Search

Widely produced and marketed worldwide, orchids suffers from several diseases that have a negative effect on production. Black rot disease is among the most common and severe disease, characterized by black spots of rot on leaves, pseudobulbs and roots, which usually lead to the plant death. The world literature lists some Phytophthora species as causal agents of the disease, but there is no advanced study on the etiology of this disease in Brazil, which makes it difficult to determine an efficient control method. This work aims to contribute to the study of the etiology of black rot on Cattleya leopoldii in Brazil. Severely diseased C. leopoldii plants with typical symptoms of black rot, collected from a commercial orchid farm in Brazil, were taken to the laboratory and used to isolate the pathogen. Based on morphological characters analysis combined with molecular data, the isolates were identified as belonging to the species Phytophthora heterospora. This is the first worldwide report of P. heterospora causing black rot disease on orchids., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have seemed to influence the work reported in this paper. Data availability: The alignment datasets generated during the study are available from the corresponding author upon request. All other data generated or analyzed during this study are included in this published article., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Globally, forests are constantly threatened by a plethora of disturbances of natural and anthropogenic origin, such as climate change, forest fires, urbanization, and pollution. Besides the most common stressors, during the last few years, Portuguese forests have been impacted by severe decline phenomena caused by invasive pathogens, many of which belong to the genus Phytophthora . The genus Phytophthora includes a large number of species that are invading forest ecosystems worldwide, chiefly as a consequence of global trade and human activities. This paper reports the results of a survey of Phytophthora diversity in natural and semi-natural forest ecosystems in Portugal along an elevation gradient. Isolations performed from 138 symptomatic plant tissues and rhizosphere samples collected from 26 plant species yielded a total of 19 Phytophthora species belonging to 6 phylogenetic clades, including P. cinnamomi (36 isolates), P. multivora (20), P. plurivora (9), P. cactorum (8), P. lacustris (8), P. pseudocryptogea (8), P. amnicola (6), P. hedraiandra (6), P. pseudosyringae (5), P. thermophila (5), P. bilorbang (4), P. inundata (4), P. asparagi (3), P. citricola (3), P. gonapodyides (3), P. rosacearum (3), P. chlamydospora (2), P. pachypleura (2), and P. syringae (1). Overall, the data obtained highlight the widespread occurrence of P. cinnamomi in natural ecosystems from sea level to mountain habitats. The results of the pathogenicity tests carried out on 2-year-old chestnut plants confirmed the key role of P. cinnamomi in the recrudescence of chestnut ink disease and the additional risk posed by P. pachypleura , P. plurivora , and P. multivora to Portuguese chestnut forests. Finally, three species, P. citricola , P. hedraiandra , and P. pachypleura , are reported for the first time in the natural ecosystems of Portugal.

Here, we present a protocol for the isolation and detection of Phytophthora oospores directly from soil samples. Our method incorporates a novel technique for isolating Phytophthora oospores using filter pouches and an improved DNA extraction procedure specifically designed for oospores. While we have primarily developed this protocol for detecting P. agathidicida oospores using end-point PCR, we believe these methods can be readily adapted for other Phytophthora species. Furthermore, the DNA extracted using this protocol is suitable as input for other DNA-based detection methods., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We describe a protocol to amplify DNA barcodes of known and unknown taxa of Phytophthora and related plant pathogenic oomycetes from a range of environments. The methods focus on sampling pathogen propagules from water using in situ sampling and filtration equipment and buffers that enable efficient storage and DNA extraction for later downstream processing., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Leaf surface microbial communities play an important role in forest ecosystems and are known to be affected by environmental and host conditions, including diseases impacting the host. Phytophthora agathidicida is a soil-borne pathogen that causes severe disease (kauri dieback) in one of New Zealand's endemic trees, Agathis australis (kauri). This research characterised the microbial communities of the A. australis phyllosphere (i.e. leaf surface) using modern molecular techniques and explored the effects of P. agathidicida on those communities. Fresh leaves were collected from trees where P. agathidicida was and was not detected in the soil and characterisation of the leaf surface microbial community was carried out via high-throughput amplicon sequencing of the internal transcribed spacer (ITS) and 16S ribosomal RNA regions. Nutrients in leaf leachates were also measured to identify other possible drivers of microbial diversity. The dominant phyllosphere microbial phylum was Proteobacteria followed by Acidobacteria. The phyllosphere microbial richness of A. agathis associated with P. agathidicida-infected soils was found to be generally lower than where the pathogen was not detected for both prokaryote (bacterial) and fungal phyla. Leaf leachate pH as well as boron and silicon had significant associations with bacterial and fungal community structure. These findings contribute to the development of a comprehensive understanding of A. australis leaf surface microbial communities and the effects of the soil pathogen P. agathidicida on those communities., (© 2024. The Author(s).)

Phytophthora cactorum is the most common causal agent of Phytophthora crown rot and leather rot of strawberry, but P. nicotianae is also responsible for the disease in Florida. Studies of P. nicotianae populations have suggested that different groups of genotypes are associated with different hosts; however, it is not yet clear how many lineages exist globally and how they are related to different production systems. The aim of this study was to determine the genetic relationships of P. nicotianae isolates from Florida strawberry with genotypes reported from other hosts, quantify the genetic variation on strawberry, and test for an association with nursery source. A total of 49 isolates of P. nicotianae were collected from strawberry plants originating from multiple nursery sources during six seasons of commercial fruit production in Florida. Microsatellite genotyping identified 28 multilocus genotypes on strawberry that were distinct among 208 isolates originating from various hosts and locations. Based on STRUCTURE analysis, two genetic groups were identified: one consisting of isolates from strawberry, and the other comprising samples from different hosts. Multilocus genotypes were shared among nursery sources, and populations defined by nursery were not differentiated. Both mating types were found among the isolates from North Carolina- and California-origin plants and in most strawberry seasons; however, a predominance of A1 was observed, and regular sexual reproduction was not supported by the data. This study reveals a unique genetic population of P. nicotianae associated with strawberry and emphasizes the vital role of nursery monitoring in mitigating disease spread., Competing Interests: The author(s) declare no conflict of interest.

Phytophthora parasitica is a highly destructive oomycete plant pathogen that is capable of infecting a wide range of hosts including many agricultural cash crops, fruit trees, and ornamental garden plants. One of the most important diseases caused by P. parasitica worldwide is black shank of tobacco. Rapid, sensitive, and specific pathogen detection is crucial for early rapid diagnosis, which can facilitate effective disease management. In this study, we used a genomics approach to identify repeated sequences in the genome of P. parasitica by genome sequence alignment and identified a 203-bp P. parasitica -specific sequence, PpM34, that is present in 31 to 60 copies in the genome. The P. parasitica genome specificity of PpM34 was supported by PCR amplification of 24 genetically diverse strains of P. parasitica , 32 strains representing 12 other Phytophthora species, one Pythium species, six fungal species, and three bacterial species, all of which are plant pathogens. Our PCR and real-time PCR assays showed that the PpM34 sequence was highly sensitive in specifically detecting P. parasitica . Finally, we developed a PpM34-based high-efficiency recombinase polymerase amplification assay, which allowed us to specifically detect as little as 1 pg of P. parasitica total DNA from both pure cultures and infected Nicotiana benthamiana at 39°C using a fluorometric thermal cycler. The sensitivity, specificity, convenience, and rapidity of this assay represent a major improvement for early diagnosis of P. parasitica infection., Competing Interests: The author(s) declare no conflict of interest.

