The development of strategies for the management and research of foliar pathogens on Eucalypt plantations : using Mycosphaerella as a case study

Foliar pathogens are capable of severely reducing the productivity and stem form of eucalypt trees in plantations. Mycosphaerella is one of the most severe defoliating pathogens to eucalypts worldwide. In Australia Mycosphaerella leaf disease (MLD) has produced episodes of severe defoliation in Tasmania, Gippsland and south-west Western Australia. Mycosphaerella is one of the most researched pathogens of eucalypts, however the majority of studies have concentrated on the taxonomy of pathogens and investigating their geographical and host ranges. In this thesis, MLD has been chosen as a model system to develop and apply new technologies to researching foliar pathogens. Research was conducted in the four main areas of disease assessment, growth impacts, molecular detection and resistance mechanisms. Acquiring accurate and repeatable damage estimates at a tree level is essential for calculating damage at plantation and estate levels; especially where data will be used for computer generated modelling using programs such as Maestra or CABALA. Repeated assessments using the Crown Damage Index (CDI) tested the suitability of the method to provide reliable, objective and repeatable results. Nine assessors, with varying levels of experience, estimated damage on three plots of fifty trees each (3-4 years old), to obtain an understanding of the subjectivity of assessing damage caused by insects (e.g. Chrysophtharta spp.) and fungal pathogens (e.g. Mycosphaerella spp.) on Eucalyptus globulus Labill. Damage levels were measured by destructive sampling to enable direct comparisons between estimates and damage levels to be made. The most experienced assessors provided the most repeatable estimates and were generally the most accurate. The incidence of foliar necrosis was the least subjective measure while defoliation was the most subjective and the least accurate of the indices measured. All assessors, regardless of experience, were able to predict the Crown Damage Index (a combined index of all damage classes) to within 12 % of measured damage levels. Further modification of the CDI in a separate study on younger trees (1-2 years old) further reduced the errors involved with damage estimates to within 4 % of actual damage levels. Despite the importance of Mycosphaerella species as significant defoliating pathogens of temperate plantation eucalypts such as E. globulus, there have been no studies to investigate the effects of Mycosphaerella damage on growth of young trees measured through to rotation length. From the results of two growth trials, one short term and one longer term (tree growth was monitored until 3 and 6 years old, respectively), the damage threshold (level of damage before there were significant growth effects) was estimated to be approximately 20 %. In both trials, losses in volume were only observed until trees changed to adult foliage at which point the growth rate returned to that of the control trees. We predicted that with less than 80 % damage, growth rates follow a type 1 growth response, i.e. after an initial growth loss damaged trees recover and have a growth rate that is parallel with control trees. Above 80 % loss of effective leaf area, it is predicted that growth rates of control trees and damaged trees are permanently divergent. To give a longterm estimate of impact, the growth of trees in the longer term trial were modelled to rotation length. After 25 years growth (rotation length) it is estimated that a loss of one year's growth will occur as a result of the MLD damage observed in this trial. One year was also the length of time that juvenile foliage was exposed to greater than 20% damage. A nested PCR detection method was applied to leaves and stems infected by MLD to detect the five most common Mycosphaerella species that occur in Tasmania. Leaf samples were taken from E. globulus and Eucalyptus nitens (Deane and Maid.) Maid. plantations in the northern regions of Tasmania and native re-growth in the north-east of Tasmania. For the first time it has been conclusively shown that in excess of five Mycosphaerella species can coexist in E. globulus leaves and four in E. nitens leaves, including a record of Mycosphaerella nubilosa (Cooke) Hansf. on E. nitens which has only been documented once before. Samples from native Eucalyptus regnans (Thum) Lindaure provided evidence that the co-existence of several Mycosphaerella species on a single lesion may occur outside the plantation environment. The molecular detection test was a rapid, reliable and cost-effective method in comparison with classical mycological methods for the identification and differentiation of species associated with MLD on eucalypts. These studies have highlighted the potential for multiple pathogenic species of Mycosphaerella to simultaneously occupy the same niche. The Mycosphaerella detection technique was also applied to determine the presence