Development of Nested Polymerase Chain Reaction Detection of Mycosphaerella spp. and Its Application to the Study of Leaf Disease in Eucalyptus Plantations.

ABSTRACT Mycosphaerella leaf disease (MLD) is a serious disease of two of the major eucalypt species grown in temperate regions worldwide, Eucalyptus globulus and E. nitens. More than 30 species of Mycosphaerella have been reported on eucalypts worldwide. Accurate, rapid, and early discrimination of Mycosphaerella spp. causing crown damage to E. globulus and E. nitens will assist the development of sustainable management strategies. This study describes the development, and incorporation in a nested polymerase chain reaction (PCR) approach, of specific primers for the detection and identification of Mycosphaerella spp. commonly reported from leaf lesions of E. globulus and E. nitens in Australia. Primer design was assisted by sequence alignment and phylogenetic analysis of 165 nonredundant sequences from the nuclear ribosomal DNA internal transcribed spacer regions of Mycosphaerella and related species. Phylo-genetic analysis revealed very high sequence similarity for two taxon groups, Mycosphaerella grandis and M. parva, and M. vespa, M. ambi phylla, and M. molleriana, and primers were designed to differentiate each of the two groups. Three other species, M. cryptica, M. nubilosa, and M. tasmaniensis, were distinct and distinguished by species-specific primers. In double-blind trials, the detection test accurately and rapidly identified Mycosphaerella spp. in cultures and discriminated against other pathogens that co-occur in or on Eucalyptus leaves, thereby verifying its reliability. The detection test has an internal amplification control in the first-round PCR with fungal-specific primers to raise confidence in test results, particularly to highlight negative results due to PCR inhibition. When applied to DNA extracted from leaf or stem samples either as multiple or single lesions, it detected and identified up to five Mycosphaerella spp. or taxon groups in both positively identified and in young (putative) MLD lesions. The samples were 20 mm(2) or larger in surface area and were collected while undertaking disease rating assessments in an experimental investigation of Eucalyptus plantations and regrowth forest. Using nested PCR detection, Mycosphaerella spp. were positively identified in 2 days, 1 to 5 months earlier than by classical methods, demonstrating the potential application of this detection test to the early discrimination of MLD components in ecological, epidemiological, and genetic investigations.

Photosynthesis of Eucalyptus globulus with Mycosphaerella leaf disease.

Mycosphaerella leaf disease (MLD) is a major cause of foliage damage in Eucalyptus globulus plantations. Our study is the first to describe the physiological effects of MLD on E. globulus leaves. It involved measurements on both field and potted plants. Changes in photosynthetic parameters in response to MLD were quantified in a study using gas exchange techniques. There was a negative linear relationship between light-saturated photosynthesis (A(max)) and leaf-level damage from MLD. Reductions in A(max) were proportionally greater than might be expected from the reduction in green leaf area as a result of the disease, indicating that asymptomatic tissue also was affected by MLD. The reductions in A(max) were not related to increases in stomatal resistance, but were a result of reduced activity of ribulose bisphosphate carboxylase (Rubisco) and changes in the capacity for ribulose bisphosphate (RuBP) regeneration. Changes in mesophyll resistance to CO2 were also implicated. The effect of MLD was similar at different sites and irrespective of tree-level infection, suggesting a general leaf-level response of E. globulus to MLD.

Precision and accuracy of pest and pathogen damage assessment in young eucalypt plantations.

Fungal pathogens, browsing mammals, birds, insects, nutrient deficiencies, drought, frost and waterlogging are all damaging agents to plantation species. The subsequent loss in leaf tissue or reduced photosynthetic potential can reduce growth and potentially lead to tree death. The Crown Damage Index (CDI) was developed in Australia to quantify damage in young eucalypt plantations. The accuracy and precision of assessing damage at a tree level were determined to ensure the reliability, objectivity and repeatability of the CDI method. Nine assessors, with varying levels of experience, estimated damage on three plots of fifty trees each, 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. 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.

Identification of hydrolysable tannins in the reaction zone of Eucalyptus nitens wood by high performance liquid chromatography--electrospray ionisation mass spectrometry.

The first detailed analysis of the phenolic constituents of the reaction zones (tissue of antimicrobial defence) from the sapwood of a Eucalyptus spp. is presented. Plantation-grown Eucalyptus nitens trees with stem decay resulting from pruning wounds were sampled and extracts were prepared from healthy sapwood and from reaction zone tissue. Analysis by HPLC with ESI-MS revealed that a diverse range of hydrolysable tannins are present in both healthy sapwood and in reaction zone extracts, including over 30 gallotannins, ellagitannins and phenols. Eight tannins were unequivocally identified, including the gallotannins tri-O-galloyl-beta-D-glucose, tetra-O-galloyl-beta-D-glucose and penta-O-galloyl-beta-D-glucose, and the ellagitannins pedunculagin, tellimagrandin I, casuarinin, casuarictin and tellimagrandin II. The phenols gallic acid, ellagic acid and catechin were also identified. The ellagitannins (particularly pedunculagin) are considerably more abundant in the reaction zone than in the healthy sapwood and may contribute to the effectiveness of the reaction zone as an antimicrobial barrier.