Volume 55, Issue 1 p. 137-144
Free Access

Verticillium longisporum and V. dahliae: infection and disease in Brassica napus

L. Zhou

L. Zhou

Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Bioinformatics Phase I, Washington Street, Blacksburg, Virginia, VA 24061–0477, USA;

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Q. Hu

Q. Hu

Institute of Oil Crops, Chinese Academy of Agricultural Sciences, 430062 Wuhan, P.R. China; and

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A. Johansson

A. Johansson

Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, PO Box 7080, 750 07 Uppsala, Sweden

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C. Dixelius

C. Dixelius

Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, PO Box 7080, 750 07 Uppsala, Sweden

E-mail: [email protected]

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First published: 20 December 2005
Citations: 55

Abstract

Verticillium wilt of oilseed rape (Brassica napus) is caused primarily by Verticillium longisporum and has become a serious problem in northern Europe. In order to evaluate whether V. longisporum and V. dahliae differ in their interaction with oilseed rape, phenotypical and molecular assessments were made. Oilseed rape plants for fungal assessments were inoculated with V. longisporum and V. dahliae via root-dipping and samples were taken from roots, stems, leaves, flowers, pods and seeds during plant development. The infection by V. longisporum was found to start mainly in lateral roots and root-hairs, followed by colonization of the xylem vessels and extensive spread in stems and leaves, whereas V. dahliae infected the main roots and remained in the region below the cotyledon node of the plants. Re-isolation studies, together with PCR analysis of samples taken from early growth stages through to fully ripe plants, showed that the onset of flowering was a critical phase for V. longisporum to colonize plants. No seeds infected with V. longisporum were found. Mycelial growth from V. dahliae but not V. longisporum was significantly reduced on media containing tissue from a low glucosinolate B. napus genotype compared with growth on media containing tissue from a high glucosinolate cultivar. The results of this study suggest that V. longisporum favours B. napus as host and that the transition from the vegetative to the generative phase is of importance for the spread of the fungus in oilseed rape plants.

Introduction

Verticillium wilt on Brassica oil crops has been observed for about 50 years in Sweden and has been recognized as an increasing problem since the 1970s (Dixelius et al., 2005). This disease is now considered to be the major threat to oilseed rape (Brassica napus) and turnip rape (B. rapa) production in Sweden. In Sweden and Germany this disease is caused by Verticillium longisporum (Karapapa et al., 1997), a vascular wilt pathogen closely related to V. dahliae and V. albo-atrum (Fahleson et al., 2004). In previous publications from these two countries on this disease, the name V. dahliae was frequently used to identify the causal agent of verticillium wilt on Brassica crops. Misidentification of the two species has also been reported from horseradish (Babadoost et al., 2004). Brassica-oil crops can occasionally host several other Verticillium species, especially on very weak plants, and V. longisporum can also infect plant species outside the Brassicaceae (Johansson et al., 2005). Hence, great care is needed to distinguish the different disease-causing pathogens, particularly since morphological characters are often misleading. In addition, crops like potato and sugar beet are hosts for V. dahliae in the same geographic areas (Pegg & Brady, 2002), which implies that microsclerotia from the two species can be present in the soil. Plant breeders have searched intensively for sources of resistance to verticillium wilt in the oilseed rape germplasm without much success. Recently, promising B. oleracea and B. rapa-genotypes with enhanced resistance were identified (Happstadius et al., 2003; Dixelius et al., 2005), but it will take many years of breeding using these gene sources before a resistant oilseed rape variety can reach the market.

Diagnosis of verticillium wilt on brassica-oil crops has proved to be very difficult, especially on winter types in early spring. Stem sections often show discoloration due to both V. longisporum and Leptosphaeria maculans (blackleg), which makes symptom differentiation difficult (Kuusk et al., 2002). Insect damage caused by Psylloides chrysocephala (cabbage stem flea beetle) larvae can, in some years, confound diagnosis of verticillium wilt further.

