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Keywords:

  • A-group/B-group Leptosphaeria maculans;
  • ascospores;
  • germination;
  • penetration;
  • temperature

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Ascospores of both A-group and B-group Leptosphaeria maculans germinated at temperatures from 5 to 20°C on leaves of oilseed rape. Germination of ascospores of both groups started 2 h after inoculation and percentage germination reached its maximum about 14 h after inoculation at all temperatures. Both the percentage of A-/B-group ascospores that had germinated after 24 h incubation and germ tube length increased with increasing temperature from 5 to 20°C. Germ tubes from B-group ascospores were longer than those from A-group ascospores at all temperatures, with the greatest difference at 20°C. Hyphae from ascospores of both groups penetrated the leaves predominantly through stomata, at temperatures from 5 to 20°C. A-group ascospores produced highly branched hyphae that grew tortuously, whereas B-group ascospores produced long, straight hyphae. The percentage of germinated ascospores that penetrated stomata increased with increasing temperature from 5 to 20°C and was greater for A-group than for B-group L. maculans after 40 h incubation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Phoma stem canker (blackleg), caused by Leptosphaeria maculans (anamorph Phoma lingam), is an important disease of oilseed rape (Brassica napus ssp. oleifera) in the UK (Fitt et al., 1997) and worldwide (West et al., 2001). Leptosphaeria maculans is a complex of at least two genetically distinct groups, described as aggressive and nonaggressive (Koch et al., 1989), Tox+ and Tox0 (Balesdent et al., 1992) or A-group and B-group (Johnson & Lewis, 1990; Williams & Fitt, 1999; West et al., 2001). These two groups are now considered different species (Williams, 1992; Williams & Fitt, 1999; West et al., 2001), named Leptosphaeria maculans (A-group) and Leptosphaeria biglobosa (B-group) on the basis of differences in pseudothecial morphology (Shoemaker & Brun, 2001). The description of L. biglobosa was based on a few isolates from pseudothecia on Brassica juncea (Indian mustard) or B. napus. The B-group is polymorphic and may include more than one species (Rouxel et al., 1994). It is not clear if the L. biglobosa description corresponds to that of the B subgroup NA1, the only subgroup isolated from oilseed rape in Europe (Ansan-Melayah et al., 1997; West et al., 2002). Therefore the pathogens are described in this paper as A-group and B-group L. maculans (Johnson & Lewis, 1990). Both A- and B-group L. maculans are present in the UK, and stem canker caused losses in excess of £20 M per season from 1993 to 2001, despite the use of fungicides (Defra survey results; www.csl.gov.uk/prodserv/cons/crop/survey/osrintro.cfm). However, the relative importance of the two groups in development of severe epidemics is not clearly understood.

In Europe, phoma stem canker epidemics on winter oilseed rape are initiated in autumn (October–December) by airborne ascospores released from infected oilseed rape debris from previous crops (Gladders & Musa, 1980; Schramm & Hoffmann, 1991; West et al., 2001). Under favourable conditions, ascospores infect leaves to produce lesions (phoma leaf spots) from which the pathogen grows down petioles into stems to initiate basal stem cankers or upper stem lesions (Hammond et al., 1985; Hammond & Lewis, 1987; Zhou et al., 1999; Sun et al., 2000). The more damaging A-group is predominant on winter oilseed rape in western Europe (Williams & Fitt, 1999; West et al., 2001; West et al., 2002), while the less damaging B-group is predominant in eastern Europe, including Poland (Jedryczka et al., 1999). After inoculation with ascospores, leaf lesions of B-group L. maculans are smaller, with fewer pycnidia than the large pale grey lesions of A-group L. maculans, in both controlled environment experiments and natural conditions (Johnson & Lewis, 1994; Ansan-Melayah et al., 1997; Brun et al., 1997; Biddulph et al., 1999; Toscano-Underwood et al., 2001). In controlled environments, many B-group infections remain as pinpoint lesions (Toscano-Underwood et al., 2001), suggesting noncompatible reactions. It is not clear mechanistically why these differences between A- and B-group lesion phenotypes occur.

Although stem canker is known to be a monocyclic disease, with epidemics initiated by airborne ascospores (Gladders & Musa, 1980; Schramm & Hoffmann, 1991; West et al., 2001), most experiments on the biology of L. maculans have been done with conidia (e.g. Hammond & Lewis, 1987; Johnson & Lewis, 1994). As conidia are not normally infective, leaves have had to be wounded before inoculation to obtain lesions, and it is not possible to study mechanisms of penetration of leaves using this methodology. Although there is no evidence that conidia play a role in the development of stem canker epidemics in the UK (West et al., 2001), it is only recently that experiments on infection criteria have been done with ascospores (Biddulph et al., 1999; Toscano-Underwood et al., 2001). It has been shown that the A-group penetrates leaves through the stomata (Hammond et al., 1985), but no comparable information is available for the B-group of L. maculans. To control stem canker epidemics effectively, differences between the two groups in the process of infection need to be better understood.

