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Summary: Phytophthora parasitica causes diseases on a broad range of host plants. It secretes numerous effectors to suppress plant immunity. However, only a few virulence effectors in P. parasitica have been characterized.Here, we highlight that PpE18, a conserved RXLR effector in P. parasitica, was a virulence factor and suppresses Nicotiana benthamiana immunity. Utilizing luciferase complementation, co‐immunoprecipitation, and GST pull‐down assays, we determined that PpE18 targeted NbAPX3‐1, a peroxisome membrane‐associated ascorbate peroxidase with reactive oxygen species (ROS)‐scavenging activity and positively regulates plant immunity in N. benthamiana. We show that the ROS‐scavenging activity of NbAPX3‐1 was critical for its immune function and was hindered by the binding of PpE18. The interaction between PpE18 and NbAPX3‐1 resulted in an elevation of ROS levels in the peroxisome.Moreover, we discovered that the ankyrin repeat‐containing protein NbANKr2 acted as a positive immune regulator, interacting with both NbAPX3‐1 and PpE18. NbANKr2 was required for NbAPX3‐1‐mediated disease resistance. PpE18 competitively interfered with the interaction between NbAPX3‐1 and NbANKr2, thereby weakening plant resistance.Our results reveal an effective counter‐defense mechanism by which P. parasitica employed effector PpE18 to suppress host cellular defense, by suppressing biochemical activity and disturbing immune function of NbAPX3‐1 during infection. [ABSTRACT FROM AUTHOR]

Oomycete, particularly Phytophthora species, causes the most devastating crop diseases, such as potato late blight, and threatens the sustainable crop production worldwide. Our previous studies identified Resistance to Phytophthora parasitica 1 (RTP1) as a negative regulator of Arabidopsis resistance to multiple biotrophic pathogens and RTP1 loss-of-function plants displayed rapid cell death and reactive oxygen species (ROS) production during early colonization of P. parasitica. In this study, we aim to decipher the mechanism of RTP1-mediated cell death, and identify a member of vaculoar processing enzymes (VPEs), γVPE, playing a role in rtp1-mediated resistance to P. parasitica and cell death occurrence. Our results showed up-regulation of the expression of γVPE as well as increased VPE/caspase 1-like protease activity in P. parasitica-infected rtp1 mutant plants. Besides, we found that the VPE/caspase 1-like protease activity was required for the cell death occurrence in Arabidopsis plants during the infection of P. parasitica as well as rtp1-mediated resistance to P. parasitica. Further pathogenicity assays on either Arabidopsis γvpe mutant plants or leaves of Nicotiana benthamiana with transient overexpression of γVPE demonstrated γVPE could positively affect plant resistance to P. parasitica. Together, our studies suggest that γVPE might function as an important regulator of plant defense and cell death occurrence in response to P. parasitica infection, and VPE/caspase 1-like protease activity is required for rtp1-mediated resistance to P. parasitica.

The phytopathogen Phytophthora parasitica, from the Oomycetes class, known to be the tobacco black shank agent, can induce devastating diseases in various crop, plant and forest ecosystems. The genus Phytophthora has been studied at the cellular level, suggesting that different developmental steps are induced by the expression of some specific genes. However, these studies have only been carried out on certain species, such as Phytophthora infestans and Phytophthora cactorum. As for Phytophthora parasitica, which can be considered as one of the top ten oomycete pathogens due to the economic impact and effect it has on food security, even less functional analyses and transcriptomics data are available. To date, little is known about the protein expression of Phytophthora parasitica, information that is essential for achieving a better understanding of this species. In this study, we aimed to gain insight into the proteomics of the mycelium of the Phytophthora parasitica strain INRA 310 by addressing the following questions: (i) how many predicted proteins can be detected on the mycelium of P. parasitica INRA 310, and (ii) what proteins can be detected? The proteomics experiments were performed on the mycelium of the strain Phytophthora parasitica INRA310, using the nanoliquid chromatography-MS/MS technique. A total of 219 proteins were identified, including ten unknown proteins and 209 proteins involved in lipid, carbohydrate, nucleotide, energy production and other metabolic pathways. This proteomics study is, to our knowledge, the first to be performed on the mycelium of Phytophthora parasitica INRA 310. It gives a brief first insight into its in vitro-expressed proteins. This work may be the first step before further, more comprehensive studies are undertaken with the aim of better understanding the biology of this species and its pathogenicity.

