CN110100685B - Method for identifying disease resistance of peanut net blotch by conidium inoculation - Google Patents
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Abstract
The invention discloses a method for identifying disease resistance of peanut net blotch by conidium inoculation, belonging to the technical field of crop disease resistance identification. According to the invention, conidia of the net blotch pathogenic bacteria are cultured under the condition of 16 hours of long illumination to generate a large number of conidia, the conidia are broken to prepare conidia suspension, peanut seedlings are inoculated by spraying, the area of the diseased leaf spot is analyzed by using a leaf diseased spot area analysis system, the disease index is calculated, and the disease resistance of the net blotch of different peanut materials is evaluated. The method develops a culture mode that the net blotch pathogenic bacteria quickly generate a large number of conidiospore devices, solves the problems of difficult spore production, slow spore production, small spore production amount, difficult spore taking and the like of the net blotch pathogenic bacteria, and the prepared conidiospore suspension has high uniformity and easy quantification, overcomes the problem of difficult quantification of hypha suspension inoculation, and is suitable for identifying the disease resistance of indoor potted peanuts.
Description
Technical Field
The invention relates to a method for identifying disease resistance of peanut net blotch by conidium inoculation, belonging to the technical field of crop disease resistance identification.
Background
Peanut (Arachis Hypogaea L.) is an economic and oil crop widely planted in the world, China is a big country for planting, consuming and exporting peanuts, and the annual yield of peanuts is at the top of the world, so that the stable yield and the high yield of peanuts are always concerned by many parties. The net blotch of peanuts is a main disease of peanut leaves, can occur in the whole growth period, and can cause a large amount of leaves to fall off in the later growth period of the peanuts, so that the yield is reduced by 10% -40%, and the disease becomes a disease to be solved urgently in production. Peanut net blotch is caused by fungal infestation, which was first discovered in 1934 and was named Mycosphaerella arachidicola by Khokhr, belonging to the genus sphaera; in 1969, Marasas et al separated different fungi from peanut leaves, and classified them into Phoma genus according to their biological morphology, named Phoma arachidicola. In 2010, Aveskamp et al used sequence information on ribosomal DNA to evolutionarily classify Phoma into 9 subgroups, and both of these pathogens clustered into the Peyronellaena species (Peyronellaea), and thus, they were named Peyronellaea arachidicola (Phoma arachidicola). The peanut stalk mold has strong cellulase activity, and most of the infection points radially extend outwards along the veins to form reticulate blotches without obvious edges, so the peanut net blotch is also called as the blotch. The peanut stem-point mould filaments are compact, white or grey white, the meristematic infusorian is semi-buried or buried, the conidium collected in the field is mostly bisporous, and the conidium artificially cultured is mostly monospore; chlamydospores, which live in the middle or top of the hypha, are unigenic or cross-living. The peanut stem-point mold generally overwintering in the residual leaf tissue by hypha and conidium devices, so that a large amount of conidia can be spread along with wind when the conditions are suitable, the drug control effect is poor, and planting of disease-resistant varieties becomes the most economic and effective mode for preventing peanut net blotch.
The breeding of disease-resistant varieties depends on disease nursery and artificial inoculation identification, while natural disease nursery identification is greatly influenced by climate and strains, and even if disease conditions are artificially created, the disease nursery area is still a limiting factor, so that the operability of artificial inoculation identification is stronger. Queen Zhegyu and the like develop a peanut net blotch hypha suspension inoculation identification method, but because the mycelium grows compactly and is not easy to disperse, the concentration of the hypha suspension cannot be quantified, the hypha sprayed on the leaves has different lengths and is not uniformly distributed, the pathogenicity has larger difference, the experimental error is larger, and the identification result is inaccurate. The method adopts conidium suspension inoculation identification method, and has the advantages that: most of the artificially cultured conidia are monospores, the concentration of the prepared suspension is easy to quantify, the spores sprayed to the leaves are uniformly distributed, the germination and growth consistency of the spores is good, and the repeatability of the identification result is good. Conidia are easier to separate than chlamydospores because chlamydospores are transformed by hyphae, and their physical linkage makes it technically challenging to separate them; in addition, conidia are produced in much higher numbers than chlamydospores, and there may be hundreds of conidia in one conidiophor. However, the method is limited in that the peanut phoma is easy to produce chlamydospores under artificial culture conditions, the production of conidiospore devices is difficult, the time is long (more than 20 days), the quantity is small, and the conidiospore devices, hyphae and chlamydospores are mixed together, so that the conidiospores are not easy to obtain. Therefore, developing a method for rapidly and conveniently obtaining a large number of conidia becomes a key for popularizing the identification method.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for identifying the disease resistance of peanut net blotch by conidium inoculation, the method develops a culture mode that pathogenic bacteria of the net blotch produce spores rapidly and massively and obtain conidia, the prepared conidium suspension has high uniformity and is easy to quantify, and the problem that hypha suspension is difficult to inoculate quantitatively is solved.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for identifying disease resistance of peanut net blotch by conidium inoculation comprises the following steps:
(1) inoculating conidia of the pathogenic bacteria of the net blotch, and culturing a conidiophore under the light-dark alternating condition;
(2) crushing a conidiophore to prepare a conidiophore suspension B;
(3) and (3) spraying and inoculating the conidium suspension B to peanut seedlings, analyzing the area of the diseased leaf spots by using a diseased leaf spot area analysis system, calculating disease indexes, and evaluating the disease resistance of the net blotches of different peanut materials.