Phytophthora citrophthora and P. syringae are currently the primary causal organisms of brown rot of citrus fruits in California. To possibly find an explanation for the prevalence of the previously minor species P. syringae , we determined the population structures of both pathogens in California using next-generation sequencing and population genomics analyses. Whole-genome sequencing and aligning with newly assembled reference genomes identified 972,266 variants in 132 isolates of P. citrophthora and 422,208 variants in 154 isolates (including 24 from noncitrus tree crops) of P. syringae originating from three major growing regions. The resulting data sets were visualized using principal component analysis, discriminant analysis of principal components, unweighted pair-group method with arithmetic mean dendrograms, fastStructure, and minimum spanning networks, and we obtained the index of association, diversity summary statistics, and genetic distance statistics values G ST , G '' ST , and Jost's D. Subpopulations of both species were mostly defined by their geographic origin indicating restricted dispersal of inoculum. Except for five isolates, the population structure of P. citrophthora (that is heterothallic and unlikely to reproduce sexually) was clonal to semi-clonal, with very little genetic diversity within and among subgroups. In contrast, the population structure of P. syringae was also clonal to semi-clonal, but isolates were placed into four main clusters of much higher diversity. Clonality in both species can be explained by a high level of asexual reproduction. The higher diversity in the homothallic P. syringae is likely due to commonly occurring sexual reproduction. One distinct cluster of P. syringae consisted solely of isolates from noncitrus hosts; therefore, the origin of P. syringae in citrus could not be resolved., Competing Interests: The author(s) declare no conflict of interest.

Chinese hickory (Carya cathayensis Sarg.) is an economically and ecologically important nut plant in China. Dieback and basal stem necrosis have been observed in the plants since 2016, and its recent spread has significantly affected plant growth and nut production. Therefore, a survey was conducted to evaluate the disease incidence at five sites in Linan County, China. The highest incidence was recorded at the Tuankou site at up to 11.39% in 2019. The oomycete, Phytophthora cinnamomi, was isolated from symptomatic plant tissue and plantation soil using baiting and selective media-based detection methods and identified. Artificial infection with the representative P. cinnamomi ST402 isolate produced vertically elongated discolorations in the outer xylem and necrotic symptoms in C. cathayensis seedlings in a greenhouse trial. Molecular detections based on loop-mediated isothermal amplification (LAMP) specific to P. cinnamomi ST402 were conducted. Result indicated that LAMP detection showed a high coherence level with the baiting assays for P. cinnamomi detection in the field. This study provides the evidence of existence of high-pathogenic P. cinnamomi in the C. cathayensis plantation soil in China and the insights into a convenient tool developed for conducting field monitoring of this aggressive pathogen., Competing Interests: The authors have declared that no competing interests exist.

Phytophthora root and stem rot caused by P. sojae is a destructive soybean soil-borne disease found worldwide. Discovery of genes conferring broad-spectrum resistance to the pathogen is a need to prevent the outbreak of the disease. Here, we show that soybean Rps11 is a 27.7-kb nucleotide-binding site-leucine-rich repeat (NBS-LRR or NLR) gene conferring broad-spectrum resistance to the pathogen. Rps11 is located in a genomic region harboring a cluster of large NLR genes of a single origin in soybean, and is derived from rounds of unequal recombination. Such events result in promoter fusion and LRR expansion that may contribute to the broad resistance spectrum. The NLR gene cluster exhibits drastic structural diversification among phylogenetically representative varieties, including gene copy number variation ranging from five to 23 copies, and absence of allelic copies of Rps11 in any of the non-Rps11-donor varieties examined, exemplifying innovative evolution of NLR genes and NLR gene clusters., (© 2021. The Author(s).)

The Phytophtora root and stem rot is a serious disease in soybean. It is caused by the oomycete pathogen Phytophthora sojae. Growing Phytophthora resistant cultivars is the major method of controlling this disease. Resistance is race- or gene-specific; a single gene confers immunity against only a subset of the P. sojae isolates. Unfortunately, rapid evolution of new Phytophthora sojae virulent pathotypes limits the effectiveness of an Rps ("resistance to Phytophthora sojae") gene to 8-15 years. The current study was designed to investigate the effectiveness of Rps12 against a set of P. sojae isolates using recombinant inbred lines (RILs) that contain recombination break points in the Rps12 region. Our study revealed a unique Rps gene linked to the Rps12 locus. We named this novel gene as Rps13 that confers resistance against P. sojae isolate V13, which is virulent to recombinants that contains Rps12 but lack Rps13. The genetic distance between the two Rps genes is 4 cM. Our study revealed that two tightly linked functional Rps genes with distinct race-specificity provide broad-spectrum resistance in soybean. We report here the molecular markers for incorporating the broad-spectrum Phytophthora resistance conferred by the two Rps genes in commercial soybean cultivars., (© 2021. The Author(s).)

Slow growing oomycete isolates with morphological resemblance to Phytophthora were obtained from forest streams during routine monitoring for the EU quarantine forest pathogen Phytophthora ramorum in Ireland and Northern Ireland. Internal Transcribed Spacer (ITS) sequence analysis indicated that they belonged to two previously unknown species of Nothophytophthora, a recently erected sister genus of Phytophthora. Morphological and temperature-growth studies were carried out to characterise both new species. In addition, Bayesian and Maximum-Likelihood analyses of nuclear 5-loci and mitochondrial 3-loci datasets were performed to resolve the phylogenetic positions of the two new species. Both species were sterile, formed chlamydospores and partially caducous nonpapillate sporangia, and showed slower growth than any of the six known Nothophytophthora species. In all phylogenetic analyses both species formed distinct, strongly supported clades, closely related to N. chlamydospora and N. valdiviana from Chile. Based on their unique combination of morphological and physiological characters and their distinct phylogenetic positions the two new species are described as Nothophytophthora irlandica sp. nov. and N. lirii sp. nov. Their potential lifestyle and geographic origin are discussed., Competing Interests: The authors have declared that no competing interests exist.

