Oilseed rape (Brassica napus L.) plants with typical club root symptoms were detected on two farms in the La Araucanía region (37°35′ to 39°37′ S), southern Chile. In 2010, affected plants were found in large areas throughout three fields on a single farm and disease incidence ranged from 30 to 100%. In 2013, plants with club root were found in one field on a different farm. Disease incidence in the affected areas was 30%. In both cases, affected plants showed root swellings or distortions, but no aerial symptoms were evident. Cross-sections from symptomatic roots were observed under light and fluorescent microscope and compared to healthy roots. The presence of plasmodia with resting spores in the root tissue pointed to Plasmodiophora brassicae Woronin as the causal agent. Pathogenicity was evaluated in the greenhouse. Inoculum was prepared by grinding 10 g of fresh galled roots in sterile water. The macerated tissue was filtered through sterile cheesecloth and the spore suspensions were adjusted to 1 × 107 spores/ml. Seeds of oilseed rape cv. Imminent were germinated and 5-day-old seedlings were transplanted in 250-ml pots (4 seedlings per pot). The soil surrounding the base of each seedling was inoculated with 1 ml of spore suspension. One pot received no inoculum and was used as a control. Pots were watered regularly. After 45 days, the inoculated plants showed root swelling similar to that observed in the field, whereas no symptoms were observed on the roots of the control plants. Specific PCR detection for P. brassicae was performed with pairs of primers TC1F/TC1R and TC2F/TC2R, according to the protocol described by Cao et al. (1). Total DNA was obtained from old galled roots collected in the field and galled roots from plants of the pathogenicity test, using the E.Z.N.A HP Plant DNA mini kit (Omega Biotek). Amplicon sizes of 548 and 519 bp, respectively, were obtained for each primer set, which is consistent with that reported by Cao et al. (1). Seed contamination with P. brassicae in the same seed lot used to sow the commercial field of 2013 was evaluated using the PCR method described above. Washing protocols to collect resting spores from seed was based on Rennie et al. (2). Total DNA was extracted from the resting spores pellet that had been ground in liquid nitrogen, using E.Z.N.A HP Plant DNA mini kit. PCR was performed on undiluted and diluted (1/10, 1/100, 1/1000, and 1/10000) DNA. Total DNA from a plant with gall roots where plasmodia were observed and a plant with healthy roots were used as positive and negative control, respectively. A 548-bp amplicon was amplified in the 1/10 and 1/100 dilutions with the TC1F/TC1R primers only indicating that the pathogen may have been present in the seed lot. In Chile, club root symptoms on B. napus were described in 2008 (3), though no indication of location or incidence was given. To our knowledge, this is the first confirmed case of club root disease in an oilseed rape field. This finding could prelude new cases and possibly an outbreak of club root disease on oilseed rape, jeopardizing this important crop of southern Chile. Oilseed rape production in Chile relies on imported seed of hybrids and parental materials. Although seed contamination with P. brassicae is thought to play a minor role in the epidemiology of the disease, we cannot ignore the possibility that the occurrence of this disease in 2013 may have been associated with the use of contaminated seed.
References: (1) T. Cao et al. Plant Dis. 91:80, 2007. (2) D. C. Rennie et al. Plant Pathol. 60:811, 2011. (3) Rina Acuña P. Compendio de Fitopatógenos de Cultivos Agrícolas en Chile. Servicio Agrícola y Ganadero, Santiago, Chile, 2008.