2 Institute of Plant Genetics, 60-749 Poznan, Poland
Phoma lesions produced on oilseed rape leaves by B-group ascospores were smaller and less distinctive than lesions produced by A-group ascospores of Leptosphaeria maculans, when leaves were inoculated with ascospore suspensions obtained from infected debris from Poland or the UK. Both Agroup ascospores and B-group ascospores of L. maculans were able to infect leaves of oilseed rape and produce lesions at temperatures from 4 to 20oC and leaf wetness durations greater than 4 h. However, the greatest number of lesions were produced with a leaf wetness duration of 48h at temperatures of 20oC for A-group and >12oC for B-group ascospores. As leaf wetness durations and temperatures decreased below the optimal values, the numbers of lesions produced decreased. For example, very few lesions were produced at 4oC (and only with a leaf wetness duration >48 h) or with a leaf wetness duration of 4h (and only at temperatures >12oC). There was no evidence that the maximum number of lesions produced in relation to number of ascospores inoculated differed between A-group and B-group L. maculans. The incubation period (from inoculation to the appearance of the first lesion) of B-group L. maculans was shorter than that of A-group L. maculans; at 20oC, lesions appeared within 2 days rather than 5 days from inoculation. As temperature decreased below 20oC, the length of the incubation period increased.
KEYWORDS: Blackleg, leaf lesions, Phoma lingam, UK, Poland
Stem canker or blackleg (Leptosphaeria maculans; anamorph Phoma lingam) is one of the most damaging diseases of winter oilseed rape in the UK (Fitt et al., 1997) and other parts of Europe. In the UK, epidemics are initiated in autumn by air-borne ascospores released from infected debris (Gladders & Musa, 1988). These ascospores infect leaves to produce phoma leaf spots and the pathogen then grows down the leaf petioles into the stem. However, in Poland, distinctive symptoms of the disease are not generally visible on leaves of crops in the autumn (Jedryczka et al., personal communication). Furthermore, the disease is caused by two genetically different groups of L. maculans; in the UK the A group predominates whereas in Poland the B group predominates. These two groups of L. maculans may even be different species (Taylor et al., 1991; Williams & Fitt, 1999). Few experiments on the disease have used ascospores as inoculum for infecting oilseed rape leaves. In most experiments, conidia have been used to inoculate leaves which have had to be wounded beforehand because conidia cannot usually infect healthy leaves. The severity of stem canker epidemics in the spring and summer may depend on when leaf spots appear in the autumn, which is affected by the occurrence of weather favourable for infection by the ascospores. The effects of temperature and leaf wetness duration on infection of winter oilseed rape leaves by UK and Polish ascospores of L. maculans are reported in this paper.
Ascospores of L. maculans were ejected from naturally infected debris obtained from Rothamsted in the UK or Cerekwica near Poznan in Poland onto agar plates. Oilseed rape plants (cv Nickel) were grown in pots filled with a peat-based compost. Mature pseudothecia of L. maculans were excised from the infected stem debris from the UK or Poland, then crushed in water with a pestle and mortar to release the ascospores. Plants with four expanded leaves (GS 1,4) were inoculated by spraying with an ascospore suspension. Treatments were temperature (growth cabinets at 4, 8, 12, 16 or 20oC) and leaf wetness duration (4, 8, 16, 24, 30, 48 or >70 h after inoculation, maintained by covering plants with polyethylene bags). Cabinets were maintained at different temperatures and leaf wetness duration treatments (six plants per treatment) were arranged randomly in each cabinet. Numbers of new phoma leaf spot lesions which appeared were counted regularly (generally daily) until no new lesions appeared.
More than 50 single ascospore colonies were obtained from each set of debris; all colonies from Polish debris produced yellow-brown pigmentation characteristic of the B group of L. maculans and colonies from UK debris did not produce the pigmentation (A group). In controlled environment experiments, lesions on oilseed rape leaves produced by B-group ascospores were smaller and less distinctive than those produced by A-group ascospores of L. maculans. Lesions produced by A-group ascospores contained numerous black pycnidia, whereas those produced by B-group ascospores contained few pycnidia. Ascospores of both A-group and B-group L. maculans were able to infect leaves and produce lesions at temperatures from 4 to 20oC and leaf wetness durations greater than 4 h (Table 1). However, the greatest numbers of lesions were produced when leaf wetness durations were at least 48 h. When plants were inoculated with A-group ascospores, most lesions were produced at >20oC, with fewer lesions at lower temperatures or at 24oC (Biddulph et al., personal communication). However, when plants were inoculated with B-group ascospores, most lesions were produced at >12oC with a 48 h leaf wetness period. As leaf wetness durations and temperatures decreased below the optimal values, the numbers of lesions produced decreased for both types of ascospore inoculum. For example, very few lesions were produced at 4oC (and only with a leaf wetness duration >48 h) or with a leaf wetness duration of 4 h (and only at temperatures >12oC). There was no evidence that the maximum number of lesions produced in relation to ascospore inoculum concentration differed between A-group and B-group ascospore inoculum.
The incubation period (from inoculation to the appearance of the first lesion) of B-group L. maculans was shorter than that of A-group L. maculans (Table 2). Lesions appeared within 2 days from inoculation with B-group ascospores, but not until nearly 5 days from inoculation with A-group ascospores, at a temperature of 20oC and a wetness duration of 48 h. The estimated length of the incubation period increased as temperature decreased below 20oC.
