¹CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601.
²NSW Agriculture Agricultural Institute, PMB Wagga Wagga 2650.

ABSTRACT

Decaying residues of Brassica crops such as canola (Brassica napus) are thought to suppress inoculum of soil-borne fungal pathogens by a process termed biofumigation. We investigated the direct suppression of four soil-borne cereal pathogens by canola root residues incorporated into the soil. Pots were prepared with a 2 cm layer of soil containing fungal inoculum mixed with various rates of freeze-dried canola roots (0 – 1.0 % w/w), between a lower 8cm and upper 2cm layer of uninoculated soil. Wheat was sown into the upper layer and the impact of the Brassica tissues on the pathogens was assessed using the development of disease symptoms on wheat seedling roots at the 4-leaf stage as a bioassay. At the lowest rates (0.15% w/w), canola root residues reduced infection severity of the fungi by 0 – 50% while at the highest rate (1% w/w) infection was reduced by 50-90%. The effectiveness of suppression differed among the different fungi. Gaeumannomyces (take-all), Rhizoctonia and Fusarium were more sensitive to the canola residues than Bipolaris. The results suggest that suppressive effects of canola root residues on fungal inoculum in the field may be significant, given that the amount of canola root tissues in the field (0-10 cm) commonly ranges from 0.1 – 0.5 % w/w.

INTRODUCTION

Canola is an excellent break crop for wheat, and its effectiveness is thought to be due in part to the suppression of soil-borne cereal pathogens by biocidal compounds released by decaying root tissues, which reduce disease infection in following crops (Angus et al. 1991, Kirkegaard et al. 1994). Previous studies have identified 2-phenylethyl isothiocyanate (2PE-ITC), a product of root 2-phenylethyl glucosinolate (2PE-GSL) degradation, as the likely compound responsible for the suppressive effects of canola roots (Sarwar et al. 1998, Potter et al. 1998). In vitro toxicity tests using both pure 2PE-ITC in agar, or re-hydrated Brassica root tissues have demonstrated suppression of a range of soil-borne cereal pathogens by ITCs (Angus et al 1994, Kirkegaard et al 1996, Sarwar et al 1998). Although these studies provide evidence for the toxicity of ITCs under laboratory conditions, their effectiveness may be reduced in soil, where losses of ITCs due to sorption and other processes may occur (Brown and Morra 1997). The efficiency of conversion of glucosinolates in Brassica tissues into ITCs can be as low as 15% in soil (Borek et al. 1997).

The aim of this experiment was to determine the effect of incorporated canola root tissues on the inoculum of soil-borne cereal pathogens. The rates of canola root tissues used in the experiment covered the range found in field-grown canola crops (Kirkegaard and Sarwar 1998), and the development of disease symptoms on wheat seedlings grown in the soil was used to assess the impact on added fungal inoculum.

MATERIALS AND METHODS

Red-earth soil was collected from the surface (0-10 cm) of a cropping paddock at Harden in southern NSW Australia, air dried and sieved to 2mm. The soil was fumigated by exposure to methyl bromide (56 g m^-3) in a pressurised chamber for 48 h, and then aired for 6 days prior to pot preparation. The canola root tissue used in the experiments (cv Tamara) was collected from a field experiment at mid-flowering, freeze dried and ground in a Wiley mill (1 mm sieve). The tissue was analysed for glucosinolates using the HPLC method described by Kirkegaard and Sarwar (1998). The concentration of the major glucosinolate (2PE-GSL) in the tissues was 22.6 μmole/g. Four fungal species (Gaeumannomyces graminis var. tritici; Rhizoctonia solani, Fusarium graminearum, Bipolaris sorokiniana) originally isolated from field-grown wheat crops in NSW were included in the experiment. Inoculum was prepared on sterile ryegrass seed by allowing plugs of fungal mycelium grown on agar to thoroughly colonise the sterile seed in large flasks over a four week period, then air-drying in a laminar flow cabinet at room temperature.