Many studies have been performed with V. dahliae to elucidate its host range and epidemiology (Schnathorst, 1981; Beckman, 1987; Gold et al., 1996; Heinz et al., 1998; Rowe & Powelson, 2002). The infection process of V. dahliae is divided into two main parts. Determinative phase I starts with the germination of microsclerotia, in response to root exudates (Mol & Scholte, 1995). The fungus enters the root and immature xylem elements by the fungal hyphae (Beckman, 1987). This is followed by phase II, which consists of multiple events including hyphal proliferation and the production of conidia (Beckman, 1987). The conidia are spread in the vascular elements where they are trapped by vessel end walls, where conidial germination and colonization occur. A new set of conidia is produced which continue the colonization of the upstream vessels (Beckman, 1987; Gold et al., 1996). As the foliage begins to senesce, the fungus leaves the xylem elements and colonizes the surrounding nonvascular tissues. Subsequently, microsclerotia form in the dying stems and leaves (Mol, 1995; Mol & Scholte, 1995) followed by an enrichment of inoculum density when debris is incorporated in the soil.

Glucosinolates are the main class of secondary metabolites in cruciferous crops and more than 100 different compounds have been identified (Louda & Mole, 1991). They have received attention since it has been shown that their breakdown products, in particular, can not only possess antifungal and antibacterial activity, but also affect insects and viruses attacking cruciferous crops (Mithen, 2001). Upon tissue disruption, glucosinolates are hydrolysed by myrosinase to release isothiocyanates, thiocyanates, nitriles or oxozolidinethiones. Isothiocyanates, in particular, have shown a high potential for suppressing a broad range of soilborne pathogens under in vitro conditions (Sarwar et al., 1998).

In order to generate more knowledge concerning verticillium wilt infection of oilseed rape, experiments were set up to assess the infection patterns of V. longisporum and V. dahliae, with the specific aim of investigating whether the two fungi differ in their interaction with B. napus as host. The potential for long-distance spread of V. longisporum via seeds and the possible influence of the degradation products of glucosinolates on fungal growth were also investigated.

Materials and methods

Cultivars of B. napus and isolates of Verticillium

Two accessions of spring type Brassica napus were used in the studies: cv. Maskot, moderately susceptible to verticillium wilt, and a highly susceptible breeding line Sv8326503 (96-326004). Three batches comprising 525 plants of each genotype were root dip-inoculated (Koike et al., 1994). Seedlings were inoculated with one of three isolates of V. longisporum, isolated from oilseed rape – CBS 110218 (VD1), CBS 110219 (VD4) and CBS 110220 (VD11) – or one of two isolates of V. dahliae: one from oilseed rape G12-1 and one from sugar beet NOVA2 (Steventon et al., 2002a; Fahleson et al., 2003). All isolates used were cultured from single-spore isolates and grown on potato dextrose agar (PDA; Difco) or malt extract agar (Difco) plates in the dark at 18–22°C. For long-term storage, fungal isolates were cultured on PDA together with cryopreservation beads (STC Technical Service Consultants Ltd) for 3 weeks. Beads coated with mycelia were incubated in the cryopreservation solution provided by the manufacturer. All bead samples were stored in vials at −70°C. Five hundred and twenty-five plants per genotype and trial were treated with water as control material. Two trials for each genotype and fungal isolate combination were performed. In total, 980 inoculated and 980 control plants were examined using diquat treatment, PCR or microscopic analysis.

Inoculation, culture conditions and sample treatments

One or two seeds of each genotype were planted in a small pot containing sterilized soil and covered with sand. After germination, the seedlings were thinned to one plant per pot. The pots were placed into plastic containers and maintained in a glasshouse with a 14-h photoperiod and a temperature of 15°C (light) and 10°C (dark). After the development of the first true leaves (approximately 14 days), plants were root dip-inoculated using a conidial suspension of each Verticillium isolate adjusted to 106 conidia mL−1 using sterile distilled water (Koike et al., 1994). The plants were replanted after inoculation into 10 × 10 cm pots and maintained in a glasshouse or culture chamber with a 16-h photoperiod and temperatures of 22°C (light) and 18°C (dark). Experiments were also performed where the conidial suspensions of CBS110218 (VD1) and G12-1 were mixed in a 1:1 ratio in the root-dipping solution, as were CBS110219 (VD4) and NOVA2. These plants were grown as described for single isolate inoculations.