The preparation method of the conidia of the net blotch pathogenic bacteria for inoculation in the step (1) comprises the following steps:
firstly, picking out pathogenic bacteria hyphae of the net blotch to inoculate on an OA culture medium, and culturing under light and dark alternation to generate sporadic conidiospore devices;
secondly, selecting conidiophores, crushing the conidiophores on a new OA culture medium, and culturing under light and dark alternation to obtain densely distributed conidiophores;
and thirdly, collecting a conidium container, crushing to prepare a conidium suspension A, and inoculating the conidium.
The molecular spores of the pathogenic bacteria of the netowski disease in the step (1) are inoculated in an OA culture medium.
The light and dark alternation is to culture under light for 16h and then culture under dark for 8h, and the above steps are carried out alternately in circulation.
The culture temperature is 25 deg.C, and the culture time is 7-10 days.
The specific method of the step (2) is as follows: collecting conidiophore, placing into ball mill sample injector with sterile water, performing sample injection crushing for 40s at 30hz vibration frequency, filtering with 3 layers of sterile gauze, adding tween-20, and mixing.
The concentration of the spore suspension in the step (3) is 1 × 106-1×107One per ml.
The invention has the beneficial effects that:
according to the invention, conidia of the net blotch pathogenic bacteria are cultured under the condition of 16 hours of long illumination to generate a large number of conidia, the conidia are broken to prepare conidia suspension, peanut seedlings are inoculated by spraying, the area of the diseased leaf spot is analyzed by using a leaf diseased spot area analysis system, the disease index is calculated, and the disease resistance of the net blotch of different peanut materials is evaluated. The method develops a culture mode that the net blotch pathogenic bacteria quickly generate a large number of conidiospore devices, solves the problems of difficult spore production, slow spore production, small spore production amount, difficult spore taking and the like of the net blotch pathogenic bacteria, and the prepared conidiospore suspension has high uniformity and easy quantification, overcomes the problem of difficult quantification of hypha suspension inoculation, and is suitable for identifying the disease resistance of indoor potted peanuts.
Drawings
FIG. 1 is a graph of indoor inoculation identification standard (disease grade) of peanut net blotch.
FIG. 2 shows the colony morphology generated by hypha inoculation under different light conditions. A: the light-dark alternation L// D is 16h/8 h; b: alternating L// D between light and dark is 12h/12 h; c: and culturing in dark for 0h/24 h.
FIG. 3 shows the colony morphology generated by conidia inoculation under different illumination conditions. A: the light-dark alternation L// D is 16h/8 h; b: alternating L// D between light and dark is 12h/12 h; c: and culturing in dark for 0h/24 h.
FIG. 4 is the percentage of lesion area of each variety to total leaf area at different conidia inoculation concentrations.
FIG. 5 shows disease indexes of various varieties at different conidia inoculation concentrations.
FIG. 6 shows the distribution of disease index and relative disease index of the recombinant inbred lines.
FIG. 7 shows the difference in disease index between the strains after inoculation with hyphal and conidia suspensions.
