Phytophthora species repeatedly introduced in Northern California through restoration projects can spread into adjacent sites

This study investigates whether Phytophthora species may have been repeatedly introduced in natural habitats through restoration projects. Six plant species across five research locations in three counties within the San Francisco Bay Area were tested for pathogen infection of stems, roots and for rhizosphere infestation at failing restoration sites. Where possible, the same hosts were evaluated in one neighboring un-restored site disturbed by the presence of culverts, drainages or trails that also intersected the restoration site, and in a naturally regenerated and undisturbed control site. Although native or endemic pathogens were isolated from all three types of sites, Phytophthora species were never isolated from control undisturbed sites. Statistical analyses confirmed that percentage of positive Phytophthora isolations was significantly higher in restoration sites and adjoining disturbed sites than in control sites. Presence of Phytophthora species was correlated with disease symptoms, plant death and lack of regeneration. Furthermore, six of eight Phytophthora species isolated in the field had previously been reported from plant production facilities providing stock for habitat restoration. To our knowledge, this is the first controlled survey linking the presence of entire Phytophthora species assemblages to failing restoration projects and to the plant production facilities that provide plant stock for restoration, while showing that Phytophthora species are absent in neighboring undisturbed sites. This study further proves that these pathogens are spreading from restoration sites through disturbance pathways.

     

Surprising low diversity of the plant pathogen Phytophthora in Amazonian forests

The genus Phytophthora represents a group of plant pathogens with broad global distribution. The majority of them cause the collar and root-rot of diverse plant species. Little is known about Phytophthora communities in forest ecosystems, especially in the Neotropical forests where natural enemies could maintain the huge plant diversity via negative density dependence. We characterized the diversity of soil-borne Phytophthora communities in the North French Guiana rainforest and investigated how they are structured by host identity and environmental factors. In this little-explored habitat, 250 soil cores were sampled from 10 plots hosting 10 different plant families across three forest environments (Terra Firme, Seasonally Flooded and White Sand). Phytophthora diversity was studied using a baiting approach and metabarcoding (High-Throughput Sequencing) on environmental DNA extracted from both soil samples and baiting-leaves. These three approaches revealed very similar communities, characterized by an unexpected low diversity of Phytophthora species, with the dominance of two cryptic species close to Phytophthora heveae. As expected, the Phytophthora community composition of the French Guiana rainforest was significantly impacted by the host plant family and environment. However, these plant pathogen communities are very small and are dominated by generalist species, questioning their potential roles as drivers of plant diversity in these Amazonian forests.     

Ectopic expression of <Emphasis Type="Italic">Dahlia merckii</Emphasis> defensin <Emphasis Type="Italic">DmAMP</Emphasis>1 improves papaya resistance to <Emphasis Type="Italic">Phytophthora palmivora</Emphasis> by reducing pathogen vigor

Phytophthora spp., some of the more important casual agents of plant diseases, are responsible for heavy economic losses worldwide. Plant defensins have been introduced as transgenes into a range of species to increase host resistance to pathogens to which they were originally susceptible. However, the effectiveness and mechanism of interaction of the defensins with Phytophthora spp. have not been clearly characterized in planta. In this study, we expressed the Dahlia merckii defensin, DmAMP1, in papaya (Carica papaya L.), a plant highly susceptible to a root, stem, and fruit rot disease caused by Phytophthora palmivora. Extracts of total leaf proteins from transformed plants inhibited growth of Phytophthora in vitro and discs cut from the leaves of transformed plants inhibited growth of Phytophthora in a bioassay. Results from our greenhouse inoculation experiments demonstrate that expressing the DmAMP1 gene in papaya plants increased resistance against P. palmivora and that this increased resistance was associated with reduced hyphae growth of P. palmivora at the infection sites. The inhibitory effects of DmAMP1 expression in papaya suggest this approach has good potential to impart transgenic resistance against Phytophthora in papaya.     