Plant growth stages were recorded using the BBCH identification key (Lancashire et al., 1991). Thirty-five inoculated and 35 control plants were collected once a week between developmental stages BBCH 13 (three leaves unfolded) and BBCH 89 (fully ripe). Levels of disease severity were evaluated using a scale of 0–5, where 0 = no symptoms and 5 = dead plants, according to the scale described by Steventon et al. (2002b). Numbers of entirely yellow leaves compared with control plants were also monitored. Additionally, 5 cm sections from the basal stem (below the cotyledonary node) and upper stem (below the first flower bud) were collected from each plant. The stem pieces were washed, surface-sterilized with 0·25% sodium hypochlorite for 1 min, followed by three rinses in sterile water, and treatment in a diquat solution of 0·3% (w/v) Reglone (Syngenta), to enhance tissue senescence (Andersson, 2003). To identify optimal conditions, 0, 1, 5 and 10 min treatments of the diquat solution were compared in combination with different incubation times under humid conditions (0, 1, 2, 5 and 10 days). For this test, basal stem samples from an additional 100 oilseed rape plants (cv. Maskot) at BBCH 65 were used. Tissue samples were incubated in Petri dishes at 20°C with 95% humidity for 24 h and left to dry for up to a month. Re-isolation of fungi was performed using PDA, augmented with 3 g L−1 of streptomycin (Astra Zeneca, Sweden) and incubated at 20°C in darkness.

Fungal growth on media containing plant tissue

Seeds of cv. Maskot (< 12 µmol glucosinolates per g defatted dry matter) and cv. Niklas (90–120 µmol glucosinolates per g defatted dry matter) (Jönsson & Uppström, 1986) were surface-sterilized by agitating in 0·52% sodium hypochlorite for 1 h. Seeds were then dipped for 1 min in 70% ethanol, followed by three washes in sterile distilled water. The seeds were germinated in Petri dishes containing sterile Murashige and Skoog (MS) medium (ICN Biomedicals Inc.) augmented with 1% (w/v) sucrose (MS-1) and solidified with 3 g of gelrite L−1 (Merck, USA). The dishes were incubated at 22°C with a 16-h photoperiod for 4 days to obtain cotyledons. These seedlings were further cultivated in 15-cm-high glass containers containing MS-1 medium using the same growing conditions for 3 weeks to obtain true leaves. Cotyledons or true leaves of equal sizes from one, five, 10 or 20 seedlings of each of the two cultivars were frozen in liquid nitrogen and ground with a mortar and pestle. The approximate weight corresponded to 25, 125, 250 and 500 mg, respectively. Each tissue sample was mixed in a final volume of 100 mL PDA at 45°C. Ten millilitres of each combination were pipetted into 9 cm Petri dishes. After solidification, plugs (4 mm in diameter) of isolates CBS 110218 (VD1), CBS 110219 (VD4), CBS 110220 (VD11), G12-1 and NOVA2 were transferred to the centre of each dish and incubated in darkness at 22°C. Fungal growth was measured as the number of days taken for mycelia to reach the edge of the Petri dish on five replicate dishes Non-amended PDA was used as controls. The experiment was repeated three times.

Statistical analysis

Means, standard deviation, Student's t-test, Tukey's test and analysis of variance were calculated using PROC GLM (SAS Institute) and utilized in the assessments of the spatial infection pattern. Differences among means were tested using Fisher's least significant difference (LSD). Tukey's test was also used to calculate the honest significant difference (HSD) in the analysis of fungal growth in response to plant tissue extracts.

Seed analysis

Ten seed samples from 10 selected fields in Östergötland, Sweden (50 g of seed per location), where incidence of verticillium wilt disease ranged from 15 to 94%, were collected. Two samples, each derived from 40 plants (cv. Maskot) inoculated with VD1 or G12-1 isolates, and seeds from water-inoculated control plants grown under glasshouse conditions were also analysed. One hundred seeds from each sample were surface-sterilized by soaking in 70% ethanol for 30 s, followed by 5 min in 0·25% sodium hypochlorite solution, and rinsed three times in distilled sterile water. The surface-sterilized seeds were incubated on wet filter paper in Petri dishes for 1·5 or 4 days, before cotyledons and seed coats were separated and used for DNA extraction. Surface-sterilized seeds and germinated seedlings were also placed on PDA in Petri dishes and incubated in darkness at room temperature (20°C) and observed for fungal growth.

