Miss Magriet Van der Nest

PhD Student

Research Interests

THESIS TITLE: Compatibility in Amylostereum areolatum

For my PhD study, I am working on the white-rot fungus Amylostereum areolatum (Basidiomycotina) and its symbiont, the Sirex noctilio woodwasp, that seriously threaten pine forestry in various countries in the Southern Hemisphere and the United States of America where they have been introduced. To control the Amylostereum-Sirex complex several biological control agents have been used in the past, with the parasitic nematode Deladenus siricidicola (= Beddingia siricidicola) that sterilizes the female wasps being the most successful.  However, despite considerable efforts to monitor and control the wasp with this agent, the pest complex continues to kill significant numbers of trees and spread to previously unaffected areas.  A possible alternative approach for managing the Amylostereum-Sirex complex is to focus control efforts on the fungus, instead of the wasp. Since this would be detailed knowledge of the biology of A. areolatum, my PhD focussed on increasing our understanding of the mechanisms that determine the outcome of intraspecific interactions of A. areolatum, as well as to determine how the association of the woodwasp influence the biology and evolution of its fungal partner.

Two independent systems regulate mycelial compatibility in A. areolatum.  The one system (referred to as sexual compatibility) regulates hyphal fusion between homokaryons.  A. areolatum has a heterothallic tetrapolar mating system, which means that sexual compatibility is controlled by the genes present on two unlinked mating type (mat) loci (mat-A and mat-B).  Only homokaryons that have different allelic specificities at both their mat loci are sexually compatible and are able to form a heterokaryon.  The second system (referred to as vegetative compatibility) that regulates mycelial compatibility in A. areolatum controls hyphal fusion or anastomosis between heterokaryons. The entire process is controlled by the genes encoded at the heterokaryon incompatibility (het) loci, where heterokaryons that have similar allelic specificities at all of their het loci are vegetatively compatible.  Heterokaryons that have different allelic specificities at one or all of their het loci are vegetatively incompatible as cell death prevents the persistence of anastomosis between these individuals.

Conducting research on the molecular genetics of homobasidiomycetes is complex.  The main obstacle is the difficulty (and sometimes impossibility) of performing controlled sexual crosses – many  species, including A. areolatum, rarely fruit in nature and do not readily produce fruiting bodies in the laboratory.  An additional hurdle associated with studying the molecular basis of fungal recognition is the variable nature of the genes controlling these systems. The uses of traditional gene finding methods such as Southern hybridization and, to some extent, the use of degenerate primers are not feasible.  As a result, the characterization of the sexual mating systems of homobasidiomycetes has almost exclusively been limited to the model homobasidiomycetes Schizophyllum commune and Coprinus cinereus.  Despite the fact that fungal vegetative incompatibility has been studied for decades, most research has focussed on the model ascomycetes Neurospora crassa and Podospora ansarina.  Almost no information is available about the het genes of basidiomycetes.  We only know that the basidiomycetes harbour apparently fewer het loci than their ascomycete relatives.
As part of my PhD, we were able to determine that there are at least two het loci present in A. areolatum using a set of homokaryons obtained from field-collected basidiocarps and their mated heterokaryons.  Portions of a putative homologue of the pheromone receptors encoded at the mat-B locus and a portion of a putative homologue of the mitochondrial intermediate peptidase (mip) gene linked to the mat-A locus were identified in A. areolatum.  We also  demonstrated that these loci are linked to mating type and that, typically to tetrapolar fungi, both the mat-A and mat-B loci is necessary for sexual compatibility and dikaryon formation.  We were able to map both the mat and het-loci on an amplified fragment length polymorphisms (AFLPs) genetic linkage map.  In order to identify markers closely linked to the recognition loci AFLP-based bulked segregant analysis (BSA) was used.  The map revealed an association between the recognition loci and the genes involved in fitness in terms of the in vitro growth rate of A. areolatum.  An association between the recognition and fitness have also been found in the homobasidiomycetes Agaricus bisporus, Pleurotus ostreatus and Heterobasidion annosum.  A possible explanation for the association between fitness and the recognition loci may be because the evolutionary forces acting on these systems differ from those acting on the rest of the genome.  Balancing selection that retains alleles at approximately equal proportions in populations may help to explain the association between the recognition loci and mycelial growth in A. areolatum, as it also maintains potentially deleterious mutations in a population that would otherwise have been lost due to negative selection.  Important signatures of balancing selection were identified in Amylostereum spp., including the presence of trans-specific polymorphisms and higher nucleotide diversity in the mat genes in comparison to housekeeping genes. 