Heat treatment induced bacterial changes in irrigation water and their implications for plant disease management

A new heat treatment for recycled irrigation water using 48 °C for 24 h to inactivate Phytophthora and bacterial plant pathogens is estimated to reduce fuel cost and environmental footprint by more than 50 % compared to current protocol (95 °C for 30 s). The objective of this study was to determine the impact of this new heat treatment temperature regime on bacterial community structure in water and its practical implications. Bacterial communities in irrigation water were analyzed before and after heat treatment using both culture-dependent and -independent strategies based on the 16S ribosomal DNA. A significant shift was observed in the bacterial community after heat treatment. Most importantly, bacteria with biological control potential—Bacillus and Paenibacillus, and Pseudomonas species became more abundant at both 48 and 42 °C. These findings imply that the new heat treatment procedure not only controls existing plant pathogens but also may make the heat-treated irrigation water a more antagonistic environment against plant pathogens, promoting sustainable disease management.     

Strategies of attack and defense in plant–oomycete interactions, accentuated for Phytophthora parasitica Dastur (syn. P. Nicotianae Breda de Haan)

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to their particular physiological characteristics, no efficient treatments against diseases caused by these microorganisms are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. Available data are scarce, and genomic approaches were mainly developed for the two species, Phytophthora infestans and Phytophthora sojae. However, these two species are exceptions from, rather than representative species for, the genus. P. infestans is a foliar pathogen, and P. sojae infects a narrow range of host plants, while the majority of Phytophthora species are quite unselective, root-infecting pathogens. To represent this majority, Phytophthora parasitica emerges as a model for the genus, and genomic resources for analyzing its interaction with plants are developing. The aim of this review is to assemble current knowledge on cytological and molecular processes that are underlying plant–pathogen interactions involving Phytophthora species and in particular P. parasitica, and to place them into the context of a hypothetical scheme of co-evolution between the pathogen and the host.     

Developing a taxonomic identification system of Phytophthora species based on microsatellites

BackgroundPhytophthora is the most important genus of the Oomycete plant pathogens. Nowadays, there are 117 described species in this genus, most of them being primary invaders of plant tissues. The different species are causal agents of diseases in a wide range of crops and plants in natural environments. In order to develop control strategies against Phytophthoraspecies, it is important to know the biology, ecology and evolutionary processes of these important pathogens.AimsThe aim of this study was to propose and validate a low cost identification system for Phytophthora species based on a set of polymorphic microsatellite (SSRs) markers.MethodsThirty-three isolates representing Phytophthora infestans, Phytophthora andina, Phytophthora sojae, Phytophthora cryptogea, Phytophthora nicotianae, Phytophthora capsici and Phytophthora cinnamomi species were obtained, and 13 SSRs were selected as potentially transferable markers between these species. Amplification conditions, including annealing temperatures, were standardized for several markers.ResultsA subset of these markers amplified in all species, showing species-specific alleles.ConclusionsThe adaptability and impact of the identification system in Colombia, an Andean agricultural country where different Phytophthora species co-exist in the same or in several hosts grown together, are discussed.     

Molecular Profiling of the Phytophthora plurivora Secretome: A Step towards Understanding the Cross-Talk between Plant Pathogenic Oomycetes and Their Hosts

The understanding of molecular mechanisms underlying host–pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora-F. sylvatica interaction.     

Plant-Pathogenic Oomycetes,Escherichia coli Strains,and Salmonella spp. Frequently Found in Surface Water Used for Irrigation of Fruit and Vegetable Crops in New York State