The phytopathogen Phytophthora parasitica, from the Oomycetes class, known to be the tobacco black shank agent, can induce devastating diseases in various crop, plant and forest ecosystems. The genus Phytophthora has been studied at the cellular level, suggesting that different developmental steps are induced by the expression of some specific genes. However, these studies have only been carried out on certain species, such as Phytophthora infestans and Phytophthora cactorum. As for Phytophthora parasitica, which can be considered as one of the top ten oomycete pathogens due to the economic impact and effect it has on food security, even less functional analyses and transcriptomics data are available. To date, little is known about the protein expression of Phytophthora parasitica, information that is essential for achieving a better understanding of this species. In this study, we aimed to gain insight into the proteomics of the mycelium of the Phytophthora parasitica strain INRA 310 by addressing the following questions: (i) how many predicted proteins can be detected on the mycelium of P. parasitica INRA 310, and (ii) what proteins can be detected? The proteomics experiments were performed on the mycelium of the strain Phytophthora parasitica INRA310, using the nanoliquid chromatography-MS/MS technique. A total of 219 proteins were identified, including ten unknown proteins and 209 proteins involved in lipid, carbohydrate, nucleotide, energy production and other metabolic pathways. This proteomics study is, to our knowledge, the first to be performed on the mycelium of Phytophthora parasitica INRA 310. It gives a brief first insight into its in vitro-expressed proteins. This work may be the first step before further, more comprehensive studies are undertaken with the aim of better understanding the biology of this species and its pathogenicity. [ABSTRACT FROM AUTHOR]

Aims The soil-borne oomycete pathogen Phytophthora parasitica can cause black shank disease in tobacco plants. The use of resistant varieties can be used to control black shank disease. The potential relationships of the composition of the rhizosphere microbiome to resistance to black shank disease are poorly understood. This work aims to compare the rhizosphere microbial community and network of the tobacco resistant variety HB202 with the susceptible variety XY3. Methods and Results Rhizospheric soils were collected from tobacco plants of HB202 and XY3 in the fields with same soil types and agricultural operations. The compositions of the rhizosphere microbial communities were revealed by Illumina sequencing of bacterial 16S rRNA genes and fungal spacer (ITS) sequences and analysed with molecular ecological network pipeline. The alpha diversity of fungal communities of the two varieties was significantly different. The structure and composition of bacterial and fungal communities in the resistant variety in the rhizosphere was different from the susceptible variety. Relative abundances of beneficial genera in the HB202 microbiota were higher than in the XY3. Conversely, the XY3 microbiota exhibited a higher abundance of deleterious genera compared to the HB202 microbiota. The resistant variety influences the topological properties and microbial interactions in the rhizosphere against the disease. The network of the HB202 was more complex and had higher connectivity compared to the XY3 network. Conclusions The rhizosphere microbial communities and networks of two tobacco varieties are very different. These changes in the microbial communities and their interactions may play an important role in tobacco resistance to black shank disease. [ABSTRACT FROM AUTHOR]

Foot and root rot caused by Phytophthora parasitica is a substantial threat to citrus cultivation, affecting both yield and quality. Thus, rapid and accurate detection of P. parasitica plays an important role in disease management. The aim of this study was to develop a simple diagnostic method to detect P. parasitica infection by combining recombinase polymerase amplification and lateral flow strips (LF-RPA). To establish the LF-RPA assay of P. parasitica, the primers and probe designed based on the Ypt1 gene were tested for specificity to P. parasitica, which showed no cross-reactivity with DNAs of other related oomycete species. The LF-RPA assay detected the amount of genomic DNA of P. parasitica which was as low as 1 pg. To make the LF-RPA assay useful in low-resource settings, four simplified DNA extraction methods were compared, after which the LF-RPA assay was applied, with no specialized equipment, to analyze a diverse range of citrus tissues by using a simplified PEG-NaOH method for DNA extraction. This method was successful in detecting P. parasitica in infected plant samples within 30 min. Combining the LF-RPA assay and a simplified DNA extraction method could be a potential detection test for P. parasitica, especially in areas with limited resources.

