| Clubroot caused by the obligate parasite Plasmodiophora brassicae Woroninis a soil-borne disease of plants in the Brassicaceae family.It has been spreading in the fields over many countries of the world,thus posting an emerging threat to the quality and yield of cruciferous crops.P.brassicae can persist in the soil for up to 15 years as resting spores by avoiding all kinds of management practices and chemical fungicides.Therefore,it is difficult to control the clubroot disease.Diagnosis of P.brassicae at the early stages is very important.This study aimed at building a practiccal clubroot disease detection system based on routine molecular methods.Meanwhile,we explored near infrared spectroscopy to assessthe disease and to detect the pathogen Moreover,we studied the antimicrobial mechanisms of the biological drug camptothecin(CPT)against P.brassicae and Acidovorax avenae subsp.avenae(Aaa)RS-2 strain.The main results were presented as follows:1.Under the light microscope,we observed clearly the morphological characteristics(shape and size)of resting spores of P.brassicae in the diseased roots of cabbages collected from the fields in Changyang County of Hubei province.Different forms of P.brassicae in the root hairs of the diseased cabbage seedlingx(cultivar: Kaixuanyou pakchoi)were observed,including primary zoospores in 2-dold seedlings,the primary plasmodium in 2 to 4-d-seedlings,and zoosporangia in 8-d-old seedlings The method is simple,rapid and economic.It can be used to detect P.brassicae infection at theearly stages of plant growing.2.By comparing the CTAB method and the potassium acetate extraction method to extract DNA from diseased plant samples and screen the PCR conditions and primers,a PCR detection method for P.brassicae-infected plants was established.The detection threshold for the template DNA reached as low as 10-4 ng DNA/?L.The optimized method showed consistent detection performance when applied to a series of different samples,including 4 Brassiceace plants from different fields,8 indoor Brassiceace seedlings vaccinated with disease soil,and clubroot disease infected soil samples.3.A novel P.brassicae's infection analysis and detection method was explored based on near infrared spectroscopy analysis over canola root(leaf)samples.By employing derivative spectra analysis and principal component analysis to examine near infrared spectra of the root(leaf)samples,useful structures(information),which are helpful to the clubroot detection,were discovered in the principal component feature space of the 1-order and 2-order derivative spectra.By using support vector machine for clubroot detection,the accuracy rate is up to 99.71% with the 0-order and 2-order derivative spectra of root samples of oilseed rape when the Sigma element was set to 0.4,and 99.28% with the 0-order and 1-order derivative spectra of leaf samples when the Sigma element was set to 3.5 to 4.0.The above-mentioned methods are helpful for detection of P.brassicae at the early stages of disease development and for management of clubroot disease.4.Camptothecin(CPT)was investigated for suppression of Aaa RS-2 strain.The results showed that the CPT at 0.50 mg/mL showed inhibition by 73.9% to growth of Aaa RS-2 strain(pH 7.5,8 h).Furthermore,inhibition of CPT against Aaa RS-2 strain was further verified by a series of related experiments such swarming motility,live/dead cell staining,transmission electron microscopy(TEM)and measurement of the secreted hcp by ELISA,and also verified with the differential expression analysis of secretion system-related(porin gene)and topoisomerase-related genes(gyrB gene).These results are helpful for application of CPT to control Aaa RS-2 on rice. |
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