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Phylogeny and molecular diagnosis of nectriaceous fungi associated with black root rot in avocado

Louisamarie Parkinson: Queensland Alliance for Agriculture and Food Innovation, The University of Queensland

<div>Root infection of avocado by some nectriaceous fungi causes black root rot, and may severely impact establishment of nursery plants in orchards. Symptoms are frequently masked in the nursery, but after transplanting include black necrotic roots, tree stunting, wilt, leaf drop, rapid decline and death within months. Nectriaceous fungi (153 isolates) from avocado and other hosts were collected from all avocado growing regions in Australia. Phylogenetic analyses of concatenated ITS, β-tubulin and histone H3 loci identified six genera, <em>Gliocladiopsis</em>, <em>Cylindrocladiella</em>, <em>Mariannaea</em>, <em>Calonectria</em>, <em>Ilyonectria</em> and <em>Dactylonectria</em>, associated with black root rot in Australia. Three new <em>Gliocladiopsis</em> species were formally classified and over 20 putative new species (and species complexes) across the genera remain to be resolved. Primers were designed from β-tubulin and histone H3 sequence data of <em>C. ilicicola</em> and <em>D. macrodidyma</em>, the two most pathogenic species, for development of a loop-mediated isothermal amplification (LAMP) assay. Specificity of detection was confirmed with 65 target, closely related and non-target species across several genera. The limit of sensitivity was 0.001 ng/µl DNA for <em>C. ilicicola</em> and 0.01 ng/µl DNA for <em>D. macrodidyma</em>. The diagnostic assay was validated using pure fungal mycelia and necrotic avocado roots from inoculated seedlings, and positive detections obtained within 12–23 min for both pathogens.</div>