FIG. 8 shows the difference in coefficient of variation between the hyphal and conidia suspension inoculation replicates.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1
A method for identifying disease resistance of peanut net blotch by conidium inoculation comprises the following steps:
(1) selecting hypha of the net blotch pathogenic bacteria, inoculating the hypha on an OA (oat agar) culture medium, culturing for 7-10 days at constant temperature of 25 ℃ under light-dark alternation (light illumination is 16h and dark illumination is 8h, namely L// D is 16h/8h), and spreading the hypha on the whole culture dish, wherein some conidiospore devices are distributed sporadically;
(2) crushing conidiophores on a new OA culture medium, and culturing at constant temperature of 25 ℃ for 7-10D under light-dark alternation (L// D is 16h/8h) to generate a large number of conidiophores which are densely distributed on the OA culture medium;
(3) scraping off a conidiophore on an OA culture medium on an ultra-clean workbench, collecting the conidiophore into a ball mill sample injector added with sterile water, performing sample injection crushing for 40s at 30hz vibration frequency, filtering by using 3 layers of sterile gauze, removing agar culture medium and hyphae, adding Tween-20, and uniformly mixing to obtain a conidiophore suspension A;
(4) coating the conidium suspension A on an OA culture medium, culturing at constant temperature of 25 ℃ for 7-10D under light-dark alternation (L// D is 16h/8h), and generating a large number of conidium devices which are radially and densely and uniformly distributed on the OA culture medium;
(5) scraping off conidium device on OA culture medium on ultra-clean bench, collecting in ball mill sample injector with sterile water, sampling and breaking at 30hz vibration frequency for 40s, filtering with 3 layers of sterile gauze, removing agar culture medium and mycelium to obtain pure conidium suspension B, adding Tween-20, and mixing until the concentration of conidium suspension B is 1 × 106-1×107Per ml;
(6) and (3) spraying and inoculating the conidium suspension B to peanut seedlings (6-8 leaf years old), analyzing the area of the leaf scabs by using a leaf scab area analysis system after 12 days, calculating disease indexes, and evaluating the disease resistance of the net blotch of different peanut materials.
Wherein, the culture conditions of the peanut seedlings are as follows: and after accelerating germination for 48 hours, planting the germinated seeds in a 10cm pot, and placing the pot in an artificial climate chamber, wherein the temperature of the climate chamber is set to be 25 ℃, the relative humidity is 90-95%, the illumination is 16 hours, and the darkness is 8 hours.
The disease index calculation formula is as follows:
disease index { [ Σ (disease-grade number of plants × disease grade) ]/(survey total number of plants × 9) } × 100
Indoor inoculation identification standards (disease grade) of the peanut net blotch are as follows (as shown in figure 1):
Example 2 optimization of culture conditions
2.1 hyphal inoculation
The bacterial strains of the netowski pathogenic bacteria are taken and put on an OA culture medium, the culture medium is cultured for 10D (25 ℃) under different illumination conditions, obvious bacterial strains are generated on the front surfaces of the bacterial colonies under light-dark alternation (L// D is 16h/8h), and convex hypha clusters are formed (figure 2A), black dots in the culture medium can be seen on the back surfaces of the bacterial colonies by naked eyes, and a large number of conidia can be seen to be scattered from a spore container by manual tabletting. Colonies under alternating light and dark (L// D12 h/12h) produced more pronounced hyphae on the front (fig. 2B), while colonies under dark culture (L// D0 h/24h) were thin on the front (fig. 2C). Data statistics shows that the number of conidiospore devices generated by culturing pathogenic bacteria hyphae for 10 days under 16h/8h light-dark alternation is large, and about 57 conidiospore devices are generated on average in each 10 dishes; the number of conidiophores generated by culturing for 10d under 12h/12h light-dark alternation is about 15 conidiophores per 10 dishes; culture in the dark for 10 days produced the least number of conidia, with an average of about 2.3 conidia per 10 dishes and more chlamydospores.
2.2 conidia inoculation
2. mu.l of each aliquot were pipetted at a concentration of about 3X 106A conidia suspension of netospora pathogenic bacteria (about 6000 conidia) was inoculated on an OA medium, and after incubation at 25 ℃ for 7 days under alternating light and dark conditions (L// D ═ 16h/8h and L// D ═ 12h/12h) and (L// D ═ 0h/24h), conidiophore formation on the medium was observed (FIG. 3). Under the light-dark alternative condition, conidiophores on the culture medium are uniform and dense, grow in a radial shape, are regular in shape and are distributed on the culture medium in a circular ring shape (fig. 3A/3B); the conidiophores grown in dark culture had no fixed shape and were not distributed substantially in a circular ring shape (FIG. 3C). Taking 10ml of sterile water as a solvent in each dish, the statistical result shows that the sporulation yield under the light-dark alternation of 16h/8h is the highest, and the average sporulation yield is 34.23 multiplied by 105Per ml; the average sporulation yield under the light-dark alternation of 12h/12h is 3.14 multiplied by 105Per ml; the average spore yield under dark culture was 5.66X 105One per ml.
