A loop-mediated isothermal amplification (LAMP) assay for the detection of Cryptotermes brevis West Indian drywood termite (Blattodea: Kalotermitidae).

Cryptotermes brevis is one of the most destructive invasive termites in the subtropics and tropics and is a common biosecurity intercept at the Australian border. Drywood termite species are cryptic and difficult to identify morphologically in situations when soldiers or imagos are unavailable. We developed a novel DNA based loop-mediated isothermal amplification (LAMP) assay to detect C. brevis and differentiate it from other drywood termites. Validated voucher specimens of 30 different drywood termite species were obtained from several insect collections from which DNA was extracted and amplified. The amplicons containing partial mitochondrial 16S rRNA were sequenced and a DNA database was created from which C. brevis LAMP primers were developed, optimized, and tested. The assay was assessed against a range of target and non-target species and found to be specific, successfully amplifying the target specimens of C. brevis in under 30 min. Amplification success was variable against C. brevis faecal pellets due to minute, unmeasurable or degraded DNA. This LAMP test is a new tool for the rapid detection of C. brevis that will enable faster and less destructive management of drywood termite infestations.

Pathways to false-positive diagnoses using molecular genetic detection methods; Phytophthora cinnamomi a case study.

Phytophthora cinnamomi is one of the world's most invasive plant pathogens affecting ornamental plants, horticultural crops and natural ecosystems. Accurate diagnosis is very important to determine the presence or absence of this pathogen in diseased and asymptomatic plants. In previous studies, P. cinnamomi species-specific primers were designed and tested using various polymerase chain reaction (PCR) techniques including conventional PCR, nested PCR and quantitative real-time PCR. In all cases, the primers were stated to be highly specific and sensitive to P. cinnamomi. However, few of these studies tested their primers against closely related Phytophthora species (Phytophthora clade 7). In this study, we tested these purported P. cinnamomi-specific primer sets against 11 other species from clade 7 and determined their specificity; of the eight tested primer sets only three were specific to P. cinnamomi. This study demonstrated the importance of testing primers against closely related species within the same clade, and not just other species within the same genus. The findings of this study are relevant to all species-specific microbial diagnosis.

Current and projected global distribution of Phytophthora cinnamomi, one of the world's worst plant pathogens.

Globally, Phytophthora cinnamomi is listed as one of the 100 worst invasive alien species and active management is required to reduce impact and prevent spread in both horticulture and natural ecosystems. Conversely, there are regions thought to be suitable for the pathogen where no disease is observed. We developed a climex model for the global distribution of P. cinnamomi based on the pathogen's response to temperature and moisture and by incorporating extensive empirical evidence on the presence and absence of the pathogen. The climex model captured areas of climatic suitability where P. cinnamomi occurs that is congruent with all available records. The model was validated by the collection of soil samples from asymptomatic vegetation in areas projected to be suitable by the model for which there were few records. DNA was extracted, and the presence or absence of P. cinnamomi was determined by high-throughput sequencing (HTS). While not detected using traditional isolation methods, HTS detected P. cinnamomi at higher elevations in eastern Australia and central Tasmania as projected by the climex model. Further support for the climex model was obtained using the large data set from south-west Australia where the proportion of positive records in an area is related to the Ecoclimatic Index value for the same area. We provide for the first time a comprehensive global map of the current P. cinnamomi distribution, an improved climex model of the distribution, and a projection to 2080 of the distribution with predicted climate change. This information provides the basis for more detailed regional-scale modelling and supports risk assessment for governments to plan management of this important soil-borne plant pathogen.

New cryptic species of Teratosphaeria on Eucalyptus in Australia.

Teratosphaeria destructans and T. viscida are serious pathogens causing leaf, bud and shoot blight diseases of Eucalyptus plantations in the subtropics and tropics of South-East Asia (T. destructans) and North Queensland, Australia (T. viscida). During disease surveys in northern Western Australia and the Northern Territory of Australia, symptoms resembling those of T. destructans were observed on young and adult leaves of native and plantation Eucalyptus spp. and its hybrids. Phylogenetic studies revealed Teratosphaeria species associated with these symptoms are new taxonomic novelties described here as T. novaehollandiae and T. tiwiana spp. nov. Isolates from previous records of T. destructans recorded in Australia were re-examined and based upon the phylogenetic evidence are reassigned to these new taxa. We conclude that T. destructans is absent from Australia.