Phytophthora nicotianae is highly pathogenic to Solanaceous crops and is a major problem in tobacco production. The tobacco cultivar Beihart1000-1 (BH) is resistant, whereas the Xiaohuangjin 1025 (XHJ) cultivar is susceptible to infection. Here, BH and XHJ were used as models to identify resistant and susceptible genes using RNA sequencing (RNA-seq). Roots were sampled at 0, 6, 12, 24, and 60 h post infection. In total, 23,753 and 25,187 differentially expressed genes (DEGs) were identified in BH and XHJ, respectively. By mapping upregulated DEGs to the KEGG database, changes of the rich factor of "plant pathogen interaction pathway" were corresponded to the infection process. Of all the DEGs in this pathway, 38 were specifically regulated in BH. These genes included 11 disease-resistance proteins, 3 pathogenesis-related proteins, 4 RLP/RLKs, 2 CNGCs, 7 calcium-dependent protein kinases, 4 calcium-binding proteins, 1 mitogen-activated protein kinase kinase, 1 protein EDS1L, 2 WRKY transcription factors, 1 mannosyltransferase, and 1 calmodulin-like protein. By combining the analysis of reported susceptible (S) gene homologs and DEGs in XHJ, 9 S gene homologs were identified, which included 1 calmodulin-binding transcription activator, 1 cyclic nucleotide-gated ion channel, 1 protein trichome birefringence-like protein, 1 plant UBX domain-containing protein, 1 ADP-ribosylation factor GTPase-activating protein, 2 callose synthases, and 2 cellulose synthase A catalytic subunits. qRT-PCR was used to validate the RNA-seq data. The comprehensive transcriptome dataset described here, including candidate resistant and susceptible genes, will provide a valuable resource for breeding tobacco plants resistant to P. nicotianae infections.

Phytophthora species cause destructive plant diseases worldwide. All Phytophthora species, except for one, are listed as plant quarantine organisms in Japan. The exception, Phytophthora nicotianae is considered to be a domestic species. The injurious pests Phytophthora ramorum, Phytophthora lateralis, and Phytophthora kernoviae are invasive pathogens that cause tree mortality worldwide, mainly in the United States and the United Kingdom. To effectively control Phytophthora diseases, we established detection methods that utilize the loop-mediated isothermal amplification (LAMP) of the genus Phytophthora and the four species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae. LAMP primers for P. ramorum, P. lateralis, and P. kernoviae were newly designed in the present study. Our multiplex assay includes the detection of plant DNA as an internal control. When the optimum ratio between plant and pathogen primers was used in multiplex LAMP assays, 1 pg to 100 fg of pathogen DNA was detected with similar sensitivity to that in simplex LAMP assays. The detection of plant DNA in the absence of pathogens enables us to check for and avoid undesirable negative results caused by enzyme inactivation or the contamination of amplification inhibitors from plant tissues. The total time from sample collection to results is approximately 120‍ ‍min, and, thus, our multiplex LAMP assay may be used as an accurate and time-saving detection method for Phytophthora pathogens.

Roots act as a biological filter that exclusively allows only a portion of the soil-associated microbial diversity to infect the plant. This microbial diversity includes organisms both beneficial and detrimental to plants. Phytophthora species are among the most important groups of detrimental microbes that cause various soil-borne plant diseases. We used a metabarcoding approach with Phytophthora-specific primers to compare the diversity and richness of Phytophthora species associated with roots of native and non-native trees, using different types of soil inocula collected from native and managed forests. Specifically, we analysed (1) roots of two non-native tree species (Eucalyptus grandis and Acacia mearnsii) and native trees, (2) roots of two non-native tree species from an in vivo plant baiting trial, (3) roots collected from the field versus those from the baiting trial, and (4) roots and soil samples collected from the field. The origin of the soil and the interaction between root and soil significantly influenced Phytophthora species richness. Moreover, species richness and community composition were significantly different between the field root samples and field soil samples with a higher number of Phytophthora species in the soil than in the roots. The results also revealed a substantial and previously undetected diversity of Phytophthora species from South Africa.

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., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

DNA extraction can be lengthy and sometimes ends up with amplification inhibitors. We present the potential of recombinase polymerase amplification (RPA) to replace plant DNA extraction. In our rapid 'RPA-PCR couple' concept, RPA is tuned to slower reaction kinetics to promote amplification of long targets. RPA primers amplify target and some flanking regions directly from simple plant macerates. Then PCR primers exponentially amplify the target directly from the RPA reaction. We present the coupling of RPA with conventional, TaqMan and SYBR Green PCR assays. We applied the concept to strawberry Phytophthora pathogens and the Phytophthora identification marker atp9-nad9 . We found RPA-PCR couple specific, sensitive and reliable. The approach may also benefit other difficult samples such as food, feces and ancient samples.

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 µm) method that captures the pathogen's spores (zoospores) and is later used to amplify the pathogen's DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 10 2 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Root and collar rot disease caused by Phytophthora capsici (Leonian) is one of the most serious diseases in pepper, Capsicum annuum L. Knowledge about resistant genes is limited in pepper accessions to P. capsici. In this study, a diverse collection of 37 commercial edible and ornamental genotypes, and implication of seven novel candidate DEGs genes (XLOC_ 021757, XLOC_021821, XLOC_012788, XLOC_011295, XLOC_021928, XLOC_015473 and XLOC_000341) were up-regulated on resistant and susceptible pepper cultivars, through real-time polymerase chain reaction (qPCR) at transplanting and maturing stages. All seven related defense-gene candidates were up-regulated in all inoculated accessions to P. capsici, but these genes were highly expressed in resistant ones, 19OrnP-PBI, 37ChillP-Paleo, and 23CherryP-Orsh. The transcriptional levels of the seven related candidate DEGs were 5.90, 5.64, 5.62, 5.18, 3.94, 3.69, 3.16 folds higher in the resistant pepper genotypes, than the control ones, non-inoculated genotypes respectively. The candidate genes expressed herein, will provide a basis for further gene cloning and functional verification studies, and also will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici., Competing Interests: Declaration of Competing Interest There is no conflict of interest relating to this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Wood and wood products can harbor microorganisms that can raise phytosanitary concerns in countries importing or exporting these products. To evaluate the efficacy of wood treatment on the survival of microorganisms of phytosanitary concern the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can become rapidly unstable after cell death, providing a proxy measure of viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of mycelial cultures of the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and offers a potential way to assess the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade., Competing Interests: The authors have declared that no competing interests exist.

The root rot causing oomycete, Phytophthora agathidicida, threatens the long-term survival of the iconic New Zealand kauri. Currently, testing for this pathogen involves an extended soil bioassay that takes 14-20 days and requires specialised staff, consumables, and infrastructure. Here we describe a loop-mediated isothermal amplification (LAMP) assay for the detection of P. agathidicida that targets a portion of the mitochondrial apocytochrome b coding sequence. This assay has high specificity and sensitivity; it did not cross react with a range of other Phytophthora isolates and detected as little as 1 fg of total P. agathidicida DNA or 116 copies of the target locus. Assay performance was further investigated by testing plant tissue baits from flooded soil samples using both the extended soil bioassay and LAMP testing of DNA extracted from baits. In these comparisons, P. agathidicida was detected more frequently using the LAMP test. In addition to greater sensitivity, by removing the need for culturing, the hybrid baiting plus LAMP approach is more cost effective than the extended soil bioassay and, importantly, does not require a centralised laboratory facility with specialised staff, consumables, and equipment. Such testing will allow us to address outstanding questions about P. agathidicida. For example, the hybrid approach could enable monitoring of the pathogen beyond areas with visible disease symptoms, allow direct evaluation of rates and patterns of spread, and allow the effectiveness of disease control to be evaluated. The hybrid LAMP bioassay also has the potential to empower local communities to evaluate the pathogen status of local kauri stands, providing information for disease management and conservation initiatives., Competing Interests: The authors have declared that no competing interests exist.

