Molecular markers have been used in a wide range of studies with plant pathogenic fungi (see earlier). Although these can be reviewed, the volume of the literature available is considerable, and so a single case study is presented here that illustrates how the molecular epidemiology of a plant pathogen can be related directly to agricultural practice.
One series of studies that has shown the range and limitations of molecular markers in following plant disease epidemiology is the investigation of basal stem rot (BSR) of oil palm by G. boninense. BSR was first recognized in West Africa in 1915, and as oil palm was distributed through out the world, it was recorded in many other countries. The first report in SE Asia was in 1931, and since that time BSR incidence has increased to the point where the lack of techniques for management of the disease is considered a major constraint to oil palm production in SE Asia [see Ariffin et al. (2000)]. Ganoderma species attack a variety of tropical perennial crops including rubber, tea, and pineapple. In these instances the Ganoderma appears to be largely transmitted through the soil, possibly in plant debris, and spreading infection patches may be evident in fields. The species G. boninense occurs as a saprophyte on dead palms, particularly coconuts, but appears to be pathogenic only to oil palm. For some years transmission of G. boninense in oil palm was believed to be through the soil, as for other species, and disease control was attempted through practices that included digging large pits around infected palms (Turner 1981). The first attempt to use molecular methods to investigate the epidemiology of BSR in oil palm was made in the 1990s, when initial studies were made with iso-enzyme profiles (Miller et al. 1995). Although some enzyme systems initially appeared useful for differentiating species, in G. boninense it was found that in general iso-enzyme profiles were either consistent, or showed considerable variation. Ganoderma is a basidiomycete that forms polyporpoid brackets on the outer surface of infected palms. The brackets are dikaryotic, and basidiospores are produced by meiosis. The mycelial form found in infected tissue is also generally dikaryotic, and some of the isoenzyme variability may therefore be due to recombination events from the original fusion of monokaryotic basidio-spores. However, pectinase isoenzyme analysis identified a characteristic enzyme profile that was consistent for nearly all Ganoderma isolates obtained from palm hosts (Miller et al. 1995; 2000). Given the known involvement of pectin and pectin degradation in plant pathology, this finding may indicate a common mode of action for all of the palm associated Ganoderma species.
The first DNA based method to be investigated in these studies was analysis of RFLPs derived from presumptive mtDNA (AT rich DNA). This relatively simple technique gave rather unusual results, in that different RFLPs were obtained from different cultures, suggesting considerable heterogeneity in the mitochondrial genome (Miller et al. 1999). The RFLP profiles proved to be consistent among single spore isolates from a single basidiome, and so were considered to provide "parental line" fingerprints, characteristic of the dikaryon. These RFLPs could therefore be used to define sibling families. This assumption was supported by monokaryon and dikaryon intercompatibility studies (Pilotti et al. 2000).
Subsequent investigation of molecular fingerprinting methods including RAPDs and AFLP supported the bulk of the iso-enzyme studies and gave different profiles for isolates derived from single spores from the same basidiome (Bridge et al. 2000; Pilotti et al. 2000). When these molecular methods were applied to isolates obtained from single plantations and planting blocks it was found that nearly all of the isolates differed from each other, including isolates obtained from adjacent palms. This finding was again supported by intercompatibility studies (Miller et al. 1999; 2000; Pilotti et al. 2000). These results could not have come about as a result of simple mycelial spread in the soil, as vegetative spread could be expected to result in at least some palms being infected by the same isolate. It was therefore concluded that infection could be due to one of two mechanisms, either singly or in combination. The first was that there might have been mycelial spread from multiple inoculum sources, with virtually no cross infection. This would account for the molecular variability recorded, but would also require each infection to be the result of different infected debris. The second possibility was that infections were due to new dikaryons formed from fusion of monokaryons from individual spores [see Sanderson and Pilotti (1997); Sanderson et al. (2000)].
Although the molecular markers studied showed sufficient variability to identify individual isolates for local epidemiology, they did not show sufficient conservation to allow the wider detection of the pathogen in the environment. BSR was considered to be due to the single species G. boninense, and although there is considerable uncertainty regarding species concepts in Ganoderma, some information is available on sequences within the rRNA gene cluster. Initial studies have shown that the ITS regions are relatively similar across the genus, with most variation being found in the 3' terminal region of the ITS2 sequence (Moncalvo et al. 1995a,b). The ITS sequences obtained from multiple isolates of G. boninense showed very little sequence variation, and a short sequence of 16 bases in the ITS2 region was found to be unique to the species. A PCR primer has been derived from this region, and the combination of this and the universal rRNA primer ITS3 allowed the specific amplification of G. boninense sequences from cultures, specimens, and infected palm material (Bridge et al. 2001). Current surveys being undertaken by the Oil Palm Research Association in Papua New Guinea with the PCR based diagnostic are detecting G. boninense in the internal tissues of recently cut frond bases of young oil palms (Bridge et al. 2000; 2001). This finding suggests that such cut surfaces may provide an entry route for spores. This mode of infection has been reported many times before for fungal pathogens of woody trees, and the initial results from the oil palm research suggest that the developmental state of the oil palm and other factors may also be important in the establishment of infection.
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