DNA isolation and PCR analysis

Plant samples were collected once a week from different parts (root, basal stem = below cotyledon node, upper stem = below first flower buds, basal leaf = leaf number 6, upper leaf = below main inflorescence, flowers, pods and seeds) of 35 inoculated and control plants of each genotype, and repeated twice as described earlier, and analysed by PCR. Seed coats and seedlings from the seed contamination investigation were also analysed using PCR. Total DNA was isolated by grinding the tissue together with glass beads (150–212 µm, Sigma-Aldrich), using the method described by Edwards et al. (1991) with modifications (Steventon et al., 2002a). Verticillium dahliae primers 5′-CACATTCAGTTCAGGACGGA-3′ and 5′-CCGAAATACTCCAGTAGAAGG-3′, and V. longisporum primers 5′-TCTCCTCTCTACGAGAACGA-3′ and 5′-CACTTTCT-AAGTATCCTTCCTAT-3′ were used in the PCR reactions (Li et al., 1999; Steventon et al., 2002a). Ten nanograms of DNA were added to 20 µL of a reaction mix containing 7·8 µL sterile distilled water, 2·5 µL 10 × PCR buffer (100 mm Tris-HCl, pH 8·3, 500 mm KCl) (Perkin Elmer Applied Biosystems), 1·25 mm MgCl2 (Perkin Elmer Applied Biosystems), 0·14 mm dNTP (Amersham Pharmacia Biotech), 7·5 units Amplitaq Gold (Perkin Elmer Applied Biosystems) and 2·6 µL of each primer (4 µm). Amplification using the V. longisporum primers was carried out under the following conditions: one cycle at 94°C for 2 min, followed by 30 cycles of 94°C for 2 min, 48°C for 2 min, 72°C for 3 min, followed by one cycle of 72°C for 10 min for final extension. For the V. dahliae primers, the following conditions were used: one cycle at 95°C for 3 min, one cycle at 37°C for 1 min, and 72°C for 1 min, followed by 40 cycles of 95°C for 1 min, 37°C for 1 min, 72°C for 1 min, followed by one cycle of 72°C for 4 min for final extension. The PCR amplifications were carried out in a Perkin-Elmer/Cetus 9600 thermocycler. The PCR products were visualized on a 1·2% agarose gel by ethidium bromide staining. A threshold level of expected PCR fragments in at least 90% of the samples in each tissue category and developmental stage was set to indicate presence of pathogen.

Staining and microscopy of inoculated B. napus tissue

Trypan blue staining was performed on inoculated samples using the method described by Koch & Slusarenko (1990), with slight modifications. Samples, approximately 2 cm long, were taken from the root, basal stem (below cotyledon node), lower stem (above the cotyledon node) and upper stem (below first flower buds). In total, samples from 35 plants per week from each genotype were used, and the experiment was repeated twice. Cross-sections of oilseed rape were wrapped with one layer of miracloth (Calbiochem, Biosciences Inc) and stained by dipping the samples into a boiling trypan blue staining mixture for 2 min, followed by de-staining in a 1 g L−1 chloral hydrate (Sigma-Aldrich) solution overnight. Before microscopic observations, the cross-sections were washed twice in 50% glycerol. Fixation in ethanol was extended to 4 days, followed by sectioning into specimens approximately 3 mm thick. Stained samples from various parts of the oilseed rape plants were examined using light microscopy. For transmission electron microscopy, samples from roots and stems were fixed in 2·5% glutaraldehyde in 0·1 m PBS (pH 7·2) and stored at 4°C before being subjected to dehydration in ethanol and acetone and embedding in TAAB 812 resin (TAAB Laboratory Equipment Ltd) (Sundberg et al., 1997). The samples were cut into 40-nm sections by using an LKB Ultratom I (LKB Products) with a Du Point diamond knife. The sections were stained with uranyl acetate and lead citrate and visualized by transmission electron microscopy using a Philips CM10 microscope.