Temperature affects the infection of winter oilseed rape leaves by L. maculans, but little is known about its effects on different stages in the process. Ascospores of A- or B-group L. maculans infected leaves (cvs Lipton or Nickel) at temperatures from 5 to 24°C, with the greatest number of lesions produced at an optimum near 20°C (Biddulph et al., 1999; Toscano-Underwood et al., 2001). The incubation period (time from inoculation to appearance of 50% of lesions, a better estimate than time to first lesions; Inman et al., 1997) was shortest at this temperature, and shorter for the B-group than the A-group. However, this estimated incubation period includes the times for spore adhesion, germination, penetration, and for the leaf colonization phase before the appearance of lesions. On water agar, the percentage of ascospores of both groups that germinated increased with temperature from 5 to 20°C, and time elapsed from inoculation until 50% of spores germinated was shorter for B-group than for A-group ascospores (Huang et al., 2001). Furthermore, A-group ascospores consistently produced branched, tortuous hyphae, while B-group ascospores produced long, straight hyphae with little or no branching, whether the ascospores came from natural populations in Australia (A) (B.J. Howlett, University of Melbourne, Australia personal communication); the UK (A, B); Poland (B); or China (B); or from crosses between defined A- or B-group isolates (Huang, 2002). Little comparative work has been done on germination of ascospores of A- and B-group L. maculans in relation to penetration of leaves at different temperatures. This paper describes effects of temperature on ascospore germination and penetration of winter oilseed rape leaves by A- and B-group L. maculans.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Effects of temperature on germination of ascospores of A- and B-group L. maculans on leaf surfaces

Three replicate experiments were done in temperature-regulated incubators (5, 10, 15 or 20°C) to investigate effects of temperature on germination of ascospores of A- and B-group L. maculans on oilseed rape leaves (cv. Lipton). Before inoculation, leaves were detached from plants and placed in 9 cm Petri dishes (one leaf per dish). Petri dishes with leaves inoculated with ascospores of A- or B-group L. maculans were incubated in darkness at constant temperatures. Petri dishes with inoculated leaves were arranged in completely randomized patterns in each incubator (temperature treatment). Temperature treatments were replicated in time and incubators were allocated randomly to temperature treatments in different replicates when possible, but some incubators could not operate at 5 or 10°C.

Plant material

Oilseed rape seeds cv. Lipton (resistance score 5 on a 1–9 scale; Anonymous, 1997) were sown in 9 cm diameter pots containing peat-based compost and a soluble fertilizer (1·5 kg PG mix m−3; Petersfield Products, Cosby, Leicester, UK). Plants were grown in a glasshouse and thinned to one plant per pot 10 days after sowing. Pots were then placed in seed trays (32 × 62 × 7 cm; six pots per tray), with adequate irrigation throughout the experiments. Trays were then transferred to a controlled-environment cabinet designed at Rothamsted (2·5 × 1·0 × 1·35 m; light intensity 210 µe m−2 s−1, 80–85% relative humidity, 12 h daylength) at 15°C. Plants were grown in the cabinet for 14 days until each had two true leaves fully expanded with the third leaf just starting to expand [growth stage (GS) 1,3; Sylvester-Bradley & Makepeace, 1985].

Production of ascospores

Pieces (≈30–50 cm long) of winter oilseed rape stem debris (cv. Lipton) with stem canker lesions were collected after harvest from fields at Rothamsted, UK and Poznan, Poland in July/August 1999. These stem pieces from Rothamsted or Poznan were expected to produce ascospores which were predominantly of A- or B-group L. maculans, respectively (Jedryczka et al., 1999). Stem pieces containing mature pseudothecia were stored dry at −5°C until required. The identity and representativeness of the ascospores used (A- or B-group) had been confirmed previously (Huang et al., 2001; Toscano-Underwood et al., 2001). UK stem pieces from the tap root and stem base (<10 cm above ground level) had produced predominantly A-group ascospores, but pieces from upper stems (10–30 cm above ground level) produced more B-group ascospores. Consequently, only the stem base and tap root regions of UK stem debris were used to produce A-group ascospores. Polish upper stem debris, previously found to produce only B-group ascospores, was used to produce B-group inoculum (as it would not have been possible to obtain only B-group ascospores from UK debris). Furthermore, these ascospores of UK A-group and Polish B-group L. maculans had been shown to germinate in the same way as ascospores produced from crosses between defined isolates of A-group and B-group L. maculans (Huang et al., 2001) or natural populations from different countries (Huang, 2002).

