RESUMO
Phytophthora species are oomycete plant pathogens that cause great economic and ecological impacts. The Phytophthora genus includes over 180 known species, infecting a wide range of plant hosts, including crops, trees, and ornamentals. We sequenced the genomes of 31 individual Phytophthora species and 24 individual transcriptomes to study genetic relationships across the genus. De novo genome assemblies revealed variation in genome sizes, numbers of predicted genes, and in repetitive element content across the Phytophthora genus. A genus-wide comparison evaluated orthologous groups of genes. Predicted effector gene counts varied across Phytophthora species by effector family, genome size, and plant host range. Predicted numbers of apoplastic effectors increased as the host range of Phytophthora species increased. Predicted numbers of cytoplasmic effectors also increased with host range but leveled off or decreased in Phytophthora species that have enormous host ranges. With extensive sequencing across the Phytophthora genus, we now have the genomic resources to evaluate horizontal gene transfer events across the oomycetes. Using a machine-learning approach to identify horizontally transferred genes with bacterial or fungal origin, we identified 44 candidates over 36 Phytophthora species genomes. Phylogenetic reconstruction indicates that the transfers of most of these 44 candidates happened in parallel to major advances in the evolution of the oomycetes and Phytophthora spp. We conclude that the 31 genomes presented here are essential for investigating genus-wide genomic associations in genus Phytophthora. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
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Phytophthora , Phytophthora/genética , Filogenia , Transferência Genética Horizontal , Genoma , Genômica , Plantas/genéticaRESUMO
There is a limited number of chemical control agents for managing Phytophthora root and collar rot diseases of avocado internationally; of these, phosphite is one of the most effective. To determine whether prolonged phosphite use in New Zealand avocado orchards has led to decreased sensitivity of Phytophthora cinnamomi to phosphite, 57 isolates were collected from phosphite-treated and -untreated avocado orchards and screened for tolerance using a mycelial growth inhibition assay. The inhibitory effect of phosphite on mycelial growth was tested in vitro using six concentrations of phosphite. Based on changes in mycelial growth using optical density measurements to calculate the effective concentration to reduce growth by 50% (EC50) estimates, three phosphite-susceptible (EC50 range = 18.71 to 29.26 µg/ml) and three tolerant (EC50 range = 81.85 to 123.89 µg/ml) isolates were selected. The effects of phosphite on the colonization of lupin (Lupinus angustifolius) seedling roots and sporangia and zoospore production of three susceptible and three tolerant isolates were determined. The three tolerant isolates colonized lupin roots more extensively than the three susceptible isolates in the presence of phosphite at 5 and 10 g/liter. The tolerant isolates were able to asymptomatically colonize further above the lesion margin in the lupin treated with phosphite at 5 g/liter relative to the phosphite-susceptible isolates but no isolates were completely resistant to phosphite. The tolerant isolates produced more sporangia and, consequently, zoospores in the presence of phosphite than the susceptible isolates. The detection of phosphite tolerance by P. cinnamomi in planta and in vivo is concerning for the future efficacy of phosphite to manage Phytophthora diseases.
Assuntos
Persea , Fosfitos , Phytophthora , Phytophthora/fisiologia , Fosfitos/farmacologia , Nova ZelândiaRESUMO
Phytophthora pluvialis is an oomycete that was first isolated from soil, water, and tree foliage in mixed Douglas-fir-tanoak forests of the U.S. Pacific Northwest (PNW). It was then identified as the causal agent of red needle cast of radiata pine (Pinus radiata) in New Zealand (NZ). Genotyping-by-sequencing was used to obtain 1,543 single nucleotide polymorphisms across 145 P. pluvialis isolates to characterize the population structure in the PNW and NZ. We tested the hypothesis that P. pluvialis was introduced to NZ from the PNW using genetic distance measurements and population structure analyses among locations between countries. The low genetic distance, population heterozygosity, and lack of geographic structure in NZ suggest a single colonization event from the United States followed by clonal expansion in NZ. The PNW Coast Range was proposed as a presumptive center of origin of the currently known distribution of P. pluvialis based on its geographic range and position as the central cluster in a minimum spanning network. The Coastal cluster of isolates were located at the root of every U.S. cluster and emerged earlier than all NZ clusters. The Coastal cluster had the highest degree of heterozygosity (Hs = 0.254) and median pairwise genetic distance (0.093) relative to any other cluster. Finally, the rapid host diversification between closely related isolates of P. pluvialis in NZ indicate that this pathogen has the potential to infect a broader range of hosts than is currently recognized.