Experimental pots, 5cm x 5 cm and 12 cm deep were prepared as follows; a lower 8 cm layer of uninoculated soil was overlain by a 2 cm treatment layer (33 g) containing soil thoroughly mixed with various rates of ground, freeze dried canola roots viz: 0 (control), 50, 100, 200, 300 and 400 mg of tissue. Eight pieces of fungal inoculum were spaced evenly on the surface of this layer and then pushed down to embed them within the layer. An upper 2cm layer of uninoculated soil was added to provide inoculum free soil in which to sow the wheat seed. Four replicate pots of each rate of canola root tissue was prepared for each fungal species. Two other treatments were included in the experiment. A treatment with no added fungal inoculum or canola root tissue was included to provide undiseased roots for use in disease symptom assessment. Finally, a treatment in which 400 mg of freeze-dried wheat root tissue was added instead of canola root tissue (using take-all inoculum only) was used to determine if the suppressive effects were specific to Brassica root residues, or could be caused by root tissues of other species or organic matter per se. The treatments are summarised in Table 1.

Table 1 Treatments (1-6) established using four different fungal species, and two additional treatments.

Treatment Tissue (mg) Inoculum

1 0 (control) +

2 50 +

3 100 +

4 200 +

5 300 +

6 400 +

7 0 -

8 400(wheat) +(take-all only)

The pots were wet from the bottom by standing in water overnight and were then placed in a growth cabinet at 16/11 ^oC and allowed to drain for 4 days prior to sowing. Seven wheat seeds (cv Janz) were then sown into the surface layer which was covered with plastic beads to reduce surface evaporation. After emergence, the wheat was thinned to 4 plants per pot and grown under 8 h photoperiod to the 4-leaf stage. The pots were periodically re-watered to their original weight using ¼ strength Hoaglands solution to ensure no water or nutrient limitations.

At the 4-leaf stage, the soil was washed from the roots which were examined for symptoms of disease. The disease severity was rated from 0-5 on the basis of the number of seminal roots with symptoms of disease. In order to compare the relative effects of canola tissues on the different pathogens, disease severity for treatments 2-6 was calculated as % of the untreated control (treatment 1). Root pieces exhibiting symptoms were removed and Kochs postulates used to confirm that the symptoms were caused by the added fungal inoculum. Unusual root symptoms unrelated to disease were also noted. The root and shoot tissues were then separated, oven dried then weighed.

RESULTS AND DISCUSSION

Characteristic symptoms developed well in the control treatments for each fungal species and these symptoms were confirmed by Kochs postulates. The impact of the canola root tissues on disease severity for each fungal species is shown in Figure 1. The canola root residues reduced the severity of all diseases and the impact increased with increasing rates of root residues. There were significant differences (ANOVA, P ≤ 0.05) in the effect of canola root tissues on different fungal pathogens. Gaeumannomyces, Rhizoctonia and Fusarium were more sensitive to the incorporated tissues than Bipolaris. These results are consistent with previous in-vitro studies using pure ITCs or rehydrated Brassica tissues (Kirkegaard et al. 1996, Sarwar et al. 1998).

Figure 1. Effect of incorporated canola root tissue on the infection severity of four fungal diseases on the roots of wheat seedlings at the four leaf stage. (s) Bipolaris, (t) Fusarium, (l) Rhizoctonia, (n) Gaeumannomyces. LSD (P=0.05: Fungal species = 28; Tissue rate = 30; Interaction = not significant).

The severity of Gaeumannomyces infection in treatment 8 (400 mg of wheat root tissue) was not significantly (ANOVA, P ≤ 0.05) different to that of the control, indicating that wheat root residue had no impact on disease severity, and that the suppression observed was specific to canola root tissues.

The levels of canola root tissues commonly found in field soils are 0.1 – 0.5 % w/w in the top 15 cm (Kirkegaard and Sarwar 1998), and the concentration of 2PE-GSL in Australian canola varieties at flowering varies from 5 – 28 μmole/g (Kirkegaard and Sarwar 1999). In this experiment, rates of 0.1 – 0.5 % w/w of canola root tissue containing 20 μmole/g 2PE-GSL reduced disease severity by 30 – 50% which suggest that significant impacts of canola residues may be expected in the field. These levels of root tissue equate to an input of 30 – 100 nmole of 2PE-GSL/g soil (Figure 1). Potter et al. (1998) have shown that these rates of 2PE-GSL added as canola root residue also reduced numbers of the cereal nematode Pratylenchus neglectus by 50%.

