Management approaches for Ceratocystis wilt and canker disease in Acacia plantations

Acacia species are exotic hardwood trees extensively planted in SE Asia. Due to their rapid growth, good wood quality and tolerance to a wide spectrum of environmental conditions, they are important economically, not only for timber products, but also as raw material for significant pulp and paper industries in Indonesia. A devastating disease of plantation Acacia mangium in SE Asia is caused by a fungal pathogen belonging to the genus Ceratocystis. This fungal pathogen, Ceratocystis manginecans, causes stem cankers and rapid wilting of trees. At the start of my doctoral studies very little was known about the disease and the search for resistance in A. mangium was proving challenging requiring a large number of different sources of germplasm to be screened and for alternative management strategies to be considered. The aim of my first investigation was to improve screening procedures for resistance to C. manginecans in Acacia species by comparing the current screening protocol (inoculation of potted plants) with two potential rapid screening procedures; inoculation of detached acacia phyllodes (using a spore suspension) and stem segments (using mycelial plugs). Inoculation procedures were tested on three species of Acacia; A. mangium, A. crassicarpa and an Acacia hybrid (A. mangium x A. auriculiformis clones). Two cultures of C. manginecans obtained from different geographic regions were used in the experiments. Lesion length on stems and necrosis length on phyllodes were assessed and used as a measure of the susceptibility of the tested plant. The relative levels of susceptibility in each protocol were compared as a basis to assess the utility of the rapid screening protocols. The stem segments were prone to contamination by other fungi and to desiccation, while results from the potted plant and phyllode protocols showed similar trends of susceptibility among the Acacia clones and species. The ease, rapidity and reproducibility of the phyllode inoculation protocol makes this a potential replacement for inoculation of potted plants as a preliminary screening protocol to identify A. mangium germplasm that is less susceptible to ceratocystis wilt and canker disease. We also evaluated whether the expression of resistance is influenced by the type of inoculum (mycelium or spore suspension). Both mycelial plugs and spore suspensions produced a similar level of disease incidence, so either can be used for inoculation assays permitting the testing of plants with spores of different isolates in the same inoculum. Acacia crassicarpa and A. auriculiformis are considered by the industry as promising sources for resistance against ceratocystis wilt and canker disease. This was also demonstrated in my study; the A. crassicarpa clones used were demonstrably more tolerant to Ceratocystis infection than A. mangium, with much smaller lesions. The disease indices for the Acacia hybrid were intermediate between A. crassicarpa and A. mangium. Bacterial endophytes living in roots, stems and phyllodes of A. mangium were cultured to investigate their biodiversity and to obtain a library of isolates to test for biological control activity against C. manginecans. Samples from trees between one and five years old were collected and their culturable endophytic bacteria were identified by sequence analysis of their 16S rDNA. In total, 278 bacterial isolates were derived from 270 samples representing 90 trees. Most bacteria were isolated from roots and more isolates were obtained from young acacia trees than from the older trees. Analysis of 16S rDNA sequences grouped the endophytic bacteria into five clusters: Firmicutes, Alphaproteobacteria, Betaproteobacteria, Gammaproteo-bacteria and Actinobacteria. Firmicutes were predominant with 66.2% of the isolates belonging to this group. Among 25 genera which were isolated successfully, Bacillus and Burkholderia were the most frequently isolated endophytic bacteria in A. mangium. Several of the genera isolated from A. mangium are those previously demonstrated to include species with potential as biological control agents (BCAs) against plant pathogens and/or the ability to enhance plant growth. In-vitro assays of the 278 isolated endophytic bacteria against C. manginecans revealed 157 isolates with an ability to inhibit the growth of this fungal pathogen. Nine isolates demonstrated an ability to produce metabolites that inhibit the growth of C. manginecans in the absence of viable bacteria. The 16S rDNA analysis of these nine isolates placed them in the genera Paenibacillus, Lysinibacillus, Staphylococcus, Pantoea, Ralstonia, Cupriavidus and Ochrobactrum. Several methods for inoculating the host tissue of A. mangium with these endophytic bacteria were evaluated. Two selected endophytic bacteria, Paenibacillus (EB-05/EB-93) and Lysinibacillus (EB-232) were introduced into A. mangium through the different methods and were positively detected in acacia tissues using specific primers. Applying liquid bacterial cultures to the root zone of germinating acacia seedlings was the most efficient inoculation method when compared with inoculation by seed treatment, or by dipping or spraying of acacia stem cuttings. The deployment of any management strategy in the field will require monitoring to assess effectiveness. At the start of this thesis there was only anecdotal knowledge as to the timing of symptom development (e.g. reduction in leaf area index and yellowing) and the appearance of signs (e.g. insect damage) associated with C. manginecans. Healthy trees neighbouring diseased trees were monitored over 19 weeks. This data (especially LAI data) will be invaluable for carrying out monitoring activities, especially early detection through aerial observation. In summary, the research in this thesis demonstrates the potential of phyllode inoculation as a rapid and low-cost method to extensively test and replicate resistance screening. Selected bacterial endophytes inhibit C. manginecans isolates in vitro and these endophytes can be effectively delivered into a germinating Acacia seedling. The application of bacterial endophytes as biocontrol agents could be part of an alternative management strategy for C. manginecans integrated with the deployment of resistance. My study is the first attempt to establish a description and potential timeline associated with the disease life cycle. Such information can be used to improve monitoring programs for screening resistance and/or alternative management strategies in field experiments or the detection of ceratocystis wilt and canker disease for inventory purposes