CN109468237B - Peanut net blotch pathogenic bacteria and application thereof - Google Patents
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Abstract
The invention provides a peanut net blotch pathogenic bacterium and application thereof, belongs to the technical field of crop disease resistance screening, and can quickly know the infection conditions of the pathogenic bacterium, so that the occurrence trend of the whole disease can be accurately predicted. The pathogenic bacteria of the peanut net blotch are named as Phoma arachidicola WB-SY, the preservation number is CGMCC No.16380, the preservation place is the China general microbiological culture Collection center, and the preservation time is 2018, 11 months and 16 days. The invention can be applied to the prediction and prevention of field diseases.
Description
Technical Field
The invention belongs to the technical field of crop disease resistance screening, and particularly relates to a peanut net blotch pathogenic bacterium and application thereof.
Background
The leaf spot is a disease which is common and seriously harmful to peanuts, and mainly comprises cercospora brown spot and cercospora black spot of peanuts and newly-developed cercospora leaf spot of peanuts in recent years. The pathogenicity identification method for the peanut net blotch pathogenic bacteria generally adopts indoor in vitro inoculation and field living inoculation, but because the field living inoculation has the defects of high requirement on environmental conditions and long test period, the test usually adopts indoor living plant inoculation substitution, and environmental conditions similar to field pathogenicity, such as temperature, humidity, illumination and the like, are regulated through a greenhouse, so that the optimal pathogenic infection conditions of the pathogenic bacteria are screened out.
At present, systematic research on the biological characteristics of peanut reticulospora has been carried out at home and abroad, and research results show that the dictyosphaera grows slowly and has extremely low spore yield, and even if various spore-promoting culture mediums are used for long-time culture, conidia cannot be produced or can only be produced to a very small extent, but the requirements on field inoculation identification are far not met. If a corresponding prevention mechanism is formulated to guide field disease control, the infection conditions of pathogenic bacteria need to be known quickly so as to accurately predict the occurrence trend of the whole disease. Therefore, the provision of a strain with high pathogen activity and pathogenicity to enable the infection conditions of the pathogen to be rapidly known is of great significance to the prediction, prediction and control of the pathogen.
Disclosure of Invention
The invention provides a peanut net blotch pathogenic bacterium and application thereof, the peanut net blotch pathogenic bacterium has high activity and high pathogenicity, and the optimum pathogenicity condition and method can be obtained by screening, so that the infection condition of the pathogenic bacterium can be rapidly known, and the occurrence trend of the whole disease can be accurately predicted.
In order to achieve the aim, the invention provides a pathogenic bacterium of peanut net blotch, which is named as Phoma arachidicola WB-SY, the preservation number is CGMCC No.16380, the preservation place is China general microbiological culture Collection center, and the preservation time is 2018, 11 and 16 days.
The invention also provides application of the peanut net blotch pathogenic bacteria Phomaarachidicola WB-SY in rapid prediction of the occurrence trend of the peanut net blotch diseases.
Preferably, the peanut net blotch pathogenic bacteria Phoma arachidicola WB-SY are prepared into spore suspensions with different concentrations, the spore suspensions are evenly inoculated on healthy peanut plants with the same growth vigor and size under different inoculation humidity, moisture retention time and different growth periods, the peanut plants are cultured at constant temperature at room temperature, and the disease condition and disease index of each plant are observed and recorded.
Preferably, the spore suspension has a concentration of 103Per m L-106And each m L.
Preferably, the inoculation humidity is 50% -100%, and the moisturizing time is 6-48 h.
Preferably, the growth period includes the pre-anthesis period, the initial anthesis period, the full bloom period and the end of development.
Preferably, the concentration of Phoma arachidicola WB-SY is 106Inoculating L spores suspension to healthy peanut plants with consistent growth and size at the terminal flowering stage of peanut, keeping moisture at 90-100% inoculation humidity for 36-48 h, and culturing at constant temperature of 25-28 deg.C.
The invention also provides a method for screening the pathogenic bacteria of the peanut net blotch by adopting an in vitro leaf inoculation method, which comprises the following steps:
preparing a hypha suspension of the peanut net blotch pathogenic bacteria, uniformly spraying the hypha suspension on the peanut compound leaves, culturing at constant temperature at room temperature under the conditions of alternating near ultraviolet light, darkness and light and dark, and observing and recording the disease condition and disease index of each plant.
Preferably, the petiole of the compound leaf is wrapped with 0.1% benzimidazole soaked sterilized cotton and then wrapped with tinfoil paper.