Phylogenetic reassessment supports accommodation of Phaeophleospora and Colletogloeopsis from eucalypts in Kirramyces.

Species of Phaeophleospora are anamorphs of Mycosphaerella and they include some of the most serious foliar pathogens of Eucalyptus spp. grown in plantations worldwide. Pathogens assigned to this genus and occurring on Eucalyptus spp. were previously treated in Kirramyces and they are also phylogenetically closely related to other anamorphs of Mycosphaerella residing in the genus Colletogloeopsis. The primary aim of this study was to consider the appropriate taxonomic placement of these species. To achieve this goal, morphological characteristics and DNA sequence data from the ITS and translation EF1-alpha gene regions were used to compare the type species P. eugeniae, Phaeophleospora spp. and Colletogloeopsis spp. occurring on eucalypts, using ex-type cultures and herbarium specimens. Phylogenetic data and morphological comparisons supported the separation of P. eugeniae from Phaeophleospora species occurring on eucalypts. The name Phaeophleospora is retained for P. eugeniae and the name Kirramyces is resurrected for the species occurring on eucalypts (genera Eucalyptus, Corymbia, and Angophora). Sequence data from the type specimens of two previously described species of Kirramyces, K. lilianiae and K. delegatensis, show they reside in a clade with other Kirramyces spp. Morphological and DNA sequence comparisons also showed that there is considerable overlap between species of Phaeophleospora and Colletogloeopsis from eucalypts. Based on these findings, Colletogloeopsis is reduced to synonymy with the older Kirramyces and the description of Kirramyces is emended to include species with aseptate, as well as multiseptate, conidia produced in acervuli or pycnidia. Two new species of Kirramyces, K. angophorae and K. corymbiae, are also described.

Multiple gene genealogies reveal important relationships between species of Phaeophleospora infecting Eucalyptus leaves.

The majority of Eucalyptus species are native to Australia, but worldwide there are over 3 million ha of exotic plantations, especially in the tropics and subtropics. Of the numerous known leaf diseases, three species of Phaeophleospora can cause severe defoliation of young Eucalyptus; Phaeophleospora destructans, Phaeophleospora eucalypti and Phaeophleospora epicoccoides. Phaeophleospora destructans has a major impact on seedling survival in Asia and has not, as yet, been found in Australia where it is considered a serious threat to the biosecurity of native eucalypts. It can be difficult to distinguish Phaeophleospora species based on symptoms and micromorphology and an unequivocal diagnostic tool for quarantine purposes would be useful. In this study, a multiple gene genealogy of these Phaeophleospora species and designed specific primers has been constructed to detect their presence from leaf samples. The phylogenetic position of these Phaeophleospora species within Mycosphaerella was established. They are closely related to each other and to other important Eucalyptus pathogens, Mycosphaerella nubilosa, Mycosphaerella cryptica and Colletogloeopsis zuluensis. The specific primers developed can now be used for diagnostic and screening purposes within Australia.

Taxonomic identity of the sterile red fungus inferred using nuclear rDNA ITS 1 sequences.

The taxonomic position and properties of an unidentified fungus isolated from wheat roots was investigated. The Sterile Red Fungus (SRF) is characterised by its fast growing habit, red pigmentation, non-sporing nature and mycelial form resembling some Laetisaria and Limonomyces species. rDNA variation was used to study the relationship of the SRF to Laetisaria spp. and Limonomyces spp. Nucleotide sequence obtained from eight Laetisaria and three Limonomyces species representing the ITS 1 region, were analysed by PCR and direct sequencing. Plant growth promoting properties of the five taxa were also determined. The SRF had closest identity (98 %) to British material of Limonomyces roseipellis. UPGMA analysis of ITS 1 DNA sequence support a close relationship between SRF and L. roseipellis. The relationship inferred by nucleotide sequence data was supported by phenotypic analysis; both L. roseipellis and the SRF were shown to promote the growth of wheat plants. Unexpectedly, Laetisaria arvalis and material named as Limonomyces roseipellis from New Zealand appeared to be closely related with 98 % rDNA sequence identity, suggesting the misidentification of the New Zealand collection.