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Phytophthora , Nova Zelândia , Noroeste dos Estados Unidos , Filogenia , Phytophthora/genética , Doenças das PlantasRESUMO
BACKGROUND AND AIMS: Biotic and abiotic stressors can cause different defoliation patterns within trees. Foliar pathogens of conifers commonly prefer older needles and infection with defoliation that progresses from the bottom crown to the top. The functional role of the lower crown of trees is a key question to address the impact of defoliation caused by foliar pathogens. METHODS: A 2 year artificial defoliation experiment was performed using two genotypes of grafted Pinus radiata to investigate the effects of lower-crown defoliation on carbon (C) assimilation and allocation. Grafts received one of the following treatments in consecutive years: control-control, control-defoliated, defoliated-control and defoliated-defoliated. RESULTS: No upregulation of photosynthesis either biochemically or through stomatal control was observed in response to defoliation. The root:shoot ratio and leaf mass were not affected by any treatment, suggesting prioritization of crown regrowth following defoliation. In genotype B, defoliation appeared to impose C shortage and caused reduced above-ground growth and sugar storage in roots, while in genotype A, neither growth nor storage was altered. Root C storage in genotype B decreased only transiently and recovered over the second growing season. CONCLUSIONS: In genotype A, the contribution of the lower crown to the whole-tree C uptake appears to be negligible, presumably conferring resilience to foliar pathogens affecting the lower crown. Our results suggest that there is no C limitation after lower-crown defoliation in P. radiata grafts. Further, our findings imply genotype-specific defoliation tolerance in P. radiata.
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Pinus , Carbono , Fotossíntese , Folhas de Planta , ÁrvoresRESUMO
The emergence of Phytophthora pluvialis as a foliar pathogen of Douglas fir in New Zealand and the Pacific Northwest United States has raised questions about its interaction with the widespread Swiss needle cast (SNC) disease. During Spring 2017, we repeatedly sampled 30 trees along an environmental gradient in each region and 292 additional trees in a longitudinal transect to assess the P. pluvialis epidemic and the association between P. pluvialis and Nothophaeocryptopus gaeumannii, which are causal agents of SNC. Both pathogens were consistently more abundant in the host's exotic environment in New Zealand. In both areas, the two pathogens co-exist in different spatial scales for regions and needles. The relative abundance of both pathogens was negatively correlated in the Pacific Northwest, where both presumably have co-existed for longer. Our findings confirmed the interaction of P. pluvialis and N. gaeumannii as foliar pathogens of Douglas fir and suggest a within-site spatial variation in the Pacific Northwest.
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Ascomicetos , Phytophthora , Pseudotsuga , Ascomicetos/fisiologia , Nova Zelândia , Noroeste dos Estados Unidos , Phytophthora/fisiologia , Doenças das Plantas/microbiologia , Pseudotsuga/microbiologiaRESUMO
Red needle cast is a significant foliar disease of commercial stands of Pinus radiata caused by Phytophthora pluvialis in New Zealand. The effect of copper, applied as a foliar spray of cuprous oxide at a range of doses between 0 and 1.72 kg ha-1, was investigated in two controlled trials with potted plants and in an operational trial with mature P. radiata. In all trials, lesions formed on needles after artificial exposure to the infecting propagules (zoospores) of P. pluvialis were used to determine treatment efficacy, with the number and/or length of lesions as the dependent variable. Results across all trials indicated that cuprous oxide was highly effective at reducing infection of P. radiata with P. pluvialis. Application rates equivalent to ≥0.65 kg ha-1 significantly reduced infection levels relative to a control treatment, with foliar surface copper levels as low as 13 to 26 mg kg-1 of needle tissue preventing infection. Greater copper content was associated with a reduction in the proportion of needles with P. pluvialis lesions, with the probability of lesions developing decreasing approximately 1% for every 1 unit (in milligrams per kilogram) increase in copper content. Over a 90-day period, surface copper content declined to 30% of that originally applied, indicating an approximate period of treatment efficacy of 3 months. Our findings highlight the potential of cuprous oxide for the control of red needle cast in P. radiata stands. Further information about the optimal field dose, timing, and the frequency of foliar cuprous oxide application is key to prevent infection and also reduce the build up of inoculum during severe outbreaks of this pathogen.