In the United States, surface water is commonly used to irrigate a variety of produce crops and can harbor pathogens responsible for food-borne illnesses and plant diseases. Understanding when pathogens infest water sources is valuable information for produce growers to improve the food safety and production of these crops. In this study, prevalence data along with regression tree analyses were used to correlate water quality parameters (pH, temperature, turbidity), irrigation site properties (source, the presence of livestock or fowl nearby), and precipitation data to the presence and concentrations of Escherichia coli, Salmonella spp., and hymexazol-insensitive (HIS) oomycetes (Phytophthora and Pythium spp.) in New York State surface waters. A total of 123 samples from 18 sites across New York State were tested for E. coli and Salmonella spp., of which 33% and 43% were positive, respectively. Additionally, 210 samples from 38 sites were tested for HIS oomycetes, and 88% were found to be positive, with 10 species of Phytophthora and 11 species of Pythium being identified from the samples. Regression analysis found no strong correlations between water quality parameters, site factors, or precipitation to the presence or concentration of E. coli in irrigation sources. For Salmonella, precipitation (≤0.64 cm) 3 days before sampling was correlated to both presence and the highest counts. Analyses for oomycetes found creeks to have higher average counts than ponds, and higher turbidity levels were associated with higher oomycete counts. Overall, information gathered from this study can be used to better understand the food safety and plant pathogen risks of using surface water for irrigation.     

Signatures of selection and host‐adapted gene expression of the Phytophthora infestans RNA silencing suppressor PSR2

Phytophthora infestans is a devastating pathogen in agricultural systems. Recently, an RNA silencing suppressor (PSR2, ‘Phytophthora suppressor of RNA silencing 2’) has been described in P. infestans. PSR2 has been shown to increase the virulence of Phytophthora pathogens on their hosts. This gene is one of the few effectors present in many economically important Phytophthora species. In this study, we investigated: (i) the evolutionary history of PSR2 within and between species of Phytophthora; and (ii) the interaction between sequence variation, gene expression and virulence. In P. infestans, the highest PiPSR2 expression was correlated with decreased symptom expression. The highest gene expression was observed in the biotrophic phase of the pathogen, suggesting that PSR2 is important during early infection. Protein sequence conservation was negatively correlated with host range, suggesting host range as a driver of PSR2 evolution. Within species, we detected elevated amino acid variation, as observed for other effectors; however, the frequency spectrum of the mutations was inconsistent with strong balancing selection. This evolutionary pattern may be related to the conservation of the host target(s) of PSR2 and the absence of known corresponding R genes. In summary, our study indicates that PSR2 is a conserved effector that acts as a master switch to modify plant gene regulation early during infection for the pathogen's benefit. The conservation of PSR2 and its important role in virulence make it a promising target for pathogen management.     

Zoosporic Tolerance to pH Stress and Its Implications for Phytophthora Species in Aquatic Ecosystems