Oomycete plant pathogens are difficult to control and routine genetic research is challenging. A major problem is instability of isolates. Here we characterize >600 field and single zoospore isolates of Phytophthora capsici for inheritance of mating type, sensitivity to mefenoxam, chromosome copy number and heterozygous allele frequencies. The A2 mating type was highly unstable with 26% of 241 A2 isolates remaining A2. The A1 mating type was stable. Isolates intermediately resistant to mefenoxam produced fully resistant single-spore progeny. Sensitive isolates remained fully sensitive. Genome re-sequencing of single zoospore isolates revealed extreme aneuploidy; a phenomenon dubbed Dynamic Extreme Aneuploidy (DEA). DEA is characterized by the asexual inheritance of diverse intra-genomic combinations of chromosomal ploidy ranging from 2N to 3N and heterozygous allele frequencies that do not strictly correspond to ploidy. Isolates sectoring on agar media showed dramatically altered heterozygous allele frequencies. DEA can explain the rapid increase of advantageous alleles (e.g. drug resistance), mating type switches and copy neutral loss of heterozygosity (LOH). Although the mechanisms driving DEA are unknown, it can play an important role in adaptation and evolution and seriously hinders all aspects of P. capsici research., Competing Interests: The authors have declared that no competing interests exist.

Aims: This study aimed to develop a random amplified polymorphic DNA (RAPD)-based sequence characterized amplified region (SCAR) marker for species-specific detection of Phytophthora nicotianae, a global plant pathogen. Another objective was to develop a multiplex PCR assay for simultaneous detection of P. nicotianae and huanglongbing-causing bacterium, Candidatus Liberibacter asiaticus (CaLas) in citrus roots using the developed SCAR marker and a previously published 16SrDNA-based CaLas-specific primer set., Methods and Results: The RAPD primer, OPA4, amplified a specific fragment of c. 400 bp only in P. nicotianae isolates. The fragment was eluted, purified, cloned and sequenced. One set of SCAR primers (SCAR4F/SCAR4R1), developed from the sequence information of the fragment, was found specific to P. nicotianae and produced an amplicon of 330 bp size, and was found non-specific to the five Phytophthora species (P. citrophthora, P. palmivora, P. lacustris, P. boehmeriae and P. insolita) and five other pathogens (Mycosphaerella citri, Alternaria alternata, Septobasidium pseudopedicillatum, Phytopythium vexans and Colletotrichum gloeosporioides) isolated from the citrus agroecosystem. The sensitivity of the primer pair was 5 pg µl -1 of mycelial DNA. Furthermore, the specific SCAR primers coupled with a previously reported CaLas-specific primer set were used effectively in developing a multiplex PCR assay to detect P. nicotianae and CaLas simultaneously in root tissues of citrus plants., Conclusions: A rapid method using a RAPD-based SCAR marker for the detection of P. nicotianae was developed. Furthermore, a multiplex PCR assay was established for simultaneous detection of P. nicotianae and CaLas in citrus roots., Significance and Impact of the Study: A RAPD-SCAR marker-based detection system and the one-step multiplex PCR method developed in this study can be applied to index citrus trees infected (individually or conjointly) with P. nicotianae and CaLas. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance., (© 2019 The Society for Applied Microbiology.)

Phytophthora cinnamomi Rands is a devastating root rot pathogen of avocado. Robust and sensitive root quantification methods are required for determining seasonal P. cinnamomi root colonization patterns and evaluating management strategies. Our study investigated four P. cinnamomi root quantification methods using a newly developed P. cinnamomi-avocado-seedling bioassay system and a P. cinnamomi-specific probe-based qPCR assay. Phytophthora cinnamomi quantification through plating of roots (root plating) or lemon leaf disks obtained from root baitings (root-baiting-plating) onto semi-selective media were the best methods. Root plating consistently yielded significant differences in P. cinnamomi quantities obtained from seedling roots inoculated with five zoospore concentrations (10-1 × 105 zoospores/ml), whereas root-baiting-plating did so less often. The two methods were comparable in yielding root quantities that were significantly correlated with the inoculated zoospore concentrations, rarely yielding false negatives and having the lowest variability between replicates of the same treatment. qPCR quantification from roots was also an effective method; however, treatment replicates were highly variable and false negatives occurred more frequently. The least effective quantification method was qPCR quantification from lemon leaf disks obtained from root baitings., (© FEMS 2019.)

Phytophthora capsici is an important oomycete pathogen that causes devastating diseases in various crops. Methods for the rapid detection of P. capsici are important for disease control and eradication programmes. Here, we developed an assay based on lateral flow strip-based recombinase polymerase amplification (LF-RPA) for the rapid and equipment-free detection of P. capsici. The specific primers and a probe were designed using the sequence of Ypt1, and the optimal assay conditions were 40°C for 20 min. The specificity of the assay was verified using closely related oomycetes and fungal species, and its detection limit was 10 pg of genomic DNA. In combination with a simple DNA extraction method, the LF-RPA assay enabled detection of P. capsici in diseased pepper samples without specialized equipment within 30 min. Consequently, the LF-RPA assay developed in this study enables rapid and equipment-free detection of P. capsici and has potential for further development as a diagnostic kit for application in the field or in resource-limited laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a novel assay based on lateral flow strip-based recombinase polymerase amplification (LF-RPA) for the rapid and equipment-free detection of Phytophthora capsici. In combination with a simple DNA extraction method, the LF-RPA assay detected P. capsici in field samples without specialized equipment within 30 min. The assay has potential for further development as a diagnostic kit for application in the field or in resource-limited laboratories., (© 2019 The Society for Applied Microbiology.)

Phytophthora root rot (PRR) causes serious annual soybean yield losses worldwide. The most effective method to prevent PRR involves growing cultivars that possess genes conferring resistance to Phytophthora sojae (Rps). In this study, QTL-sequencing combined with genetic mapping was used to identify RpsX in soybean cultivar Xiu94-11 resistance to all P. sojae isolates tested, exhibiting broad-spectrum PRR resistance. Subsequent analysis revealed RpsX was located in the 242-kb genomic region spanning the RpsQ locus. However, a phylogenetic investigation indicated Xiu94-11 carrying RpsX is distantly related to the cultivars containing RpsQ , implying RpsX and RpsQ have different origins. An examination of candidate genes revealed RpsX and RpsQ share common nonsynonymous SNP and a 144-bp insertion in the Glyma.03g027200 sequence encoding a leucine-rich repeat (LRR) region. Glyma.03g027200 was considered to be the likely candidate gene of RpsQ and RpsX . Sequence analyses confirmed that the 144-bp insertion caused by an unequal exchange resulted in two additional LRR-encoding fragments in the candidate gene. A marker developed based on the 144-bp insertion was used to analyze the genetic population and germplasm, and proved to be useful for identifying the RpsX and RpsQ alleles. This study implies that the number of LRR units in the LRR domain may be important for PRR resistance in soybean.