Assuntos
Cobre , Phytophthora , Pinus , Doenças das Plantas , Antiparasitários/farmacologia , Cobre/análise , Cobre/farmacologia , Nova Zelândia , Phytophthora/efeitos dos fármacos , Phytophthora/fisiologia , Pinus/parasitologia , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controleRESUMO
Contents 1314 I. 1315 II. 1316 III. 1322 IV. 1323 V. 1325 VI. 1326 VII. 1326 VIII. 1327 1328 References 1328 SUMMARY: Invasions of alien plants are typically studied as invasions of individual species, yet interactions between plants and symbiotic fungi (mutualists and potential pathogens) affect plant survival, physiological traits, and reproduction and hence invasion success. Studies show that plant-fungal associations are frequently key drivers of plant invasion success and impact, but clear conceptual frameworks and integration across studies are needed to move beyond a series of case studies towards a more predictive understanding. Here, we consider linked plant-fungal invasions from the perspective of plant and fungal origin, simplified to the least complex representations or 'motifs'. By characterizing these interaction motifs, parallels in invasion processes between pathogen and mutualist fungi become clear, although the outcomes are often opposite in effect. These interaction motifs provide hypotheses for fungal-driven dynamics behind observed plant invasion trajectories. In some situations, the effects of plant-fungal interactions are inconsistent or negligible. Variability in when and where different interaction motifs matter may be driven by specificity in the plant-fungal interaction, the size of the effect of the symbiosis (negative to positive) on plants and the dependence (obligate to facultative) of the plant-fungal interaction. Linked plant-fungal invasions can transform communities and ecosystem function, with potential for persistent legacies preventing ecosystem restoration.
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Fungos/patogenicidade , Plantas/microbiologia , Ecossistema , Fungos/fisiologia , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno/fisiologia , SimbioseRESUMO
Phytophthora pluvialis is associated with early defoliation and shoot dieback in Douglas-fir in Oregon and New Zealand. In 2013, P. pluvialis was described from mixed tanoak-Douglas-fir forests in the Pacific Northwest and concurrently recognized as the main causal agent of red needle cast (RNC) in New Zealand radiata pine plantations. Little is known about its infection cycle and impact on host physiology. P. pluvialis studies in Douglas-fir are challenging due to the ubiquitous presence of the endophyte Phaeocryptopus gaeumannii, which produces similar symptoms and premature defoliation with persistent needle wetness, known as Swiss needle cast (SNC). Nonetheless, our study showed P. pluvialis infection in the presence of SNC. Exclusive expression of P. pluvialis is difficult to achieve as both diseases are promoted by high humidity. Here we established a 'dry leaf' strategy to suppress SNC when inoculating Douglas-fir needles for RNC studies. Sheltering plants along with drip irrigation to avoid needle wetness during the P. gaeumannii sporulation period suppressed its development in the new season flush. The diminished endophyte inoculum enabled bias-reduced studies of P. pluvialis impacts on Douglas-fir without the confounding effects of stomatal blockage and premature defoliation caused by P. gaeumannii.
RESUMO
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, Fv /Fm ; effective quantum yield of photosystem II, ΦPSII ) were assessed. In addition, stomatal conductance (gs ) 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, Fv /Fm , ΦPSII and gs 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.
Assuntos
Magnoliopsida/efeitos dos fármacos , Fosfitos/farmacologia , Doenças das Plantas/prevenção & controle , Traqueófitas/efeitos dos fármacos , Clorofila/metabolismo , Clorofila A , Fluorescência , Magnoliopsida/fisiologia , Fosfitos/efeitos adversos , Complexo de Proteína do Fotossistema II/metabolismo , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/fisiologia , Transpiração Vegetal , Traqueófitas/fisiologia , ÁrvoresRESUMO
Phytophthora species are notorious plant pathogens, with some causing devastating tree diseases that threaten the survival of their host species. One such example is Phytophthora agathidicida, the causal agent of kauri dieback - a root and trunk rot disease that kills the ancient, iconic and culturally significant tree species, Agathis australis (New Zealand kauri). A deeper understanding of how Phytophthora pathogens infect their hosts and cause disease is critical for the development of effective treatments. Such an understanding can be gained by interrogating pathogen genomes for effector genes, which are involved in virulence or pathogenicity. Although genome sequencing has become more affordable, the complete assembly of Phytophthora genomes has been problematic, particularly for those with a high abundance of repetitive sequences. Therefore, effector genes located in repetitive regions could be truncated or missed in a fragmented genome assembly. Using a combination of long-read PacBio sequences, chromatin conformation capture (Hi-C) and Illumina short reads, we assembled the P. agathidicida genome into ten complete chromosomes, with a genome size of 57 Mb including 34% repeats. This is the first Phytophthora genome assembled to chromosome level and it reveals a high level of syntenic conservation with the complete genome of Peronospora effusa, the only other completely assembled genome sequence of an oomycete. All P. agathidicida chromosomes have clearly defined centromeres and contain candidate effector genes such as RXLRs and CRNs, but in different proportions, reflecting the presence of gene family clusters. Candidate effector genes are predominantly found in gene-poor, repeat-rich regions of the genome, and in some cases showed a high degree of duplication. Analysis of candidate RXLR effector genes that occur in multicopy gene families indicated half of them were not expressed in planta. Candidate CRN effector gene families showed evidence of transposon-mediated recombination leading to new combinations of protein domains, both within and between chromosomes. Further analysis of this complete genome assembly will help inform new methods of disease control against P. agathidicida and other Phytophthora species, ultimately helping decipher how Phytophthora pathogens have evolved to shape their effector repertoires and how they might adapt in the future.
