Acknowledgements
7.8. Variability Within G. xylarioides
7.8.2. Genetic variability and the role of the sexual cycle
The presence of a perfect state in G. xylarioides implies at least some degree of variability within the coffee wilt pathogen, but until recently, little was known about the underlying genetics of the fungus, the extent of genetic variability (if any) that exists or how it relates to other attributes, including field behaviour. This was partly a consequence of limited expertise and meth- odology to investigate genetic diversity. However, the recent re-emergence and devastating effects of the disease in Africa, coupled with development and improved access to modern molecular approaches, has renewed inter- est in the fungus. As a consequence, a number of research centres across the world have now completed comprehensive studies to investigate ge- netic variability within G. xylarioides and to explore the relationship between G. xylarioides and other fusaria (namely from coffee) from a diagnostic and taxonomic perspective.
As facilities and expertise required to undertake molecular research in Africa remain limited, the majority of such work has been undertaken at CABI, CIRAD and the Université catholique de Louvain (UCL) in the UK, France and Belgium, respectively. A broad range of primarily PCR-based ap- proaches — e.g. analyses of microsatellite loci, presumptive mitochondrial DNA restriction fragment length polymorphisms, amplified fragment length polymorphisms, inter-simple sequence repeat [ISSR] anchored sequences, nuclear housekeeping genes [translation elongation factor, tef, calmodu- lin, CL, histone H3] and intergenic spacer and internal transcribed spacer regions — were applied to explore intraspecific genetic variability within des- ignated regions of the G. xylarioides genome and within specific cell structures (e.g. nuclei, mitochondria, ribosomes). Analysis of internal transcribed spacer, for example, has been successfully used to delimit species, whereas analysis of ISSR, intergenic spacer and microsatellites can reveal interspecific and in- traspecific variability. ISSR analysis in particular can reveal genetic variability within fungi in a similar manner to random amplified polymorphic DNA but is deemed to provide more reliable discrimination of individual fungal strains.
As may be expected, the various molecular approaches differed in the manner and extent to which they exposed genetic variability. Nevertheless, the findings of these studies, which examined several hundred strains of G. xylarioides, were generally consistent and provided an intriguing in- sight into genetic diversity and how it relates to other recognized traits.
Although overall variability within the pathogen would appear to be lim- ited, the research suggests that two major genetically distinct populations are responsible for current outbreaks across DRC, Uganda, Tanzania and Ethiopia (Girma, 2004; Tshilenge-Djim et al., 2004; Adugna et al., 2005; Lep- oint, 2006; Rutherford, 2006; Bieysse, 2007). The first of these consists of G. xylarioides obtained from C. canephora affected by CWD in Uganda, DRC and Tanzania since re-emergence of the disease. Interestingly, this group also includes a single strain obtained from excelsa in Uganda. The second population consists of G. xylarioides obtained from C. arabica affected by CWD in Ethiopia. Despite the level of resolution possible with some of the molecular approaches applied, genetic diversity is not apparent within ei- ther of the two populations. As yet, therefore, it has not been possible to more precisely define the origin of strains in each population either geo- graphically or in terms of the coffee species or cultivar of origin. This is perhaps surprising, given that production of perithecia by the fungus is frequently observed on coffee trees under suitable conditions in the field (Van der Graaff and Pieters, 1978; Girma et al., 2001) and assuming, of course, that the perithecia so observed are a reliable indicator of sexual reproduction.
Genetic variability also exists among G. xylarioides strains recovered from C. canephora and C. excelsa during earlier CWD epidemics in CAR, Guinea and Ivory Coast, all of which differ genetically to the two populations re- sponsible for current outbreaks. Unfortunately, few strains are available for study from these earlier outbreaks, and these may have become altered due to routine sub-culturing on nutrient-rich culture media since their isolation.
Uncertainty therefore remains as to how and why this variability may have arisen and how it relates to field behaviour. Indeed, whether any of these strains were responsible for the earlier outbreaks, and to what extent, re- mains unknown. Furthermore, we do not know precisely how these strains relate to those currently affecting C. canephora, C. excelsa and C. arabica in DRC, Uganda, Tanzania and Ethiopia. It is feasible that the limited genetic diversity observed in current strains may have arisen as a consequence of (i) selection pressure imposed by widespread cultivation of ‘resistant’ coffee genotypes (species or varieties) to counteract the earlier epidemics and/or (ii) the relative fitness and rapid spread of a single or small number of survi- vors or variants, perhaps in response to cultivation of resistant germplasm.
Apparent sexual incompatibility observed in the fungus (see below) may have also played a role in emergence of the genetic variants encountered to date.
