prevented, and the conditions of falling off and uneven inoculation of an inoculation microbial inoculum can be avoided; the inoculation auxiliary agent and the strain can be preserved at low temperature after being mixed into an inoculation microbial inoculum, and the inoculation auxiliary agent and the strain have little influence on the activity of pathogenic bacteria. The inoculating auxiliary agent is suitable for screening and identifying the resistance of wheat varieties to root rot, and is beneficial to reducing the workload.
Description
Technical Field
The invention belongs to the field of agriculture, and relates to an inoculating aid for promoting wheat root rot infection.
Background
Wheat root rot (white root rot), also known as root rot leaf spot or black embryo, is caused by various parasitic bacteria and saprophytic bacteria such as vermiculella longa, fusarium and the like, can occur in the whole growth period of wheat, can harm the root and stem parts of the wheat to cause root rot, can harm the leaf parts of the wheat to cause leaf spots, can also harm the wheat seeds to cause black embryos. The root rot is extremely widely distributed and occurs in wheat planting countries, and belongs to worldwide diseases. China mainly occurs in northeast, northwest, north China, inner Mongolia and other areas, and is continuously expanded in recent years, and Guangdong and Fujian wheat areas are also found. In recent years, wheat root rot is aggravated year by year in Huang-Huai wheat areas due to straw returning and other reasons. The investigation result shows that the incidence rate of the wheat root rot general disease field is 1% -5%, the incidence rate of the heavier disease field is about 10%, and the seed bacteria carrying rate is higher. The root rot of wheat has a great influence on the wheat yield, and is generally reduced by 10% -30% and the serious land mass is reduced by 30% -70%. Therefore, wheat root rot has become an important issue in wheat production.
The utilization of disease-resistant varieties is one of the main measures for preventing and treating wheat root rot. Research on root rot resistance of wheat varieties at home and abroad jointly shows that most of the production-promoted blind and purchased varieties show disease or high sense, and only a small amount of the varieties belong to disease-resistant varieties and high-resistance varieties are few. Therefore, screening and identifying resistance of wheat varieties becomes an important issue in wheat breeding.
At present, the dominant pathogen of wheat root rot in Jiangsu, anhui, shandong, henan and Hebei is wheat root rot and Helminthosporum graminearumBipolaris sorokiniana). The common inoculation mode is puncture inoculation at seedling stage or spraying bacterial suspension for 3-4 times at flowering stage of wheat. Although the inoculation rate of puncture inoculation is high, the resistance of wheat in actual production cannot be simulated, and the method is only suitable for indoor research; and the evaluation difference of the disease level is also larger. The flowering period of wheat is short, only 3-4 days, and repeated spraying of bacterial suspension is easy to cause excessive humidity, excessive swelling and cracking of pollen grains due to water absorption, influence fertilization, lead to yield reduction and influence on resistance judgment. The surfaces of the parts such as the wheat leaves are provided with wax layers or fuzz, so that the wheat leaves have a strong water-repellent effect, and bacterial suspension is gathered and rolled off; when the spore suspension is actually used, the spore suspension gradually subsides along with the extension of time, and the poor inoculation effect or uneven inoculation can be caused, so that the statistics of the morbidity is affected. Therefore, developing an inoculating aid that promotes the onset of wheat root rot is of great importance in screening and identifying wheat varieties for resistance to root rot.
Disclosure of Invention
Aiming at the problems of uneven inoculation and low morbidity of the existing wheat root rot, the invention provides an inoculation auxiliary agent for promoting the infection of the wheat root rot, which can enhance the inoculation efficiency of artificially inoculating the wheat root rot.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
An inoculating auxiliary agent for promoting wheat root rot infection comprises the following raw materials in parts by weight:
。
the wheat leaf is a seedling stage wheat leaf.
The cellulase activity is not less than 1U/mg; the lignin enzyme activity is not less than 0.1U/mg.
The surfactant is a nonionic surfactant; preferably at least one of sorbitan fatty acid ester and laurinol polyoxyethylene ether.
The filler is one or more selected from talcum powder, kaolin, mica powder, white carbon black, starch or titanium dioxide. Preferably a combination of talc and titanium dioxide.
The lubricant is one or more selected from magnesium stearate or zinc stearate.
