CN116606745A - Wood rot fungus composite microbial inoculant and application thereof in preventing and treating pine wood nematode disease - Google Patents
Wood rot fungus composite microbial inoculant and application thereof in preventing and treating pine wood nematode disease Download PDFInfo
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Abstract
The invention discloses a wood rot fungi composite microbial inoculant and application thereof in preventing and treating pine wood nematode diseases. According to the invention, screening from a plurality of wood rot fungi composite microbial inoculant combinations, the combination of the phellinus linteus and the chlamydomonas reinhardtii completely inhibits the reproduction of pine wood nematodes; the wood block decomposition test shows that the composite microbial inoculum has a corrosion rate on the Korean pine wood block which is obviously higher than that of other microbial inoculum combinations, and can effectively kill pine wood nematodes in the stump and decompose the stump efficiently. The wood rot fungi composite microbial inoculum consisting of the inonotus obliquus and the phellinus linteus provided by the invention can be applied to preventing and treating pine wood nematode diseases. The wood rot fungi composite microbial inoculum composed of the inonotus obliquus and the phellopterium erythrorhizon has higher corrosion rate on wood blocks, can decompose stumps efficiently, kills pine wood nematodes in the stumps of pine wood nematode epidemic, has the advantages of high efficiency, no pollution, rapidness, simplicity and the like, and has application prospects in the aspect of preventing and controlling pine wood nematodes or infection sources in the stumps of pine wood nematode epidemic.
Description
Technical Field
The invention relates to a wood rot fungus composite microbial inoculum, in particular to a composite microbial inoculum composed of phellinus linteus and application thereof in preventing and controlling pine wood nematodes, and belongs to the field of biological prevention and control of pine wood nematode diseases.
Background
Pine wood nematode disease is also called pine wilt disease, belongs to a type of destructive disease of pine tree, and is called "cancer" of pine tree. Pine wood nematode disease was first developed in China and introduced by Japan in 1982. Once the pine tree is damaged, the pine tree can die after about 6 weeks, and if the pine tree cannot be timely prevented and controlled, the large-area pine tree forest can be destroyed in 3-5 years. Therefore, the prevention and control of pine wood nematode disease is being increased (in blue, research on hazard and comprehensive prevention and control countermeasures for pine wood nematode disease [ J ], agricultural development and equipment 2020 (7): 233).
The biological control is relatively good in environmental protection, and the biological control on pine wood nematodes is mainly realized by releasing natural enemies of propagation medium insects such as monochamus alternatus in forests so as to control the density of the propagation medium insects.
The pine wood nematode disease wood-stump contains a certain amount of pine wood nematodes and monochamus alternatus larvae, which are one of important infection sources of the pine wood nematode disease (Zhu Kegong, yao Shiyi, zhang Jingyi. Research on infection sources of the pine wood nematode disease [ J ]. Shandong forestry science and technology, 1992 (4): 45-48)), and the existing biological control method has poor control effect on the pine wood nematode disease wood-stump, so that a more effective control method for the pine wood nematode disease wood-stump needs to be developed.
Disclosure of Invention
The invention aims to provide a wood rot fungi composite microbial inoculum capable of decomposing wood and killing pine wood nematodes;
the second purpose of the invention is to apply the wood rot fungi composite microbial inoculum to decompose the wood-worm disease wood-stump or kill the pine wood nematodes;
the above object of the present invention is achieved by the following technical solutions:
the invention provides a wood rot fungus compound microbial inoculum which consists of inonotus obliquus (Lenzites betulina) and inonotus erythroedge (Fomitopsis pinicola).
In order to achieve better technical effect, the ratio of the inonotus obliquus to the inonotus erythropolis is (0.5-3) according to the volume ratio: (0.5-3); more preferably, the ratio of Inonotus obliquus to Phellinus linteus is 2:1.
firstly, a plurality of white rot fungi and brown rot fungi are paired and analyzed to obtain a strain combination which does not produce antagonistic effect; the strain combination which does not produce antagonistic effect is further verified to influence the propagation of the pine wood nematodes, and test results show that the pine wood nematodes are not separated from the colony of the combination of the strain phellopterium erythrorhizon and the chlamydia, that is, the propagation of the pine wood nematodes is completely inhibited by the combination of the phellopterium erythrorhizon and the chlamydia. According to the invention, the decomposition test of the wood blocks is further carried out on the combination of various microbial agents, and the result shows that the corrosion rate of the composite microbial agent combining the phellinus linteus and the chlamydia trachomatis to the red pine wood blocks reaches 26.71 percent, which is obviously higher than that of other microbial agent combinations to the red pine wood blocks; according to the invention, the composite microbial inoculum formed by combining the trichoderma reesei and the chlamydia trachomatis is inoculated on the stump for treating the pine wood nematode disease, the number of the pine wood nematodes in the stump and the decomposition rate of the stump are measured after 60 days of inoculation treatment, and as a result, the composite microbial inoculum formed by combining the trichoderma reesei and the chlamydia trachomatis can effectively kill the pine wood nematodes in the stump and effectively decompose the stump.
