CN102037965B - Application of chitosan in prevention and treatment of rice bacterial leaf streak - Google Patents
Application of chitosan in prevention and treatment of rice bacterial leaf streak Download PDFInfo
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- CN102037965B CN102037965B CN2011100073121A CN201110007312A CN102037965B CN 102037965 B CN102037965 B CN 102037965B CN 2011100073121 A CN2011100073121 A CN 2011100073121A CN 201110007312 A CN201110007312 A CN 201110007312A CN 102037965 B CN102037965 B CN 102037965B
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Abstract
The invention discloses an application of chitosan in prevention and treatment of rice bacterial leaf streak. A chitosan preparation provided by the invention can significantly reduce the incidence of the rice bacterial leaf streak and the size of disease spots, significantly improve the activities of phenylalanine ammonia lyase, peroxidase and polyphenol oxidase in rice seedlings and further enhance the resistance against diseases of rice. The chitosan preparation is simple and easy for application in the prevention and treatment of the rice bacterial leaf streak, and the cost is lower.
Description
Technical field
The present invention relates to the biological control field of plant protection technology, relate in particular to the application of shitosan in the control bacterial leaf streak of rice.
Background technology
The slice disease is also a kind of important bacterial disease on paddy rice, is takeed for paddy bacterial stripe disease (Bacterail stripe of rice) by Philippine's reported first and quilt and reaches the several years in 1918.Bacterial leaf streak of rice is at first with Xanthomonas translucents f.sp.oryzea, and mistake is thought bacterial leaf-blight with it.Thereafter, Goto contrasts to several mutation of this germ and paddy class blackhull disease bacterium, and research is rear to be proposed with Xanthomonas translucents f.sp.oryzea replacement Xanthomonas oryzicola. subsequently, it is final that Bradbury (1971) is modified to it again Xanthomonas translucents f.sp.oryzicola., and Dye renames the pv.oryzicola into Xanthomonas camperstris.Nineteen ninety Swing etc. admits this kind of rice Xanthomonas (Xanthomonas oryzae) again according to phenotype, genotype, the chemical classification data of cause of disease, and this kind comprised and cause respectively the sick Xanthomonas campestris bacterial leaf spot pathological form (Xanthomonas oryzae pv.oryzae) of bacterial leaf-blight and slice and 2 mutation of bacterial stripe pathological form (Xanthomonas oryzae pv.oryzicola).So far the status of this bacterium just is able to firmly.
Being the fourth-largest rice disease of China after rice blast, banded sclerotial blight and bacterial leaf-blight at present, is also quarantine disease unique on China paddy rice.Due to susceptible in recent years hybrid rice area popularization and southern numerous, the allocation and transportation of seed rice, cause this sick rapid spread, lesion constantly enlarges, and area occurs throughout the year more than 4,600,000 hectares.Now become the particularly great disease on long-grained nonglutinous rice of paddy rice.Owing to lacking good disease-resistant variety, this disease has surpassed bacterial blight of rice in the harm on the ground such as China Hunan, Guangdong, Guangxi and Hainan.Up to the present, also do not find that effective chemicals prevents and treats bacterial leaf streak of rice, apply wider copper agent bad to the effect of this disease yet.Therefore inventing new effective biochemical reagents resists this disease and is necessary very much.
Shitosan is by the product of chitin after deacetylated processing; usually deacetylation is called shitosan greater than 55% chitin; its formal name used at school is poly glucosamine; chemical name (1; 4)-2-amino-2-deoxidation-β-D-Glucose, extensively be present in the cell wall of the animal shells such as shrimp, crab and algae, fungi.
Shitosan is the wider natural materials of a kind of antimicrobial spectrum, and bacterium, saccharomycete and fungi etc. are all had in various degree inhibitory action.Shitosan has good Bc, biodegradable and having no side effect, its molecule contain-OH and-NH
2-active group, easy and gas chromatography reacts, and is widely used at present the fields such as industry, agricultural, medicine, chemical industry, environmental protection and water treatment.