The binder is selected from one or more of sugar powder and polyethylene glycol. Preferably, the sugar is selected from glucose and/or sucrose. The average relative molecular mass of the polyethylene glycol is 1000-2000.
Preferably, the inoculation auxiliary agent for promoting the root rot infection of the wheat comprises the following raw materials in parts by weight:
。
the fineness of the inoculating auxiliary agent is 200-250 meshes.
Preferably, the inoculating aid further comprises a colorant; the content of the colorant is 0.001-0.1wt%.
The inoculation auxiliary agent can be prepared by directly mixing raw materials; preferably, the preparation method comprises the following steps:
(1) Uniformly mixing wheat leaf powder, cellulase and lignin enzyme, and adding a small amount of lubricant to mix into powder 1;
(2) After the microcrystalline wax and the surfactant are uniformly mixed, a small amount of lubricant is added to be mixed into powder 2;
(3) Mixing the rest raw materials uniformly, adding the powder 2, mixing uniformly, adding the powder 1, and mixing to obtain the inoculation additive.
The application of the inoculating additive in inoculating wheat root rot.
Specifically, the application comprises the following steps:
(1) Inoculating the wheat root rot into the fermentation liquor, and fermenting and culturing to obtain the wheat root rot fermentation liquor;
(2) Lyophilizing to obtain lyophilized powder for wheat root rot;
(3) Uniformly mixing the wheat root rot freeze-dried powder and an inoculation additive according to the mass ratio of 1:20-1000 to obtain an inoculation microbial inoculum;
(4) And (3) spraying powder to inoculate the inoculant in the flowering phase of the wheat.
In the step (3), the mass ratio of the wheat root rot freeze-dried powder to the inoculation auxiliary agent is preferably 1:100-500.
The synergy mechanism of the invention is as follows:
the inoculating auxiliary agent contains microcrystalline wax and surfactant, so that the wax on the surface of the wheat leaf can be dissolved, the enzyme is assisted to form an invasion opening on the surface of the leaf, and the type and the dosage of the surfactant are small in toxicity to strains, so that the activity is not influenced; the wheat leaf components and the saccharides in the inoculating auxiliary agent can provide nutrition, are beneficial to improving the germination rate of wheat root rot spores, and can also stimulate the germination of spores and the elongation of bud tubes and the fixation of the spores on the surface of a host together with the enzymolysis components; the titanium dioxide in the inoculating auxiliary agent can improve the activity of wheat root rot; the binder in the inoculating auxiliary agent can absorb moisture to promote spore germination, and can also enhance the adhesiveness of the inoculating auxiliary agent and prevent falling off; in addition, the solid powdery inoculation auxiliary agent does not need to be added with water for use, so that the conditions of shedding and uneven inoculation of an inoculation microbial inoculum are effectively avoided, and the inoculation is generally only needed once; the inoculation microbial inoculum prepared by the inoculation auxiliary agent can be preserved at low temperature, and has small influence on activity.
The invention has the following advantages:
the inoculation auxiliary agent provided by the invention is beneficial to the infection incidence rate of pathogenic bacteria of the wheat root rot, water is not needed when the inoculation auxiliary agent is used, the yield reduction of wheat caused by other non-disease factors is prevented, and the conditions of falling off and uneven inoculation of an inoculation microbial inoculum can be avoided; the inoculation auxiliary agent and the strain can be preserved at low temperature after being mixed into an inoculation microbial inoculum, and the inoculation auxiliary agent and the strain have little influence on the activity of pathogenic bacteria. The inoculating auxiliary agent is suitable for screening and identifying the resistance of wheat varieties to root rot, and is beneficial to reducing the workload.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
EXAMPLE 1 preparation of the seed Strain
Taking out the wheat root rot and the navel-flattening vermicular spore preserved in a refrigerator at the temperature of 4 DEG CBipolaris sorokiniana) Bacteria are inoculated on a PDA flat plate, after being cultured in the dark for 5 days in a constant temperature incubator at 28 ℃, bacteria blocks are taken out by a 5mm puncher, and inoculated in PDB culture fluid under the aseptic condition. Inoculating 5 fungus blocks into each bottle of culture solution (100 mL/bottle), inoculating 20 bottles, shaking at 28deg.C for 7d, sterile filtering, and lyophilizing mycelium pellet.