To increase our understanding of the molecular mechanisms underlying vegetative incompatibility in A. areolatum, we identified genes that are selectively expressed during incompatibility by making use of suppression subtractive hybridization (SSH), 454-based pyrosequencing and quantitative reverse transcription PCR (qRT-PCR). To infer the possible pathways and processes underlying vegetative incompatibility, all selectively expressed transcripts were assigned putative functions and used to reconstruct the possible chain of events occurring during vegetative incompatibility A. areolatum and other fungi.  Apart from considerably increasing the existing body of knowledge regarding the molecular basis of vegetative incompatibility fungi, the findings of this study may also have potential applications in terms of management of the Amylostereum-Sirex complex as it may allow for the identification of targets in the fungus that could be used for chemical control. A better understanding of the mechanism of recognition involved in intraspecific recognition in A. areolatum may help us in the future to understanding the mechanism involved in recognition between A. areolatum and its insect partner, as well as between A. areolatum and the biological control agent D. siricidicola.

Posters and Presentations

• van der Nest MA; Slippers B; Wilken M; van Zyl K; De Vos L; Stenlid J; Wingfield MJ; Wingfield BD. 2009. Genetic linkage map for Amylostereum areolatum reveals an association between vegetative growth and sexual and self recognition. Mycological Society of America Meeting, Snowbird, Utah, United States of America.

• van der Nest MA; Slippers B; van Zyl K; Stenlid J; Wingfield MJ; Wingfield BD. 2009. Identification and characterization of selectively induced genes expressed during vegetative incompatibility in Amylostereum areolatum.  46th Congress of the Southern African Society for Plant Pathology, Gordon’s Bay.

• van der Nest MA; Slippers B; Wilken M; van Zyl K; De Vos L; Stenlid J; Wingfield MJ; Wingfield BD. 2008. Location of sexual and vegetative compatibility loci on an AFLP-based genetic linkage map of Amylostereum areolatum. 9^th International Congress of Plant Pathology, Torino, Italy.

• van der Nest MA; Slippers B; Wilken M; van Zyl K; De Vos L; Stenlid J; Wingfield MJ; Wingfield BD. 2008. Placement of Amylostereum areolatum compatibility genes and QTLs for homokaryon vigour on an AFLP linkage map.  20^th South African Genetics Society Congress, Pretoria.

• van der Nest MA; Slippers B; Stenlid J; Wingfield MJ; Wingfield BD. 2006. The effect of selection against sexual recombination on the diversity of A. areolatum mating-type genes. 8^th International Mycological Congress, Cairns, Australia.

• van der Nest MA; Wilken PM; Slippers B; Stenlid J; Wingfield BD; Wingfield MJ. 2006. Sexual compatibility in Amylostereum areolatum. 44^th Congress of the South African Society for Plant Pathology, Magalies Park Country Club.

• van der Nest MA; Slippers B; Stenlid J; Wingfield MJ; Wingfield BD. 2005. Vegetative incompatibility in Amylostereum areolatum. Mycological Society of America Meeting. Hilo, Hawaii USA

• Wingfield BD; Van der Nest MA; Blakeway FC; Edwards N. 2000. Microsatellite fingerprinting of Eucalyptus in South Africa. Proceedings of the XX IUFRO meeting, University of Oxford, Oxford, United Kingdom.

• Van der Nest MA; Steenkamp ET; Wingfield BD; Wingfield MJ. 2001. Determining the genetic background of Eucalyptus grandis using DNA sequence information. Proceedings of the 27th Annual Conference of the South African Association of Botanists, Rand Afrikaans University, Aukland Park, South Africa.

• Van der Nest, MA; Steenkamp ET; Wingfield BD; Wingfield MJ. 2000. Development of simple sequence repeat (SSR) markers in Eucalyptus. Proceedings of the 37th Congress of the Southern African Society for Plant Pathology, Combined Millennium Meeting, Grahams Town, South Africa.
  

• Van der Nest, MA; Steenkamp ET; Wingfield BD; Wingfield MJ. 2000. Evaluation of microsatellite markers for Eucalyptus fingerprinting. Proceedings of the 17th Congress of the South African Genetics Society, Pretoria, South Africa.