Preferably, the criteria for determining the disease condition and disease index are:
level 0: the whole plant is disease-free;
level 1: the area of the lesion spots accounts for less than 1/10 of the whole area;
and 2, stage: 1/10-1/4 of the area of the lesion spots in the whole patch;
and 3, level: 1/4-1/2 of the area of the lesion spots in the whole patch;
4, level: 1/2-3/4 of the area of the lesion spots in the whole patch;
and 5, stage: the area of the diseased spots accounts for more than 3/4 of the whole area, the diseased leaves fall off seriously, and the plants die.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the invention provides peanut net blotch pathogenic bacteria with high activity and high pathogenicity, and the optimum pathogenicity condition and method are obtained by screening, so that the infection condition of the pathogenic bacteria can be rapidly known, and the occurrence trend of the whole disease can be accurately predicted;
2. the method for directly detecting the pathogenicity of the isolated leaf by adopting the isolated leaf inoculation method has the advantages of rapidness, accuracy, simple operation, large screening amount, no seasonal limitation and the like;
3. the indoor living plant inoculation method has the advantages of direct, simple and stable method, quick disease occurrence, short test period and the like.
Drawings
FIG. 1 is a schematic diagram of the disease onset of different strains inoculated for different days according to the embodiment of the invention, wherein A is WB-SY inoculation 7D, B is WB-L X inoculation 9D, C is WB-PD inoculation 9D, and D is WB-SY inoculation 9D;
FIG. 2 is a schematic diagram of the disease onset of different strains inoculated for different days, wherein A is WB-L X14D, B is WB-PD 14D, C is WB-SY 14D, and D is WB-SY 21D;
FIG. 3 is a diagram showing the disease onset of different strains inoculated for different days according to the example of the present invention, wherein A is WB-L X inoculation 21D, B is WB-PD inoculation 21D, C is WB-YN1 inoculation 21D, and D is WB-YN2 inoculation 21D;
FIG. 4 is a graph showing the effect of various strains on index of disease of peanut net blotch under different illumination conditions at 28 days according to the embodiment of the present invention;
FIG. 5 is a graph showing the effect of spore suspensions of different concentrations on index of net blotch disease according to an embodiment of the present invention;
FIG. 6 is a graph showing the effect of different inoculation humidities on index of net blotch;
FIG. 7 is a graph showing the effect of different moisturizing times on index of network blotch;
FIG. 8 is a graph showing the effect of different growth periods on index of net blotch disease, as provided by an example of the present invention.
Detailed Description
In order to more clearly and fully describe the alternaria arachnoidis pathogenic bacteria and the application thereof provided by the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 in vitro leaf inoculation method
1.1 test materials
The tested peanut variety is Luhua No. 8 susceptible to net blotch, and is potted in a greenhouse for later use. During measurement, healthy peanut compound leaves with consistent growth vigor, size and growth position are selected, and the peanut compound leaves are washed by tap water and then washed by sterile water for 5 times on an ultra-clean workbench.
The test strains are 9 separated strains of WB-L X, WB-PD, WB-SY, WB-YN1, WB-YN2, WB-YN3, WB-BS, WB-YM and WB3, the strains are cultured in the dark at 25 ℃ for 25 days, hyphae on a flat plate are scraped, the hyphae are soaked in water for 30min and then are shaken in a test tube or are ground by adding water, and hypha suspension is obtained after filtration.
1.2 inoculation method
The sterilized filter paper was placed in a sterile petri dish (16cm), and sterile water was sprayed on the filter paper to preserve moisture. Under the aseptic condition, the sterile cotton is soaked by 0.1 percent benzimidazole to wrap the petiole and then wrapped by tinfoil paper to play the role of fresh keeping. One peanut leaf is placed in each culture dish. Spraying the prepared mycelium suspension onto the compound leaf uniformly in equal amount, and spraying sterile water as control. The culture dish is respectively placed under the conditions of near ultraviolet light, darkness and light-dark alternation, and cultured at the room temperature of 25-28 ℃, and the morbidity and disease index are observed and recorded. Each treatment was repeated 3 times for a total of 3 replicates.
1.3 pathogenicity of the respective test strains on in vitro leaves
After the isolated leaves are inoculated with the peanut network spot disease, observing and recording the disease condition, and calculating the disease index according to the hazard classification standard, wherein the classification standard and the formula are as follows:
the conversion formula of disease level and disease index is as follows:
as shown in A-D in figure 1, the leaf inoculated with WB-SY 7D is firstly attacked, the leaf inoculated with WB-SY 7D is not attacked under the non-near ultraviolet light conditions of the other strains and WB-SY inoculation, when 9D is inoculated, the leaf inoculated with WB-L X, WB-PD under the ultraviolet condition can observe small disease spots, as shown in A-D in figure 2, when 14D is inoculated, the leaf inoculated with WB-L X, WB-PD, WB-SY, WB-YN1 and WB-YN2 under the ultraviolet condition is attacked, but the disease degree is different, the disease indexes are respectively 10.2, 9.8, 12.6, 3.5 and 4.2, when the leaf inoculated with WB-L X, WB-PD and WB-SY under the alternating light and dark conditions, the disease is not serious, the small disease spots can be observed in the initial stage in the darkness, when the disease indexes are not attacked under the alternating light and the disease indexes are respectively 10.2, 9.8, 12.6, 3.5 and 4.2, when the disease indexes are respectively inoculated with alternating light and dark spots, when the disease indexes are respectively inoculated with WB-11, 19, 19.8, 19.6, 19, 19.8, 19, 6, 4.5 and 6, 4.2, 6, 9.2, 6, and 4.2, 9.2, 9.
