INVESTIGATING RESISTANCE AND EPIDEMIOLOGY OF BLACKLEG (LEPTOSPHAERIA MACULANS) IN CANOLA M. SosnowskiA,B, E. ScottA and M. RamseyC ADepartment of Applied and Molecular Ecology, Adelaide University, Waite Campus, Glen Osmond 5064, South Australia BSouth Australian Research and Development Institute (SARDI), GPO Box 397, Adelaide 5001, South Australia CAnimal and Plant Control Commission, GPO Box 1671, Adelaide 5001, South Australia E-mail: [email protected] ABSTRACT The pathway of colonisation by Leptosphaeria maculans through canola (Brassica napus) plants was visualised using scanning electron microscopy. L. maculans hyphae were responsible for damaging the leaf cuticle allowing penetration through to the epidermis and into the mesophyll. It seems that mechanisms responsible for stem resistance occur at the petiole-stem junction. Cultivars, which were resistant to cotyledon and stem infection, were susceptible to leaf infection in field trials. Ascospore-derived infection was greatest early in the season, when canola plants were undergoing early leaf growth and were therefore most vulnerable. By the time flowering occurred, the supply of ascospores was exhausted. Rainfall, temperature and wind were correlated to the amount of infection on plants. KEYWORDS Brassica napus, Phoma lingam, microscopy, ascospore, pycnidiospore INTRODUCTION Blackleg disease of canola (Brassica napus) is caused by the fungus Leptosphaeria maculans (anamorph Phoma lingam). This fungus is the most important pathogen of canola in Australia. It has the potential to devastate crops (Bokor et al. 1975) in an industry which was estimated to be valued at 500 million dollars in Australia in 2000/2001 (Anon. 2001). The area sown to canola in Australia has expanded tenfold over the last decade, for several reasons; its value as a cash crop, the development of cultivars better adapted to Australian conditions, its role as a crop between cereals and the difficulties experienced with pulse crops. This expansion has brought rotations closer together and hence has increased disease pressure on the canola crop. Blackleg control relies on the use of resistant canola cultivars along with stubble management, longer rotations and fungicide treatment (Barbetti and Khangura 2000). L. maculans is a genetically diverse fungus that has the potential to overcome resistance (Pongam et al. 1999, Sosnowski et al. 2001). Therefore it is important to seek new sources of resistance by identifying additional mechanisms by which canola plants can resist or contain infection by the pathogen. Some commercial cultivars have good resistance to the stem canker phase of blackleg, but may suffer yield or quality penalties and may not be suited to certain environments due to their time to maturity. Environmental factors can determine the extent of blackleg development in the field (West et al. 2001), so disease pressure will vary between regions. Therefore it is important to select the most appropriate canola cultivar as part of a management strategy for a particular area. This paper reports on findings from the PhD project “Investigating resistance and management of blackleg (Leptosphaeria maculans) in canola” in which we are investigating (i) the mechanisms of blackleg resistance in the stem and petiole of canola using microscopy and (ii) the effects of environment and canola cultivar on the development of blackleg. MATERIALS AND METHODS Microscopy The process by which the hyphae of the blackleg fungus grow through the leaves and petiole of the canola plant was visualised using the Field Emission Scanning Electron Microscope (FESEM), at the Centre for Electron Microscopy and Microstructure Analysis (CEMMSA), University of Adelaide, South Australia. Doubled haploid plants, supplied by Mr Neil Wratten (canola breeder, New South Wales Agriculture) were used to eliminate the effects of genetic variation within cultivars. Plants, at the 3-leaf stage, were inoculated by placing 10l droplets of pycnidiospore suspension (106 spores/ml sterile distilled water) of the virulent (A-group) isolate 66/97 of L. maculans on the adaxial surface of leaves. Controls were inoculated with sterile distilled water only. Samples were prepared for the FESEM using a standard protocol developed by CEMMSA. Infection processes in cv. Scoop (resistant to stem cankering) and cv. Westar (susceptible) were compared. Epidemiology Field trials were conducted at Kingsford Research Station, near Gawler, South Australia and Charlick Research Station, near Strathalbyn, South Australia during 2000. The trials, sown in May, comprised eight cultivars and three replications. The cultivars included; Dunkeld, Scoop and Mystic (resistant), Pinnacle (moderately resistant), Monty and Karoo (moderately susceptible), Hyola 42 