Therefore, the wood rot fungi composite bacterial agent composed of the inonotus obliquus and the phellinus linteus can be applied to preventing and treating pine wood nematode diseases, and comprises the following components: inoculating the wood rot fungi composite microbial inoculum into the pine wood nematode epidemic wood stump, and killing the pine wood nematodes in the stump while decomposing the stump; wherein, inoculation can be carried out on the stump according to the culture characteristics of each wood rot fungus, and the microbial inoculum prepared in advance is smeared on the surface of the stump during inoculation, and then a breathable film is covered on the stump for facilitating moisture preservation.
The wood rot fungi composite microbial inoculum composed of the inonotus obliquus and the phellopterium erythrorhizon has higher corrosion rate on wood blocks, can decompose the stump efficiently, kills pine wood nematodes in the stump while decomposing the stump, has the advantages of high efficiency, no pollution, rapidness, simplicity and the like, and has important application prospect in the aspect of preventing and controlling the residual pine wood nematodes or infection sources in the stump of the pine wood nematode epidemic disease.
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FIG. 1 results of complex colony planar culture; culturing results of S.sanguinolentum and T.pubiscens; culturing results of P.beta.and P.sordida; c: culture results of adicosa and p.chrysosporium; d: S.Ostrea and P.betulinu culture results; e: culture results of adicosa and t.biforme; f: culture results of adicosa and t.pubiscens; g: culture results of adiplosa and p.sordida; h: culture results of subvertedspora and p.betulinus; i: culturing results of Ostrea and P.osteatus; j: cultivation results of P.osteatus and B.Adusta; k: culture results of adiplosa and b.adiusta; l: culture results of l.betulina and f.pinicola; m: culturing results of the l.betulina and l.edodes; n: culture results of t.orientalis and p.planta; o: culture results of planta and p.sordida; p: culture results of beta and P. Chrysosporium; q: culturing results of l.betulina and p.ostreatus; r: culture results of placeta and t.biforme.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Bacterial source and culture method
1 strain
Strain name: lenzites betulina (Inonotus obliquus, accession number CFCC 5473, accession number LB 3) and Fomitopsis pinicola (Fomitopsis pinicola, accession number CFCC 80995, accession number FP 5);
strain sources: provided by China forestry microbiological culture collection center (CFCC);
the best proportion is as follows: 2:1.
other white rot fungi and brown rot fungi strains used in the invention are provided by China center for type culture Collection (CFCC) of forestry microorganisms.
2 culture method
The test was performed in the second half of 2021 in the "plant pathogen and disease management" subject group laboratory of the national institute of forestry science, sensen, environmental protection agency.
About 0.5cm of activated wood rot fungus strain mycelium block 2 Inoculating to the center of the plate culture medium, and culturing in a biochemical incubator at 25 ℃ for 7d.
Test example 1 antagonistic screening test of white rot fungi and brown rot fungi in combination
White rot fungi and brown rot fungi are inoculated on two sides of a culture dish in a pairing mode, the diameter of hyphae is counted every day until the hyphae grow to be full of plates, and the test strains are shown in table 1.
TABLE 1 test strains
34 species were combined, which were analyzed to be non-producing or weakly antagonistic, as shown in Table 2, and the morphology of the composite culture colonies was shown in FIG. 1.
TABLE 2 antagonistic-free colonies
Test example 2 test of Effect of Complex microbial inoculant combinations on pine wood nematode propagation
The number of pine wood nematodes isolated after 8d of cultivation on colonies of pure and composite cultures of pine wood nematodes inoculated with brown rot fungi and white rot fungi strains is shown in Table 3 (section).
TABLE 3 number of 8d culture of pine wood nematodes on wood rot colonies
The test result shows that the 12 groups of compound cultured colonies have certain inhibition effect on the reproduction of the pine wood nematodes. Wherein no pine nematodes were isolated at all from colonies of the combination of Phellinus linteus and Phellinus linteus, indicating that the proliferation of pine nematodes was completely inhibited by the combination of the strains of Phellinus linteus and Phellinus linteus.
