GENETIC ANALYSIS OF THE BLACKLEG FUNGUS, LEPTOSPHAERIA MACULANS

Barbara J Howlett, Anton J Cozijnsen, Kerryn Popa and Agus Purwantara

School of Botany, the University of Melbourne, Parkville VIC 3052, Australia

ABSTRACT

Leptosphaeria maculans causes blackleg disease of oilseed Brassica crops including canola (Brassica napus) worldwide.  Indian mustard (Brassica juncea) varieties are generally resistant to blackleg but recently L. maculans isolates that attack Indian mustard have been reported in Australia.  These fungal isolates are of concern as Australian breeding programs are developing Indian mustard for low rainfall areas, as this crop is better suited to such conditions than canola.  We are mapping a virulence gene that enables L. maculans to attack Indian mustard.  L. maculans is amenable to genetic analysis as it is haploid, outcrossing, can be transformed and has a relatively small genome size (35 Mb).  Its chromosomes are of a size range (0.7 to 3.5 Mb) and number (15) for optimal resolution by electrophoretic karyotyping.  We are developing a genetic map using Amplified Fragment Length Polymorphic (AFLP) markers.  We have scored more than 145 markers in 58 F₁ progeny.  Currently AFLP markers are being hybridised to blots of pulsed field gels of L. maculans chromosomal DNA to identify anchored markers so that chromosomes can be assigned to particular linkage groups. So far six markers have been anchored to chromosomes and the closest marker to the virulence locus is 30 cM.

KEYWORDS

Genetic map, Amplified Fragment Length Polymorphisms, electrophoretic karyotyping, Brassica juncea

INTRODUCTION

Leptosphaeria maculans causes blackleg disease of oilseed Brassica crops including canola (Brassica napus) worldwide.  Indian mustard (Brassica juncea) varieties are generally resistant to blackleg but recently L. maculans isolates that attack B. juncea have been reported in Australia (Ballinger and Salisbury, 1996; Purwantara et al., 1999). These isolates are of concern as the Australian Grains Research and Development Corporation breeding program is developing Indian mustard for low rainfall areas, as this crop is better suited to such conditions than canola.

We have set up crosses between blackleg isolates to determine the genetic basis of virulence on B. juncea.  In a cross between a virulent blackleg isolate (attacks) and an avirulent isolate (cannot attack), the F₁ and backcross progeny segregate in a 1:1 ratio, suggesting the presence of a single gene (Chen et al. 1996).  We plan to isolate this host specificity gene by positional (map-based) cloning.  L. maculans is amenable to such molecular genetic analysis as it is haploid, outcrossing and can be transformed.  Furthermore, its genome size (35 Million base pairs (Mb)) is about thirty times smaller than that of Brassicas.  Its chromosomes are of a size range (0.7 to 3.2 Mb) and number (15) for optimal resolution for electrophoretic karyotyping (Howlett 1997).  In spite of the economic importance of blackleg disease and the amenability of L. maculans to genetic manipulation, little molecular analysis has been carried out on this fungus.  We are mapping the host specificity gene and developing a genetic map of this fungus using Amplified Fragment Length Polymorphic (AFLP) analysis.  Our progress is presented below.

Materials and methods

L. maculans isolates and electrophoretic karyotyping  Crosses were set up between two L. maculans isolates collected from blackleg-infested canola stubble (M1, virulent and C13, avirulent) and resultant random sexual spores (ascospores) and tetrads (group of eight spores derived from a single meiotic event) were isolated from individual sexual fruiting bodies as described by Plummer and Howlett (1995).  Occasionally it was difficult to remove contaminating parental vegetative spores (pycnidiospores) from ascospores.  Accordingly before any of the progeny was analysed further, its karyotype was determined by pulsed field gel electrophoresis, as this technique can ‘fingerprint’ isolates.  The only L. maculans isolates that have identical karyotypes are clonal (Howlett 1997).

Virulence testing  Virulence of isolates was tested on cotyledons and stems of B. juncea cv. Stoke and B. napus cv. Midas as described by Chen et al. (1996).  The latter cultivar is susceptible to all Australian isolates tested so far and is used as a positive control to check that pycnidiospores unable to attack B. juncea, are capable of infecting susceptible hosts.