SUMMARY: Oomycetes are diploid eukaryotic microorganisms that seriously threaten sustainable crop production. MicroRNAs (miRNAs) and corresponding natural antisense transcripts (NATs) are important regulators of multiple biological processes. However, little is known about their roles in plant immunity against oomycete pathogens. In this study, we report the identification and functional characterization of miR398b and its cis‐NAT, the core‐2/I‐branching beta‐1,6‐N‐acetylglucosaminyltransferase gene (AtC2GnT), in plant immunity. Gain‐ and loss‐of‐function assays revealed that miR398b mediates Arabidopsis thaliana susceptibility to Phytophthora parasitica by targeting Cu/Zn‐Superoxidase Dismutase1 (CSD1) and CSD2, leading to suppressed expression of CSD1 and CSD2 and decreased plant disease resistance. We further showed that AtC2GnT transcripts could inhibit the miR398b–CSDs module via inhibition of pri‐miR398b expression, leading to elevated plant resistance to P. parasitica. Furthermore, quantitative reverse transcription PCR, RNA ligase‐mediated 5′‐amplification of cDNA ends (RLM‐5′ RACE), and transient expression assays indicated that miR398b suppresses the expression of AtC2GnT. We generated AtC2GnT‐silenced A. thaliana plants by CRISPR/Cas9 or RNA interference methods, and the Nicotiana benthamiana NbC2GnT‐silenced plants by virus‐induced gene silencing. Pathogenicity assays showed that the C2GnT‐silenced plants were more susceptible, while AtC2GnT‐overexpressing plants exhibited elevated resistance to P. parasitica. AtC2GnT encodes a Golgi‐localized protein, and transient expression of AtC2GnT enhanced N. benthamiana resistance to Phytophthora pathogens. Taken together, our results revealed a positive role of AtC2GnT and a negative regulatory loop formed by miR398b and AtC2GnT in regulating plant resistance to P. parasitica. [ABSTRACT FROM AUTHOR]

Oomycetes represent a unique group of plant pathogens that are phylogenetically distant from true fungi and cause significant crop losses and environmental damage. Understanding of the genetic basis of host plant susceptibility facilitates the development of novel disease resistance strategies. In this study, we report the identification of an Arabidopsis thaliana T‐DNA mutant with enhanced resistance to Phytophthora parasitica with an insertion in the Raf‐like mitogen‐activated protein kinase kinase kinase gene Raf36. We generated additional raf36 mutants by CRISPR/Cas9 technology as well as Raf36 complementation and overexpression transformants, with consistent results of infection assays showing that Raf36 mediates Arabidopsis susceptibility to P. parasitica. Using a virus‐induced gene silencing assay, we silenced Raf36 homologous genes in Nicotiana benthamiana and demonstrated by infection assays the conserved immune function of Raf36. Mutagenesis analyses indicated that the kinase activity of Raf36 is important for its immune function and interaction with MKK2, a MAPK kinase. By generating and analysing mkk2 mutants and MKK2 complementation and overexpression transformants, we found that MKK2 is a positive immune regulator in the response to P. parasitica infection. Furthermore, infection assay on mkk2 raf36 double mutant plants indicated that MKK2 is required for the raf36‐conferred resistance to P. parasitica. Taken together, we identified a Raf‐like kinase Raf36 as a novel plant susceptibility factor that functions upstream of MKK2 and directly targets it to negatively regulate plant resistance to P. parasitica. [ABSTRACT FROM AUTHOR]

Aim: The main aim of the present study was to develop nanotechnology‐based solutions for the management of a fungus, Phytophthora parasitica causing gummosis in citrus. Methods and Results: Biogenic copper nanoparticles (CuONPs) were synthesized using two different biocontrol agents, Pseudomonas fluorescens and Trichoderma viride and characterized using different analytical techniques. Furthermore, in vitro (at the concentrations of 10, 15, 30, 50, 70, 100 and 150 mg/L) and in vivo (at the concentration of 100 mg/L) activities of these nanoparticles were evaluated for their antifungal efficacy against P. parasitica. The results obtained confirmed the synthesis of irregular‐shaped CuONPs having a size in the range 40–100 nm in case of P. fluorescens, whereas, spherical CuONPs in the size range 20–80 were recorded in case of T. viride. As far as the in vitro antifungal efficacies of both these CuONPs is concerned, the maximum percent growth inhibition was observed in case of CuONPs synthesized from T. viride compared to CuONPs from P. fluorescens. However, in case of in vivo antifungal efficacies, CuONPs synthesized from T. viride showed the activity significantly higher than the conventionally used Bordeaux mixture. Conclusions: It can be concluded that biosynthesized CuONPs can be effectively used as a potential fungicide against P. parasitica. Significance and Impact of the Study: The application of nanoparticles having antifungal activities can be used as alternative fungicides to the conventional chemical fungicides. It has the potential to revolutionize the existing management strategies available for plant pathogenic fungi. [ABSTRACT FROM AUTHOR]