of species associated with MLD in leaf lesions of varying development, including asymptomatic tissue. Symptoms characteristic of putative Mycosphaerella lesions, collected from commercial E. globulus and E. nitens plantations, were categorised into five stages of development with asymptomatic tissue designated as the stage prior to any symptom expression. Lesions in all categories, including some asymptomatic leaf tissue, tested positive for the presence of up to four ycosphaerella species. The number and composition of species within a lesion varied between early and late stage lesions, with trends occurring for the most pathogenic species to occupy necrotic and reproductive lesions exclusively (E. nitens) or with only one other species (E. globulus). Early detection of Mycosphaerella species in asymptomatic leaves and at any stage of lesion maturity will facilitate more accurate, rapid and broad scale screening of plantations for ecological and epidemiological investigations at earlier stages of disease development. Effective and reproducible artificial inoculation techniques for MLD have not been developed; the confirmation of Mycosphaerella species in naturally infected early lesions using the nested PCR detection system enables the study of field infected leaves to determine the effects of infection on host physiology and resistance. The timing and strength of necrophylactic periderm formation, deposition of defence chemicals and accumulation/retention of photosynthetic pigments were compared between MLD susceptible E. globulus and the more MLD resistant E. nitens after infection of the leaves with Mycosphaerella species. Resistance of E. nitens, as observed in southern Australia, was attributed to the speed of necrophylactic periderm formation, which was directly related to the amount and type of cell division occurring in the mesophyll cells. In E. nitens the necrophylactic periderm is formed early in lesion development by cellular division of mesophyll cells which were quickly reinforced with lignin, suberin and other polyphenolics. It is suggested that the rapid nature of necrophylactic periderm formation in leaves of E. nitens was due to the presence of isobilateral palisade layers and the need for less cellular division to fill intracellular spaces and form a continuous barrier of cells. In E. globulus, which has only one adaxial palisade layer, the necrophylactic periderm was formed more slowly and in a distorted fashion. It was primarily formed through hypertrophic changes to existing cells and limited cell divisions. Deposits of lignin and suberin in the cells of the necrophylactic periderm did not occur in E. globulus until later stages of lesion development, and in many cases the necrophylactic periderm appeared to be ineffective in preventing further disease development. From this study of necrophylactic periderm formation, it was suggested that increased mesophyll density within a leaf may be linked with the speed and shape of necrophylactic periderms that are formed after infection by Mycosphaerella species; thus the more resistant species/families are able to restrict pathogen spread more effectively than susceptible species/families. Under the same environmental conditions and inoculum load, northern NSW provenances of E. nitens have been observed to be more resistant to MLD than southern NSW provenances. Using histological methods, one provenance from each distribution were investigated with respect to constitutive anatomy. The cellular and histochemical changes after infection by Mycosphaerella species that led to barrier zone formation, including accumulation of defence compounds such as suberin, lignin and flavanoids were also compared. Leaves of resistant provenances were significantly thinner, had a higher proportion of palisade mesophyll and reduced intracellular airspace compared with those from the susceptible provenance. After infection, more cellular division was observed in sections from resistant leaves and the necrophylactic periderm formed was more organised, continuous, suberised and lignified than necrophylactic periderms formed in susceptible leaves. It is suggested that higher constitutive proportions of cell-dense palisade layers and thinner leaves can reduce the cellular division required to form of necrophylactic periderms after injury and compartmentalise pathogens more rapidly. More compact palisade layers may also play a role in the slowing or prevention of infection as some Mycosphaerella species may not be able to penetrate tightly packed mesophyll cells. Resistance of E. globulus juvenile foliage to MLD has been shown to be under high genetic control. Differences between pairs of resistant and susceptible families, in constitutive traits of juvenile leaves such as stomatal density (counted with wax on and with wax removed), leaf density, total phenolics and total leaf wax was assessed on juvenile leaves. Four resistant and susceptible pairs of families were compared including one inter-provenance, one intra-provenance and two wi...