AFLP analysis   AFLP analysis was performed using a kit from Gibco-BRL and the protocol of Vos et al. (1995).  Genomic DNA from individual fungal isolates was digested with Mse 1 and Eco R1, appropriate adaptors were ligated to the restriction fragments and the mixture was pre-amplified.  In preliminary experiments, amplification was carried out with Mse 1 and Eco R1 primers containing two selective nucleotides at the 3’ ends (M+2, E+2), with the Eco R1 primer labeled with [g ³³P] ATP.  In later experiments, one selective nucleotide at the 3’ end of the Eco R1 primer (M+2, E+1) was used; this gave rise to more bands per lane, and consequently more polymorphic bands between parents.  A portion of the amplification products was separated on a denaturing 5.5% acrylamide gel containing 7.5 M urea.  After electrophoresis the gel was dried and exposed against Xray film overnight.

RESULTS AND DISCUSSION

Amongst the progeny there were two classes of isolates based on their reactions on B. juncea cv. Stoke.  Virulent isolates formed cotyledonary lesions ranging in size from 0.6 to 1.1 cm in length, whilst avirulent isolates generally caused necrotic spots (less than 0.25 cm in diameter).  F₁ progeny showed 35:34 inheritance for virulence:avirulence which indicates the presence of a single gene.  This was supported by tetrad analysis; four tetrads each had four virulent and four avirulent progeny.  All isolates tested formed lesions on B. napus cv. Midas.  In all cases, isolates displayed the same phenotype (avirulence or virulence) with cotyledonary or stem inoculations.  Electrophoretic karyotyping successfully discriminated between parents and progeny.  A high degree of chromosomal length polymorphisms was obvious in both parents and progeny as is seen in other L. maculans crosses (Plummer and Howlett 1995).  Chromosomes ranged in size from 0.6 to 3.6 Mb and the smallest chromosome in isolate M1 appeared to be a B type chromosome as it is not inherited in a Mendelian fashion.

AFLP analysis generated a large number of bands polymorphic between the parents.  One hundred and fifty six markers were scored for 58 F₁ progeny.  Analysis using MAPMAKER version 3.0 (Lander et al. 1987) with a LOD score of 3.0 and Maximum distance of 50 cM put 124 markers into 20 linkage groups; there were also 12 pairs of markers and 32 were unlinked.  The segregation ratios of nearly all markers were 1:1, except one that was present in one parent and all progeny.  This marker is probably mitochondrial.

In theory, the host specificity gene being sought encodes either a virulence or avirulence function.  Since L. maculans is haploid, inheritance studies cannot distinguish between these two possibilities; the only way to resolve this issue is by mutational analysis.  When the host specificity locus was treated as an avirulence locus, MAPMAKER analysis did not assign it to a linkage group.  When it was treated as a virulence locus, it was assigned to linkage group 4 where the nearest marker is 28 cM.  Currently anchored markers are being identified so that chromosomes can be assigned to particular linkage groups.  AFLP markers are being hybridised to blots of pulsed field gels of L. maculans chromosomal DNA.  When the chromosome containing the host specificity gene is identified, a DNA library will be made from it and used to isolate clones linked to this gene by chromosome walking.  Such clones will be tested for host specificity by transformation of isolates lacking this gene.

REFERENCES

Ballinger, D.J. and Salisbury, P.A. (1996). Seedling and adult plant evaluation of race variability in Leptosphaeria maculans on Brassica species in Australia. Australian Journal of Experimental Agriculture 36, 485-8.

Chen, C.Y., Plummer, K.M. and Howlett, B.J. (1996). Ability of an Leptosphaeria maculans isolate to form stem cankers on Indian mustard segregates as a single locus. European Journal of Plant Pathology 102, 349-52.

Howlett, B.J. (1997). Genome analysis of the fungal plant pathogen, Leptosphaeria maculans using pulsed field gel electrophoresis.  Electrophoresis 18, 1544-7.

Lander, E.S., Green, P., Abrahamson, J., Barlow, A. and Daly, M.J. (1987). MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations.  Genomics 1, 174-81.

Plummer, K.M. and Howlett, B.J. (1995). Inheritance of chromosomal length polymorphisms in the ascomycete, Leptosphaeria maculans. Molecular and General Genetics 247, 416-22.

Purwantara A, Burton WA, Salisbury PA and Howlett BJ (1998) Reaction of Brassica juncea (Indian mustard) lines to Australian isolates of Leptosphaeria maculans under glasshouse and field conditions. European Journal of Plant Pathology 105: in press.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kupier, M. and Zabeau, M. (1995). AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23, 4407-14.