1.4 conclusion
Under the same illumination condition, among the 9 screened strains, the strain with the strongest pathogenicity is WB-SY, and the same strain has different pathogenicity under different illumination conditions, so that the WB-SY with the strongest pathogenicity is screened out to be used as the strain inoculated to the next living body. The pathogenic bacteria is named as Phoma arachi dicola WB-SY, the preservation number is CGMCC No.16380, the preservation place is the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation time is 11 months and 16 days in 2018.
Because the strains with high pathogenic bacteria activity and pathogenicity need to be applied in the processes of biological characteristic culture of pathogenic bacteria and screening of a disease-resistant inoculation method, the requirements on the preservation method and conditions of the strains are higher. The pathogenic bacteria WB-SY screened by the embodiment has higher hypha growth rate and spore production activity, so pathogenic bacteria are connected back to host plants at intervals, and are separated again under the condition of definite morbidity and no infectious microbes, so that the activity of the pathogenic bacteria is ensured.
Example 2 in vivo plant inoculation method
2.1 test materials
The tested peanut variety is Luhua No. 8 susceptible to net blotch, and is potted in a greenhouse for later use. The test strain selects the strong pathogenic strain WB-SY screened from the in vitro leaf pathogenicity test to screen the living plant inoculation condition.
2.2 inoculation method
Selecting healthy peanut plants with consistent growth and size, preparing spore suspensions with different concentrations, inoculating at different inoculation humidity, moisture retention time and different growth periods, culturing in a greenhouse at 25-28 ℃, and observing and recording the morbidity and disease index.
2.3 Effect of concentration of Dictyophora grisea spore suspension on index of disease of Neurospora
Inoculating the sporule suspension of the alternaria mali with different concentrations on the peanuts with the same inoculation humidity, moisture retention time and growth period, wherein the concentration of the sporule suspension of the alternaria mali for inoculation is 10 respectively3Each/ml, 104Each/ml, 105Each/ml, 106The disease indexes of the peanut net blotch disease after 30 days of inoculation are 49.1, 50.8, 56.0 and 68.2 as shown in figure 5. The result of the disease index anova shows that the disease index is worsened with the increase of the concentration of the spore suspension, and the concentration of the spore suspension is 106The index of the disease of the net blotch reaches a remarkable level (P) with other concentrations at each ml<0.05)。
2.4 Effect of inoculation humidity on index of disease of Net blotch
Under the conditions of the same growth period, spore concentration and moisture retention time, the inoculation humidity of the inoculated maculopathy bacteria is respectively controlled at 50%, 60%, 70%, 80%, 90% and 100%, and as shown in fig. 6, disease indexes of the maculopathy disease after 30d of inoculation are respectively 0, 10.8, 31.2, 46.3, 48.8 and 49.6. Disease index anova results showed that with increasing inoculation humidity, the index of disease of net blotch increased, with no significant difference between humidity 100 and 90, but with a significant difference from the remaining humidity (P < 0.05). Therefore, the inoculation humidity is selected to be between 90% and 100%.
2.5 Effect of moisturizing time on index of disease of Net blotch
Under the conditions of the same growth period, spore concentration and inoculation humidity, the moisture retention time after inoculation of the dictyosphaera is controlled to be 6h, 12h, 24h, 36h and 48h respectively, as shown in fig. 7, and the disease indexes of the disease of the dictyosphaera after inoculation for 30d are 11.3, 13.6, 34.6, 48.5 and 52.1 respectively. The disease index analysis result shows that the disease index of the net blotch is increased along with the increase of the moisturizing time, the disease indexes are not obviously different when the moisturizing time is 36h and 48h, and the moisturizing time is obviously higher than the rest moisturizing time (P <0.05), so the moisturizing time is at least 36 h.
2.6 Effect of different growth periods on index of network leaf blight disease
The alternaria alternata is inoculated in different growth periods of the peanuts, the spore concentration, the inoculation humidity and the moisture retention time are kept consistent, as shown in figure 8, disease indexes of four growth periods of early flowering period, initial flowering period, full flowering period and late flowering period are respectively 21.1, 35.0, 44.6 and 51.4 after inoculation for 30 d. Disease index anova results showed that the disease index of net blotch inoculated at the end of flowering was significantly higher than that of other growth stages (P < 0.05).