Fresh ascospore suspensions were used as inoculum. To obtain ascospores, pieces of stem debris bearing mature L. maculans pseudothecia were attached to the underside of Petri dish lids and sprayed with distilled water to induce release of ascospores. The lids were placed over the Petri dish bases. After 3 h at ≈20°C, large numbers of ascospores had been discharged into the dishes (Huang et al., 2001). Ascospore suspensions from several dishes were combined and the concentration adjusted to 5 × 103 ascospores mL−1 using a haemocytometer slide.

Inoculation of surfaces of detached leaves

The first and second true leaves from potted plants were excised by cutting at the base of petioles with surgical scissors. Excised leaves were immediately placed in a 500 mL beaker with their petioles immersed in 250 mL water for ≈15 min. Petioles were cut off and leaf laminas were transferred to Petri dishes (diameter 9 cm) lined with two layers of filter paper (Whatman no. 1; Whatman International Ltd, Maidstone, UK) moistened with distilled water (one leaf per dish) and preconditioned overnight at the desired temperatures before inoculation. Six sites on the adaxial surface of each leaf were rubbed gently (with an eraser, diameter ≈5 mm). Each site was inoculated by applying one drop (c. 30 µL) of fresh ascospore suspension (5 × 103 ascospores mL−1) with a Gilson pipette (Gilson Medical Electronics SA, Villiers-le-Bel, France). The insides of the lids were sprayed with distilled water to maintain a high relative humidity. The dishes were then placed in incubators at 5, 10, 15 or 20°C, and germination parameters assessed after incubation times of 2, 4, 6, 8, 10, 12, 14 or 24 h. One leaf with six inoculated sites was used for each temperature/incubation time assessment. In total, 32 leaves (192 sites) were inoculated for each experiment.

Assessment of ascospore germination

After the designated incubation time, leaves were removed from incubators and one drop of trypan blue (0·1% w/v in lactophenol; BDH Microscopical Reagents, Poole, UK) placed on each inoculated site to stop germination. After 2 min, the excess dye was removed with absorbent paper and the inoculated sites dried using a hair-drier. A film of collodion (30% in absolute ethyl alcohol; Fisher Scientific Ltd, Loughborough, UK) was applied to each inoculated site using a sable brush and left to dry for 2 min. Collodion membranes were then peeled off with forceps and placed in drops of distilled water on glass slides (75 × 25 mm), with two membranes (two inoculated sites) per slide. The percentages of ascospores of A- or B-group L. maculans that had germinated were assessed by scanning the length of each slide under a light microscope (Olympus Optical Co., London, UK) at ×200 magnification (300–500 ascospores were assessed at each temperature/incubation time test). An ascospore was considered to have germinated if the length of the germ tube exceeded the width of the ascospore (≈5 µm). The lengths of the germ tubes from 20 A-group and 20 B-group ascospores (from the three slides for each temperature/incubation time) were measured. If the ascospore had produced more than one germ tube, only the length of the longest was measured.

Statistical analysis

The maximum percentage (Go) of ascospores that germinated for each experiment and treatment was obtained from the observed data. The observed data were then normalized as a percentage (Gn) of Go. Analyses of variance were used to assess the effects of temperature and L. maculans group on ascospore germination, using the genstat statistical software (Payne et al., 1993). To estimate the time to germination of 50% of viable ascospores (Ve50) and rate of germination at Ve50 (re50) for each temperature and L. maculans group, a generalized linear model with a binomial distribution and a logit link function {loge[Gn/(100 − Gn)]} (logistic regression) with time as the explanatory variable was fitted to the data. Linear regressions of observed germ tube length on time were calculated separately for each temperature and L. maculans group, and analyses of position and parallelism were used to assess whether the data were best fitted by a single line or a series of parallel lines or nonparallel lines for different temperatures and groups.

Penetration of leaf surfaces by A- or B-group L. maculans

Five experiments were done in controlled-environment cabinets (Table 1). Experiments 1, 2 and 3 investigated the mode of penetration of leaf surfaces by hyphae of A- and B-group L. maculans. Experiments 4 and 5 were two replicates in time investigating the effects of temperature on percentage of germinated ascospores of A- and B-group L. maculans which penetrated stomata. In all experiments on penetration of leaves, ascospore suspensions were inoculated onto attached leaves, which had received no prior treatment, e.g. mechanical abrasion.

Table 1.  Temperatures, cabinets and methods used at different times in each of five experiments to examine the penetration of stomata by hyphae from ascospores of A- and B-group Leptosphaeria maculans (produced on debris of oilseed rape cv. Lipton) on surfaces of attached oilseed leaves (cv. Lipton)
ExperimentObservationAssessment methodaTemperature (°C)Assessment times (h)Cabinetb
  • a

    LM, light microscope; FL, fresh leaf tissue; SEM, scanning electron microscope; DL, decolorized leaf tissue.