RESUMO
Plant pathogens are introduced to new geographical regions ever more frequently as global connectivity increases. Predicting the threat they pose to plant health can be difficult without in-depth knowledge of behaviour, distribution and spread. Here, we evaluate the potential for using biological traits and phylogeny to predict global threats from emerging pathogens.We use a species-level trait database and phylogeny for 179 Phytophthora species: oomycete pathogens impacting natural, agricultural, horticultural and forestry settings. We compile host and distribution reports for Phytophthora species across 178 countries and evaluate the power of traits, phylogeny and time since description (reflecting species-level knowledge) to explain and predict their international transport, maximum latitude and host breadth using Bayesian phylogenetic generalised linear mixed models.In the best-performing models, traits, phylogeny and time since description together explained up to 90%, 97% and 87% of variance in number of countries reached, latitudinal limits and host range, respectively. Traits and phylogeny together explained up to 26%, 41% and 34% of variance in the number of countries reached, maximum latitude and host plant families affected, respectively, but time since description had the strongest effect.Root-attacking species were reported in more countries, and on more host plant families than foliar-attacking species. Host generalist pathogens had thicker-walled resting structures (stress-tolerant oospores) and faster growth rates at their optima. Cold-tolerant species are reported in more countries and at higher latitudes, though more accurate interspecific empirical data are needed to confirm this finding. Policy implications. We evaluate the potential of an evolutionary trait-based framework to support horizon-scanning approaches for identifying pathogens with greater potential for global-scale impacts. Potential future threats from Phytophthora include Phytophthora x heterohybrida, P. lactucae, P. glovera, P. x incrassata, P. amnicola and P. aquimorbida, which are recently described, possibly under-reported species, with similar traits and/or phylogenetic proximity to other high-impact species. Priority traits to measure for emerging species may be thermal minima, oospore wall index and growth rate at optimum temperature. Trait-based horizon-scanning approaches would benefit from the development of international and cross-sectoral collaborations to deliver centralised databases incorporating pathogen distributions, traits and phylogeny.
RESUMO
Phosphite, an analog of phosphate is used to control oomycete diseases on a wide range of horticultural crops and in native ecosystems. In this study, we investigated morphological and transcriptional changes induced in Phytophthora cinnamomi by phosphite. Cytological observations revealed that phosphite caused hyphal distortions and lysis of cell walls and had an adverse effect on hyphal growth. At the molecular level, the expression levels of 43 transcripts were changed. Many of these encoded proteins involved in cell wall synthesis, or cytoskeleton functioning. The results of both the microscopic and molecular investigations are consistent with phosphite inhibiting the function of the cytoskeleton and cell wall synthesis.
Assuntos
Fosfitos/farmacologia , Phytophthora/efeitos dos fármacos , Phytophthora/genética , Sequência de Bases , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Primers do DNA/genética , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Phytophthora/crescimento & desenvolvimento , Phytophthora/metabolismo , Doenças das Plantas/parasitologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing-based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant-pathogen interactions in gymnosperm forest trees, including kauri.
