AOBPreview originally published online on March 13, 2006
Annals of Botany 2006 97(6):1151-1156; doi:10.1093/aob/mcl062
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Enhanced Pathogenicity of Leptosphaeria maculans Pycnidiospores from Paired Co-inoculation of Brassica napus Cotyledons with Ascospores
1 School of Earth and Geographical Sciences, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia and 2 School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
* For correspondence. E-mail hli{at}cyllene.uwa.edu.au
Received: 15 November 2005 Returned for revision: 1 January 2006 Accepted: 6 January 2006 Published electronically: 13 March 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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• Background and Aims Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus) worldwide, including Australia. In most cases, the severity of the disease in the field is related to infections caused by airborne ascospores. In contrast, pycnidiospores originating from leaf and stem lesions and stubble are widely assumed to play only a relatively minor role in the epidemiology of blackleg. It is not clear whether, under certain conditions, pycnidiospores can cause severe disease in the field. The aim of the work reported was to determine if the pathogenicity of pycnidiospores is enhanced by paired co-inoculation of B. napus cotyledons with ascospores.
• Methods Three investigations were carried out under controlled-environment conditions using various L. maculans isolates and B. napus cultivars with different levels of host resistance to blackleg.
• Key Results In all three experiments, co-inoculation with ascospores increased the ability of pycnidiospores to cause more disease on B. napus than when inoculations consisted of pycnidiospores alone. This effect was significantly influenced by the host resistance of the cultivar, but overall was independent of the L. maculans isolate used in the different experiments. This effect was also independent of timing of inoculation with the ascospores, with increased disease from pycnidiospores occurring on the cotyledon of the seedling in situations where inoculations with ascospores were carried out 0, 1 or 2 d after pycnidiospore inoculation. This enhanced pathogenicity of pycnidiospores was evident even when low concentrations of pycnidiospores were applied to the other cotyledon of the same seedling.
• Conclusions These results may explain continuing severe blackleg disease cycles throughout the cropping season even when ascospore fallout was low or constrained only to a brief period or phase of the cropping season, and suggest that disease epidemics may be polycyclic rather than monocyclic.
Key words: Brassica napus, Leptosphaeria maculans, pycnidiospores, ascospores, co-inoculation, pathogenicity, blackleg disease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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The ascomycete fungus, Leptosphaeria maculans (anamorph: Phoma lingam), commonly known as the blackleg pathogen, has the ability to infect a number of cruciferous crops. Blackleg disease causes severe yield losses of oilseed rape (Brassica napus) worldwide (Barbetti, 1975a
; Gugel and Petrie, 1992; West et al., 2001). The pathogen cycle is generally considered to be monocyclic (West et al., 2001), with the windborne ascospores (sexual) released from infested stubble being the primary source of inoculum for the disease (Brunin and Lacoste, 1970; Bokor et al., 1975). Pycnidia, produced on infested stubble and on cotyledon, leaf and stem lesions on infected plants during the growing season, produce pycnidiospores (asexual) that can be dispersed via direct contact or rain splash (Hall, 1992). Pycnidiospores are considered to be the secondary source of inoculum (Hammond et al., 1985), and are usually required in higher concentrations than ascospores to ensure lesions are produced (Barbetti, 1976; Wood and Barbetti, 1977). Although attempts have been made to define the role of ascospores and pycnidiospores in the epidemiology of blackleg disease (e.g. Brunin and Lacoste, 1970; Barbetti, 1976, respectively), the role of pycnidiospores still remains unclear. In Australia, pycnidiospores have been implicated to play an important role (Barbetti, 1975b, 1976), and this may be directly related to weather conditions and timing of release of these spores. However, outside Australia, the role of pycnidiospores has generally been assumed to be minor in disease epidemics (West et al., 2001). One of the reasons for this is that, under controlled conditions, high concentrations of pycnidiospores are needed for reliable infection compared with reliable and severe infection produced even with relatively low concentrations of ascospores (Wood and Barbetti, 1977). The role of pycnidiospores in disease epidemics clearly warrants investigation, especially in relation to epidemics which are not closely associated with heavy ascospore showers (Barbetti, 1975b). Here we report the results of three controlled environment experiments to determine the potential of pycnidiospores to cause severe disease in the presence of ascospores. This was evaluated taking into consideration the time of co-inoculation with ascospores, the resistance level of cultivars and the concentration of pycnidiospores. To avoid additive effects, the two spore types were inoculated on separate cotyledons of each seedling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Plant material
Three separate experiments were undertaken, each using one or more different spring-type oilseed rape B. napus L. cultivar. In experiment 1, the historical cultivar Oro, a cultivar that had been utilized commercially in Australia in the 1970s when the oilseed rape industry collapsed following severe blackleg epidemics, was used. In experiment 2, the commercial cultivar Surpass 400, released commercially in 2000 and containing single dominant gene-based resistance derived from B. rapa ssp. sylvestris, was used. In experiment 3, two cultivars were used, i.e. Westar, a highly susceptible Canadian cultivar, and Dunkeld, an Australian commercial cultivar expressing polygenic resistance at seedling (including cotyledon) and/or adult plant stages depending upon the environment (Hua Li et al., 2006
).
Fungal isolates
In experiment 1, the historical isolate WAC2244 of L. maculans (Desm.) Ces. et de Not. (anamorph: Phoma lingam), isolated in 1972 from an unknown cultivar of B. napus oilseed rape growing in south-western Western Australia (Hua Li et al., 2005), was used. This isolate is known to be able to overcome the single dominant gene-based resistance, derived from B. rapa ssp. sylvestris, in cultivar Surpass 400 (Hua Li et al., 2005). In experiment 2, isolate UWA S4, reported as an isolate that can overcome the single dominant gene-based resistance, derived from B. rapa ssp. sylvestris, in cultivar Surpass 400 (Li et al., 2003; Hua Li et al., 2003, 2004b), was used. This isolate was obtained from a crown lesion on cultivar Surpass 400 from Mt Barker, Western Australia in 2001. In experiment 3, isolate UWA A 19-1, recovered in 2004 from Surpass 501TT stubble from the 2003 season at Mt Barker, Western Australia, was used. Isolate UWA A19-1 is not known to overcome the resistance in cultivar Surpass 400. Although the nature of the L. maculans strains used for pycnidiospore production were known, the strains used for ascospore inoculation were directly obtained from field samples and could be a mixture of strains.
Spore treatments
Experiment 1
For this experiment, a single concentration each of pycnidiospores (1 x 103 mL–1) and ascospores (5·0 x 102 mL–1) was used. Treatments consisted of either ascospores alone applied to both cotyledons, pycnidiospores alone applied to both cotyledons or ascospores applied to one cotyledon and pycnidiospores applied to the remaining cotyledon of each inoculated plant (10 d old) at the same time. Spores were applied as a 10 µL drop per cotyledon, without wounding, using a micro-syringe.
Experiment 2
For this experiment, a single concentration each of pycnidiospores (1 x 106 mL–1) and ascospores (5·0 x 103 mL–1) was used. Treatments consisted of either ascospores alone applied to both cotyledons, pycnidiospores alone applied to both cotyledons or ascospores applied to one cotyledon and pycnidiospores applied to the remaining cotyledon of each inoculated plant (10 d old) at the same time. Spores were applied as a 10 µL drop per cotyledon, without wounding, using a micro-syringe.
Experiment 3
There were five categories of treatments for each of the two cultivars in this experiment. Within each category, there were different concentrations of pycnidiospores used (details given in Fig. 1). The first two categories were controls, in which one cotyledon of each plant was inoculated with pycnidiospores (in category 1) at concentrations of 1 x 106, 1 x 105, 1 x 104, 1 x 103 and 1 x 102 spores mL–1, or ascospores (in category 2) at a concentration of 1 x 104 spores mL–1. Categories 3, 4 and 5 consisted of both cotyledons being inoculated; one with pycnidiospores and the other with ascospores. In these three categories, one cotyledon of each plant was inoculated with the different concentrations of pycnidiospores (1 x 105, 1 x 104, 1 x 103 and 1 x 102 spores mL–1) on day 0 (when the plants were 10 d old). In category 4, the alternative cotyledons were also inoculated with ascospores on day 1, and in category 5, the alternative cotyledons were inoculated with ascospores on day 2.
