B. G. Lewis

The use of AFLP fingerprinting for the detection of genetic variation in fungi

A new PCR-based technique for the detection of inter- and intraspecific genetic variation has been tested on isolates of the fungal phytopathogens Cladosporium fulvum and Pyrenopeziza brassicae . The method is based on the selective PCR amplification of restriction fragments from digests of genomic DNA. We show that the technique is very efficient at detecting polymorphisms, even in species where very little variation could previously be found by RFLP analysis. 21 primer combinations were used on four isolates of P. brassicae , detecting a total of 162 polymorphisms (mean = 4·1 polymorphisms per primer combination per pair of isolates). Four primer combinations were used on eight isolates of C. fulvum , detecting a total of 32 polymorphisms (mean = 3·3 polymorphisms per primer combination per pair of isolates). Primer combinations varied in their ability to detect variation, ranging from 0 to 24 polymorphisms between P. brassicae isolates and 0 to 10 polymorphisms between C. fulvum isolates. AFLP fingerprints were highly reproducible and have great potential as a tool for evaluating genetic diversity of fungal pathogens.

Glucosinolates of wild and cultivated Brassica species

Abstract Major differences were found in the occurrence of glucosinolates in the leaves of different wild species of the Brassica oleracea (2n = 18) group. The distribution of glucosinolates supports the present classification with the exception of the inclusion of both Tunisian and Sardinian populations within B. insuloris . The results also support the hypothesis of a multiphyletic origin of cultivated forms of B. oleracea from a number of wild species. In contrast, wild populations of B. rapa had very similar glucosinolate contents. Wild populations had a considerably higher total glucosinolate content than cultivars which may limit their usefulness in breeding programmes.

Resistance of leaves of Brassica species to Leptosphaeria maculans

Under controlled conditions, the range of responses in Brassica napusssp. oleiferaand its two putative parents, B. rapa and B. oleracea, were tested by inoculating leaves with a mixture of eight isolates or two separate isolates of Leptosphaeria maculons. Infection resulted in a hypersensitive reaction, or localized lesions of various sizes, or systemic colonization of the lamina and petiole. Although not apparently associated with hypersensitive resistance, levels of alkenyl glucosinolates in leaves were inversely correlated with lesion extension. The value and limitations of these various sources of resistance in breeding lines of B. napus ssp. oleifera are discussed.

DNA polymorphism in Leptosphaeria maculans

Abstract DNA polymorphism in Leptosphaeria maculans is described for the first time. Two distinct genetic groups, differing in repetitive DNA sequences, were detected by gel electrophoresis and by probing Southern blots with mitochondrial, ribosomal or random genomic DNA sequences. These two genetic groups correspond with groupings based on ability to accumulate extra-cellular pigments in culture but do not give a perfect correlation with stem canker potential on oilseed rape. It is concluded that there are major genetical differences between the two sub-groups within the population of L. maculans.

Infection of pea epicotyls by Mycosphaerella pinodes

Infection of pea epicotyls by Mycosphaerella pinodes is described for the first time. The pathogen penetrated the cuticle with appressoria and infection pegs then grew through the outer wall of the epidermis, occasionally penetrating cells without causing necrosis. This phase of the disease, which is probably biotrophic, is followed by a typical necrotrophic phase involving progressive necrosis; the resulting macroscopic lesion often girdles the epicotyl. Epicotyl infection is particularly damaging and resistant cultivars are urgently needed.

Discovery of the teleomorph of Phoma medicaginis var. pinodella in culture

The teleomorph of Phoma medicaginis var. pinodella is reported for the first time. A single isolate, collected from infected plant material in Australia, produced asci and ascospores in culture. The anamorph was identified as Phoma medicaginis var. pinodella on the basis of axenic colony morphology, symptoms on the foliage of a susceptible pea line and characteristic hybridization banding patterns observed during RFLP analysis. The ascospores and asci, although similar in morphology to those produced by Mycosphaerella pinodes, were considerably larger. Therefore, the binomial assignment of the teleomorph remains open to speculation. The potential impact on disease dissemination and effectiveness of control measures against P. medicaginis var. pinodella are discussed.

Ultrastructural studies of the limitation of lesions caused by Leptosphaeria maculans in stems of Brassica napus var. oleifera

Reactions of Brassica napus var. oleifera stems to two isolates of Leptosphaeria maculans , differing in aggression, were studied by TEM, SEM X-ray microanalysis and flame spectrophotometry. The common reactions associated with resistance were lignification of walls and, rapid, specific accumulation of calcium. Injection—infiltration of calcium accelerated delimitation of lesions, Calcium accumulated mainly at the surface of secondary cell walls within and around the perimeter of lesions in association with previously induced lignin co-polymers. It is suggested that this event is involved in resistance and its likely general significance is discussed.

Classification of races by DNA polymorphism analysis and vegetative compatibility grouping in Fusarium oxysporum f.sp. pisi

Abstract The race structure of a collection of Fusarium oxysporum f.sp. pisi isolates from the U.K. and the U.S.A. was investigated. Isolates were assigned to races 1,2,5 or 6 by pathogenicity tests on a series of host differentials. The relationships between isolates within each race and between races was then examined by restriction fragment length polymorphism analysis and vegetative compatibility grouping. For the restriction fragment length polymorphism analysis three clones, containing DNA from Race 1, were constructed and used as probes; pDG102 and pDG106 contained moderately repetitive DNA and pDG312 contained a ribosomal DNA repeat. Vegetative compatibility was studied by complementation tests of chlorate resistant mutants. Both restriction fragment length polymorphism analysis and vegetative compatibility grouping were able to distinguish the four races and revealed that Race 2 comprised two separate groups, termed 2A and 2B. The evolutionary implications of the findings are discussed.

Differential responses of oilseed rape leaves to Leptosphaeria maculans

Under controlled environmental conditions, leaves of winter oilseed rape, cv. Rapora, responded differentially to three different isolates of Leptosphaeria maculans. Inoculation with pycnidiospores or ascospores caused either symptomless localized infections or a mixture of localized lesions and systemic infections. Susceptibility increased with leaf age. An unusual phenomenon involving secondary infection from primary localized lesions is described. Since the isolates tested represent the main aggression groups amongst indigenous populations of L. maculans and caused qualitatively different reactions, cv. Rapora may provide a useful host for differentiating the groups.

Factors affecting systemic infection of Pisum sativum by Peronospora viciae

Glasshouse experiments with a series of near-isogenic lines showed that the st gene, which reduces stipule area, and the tl gene, which converts tendrils to leaflets, considerably increased susceptibility to systemic infection with Peronospora viciae . Similar effects were observed with these pea lines when naturally infected in the field, indicating that direct infection of the apex is an important route for systemic infection and that its incidence is influenced by morphology of the apical primordia. In a growth room experiment at 25 °C where leaf necrosis was associated with inhibited sporulation, symptomless invasion of the stem tissues resulted in systemic infection, indicating that some apical infection may originate from leaf lesions under high temperature conditions.