Assuntos
Araucariaceae/parasitologia , Genoma/genética , Interações Hospedeiro-Patógeno , Phytophthora/genética , Doenças das Plantas/parasitologia , Proteínas/metabolismo , Araucariaceae/imunologia , Cycadopsida/imunologia , Cycadopsida/parasitologia , Nova Zelândia , Filogenia , Phytophthora/fisiologia , Doenças das Plantas/imunologia , Imunidade Vegetal , Folhas de Planta/imunologia , Folhas de Planta/parasitologia , Proteínas/genética , Interferência de RNA , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/parasitologiaRESUMO
Species of the genus Phytophthora are arguably the most destructive plant pathogens causing widespread damage to many horticultural and ornamental species, and to native ecosystems throughout the world. Globalization has increased the volume of plants being transported over long distances and has increased the spread of Phytophthora species. As traditional detection methods such as baiting or direct isolation are incapable of handling the large volume of material to be tested, researchers have developed more rapid and specific antibody and DNA based tests. This review compares the performance of the different types of tests used for detection of Phytophthora.
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Técnicas Microbiológicas/métodos , Phytophthora/isolamento & purificação , Doenças das Plantas/microbiologia , Plantas/microbiologia , Proteínas de Algas/isolamento & purificação , DNA de Algas/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Phytophthora/química , Phytophthora/genética , Reação em Cadeia da Polimerase/métodosRESUMO
Understanding the epidemiology of infectious diseases in a host population is a major challenge in forestry. Radiata pine plantations in New Zealand are impacted by a foliar disease, red needle cast (RNC), caused by Phytophthora pluvialis. This pathogen is dispersed by water splash with polycyclic infection affecting the lower part of the tree canopy. In this study, we extended an SI (Susceptible-Infectious) model presented for RNC to analyse the key epidemiological drivers. We conducted two experiments to empirically fit the extended model: a detached-needle assay and an in vivo inoculation. We used the detached-needle assay data to compare resistant and susceptible genotypes, and the in vivo inoculation data was used to inform sustained infection of the whole plant. We also compared isolations and real-time quantitative PCR (qPCR) to assess P. pluvialis infection. The primary infection rate and the incubation time were similar for susceptible and resistant genotypes. The pathogen death rate was 2.5 times higher for resistant than susceptible genotypes. Further, external proliferation of mycelium and sporangia were only observed on 28% of the resistant ramets compared to 90% of the susceptible ones. Detection methods were the single most important factor influencing parameter estimates of the model, giving qualitatively different epidemic outputs. In the early stages of infection, qPCR proved to be more efficient than isolations but the reverse was true at later points in time. Isolations were not influenced by the presence of lesions in the needles, while 19% of lesioned needle maximized qPCR detection. A primary infection peak identified via qPCR occurred at 4 days after inoculation (dai) with a secondary peak observed 22 dai. Our results have important implications to the management of RNC, by highlighting the main differences in the response of susceptible and resistant genotypes, and comparing the most common assessment methods to detect RNC epidemics.
Assuntos
Phytophthora/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Epidemias , Regulação da Expressão Gênica de Plantas/genética , Genótipo , Micélio/genética , Agulhas , Nova Zelândia , Pinus/genética , Pinus/parasitologia , Esporângios/genéticaRESUMO
Genome sequences were generated for six oomycete isolates collected from forests in Valdivia, Chile. Three of the isolates were identified morphologically as Phytophthora kernoviae, whereas two were similar to other clade 10 Phytophthora species. One isolate was tentatively identified as Nothophytophthora valdiviana based on nucleotide sequence similarity in the cytochrome oxidase 1 gene. This is the first genome sequence for this recently described genus. The genome assembly was more fragmented and contained many duplicated genes when compared with the other Phytophthora sequences. Comparative analyses were performed with genomic sequences of the P. kernoviae isolates from the UK and New Zealand. Although the potential New Zealand origin of P. kernoviae has been suggested, new isolations from Chile had cast doubt on this hypothesis. We present evidence supporting P. kernoviae as having originated in New Zealand. However, investigation of the diversity of oomycete species in Chile has been limited and warrants further exploration. We demonstrate the expediency of genomic analyses in determining phylogenetic relationships between isolates within new and often scantly represented taxonomic groups, such as Phytophthora clade 10 and Nothophytophthora. Data are available on GenBank via BioProject accession number PRJNA352331.