The pattern of ITC release from decaying root residues in the field is likely to be different from that achieved in this experiment. The instantaneous rehydration of ground, freeze-dried material is likely to cause a rapid release of ITCs from the tissues, and the thorough mixing of the tissue throughout the soil would maximise its contact with the fungal inoculum. In comparison, canola root residues in the field would decay over a period of days or weeks, and the distribution of ITCs would presumably be concentrated close to roots and crop rows. In addition there is a 4-month fallow period following canola before wheat would be sown in the field, while the impacts in this experiment were measured immediately after tissue incorporation. Also, the use of artificial inoculum in sterilised soil is not representative of the biological environment in the field. Nevertheless, the experiment demonstrates the variable responses of the cereal pathogens tested to the suppressive effects of canola root residues, at rates which could be expected to occur in the field. Further studies are in progress to determine the impact of canola root residues under field conditions.

Interestingly, there was evidence of direct phytotoxic effects of the canola root tissues on wheat roots at higher rates (300 – 400 mg). Allelopathic effects of 2PE-ITC to wheat have been reported previously (Bialy et al. 1990). The symptoms we observed included general browning of the roots and a lack of secondary branching or root hair development, and in some cases shoot growth was reduced by up to 20%. The symptoms occurred irrespective of the fungal species present, no disease could be isolated from the roots, and the incidence of the symptoms increased with increasing rated of added tissues. The importance of canola allelopathy to wheat is unclear, as the rates of canola tissue at which effects occurred were higher than those found under field conditions. Also, under field conditions, allelopathy of residual canola tissues would probably decline with 2PE-ITC during the fallow period between crops.

ACKNOWLEDGEMENTS

The technical assistance of Mr A. Ingram and Ms S. Hely, and identification of pathogens isolated from roots by Dr S. Simpfendorfer is gratefully acknowledged. Funding was provided by the Grains Research and Development Corporation.

REFERENCES

Angus J.F., van Herwaaden A.F. and Howe G.N. (1991). Productivity and break-crop effect of winter growing oilseeds. Australian Journal of Experimental Agriculture 31, 669-677.

Angus J.F., Gardner P.A., Kirkegaard J.A. and Desmarchelier J.M. (1994). Biofumigation: Isothiocyanates released from Brassica roots inhibit the growth of the take-all fungus. Plant and Soil 162, 107-112.

Bialy Z., Oleszek W., Lewis J. and Fenwick G.R. (1990). Allelopathic potential of glucosinolates (mustard oil glycosides) and their degradation products against wheat. Plant and Soil 129, 277 – 281.

Borek V., Elberson L.R., McCaffry J.P. and Morra M.J. (1997). Toxicity of rapeseed meal and methyl isothiocyanate to larvae of the black vine weevil (Coleoptera: Curculionidae). Journal of Economic Entomology 90, 109-112.

Brown P.D. and Morra M.J. (1997). Control of soil-borne plant pests using glucosinolate containing plants. Advances in Agronomy 61, 167-231.

Kirkegaard J.A., Gardner P.A., Angus J.F. and Koetz E. (1994). Effect of Brassica crops on the growth and yield of wheat. Australian Journal of Agricultural Research 45, 529-545.

Kirkegaard J.A., Wong P.T.W. and Desmarchelier J.M. (1996). In-vitro suppression of fungal root pathogens of cereals by Brassica tissues. Plant Pathology. 45, 593-603.

Kirkegaard J.A. and Sarwar M. (1998). Biofumigation potential of brassicas. I. Variation in glucosinolate profiles of diverse field-grown brassicas. Plant and Soil 201, 71 - 89.

Kirkegaard J.A. and Sarwar M (1999). Glucosinolate profiles of Australian canola (Brassica napus annua L.) and Indian mustard (Brassica juncea L.) cultivars: implications for biofumigation. Australian Journal of Agricultural Research 50, 315-24.

Potter M.J., Davies K. and Rathjen A.J (1998). Suppressive impact of glucosinolates in Brassica vegetative tissues on root lesion nematode Pratylenchus neglectus. Journal of Chemical Ecology 24, 67-80.

Sarwar M., Kirkegaard J. A., Wong P. T. W. and Desmarchelier J. M. (1998). Biofumigation potential of brassicas. III In-vitro toxicity of isothiocyanates to soil-borne fungal pathogens. Plant Soil 201, 103 - 112.