Results

Infection studies

Observations of roots and vertical stem sections of oilseed rape plants stained by trypan blue indicated that infection by V. longisporum was primarily initiated from lateral roots or root hairs (Fig. 1A) whereas V. dahliae infected via the main root. After hyphae of V. longisporum entered the root, the xylem tissue was targeted (Fig. 1B) followed by colonization of upper portions of the vessel elements (Fig. 1C). Establishment in vessel elements and invasion of mycelia via plasmodesmata between adjacent xylem cells thereafter became obvious (Fig. 1H and I). Upon sectioning the plants at BBCH 61, visible fungal structures from V. longisporum with and without trypan blue staining were found in upper primary root, basal, lower and upper stem samples (Fig. 1D–F). In contrast, only a few fungal structures of V. dahliae were occasionally visible in the basal stem region (Fig. 1G). No differences in colonization patterns between isolates of the same species, or between the two B. napus genotypes, were observed when assessing stained explants. These microscopic observations can be compared with external plant assessments where stunting is initiated at the beginning of stem elongation (BBCH 30), chlorosis of leaves followed by wilting when flower buds are present but still enclosed by leaves (BBCH 50), and microsclerotia formation when 50% of pods are ripe (BBCH 85).

Details are in the caption following the image

The development of infection by Verticillium longisporum in Brassica napus cv. Maskot. (A) Mycelial growth of V. longisporum (VD1) in a lateral root. (B, C) Discoloration and establishment in xylem vessels. Trypan blue-stained vertical sections. Transverse sections of V. longisporum-inoculated plants at BBCH 61: (D) trypan blue-stained lower stem; microsclerotia developing in the phloem; (E) upper root and (F) upper stem samples. Arrested microsclerotia development of V. dahliae (G12-1) at BBCH 61: (G) lower stem. Transmission electron micrographs of V. longisporum inoculated plants, transverse sections: (H) presence of mycelium in xylem tissue; (I) hyphal growth through plasmodesmata. Arrows indicate mycelial growth in (A)–(C); proliferation of microsclerotia in (D) and (E).

Diquat is a contact herbicide, which is referred to as a desiccant because it causes entire plants or plant parts to dry out very quickly (Hayes & Laws, 1990). Experiments during the early 1990s at the Department of Plant Pathology, SLU, Uppsala revealed that diquat treatments could trigger development of Verticillium spp. (particularly microsclerotia) on collected material and it was used here to facilitate assessment of incidence and severity of disease in inoculated plants. Initial tests to optimize the procedure showed that a 1 min treatment with the diquat solution in combination with 1 day of incubation under humid conditions gave the best results (data not shown). The results showed that the initiation of flowering (BBCH 60, first flowers open) is a very critical phase where V. longisporum could be found both at the base and in upper parts of the plant stems (Table 1). The disease was further accentuated at full flowering (BBCH 65). At this stage, approximately four additional yellow leaves were present on the inoculated plants compared with control plants. No obvious early defoliation was noted on the inoculated plants.

Table 1. Percentage of samples from Verticillium longisporum (VD1)-inoculated oilseed rape cv. Maskot with microsclerotia and disease severity ratings of plants at different growth stages
Growth stagea Upper stemb (% infected) Basal stemc (% infected) Disease severity ratingd
13 NPe 10 0
30 NP 20 0·5
50 NP 30 1·4
60 45 90 2·7
65 50 100 3·6
69 80 80 3·7
79 85 70 3·6
89 90 90 3·6
LSDf 0·4
  • a Growth stages according to the BBCH identification key. BBCH 13, three leaves unfolded; BBCH 30, beginning of stem elongation, no internodes; BBCH 50, flower buds present, still enclosed by leaves; BBCH 60, first flowers open; BBCH 65, full flowering, 50% of flowers on main raceme open; BBCH 69, end of flowering; BBCH 79, nearly all pods had reached final size; BBCH 89, fully ripe.
  • b Upper stem = samples taken below the first flower buds.
  • c Basal stem = samples taken below the cotyledon node.
  • d Disease severity was based on both the percentage of wilted leaves and the percentage of stunting (cm) when compared with noninoculated plants. 0,no symptoms; 1,wilting and stunting < 25%; 2,wilting and stunting > 25%; 3,wilting and stunting > 50%; 4,wilting and stunting > 90%; 5,dead plant.
  • e NP, upper stem sampling sites not present at the particular BBCH stage.
  • f Least significant difference (LSD) was calculated at P≤ 0·05, d.f. = 5, and mean of 35 plants.