Oomycetes represent a unique group of plant pathogens that are destructive to a wide range of crops and natural ecosystems. Phytophthora species possess active small RNA (sRNA) silencing pathways, but little is known about the biological roles of sRNAs and associated factors in pathogenicity. Here we show that an AGO gene, PpAGO3 , plays a major role in the regulation of effector genes hence the pathogenicity of Phytophthora parasitica. PpAGO3 was unique among five predicted AGO genes in P. parasitica , showing strong mycelium stage-specific expression. Using the CRISPR-Cas9 technology, we generated PpAGO3ΔRGG1-3 mutants that carried a deletion of 1, 2, or 3 copies of the N-terminal RGG motif (Q RGG YD) but failed to obtain complete knockout mutants, which suggests its vital role in P. parasitica. These mutants showed increased pathogenicity on both Nicotiana benthamiana and Arabidopsis thaliana plants. Transcriptome and sRNA sequencing of PpAGO3ΔRGG1 and PpAGO3ΔRGG3 showed that these mutants were differentially accumulated with 25–26 nt sRNAs associated with 70 predicted cytoplasmic effector genes compared to the wild-type, of which 13 exhibited inverse correlation between gene expression and 25–26 nt sRNA accumulation. Transient overexpression of the upregulated RXLR effector genes, PPTG_01869 and PPTG_15425 identified in the mutants PpAGO3ΔRGG1 and PpAGO3ΔRGG3 , strongly enhanced N. benthamiana susceptibility to P. parasitica. Our results suggest that PpAGO3 functions together with 25–26 nt sRNAs to confer dynamic expression regulation of effector genes in P. parasitica , thereby contributing to infection and pathogenicity of the pathogen. [ABSTRACT FROM AUTHOR]

Oomycetes represent a unique group of plant pathogens that are destructive to a wide range of crops and natural ecosystems. Phytophthora species possess active small RNA (sRNA) silencing pathways, but little is known about the biological roles of sRNAs and associated factors in pathogenicity. Here we show that an AGO gene, PpAGO3, plays a major role in the regulation of effector genes hence the pathogenicity of Phytophthora parasitica. PpAGO3 was unique among five predicted AGO genes in P. parasitica, showing strong mycelium stage-specific expression. Using the CRISPR-Cas9 technology, we generated PpAGO3ΔRGG1-3 mutants that carried a deletion of 1, 2, or 3 copies of the N-terminal RGG motif (QRGGYD) but failed to obtain complete knockout mutants, which suggests its vital role in P. parasitica. These mutants showed increased pathogenicity on both Nicotiana benthamiana and Arabidopsis thaliana plants. Transcriptome and sRNA sequencing of PpAGO3ΔRGG1 and PpAGO3ΔRGG3 showed that these mutants were differentially accumulated with 25–26 nt sRNAs associated with 70 predicted cytoplasmic effector genes compared to the wild-type, of which 13 exhibited inverse correlation between gene expression and 25–26 nt sRNA accumulation. Transient overexpression of the upregulated RXLR effector genes, PPTG_01869 and PPTG_15425 identified in the mutants PpAGO3ΔRGG1 and PpAGO3ΔRGG3, strongly enhanced N. benthamiana susceptibility to P. parasitica. Our results suggest that PpAGO3 functions together with 25–26 nt sRNAs to confer dynamic expression regulation of effector genes in P. parasitica, thereby contributing to infection and pathogenicity of the pathogen.

Abstract Diseases caused by Phytophthora species seriously affect global crop production and food security. Identification of key factors involved in plant resistance is valuable for disease management. Previously, we characterized the transcriptome of Nicotiana benthamiana which was infected with Phytophthora parasitica. Here, we selected NbERF173, one of the most strongly up-regulated genes of N. benthamiana in response to P. parasitica infection, for further investigation. First, NbERF173 encodes a conserved transcription factor in our tested plant species. Second, overexpression of NbERF173 in N. benthamiana enhanced its resistance to P. parasitica, and silencing of NbERF173 significantly promoted the infection of both P. parasitica and Botrytis cinerea. Additionally, we demonstrated that NbERF173 can participate in reprograming of gene expression during P. parasitica infection and manipulate expression patterns of many defense-related genes, including two proteinase inhibitors encoding genes (PI1-B and KTI1). Furthermore, overexpression of PI1-B and KTI1 strengthened plant resistance to P. parasitica and partially restored the deficiency in resistance of NbERF173-silenced N. benthamiana plants. Finally, we found that NbERF173 could not bind to the promoters of PI1-B and KTI1 using yeast one-hybrid assay. Together, our results suggest that NbERF173 positively regulate the disease resistance, probably by reprograming of defense-related genes.