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Pycnidiospore inoculum preparation
Pycnidiospore suspensions for inoculation were prepared as follows. A small agar strip (approx. 0·5 x 1 cm) containing pycnidia was transferred to a sterile endorphin tube containing 300 µL of sterile DI water, and left to sit for 10 min until the spores were released into the water. Aliquots of 100 µL were then spread over V8 agar plates, which were then sealed with Parafilm and incubated at 22 °C under a single cool-white fluorescent light tube and a single black light tube (Phillips TL 40W/80RS F40 BLB) for a further 7 d, or until the cultures sporulated. The culture was then flooded with approx. 10 mL of sterile DI water, and gently rubbed with a sterile bent glass rod to release the spores into the water. This suspension was then strained through Miracloth (Calbiochem, La Jolla, CA, USA) into a conical flask and the spore concentration was adjusted to 1 x 107 spores mL–1 with the aid of a haemocytometer. Serial dilutions were then performed so there was 9 mL of each concentration: i.e. 1 x 106, 1 x 105, 1 x 104, 1 x 103 and 1 x 102 spores mL–1. The spore suspensions were transferred to sterile 20 mL tubes and frozen at –20 °C until they were required. Inoculum stored in this way remains viable for at least 2 years (Somda et al., 1998
) but, in this case, it was used about 3 weeks later.
Ascospore inoculum preparation
Oilseed rape residues containing mature pseudothecia of L. maculans were placed in a tube of sterile DI water (approx. 10 mL) and incubated for 1 h at 4 °C. The ascospores were discharged into the water during this time. This suspension was then adjusted to 5 x 102 (experiment 1), 5 x 103 (experiment 2) or 1 x 104 (experiment 3) spores mL–1 using a haemocytometer, and utilized immediately to avoid the risk of premature germination of the ascospores.
Inoculation of oilseed rape seedlings
All experiments were conducted in artificially lit controlled-environment rooms with the air temperature maintained at 15 °C during the night and 20 °C during the day. The light intensity was 150 µE m–2 s–2 with a 12 h photoperiod.
While plants were 10 d old at day 0 inoculation, they were 11 d old in category 4 (i.e. day 1) in terms of ascospore inoculation and 12 d old in category 5 (i.e. day 2) in terms of ascospore inoculation. A 2 µL aliquot of wetting agent (10 % Tween-20) was added to each 10 mL of spore suspension to ensure the inoculation droplets remained on the cotyledon surface. At the time of inoculation, 10 µLof the appropriate suspension was pipetted onto the centre of each cotyledon to be inoculated. Where ascospores were applied, the cotyledons were marked on the margin with black text marker pen. Following inoculation, the plants were encased within polyethylene bags for 4 d to maintain high humidity in order to encourage infection and disease development.
Disease assessments
Separate assessments were made of disease caused by pycnidiospores or ascospores at 17 (experiments 2 and 3) or 18 d (experiment 1) post-inoculation. In experiment 1, the numbers of cotyledons with blackleg lesions were recorded. For experiment 2, the diameter of blackleg lesions was measured. In experiment 3, disease severity was scored using a 0–9 scale modified from Williams (1985), where: 0 = no visible symptoms; 1 = necrotic hypersensitive; 2 = grey-green tissue collapse (1 mm diameter); 3 = collapsed spots (2 mm diameter); 4 = collapsed spots (2–3 mm diameter); 5 = collapsed spots (3–4 mm diameter); 6 = collapsed spots (5–6 mm diameter); 7 = collapsed spots (>6 mm diameter) with a few pycnidia; 8 = collapsing of cotyledon tissue with a few pycnidia; and, 9 = collapsing of cotyledon tissue with masses of pycnidia.