PCR analysis of collected plant samples enhanced the sensitivity, speed and specificity of the assessments. Verticillium longisporum was recorded by PCR in roots when three leaves of the plants were unfolded (BBCH 13) and then in stem samples taken below the cotyledon node just before the first side shoot was detectable (BBCH 21). In contrast, presence of V. dahliae in roots was not recorded using PCR until full flowering (BBCH 65). DNA from V. longisporum could be detected in roots, stems and leaves in more than 90% of the plants, but this incidence was reduced to 42% in pods. The presence of V. dahliae, however, was detected in roots of more than 90% of the plants examined, but the incidence revealed by PCR was 67% in the lower stem sections, in samples taken at BBCH 79 (when nearly all pods had reached their final size) (Fig. 2). Inoculation experiments where 1:1 mixtures of V. longisporum and V. dahliae isolates were used did not show any difference in patterns of infection compared with using each isolate alone (data not shown).

Details are in the caption following the image

PCR analysis of fungal DNA present in various parts of inoculated plants. (a) Presence of Verticillium longisporum (VD1) results in a 350-bp amplified fragment: lane 1, 1 kb DNA ladder (MBI, Fermentas, Lithuania); lane 2, upper root; lane 3, basal stem; lane 4, upper stem; lane 5, upper leaf; lane 6, flower; lane 7, seed; lane 8, pod. (b) Presence of V. dahliae (G12-1) gives a 400-bp fragment: lane 1, 1 kb DNA ladder; lane 2, upper root; lane 3, basal stem; lane 4, upper stem; lane 5, upper leaf; lane 6, flower; lane 7, seed; lane 8, pod.

Fungal growth on media containing plant tissue

The growth of both V. longisporum and V. dahliae isolates varied with the amount of added plant tissue (Fig. 3). Potato dextrose agar containing extracts from cotyledons from five, 10 or 20 seedlings significantly enhanced the fungal growth compared with PDA with cotyledon extract from one plant or PDA alone. No significant differences were found in fungal growth between the sources of plant material (cotyledons vs. true leaves, data not shown). However, the two V. dahliae isolates grew significantly more slowly (P ≤ 0·01) on PDA containing cotyledon extracts or true leaves (data not shown) from five, 10 or 20 plants of cv. Maskot (Fig. 3b) compared with cv. Niklas (Fig. 3a), whereas no such growth differences were found with V. longisporum plates.

Details are in the caption following the image

Growth of Verticillium longisporium (isolates VD1, VD4, VD11) and V. dahliae (isolates G12-1, NOVA2) on MS-1 agar containing plant extract of zero, one, five, 10 or 20 cotyledons (c) from seedlings of high glucosinolate oilseed rape cv. Niklas (a) and low glucosinolate cv. Maskot (b). Growth was measured as the mean number of days taken by the mycelium to reach the perimeter of agar in 9 cm Petri dishes. Tukey's test was used to calculate the honest significant difference (HSD) = 3·1 days at P ≤ 0·05.

Seed analysis

A clear difference in seed size was found between the 10 field samples. Seeds from fields with an incidence of disease higher than 60% had a diameter of less than 2 mm, compared with a diameter > 2·5 mm when disease incidence was lower. Seed coats or seedlings did not show signs of fungal contamination from Verticillium spp. when analysing field or inoculated material via PCR or monitoring the samples for fungal growth or microsclerotia development.

Discussion

Clear differences in the colonization of B. napus plants by V. longisporum and V. dahliae were found. Both species entered roots, but V. longisporum colonized the entire plant whereas V. dahliae remained in the basal regions of the plants. The initiation of flowering seems to be a very important plant developmental phase for V. longisporum to spread in oilseed rape plants. Similarly, accelerated flowering has also been linked to a susceptible response to V. dahliae in Arabidopsis thaliana (Veronese et al., 2003). This observation might be related to changes in host vascular tissues that take place during the switch from vegetative to reproductive development. Most physiological studies of the transition from vegetative to reproductive stages are performed on Brassica crops of horticultural importance. In contrast, fundamental developmental information from studies of Arabidopsis is extensive (Fukuda, 2004; Henderson & Dean, 2004). In stem tissue associated with inflorescences, the vascular elements differentiate in a complex manner regulated by auxins and cytokinins, and after cell elongation, vessel elements deposit a large amount of cellulose in their secondary walls before cell death is initiated (Ye et al., 2002). Cytokinins, for example, in conjunction with other plant hormones, not only control cell division, but also affect many physiological and developmental functions such as leaf senescence, the formation and activity of shoot apical meristems, and floral transition (Mok & Mok, 2001). Infection by pathogens also commonly upregulates extracellular invertase, which is a key enzyme involved in carbohydrate partitioning between source and sink tissue (Roitsch et al., 2003). How physiological changes and the vascular patterning in plants affect the infection pattern of verticillium wilt disease remains to be elucidated. Nevertheless, the overall colonization pattern of V. longisporum in oilseed rape seems to be in agreement with earlier reports concerning V. dahliae in a range of host species (Beckman, 1987; Mol, 1995; Rowe & Powelson, 2002). However, the observation that V. longisporum invades adjacent xylem cells via plasmodesmata has hitherto not been shown in any V. dahliae systems.

