Development of Nested Polymerase Chain Reaction Detection ofMycosphaerellaspp. and Its Application to the Study of Leaf Disease inEucalyptusPlantations

Abstract

Glen, M., Smith, A. H., Langrell, S. R. H., and Mohammed, C. L. 2007. Development of nested polymerase chain reaction detection of Mycosphaerella spp. and its application to the study of leaf disease in Eucalyptus plantations. Phytopathology 97:132-144.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. Phylogenetic analysis revealed very high sequence similarity for two taxon groups, Mycosphaerella grandis and M. parva, and M. vespa, M. ambiphylla, 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.

A latitudinal cline in disease resistance of a host tree

“…Morphological assignment of Teratosphaeria species from typical necrotic leaf lesions on leaves collected from each trial indicated that T. cryptica was the principal lesion-causing species in the studied trials, followed by T. nubilosa. Nested polymerase chain reaction detection using infected leaves (Glen et al, 2007) confirmed this and revealed the additional presence of T. vespa, T. parva and T. tasmaniensis in most trials.…”

“…The most obvious symptoms of MLD are necrotic leaf spots that grow in size and can coalesce to form large blotches. Such lesions may be occupied, and/or caused by, more than one Teratosphaeria species (Glen et al, 2007). MLD lesions reduce the photosynthetic capacity of trees, can lead to premature leaf abscission and, in extreme cases, severe defoliation, affecting both growth and tree form (Mohammed et al, 2003).…”

A Species-Specific Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of Colletotrichum capsici

“…The nuclear rDNA region, b-tubulin gene, and an ascomycete mating-type gene are the most common DNA sequences used for these purposes as their characteristics allow the identification of pathogens at species level. The ITS region is the most widely sequenced region and has been used to design speciesspecific primers to unequivocally detect the presence of the species of interest, as reported for some fungal species [17][18][19][20] as well as Colletotrichum acutatum, C. gloeosporioides, and C. coccodes [21,22]. In this study, speciesspecific primers were designed with the aim of developing a reliable, rapid, and sensitive method to specifically detect C. capsici.…”

Teratosphaeria pseudonubilosa sp. nov., a serious Eucalyptus leaf pathogen in the Teratosphaeria nubilosa species complex

“…It will, therefore, be necessary to reexamine all previous disease situations and tree improvement studies (e.g., Carnegie and Ades 2003;Carnegie et al 1994Carnegie et al , 1998 where T. nubilosa sensu lato has been implicated and to determine whether there might be differences in the host range and ecology of the two pathogens. This should not be difficult because the four fixed nucleotide characters in the ITS gene region are diagnostic and for many previous studies, sequence data are available in GenBank (Crous et al 2004;Glen et al 2007;Hunter et al 2004bHunter et al , 2009Kularatne et al 2004;Maxwell et al 2001;Pérez et al 2009a, b). Likewise, microsatellite data used on population studies (Pérez et al 2012;Hunter et al 2008Hunter et al , 2009 should also make it possible to determine which species was treated in previous studies.…”

“…Countries or continents where only T. pseudonubilosa was found include New Zealand, Western Australia and Tasmania (Crous et al 2004;Glen et al 2007;Hunter et al 2009;Kularatne et al 2004;Maxwell et al 2001;Pérez et al 2012). Likewise, areas where only T. nubilosa was found include New South Wales, Africa, Europe and South America (Carnegie 2007a;Crous et al 2004;Hunter et al 2004bHunter et al , 2009Pérez et al 2009aPérez et al , b, 2012.…”