Microscopic analysis of oil palm (Elaeis guineensis) infection by Ganoderma boninense

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Oil Palm (Elaeis guineensis Jacq.) is an important agricultural commodity for many developing countries in South East Asia including Papua New Guinea and the Solomon Islands. Currently basal stem rot (BSR) disease caused by Ganoderma boninense is the major disease of economic importance causing severe crop losses. BSR disease has been known for over 100 years however, progress in disease control and management has been hampered by lack of understanding of Ganoderma-oil palm interaction which is essential in developing better strategies to control BSR incidence. To date, there are no effective control methods to manage BSR disease. The viable long term alternative would be to breed for resistant germplasm. However, this strategy is dependent on improved understanding of the factors associated with resistance/tolerance and susceptibility. These include genetic, molecular, physiological and microscopic factors. Development of suitable screening trials will also facilitate Ganoderma-oil palm interaction studies and to better screen germplasm in breeding studies. A reliable pathological test depends on well-characterised pathogen strains. For this reason a Ganoderma isolate (3039) used routinely in nursery pathological testing by OPRA (Oil Palm Research Association) was imported from PNG. However, this meant that under quarantine conditions experimental design would be restricted. Thus additional Australian isolates were sought for this study. Brackets of Ganoderma-like fungi were collected from around Brisbane and Cairns, Queensland. All new Australian isolates were described based on bracket morphology, basidiospore characteristics (where these were available), vegetative growth rates and sequencing of the ITS regions. The two isolates RD1 and RD2 which were sampled from golden cane (Dypsis lutescens) palms in Cairns, were found to have identical ITS sequences to the PNG isolate 3039 and a G. boninense reference sequence, thus these three isolates were considered to be G. boninense. Both RD1 and RD2 were tested for pathogenicity on oil palm. G. boninense was re-isolated from roots of oil palm seedlings inoculated with RD1, thus indicating that it was pathogenic on oil palm. At this stage G. boninense was unable to be re-isolated from RD2 inoculated oil palm seedlings. PNG isolate 3039 was used for pathogenicity assays under controlled environmental conditions in growth cabinets on oil palm seedlings. It was found to infect roots as early as two months post inoculation, indicating that it followed a similar pattern of infection in a growth cabinet as in a nursery environment. G. boninense does not readily produce spores in artificial media, thus it is first grown on a substrate which is then used as inoculum. Traditionally rubber wood blocks are used, but since these are not readily available in Brisbane, various other substrates were tested. Of the substrates tested the two best and most reliable proved to be potato dextrose agar and sorghum grains. Thus inoculum delivery tests were carried out on oil palm seedlings under growth cabinet conditions using agar plugs and sorghum grains colonised with 3039. BSR symptoms usually appear 4 mpi in nursery inoculated plants. Microscopic examination under fluorescence microscopy revealed that both inoculum delivery methods caused colonisation, however earlier infection by 3039 using sorghum grain inoculum as compared to using PDA plugs was observed. Sorghum grain inoculations were found have better infection potential than agar plugs, possibly due to higher Ganoderma biomass in the grains. Bright field and fluorescence microscopy was used to follow Ganoderma infection of oil palm seedlings. Microscopy revealed that Ganoderma appears to have a preference for colonising root tissue as opposed to bole tissue, even though the bole was the sight of inoculation. Therefore Ganoderma’s survival and spread in soil was tested, whereby PDA and sorghum grains colonised with 3039 were placed in soil for two weeks and re-isolation from soil up to 2cm away from original site was attempted. Live Ganoderma was not found away from inoculation site indicating that this fungus does not survive away from a food source, whether that be a live host or artificial medium. Hyphae were observed in the sub epidermal highly lignified corky cell layer found in primary and lateral roots. Other plant tissues colonised by Ganoderma included lateral roots emerging from primary roots, and the root-bole interface region and dead tissue areas associated with roots and bole. The tissue regions where Ganoderma has been observed did not fluoresce under fluorescence microscopy when stained with calcofluor white indicating that these were either dead or so highly lignified that these could not be stained. Furthermore, this may also suggest that initial establishment of Ganoderma in oil palm is more saprophytic than pathogenic. No specialized hyphal structures were observed during this early infection study. This study describes for the first time an account of early processes of Ganoderma colonisation and infection on oil palm seedlings.