  • b

    Cabinets are in order of increasing temperature. Temperature treatments were done in controlled environment cabinets with 12 h light/12 h darkness. Cabinets were allocated randomly to temperature treatments whenever it was possible, but growth cabinets L and M could not be operated at temperatures of 10°C or less. N was a Sanyo PG 660 growth cabinet; R was a Gallenkamp Fi-totron 600H growth cabinet; L and M were Rothamsted-designed growth cabinets.

  • c

    Observations were started 16 h after inoculation and continued at intervals until 72 h after inoculation.

  • d

    Percentage of germinated ascospores producing germ tubes/hyphae that penetrated surfaces of oilseed rape leaves (cv. Lipton) through stomata.

  • e

    Four-hourly assessments were started at 16 h and stopped at 72 h after inoculation at 5 and 10°C, and were started at 12 h and stopped at 52 h after inoculation at 15 and 20°C.

1Mode of penetrationLM, FL1516–72cL
2Mode of penetrationSEM, FL1516–72L
3Mode of penetrationSEM, FL1516–72L
4% stomatal penetrationdLM, DL5,10,15,2012–72eR,N,M,L
5% stomatal penetrationLM, DL5,10,15,2012–72R,N,M,L
Preparation of plants and ascospore suspensions

Oilseed rape plants (cv. Lipton) were grown in a glasshouse for 10 days and then transferred to a Rothamsted-designed controlled environment cabinet at 15°C for 14 days until they reached GS 1,3. In experiments 1–3, plants were kept in the same growth cabinet before and after inoculation. In experiments 4 and 5, plants were transferred to treatment growth cabinets (5, 10, 15 or 20°C) overnight before inoculation. The cabinets used were a Sanyo PG 660 cabinet and a Gallenkamp Fi-totron 600H growth cabinet, both with dimensions 1·2 × 0·6 × 0·86 m, fluorescent lighting giving a light intensity of 200 µe m−2 s−1and a relative humidity of 70–85%, and two of the Rothamsted-designed cabinets. A daylength of 12 h was maintained during experiments. Cabinets for temperature treatments were allocated randomly when possible, but some cabinets could not operate at 5 and 10°C. Ascospores of A- and B-group L. maculans for all five experiments were obtained from stem debris (cv. Lipton) collected in July/August 2001 from Rothamsted, UK and Poznan, Poland, respectively, and ascospore suspensions were prepared.

Inoculation of surfaces of attached leaves

The first and second true leaves of plants at GS 1,3 were inoculated by placing 10 µL fresh ascospore suspension (containing 5 × 103 or 104 ascospores mL−1, experiments 1–3 or 4 and 5, respectively) on each of 10–12 sites on their adaxial surfaces with a Gilson pipette. In experiments 1–3, 12 plants (one plant per pot) were inoculated with each ascospore group. In experiment 1, sites on two plants per group were wounded with a sterile pin before inoculation. In experiments 4 and 5, 12 plants per temperature (5, 10, 15 or 20°C) were inoculated with ascospores of A- or B-group L. maculans. Two oilseed rape leaves with 10 inoculated sites each were used for each temperature/incubation time tested. Inoculated plants were placed in seed trays (22 × 36 × 7 cm; four pots per tray), which were arranged in a randomized design in the growth cabinet. Trays were covered with polyethylene bags sprayed inside with distilled water to maintain a high relative humidity and kept filled with water (depth 1 cm) for the duration of the experiments.

Assessment of hyphal penetration

In experiment 1, the mode of penetration of leaf surfaces by A- or B-group L. maculans was investigated by light microscopy at ×400 magnification. Randomly selected inoculated leaves (two leaves per assessment) were detached by cutting at the base of the petiole with surgical scissors at times from 16 to 72 h after inoculation. Inoculated sites on the leaf were excised (if the leaf was wounded, the excised section included the wound) with a scalpel and placed on microscope slides. Leaf pieces were stained with trypan blue (0·1 w/v in lactophenol) and covered with cover slips. The slides were gently heated over a flame before examination. Penetration through stomata or wounds was recorded.