Phytophthora species, a group of destructive plant pathogens, are commonly referred to as water molds, but little is known about their aquatic ecology. Here we show the effect of pH on zoospore survival of seven Phytophthora species commonly isolated from irrigation reservoirs and natural waterways and dissect zoospore survival strategy. Zoospores were incubated in a basal salt liquid medium at pH 3 to 11 for up to 7 days and then plated on a selective medium to determine their survival. The optimal pHs differed among Phytophthora species, with the optimal pH for P. citricola at pH 9, the optimal pH for P. tropicalis at pH 5, and the optimal pH for the five other species, P. citrophthora, P. insolita, P. irrigata, P. megasperma, and P. nicotianae, at pH 7. The greatest number of colonies was recovered from zoospores of all species plated immediately after being exposed to different levels of pH. At pH 5 to 11, the recovery rate decreased sharply (P ≤ 0.0472) after 1-day exposure for five of the seven species. In contrast, no change occurred (P ≥ 0.1125) in the recovery of any species even after a 7-day exposure at pH 3. Overall, P. megasperma and P. citricola survived longer at higher rates in a wider range of pHs than other species did. These results are generally applicable to field conditions as indicated by additional examination of P. citrophthora and P. megasperma in irrigation water at different levels of pH. These results challenge the notion that all Phytophthora species inhabit aquatic environments as water molds and have significant implications in the management of plant diseases resulting from waterborne microbial contamination.Phytophthora species, a group of oomycetes in the kingdom of Stramenopila and well-known plant pathogens, were first listed as “water molds” by Blackwell in 1944 (5), and this notion has since been generally accepted. These species are phylogenetically close to golden-brown algae, although morphologically and physiologically, they resemble fungi. Most algae are aquatic in nature. Phytophthora species produce flagellate zoospores as their primary dispersal structure (35-37, 39). Zoospores can travel in aquatic environments actively on their own locomotion and passively through water movement (12, 13, 41).More than 20 species of Phytophthora, including P. ramorum, the sudden oak death pathogen, have been isolated from irrigation reservoirs and natural waterways (20-22, 30, 40, 43), and a number of previously unknown taxa also have been documented in aquatic environments (8, 24). These pathogens pose a threat to agricultural sustainability and natural ecosystems, as agriculture increasingly depends on recycled water for irrigation in light of rapidly spreading global water scarcity (19, 22). Recycling irrigation systems provide an efficient means of pathogen dissemination from a single point of infection to an entire farm and from a single farm to other farms sharing the same water resources (22, 24).A search of science-based solutions to this crop health issue reveals a surprising lack of information on the aquatic ecology of Phytophthora species. For instance, hydrogen ion concentration (pH) is among the most important water quality parameters which influence sporangium production and germination (1-3, 6, 32, 34, 38), survival of thick-walled chlamydospores and oospores in the soil environment, and disease development (2, 4, 33, 44). However, the effect of pH on the survival of zoospores and growth of germlings in aquatic environments is not known. As motile zoospores lack cell walls and encysted spores or cysts have thin walls, they are presumably more vulnerable to pH stress than chlamydospores and oospores are. On the other hand, the pH level is likely to fluctuate more regularly and at a greater range in aquatic systems, such as irrigation reservoirs, than in soil systems. pH can change diurnally due to respiration of aquatic plants and seasonally due to rain, oxidation of sulfide-containing sediments through the production of sulfuric acid, algal blooms, and released bases or acids from residues of fertilizer and pesticides. Thus, zoospores and aquatic systems are more prone to the influence of wide pH changes than chlamydospores/oospores in soil systems are. The aim of this study was to determine the impact of pH on zoospore survival and understand the aquatic ecology of different Phytophthora species.     

Predictors of Phytophthora diversity and community composition in natural areas across diverse Australian ecoregions

Comprehensive understanding of the patterns and drivers of microbial diversity at a landscape scale is in its infancy, despite the recent ease by which soil communities can be characterized using massively parallel amplicon sequencing. Here we report on a comprehensive analysis of the drivers of diversity distribution and composition of the ecologically and economically important Phytophthora genus from 414 soil samples collected across Australia. We assessed 22 environmental and seven categorical variables as potential predictors of Phytophthora species richness, α and β diversity, including both phylogenetically and non‐phylogenically explicit methods. In addition, we classified each species as putatively native or introduced and examined the distribution with respect to putative origin. The two most widespread species, P. multivora and P. cinnamomi, are introduced, though five of the ten most widely distributed species are putatively native. Introduced taxa comprised over 54% of Australia's Phytophthora diversity and these species are known pathogens of annual and perennial crop habitats as well as urban landscapes and forestry. Patterns of composition were most strongly predicted by bioregion (R² = 0.29) and ecoregion (R² = 0.26) identity; mean precipitation of warmest quarter, mean temperature of the wettest quarter and latitude were also highly significant and described approximately 21, 14 and 13% of variation in NMDS composition, respectively. We also found statistically significant evidence for phylogenetic over‐dispersion with respect to key climate variables.This study provides a strong baseline for understanding biogeographical patterns in this important genus as well the impact of key plant pathogens and invasive Phytophthora species in natural ecosystems.     

