Screening And Evaluation Of Antagonistic Strains To Clubroot Of Cabbage

Plasmodiophora brassicae Woron. is a soil-borne obligate plant pathogen causing the economically important clubroot disease of crucifers, the disease epdemic can result in yield decline or no yield. In recent decades, clubroot become more serious due to scale planting of cruciferous crops.There are very few effective ways to control this disease. Chemical control could cause environmental pollution, finding one or more safe and efficient ways to prevent and control clubroot has important significance for crop production. Because biological control because has widely source and friendly to environment, so researchers pay great attention to it.P. brassicae is an obligate parasitic fungus, traditional method of screening antagonistic strains is unsuitable for it, hence more researchers screen it in the field or greenhouse, but the cycle is long.Based on the traditional screening method, this study applied the solution culture technology to observe root hair infection on cabbage roots, using antagonistic strains to inhibite the infection of root hair directly as the evaluation indicators for screening. At the same time, through inhibiting the germination of resting sporangia experiment, detecting the activity of resting sporangia and the activity test of chitinase, two excellent strains were chosen to greenhouse pot experiment to evaluate the control effect, finally making sure the above methods were scientific.1. Isolation and screening of antagonistic strains to P. brassicaeIsolation of antagonistic strains used soil dilution method. The soil was collected from different mustard fields of serious disease in Fuling, Chongqing. Total94actinomycetes and111bacterias were isolated. Streaked plate method was applied for the screening of antagonistic strains. Due to the obligate characteristic of P. brassicae, in this study, we chose Fusarum oxysporum as the indicated fungus, because the main elements of its cell wall are similar to P. brassicae’s.15antagonistic strains were obtained, their inhibition zone were from8mm to17mm, and strains SYZ2-6、 YYD3-1、GAS1-9、FLD17-6and FLB2-2can produce chitinase.2. Inhibiting the germination of resting sporangia and detecting the activity of resting sporangiaResting spores of P. brassicae obtained from mustard galls were washed and put at24℃to thaw. The soft out layer of the galls was sliced while the hard part was discarded, and then intermittently agitating the shredded gall in distilled water at high speed for7min and filed through2,8layers of Gauze Wrap, then got centrifuged. Surface sterilization of resting spores was performed according to Asano’s method. The spore suspension was prepared with sterile distilled water and stored at4℃. Through the solution culture to collect root exudates, fermented liquid of antagonistic strains was collected through liquid cultivation with the agitator.The resting spores were cultured at pH6.2,24℃in the nutrient solution containing root secretion, add the fermented liquid on scale. As the time going, different treatments’germination rate increase gradually, but only treatments GAS1-9YYD3-1、 FLD17-6、Itb295and XF-1show significant difference.At48hours after mixing up the resting spore suspension and fermented liquid, using Evan’s Blue to dyeing the mixing liquid, treatments GASl-9、 YYD3-1、 FLD17-6and SYZ2-6made the resting spores lost activity.Using Evan’s Blue to dyeing the saved resting spores at4℃.As the time going, more and more resting spores lost their activities, in the first10days, there was no significant difference, but after15days, the activity decline got much significant.3. Antagonistic strains inhibiting the infection of root hairCabbage seeds after4days accelerating germination, seedings were transplanted to10mL centrifuge tubes containing Hoagland nutrient solution, and the tubes were rapped with black tape to block light and prevent algal growth. After5days, mixing resting spore suspension and fermented liquid, the optimal conditions was pH5.5, temperature was from20to24℃, and an inoculation concentration of1×107spores/mL. At the days7th,10th and15th, the hair infection rate of seedings was investigated randomly. It was found in the study that treatments GAS1-9、 YYD3-1、FLD17-6、SYZ2-6and XF-1 could inhibit the infection of root hair and showed significant difference.4. The experiment of greenhouse potStrains GAS1-9and FLD17-6were chosen in the study. In the experiment of greenhouse pot, the soil with the resting spores of pathogen to get dieases cabbage.Resting spores of P. brassicae obtained from mustard galls got rotted for5days at24℃, and then the shredded gall was intermittently agitated in distilled water at high speed for7min, regulating the pH6.2, then mixing it with sterile soil, making the soil concentration reach8×10spores/g. Seedings were prepared for transplanting to soil with pathogens, watering fermented liquid at the same time.At the day of60th, all cabbages plants were pulled out to investigate the club and the disease incidence. The test showed that when watering a small amount of fermented liquid, it could only reduce the disease index, but when a large number of fermented liquid was watered, the dieases rate and disease index both decreased much significantly.