Summary of the invention
The invention provides the application of a kind of shitosan in the control bacterial leaf streak of rice, solved the problem of few, the anti-heterodyne of existing medicament.
The application of shitosan in the control bacterial leaf streak of rice.
The present invention also provides the application of shitosan in improving Rice Resistance bacterial stripe performance, reduce the bacterial leaf streak of rice incidence of disease and degree of disease and suppressing bacterial leaf streak of rice pathogen (Xanthomonas oryzae pv.oryzicola) growth.
The present invention also provides a kind of medicament of preventing and treating the vegetative bacteria cecospora spot, and this kind medicament, respectively take shitosan as Main Ingredients and Appearance, adds conventional pharmaceutical excipient,, as water, acetic acid etc., makes the formulations such as aqua, pulvis.
The deacetylation of described shitosan is preferably greater than 85%, easily obtains, and cost is low.
The present invention also provides a kind of preparation method of above-mentioned medicament, comprises the following steps:
It is in 1% acetic acid solution that the shitosan powder is dissolved in percent by volume, then adds water and is mixed with the chitosan solution that concentration is 5mg/mL.NaOH regulates pH to 6.0, makes shitosan dissolve, and its consumption and concentration can be adjusted as required.
Adopt above-mentioned chitosan solution not only in vitro, can suppress the growth of Xanthomonas oryzae pv.oryzicola (Xoc), and can effectively reduce incidence and the order of severity of cecospora spot on rice living body.
Shitosan is to paddy rice nonhazardous effect, and can significantly reduce the incidence of disease and the scab diameter of bacterial leaf streak of rice, can also strengthen the performance of its bacteria resistance cecospora spot simultaneously.
Description of drawings
Fig. 1 is the impact of the chitosan solution of variable concentrations in embodiment 3 on Xoc BLS-01 growth;
Fig. 2 is that in embodiment 5, concentration is the impact of the shitosan of 0.20mg/mL on the rice disease incidence;
Fig. 3 is that in embodiment 5, concentration is the impact of the shitosan of 0.20mg/mL on paddy rice scab diameter;
Embodiment
Embodiment 1 prepares chitosan solution
It is in 1% acetic acid that the deacetylation shitosan powder that is 85% is dissolved in percent by volume, is mixed with the chitosan solution that concentration is 5mg/mL, with NaOH, regulates pH to 6.0, at room temperature shakes 24 hours, with sterilizing 20 minutes of 121 ℃ of high-pressure sterilizing pots, stand-by.The contrast that the aseptic deionized water of pH as 6.0 processed as shitosan is used in experiment.
Embodiment 2 in vitros detect the impact of shitosan on Xoc BLS-01 growth
The strain X oc BLS-01 of the present embodiment is provided by Zhejiang University's biotechnology research, and this bacterial strain is test material, and its characteristic is on not impact of result of the test.The counting method that adopts the present embodiment detects the impact of shitosan on Xoc BLS-01 growth, and concrete grammar is as follows:
Xoc BLS-01 in agar medium, cultivated 48 hours, and with sterile water, made bacteria suspension (10 for 28 ℃
9CFU/mL).10 times of gradient dilutions, draw the Xoc bacteria suspension 10 μ L of each concentration to flat board, and the flat board after processing is placed in 28 ℃ of incubators and cultivated 48 hours.The contrast that the aseptic deionized water of pH as 6.0 processed as shitosan is used in experiment, and six repetitions are established in each processing.Experiment repeats twice.
Bacteriostatic activity when embodiment 3 detects the shitosan variable concentrations
Adding aseptic deionized water to be mixed with concentration in the chitosan solution of the 5mg/mL that embodiment 1 makes is 0.05,0.10, and the chitosan solution of 0.20mg/mL.Inoculate throughout Xoc bacteria suspension (10 in the reason
8CFU/mL), under 160r/min, 28 ℃ of conditions, shaken cultivation is 4 hours, collects sample and press embodiment 2 detection thalline numbers.The contrast that the aseptic deionized water of pH as 6.0 processed as shitosan is used in experiment.