and Q2 (susceptible) (Stanley and Potter 2001). The trials were inoculated late in May with barley grain, which was autoclaved and colonised with L. maculans and we relied on natural inoculum for the rest od the season. Ten seedlings were randomly marked in each plot. Cotyledon, leaf/petiole and stem infection was rated separately on a weekly basis throughout the growing season. Climatic data were collected using automated weather stations. Trays, containing 18 plants (cv. Hyola 42) at the 3-leaf stage, were placed 20m from each trial every week, throughout the season, and then returned to the glasshouse a week later. The number of leaf lesions on each plant was counted after incubation for 10 days. RESULTS Microscopy FESEM images of the infection process showed the germination of pycnidiospores of L. maculans and subsequent penetration of the leaf by hyphae (Fig. 1A). It was also noticed that some damage to the cuticle was associated with the hyphae (Fig. 1B). Hyphae were observed in the xylem vessels in petioles of both cultivars (Fig. 1C), and were growing toward the stem of the plant. In the stem, hyphae could be located only in the vascular tissue of the susceptible cultivar, Westar. The hyphae grew through the walls of the vascular tissue and colonised the cortex (Fig. 1D). Epidemiology Figure 2 shows the incidence of blackleg infection throughout the season at the Kingsford trial site in 2000. Results were similar at Charlick (not shown). Cotyledon infection varied between cultivars, with 40-50% infection occurring in Dunkeld, Hyola42, Monty and Q2 (a Canadian cultivar). Leaf/petiole infection was first observed on June 15 and reached 100% incidence within 3 weeks for all cultivars. The incidence of stem infection was greater than 80% for the cultivars Q2, Monty, Hyola 42 and Karoo by November. Pinnacle had only 40% stem infection compared to the cultivars Mystic, Dunkeld and Scoop, which had 65-75% infection. Infection of the spore trap plants was greatest during June and July, with a maximum of 37 lesions/plant in the last week of June (Fig. 6). There was no infection after mid-September. Weeks in which total rainfall, mean temperature and mean wind speed were high correlated with weeks in which the number of lesions on the trap plants was greatest, until mid-September. Figure 1 A. Hyphae growing from spores on the surface of a canola leaf (cv. Scoop). Arrows indicate hyphae entering stomata (bar=25m). B. Wounded leaf epidermis associated with L. maculans hyphae (cv. Westar) (bar=5m). C. Hyphae growing through the xylem vessel in the petiole (cv. Scoop) (bar=20m). D. Hyphae (H) grow down through the vascular tissue (Vt) and then colonise the cortex (Co) in the lower stem region (cv. Westar) (bar=100m). DISCUSSION Microscopy The damage to the cuticle associated with hyphae (see figure 1B) eventually resulted in large wounds, through which hyphae entered the epidermis and mesophyll region. No such wounding was observed in the controls. This suggests that the fungus may produce compounds which damage the leaf surface to facilitate penetration, as the only documented entry points are stomata and wounds (Chen and Howlett 1996). L. maculans hyphae were found in the petioles of both the resistant and susceptible cultivars, and in the stem of the susceptible cv. Westar. This suggests that the passage of hyphae from petiole to stem appears to be restricted in cv. Scoop. Hammond and Lewis (1987) showed that earlier and more intense lignification was associated with the stem xylem vessels in a resistant rapeseed (B. napus) cultivar compared to a susceptible cultivar. Histochemical methods are now being employed to determine if compounds such as lignin, suberin and callose are associated with resistance of canola to pathogen invasion at the petiole/stem region. Epidemiology From the field trial at Kingsford, it appeared that some cultivars had resistance to cotyledon and stem infection, but all cultivars were highly susceptible to leaf infection, suggesting that leaf resistance does not have a role in the overall resistance of current cultivars. This is most likely because infected seedlings do not survive, hence are not selected, and current resistant cultivars are selected on the basis of stem canker resistance (Salisbury et al. 1995). Pinnacle (moderately resistant) had less stem infection than the resistant cultivars, Dunkeld and Scoop. This observation corresponded with observations in some commercial crops in 2000, which showed that stem infection in Dunkeld and Charlton was more severe than in previous years (Stanley and Potter throughout thegrowingseason. (km/h) and wind direction, and number of leaf lesions on Hyola42 trap plants at Kingsford greatest infectionperiods maybeapossiblemanagementstrategytoreduce