Establishment Of Plasmodiophora Brassicae Single-spore System

Over the last hundred years,Crucifer clubroot,which can cause serious injuries and exert significant influences on the yield of cruciferae,has been the main soil-borne disease of cruciferae.In our country,with a high spreading speed and a wide distribution,clubroot needs overall effective prevention and control without delay.Nowadays,the common prevention methods of cruciferae disease mainly include chemical prevention,biological control,control in the field,breeding for disease resistance and so on.Among those methods,the most convenient and effective one is the breeding of resistant varieties.But clubroot is a kind of bacteria characterized with the typical feature of physiological race,and blended race exists in Plasmodiophora brassicae tumor,therefore the physiological race identified with the Plasmodiophora brassicae as inoculums source is "pathotype",not the physiological race.Physiological races vary in different distributes and environments,leading to a great difficulty in the application of breeding for disease resistance and disease resistant cultivar.Aiming to establish Plasmodiophora brassicae single-spore system,the research tries to acquire the single-spore system through single-spore isolation,host propagation,etc.,verified by protocols in Molecular Biology.Meanwhile,some related researches are carried out:exploring the best way to preserve resting spores,the time it takes to be infected and the forming process of resting spores through the observation of the effectiveness of Plasmodiophora brassicae under laboratory conditions;increasing the resting spores' germination rate in the process of inoculation through the study of non-host plant;cultivated the single-spore system by improving the technology of single spore separation process,and detected the propagation clubroot by PCR amplification.1.The observation of the inoculation concentration and preserve observation of crucifer clubroot under laboratory conditionsThe test reveals that spore germination still takes place in the 4? surroundings.Thus the clubroot samples collected in the field are best preserved in the form of root nodule in the refrigerator with a constant temperature of-20?.In proper seasons,the stages of infection of inoculated plants in the lab are supposed to conform to the field infection;the over-high concentration of the inoculation can affect the plants' early growth thus failing to supply sufficient nutrition to clubroot.The inoculation concentration forms a negative correlation with root nodule's size.In order to get the complete resting spores,the most suitable concentration should be 1×106.2.The exploration of the role non-host plants play in the resting spore germinationImmersing the resting spores of Plasmodiophora brassicae in the root solutions of Chinese cabbage,Lolium perenne,Allium porrum,Capsicum annuum.and Raphanus sativus,we got the respective germination rates 7 days later:93.00%,75.33%,50.67%,78.67%and 69.00%,with the Lolium perenne ranking the top,proving that some non-host plants' root solutions can facilitate the germination of resting spores.Therefore,the root solutions of Lolium perenne can replace those of host-plants to work as germination accelerator in isolating single spore.3.Establishment of Plasmodiophora Brassicae Single-spore systemThrough the improvement of single-spore isolation technique,spore suspension procession,cultivating inoculated plants and other aspects,22 single-spore tumors of cruciferous root disease are acquired from different places;with three times propagation of one of these single spore tumors,65 root tumors are finally achieved,successfully forming a single-spore system.4.Plasmodiophora brassicae PCR amplification of the single-spore tumor and the sequence analysisBy using specific primer for PCR amplification of the single spore tumor derived from two propagations and electrophoresis,we got the result that the root tumor consists of Plasmodiophora brassicae resting spore.The amplification products are sequenced and are found that the two sequences are almost identical,which indicates that the process of single-spore propagation did not polluted by other Plasmodiophora brassicae.