EXAMPLE 2 inoculating aid component
Preparation of an seeding aid
Weighing 0.10g of 3U/mg cellulase and 0.1U/mg lignin enzyme, adding different components or contents into the raw materials according to the table 1, adding talcum powder to complement to 100g to obtain different inoculation auxiliary agents, and then adding 1.00g of bacterial powder in the example 1 to obtain the inoculation bacterial agent.
Table 1 inoculating aid composition
。
Morbidity and disease progression statistics for indoor vaccination
Selecting wheat seeds, weighing 5g of seeds respectively, broadcasting the seeds into each planting pot, and culturing until the tillering stage. Wheat seedlings (variety: jinan 15) with 5 leaves and 1 core of main stem and leaf age were inoculated with the inoculant powder spray of the composition according to Table 1, 1.00g of the inoculant was inoculated per pot with 1g of the inoculant powder +99.00g of talcum powder as a control, and 10 pots were repeated 3 times per treatment. Culturing in artificial climate chamber at 25deg.C, lighting for 8 hr and darkness for 16 hr, and relative humidity of 75% + -5%. The time required for inoculation to onset of disease and the disease index 5 days after onset of disease were counted, and the effect of different components and contents on disease time and disease development compared with control was analyzed by Ducan's new bipolar assay.
The morbidity is calculated according to the following formula:
incidence (%) = number of diseased plants/total inoculated plants x 100%;
grading is performed according to the following criteria:
level 0: no disease spots;
stage 1: the area of the disease spots accounts for less than 5% of the basal part of the stem;
3 stages: the area of the lesion accounts for 6% -10% of the basal part of the stem;
5 stages: the area of the lesion accounts for 11% -20% of the basal part of the stem;
7 stages: the area of the lesion accounts for 21% -50% of the basal part of the stem;
stage 9: the area of the disease spots accounts for more than 50% of the basal part of the stem;
disease index (%) =Σ (number of disease plants at each stage×relative number)/(total number of investigation×9) ×100%.
TABLE 2 onset time of different vaccination aids and disease index 5 days after onset
。
As can be seen from the data in table 2, in the experimental range, the mixing of the wheat leaf powder, the sucrose powder, the polyethylene glycol-1000 and the microcrystalline wax with span-60 all accelerated the infection of the germs, which is manifested by a shortened time from inoculation to onset of disease; the microcrystalline wax has the greatest influence on the attack time, and the sucrose powder and the polyethylene glycol are the second most, so that the influence of the wheat leaf powder on the attack time is smaller than that of other auxiliary agents. The mixing of the wheat leaf powder, the sucrose powder, the polyethylene glycol-1000 and the microcrystalline wax and span-60 has larger difference in the expansion influence on the disease course development, namely the disease spots; the sucrose powder has the greatest effect on the spreading of the disease spots, the microcrystalline wax is mixed with span-60, the wheat leaf powder is then mixed, and the polyethylene glycol-1000 has less effect on the spreading of the disease spots after infection.
EXAMPLE 3 screening of the amount of seeding aid
Preparation of seeding aid
Inoculating aids of different compositions were prepared according to the components in the following table:
the preparation method comprises the following steps:
(1) Uniformly mixing wheat leaf powder, cellulase and lignin enzyme, and adding about 1/3 of magnesium stearate to mix into powder 1;
(2) After the microcrystalline wax and the surfactant are uniformly mixed, about 1/3 of magnesium stearate is added to be mixed into powder 2;
(3) Mixing the rest raw materials uniformly, adding the powder 2, mixing uniformly, adding the powder 1, and mixing to obtain the inoculation additive.
Influence of different inoculation auxiliary agents and bacterial powder mass ratios on disease time
Wheat seedlings were obtained as per example 2, following the procedure of wheat flour: the mass ratio of the auxiliary agent is 1:20-1000, the inoculant is prepared and inoculated, and then the time required for starting the inoculation is counted, and the result is shown in table 3.
TABLE 3 time to onset of different vaccination ratios (means.+ -. S.E.)