As shown in Figure 1, chitosan solution shows significant bacteriostatic activity to Xoc BLS-01 when variable concentrations, and along with the increase bacteriostatic activity of concentration strengthens gradually.Compared with the control, chitosan solution cell quantity when concentration 0.05mg/mL 1.88Log CFU/mL that descended, the bacterial number 2.05Log CFU/mL that descended when concentration 0.10mg/mL, yet bacterial number has descended 3.96 when 0.20mg/mL, so chitosan solution bacteriostatic activity when 0.2mg/mL is the highest.
Embodiment 4 detects shitosan at the bacteriostatic activity of different vaccination under the time
The chitosan solution 100 μ L of the 5mg/mL that absorption embodiment 1 makes, adding 4.9mL aseptic deionized water compound concentration is the chitosan solution of 0.20mg/mL., with embodiment 3, inoculate throughout Xoc bacteria suspension (10 in the reason
8CFU/mL), shaken cultivation 0,2,4,6 under 160r/min, 28 ℃ of conditions, detect the growing state of Xoc BLS-01 after 12,24 hours.The contrast that the aseptic deionized water of pH as 6.0 processed as shitosan is used in experiment.
Result shows, inoculation Xoc cell quantity 0.06Log CFU/mL that descended after BLS-012 hour in aseptic deionized water in contrast, and next the bacterium amount keeps stable.And in the processed group chitosan solution, the Xoc bacterial number significantly descends, and reduces gradually in 0～12 hour, and bacterial number increased to some extent than 12 hours in 24 hours.Compared with the control, inoculate after 2 hours, the cell quantity in chitosan solution has reduced 2.74Log CFU/mL, and after inoculation 12 hours, the cell quantity in chitosan solution has reduced 5.87Log CFU/mL (in Table 1).
The bacteriostatic activity of table 1 chitosan solution different vaccination time to Xoc BLS-01
Inoculation Xoc BLS-01 on embodiment 5 shitosan treated water rice seedling
Measured shitosan to the incidence of rice streak and the inhibitory action of the order of severity under greenhouse experiment.Concrete grammar is as follows:
1, rice paddy seed is seeded in the plastic basin (diameter 8.5cm, high 7.5cm) that fills paddy soil, 10 seeds of every basin, every processing repeats 4 times;
2, Xoc bacterial strain BLS-01 incubated overnight in the nutrient agar liquid nutrient medium, and regulate concentration to 10
8CFU/mL;
3, after 5 week, with the chitosan solution that concentration is 0.20mg/mL, be sprayed onto rice seedling, every basin sprays 5ml, with the equivalent sterile purified water, sprays and does check experiment;
4, after air-dry, the bacterium liquid of above-mentioned steps 2 is sprayed on the rice seedling that is inoculated in step 3, every basin sprays 5mL;
5, cover all seedling 24h that inoculated to keep high humidity environment with plastic sack, cultivate in the growth case, temperature setting is set to 28 ℃, relative moisture 90%, and every day, light dark replaced, illumination 16h, dark 8h;
6, inoculate rear 7d, observe and record the average diameter size of disease incidence and scab, whole test repeats 2 times.
Result of the test shows: in the control treatment of not spraying shitosan, rice seedling shows the disease symptom after inoculation Population of Xanthomonas Oryzae Pv 5d, and after shitosan was processed, 1d appearred having postponed in the disease symptom.In control treatment, rice seedling 7d disease incidence of disease after inoculation reaches 81.46%, and average scab diameter is 29.6mm (seeing Fig. 2 and Fig. 3); Yet after shitosan was processed, after rice seedling inoculation 7d, the disease incidence reduced 43.80%, and the scab diameter reduces by 53.61% (seeing Fig. 2 and Fig. 3), significant difference compared with the control.And after shitosan was processed, the incidence of rice streak and scab diameter kept stable after inoculation 7d.Rice seedling without inoculation cecospora spot germ does not show the disease symptom.
Embodiment 6 shitosans are processed the impact on the rice seedlings enzymic activity
The mensuration concrete grammar of rice seedling enzymic activity is as follows:
According to embodiment 5 rice leafs at inoculation 0,12,24,48,72,96 after Population of Xanthomonas Oryzae Pv, gather after 120h, every basin rice seedling gathers 1g blade (fresh weight) ,-70 ℃ of preservations.