Pathogenicity tests using low- and high-glucosinolate B. napus genotypes have not revealed any correlation between resistance to V. longisporum and glucosinolate levels (Heale & Karapapa, 1999). Similarly, in the present study, no differences were observed in fungal growth due to the origin of plant tissue. Thus, the difference in total glucosinolate content between cotyledons, which reflects the content of seeds, compared with vegetative tissue, in this case true leaves, plays a minor role. However, the presence of a specific glucosinolate or breakdown product, or other compounds in the two plant genotypes used in the study, might be of importance and could explain the difference in mycelia growth between V. longisporum and V. dahliae. Differential responses of volatiles from the true leaves of broccoli, cauliflower and Indian mustard (B. juncea) on mycelia growth and germination of microsclerotia of V. longisporum have been reported (Debode et al., 2005). In this in vitro assay, Indian mustard showed the most antifungal effects, especially on microsclerotia germination, compared with the two B. oleracea genotypes. Evidently, different Brassica species and genotypes contain compounds that can affect the growth of V. longisporum, at least under in vitro conditions. It is speculated that the restricted numbers of genotypes used for introducing the low glucosinolate trait into oilseed rape cultivars are the source of some less favourable traits in this respect. The first B. napus cultivar with low erucic and glucosinolate contents was launched on the Swedish market in 1981 (Jönsson & Uppström, 1986). This introduction may have encouraged V. longisporum in soils and partly explain the increased occurrence of the disease, in combination with the short crop rotation practices previously applied (Dixelius et al., 2005). Microsclerotia of both V. longisporum and V. dahliae are present in variable density in Swedish soils (Johansson et al., 2005). However, impact on soil populations by different plant species is difficult to elucidate, since several species outside the Brassicaceae seems to have the potential to act as reservoirs of V. longisporum inoculum, and barley can act as a potential host of V. dahliae. The only evident link that could be found between cropping history and presence of Verticillium is between brassica-oil crops and V. longisporum. (Johansson et al., 2005).However, many plant species seem to have the capacity to act as bridging hosts, and thereby contribute to maintaining various soil populations at low levels.

The use of diquat treatment to promote proliferation of microsclerotia is a simple and useful approach to demonstrate infection. However, if the infection level in the plant is low, a long incubation time is needed for the fungus to proliferate and develop microsclerotia in the herbicide-treated tissue. In comparison, PCR analysis was very rapid, sensitive and species-specific, and revealed the presence of V. longisporum in roots at growth stage BBCH 13 within 24 h. However, plant sample size, especially length of stem pieces, can be of importance for the outcome of both methods, since the fungus can be discontinuously present in the plant tissue.

In conclusion, the assessments of the inoculated plants in this study suggest that B. napus is more susceptible to selected isolates of V. longisporum compared with V. dahliae, and the spread of V. longisporum in the plant is most evident at the time of the onset of flowering. Although many questions linked to the verticillium wilt–oilseed rape interaction remain to be answered, one important finding in this work is that transmission of the disease via seeds is not a likely scenario, although external fungal contamination upon harvesting cannot be excluded.

Acknowledgements

We thank G. Berg at the Plant Protection Service, SJV, Alnarp and I. Happstadius, Svalöf Weibull AB for material and valuable discussions, and the initial work done by C. Andersson and L. Steventon. We are also grateful for technical assistance by G. Swärdh, G. Rönnqvist and A. Axén, Uppsala University, for help with TEM analysis. The work was supported by grants from the Swedish Seed and Oilseed Growers Association, Carl Tryggers Foundation and the Swedish Farmers Foundation for Agricultural Research.