Key Message: Major QTL for Phytophthora root rot resistance have been identified in three mapping populations with independent sources of resistance contributed by C. echinospermum and C. arietinum. Phytophthora root rot (PRR) caused by the oomycete Phytophthora medicaginis is a major soil-borne disease of chickpea in Australia. With no economic in-crop control of PRR, a genetic approach to improve resistance is the most practical management option. Moderate field resistance has been incorporated in the cultivated C. arietinum variety, Yorker, and a higher level of resistance has been identified in a derivative of wild chickpea (C. echinospermum, interspecific breeding line 04067-81-2-1-1). These genotypes and two other released varieties were used to develop one intra-specific and two interspecific F 6 -derived recombinant inbred line mapping populations for genetic analysis of resistance. The Yorker × Genesis114 (YG), Rupali × 04067-81-2-1-1 (RB) and Yorker × 04067-81-2-1-1 (YB) populations were genotyped using genotyping-by-sequencing and phenotyped for PRR under three field environments with a mixture of 10 P. medicaginis isolates. Whole-genome QTL analysis identified major QTL QRBprrsi01, QYBprrsi01, QRBprrsi03 and QYBprrsi02 for PRR resistance on chromosomes 3 and 6, in RB and YB populations, respectively, with the resistance source derived from the wild Cicer species. QTL QYGprrsi02 and QYGprrsi03 were also identified on chromosomes 5 and 6 in YG population from C. arietinum. Aligning QTL regions to the corresponding chickpea reference genome suggested that the resistance source from C. arietinum and C. echinospermum may be different. The findings from this study provide tools for marker-assisted selection in chickpea breeding and information to assist the development of populations suitable for fine-mapping of resistance loci to determine the molecular basis for PRR resistance in chickpea.

Bud rot disease is a damaging disease of oil palm in Colombia. The pathogen responsible for this disease is a species of oomyctes, Phytophthora palmivora which is also the causal pathogen of several tropical crop diseases such as fruit rot and stem canker of cocoa, rubber, durian and jackfruit. No outbreaks of bud rot have been reported in oil palm in Malaysia or other Southeast Asian countries, despite this particular species being present in the region. Analysis of the genomic sequences of several genetic markers; the internal transcribe spacer regions (ITS) of the ribosomal RNA gene cluster, beta-tubulin gene, translation elongation factor 1 alpha gene (EF-1α), cytochrome c oxidase subunit I & II (COXI and COXII) gene cluster along with amplified fragment length polymorphism (AFLP) analyses have been carried out to investigate the genetic diversity and variation of P. palmivora isolates from around the world and from different hosts in comparison to Colombian oil palm isolates, as one of the steps in understanding why this species of oomycetes causes devastating damage to oil palm in Latin America but not in other regions. Phylogenetic analyses of these regions showed that the Colombian oil palm isolates were not separated from Malaysian isolates. AFLP analysis and a new marker PPHPAV, targeting an unclassified hypothetical protein, was found to be able to differentiate Malaysian and Colombian isolates and showed a clear clade separations. Despite this, pathogenicity studies did not show any significant differences in the level of aggressiveness of different isolates against oil palm in glasshouse tests.

Plant deaths had been observed in the sub-alpine and alpine areas of Australia. Although no detailed aetiology was established, patches of dying vegetation and progressive thinning of canopy suggested the involvement of root pathogens. Baiting of roots and associated rhizosphere soil from surveys conducted in mountainous regions New South Wales and Tasmania resulted in the isolation of eight Phytophthora species; Phytophthora cactorum, Phytophthora cryptogea, Phytophthora fallax, Phytophthora gonapodyides, Phytophthora gregata, Phytophthora pseudocryptogea, and two new species, Phytophthora cacuminis sp. nov and Phytophthora oreophila sp. nov, described here. P. cacuminis sp. nov is closely related to P. fallax, and was isolated from asymptomatic Eucalyptus coccifera and species from the family Proteaceae in Mount Field NP in Tasmania. P. oreophila sp. nov, was isolated from a disturbed alpine herbfield in Kosciuzsko National Park. The low cardinal temperature for growth of the new species suggest they are well adapted to survive under these conditions, and should be regarded as potential threats to the diverse flora of sub-alpine/alpine ecosystems. P. gregata and P. cryptogea have already been implicated in poor plant health. Tests on a range of alpine/subalpine plant species are now needed to determine their pathogenicity, host range and invasive potential., (Copyright © 2018 British Mycological Society. All rights reserved.)

Anthropogenic activities have a major impact on the global environment. Canada's natural resources are threatened by the spread of fungal pathogens, which is facilitated by agricultural practices and international trade. Fungi are introduced to new environments and sometimes become established, in which case they can cause disease outbreaks resulting in extensive forest decline. Here, we describe how a nationwide sample collection strategy coupled to next-generation sequencing (NGS) (i.e., metagenomics) can achieve fast and comprehensive screening for exotic invasive species. This methodology can help provide guidance to phytopathology stakeholders such as regulatory agencies. Several regulated invasive species were monitored by processing field samples collected over 3 years (2013 to 2015) near high-risk areas across Canada. Fifteen sequencing runs were required on the Ion Torrent platform to process 398 samples that yielded 45 million reads. High-throughput screening of fungal and oomycete operational taxonomic units using customized fungi-specific ribosomal internal transcribed spacer 1 barcoded primers was performed. Likewise, Phytophthora-specific barcoded primers were used to amplify the adenosine triphosphate synthase subunit 9-nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer. Several Phytophthora spp. were detected by NGS and confirmed by species-specific quantitative polymerase chain reaction (qPCR) assays. The target species Heterobasidion annosum sensu stricto could be detected only through metagenomics. We demonstrated that screening target species using a variety of sampling techniques and NGS-the results of which were validated by qPCR-has the potential to increase survey capacity and detection sensitivity, reduce hands-on time and costs, and assist regulatory agencies to identify ports of entry. Considering that early detection and prevention are the keys in mitigating invasive species damage, our method represents a substantial asset in plant pathology management.

45 bacterial isolates from potatoes and wheat rhizospheres near Sétif (Algeria) pre-selected for their antagonistic activity against three fungal plant pathogens, two necrotrophic Fusarium solani var. coeruleum and Phytophtora infestans, and a systemic F. oxysporum f. sp. albedinis. Molecular typing of the isolates showed abundance of Bacillus compared to Pseudomonas. Some of the tested strains have shown very high biofilm formation. Among the 24 Gram-positive bacilli screened for four cyclic lipopeptides genes, some isolates harbor two or more genes, while others have a single gene or have none. Four selected isolates were able to regulate the expression of six defense-related genes in Arabidopsis and produce salicylic acid. Upon the features assessed in this study, strain B. amyloliquefaciens A16 was selected for a subsequent use as seed treatment and biocontrol agent in semi-arid region fields. This strain showed important biofilm formation, regulation of Arabidopsis defenses, and harbored three cLPs genes.

