Department of Biological and Nutritional Sciences, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK

Background and objectives
Cochliobolus sativus is an important pathogen of wheat and barley, causing a broad range of symptoms from root rot to black point on seeds. Several other species of Cochliobolus are also pathogens of grasses, but tend to be more specialized for leaves. It is of interest to determine why different members of this genus have different modes of pathogenesis. Because C. sativus has a particular ability to cause rotting, production of cell wall-degrading enzymes (CWDEs) is likely to be especially important for its pathogenicity compared to other Cochliobolus species. Monocot primary cell walls have a high content of xylans, so that xylanases are among the more important CWDEs of cereal pathogens. The goal of our research is to characterize the xylanase genes of C. sativus (CS) and determine their role in pathogenesis. Three genes coding for xylanases of glycan hydrolase family 11 [1], XYL1, XYL2, and XYL3, have been identified in the related fungus C. carbonum (CC) [2]. We have determined the complete sequence of the CS counterpart of XYL2, and shown that it is expressed in infected plants. Here we report on the identification of a CS XYL1 gene and compare xylanase genes in three different CS isolates, two from barley and one from wheat, with those of the maize pathogens CC and C. heterostrophus (CH), by gel analysis, sequencing of PCR products and Southern hybridization.

Materials and methods
One C. sativus isolate (SB) is from Scottish barley, one (YB) from Yemeni barley with black point, and one (YW) from Yemeni wheat with black point. C. heterostrophus is a laboratory strain from Dr C. Bronson, Iowa State University. Genomic DNA was amplified by PCR using primers corresponding to sequences conserved between CC XYL1 and CC and CS XYL2, with an annealing temperature of 58C. For sequencing, PCR products were cloned in a 'TA' cloning vector (Invitrogen).

Results and conclusions
C. carbonum XYL1 differs in size from CC and CS XYL2 because XYL1 has one intron and XYL2 has two. When products of PCR using primers expected to amplify both XYL1 and XYL2 were examined on 2% agarose gels, genomic DNA of all isolates except CH gave a band of the size expected for XYL2. Genomic DNA of all isolates except CS isolate YB gave a band of the size expected for XYL1; and DNA of CH and CS isolate YW gave a third, smaller band. DNA sequencing of the PCR products gave: (i) sequences with two introns, resembling XYL2, for all CS isolates but not CH; (ii) sequences with one intron, resembling XYL1, for all isolates except YB; (iii) sequences with no introns, for CS isolate YW and CH (these might originate from contaminating cloned cDNA). The previously determined CS XYL2 sequence differs from its CC counterpart in possessing a HindIII site. All XYL2-type sequences in this study were completely digested by HindIII except that of CS isolate SB, which gave two types of XYL2 product, one with the HindIII site and the other lacking it, indicating that it has two XYL2 isoforms. Xylanase genes of CH and of CS isolates SB and YW were also compared by Southern blotting and hybridization of DNA. Each gave a distinct pattern.

The results show that all the tested CS isolates have at least one XYL2-type gene, at least some isolates of both CS and CH possess XYL1-type genes, and CH does not have a XYL2-type gene that is amplified with the primers tested. Thus there is apparent variation in number and type of xylanase genes not only among but within Cochliobolus species. Two different isolates from the same host but different geographical regions (barley from Scotland and Yemen) differ from each other as well as from an isolate from a different host and region (Yemeni wheat). All the CS isolates have similar ability to cause rotting of greenhouse-grown barley and wheat seedlings, but the differences in enzymes could be related to more subtle differences in pathogenicity. Alternatively, since xylanases of family 11 are widespread among microorganisms, they may be more important for the general ability of the fungus to use cell-wall material for growth than for the early stages in pathogenesis before tissue is killed.

1. Henrissat B, 1991. Biochemical Journal 280, 309-316.
2. Apel-Birkhold PC, Walton JD, 1996. Applied and Environmental Microbiology 62, 4129-4135.