As can be seen from the data in table 3, in the experimental range, following the bacterial powder: the mass ratio of the inoculating auxiliary agent is improved, the disease time is gradually reduced, and the bacterial powder is selected in combination with the cost: and (3) carrying out field inoculation experiments according to the mass ratio of the inoculation auxiliary agent of 1:100-500.
EXAMPLE 4 preservation test
The inoculant prepared by the fungus powder and the inoculation auxiliary agent 6 according to the mass ratio of 1:500 is freeze-dried, stored in a refrigerator at-20 ℃ for 1 month and 3 months, taken out, and unsealed after being warmed to room temperature, an inoculation test is carried out according to the method of the example 2, the inoculant with the same newly prepared formula is used as a control, the time required for starting the disease and the disease index at 5 days after the disease start are counted, the influence on the disease time and the disease development after the storage is analyzed by a Ducan's new complex polar difference method, and the result is shown in a table 4.
TABLE 4 Effect of preservation time on vaccination (means.+ -. S.E.)
As can be seen from the data in table 4, compared with the freshly prepared inoculant, the inoculant has no significant effect on the initial infection capacity of the inoculant after 1 month and 3 months of preservation, and the time from inoculation to morbidity is not affected; the growth and the expansion of pathogenic bacteria are not obviously affected, and the disease finger after 5 days of disease occurrence is not affected; this indicates that the inoculant after preparation can be stored at-20℃for at least 3 months. In practical use, the seed can be prepared in large quantity in advance, stored for a period of time and then subjected to inoculation test when the seed is proper.
Example 5 field inoculation test
Preparation of inoculating aid and inoculating microbial inoculum
An inoculating aid was prepared according to the following formulation and method in example 3:
(1) Wheat leaf powder 10 wt%; 0.1wt% of cellulase; lignin enzyme 0.05 wt%; microcrystalline wax 2.5. 2.5 wt%; span-60.25wt%; 0.75wt% of magnesium stearate; sugar powder 2 wt%; polyethylene glycol 3wt%; the talcum powder is added to 100 percent;
(2) Wheat leaf powder 10 wt%; 0.1wt% of cellulase; lignin enzyme 0.05 wt%; microcrystalline wax 2.5. 2.5 wt%; span-60.25wt%; 0.75wt% of magnesium stearate; sugar powder 2 wt%; polyethylene glycol 3wt%; 0.1wt% of titanium dioxide; the talcum powder is added to 100 percent;
(3) Wheat leaf powder 10 wt%; 0.1wt% of cellulase; lignin enzyme 0.05 wt%; microcrystalline wax 2.5. 2.5 wt%; span-60.25wt%; 0.75wt% of magnesium stearate; sugar powder 2 wt%; polyethylene glycol 3wt%; 0.5wt% of titanium dioxide; the talcum powder is added to 100 percent;
(4) Wheat leaf powder 10 wt%; 0.1wt% of cellulase; lignin enzyme 0.05 wt%; microcrystalline wax 2.5. 2.5 wt%; span-60.25wt%; 0.75wt% of magnesium stearate; sugar powder 2 wt%; polyethylene glycol 3wt%; titanium dioxide 1wt%; the talcum powder is added to 100 percent;
(5) Talcum powder 100%.
The bacterial powder is obtained according to the method of the example 1, and then the bacterial powder and the inoculation auxiliary agent are uniformly mixed according to the mass ratio of 1:200 to prepare different inoculation bacterial agents, and the serial numbers are respectively 1-5.
Field inoculation test
As the current wheat seed coating agent is popularized, the incidence rate and harm of seedling stage are reduced, and the wheat before and after heading has no need of root rot of wheat and has no harm to the navel-flattening vermicular sporeBipolaris sorokiniana) Is more important to the influence of the wheat yield and quality, so the field inoculation test is carried out on a test base in 2019: wheat (variety: dwarf 58) was subjected to a back-pack type motorized powder blower in the order of (5) - (1) - (2) - (3) - (4) at the flowering stage at 60g/m 2 Carrying out powder spraying inoculation test on the amount of the seed; 6 replicates per treatment, 50m per replicate 2 The method comprises the steps of carrying out a first treatment on the surface of the Wherein, 3 times are repeated after 3 days of inoculation for each treatment, and the inoculation amount is 60g/m 2 . Counting the disease rate of each treatment after 7 days of inoculation, and carrying out multiple comparison by a Ducan's new complex pole difference method; randomly sampling at 5 points, 0.25m each 2 。
TABLE 5 disease index 7 days after inoculation of different microbial inoculum
The different letters indicate significant differences in disease rates or disease fingers between different bacteria at the 0.05 level.