In experiments 2 and 3, the mode of leaf penetration by hyphae from germinated ascospores was examined using a scanning electron microscope. Inoculated leaves were detached randomly from plants (one leaf per assessment) at times from 16 to 72 h after inoculation. Inoculated sites were excised, attached to aluminium stubs (10 mm in diameter, 3 mm thick with no spigot) with Tissue-Tek (Sakura, Finetek, Torrance, CA, USA) and secured into a stub holder. The stub holder with the sample was attached to a vacuum transfer device secured onto a liquid nitrogen chamber, which was part of a CT 1500 cryo-transfer system (Oxford Instruments, Eynsham, Oxfordshire, UK). The stub holder was held in the vapour above the liquid nitrogen for ≈30 s before the air in the chamber was pumped out to produce a vacuum. When the liquid nitrogen had begun to solidify, the tip of the stub holder was held in the solidifying nitrogen. Once the nitrogen had solidified, the stub holder was taken out and enclosed (under vacuum) in the inner chamber of the vacuum transfer device. This device was then disconnected from the chamber containing the nitrogen and connected onto the preparation chamber, where the sample was coated with 15 nm of gold before examination with a Phillips XL40 scanning electron microscope, which stored the images digitally.

In experiments 4 and 5, penetration by hyphae through stomata was assessed at 4 h intervals 16–72 h (5 and 10°C) or 12–52 h (15 and 20°C) after inoculation (Table 1). Two inoculated leaves (with 10 inoculated sites each) from each temperature/incubation assessment were randomly selected and detached. Inoculated sites on the leaves were excised, decolorized in a 3 : 1 (v/v) mixture of ethanol and chloroform for 24 h, and stained in 0·025% trypan blue in lactophenol for 4 h. Stained tissue was mounted in 70% glycerol in distilled water on glass slides. Penetration assessments were done using a light microscope at ×400 magnification (200–300 germinated ascospores were assessed at each temperature/incubation assessment).

Statistical analysis

The effects of temperature, time and L. maculans group, and the interaction between them, were analysed by restricted maximum likelihood estimation (REML) using genstat. REML was used to estimate variability due to plants and cabinets within the unbalanced experimental structure (unequal treatment replication over time). Wald tests were used to assess treatment effects.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Effects of temperature on germination of ascospores of A- and B-group L. maculans on leaf surfaces

Ascospores of both A- and B-group L. maculans germinated at temperatures from 5 to 20°C on leaves of oilseed rape by producing germ tubes. After 2 h incubation, A-group ascospores had started to germinate (>5% of spores had germinated) at 15 and 20°C, and B-group ascospores had started to germinate at 5–20°C (Fig. 1). At all temperatures, the percentage germination had almost reached its maximum after 14 h incubation. Percentage germination of both A- and B-group ascospores was greatest at 20°C after 24 h incubation (82 and 61% of the A- and B-group ascospores, respectively, had germinated). The mean maximum percentage germination (Go) was generally greater for A-group ascospores (74%) than for B-group ascospores (54%) (SED 1·87; P < 0·001; 14 df). The percentage of both A- and B-group ascospores that germinated after 24 h increased with increasing temperature from 60% (A-group) or 45% (B-group) at 5°C to 82% (A-group) or 61% (B-group) at 20°C (Fig. 1; Table 2).

image

Figure 1. Changes with time after inoculation in the percentage of ascospores of A-group (•) or B-group (○) Leptosphaeria maculans that had germinated on oilseed rape leaves (cv. Lipton) in darkness at 5°C (a), 10°C (b), 15°C (c) or 20°C (d). Data points illustrated are averages from three experiments.

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Table 2.  Maximum percentage germination, time to 50% germination and rate of germination of ascospores of A- or B-group Leptosphaeria maculans germinating on surfaces of detached oilseed rape leaves (cv. Lipton) in darkness at 5, 10, 15 or 20°C under continuous leaf wetness
Temperature (°C)L. maculans groupGermination parametera
GoVe50re50
  • a

    Three experiments were done. Go is the mean observed maximum percentage of ascospores that germinated at each temperature (Fig. 1). The parameters Ve50, the estimated time (h) for the germination of 50% of viable ascospores and re50, the rate of germination at Ve50, were estimated from the curves for regression of percentage germination on incubation time (Fig. 2).

  • b

    Approximate maximum SED.

5A59·56·510·5
B44·55·9 9·9
10A75·86·011·7
B53·15·7 8·2
15A78·44·716·4
B57·34·010·4
20A81·63·317·8
B60·92·718·3
SED (df) 3·74 (14)0·43b (144)2·11b (144)

Regression curves fitted well to the normalized data for changes in the percentage of ascospore germination on leaf surfaces with time (h) after inoculation with A- or B-group ascospores (Fig. 2). The curves differed significantly (deviance ratio 63·9; 47 and 144 df; P < 0·001) between the A-group and B-group, and between different temperatures. The estimated times from inoculation to 50% germination (Ve50) generally decreased with increasing temperature for both A- and B-group ascospores. The estimated rate of germination at 50% (re50) generally increased with increasing temperature for both A- and B-group ascospores, but re50 was smaller at 10 than 5°C for B-group ascospores (Table 2).

image

Figure 2. Changes with time after inoculation in the percentage of ascospores of A-group (•) or B-group (○) Leptosphaeria maculans germinating on oilseed rape leaves (cv. Lipton) in darkness at 5°C (a), 10°C (b), 15°C (c) or 20°C (d). Observed data from three experiments (•,○) were normalized as a percentage (Gn) of the maximum percentage (Go) of ascospores which germinated for each experiment and treatment, and analysed by anova (Table 2). A generalized linear model with a binomial distribution and logit link function {loge[Gn/(100 − Gn)]} was used to calculate the regression curves. Back-transformed curves are illustrated.