Phytophthora capsici is a highly destructive plant pathogen that has spread worldwide. To date, the quinone outside inhibitor (QoI) azoxystrobin has been the choice of farmers for managing this oomycete. In this study, the sensitivity of 90 P. capsici isolates collected from Yunnan, Fujian, Jiangxi, Zhejiang, and Guangdong in southern China to azoxystrobin was assessed based on mycelial growth, sporangia formation, and zoospore discharge. Furthermore, the mitochondrial cytochrome b (cytb) gene from azoxystrobin-sensitive and -resistant P. capsici isolates was compared to investigate the mechanism of QoI resistance. The high values for effective concentration to inhibit 50% of mycelial growth and large variation factor obtained provide strong support for the existence of azoxystrobin-resistant subpopulations in wild populations. The resistance frequency of P. capsici to azoxystrobin was greater than 40%. Sensitive P. capsici isolates were strongly suppressed on V8 medium plates containing azoxystrobin supplemented with salicylhydroxamic acid at 50 µg ml -1 , whereas resistant isolates grew well under these conditions. Multiple alignment analysis revealed a missense mutation in the cytb gene that alters codon 137 (GGA to AGA), causing an amino acid substitution of glycine to arginine (G137R). The fitness of the azoxystrobin-sensitive isolate is similar to that of the G137R mutant. Additionally, the P. capsici isolates used in this study exhibited decreased sensitivity to two other QoI fungicides (pyraclostrobin and famoxadone). Necessary measures should be taken to control this trend of resistance to QoI that has developed in P. capsici in southern China.

Chili pepper (Capsicum annum L.) is an important economic crop that is severely destroyed by the filamentous oomycete Phytophthora capsici. Little is known about this pathogen in key chili pepper farms in Punjab province, Pakistan. We investigated the genetic diversity of P. capsici strains using standard taxonomic and molecular tools, and characterized their colony growth patterns as well as their disease severity on chili pepper plants under the greenhouse conditions. Phylogenetic analysis based on ribosomal DNA (rDNA), β-tubulin and translation elongation factor 1α loci revealed divergent evolution in the population structure of P. capsici isolates. The mean oospore diameter of mating type A1 isolates was greater than that of mating type A2 isolates. We provide first evidence of an uneven distribution of highly virulent mating type A1 and A2 of P. capsici that are insensitive to mefenoxam, pyrimorph, dimethomorph, and azoxystrobin fungicides, and represent a risk factor that could ease outpacing the current P. capsici management strategies.

Population genetics is a powerful tool to understand patterns and evolutionary processes that are involved in plant-pathogen emergence and adaptation to agricultural ecosystems. We are interested in studying the population dynamics of Phytophthora rubi, the causal agent of Phytophthora root rot in raspberry. P. rubi is found in the western United States, where most of the fresh and processed raspberries are produced. We used genotyping-by-sequencing to characterize genetic diversity in populations of P. rubi sampled in the United States and other countries. Our results confirm that P. rubi is a monophyletic species with complete lineage sorting from its sister taxon P. fragariae. Overall, populations of P. rubi show low genetic diversity across the western United States. Demographic analyses suggest that populations of P. rubi from the western United States are the source of pathogen migration to Europe. We found no evidence for population differentiation at a global or regional (western United States) level. Finally, our results provide evidence of migration from California and Oregon into Washington. This report provides new insights into the evolution and structure of global and western United States populations of the raspberry pathogen P. rubi, indicating that human activity might be involved in moving the pathogen among regions and fields.

Proper isolation and identification of Phytophthora species is critical due to their broad distribution and huge impact on natural ecosystems throughout the world. In this study, five different sites were sampled and seven methods were compared to determine the Phytophthora community. Three traditional isolation methods were conducted (i) soil baiting, (ii) filtering of the bait water and (iii) isolation from field roots using Granny Smith apples. These were compared to four sources of eDNA used for metabarcoding using Phytophthora-specific primers on (i) sieved field soil, (ii) roots from field, (iii) filtered baiting water and (iv) roots from bait plants grown in the glasshouse in soil collected from these sites. Six Phytophthora species each were recovered by soil baiting using bait leaves and from the filtered bait water. No Phytophthora species were recovered from Granny Smith apples. eDNA extracted from field roots detected the highest number of Phytophthora species (25). These were followed by direct DNA isolation from filters (24), isolation from roots from bait plants grown in the glasshouse (19), and DNA extraction from field soil (13). Therefore, roots were determined to be the best substrate for detecting Phytophthora communities using eDNA.

Oil palm (Elaeis guineensis Jacq.) is an excellent source of vegetable oil for biodiesel production; however, there are still some limitations for its cultivation in Brazil such as Fatal Yellowing (FY) disease. FY has been studied for many years, but its causal agent has never been determined. In Colombia and nearby countries, it was reported that the causal agent of Fatal Yellowing (Pudrición del Cogollo) is the oomycete Phytophthora palmivora, however, several authors claim that Fatal Yellowing and Pudrición del Cogollo (PC) are different diseases. The major aims of this work were to test, using molecular biology tools, Brazilian oil palm trees for the co-occurrence of the oomycete Phytophthora and FY symptoms, and to characterize the fungal diversity in FY diseased and healthy leaves by next generation sequencing. Investigation with specific primers for the genus Phytophthora showed amplification in only one of the samples. Analysis of the fungal ITS region demonstrated that, at the genus level, different groups predominated in all symptomatic samples, while Pyrenochaetopsis and unclassified fungi predominated in all asymptomatic samples. Our results show that fungal communities were not the same between samples at the same stage of the disease or among all the symptomatic samples. This is the first study that describes the evolution of the microbial community in the course of plant disease and also the first work to use high throughput next generation sequencing to evaluate the fungal community associated with leaves of oil palm trees with and without symptoms of FY.

Phytophthora cinnamomi is the causal agent of root rot, canker and dieback of thousands of plant species around the globe. This oomycete not only causes severe economic losses but also threatens natural ecosystems. In South Africa, P. cinnamomi affects eucalyptus, avocado, macadamia and indigenous fynbos. Despite being one of the most important plant pathogens with a global distribution, little information is available regarding origin, invasion history and population biology. This is partly due to the limited number of molecular markers available for studying P. cinnamomi. Using available genome sequences for three isolates of P. cinnamomi, sixteen polymorphic microsatellite markers were developed as a set of multiplexable markers for both PCR and Gene Scan assays. The application of these markers on P. cinnamomi populations from avocado production areas in South Africa revealed that they were all polymorphic in these populations. The markers developed in this study represent a valuable resource for studying the population biology and movement of P. cinnamomi and will aid in the understanding of the origin and invasion history of this important species.