As can be seen from the data in table 5, the addition of titanium dioxide can significantly improve the disease rate of the inoculant compared with the addition of no titanium dioxide; compared with the microbial inoculum added with the filler, the microbial inoculum added with 0.5-1% of titanium dioxide can achieve the disease strain rate of 2 times of inoculation.
Claims (8)
1. An inoculating aid for promoting wheat root rot infection, which is characterized by comprising the following raw materials in parts by weight:
wheat leaf powder 1-50 wt%;
cellulase 0.01-0.2 wt%;
lignin enzyme 0.01-0.1 wt%;
microcrystalline wax 1-5 wt%;
sorbitan fatty acid ester 0.05-0.5 wt%;
0.25-1 wt% of lubricant;
sugar powder 0.25-4.75 wt%;
polyethylene glycol 0.25-4.75 wt%;
titanium dioxide 0.1-1 wt%;
the talcum powder is added to 100 percent;
the cellulase activity is not less than 1U/mg; the lignin enzyme activity is not less than 0.1U/mg;
the sugar is selected from glucose and/or sucrose; the average relative molecular mass of polyethylene glycol is 1000-2000.
2. The inoculating aid of claim 1, wherein the lubricant is selected from one or more of magnesium stearate or zinc stearate.
3. The inoculating aid of claim 1, further comprising a colorant; the content of the colorant is 0.001-0.1wt%.
4. The inoculating aid of claim 1, wherein the fineness of the inoculating aid is 200-250 mesh.
5. A process for the preparation of an inoculating aid according to any one of claims 1 to 4, comprising the steps of:
(1) Uniformly mixing wheat leaf powder, cellulase and lignin enzyme, and adding a small amount of lubricant to mix into powder 1;
(2) Uniformly mixing microcrystalline wax and sorbitan fatty acid ester, and adding a small amount of lubricant to mix into powder 2;
(3) Mixing the rest raw materials uniformly, adding the powder 2, mixing uniformly, adding the powder 1, and mixing to obtain the inoculation additive.
6. Use of an inoculating aid according to any one of claims 1-4 for inoculating wheat root rot.
7. The use according to claim 6, characterized by the steps of:
(1) Inoculating wheat root rot fungi into the fermentation liquor, and fermenting and culturing to obtain the wheat root rot fungi fermentation liquor;
(2) Lyophilizing to obtain lyophilized powder of wheat root rot fungus;
(3) Uniformly mixing the wheat root rot fungus freeze-dried powder and an inoculating auxiliary agent according to the mass ratio of 1:20-1000 to obtain an inoculating microbial inoculum;
(4) And (3) spraying powder to inoculate the inoculant in the flowering phase of the wheat.
8. The use according to claim 7, wherein in the step (3), the mass ratio of the wheat root rot fungus freeze-dried powder to the inoculating auxiliary agent is 1:100-500.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH05213707A (en) * | 1991-09-06 | 1993-08-24 | Osaka Gas Co Ltd | Agent for inoculating microorganism |
| CN1723780A (en) * | 2005-07-06 | 2006-01-25 | 西南大学 | Fungi spore wettable dust |
| CN202786246U (en) * | 2012-09-21 | 2013-03-13 | 河北农业大学 | Wheat rust uredispore inoculator |
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2019
- 2019-12-13 CN CN201911281437.6A patent/CN110982872B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05213707A (en) * | 1991-09-06 | 1993-08-24 | Osaka Gas Co Ltd | Agent for inoculating microorganism |
| CN1723780A (en) * | 2005-07-06 | 2006-01-25 | 西南大学 | Fungi spore wettable dust |
| CN202786246U (en) * | 2012-09-21 | 2013-03-13 | 河北农业大学 | Wheat rust uredispore inoculator |
Non-Patent Citations (1)
| Title |
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| 张眉等.山东小麦蠕孢根腐病病原鉴定及致病力分析.山东农业科学.2016,第第48卷卷(第第48卷期),材料与方法1.3. * |
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