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Effects of temperature on germ tube elongation on leaf surfaces

Germ tube length increased with increasing temperature from 5 to 20°C for both A- and B-group ascospores (Fig. 3). After 10 h, germ tubes from A-group ascospores reached average lengths of c. 15, 17, 29 and 38 µm at 5, 10, 15 and 20°C, respectively. After 10 h, germ tubes from B-group ascospores reached lengths of c. 18, 26, 41 and 76 µm at 5, 10, 15 and 20°C, respectively. At 15 and 20°C, after 12 h incubation, germ tubes of A-group L. maculans had branched extensively, forming tortuous hyphae for which lengths were difficult to measure, and B-group germ tubes were too long to be measured. Linear regressions of germ tube length on time after inoculation fitted well to the data and accounted for >95% of the variance. The combined linear regression analyses of position and parallelism suggested the data were fitted best by a series of different lines. Subsequent analyses were therefore done separately for each temperature. Percentages of the variance accounted for were 96, 98, 98 and 97% for 5, 10, 15 and 20°C, respectively. Data for 5, 10, 15 or 20°C were fitted best by pairs of nonparallel lines for A- and B-group ascospores. Germ tubes from B-group ascospores were longer than those from A-group ascospores at all temperatures tested (P < 0·001), with the difference greatest at 20°C.

image

Figure 3. Changes with time after inoculation (t) in the length of germ tubes (l) produced from ascospores of A-group (•) or B-group (○) Leptosphaeria maculans germinating on oilseed rape leaves (cv. Lipton) in darkness at 5°C (a), 10°C (b), 15°C (c) or 20°C (d). Data points illustrated are averages from three experiments. Regression lines for A- and B-group are: l = 0·75 + 1·47t and l = 1·77 + 1·76t at 5°C (two nonparallel lines); l = 1·65 + 1·61t and l = 2·07 + 2·42t at 10°C (two nonparallel lines); l = −1·50 + 2·94t and l = 0·47 + 3·97t at 15°C (two nonparallel lines); and l = 1·64 + 3·76t and l = −10·73 + 9·13t at 20°C (two nonparallel lines).

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Mode of penetration of leaf surfaces by hyphae from germinated ascospores of A- and B-group L. maculans

In all three experiments, ascospores of A-group L. maculans germinated to produce germ tubes that grew tortuously and branched extensively (Fig. 4c). These hyphae predominantly penetrated the leaf surface through stomata near the ascospore (Figs 4a and 5a), but occasionally formed appressorium-like structures (Figs 4c and 5b). Germinated ascospores of B-group L. maculans produced long germ tubes that grew in almost straight lines (Fig. 4b). The predominant mode of penetration of leaf surfaces was by hyphae entering through stomata (Figs 4b,d, 5c,d) and no appressorium-like structures were observed. The hyphae usually penetrated stomata further from the ascospore than those of A-group L. maculans. No directional growth towards stomata was observed for hyphae of either group. Hyphae from germinated ascospores of both A- and B-group L. maculans also penetrated the leaf surfaces through wounds (experiment 1).

image

Figure 4. Patterns of germination, hyphal growth and stomatal penetration on attached oilseed rape leaves (cv. Lipton) inoculated with ascospores of A-group (a,c) or B-group (b,d) Leptosphaeria maculans obtained from oilseed rape debris (cv. Lipton). (a) Germ tube from A-group ascospore penetrating through stomata 22 h after inoculation at 15°C; (b) hyphae from B-group ascospore penetrating stomata 20 h after inoculation at 20°C; (c) appressorium-like structure (AP) from A-group ascospore 72 h after inoculation at 5°C; (d) hyphae from B-group ascospores penetrating stomata and the collapse of guard cells (arrow) 42 h after inoculation at 15°C (all bars = 20 µm).

