Studies on the Role of the Glucosinolate-Myrosinase System in Resistance of Oilseed Rape to 'Sclerotinia sclerotiorum'

The glucosinolate-myrosinase (GSL-M) system in oilseed rape and other members of the family Brassicaceae produces toxic products which can limit fungal pathogen attacks on the host. The role of this system in resistance of oilseed rape to 'Sclerotinia sclerotiorum', causal agent of stem rot, was investigated. Mustard powder was used as a GSL and myrosinase source in bioassays. The effect of toxic volatiles derived from hydrolysis of glucosinolates was observed as inhibition of fungal growth. Oxalic acid, a pathogenicity factor of the pathogen, did not affect production of toxic volatiles and inhibition occurred only at very acidic pH levels, regardless of the presence of oxalic acid. This indicated that oxalic acid at physiological concentrations and pH did not affect the GSL-M defense system. Exposure of 'S. sclerotiorum' colonies to inoculated leaves or leaf discs of host species or cultivars revealed that volatiles derived from infected leaf tissues have a toxic effect. This suggested that the GSL-M system is activated during infection of leaves and disease development. Freeze-dried powders of shoot parts of brassica species and cultivars including leaf, petiole, and stem demonstrated significant differences in producing toxic volatiles through their inhibitory effects on 'S. sclerotiorum' mycelial growth 'in vitro', indicating that GSL contents in brassica species and even cultivars have different potentials for toxic products. Tolerance of 'S. sclerotiorum' to toxic volatiles derived from mustard powder and also synthetic isothiocyanates developed during repeated exposure of mycelium to these biocidal chemicals

Toxicity of hydrolysis volatile products of 'Brassica' plants to 'Sclerotinia sclerotiorum', in vitro

Oilseed rape stem rot disease caused by 'Sclerotinia sclerotiorum' causes serious yield losses worldwide. Glucosinolates as specific secondary metabolites of 'Brassicaceae' are produced in various parts of the host plants. Their enzymatic hydrolysis releases chemical components, particularly isothiocyanates, with fungitoxic activity and volatile characteristics. To investigate the effect of volatiles derived from 'Brassica' tissues, the pathogen was exposed to hydrolysis products of 'Brassica' shoot parts as sources of glucosinolates including oilseed rape varieties and two species, black and white mustard. The results showed significant differences in inhibition of 'S. sclerotiorum' growth between varieties and species. All tissues of black mustard inhibited completely the exposed colonies of the pathogen and oilseed rape varieties Dunkeld, Oscar and Rainbow had significant inhibitory effect on the fungus. The genotypes demonstrated significant differences for the production of toxic volatiles, indicating that GSL contents in Brassica species and even cultivars have different potentials for toxic products

Reaction of glucosinolate-myrosinase defence system in 'Brassica' plants to pathogenicity factor of 'Sclerotinia sclerotiorum'

The glucosinolate-myrosinase defence system, specific to Brassicales plants, produces toxic volatile compounds during mechanical injury or pathogen attack. The reaction of this system to oxalic acid, known as a pathogenicity factor of 'Sclerotinia sclerotiorum', is not fully understood. The hydrolysis of glucosinolates was studied at varying conditions in the presence of oxalic acid in the substrate. In a bioassay, colonies of the pathogen were exposed to volatiles from hydrated mustard powder used as a myrosinase and glucosinolate source. The glucosinolate-myrosinase (GSL-M) system was activated in the presence of oxalic acid at a concentration and pH similar to that expected in vivo. Volatile production was inhibited only when the pH fell to 3 or below. It is unlikely that oxalic acid plays a significant role in disarming the GSL-M system during infection of 'Brassica' hosts

Reaction of Brassica species to 'Sclerotinia sclerotiorum' applying inoculation techniques under controlled conditions

Oilseed rape is economically affected by stem rot caused by 'Sclerotinia sclerotiorum' worldwide. Glucosinolates are the specific secondary metabolites of Brassica plants that appear in different profiles of each species. Their hydrolysis products have biocidal activity and may play a role in resistance against plant pathogenic fungi. The resistance of oilseed rape ('Brassica napus') cultivars and two other Brassica species ('B. nigra' and 'Sinapis alba') was evaluated employing leaf disc inoculation, and oxalic acid and fungal inoculums on leaves of intact plants under controlled conditions. By using leaf disc inoculation, three plant ages were used to compare their reactions against the pathogen. No significant differences between genotypes were observed in this method. However, results demonstrated significant differences in main effects of wounding and plant age. The two intact plant inoculation techniques (oxalic acid and fungal mycelium) resulted in significant differences between genotypes in reaction to the disease. Furthermore, the oxalic acid assay followed the same pattern as fungal inoculations. Among the oilseed rape cultivars, AV-Sapphire and AG-Castle were the most resistant and susceptible genotypes, respectively. Brassica species differed significantly in their reaction to disease, in both wounded and non-wounded leaves with fungal mycelium inoculation and oxalic acid. Overall, non-significant differences between Brassica genotypes showed the unreliability of the leaf disc assay, whereas leaf inoculation of intact plants by means of either oxalic acid or fungal mycelium demonstrated significant differences in lesion size among Brassica cultivars and species

Induced tolerance of 'Sclerotinia sclerotiorum' to isothiocyanates and toxic volatiles from 'Brassica' species

The response of 'Sclerotinia sclerotiorum' , the causal agent of stem rot of oilseed rape ('Brassica napus'), to toxic volatiles produced by the glucosinolate-myrosinase system was studied. Mycelium plugs were exposed to inoculated leaf discs of oilseed rape cultivars and two related species, black mustard ('Brassica nigra') and white mustard ('Sinapis alba'). Growth of exposed colonies was inhibited by more than 87% compared with controls. Despite inhibition of exposed fungal colonies, the fungus continued to grow in infected tissue. Repeated exposure of the fungus to hydrated mustard powder (which contains both glucosinolates and myrosinase) or synthetic isothiocyanates (ITCs) resulted in growth inhibition decreasing from initial levels of up to 80% to insignificant levels after 2–3 days, suggesting that 'S. sclerotiorum' has the ability to adapt to volatiles during the infection progress. This adaptation was studied by investigating induction of glutathione S-transferase-like genes identified from the 'S. sclerotiorum' genome. Three genes, with locus numbers SS1G_07195.1, SS1G_01918.1 and SS1G_10295.1, appeared to be up-regulated following exposure of 'S. sclerotiorum' to mustard powder or allyl ITC. A fourth gene, SS1G_07319.1, appeared to be down-regulated. In addition, glutathione S-transferase catalytic activity in crude mycelium extracts was doubled following 48 h of exposure to mustard powder volatiles. This adaptation could allow 'S. sclerotiorum' to parasitize tissues of 'Brassica' species despite the production of toxic metabolites