Development of Inoculation techniques of Plasmodiophora brassicae

The Effect ff off Inoculation i Methods on Growth G Stages S g off Plasmodiophora brassicae Causing Club Root of Broccoli Md Nazrul IslamA & David I. GuestB A Postgraduate Research Student and BProfessor, Department of Plant Pathology, Faculty of Agriculture, Food and Natural Resources, The University of Sydney, Australia. Background Clubroot, an important worldwide root disease of cruciferous crops, is caused by the protist, Plasmodiophora brassicae Woron. - a microscopic, obligate endoparasite (Woronin, 1878). The disease cycle of the pathogen starts with infection of root hairs by primary zoospores, which have germinated from resting spores. Subsequently plasmodia and zoosporangia are formed in the root hairs, followed by the production of secondary zoospores (Ingram and Tommerup, 1972). At later stages, infection of the root cortex causes galls formation and resting spores production. However many questions about the infection process remain unanswered. The aim of this project is to develop a reliable in vitro inoculation method to facilitate further study of this pathogen. Materials and Methods Hybrid Broccoli (Brassica oleraceae Var. italica) cv. Marathon F1 was grown in UC potting mix. P. brassicae was obtained from frozen clubroots stored at -20°C. Inoculation method method-1:1: Resting spore suspension was prepared by macerating club roots, roots then filtering the homogenate and centrifuging. centrifuging Twenty μL spore suspension p ((107/ml)) was injected j into the root zone of seedlings. g Inoculation method-2: UC potting mix was mixed with inoculum slurry by blending infected club roots and distilled water (3:1) 24 hours prior to seed sowing. i Microscopic p observation of aniline-blue stained roots was p performed every y 4 days y until 36 days y after inoculation to determine % root hair infection,, % of different zoosporangial stages (primary plasmodia, mature zoosporangia, partially evacuated zoosporangia and fully evacuated zoosporangia) and to assess the secondary invasion in the cortex region, time of gall initiation and finally weighted club roots. 1 3 4 Results and Discussion Spore p Suspension p Method Soil Inoculation Method 100 %) ed roott hair (% Higher rates of infection resulted when inoculum was 90 80 mixed i d with ith potting tti mix. i The Th infection i f ti process followed f ll d 70 60 the published sequence. sequence mber of infecte 50 40 30 Four days after inoculation, inoculum mixed with soil 20 resulted lt d in i 45% infected i f t d hair h i rootst while hil only l 20% off 10 Num 0 hairy roots became infected when the inoculum was 4 8 12 16 Days After Inoculation (DAI) injected into root zone. After 16 days, 89% and 76% of hairy y roots were infected with the p primaryy zoospores p by y Figure 11. R Fi Roott hhair i iinfection f ti ffollowing ll i 2 inoculation i l ti 5 6 soil inoculation and injection of spore suspension, techniques. respectively (Figure 1 & 2). Eight g days y after inoculation, inoculum mixed with soil Spore Suspension Method Soil Inoculation Method 120 resulted in 56% matured sporangia while only 45% of %) 100 cal Cell Infecttion (% sporangia matured when the inoculum was injected into 80 the h root zone (Figure (Fi 3) After 3). Af 16 days, d 82% off 60 sporangia were completely evacuated in mixed- mixed 40 inoculum while 48% of sporangia had evacuated when inoculum, Cortic 20 Figures g -3. Matured zoosporangia p g in root hair, 4. Cortical cell infection with inoculated by y injection. j secondary plasmodia, 5. Multinucleate secondary plasmodium infected in 100% 0 cortical cells and 6. 6 Typical gall symptom at 36 Days after soil inoculation. inoculation One hundred percent cortical cell infection was 12 16 20 24 28 32 36 observed 28 days after soil inoculation, inoculation while only 20% Days After Inoculation (DAI) cortical cell infection resulted 28 days y after inoculum Figure 22. C Fi Cortical ti l cell ll iinfection f ti ffollowing ll i 2 iinoculation l ti injection and 90% after 36 days (Figures 4 & 5). Club techniques. roots are evident 36 days after inoculation in the i inoculum l i mixed is i d with i h the h potting i mix i (Figure ( i 6). ) Conclusion Soil inoculation with infected root slurry is more efficient than inoculum injection into the root zone. Root infection and symptom development is faster. F h studies Further di will ill examine i details d il off sequential i l developmental d l l stages off P. P brassicae b i b h in both i glass l house h and d field fi ld conditions. di i References Ingram, D. C. & Tommerrup, I. C., 1972. The life history of Plasmodiophora brassicae Woronin. Proc.R. Soc., London Ser.B 180: 103-112. Woronin M. Woronin, M S. S 1878. 1878 Plasmodiophora brassicae. brassicae Urheber der Kohlpflanzen-Hernie. Kohlpflanzen Hernie Jarhrb.Wiss.Bot.11:548-574. Jarhrb Wiss Bot 11:548 574 (English transilation by Charles Chupp Chupp. Phytopathological Classics No No. 4 American Phytopathological Society,Ithaca, New York.1934).