Study On The Standardization Of Cruciferae Clubroot Inoculation Indoor

Cruciferae clubroot is one of important worldwide diseases, caused by Plasmodiophora brassicae Woron., a kind of obligate parasite. In recent years, the occurrence of clubroot gets more and more serious, and the control difficulty increases much. Application of resistant varieties is regarded as an effective way for clubroot disease control. Nevertheless, the problems for artificial inoculation methods indoor and standards of disease grading must be considered adequately. Based on the current situation that there is no agreed system for inoculation methods and grading standards, the follwing researches were carried out for the purpose to establish one scientific system about cruciferae seedlings inoculation indoor, which would be probably adopted in related studies.1. Identification of resting spore germinationStructural improvements have been made for ordinary microscope, it has a differential interference effect, and made comparisons between the effect and3kinds of staining methods for differential effects on germination of resting spores, the results showed that the improved ordinary microscope could distinct better between germinated and ungerminated resting spores, which is important for researching germination conditions and standardization of inoculation.2. Preparation of resting spore suspensionThe test was conducted to detect the impact of different rot temperature and time on mustard pathogenesis, the results showed the inoculated mustard got the highest disease incidence with the clubroot rotted for5days at25℃, which indicated that this rot process could strengthen resting spore’s virulence.The test was conducted to detect the effect of diluted concentration and mixing time on the concentration of resting spores suspension. The results showed that the ratio of clubroots and sterile water was at1:5, mixing7min by winepress, filting with a layer of cheesecloth firstly, and then with4layers got the lowest loss rate of the spores, and could significantly reduce tissue residue of host plants. The final concentration after filtration could be measured precisely, and the concentration of resting spores got to the top quantity.3. Comparison and optimization of inoculation methodsTwo methods, different concentrations, weights, diameters of soil cores and growth stages for inoculation, were combined for studying the impact on disease incidence. Results indicated that the morbidity of soil mixing with the resting spores of pathogen (77.50%) was lower than root dipping with P. brassicae suspension (87.50%), but more stability was found in the former. The optimal concentration of soil core was8×107spores/g soil. The optimal weight was20g, the optimal diameter was25mm. Growth stages for inoculation at the first day (73.33%) was significantly lower than the incidence of inoculation at15days seedlings (90.00%).Comparison of disease incidence in the period of40,50,60,70days after inoculation, the result showed that the morbidity at the50th day was much higher and more stable, which could be the best time to investigate disease after inoculation.4. Disease investigation and establishment of grading standard indoorThe indicators for reflecting the disease severity was selected and measured. The correlation was compared between the fresh weight of overground part, whole-plant fresh weight, and their ratio with four types of refered grading standards. The results of the study showed that ratio between the overground part and whole-plant fresh weight and the classification standard was significantly relevant. The weight ratio of two parts could accurately reflect the severity of seedling diseases.Several groups were divided according to the ratio between overground part and whole plant fresh weight of100mustard seedlings inoculated. The symptom of mustard seedlings was described in each group, and two grading standards Li Yan and Guo Xianghua were analyzed in the study. The grading standard was modified, which was as follows. Level0:no clubroot. Level1:clubs on lateral roots. Level3:the diameter of main root is enlarged two times less than the stem, the ratio of neoplasm lateral roots less than50%. Level5:the diameter of main root is enlarged three times less than the stem, the ratio of neoplasm lateral roots is50%-75%. Level7:the diameter of main root is3-4times larger than the stem or the ratio of neoplasm lateral roots is over75%. Level9:the diameter of main root is4times more than the stem of host seedlings.The adaption analysis was conducted for comparison of the four kinds of common grading standard and the new standard proposed in the research. The results indicated: the most significant correlation (R=-0.641) was found in the new standard with mustard growing15days, soil inoculation mixing with pathogen suspension. The grading standards proposed and refered had highly relevant. It suggested that new classification standard under the above inoculation conditions could mostly reflect the change of disease and also be applicable to most of cruciferae crops.