Phytophthora austrocedri is causing widespread mortality of Austrocedrus chilensis in Argentina and Juniperus communis in Britain. The pathogen has also been isolated from J. horizontalis in Germany. Isolates from Britain, Argentina, and Germany are homothallic, with no clear differences in the dimensions of sporangia, oogonia, or oospores. Argentinian and German isolates grew faster than British isolates across a range of media and had a higher temperature tolerance, although most isolates, regardless of origin, grew best at 15°C and all isolates were killed at 25°C. Argentinian and British isolates caused lesions when inoculated onto both A. chilensis and J. communis; however, the Argentinian isolate caused longer lesions on A. chilensis than on J. communis and vice versa for the British isolate. Genetic analyses of nuclear and mitochondrial loci showed that all British isolates are identical. Argentinian isolates and the German isolate are also identical but differ from the British isolates. Single-nucleotide polymorphisms are shared between the British and Argentinian isolates. We concluded that British isolates and Argentinian isolates conform to two distinct clonal lineages of P. austrocedri founded from the same as-yet-unidentified source population. These lineages should be recognized and treated as separate risks by international plant health legislation.

A new species of the genus Phytophthora was isolated from stream water in the subtropical forests of China during a survey of forest Phytophthora from 2011 to 2013. This new species is formally described here and named Phytophthora pseudopolonica sp. nov. This new homothallic species is distinct from other known Phytophthora species in morphology and produces nonpapillate and noncaducous sporangia with internal proliferation. Spherical hyphal swellings and thin-walled chlamydospores are abundant when the species is kept in sterile water. The P. pseudopolonica sp. nov. forms smooth oogonia with paragynous and sometimes amphigynous antheridia. The optimum growth temperature of the species is 30 °C in V8-juice agar with β-sitosterol, yet it barely grows at 5 °C and 35 °C. Based on sequences of the internal transcribed spacer and the combined β-tubulin and elongation factor 1α gene sequence data, isolates of the new species cluster together into a single branch and are close to Phytophthora polonicabelonging to clade 9.

The reactions of isolates of Phytophthora cactorum, P. nicotianae and P. × pelgrandis to metalaxyl, mancozeb, dimethomorph, streptomycin and chloramphenicol were tested to obtain information about the variability of resistance in these pathogens. Distinct genetic groups showed significant differences in resistance to all tested substances except streptomycin. In response to streptomycin, the growth inhibition rates of distinct groups did not differ significantly. The most remarkable differences were detected in the reactions to chloramphenicol and metalaxyl. Discriminant analysis evaluating the effect of all substances confirmed the differences among the groups, which are in agreement with the differences revealed by earlier DNA analyses.

Genetic diversity of two Phytophthora spp.-P. cinnamomi (102 isolates), commonly encountered in Maryland nurseries and forests in the Mid-Atlantic United States, and P. plurivora (186 isolates), a species common in nurseries-was characterized using amplified fragment length polymorphism. Expected heterozygosity and other indices suggested a lower level of diversity among P. cinnamomi than P. plurivora isolates. Hierarchical clustering showed P. cinnamomi isolates separated into four clusters, and two of the largest clusters were closely related, containing 80% of the isolates. In contrast, P. plurivora isolates separated into six clusters, one of which included approximately 40% of the isolates. P. plurivora isolates recovered from the environment (e.g., soil and water) were genotypically more diverse than those found causing lesions. For both species, isolate origin (forest versus nursery or among nurseries) was a significant factor of heterozygosity. Clonal groups existed within P. cinnamomi and P. plurivora and included isolates from both forest and nurseries, suggesting that a pathway from nurseries to forests or vice versa exists.

Phytophthora root rot of soybean [Glycine max (L.) Merr.] is caused by the oomycete Phytophthora sojae (Kaufm. & Gerd.). P. sojae has a narrow host range, consisting primarily of soybean, and it is a serious pathogen worldwide. It exists in root and stem tissues as mycelium, wherein it can form oospores which subsequently germinate to release motile, infectious zoospores. Molecular assays detecting DNA of P. sojae are useful in disease diagnostics, and for determining the presence of the organism in host tissues, soils, and runoff or ponded water from potentially infested fields. Such assays as published have utilized ITS sequences from the nuclear ribosomal RNA genes in conventional PCR or dye-binding quantitative PCR (Q-PCR) but are not amenable to multiplexing, and some of these assays did not utilize control strategies for type I or type II errors. In this study, we describe primers and a bifunctional probe with specificity to a gypsy-like retroelement in the P. sojae genome to create a fluorogenic 5'-exonuclease linear hydrolysis assay, with a multiplexed internal control reaction detecting an exogenous target to validate negative calls, and with uracil-deglycosylase-mediated protection against carryover contamination. The assay specifically detected 13 different P. sojae isolates, and excluded 17 other Phytophthora species along with 20 non-Phytophthora fungal and oomycete species pathogenic on soybean. A diagnostic limit of detection of 34 fg total P. sojae DNA was observed in serial dilutions, equivalent to 0.3 genome, and a practical detection sensitivity of four zoospores per sample was achieved, despite losses during DNA extraction.

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., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phytophthora pathogens secrete effectors to manipulate host innate immunity, thus facilitating infection. Among the RXLR effectors highly induced during Phytophthora sojae infection, Avh238 not only contributes to pathogen virulence but also triggers plant cell death. However, the detailed molecular basis of Avh238 functions remains largely unknown. We mapped the regions responsible for Avh238 functions in pathogen virulence and plant cell death induction using a strategy that combines investigation of natural variation and large-scale mutagenesis assays. The correlation between cellular localization and Avh238 functions was also evaluated. We found that the 79 th residue (histidine or leucine) of Avh238 determined its cell death-inducing activity, and that the 53 amino acids in its C-terminal region are responsible for promoting Phytophthora infection. Transient expression of Avh238 in Nicotiana benthamiana revealed that nuclear localization is essential for triggering cell death, while Avh238-mediated suppression of INF1-triggered cell death requires cytoplasmic localization. Our results demonstrate that a representative example of an essential Phytophthora RXLR effector can evolve to escape recognition by the host by mutating one nucleotide site, and can also retain plant immunosuppressive activity to enhance pathogen virulence in planta., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Soybean (Glycine max (L.) Merr.) is produced across a vast swath of North America, with the greatest concentration in the Midwest. Root rot diseases and damping-off are a major concern for production, and the primary causal agents include oomycetes and fungi. In this study, we focused on examination of oomycete species distribution in this soybean production system and how environmental and soil (edaphic) factors correlate with oomycete community composition at early plant growth stages. Using a culture-based approach, 3,418 oomycete isolates were collected from 11 major soybean-producing states and most were identified to genus and species using the internal transcribed spacer region of the ribosomal DNA. Pythium was the predominant genus isolated and investigated in this study. An ecology approach was taken to understand the diversity and distribution of oomycete species across geographical locations of soybean production. Metadata associated with field sample locations were collected using geographical information systems. Operational taxonomic units (OTU) were used in this study to investigate diversity by location, with OTU being defined as isolate sequences with 97% identity to one another. The mean number of OTU ranged from 2.5 to 14 per field at the state level. Most OTU in this study, classified as Pythium clades, were present in each field in every state; however, major differences were observed in the relative abundance of each clade, which resulted in clustering of states in close proximity. Because there was similar community composition (presence or absence) but differences in OTU abundance by state, the ordination analysis did not show strong patterns of aggregation. Incorporation of 37 environmental and edaphic factors using vector-fitting and Mantel tests identified 15 factors that correlate with the community composition in this survey. Further investigation using redundancy analysis identified latitude, longitude, precipitation, and temperature as factors that contribute to the variability observed in community composition. Soil parameters such as clay content and electrical conductivity also affected distribution of oomycete species. The present study suggests that oomycete species composition across geographical locations of soybean production is affected by a combination of environmental and edaphic conditions. This knowledge provides the basis to understand the ecology and distribution of oomycete species, especially those able to cause diseases in soybean, providing cues to develop management strategies.