Foliar phosphite application has minor phytotoxic impacts across a diverse range of conifers and woody angiosperms

Phytophthora plant pathogens cause tremendous damage in planted and natural systems worldwide. Phosphite is one of the only effective chemicals to control broad‐scale Phytophthora disease. Little work has been done on the phytotoxic effects of phosphite application on plant communities especially in combination with plant physiological impacts. Here, we tested the phytotoxic impact of phosphite applied as foliar spray at 0, 12, 24 and 48 kg a.i. ha^?1. Eighteen‐month‐old saplings of 13 conifer and angiosperm species native to New Zealand, and two exotic coniferous species were treated and the development of necrotic tissue and chlorophyll‐a‐fluorescence parameters (optimal quantum yield, F_v/F_m; effective quantum yield of photosystem II, Φ_PSII) were assessed. In addition, stomatal conductance (g_s) was measured on a subset of six species. Significant necrosis assessed by digital image analysis occurred in only three species: in the lauraceous canopy tree Beilschmiedia tawa (8–14%) and the understory shrub Dodonaea viscosa (5–7%) across phosphite concentrations and solely at the highest concentration in the myrtaceous pioneer shrub Leptospermum scoparium (66%). In non‐necrotic tissue, F_v/F_m, Φ_PSII and g_s remained unaffected by the phosphite treatment. Overall, our findings suggest minor phytotoxic effects resulting from foliar phosphite application across diverse taxa and regardless of concentration. This study supports the large‐scale use of phosphite as a management tool to control plant diseases caused by Phytophthora pathogens in plantations and natural ecosystems. Long‐term studies are required to ascertain potential ecological impacts of repeated phosphite applications.     

Ecological roles of saprotrophic Peronosporales (Oomycetes,Straminipila) in natural environments

The fungus-like Peronosporales are composed of several lineages of mainly biotrophic and hemibiotrophic representatives. Saprotrophic species of Peronosporales are limited to the genera Halophytophthora and Salisapilia, and to some species in Phytopythium and Phytophthora Clades 6 and 9, which inhabit terrestrial and all types of aquatic ecosystems. The recent discovery of species of Phytophthora in marine habitats and of Halophytophthora in freshwater indicated that these genera are not only morphologically but also ecologically poorly delineated. In addition, half of these genera are not monophyletic. They play key ecological roles by upgrading nutrients to higher trophic levels through colonization of plant debris, which makes substrata more palatable for detritivores or through zoospore grazing by zooplankton, although their role as saprotrophs is still largely neglected. Some species of Phytophthora can be aggressive opportunistic pathogens of riverine forests in the presence of susceptible hosts and favourable environmental conditions and, as a consequence, most studies have focused on their role as pathogens. Identification of species is challenging due to hybridization and species complexes that harbour multiple cryptic species and, therefore, is not reliable without DNA sequencing tools.     

Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas: a review

Plant pathogens are responsible for many crop plant diseases, resulting in economic losses. The use of bacterial agents is an excellent option to fight against plant pathogens and an excellent alternative to the use of chemicals, which are offensive to the environment and to human health. Two of the most common biocontrol agents are members of the Bacillus and Pseudomonas genera. Both bacterial genera have important traits such as plant growth-promoting (PGP) properties. This review analyzes pioneering and recent works and the mechanisms used by Bacillus and Pseudomonas in their behaviour as biocontrol and PGP agents, discussing their mode of action by comparing the two genera. Undoubtedly, future integrated research strategies for biocontrol and PGP will require the help of known and novel species of both genera.     

Occurrence of Pythium spp. and Phytophthora spp. in Norwegian Greenhouses and their Pathogenicity on Cucumber Seedlings

Samples of tomato, lettuce and cucumber submitted for diagnosis to the Plant Protection Centre at the Norwegian Crop Research Institute and samples of soil, water and cucumber collected from greenhouses employing hydroponic cultures were examined for the occurrence of Pythium spp. and Phytophthora spp. Two species of Phytophthora and 16 species of Pythium were identified. Phytophthora cryptogea was found on tomato and lettuce. Phytophthora nicotianae was found on tomato fruit. Phytophthora was not found on cucumbers. Pythium irregulare and Pythium group F were the two most commonly found Pythium species in hydroponically cultivated cucumbers. A pathogenicity test with 56 isolates was performed on cucumber seedlings. The most aggressive species were Pythium aphanidermatum, P. irregulare, Pythium paroecandrum and Pythium ultimum.     