The existence of a functioning sexual cycle in G. xylarioides, as in any organism, can play a significant role in the exchange of genetic informa- tion and hence the ability of the pathogen to adapt to changing conditions,
including those brought about by modification of crop management prac- tices. Booth (1971) considered G. xylarioides to be a heterothallic fungus com- posed of sex-linked male and female forms that could be distinguished by their morphological features. As described below, Booth’s inclusion of the male form within G. xylarioides now appears inappropriate, as genetic studies show it to be more closely related to Fusarium lateritium than to G. xylarioides (Geiser et al., 2005; Lepoint et al., 2005). Booth’s original de- scription may in some ways be understandable, given that F. lateritium is also a recognized pathogen of coffee, and simultaneous colonization of CWD affected plants by several fusaria is observed (Tshilenge-Djim et al., 2004, Lepoint 2006).
Recent research nevertheless confirms that G. xylarioides is indeed het- erothallic. At UCL, the teleomorph of the fungus was successfully gener- ated when two tester strains of opposing mating type, each derived from a single conidium or ascospore, were paired in vitro (Lepoint et al., 2005). Pair- ing strains obtained from different geographic locations and coffee species enabled discrimination of three BS, BS1, BS2 and BS3, within which strains were sexually compatible (Plate 14). A sterility group (SG4), composed of a number of reproductively sterile strains, was also identified. The latter are sexually incompatible with each other and with strains in each of the BS, whereas strains belonging to any one BS are sexually incompatible with those of another. It should be noted that in these mating tests, teleomorph production and sexual compatibility are confirmed by production of fertile ascospores and not merely fusion of fungal mycelium or production of empty perithecia (‘protothecia’) (Plate 15). To date, morphological discrimination of mating types has not been possible, all strains exhibiting the characteristics previously described for Booth’s female form.
In these studies, a clear relationship between the genetic and bio- logical attributes of the pathogen was observed, in that BS1 and BS2 com- prised strains associated, respectively, with C. arabica in Ethiopia and with C. canephora in DRC, Uganda and Tanzania (Lepoint, 2006). BS3 comprised the small number of strains associated with CWD during the earlier and very damaging outbreaks in Central and West Africa. Of importance, and given the time required to perform in vitro mating assays, molecular characteriza- tion (PCR amplification and sequencing) of the mating type gene (MAT), based on primer pairs previously developed for F. oxysporum and the Gibber- ella fujikuroi species complex (GFC), enabled the identification of mating type idiomorphs, MAT1-1 and MAT1-2, thereby supporting the hypothesis that G. xylarioides sensu lato is heterothallic (Lepoint et al., 2005; Lepoint, 2006).
This now enables mating types to be identified before confirmatory crosses are performed or where these are not successful. Taxonomically, and although there are still differing opinions as to their suitability and relevance, sequenc- ing and phylogenetic analysis of MAT loci also place the fungus within the African clade of the GFC, an important taxon comprising pathogenic fusaria, and resolves four distinct phylogenetic species corresponding to the BS and SG revealed in the mating tests. These lineages were also resolved by random amplified polymorphic DNA analysis and amplification and sequencing of a
combination of non-MAT nuclear genes (Fig. 7.2). Of note, infertility of mat- ing crosses between G. xylarioides and recognized GFC mating populations suggests the coffee wilt pathogen to be a new BS within the GFC. Mating type tester strains identified for the four lineages have been deposited for secure, long-term storage at MUCL/BCCM.
The findings concerning the reproductive cycle may, at least in part, pro- vide an explanation as to why perithecia production readily occurs in nature but (to the authors’ knowledge) has never been observed on coffee plants inoculated with fungal cultures derived from a single conidiospore. The abil- ity to cross representative strains of G. xylarioides in such mating tests also offers new possibilities to further explore the nature, origins and transfer
Fig. 7.2. Maximum-parsimony phylograms based on the combined autosomal (tef + CL + H3) data set of representative strains of the G. xylarioides complex (GxC) and Gibberella indica com- plex from diverse geographical and host origins. BS and SG defined in carrot agar crosses are indicated by a coloured box for the GxC and by a dotted line for the G. indica complex next to terminal clades resolved. Trees were generated with PAUP v.4.0 b10 (Swofford, 2001) using F. oxysporum as outgroup and available National Center for Biotechnology Information sequenc- es for closely related species belonging to the GFC African clade (O’Donnell et al., 1998). Boot- strap values based on 1000 replications are indicated in percentages at internodes when replica- tion frequencies exceed 50%. (Courtesy of P. Lepoint and H. Maraite, UCL, Belgium.)
of important characteristics of the CWD pathogen and to more accurately assess the likelihood of new variants emerging and implications for future disease management.