Test example 3 decomposition test of wood block by wood rot fungi combination
1. Test method
1.1, preparing a thick bottom PDA culture medium;
1.2 preparing a plurality of Korean pine blocks with the size of 20x60x2mm, putting each block into an oven with the temperature of 103+/-2 ℃ to be baked to constant weight before inoculating wood rot fungi combination, weighing each block (accurate to 0.01 g), wrapping by using absorbent paper (or multi-layer gauze), keeping the blocks in a steam sterilizer under the normal pressure condition for about 30 minutes, and cooling to obtain the water content of the sample.
1.3 inoculation: placing wood blocks in the middle of a culture dish, transversely connecting white rot fungi and brown rot fungi blocks on two sides of the wood blocks respectively, wherein three combination modes are adopted: 11. 22, 12 (1, 2 represent two bacteria), and a pure culture control group is arranged, placed in an incubator, taken out after 2 weeks of infection, gently scraped to remove surface hyphae and impurities, and baked to constant weight in an oven at 103+/-2 ℃, and each sample is weighed respectively.
1.4 calculating the mass loss rate after decay:
calculating the mass loss rate after each sample decays, wherein the mass loss rate is expressed as a percentage, and the mass loss rate is shown in a formula (1)
Wherein:
W 1 -full dry mass of the sample prior to testing;
W 2 full dry mass after test of the test specimen.
1.5 wood rot fungi after treating the Korean pine block 60D, the mass loss rate of the wood block sample was calculated.
2. Test results
The mass loss rate of the wood block samples is shown in Table 4.
TABLE 4 Corrosion Rate of wood rot of Korean pine blocks after 60 days treatment
| Type of microbial inoculum | Corrosion rate of |
| Fomitopsis pinicola (Fr.) Kuntze | 12.75％ |
| Phellinus linteus | 10.14％ |
| Fomitopsis pinicola and Acremodella leucovora | 11.89％ |
| Fomitopsis pinicola+Phellinus linteus | 26.71％ |
| Phellinus linteus + Phanerochaete opalatinosus | 11.53％ |
| Fomitopsis pinicola+Lasiosphaera maeri | 10.78％ |
| Fomitopsis pinicola+Phanerochaete | 11.12％ |
| Fomitopsis pinicola and Stropharia rugoso-annulata | 9.98％ |
| Fomitopsis pinicola and Maota pubescens | 11.92％ |
| Fomitopsis pinicola+Orientia | 10.25％ |
| Fomitopsis pinicola and trametes versicolor | 9.89％ |
| Fomitopsis pinicola+trametes rubra | 12.78％ |
| Fomitopsis pinicola+Coriolus versicolor | 12.76％ |
| Fomitopsis pinicola+Fusarium roseum | 10.89％ |
As can be seen from the test results in Table 4, the corrosion rate of the composite microbial inoculum combining the Phellinus linteus and the Phellinus linteus on the Korean pine blocks reaches 26.71%, which is significantly higher than that of the other 11 groups of microbial inoculum combinations on the Korean pine blocks.
Test example 4 application test of Combined wood-rotting fungus inoculant in decomposing stumps and killing pine wood nematodes in stumps
1 test method
1.1 preparation of the seed Material
Bagging with polypropylene stock of 12cm diameter and 22cm length, each bag containing 100g of wood chip or 300g of talcum powder, 4g of glucose, 4g of corn flour, 1g of peptone and 0.6g of KH 2 PO 4 、0.3g MgSO 4 ·7H 2 O, 4g pectin and 4g L-sodium glutamate, bagging, mixing the materials uniformly and sealing. The sterilization was repeated 2 times in autoclave (121 ℃) for 40min each. After the temperature of the inoculated material is cooled down, the prepared bacterial liquid (the phellinus linteus and the chlamydia trachomatis) is added into each bag according to the inoculation amount of 100mL of each bag on a sterile operation table, and the air in the bag is discharged and sealed. The inoculum was turned over every day for the first 3 weeks, and then 2 times per week, and such inoculum could be stored at room temperature for at least 1 year without destroying the activity of its species. Before the field inoculation test is carried out, the activity of the strain in the bag is detected, a small amount of material in the bag is taken on a sterile operation table to see whether the material grows well on a PDA culture medium, the good hypha growth indicates that the inoculation material still has activity, and the strain can be subjected to the field test. Before wood rot fungi are inoculated on the stump, 200mL of glucose solution with the concentration of 1%, 100mL of vegetable oil and a little egg are added into each bag, and the mixture is uniformly mixed to perform inoculation.