What does it take to be a plant pathogen: genomic insights from <Emphasis Type="Italic">Streptomyces</Emphasis> species

Plant pathogenicity is rare in the genus Streptomyces, with only a dozen or so species possessing this trait out of the more than 900 species described. Nevertheless, such species have had a significant impact on agricultural economies throughout the world due to their ability to cause important crop diseases such as potato common scab, which is characterized by lesions that form on the potato tuber surface. All pathogenic species that cause common scab produce a family of phytotoxins called the thaxtomins, which function as cellulose synthesis inhibitors. In addition, the nec1 and tomA genes are conserved in several pathogenic streptomycetes, the former of which is predicted to function in the suppression of plant defense responses. Streptomyces scabies is the oldest plant pathogen described and has a world-wide distribution, whereas species such as S. turgidiscabies and S. acidiscabies are believed to be newly emergent pathogens found in more limited geographical locations. The genome sequence of S. scabies 87-22 was recently completed, and comparative genomic analyses with other sequenced microbial pathogens have revealed the presence of additional genes that may play a role in plant pathogenicity, an idea that is supported by functional analysis of one such putative virulence locus. In addition, the availability of multiple genome sequences for both pathogenic and nonpathogenic streptomycetes has provided an opportunity for comparative genomic analyses to identify the Streptomyces pathogenome. Such genomic analyses will contribute to the fundamental understanding of the mechanisms and evolution of plant pathogenicity and plant-microbe biology within this genus.     

Nonneutral GC3 and Retroelement Codon Mimicry in Phytophthora

Phytophthora is a genus entirely comprised of destructive plant pathogens. It belongs to the Stramenopila, a unique branch of eukaryotes, phylogenetically distinct from plants, animals, or fungi. Phytophthora genes show a strong preference for usage of codons ending with G or C (high GC3). The presence of high GC3 in genes can be utilized to differentiate coding regions from noncoding regions in the genome. We found that both selective pressure and mutation bias drive codon bias in Phytophthora. Indicative for selection pressure is the higher GC3 value of highly expressed genes in different Phytophthora species. Lineage specific GC increase of noncoding regions is reminiscent of whole-genome mutation bias, whereas the elevated Phytophthora GC3 is primarily a result of translation efficiency-driven selection. Heterogeneous retrotransposons exist in Phytophthora genomes and many of them vary in their GC content. Interestingly, the most widespread groups of retroelements in Phytophthora show high GC3 and a codon bias that is similar to host genes. Apparently, selection pressure has been exerted on the retroelement’s codon usage, and such mimicry of host codon bias might be beneficial for the propagation of retrotransposons. Reviewing Editor: Dr. Yves van de Peer     

Absence of evidence is not evidence of absence: Feral pigs as vectors of soil‐borne pathogens

Invasive soil‐borne pathogens are a major threat to forest ecosystems worldwide. The newly discovered soil pathogen, Phytophthora ‘taxon Agathis’ (PTA), is a serious threat to endemic kauri (Agathis australis: Araucariaceae) in New Zealand. This study examined the potential for feral pigs to act as vectors of PTA. We investigated whether snouts and trotters of feral pigs carry soil contaminated with PTA, and using these results determined the probability that feral pigs act as a vector. We screened the soil on trotters and snouts from 457 pigs for PTA using various baiting techniques and molecular testing. This study detected 19 species of plant pathogens in the soil on pig trotters and snouts, including a different Phytophthora species (Phytophthora cinnamomi). However, no PTA was isolated from the samples. A positive control experiment showed a test sensitivity of 0–3% for the baiting methods and the data obtained were used in a Bayesian probability modelling approach. This showed a posterior probability of 35–90% (dependent on test sensitivity scores and design prevalence) that pigs do vector PTA and estimated that a sample size of over 1000 trotters would be required to prove a negative result. We conclude that feral pigs cannot be ruled out as a vector of soil‐based plant pathogens and that there is still a high probability that feral pigs do vector PTA, despite our negative results. We also highlight the need to develop a more sensitive test for PTA in small soil samples associated with pigs due to unreliable detection rates using the current method.