In summary, after the hyphae are inoculated to the culture medium, the hyphae grow more vigorously and the conidiophores produce less hyphae regardless of the light-dark alternate condition or the dark culture condition. After the conidia are inoculated to the culture medium, the production amount of the conidia is far higher than that of the hypha inoculation test regardless of the light-dark alternate condition or the dark culture condition (see fig. 2 and fig. 3). Under the condition of 12h/12h light-dark alternation, the average spore yield of each vessel inoculated with conidia is equivalent to that under the condition of dark culture, and under the condition of 16h/8h light-dark alternation, the average spore yield of each vessel inoculated with conidia is more than 6 times that under other culture conditions. Therefore, under the condition of constant temperature of 25 ℃ and alternating light and dark (L// D is 16h/8h), the conidia are inoculated to obtain the most conidia.
Example 3 identification of disease resistance of different peanut varieties by inoculation with conidium suspensions of different concentrations
Scraping off conidiophore on culture medium on clean bench, collecting in ball mill sample injector with sterile water, sampling and crushing at 30hz frequency for 40s, filtering with 3 layers of sterile gauze, removing agar culture medium and mycelium to obtain pure spore suspension, adding Tween-20, mixing to obtain 1 × 10 spore suspension7Division of one/mlThe spore suspension was diluted 5, 10, 15, 20 and 25 times to obtain a concentration of 2X 106、1×106、6.7×106、5×105And 4X 105And (4) diluting each/ml of the solution for later use.
The number of peanut varieties to be inoculated is 5, namely Zheng 8903, Yuhua No. 4, Yuhua No. 22, Yuhua No. 15 and NC 94022. The seeds used in the experiment are strictly selected, the size and the color of the seeds are uniform, and the seeds are sterilized. The peanut seeds are put in a culture dish containing sterilized water for accelerating germination, and the culture dish is placed in an incubator at 30 ℃ during accelerating germination. And after accelerating germination for about 48 hours, transplanting the germinated seeds into a 10cm pot which is prepared in advance and contains sterilized vermiculite when the peanut roots grow to about 5cm, so that the peanut plants grow normally. The pot is placed in an illumination incubator/artificial climate chamber, the temperature of the climate chamber is set to be 25 ℃, the relative humidity is 90-95%, the illumination is carried out for 16 hours, and the darkness is carried out for 8 hours.
The experiment had a total of 6 inoculation concentrations, and the inoculated varieties were replicated 3 times, 5 pots each, and 1 strain per pot. Peanut seedlings (6-8 leaves old) with consistent growth are selected, tying ropes of newly-unfolded compound leaves are marked, the whole plant is sprayed and inoculated, and sterile water is sprayed in contrast. After the treatment is finished, observing and recording the incidence of diseases of the inoculated peanut varieties under different treatment every 3 days, collecting and scanning two compound leaves (3 compound leaves in total) below the tether on the stem after the disease state is stable (about 12 d), and analyzing the area of the leaf scab by using a leaf scab area analysis system (ten thousand depth) and calculating the disease index.
The areas of the disease spots of 5 varieties inoculated with conidium suspensions with different concentrations are subjected to variance analysis, and the result shows that the difference among 3 repeats of each variety is not significant (P is 0.793), which indicates that the concentration uniformity of the conidium suspension is higher, the conidia sprayed to the leaves are uniformly distributed, and the difference of the disease spots generated after infection is not significant. And 6 inoculation concentrations were analyzed for variance, with multiple comparisons: 4X 105Each/ml and 5X 105The difference of the lesion areas of the same variety is not obvious under the two inoculation concentrations of each/ml; 1X 106One/ml and 6.7X 106The disease spots of the same variety under the two inoculation concentrations of one seed/mlThe area difference is not significant; and 2 x 106Each/ml and 1X 107The two inoculum concentrations per ml differed significantly from the four inoculum concentrations described previously (P)<0.01). The lesion area becomes larger with the increase of the inoculation concentration, which indicates that the effect of the conidium suspension inoculation is stable. The areas of the disease spots of 5 varieties are subjected to variance analysis, and the differences among the varieties are extremely obvious under the same inoculation concentration (P)<0.01), indicating that conidia suspension inoculation can be used for resistance identification of different varieties (FIG. 4).