Assuntos
Análise de Sequência de DNA/métodos , Chile , Nova Zelândia , Oomicetos/genética , Oomicetos/patogenicidade , Filogenia , Phytophthora/genética , Phytophthora/patogenicidade , Reino UnidoRESUMO
Many plant tissues fluoresce due to the natural fluorophores present in cell walls or within the cell protoplast or lumen. While lignin and chlorophyll are well-known fluorophores, other components are less well characterized. Confocal fluorescence microscopy of fresh or fixed vibratome-cut sections of radiata pine needles revealed the presence of suberin, lignin, ferulate, and flavonoids associated with cell walls as well as several different extractive components and chlorophyll within tissues. Comparison of needles in different physiological states demonstrated the loss of chlorophyll in both chlorotic and necrotic needles. Necrotic needles showed a dramatic change in the fluorescence of extractives within mesophyll cells from ultraviolet (UV) excited weak blue fluorescence to blue excited strong green fluorescence associated with tissue browning. Comparisons were made among fluorophores in terms of optimal excitation, relative brightness compared to lignin, and the effect of pH of mounting medium. Fluorophores in cell walls and extractives in lumens were associated with blue or green emission, compared to the red emission of chlorophyll. Autofluorescence is, therefore, a useful method for comparing the histology of healthy and diseased needles without the need for multiple staining techniques, potentially aiding visual screening of host resistance and disease progression in needle tissue.
RESUMO
Chemical treatments are used widely in agricultural and natural settings to protect plants from diseases; however, they may exert an important selection pressure on plant pathogens, promoting the development of tolerant isolates through adaptive evolution. Phosphite is used to manage diseases caused by Phytophthora species which include a large number of the most economically damaging plant pathogens worldwide. Phosphite controls the growth of Phytophthora species in planta without killing it; as a result, isolates can develop tolerance to phosphite after prolonged exposure. We investigated the inter- and intra-specific variability in phosphite tolerance of eleven Phytophthora species, including P. ramorum, an internationally important, highly regulated pathogen. Phytophthora ramorum is a good model system because it is comprised of multiple genetically homogeneous lineages. Seven species were found to be consistently sensitive to phosphite based on the low Effective Concentration (EC) 50 values of all isolates tested (amount of phosphite required to inhibit mycelial growth by 50% relative to growth in the absence of phosphite). However, P. ramorum, P. lateralis, P. crassamura and P. cambivora showed intraspecific variability in sensitivity to phosphite, with at least one isolate showing significantly higher tolerance than the other isolates. Within the three P. ramorum evolutionarily divergent lineages tested, NA1 was the most susceptible to phosphite, the NA1 and EU1 lineages showed intralineage variability and the NA2 lineage showed a decreased sensitivity to phosphite overall as all isolates were relatively tolerant. This finding is relevant because NA1 is dominant in the wild and can be controlled using phosphite, while the EU1 lineage has recently been identified in the wild and is phosphite-tolerant, making the treatment approach potentially less effective. Phytophthora ramorum, P. lateralis and P. crassamura are either selfing, homothallic species, or are known to reproduce exclusively clonally, indicating tolerance to phosphite can emerge even in the absence of sexual recombination.
Assuntos
Fosfitos/farmacologia , Phytophthora/genética , Doenças das Plantas/genética , Seleção Genética/efeitos dos fármacos , Variação Genética , Genótipo , Fosfitos/toxicidade , Filogenia , Phytophthora/efeitos dos fármacos , Phytophthora/crescimento & desenvolvimento , Doenças das Plantas/prevenção & controleRESUMO
A high-throughput assay was developed to screen Phytophthora species for fungicide sensitivity using optical density measurements for unbiased, automated measurement of mycelial growth. The efficacy of the optical density assay (OD) to measure phosphite sensitivity in Phytophthora species was compared to two widely used methods, radial growth (RG) and dry weight (DW) assays. Three isolates of each of Phytophthora cinnamomi, P. multivora and P. pluvialis, with known phosphite exposure and three isolates of each species with no prior phosphite exposure, were screened for phosphite sensitivity using the three assays. Mycelial growth measurements were taken after culturing for 6, 14 and 15â¯days for the OD, DW and RG assays respectively. Mycelial growth inhibition at 15, 80, 200 and 500⯵g/mL phosphite relative to growth on control media was used to determine effective concentration values for 50% growth reduction (EC50). The species varied in their tolerance to phosphite with P. cinnamomi being the least sensitive followed by P. multivora and P. pluvialis. No significant differences in tolerance were found between isolates within the same species using any method. The OD assay produced comparable EC50 values to the RG and DW assays. The growth of the three species was more sensitive to phosphite in the DW than the RG and OD assays, however limited sample throughput and greater variation in measuring small amounts of mycelia in the dry weight assessment increase variability and limits throughput. The OD assay offers a fast method to enable an inventory of chemical resistance and is particularly advantageous for slow growing species as it requires less time and offers greater throughput than existing RG and DW methods.