Oomycete pathogens are commonly associated with soybean root rot and have been estimated to reduce soybean yields in the United States by 1.5 million tons on an annual basis. Limited information exists regarding the frequency and diversity of oomycete species across the major soybean-producing regions in North America. A survey was conducted across 11 major soybean-producing states in the United States and the province of Ontario, Canada. In 2011, 2,378 oomycete cultures were isolated from soybean seedling roots on a semiselective medium (CMA-PARPB) and were identified by sequencing of the internal transcribed spacer region of rDNA. Sequence results distinguished a total of 51 Pythium spp., three Phytophthora spp., three Phytopythium spp., and one Aphanomyces sp. in 2011, with Pythium sylvaticum (16%) and P. oopapillum (13%) being the most prevalent. In 2012, the survey was repeated, but, due to drought conditions across the sampling area, fewer total isolates (n = 1,038) were collected. Additionally, in 2012, a second semiselective medium (V8-RPBH) was included, which increased the Phytophthora spp. isolated from 0.7 to 7% of the total isolates. In 2012, 54 Pythium spp., seven Phytophthora spp., six Phytopythium spp., and one Pythiogeton sp. were recovered, with P. sylvaticum (14%) and P. heterothallicum (12%) being recovered most frequently. Pathogenicity and virulence were evaluated with representative isolates of each of the 84 species on soybean cv. Sloan. A seed-rot assay identified 13 and 11 pathogenic species, respectively, at 13 and 20°C. A seedling-root assay conducted at 20°C identified 43 species as pathogenic, having a significantly detrimental effect on the seedling roots as compared with the noninoculated control. A total of 15 species were pathogenic in both the seed and seedling assays. This study provides a comprehensive characterization of oomycete species present in soybean seedling roots in the major production areas in the United States and Ontario, Canada and provides a basis for disease management and breeding programs.

Two distinct Phytophthora taxa were found to be associated with brown rot of pomelo (Citrus grandis), a new disease of this ancestral Citrus species, in the Vinh Long province, Mekong River Delta area, southern Vietnam. On the basis of morphological characters and using the ITS1-5.8S-ITS2 region of the rDNA and the cytochrome oxidase subunit 1 (COI) as barcode genes, one of the two taxa was provisionally named as Phytophthora sp. prodigiosa, being closely related to but distinct from P. insolita, a species in Phytophthora Clade 9, while the other one, was closely related to but distinct from the Clade 2 species P. meadii and was informally designated as Phytophthora sp. mekongensis. Isolates of P. sp. prodigiosa and P. sp. mekongensis were also obtained from necrotic fibrous roots of Volkamer lemon (C. volkameriana) rootstocks grafted with 'King' mandarin (Citrus nobilis) and from trees of pomelo, respectively, in other provinces of the Mekong River Delta, indicating a widespread occurrence of both Phytophthora species in this citrus-growing area. Koch's postulates were fulfilled via pathogenicity tests on fruits of various Citrus species, including pomelo, grapefruit (Citrus x paradisi), sweet orange (Citrus x sinensis) and bergamot (Citrus x bergamia) as well as on the rootstock of 2-year-old trees of pomelo and sweet orange on 'Carrizo' citrange (C. sinensis 'Washington Navel' x Poncirus trifoliata). This is the first report of a Phytophthora species from Clade 2 other than P. citricola and P. citrophthora as causal agent of fruit brown rot of Citrus worldwide and the first report of P. insolita complex in Vietnam. Results indicate that likely Vietnam is still an unexplored reservoir of Phytophthora diversity.

Following recent discovery of Phytophthora lateralis on native Chamaecyparis obtusa in Taiwan, four phenotypically distinct lineages were discriminated: the Taiwan J (TWJ) and Taiwan K (TWK) in Taiwan, the Pacific Northwest (PNW) in North America and Europe and the UK in west Scotland. Across the four lineages, we analysed 88 isolates from multiple sites for microsatellite diversity. Twenty-one multilocus genotypes (MLGs) were resolved with high levels of diversity of the TWK and PNW lineages. No alleles were shared between the PNW and the Taiwanese lineages. TWK was heterozygous at three loci, whereas TWJ isolates were homozygous apart from one isolate, which exhibited a unique allele also present in the TWK lineage. PNW lineage was heterozygous at three loci. The evidence suggests its origin may be a yet unknown Asian source. North American and European PNW isolates shared all their alleles and also a dominant MLG, consistent with a previous proposal that this lineage is a recent introduction into Europe from North America. The UK lineage was monomorphic and homozygous at all loci. It shared its alleles with the PNW and the TWJ and TWK lineages, hence a possible origin in a recent hybridisation event between a Taiwan lineage and PNW cannot be ruled out., (Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance., Competing Interests: The authors have declared that no competing interests exist.

Phytophthora species are widespread and diverse in forest ecosystems, but little is known about their ecology. We explore ecological attributes of the closely related clade 3 species that occur sympatrically in western North American forests. We address the population structure, pathology, and epidemiology of P. ilicis, P. nemorosa, P. pluvialis, P. pseudosyringae, and P. psychrophila. Phytophthora species were isolated from plant tissues, rainwater falling through the forest canopy, streams, and soils in forests in western Oregon. Species identifications were based on morphology in culture with molecular confirmation using COX spacer and internal transcribed spacer (ITS) sequences. All five clade 3 Phytophthora species are present in western Oregon forests, although P. ilicis (only 1 forest isolate) and P. psychrophila (only 12 isolates) are apparently rare. P. ilicis is known only from holly in horticultural situations and once from a naturalized seedling in an urban forest. The known distribution of P. nemorosa in forest settings coincides with the ranges of its principle hosts, tanoak and myrtlewood, in Oregon and California. Although it is regularly identified from streams within the tanoak range, it has not been recovered from streams beyond that range. P. pluvialis is primarily associated with Douglas-fir canopies. It was identified from scattered locations throughout western Oregon in rain traps beneath Douglas-fir plantations and from diseased needles. P. pseudosyringae is also isolated from tanoak and myrtlewood in southwest Oregon and California, but its distribution, in streams at least, extends throughout much of western Oregon. P. psychrophila in Oregon is known only from rain traps beneath tanoak trees. Little intraspecific variation was detected in the nuclear rDNA ITS of clade 3 species. Variation in the mitochondrial COX spacer region was more frequent, with 2 to 10 haplotypes identified in the clade 3 species, for which we had multiple isolates.