And calculating disease indexes of 5 varieties under different inoculation concentrations according to the lesion area and the number of inoculated plants. The disease indexes of 5 varieties inoculated with conidium suspensions with different concentrations are subjected to variance analysis, the difference between 3 repeats of each variety is not significant (P is 0.275), and the result is consistent with the analysis result of lesion area, which indicates that the conidium quantity in quantitative conidium suspensions is high in consistency. The difference between different varieties under the same inoculation concentration is extremely obvious (P <0.01), the result is consistent with the analysis result of the lesion area, and the difference between different inoculation concentrations of the same variety (P <0.01) is extremely obvious, and the difference is different from the analysis result of the lesion area, which shows that the disease index can be calculated more accurately to present resistance difference. As the inoculation concentration of conidia increases, the disease index of each variety also increases, which indicates that the disease stability and uniformity of the conidia are higher, and the more conidia are inoculated, the larger the lesion area of the leaf is, and the higher the disease index is (FIG. 5). The disease resistance of 5 varieties is expressed as Zheng 8903> Yuhua No. 15 > NC94022> Yuhua No. 22> Yuhua No. 4, wherein Zheng 8903 is a disease-resistant variety, Yuhua No. 15 is an anti-resistant variety, NC94022 is a disease-susceptible variety, and Yuhua No. 4 and Yuhua No. 22 are high-susceptible varieties.
Example 4 identification of disease resistance of recombinant inbred lines Using conidia suspension inoculation
Randomly selecting Zheng 8903 and Yuhua No. 4 hybrid F1198 generations of recombinant inbred line materials with the concentration of 2 multiplied by 106Inoculation of conidia suspension per ml identified disease resistance of these materials. Wherein the disease index is less than or equal to 20% and the resistant family accounts for 59.18%, the disease index is more than 20% and less than or equal to 50% and the family accounts for 20.41%, and the disease index is more than 50%The pedigree accounts for 17.35% when the disease index is more than 80% and less than or equal to 100%, and the pedigree accounts for 3.06% (figure 6). Therefore, the conidium inoculation method can be applied to the resistance identification of the net blotch of multiple materials.
Example 5 comparison of the conidia suspension inoculation method with the hyphal suspension inoculation method
Inoculating the hypha of the peanut net blotch pathogenic bacteria to a PDA solid culture medium, and culturing for 7-10 days at constant temperature of 25 ℃ under light-dark alternation (16h light/8 h dark) until the hypha is fully paved on a culture dish. Gently scraping off mycelium from the culture dish, weighing 0.5g in a sterilized food processor, adding 200ml sterile water for mycelium rupture, microscopic detecting mycelium length of about 100--3g/ml. The concentration is 2.5 multiplied by 10- 3g/ml hypha suspension and concentration of 2X 106And (3) inoculating 5 varieties respectively by using each conidium suspension per ml, wherein each variety is provided with 3 times of repetition, 5 pots are repeated, and 1 strain is placed in each pot. And collecting the leaves 12d after inoculation, and carrying out lesion area analysis and calculating disease index.
The disease indexes of 5 varieties inoculated by the hyphal suspension are subjected to variance analysis, and the difference between the varieties is extremely remarkable (P < 0.01). This result is consistent with the analysis of variance of disease indexes inoculated by conidium suspension, and disease indexes of 5 varieties are Yuhua No. 4, Yuhua No. 22, NC94022, Yuhua No. 15 and Zheng 8903 in turn from top to bottom, which indicates that both inoculation methods can be used for the identification of the nethike disease resistance of peanut material (FIG. 7). However, the coefficient of variation of the three replicates of each variety inoculated by both methods was analyzed, and it was found that the coefficient of variation of the three replicates of each variety was higher than that of the conidium inoculation method (fig. 8), indicating that the stability and consistency of the hyphal inoculation method were relatively poor. Hyphae are not easily dispersed due to the structure and the like, so when the hyphae suspension is inoculated on the leaves, the distribution is uneven, the hyphae are distributed in a plurality of places, and more scabs are generated. And the hyphae distributed in a conglobation way cause the hyphae suspension to have poor concentration uniformity, and the conidium inoculation method perfectly overcomes the disadvantages of the hyphae inoculation method. The concentration of the conidium suspension is easy to quantify, the conidia inoculated to the leaves are uniformly distributed, and the homogeneity of lesion spots is better, so that the conidium inoculation method has stronger advantages compared with a hypha inoculation method.