2. enzyme liquid extracts: every part, the sample that gathers in 1 is taken the 0.5g rice leaf, add 3ml phosphate buffer (50mmol/L; PH 7.8; Contain 0.2mol/L EDTA, 1%PVP) grind evenly.The centrifugal 20min of 12000rpm.Get supernatant and be used for measuring phenylalnine ammonialyase (PAL), the activity of peroxidase (POD) and polyphenol oxidase (PPO).
3.PAL active mensuration: add successively borate buffer (pH8.8) 1mL of 0.05mol/L in test tube, distilled water 1mL, the enzyme liquid 1mL in above-mentioned steps 2.The not enzyme-added liquid of blank, the enzyme liquid of crossing with boiling water boiling replaces.Evenly mix, add the HCl cessation reaction of 1mL 6mol/L after 30 ℃ of water-bath 30min.Survey the light absorption value OD under the 290nm wavelength
290OD in per minute take every gram fresh weight changes 0.01 as a unit of enzyme activity.
4.POD active mensuration: add successively 0.05mol/L phosphate buffer 2.9mL in test tube, 2% hydrogen peroxide 1mL, the enzyme liquid 1mL in above-mentioned steps 2,0.05mol/L guaiacol 1mL.The not enzyme-added liquid of blank, the enzyme liquid of crossing with boiling water boiling replaces.Colorimetric under the 470nm wavelength immediately after evenly mixing, measure 2min.
5.PPO active mensuration: add successively 0.05mol/L phosphate buffer 1.5mL in test tube, catechol 1.5mL, the enzyme liquid 20 μ L in above-mentioned steps 2.The not enzyme-added liquid of blank, the enzyme liquid of crossing with boiling water boiling replaces.Evenly mix, add the HCl cessation reaction of 1mL 6mol/L after 30 ℃ of water-bath 30min.Survey the light absorption value OD under the 420nm wavelength
420OD in per minute take every gram fresh weight changes 1 as a unit of enzyme activity.
Experimental result shows: through shitosan treated water rice seedling, starting stage and the contrast of the activity of three kinds of enzymes after the inoculation Population of Xanthomonas Oryzae Pv do not have obvious difference, but after inoculation Population of Xanthomonas Oryzae Pv 24h all significantly higher than contrast, and reach top when 48h, at this moment, shitosan treated water rice seedling PAL, POD and PPO activity have improved 21.02%, 6.74% and 19.71% (in Table 2) than contrast respectively.
Table 2 shitosan is processed the impact on the paddy rice enzymic activity
Reach a conclusion thus: shitosan can significantly improve PAL in Rice Seedling Leaves, the activity of POD and three kinds of enzymes of PPO, thus paddy rice is strengthened the resistant function of cecospora spot, reach the effect of control bacterial leaf streak of rice.
Claims (4)
1. the application of shitosan in the control bacterial leaf streak of rice.
2. the application of shitosan in improving Rice Resistance bacterial stripe performance.
3. the application of shitosan in reducing the bacterial leaf streak of rice incidence of disease and degree of disease.
4. the application of shitosan in suppressing bacterial leaf streak of rice pathogen (Xanthomonas oryzae pv.oryzicola) growth.
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| CN101177671A (en) * | 2007-12-05 | 2008-05-14 | 南京农业大学 | Biocontrol bacteria strain preventing and curing plant disease |
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| CN101177671A (en) * | 2007-12-05 | 2008-05-14 | 南京农业大学 | Biocontrol bacteria strain preventing and curing plant disease |
Non-Patent Citations (2)
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| 壳聚糖对植物病害的抑制作用研究进展;马鹏鹏等;《天然产物研究与开发》;20011230;第13卷(第6期);82-86 * |
| 马鹏鹏等.壳聚糖对植物病害的抑制作用研究进展.《天然产物研究与开发》.2001,第13卷(第6期),82-86. |
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