1.2 preparation of stumps
15 stumps with the diameter of more than 15cm and no worm damage and peeling rot are selected and marked. No signs of bacterial breakdown were found in the stump woody part prior to inoculation. The surface of the stump is smeared with 20% lime water one week before the inoculation of the compound microbial inoculum to prevent the growth of mixed bacteria, and termite killing liquid medicine (trade name perphenna) is sprayed in the range of 1m around the stump.
1.3 determination of the amount of pine wood nematodes in the stump
The samples of the cut-back stakes were quantitatively (30 g) harvested as controls prior to inoculation. After 60d of inoculation treatment, a stump sample is quantitatively (30 g) collected at each stump inoculation position, and pine wood nematodes are separated and counted according to the Bellman funnel method.
1.4 determination of the decomposing Capacity of wood rot fungi
Prior to inoculation, 30g of samples were taken for each stump as a control. The composite microbial inoculum is selected to be carried out in the afternoon or in the cloudy day, and is inoculated on the stump according to the culture characteristics of each wood rot fungus, and the inoculation material prepared in advance is smeared on the surface of the stump during inoculation, and then a breathable film is covered on the stump for facilitating moisture preservation. Inoculating 15 stumps with the composite microbial inoculum (comprising Phellinus linteus and Phellinus linteus in a ratio of 2:1), and inoculating 60d without inoculating 15 stumps. Each stump sample is prepared into 3 regular wood block samples (2 cm multiplied by 1 cm), the wood block samples are numbered and then put into a drying oven with the temperature of 100+/-5 ℃ to be dried to constant weight, and the weight of each wood block is weighed. And comparing the quality changes before and after treatment, and analyzing the decomposition condition of each wood rot fungus strain on the stump. The weight of each stump wood block is used for measuring the decomposing capacity of the wood rot fungi on the stumps.
2 test results
2.1 stump sample weight
The weight measurement results of 15 stump samples and control samples after 60d of inoculation treatment are shown in Table 5; as can be seen from Table 5, the 15 stump samples after 60d treatment with the composite inoculant (consisting of Phellinus linteus and Phellinus linteus in a ratio of 2:1) all weighed less than the control stump samples.
TABLE 5 weight of stump samples
2.2 number of pine wood nematodes in stumps
After 60d of inoculation treatment on the stumps of the dead tree of the pine wood nematode disease, the quantity of the pine wood nematodes separated from each stump is counted, and the counting result is shown in Table 6.
TABLE 6 variation of the amount of pine wood nematodes in stump samples
According to the statistical results of Table 6, it can be seen that the pine wood nematodes in the stump of the dead tree of the pine wood nematode disease can be effectively killed after the treatment of inoculating the composite microbial inoculum on the stump of the dead tree of the pine wood nematode disease for 60 days.
Claims (10)
1. A wood rot fungus compound fungus agent is characterized by comprising inonotus obliquus (Lenzites betulina) and phellinus linteus (Fomitopsis pinicola).
2. The wood rot fungi composite microbial inoculant according to claim 1, wherein the ratio of the inonotus obliquus to the inonotus erythroedge is (0.5-3) in terms of volume ratio: (0.5-3).
3. The wood rot fungi composite microbial inoculant according to claim 1, wherein the ratio of the inonotus obliquus to the inonotus erythroedge is 2:1.
4. use of the wood rot fungi composite microbial inoculant according to any one of claims 1-4 for controlling nematodes.
5. The use according to claim 4, wherein the nematodes are pine wood nematodes.
6. The use according to claim 4, characterized in that it comprises: the wood rot fungi composite microbial inoculum is inoculated into the pine wood nematode epidemic wood stump, and the pine wood nematodes in the stump are killed while the stump is decomposed.
7. The method according to claim 6, wherein the wood rot fungi are inoculated on the stumps of the pine wood nematode disease according to the culture characteristics of the wood rot fungi, and the microbial inoculum is smeared on the surfaces of the stumps and then covered with the breathable films during the inoculation.
8. Use of the wood rot fungi composite microbial inoculant according to any one of claims 1-4 for decomposing or corroding wood.
9. The use according to claim 8, wherein the wood is pine wood nematode disease wood stump.
10. The use according to claim 9, characterized in that it comprises: inoculating the wood rot fungi composite microbial inoculant of claim 1 into pine wood nematode epidemic wood stump.
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