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image

Figure 5. Scanning electron micrographs showing penetration of attached oilseed rape leaves (cv. Lipton) inoculated with ascospores of A-group (a,b) or B-group (c,d) Leptosphaeria maculans obtained from oilseed rape debris (cv. Lipton). (a) hyphae from A-group ascospores penetrating stomata 42 h after inoculation at 15°C (bar = 10 µm); (b) appressorium-like structure formed by hyphae from A-group ascospores 48 h after inoculation at 15°C (bar = 10 µm); (c,d) hyphae from B-group ascospores penetrating stomata 42 h (c, bar = 20 µm) or 72 h after inoculation at 15°C (d, bar = 20 µm).

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Effects of temperature on penetration of leaf surfaces by hyphae of A- and B-group L. maculans

Hyphae of A- or B-group L. maculans penetrated stomata on surfaces of attached leaves of oilseed rape (cv. Lipton) at temperatures from 5 to 20°C (Fig. 6). Penetration of leaf surfaces by hyphae of A-group L. maculans was first observed 24 and 16 h after inoculation at 5 and 10°C, respectively. At the same temperatures, penetration by hyphae of B-group was first observed 20 and 16 h after inoculation, respectively. At 15 and 20°C, penetration by hyphae from ascospores of both groups was first observed 12 h after inoculation. Temperature, ascospore group and time after inoculation affected the percentage of stomata penetrated, and there was a significant interaction between these factors (Wald statistic 533·2; 33 df; P < 0·001). The percentage of germinated ascospores that penetrated through stomata (averaged over times after inoculation) increased with increasing temperature from 6% (A-group, B-group) at 5°C to 18% (A-group) or 14% (B-group) at 20°C; it was greater for A- than for B-group L. maculans at 10, 15 and 20°C (Wald statistic 1210; 1 df; P < 0·001). Percentages of germinated A-group ascospores that penetrated were greatest 72 h (5 and 10°C) and 52 h (15 and 20°C) after inoculation. For the B-group, penetration reached maxima 48 h (5 and 10°C) and 36 h (15 and 20°C) after inoculation; the differences between the two groups were less at the higher temperatures. The subsequent decreases observed for B-group L. maculans may have been artefacts; because the first lesions were being formed then, visual assessments of hyphal penetration were less accurate.

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Figure 6. Changes with time after inoculation in the percentage of germinated ascospores of A-group (•) or B-group (○) Leptosphaeria maculans which produced hyphae that had penetrated through stomata of attached oilseed rape leaves (cv. Lipton) under continuous wetness at temperatures of 5°C (a), 10°C (b), 15°C (c) or 20°C (d). Data points illustrated are averages from two experiments. SED (15 df) = 0·98.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Results from these controlled environment experiments suggest that ascospores of A- and B-group L. maculans can germinate and penetrate leaves of winter oilseed rape through stomata over a wide range of temperatures (5–20°C) (Table 3) and that the optimum may be the same as the optimum for formation of lesions (i.e. near 20°C; Biddulph et al., 1999; Toscano-Underwood et al., 2001). As the temperature increased from 5 to 20°C, the percentage of ascospores of both groups that germinated, length of germ tubes and rate of germination (re50) increased and the time to 50% germination decreased, with results on leaf surfaces similar to those on water agar (Huang et al., 2001). Furthermore, over this range the frequency of stomatal penetration increased and the time from inoculation to penetration decreased with increasing temperature. These results show how effects of temperature on germination and penetration times contribute to observed effects on times from inoculation to lesion formation for both groups, which decrease with increasing temperature from 5 to 20°C (Toscano-Underwood et al., 2001). Furthermore, by subtraction it is possible to estimate the effects of temperature on the time from penetration to lesion formation. This time (the leaf colonization phase), although difficult to measure, is theoretically a more appropriate measurement of the incubation period of a pathogen than the time from inoculation to lesion formation.

Table 3.  Effects of temperature on progress of ascospore germination, penetration and lesion formation by A- and B-group Leptosphaeria maculans on oilseed rape (cv. Lipton) leaves
ParameterTemperature (°C)SED (df)
5101520
ABABABAB
  • a

    Data from Huang et al. (2001).

  • b

    50% of maximum penetration with 48 h leaf wetness.

  • c

    Data from Toscano-Underwood et al. (2001) for 48 h leaf wetness; SED estimated by restricted maximum likelihood analysis – it is not possible to give df.

  • d

    Estimated by subtracting time to 50% penetration from time to 50% lesions; data from different experiments so SED cannot be calculated.