Table 1. Effects of temperature and wetness duration on maximum number of phoma leaf spot lesions produced on leaves of oilseed rape inoculated with A-group or B-group ascospores of Leptosphaeria maculans.
|
|
|
Maximum number of lesions1 |
||||
|
Wetness duration (h) |
Temperature (oC) |
4 |
8 |
12 |
16 |
20 |
|
|
|
A-group ascospores |
||||
|
4 |
|
*2 |
* |
* |
6 |
2 |
|
8 |
|
* |
6 |
6 |
11 |
9 |
|
16 |
|
* |
8 |
9 |
54 |
62 |
|
24 |
|
03 |
22 |
22 |
104 |
92 |
|
30 |
|
0 |
24 |
34 |
231 |
144 |
|
48 |
|
2 |
74 |
111 |
236 |
397 |
|
72 |
|
4 |
117 |
189 |
169 |
410 |
|
|
|
B-group ascospores4 |
||||
|
4 |
|
* |
0 |
4 |
0 |
8 |
|
8 |
|
* |
0 |
0 |
36 |
12 |
|
16 |
|
* |
8 |
18 |
152 |
32 |
|
24 |
|
* |
14 |
98 |
176 |
120 |
|
30 |
|
* |
2 |
82 |
100 |
88 |
|
48 |
|
48 |
60 |
440 |
304 |
248 |
|
78 |
|
62 |
* |
* |
* |
* |
1 Total on six plants with four inoculated leaves per plant
2 These treatment combinations were not tested
3 No lesions developed with these treatments
4 Values for 4, 8 and 12oC treatments (inoculated with 500 spores/ml) have been multiplied by two; values for 16 and 20oC treatments (inoculated with 250 spores/ml) have been multiplied by four, to make them more easily comparable with values for UK L. maculans (inoculated with 1000 spores/ml)
The observation that B-group ascospores of L. maculans from Poland produced smaller, less distinctive lesions on oilseed rape leaves than A-group ascospores for the UK, with fewer pycnidia developing in them, supports the conclusions of Brun et al. (1997), who observed similar differences in lesions type between French A and B groups of L. maculans, and confirmed their results by starch gel electrophoresis of lesion extracts, which produced distinctive patterns of alloenzymes. Similar differences in the type of lesions produced by the A group and B group were observed in the UK by Johnson & Lewis (1994) and in Germany by Thürwachter et al. (1999). Genetic analysis of L. maculans isolates from the UK and Poland, using the polymerase chain reaction (PCR), which was done as part of the European Union IMASCORE project (Balesdent & Rouxel, 1998), confirmed that UK populations of L. maculans were predominantly of the A group, whereas Polish populations were predominantly of the B group (Jedryczka et al., personal communication).
Table 2. Effects of temperature and wetness duration on the incubation period (time from inoculation to appearance of the first lesions1) of A-group or B-group Leptosphaeria maculans on oilseed rape leaves inoculated with ascospore suspensions.
|
|
|
Incubation period (days) |
||||
|
Wetness duration (h) |
Temperature(oC) |
4 |
8 |
12 |
16 |
20 |
|
|
|
A-group ascospores |
||||
|
4 |
|
*2 |
* |
* |
12.5 |
4.6 |
|
8 |
|
* |
15.3 |
15.9 |
8.9 |
6.6 |
|
16 |
|
* |
15.0 |
13.9 |
6.9 |
6.2 |
|
24 |
|
03 |
14.9 |
12.9 |
6.3 |
5.0 |
|
30 |
|
0 |
14.2 |
11.6 |
6.2 |
4.6 |
|
48 |
|
25.0 |
14.0 |
11.8 |
6.3 |
4.6 |
|
72 |
|
25.0 |
13.9 |
10.8 |
5.7 |
5.3 |
|
|
|
B-group ascospores |
||||
|
4 |
|
* |
0 |
9.5 |
0 |
8.0 |
|
8 |
|
* |
0 |
0 |
2.5 |
6.5 |
|
16 |
|
* |
8.0 |
8.0 |
2.5 |
4.5 |
|
24 |
|
* |
8.0 |
8.0 |
2.5 |
2.5 |
|
30 |
|
* |
8.0 |
8.0 |
2.5 |
4.5 |
|
48 |
|
8.0 |
8.0 |
8.0 |
1.5 |
1.5 |
|
78 |
|
8.0 |
* |
* |
* |
* |
1 Calculated as the mean of the last time when no lesions were present and the
first time when lesions were present
2 These treatment combinations were not tested
3 No lesions developed with these treatments
These controlled environment experiments provided no evidence that the infectivity of B-group ascospores differed from that of A-group ascospores. This suggests that the differences in lesion development between the A-group and B-group ascospores were not related to differences between them in the process of infection. To confirm this observation, these comparisons need to be repeated using ascospore inoculum with the same concentrations for A-group and B-group ascospores. It has often been suggested that B-group isolates of L. maculans, which are predominant in Poland, are less damaging to oilseed rape than A-group isolates which are predominant in the UK (Williams & Fitt, 1999); these differences appear not to be related to differences in infectivity of ascospores on leaves.
Furthermore, these controlled environment experiments suggested that the incubation period of B- group L. maculans was shorter than that of A-group L. maculans. The experiments also suggested that the optimum temperature for infection by B-group ascospores might have been lower than that for infection by A-group ascospores. However, there appeared to be some temperature/wetness duration interaction, with more lesions produced by B-group ascospores at 16oC than 12oC when the leaf wetness duration was shorter than 48 h. These interactions require further investigation before epidemiological differences between B-group and A-group L. maculans can be confirmed.
This work was funded by the UK Ministry of Agriculture, Fisheries and Food and the European Union (FAIR contract CT96-1669; coordinator M.H. Balesdent).
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