Claims (3)
1. A method for identifying disease resistance of peanut net blotch by conidium inoculation is characterized by comprising the following steps:
(1) inoculating conidia of the pathogenic bacteria of the net blotch, and culturing a conidiophore under the light-dark alternating condition;
(2) crushing a conidiophore to prepare a conidiophore suspension;
(3) spraying and inoculating the conidium suspension to peanut seedlings, analyzing the area of the diseased leaf spots by using a diseased leaf spot area analysis system, calculating disease indexes, and evaluating the disease resistance of the net blotches of different peanut materials;
the preparation method of the conidia of the net blotch pathogenic bacteria for inoculation in the step (1) comprises the following steps:
firstly, picking out pathogenic bacteria hyphae of the net blotch to inoculate on an OA culture medium, and culturing under light and dark alternation to generate sporadic conidiospore devices;
secondly, selecting conidiophores, crushing the conidiophores on a new OA culture medium, and culturing under light and dark alternation to obtain densely distributed conidiophores;
collecting and crushing a conidium device to prepare a conidium suspension for conidium inoculation;
the light-dark alternation is to perform light culture for 16 hours first and then perform dark culture for 8 hours, and the above steps are performed in a circulating and alternating manner; the culture temperature is 25 ℃, and the culture time is 7-10 d;
the specific method of the step (2) is as follows: collecting conidiophore, placing into ball mill sample injector with sterile water, performing sample injection crushing at 30Hz frequency for 40s, filtering with 3 layers of sterile gauze, adding tween-20, and mixing.
2. The method according to claim 1, wherein the molecular spores of the pathogenic bacterium of nethike disease in step (1) are inoculated on OA medium.
3. The method according to claim 1, wherein the concentration of the spore suspension in step (3) is 1 x 106-1×107One per ml.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1664094A (en) * | 2004-03-04 | 2005-09-07 | 北京市农林科学院蔬菜研究中心 | Pathogenic bacteria of gummy stem blight of melon, induction method for its conidiophore and use thereof |
| KR20060047000A (en) * | 2004-11-12 | 2006-05-18 | 주식회사 그린바이오텍 | Method for preparing conidia of ampelomyces quisqualis, formulation method comprising the conidia and the composition thereof |
| UA39411U (en) * | 2008-09-25 | 2009-02-25 | Інститут Агроекології Уаан | Method for determining effect of cereal varieties on number of phytopathogens |
| CN104480189A (en) * | 2014-11-21 | 2015-04-01 | 商丘师范学院 | In vitro identification method for tomato resistance to verticillium wilt |
| CN105191682A (en) * | 2015-10-27 | 2015-12-30 | 河南省农业科学院植物保护研究所 | Disease resistance identification method for phoma arachidicola |
| CN109468237A (en) * | 2018-12-29 | 2019-03-15 | 山东省花生研究所 | Peanut Web Blotch Disease pathogen and its application |
-
2019
- 2019-05-24 CN CN201910441671.4A patent/CN110100685B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1664094A (en) * | 2004-03-04 | 2005-09-07 | 北京市农林科学院蔬菜研究中心 | Pathogenic bacteria of gummy stem blight of melon, induction method for its conidiophore and use thereof |
| KR20060047000A (en) * | 2004-11-12 | 2006-05-18 | 주식회사 그린바이오텍 | Method for preparing conidia of ampelomyces quisqualis, formulation method comprising the conidia and the composition thereof |
| UA39411U (en) * | 2008-09-25 | 2009-02-25 | Інститут Агроекології Уаан | Method for determining effect of cereal varieties on number of phytopathogens |
| CN104480189A (en) * | 2014-11-21 | 2015-04-01 | 商丘师范学院 | In vitro identification method for tomato resistance to verticillium wilt |
| CN105191682A (en) * | 2015-10-27 | 2015-12-30 | 河南省农业科学院植物保护研究所 | Disease resistance identification method for phoma arachidicola |
| CN109468237A (en) * | 2018-12-29 | 2019-03-15 | 山东省花生研究所 | Peanut Web Blotch Disease pathogen and its application |
Non-Patent Citations (1)
| Title |
|---|
| 河南省花生网斑病的初步研究;王振跃等;《河南农业科学》;19930730(第7期);第23-25页 * |
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