A qPCR Assay for the Detection of Phytophthora cinnamomi Including an mRNA Protocol Designed to Establish Propagule Viability in Environmental Samples.

Phytophthora cinnamomi causes root and collar rot in many plant species in natural ecosystems and horticulture. A species-specific primer and probe PCIN5 were designed based on a mitochondrial locus encoding subunit 2 of cytochrome c oxidase (cox2). Eight PCR primers, including three forward and five reverse, were designed and tested in all possible combinations. Annealing temperatures were optimized for each primer pair set to maximize both specificity and sensitivity. Each set was tested against P. cinnamomi and two closely related clade 7 species, P. parvispora and P. niederhauseri. From these tests, five primer pairs were selected based on specificity and, with a species-specific P. cinnamomi probe, used to develop quantitative real-time PCR (qPCR) assays. The specificity of the two most sensitive qPCR assays was confirmed using the genomic DNA of 29 Phytophthora isolates, including 17 isolates of 11 species from clade 7, and representative species from nine other clades (all except clade 3). The assay was able to detect as little as 150 ag of P. cinnamomi DNA and showed no cross-reaction with other Phytophthora species, except for P. parvispora, a very closely related species to P. cinnamomi, which showed late amplification at high DNA concentrations. The efficiency of the qPCR protocol was evaluated with environmental samples including roots and associated soil from plants artificially infected with P. cinnamomi. Different RNA isolation kits were tested and evaluated for their performance in the isolation of RNA from environmental samples, followed by cDNA synthesis, and qPCR assay. Finally, a protocol was recommended for determining the presence of P. cinnamomi in recalcitrant environmental samples.

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.

An overview of Australia's Phytophthora species assemblage in natural ecosystems recovered from a survey in Victoria.

Although Phytophthora species cause serious diseases worldwide, until recently the main focus on disease in natural ecosystems in southern Australia has been on the distribution and impact of P. cinnamomi. However, new Phytophthora pathogens have emerged from natural ecosystems, and there is a need to better understand the diversity and distribution of these species in our natural forests, woodlands and heathlands. From a survey along a 70 km pipeline easement in Victoria, Phytophthora species were isolated from 249 rhizosphere samples and 25 bait bags deployed in 21 stream, river, or wetland locations. Of the 186 Phytophthora isolates recovered, 130 were identified to species based on ITS sequence data. Ninety-five isolates corresponded to 13 described Phytophthora species while additionally 35 isolates were identified as Clade 6 hybrids. Phytophthora cinnamomi was the most common species isolated (31 %), followed by P. elongata (6 %), both species were only recovered from soil. Samples from sites with the highest soil moisture at the time of sampling had the highest yield of isolates. Consistent with other studies throughout the world, Clade 6 species and their hybrids dominated water samples, although many of these species were also recovered less frequently from soil samples. Many of the species recovered in this study have not previously been reported from eastern Australia, reinforcing that Phytophthora species are widespread, abundant and diverse in natural ecosystems. We have probably been underestimating Phytophthora diversity in Australia.

Fungal biogeography. Global diversity and geography of soil fungi.

Fungi play major roles in ecosystem processes, but the determinants of fungal diversity and biogeographic patterns remain poorly understood. Using DNA metabarcoding data from hundreds of globally distributed soil samples, we demonstrate that fungal richness is decoupled from plant diversity. The plant-to-fungus richness ratio declines exponentially toward the poles. Climatic factors, followed by edaphic and spatial variables, constitute the best predictors of fungal richness and community composition at the global scale. Fungi show similar latitudinal diversity gradients to other organisms, with several notable exceptions. These findings advance our understanding of global fungal diversity patterns and permit integration of fungi into a general macroecological framework.