Experimental design and statistical analyses
Experiment 1 consisted of six replications, each treatment replicate consisting of six plants in a single pot, arranged in a fully randomized design. A 
(x + 0·375) transformation was applied to experiment 1 as raw data were not normally distributed. Experiment 2 consisted of 40 replications, each treatment replicate consisting of a single plant, arranged in a randomized block design. Experiment 3 consisted of ten replications arranged in a fully randomized design, each treatment replicate consisting of three plants in a pot, except for the pycnidiospore-only control treatments that consisted of only eight replicates. Data were analysed using GenStat® (Lawes Agricultural Trust, Rothamsted Experimental Station). Significant differences among means were assessed by least significant difference (l.s.d) tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Experiment 1
There were significantly more (P
0·05) cotyledons with blackleg disease from pycnidiospores in the presence of ascospores than where only pycnidiospores alone had been applied (Table 1). There were 3·4 times more cotyledons with lesions from pycnidiospores in the presence of ascospores compared with the absence of ascospores.
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Experiment 2
There were significantly larger (P
0·05) lesions on the cotyledons from blackleg disease from pycnidiospores in the presence of ascospores than where only pycnidiospores alone had been applied (Table 2). The mean lesion size from pycnidiospore infection was 5·6 mm in the presence of ascospores, while the lesion size was 3·6 mm in the absence of ascospores.
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Experiment 3
There were overall significant effects of spore treatments (P < 0·001) and cultivars (P < 0·001) and a significant interaction between spore treatments and cultivars (P < 0·001) (Table 3). On their own, pycnidiospores, even at high concentrations, were generally unreliable in producing significant disease symptoms. For example, even at a concentration of 1 x 106 pycnidiospores mL–1, the disease scores produced were only 2·16 on cultivar Dunkeld, and 2·10 on cultivar Westar.
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For pycnidiospores at a concentration of 1 x 105 spores mL–1, there was significantly more severe disease in cultivar Westar, but only in the presence of ascospores added on day 2, compared with when ascospores were absent. For pycnidiospores at a concentration of 1 x 104 spores mL–1, in the presence of ascospores on day 0 or day 1, pycnidiospores produced significantly more disease on cultivar Dunkeld compared with when ascospores were absent. For pycnidiospores at a concentration of 1 x 103 spores mL–1, more severe disease occurred where ascospores were added on day 0 or day 2 for cultivar Westar, and for day 0 for cultivar Dunkeld, compared with where ascospores were absent. At a concentration of 1 x 102 spores mL–1, pycnidiospores caused significantly more disease on cultivar Westar in the presence of ascospores when they were added on day 0, compared with when ascospores were absent. Pycnidiospores also caused significantly more disease on cultivar Dunkeld at a concentration of 1 x 102 spores mL–1 in the presence of ascospores when they were added on day 0 or day 1, compared with when ascospores were not present. The susceptibility of cultivar Westar and the relative tolerance of cultivar Dunkeld were evident in the disease scores throughout these treatments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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The clear outcome of these studies is that pycnidiospores, even at relatively low concentrations, can reliably cause severe disease providing the same seedling, but on different cotyledons, is co-inoculated with ascospores. This is a significant finding and at least partly explains the occurrence of severe epidemics at times in the oilseed rape cropping season when ascospore showers have been shown to be relatively low (e.g. Barbetti, 1975b
). It may also explain why field epidemics initiated by pycnidiospores alone develop relatively slowly (Barbetti, 1976).