Time (h)
50% germination (agar)a  8·9  7·3  7·7  5·0  5·7  3·8  6·8 3·30·83 (14)
50% germination (leaf)  6·2  5·8  5·8  5·9  4·7  4·4  3·2 3·30·41 (14)
50% penetration (leaf)b 37·0 35·3 36·0 31·1 32·8 26·9 29·924·80·75 (4)
50% lesions (leaf)c523·6449·8374·7281·8235·5133·8173·988·743·6 (max)
Incubation periodd486·6414·5338·7250·7202·7106·9144·063·9
Thermal time (degree-days)
50% germination (agar)  1·9  1·5  3·2  2·1  3·3  2·4  5·6 2·70·40 (14)
50% germination (leaf)  1·3  1·2  2·4  2·5  2·9  2·8  2·7 2·70·25 (14)
50% penetration (leaf)  7·7  7·4 15·0 13·0 20·5 16·8 24·920·70·48 (4)
50% lesions (leaf)c109·1 93·7156·1117·4147·2 83·6144·973·915·3 (max)
Incubation periodd101·4 86·3141·1104·4126·7 66·8120·053·2

Furthermore, these results show how the penetration of additional stomata after 40 h from inoculation by A-group but not B-group L. maculans can explain how the time from inoculation to the appearance of 50% of the lesions is shorter for B-group than for A-group L. maculans (Toscano-Underwood et al., 2001). There was little evidence for an interaction between temperature and L. maculans group in the time to 50% penetration (with ratios B-/A-group of 95, 86, 82 and 83% for 5, 10, 15 and 20°C, respectively). However, there was evidence for an interaction in the leaf colonization phase, with B/A ratios of 85, 74, 53 and 44% for 5, 10, 15 and 20°C, respectively. If results are expressed using the degree-day (above 0°C) approximation, the longest incubation periods are at 10°C for both groups, with the times to 50% of lesions for the A-group in the same range (130–160 degree-days) for all temperatures from 8 to 20°C (Biddulph et al., 1999; Toscano-Underwood et al., 2001).

Results from this study indicate that both A- and B-group L. maculans penetrate leaves predominantly through stomata, although they may also penetrate through wounds. However, they suggest that there may be differences between the two groups in infection strategy, as there were noticeable differences between them in the mode of germination and pattern of hyphal growth. Differences between A- and B-group L. maculans in the pattern of ascospore germination (short, highly branched A-group hyphae and long, straight B-group hyphae) were the same on leaf surfaces as on water agar (Huang et al., 2001). Hence, chemical signals or leaf surface topography do not appear to be involved in germ tube extension and differentiation in the oilseed rape/L. maculans pathosystem. Furthermore, appressorium-like structures were observed on leaf surfaces colonized by A-group hyphae but not on those colonized by B-group hyphae; Boytrytis cinerea has been observed to produce similar structures (Backhouse & Willetts, 1987). The differences observed in the pattern of hyphal growth suggest that the two groups may have different prepenetration phases, which may explain why penetration by hyphae from A-group ascospores can continue for longer than penetration by hyphae from B-group ascospores.

The maximum percentage of germinated ascospores that penetrated stomata was less for B-group than for A-group L. maculans, suggesting that B-group ascospores were less efficient than A-group ascospores in infecting oilseed rape leaves under these conditions. This may explain differences in numbers of lesions produced per ascospore between A-group L. maculans (infection efficiency 12 or 25%) and B-group L. maculans (infection efficiency 7%) (Biddulph et al., 1999; Toscano-Underwood et al., 2001). However, the differences in the appearance of lesions (Johnson & Lewis, 1994; Biddulph et al., 1999; Toscano-Underwood et al., 2001) suggest there are also differences in the postpenetration phases between the two groups. In controlled environment experiments of Johnson & Lewis (1994), isolates of both A- and B-group L. maculans, which had penetrated the leaves through wounds, grew intercellularly through the mesophyll to cause necrosis. However, A-group hyphae grew ahead of the necrotic regions, whereas necrosis caused by B-group isolates was more rapid and often spread ahead of the hyphal front. The differences between the two groups in their infection strategies observed in these controlled environment experiments suggest that different control strategies are needed in areas with different proportions of A-group and B-group in L. maculans populations. The timing of sprays is more critical in areas with a high proportion of A-group L. maculans than in areas with a high proportion of B-group L. maculans, because these results suggest that, under the same conditions, more stomata can be penetrated by the A-group than the B-group. Furthermore, leaf lesions caused by A-group expanded more quickly than those caused by B-group L. maculans (Toscano-Underwood et al., 2001). Previous studies indicate that fungicides do not control the disease once the pathogen has reached the stem (Gladders et al., 1998; West et al., 1999). Therefore, combined with information about L. maculans population structure, these results could help improve strategies for effective control of serious stem canker epidemics.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank the Perry Foundation, the UK Department of the Environment, Food and Rural Affairs, the Biotechnology and Biological Sciences Research Council, the China Scholarship Council and the British Council for supporting the work. We thank M. Jedryczka for supplying oilseed rape debris from Poland, A.D. Todd for statistical analyses of the data and A.O. Latunde-Dada and J.S. West for advice.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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