2.7 conclusion
The results of in-vivo plant inoculation tests show that the influences of different peanut growth periods, inoculation concentrations, inoculation humidity and moisturizing time on the infection of the peanut dictyosphaera are different. The higher the inoculation concentration, the higher the pathogen infection probability. Under the same inoculation condition, the infection probability of pathogenic bacteria is gradually increased along with the prolongation of the growth period of the peanuts, namely the early flowering stage is less than the initial flowering stage and less than the full flowering stage, and the phenomenon is particularly obvious when the inoculation concentration is high and the infection condition is proper. Under the same inoculation condition, the pathogenic bacteria infection probability is increased along with the extension of the leaf surface humidity and the moisture retention time, which is consistent with the phenomenon that the field peanut net blotch is serious after rain. The humidity is an important factor for pathogenic bacteria infection, and the higher the humidity is, the more sufficient the spore germinates, namely the higher the probability that the spore successfully infects plants is.
EXAMPLE 3 optimal inoculation conditions
Preparing a strong pathogenic strain WB-SY into a high-concentration spore suspension (106 spores per ml), inoculating the suspension on a peanut plant at the terminal flowering stage of peanuts, keeping the inoculation humidity between 90% and 100%, continuously preserving moisture for over 36 hours, and culturing at a constant temperature between 25 ℃ and 28 ℃. During inoculation, the seeds are preferably treated in the dark for 24-48h and then under near ultraviolet light, so that the diseases are favorably developed.
The occurrence of field diseases has mixed occurrence, the occurrence and epidemic conditions of most fungal diseases are similar, and the relationship and the influence among the mixed occurrence of various diseases are considered in the infection research of pathogenic bacteria. Pathogen invasion is a critical component of the disease process. Based on results, the peanut net blotch is easy to occur in high-concentration spore suspension, high humidity, long leaf surface moisturizing time and middle and later peanut growth periods, and factors have interaction with each other, so long as conditions are suitable, diseases can be outbreaked and prevailed in a short period.
On the basis of the conclusion, according to the application of the occurrence and prevalence rules of diseases and monitoring data in practice, disease-resistant varieties are bred, the cultivation management technology is improved, meanwhile, the relationship among factors such as the quantity of field bacteria sources, the temperature, the leaf surface humidity, the crop growth period and the like is grasped, the infection of the diseases is accurately estimated, and the occurrence trend of the whole diseases is accurately predicted so as to establish a corresponding prevention mechanism to guide the field disease control.
Claims (7)
1. The application of the pathogenic bacteria Phoma arachidicola WB-SY for rapidly predicting the occurrence trend of the peanut reticulospora disease is characterized in that the preservation number of the pathogenic bacteria Phoma arachidicola WB-SY is CGMCC No.16380, the preservation place is the China general microbiological culture Collection center, and the preservation time is 11 months and 16 days in 2018.
2. The use as claimed in claim 1, wherein the pathogenic bacteria of peanut web spot disease, Phoma arachidicola WB-SY, are prepared into spore suspensions of different concentrations, and are uniformly inoculated on healthy peanut plants with consistent growth and size under different inoculation humidity, moisture retention time and different growth periods, and are cultured at constant temperature at room temperature, and the disease incidence and disease index of each plant are observed and recorded.
3. Use according to claim 2, wherein the spore suspension has a concentration of 103Per m L-106And each m L.
4. The use according to claim 2, characterized in that the inoculation humidity is 50% -100% and the moisturizing time is 6-48 h.
5. The use of claim 2, wherein the growth period comprises pre-anthesis, beginning-anthesis, full-bloom and end-of-development.
6. Use according to claim 2, characterized in that the pathogenic bacterium Phoma arachidicola WB-SY is made to a concentration of 106Inoculating L spores suspension to healthy peanut plants with consistent growth and size at the terminal flowering stage of peanut, keeping moisture at 90-100% inoculation humidity for 36-48 h, and culturing at constant temperature of 25-28 deg.C.
7. The use of claim 2, wherein the criteria for determining the morbidity and disease index are:
level 0: the whole plant is disease-free;
level 1: the area of the lesion spots accounts for less than 1/10 of the whole area;
and 2, stage: 1/10-1/4 of the area of the lesion spots in the whole patch;
and 3, level: 1/4-1/2 of the area of the lesion spots in the whole patch;
4, level: 1/2-3/4 of the area of the lesion spots in the whole patch;
and 5, stage: the area of the diseased spots accounts for more than 3/4 of the whole area, the diseased leaves fall off seriously, and the plants die.
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