The current consideration of the non-significant involvement of pycnidiospores in field epidemics is based on the fact that large doses of pycnidiospore inoculum are required for the development of reliably severe disease symptoms (e.g. Wood and Barbetti, 1977) and also because severe epidemics are frequently associated with periods of release of ascospores (e.g. Brunin and Lacoste, 1970; Barbetti, 1975a; Salam et al., 2003), especially during the seedling phase when the host is particularly susceptible (Barbetti, 1975a; Hua Li et al., 2004a, b, 2005). It is noteworthy that pycnidiospore concentrations as low as 1 x 102 mL–1 (approx. 1 spore per inoculation site) still increased the severity of the disease in the presence of ascospores. This phenomenon has never been reported before for this disease. There have been, however, suggestions of the potential for pycnidiospores to cause significant disease in field crops in Australia (Barbetti, 1975b, 1976) and in Canada (Guo and Fernando, 2005), although no attempt was made by the authors to determine the factors affecting pycnidiospore pathogenicity. This pycnidiospore–ascospore interaction is, however, not fully understood. For example, in certain studies (N. Dean and Hua Li, unpubl. res.), it was found that when pycnidiospores and ascospores are co-inoculated onto different lobes of the same cotyledon, this enhancement of disease caused by pycnidiospores, at least in terms of lesion size, was not evident.
Overall, the time of introduction of ascospores did not appear to have a major influence on the pathogenicity of pycnidiospores. It therefore still remains unclear whether the expression of host susceptibility to pycnidiospores occurred either before or after host penetration by the germ tube(s) of ascospores. Ascospores germinate as early as 2 h after inoculation and approx. 2 d earlier than pycnidiospores (Hua Li et al., 2004a). The importance of this differential germination of the two spore types in relation to timing of expression of host susceptibility to pycnidiospores requires further definition.
The strains used in these studies varied in their virulence on cultivar Surpass 400 used in experiment 2. For example, the historical isolate WAC2244 and the isolate UWA S4 are both known to overcome the single dominant gene-based resistance derived from B. rapa ssp. sylvestris that is found in cultivar Surpass 400 (Li et al., 2003; Hua Li et al., 2003, 2005), while isolate UWA A19-1 is not known to overcome this resistance in cultivar Surpass 400 (Hua Li, unpubl. res.). Although these host responses to these pathogen isolates were predictable (e.g. Hua Li et al., 2003, 2004a, b, 2005), there still remained significant interaction between ascospores and pycnidiospores in relation to pathogenicity on all cultivars tested, even on the polygenically resistant cultivar Dunkeld. This is despite the fact that pre-infection and early infection processes in cultivars with different levels of host resistance are similar, with the host resistant response to the pathogen only occurring after penetration has occurred (Hua Li et al., 2004a). The significant cultivar x spore treatment interaction in experiment 3 indicates that a noticeable host–pathogen interaction could be involved, and this clearly needs to be evaluated in the future.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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Although these experiments were not carried out in the field, they provide clear evidence that as few as five ascospores per cotyledon can allow pycnidiospores to initiate severe disease, even when pycnidiospore levels on the other cotyledon are also as low as approx. 1 spore per cotyledon. Reports in the literature indicate that while a large proportion of ascospore showers can last from a 2- to a 4-month period (e.g. Salam et al., 2003
), there are situations where ascospore showers can last for as little as a 1-month period of the growing season (e.g. Barbetti, 1975b). In contrast, pycnidiospore discharge can continue for a longer period (Barbetti, 1975b, 1976; Guo and Fernando, 2005). Provided that the environment is conducive to the discharge and dispersal of pycnidiospores, plant tissues of susceptible hosts can be infected and therefore initiate further cycles of the disease in the field. In such instances, it could be assumed that blackleg can indeed be a polycyclic disease. This clearly requires further field studies involving artificial inoculation with ascospores. This may explain the incidence of severe disease in crops where ascospore fallout has been low or constrained to a very short part of the growing season (e.g. Barbetti, 1975b). This could also have significant implications in the field management of the disease, for example involving the need for extended periods of application or activity of fungicides and/or utilization of high levels of adult host resistance. |
ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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The authors would like to thank Dr Guijin Yan and Dr Ming Pei You for help with statistical analyses.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSLITERATURE CITED |
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