Phytophthora species can infect hundreds of different plants, including many important crops, causing a number of agriculturally relevant diseases. A key feature of attempted pathogen infection is the rapid production of the redox active molecule nitric oxide (NO). However, the potential role(s) of NO in plant resistance against Phytophthora is relatively unexplored. Here we show that the level of NO accumulation is crucial for basal resistance in Arabidopsis against Phytophthora parasitica. Counterintuitively, both relatively low or relatively high NO accumulation leads to reduced resistance against P. parasitica. S‐nitrosylation, the addition of a NO group to a protein cysteine thiol to form an S‐nitrosothiol, is an important route for NO bioactivity and this process is regulated predominantly by S‐nitrosoglutathione reductase 1 (GSNOR1). Loss‐of‐function mutations in GSNOR1 disable both salicylic acid accumulation and associated signalling, and also the production of reactive oxygen species, leading to susceptibility towards P. parasitica. Significantly, we also demonstrate that secreted proteins from P. parasitica can inhibit Arabidopsis GSNOR1 activity. [ABSTRACT FROM AUTHOR]

The receptor-like kinase Suppressor of BIR1 (SOBIR1) binds various receptor-like proteins (RLPs) that perceive microbe-associated molecular patterns (MAMPs) at the plasma membrane, which is thought to activate plant pattern-triggered immunity (PTI) against pathogen invasion. Despite its potentially crucial role, how SOBIR1 transmits immune signaling to ultimately elicit PTI remains largely unresolved. Herein, we report that a Nicotiana benthamiana gene Nb RLP1, like Nb SOBIR1, was highly induced upon Phytophthora parasitica infection. Intriguingly, Nb RLP1 is characterized as a receptor-like protein localizing to the endoplasmic reticulum (ER) membrane rather than the plasma membrane. Using bimolecular fluorescence complementation and affinity purification assays, we established that Nb RLP1 is likely to associate with Nb SOBIR1 through the contact between the ER and plasma membrane. We further found that Nb SOBIR1 at the plasma membrane partitions into mobile microdomains that undergo frequent lateral movement and internalization. Remarkably, the dynamics of Nb SOBIR1 microdomain is coupled to the remodeling of the cortical ER network. When Nb SOBIR1 microdomains were induced by the P. parasitica MAMP ParA1, tobacco cells overexpressing Nb RLP1 accelerated Nb SOBIR1 internalization. Overexpressing Nb RLP1 in tobacco further exaggerated the ParA1-induced necrosis. Together, these findings have prompted us to propose that ER and the ER-localized Nb RLP1 may play a role in transmitting plant immune signals by regulating Nb SOBIR1 internalization. [ABSTRACT FROM AUTHOR]

Protection of crop plants from phytopathogens through endophytic bacteria is a newly emerged area of biocontrol. In this study, endophytic bacteria were isolated from the rhizosphere of Cannabis sativa. Based on initial antimicrobial screening, three (03) bacteria Serratia marcescens MOSEL-w2, Enterobacter cloacae MOSEL-w7, and Paenibacillus MOSEL-w13 were selected. Antimicrobial assays of these selected bacteria against Phytophthora parasitica revealed that E. cloacae MOSEL-w7 and Paenibacillus sp. MOSEL-w13 possessed strong activity against P. parasitica. All these bacterial extracts showed strong inhibition against P. parasitica at different concentrations (4–400 µg mL−1). P. parasitica hyphae treated with ethyl acetate extract of E. cloacae MOSEL-w7 resulted in severe growth abnormalities compared to control. The extracts were further evaluated for in vivo detached-leaf assay against P. parasitica on the wild type tobacco. Application of 1% ethyl acetate bacterial extract of S. marcescens MOSEL-w2, E. cloacae MOSEL-w7, and Paenibacillus sp. MOSEL-w13 reduced P. parasitica induced lesion sizes and lesion frequencies by 60–80%. HPLC based fractions of each extract also showed bioactivity against P. parasitica. A total of 24 compounds were found in the S. marcescens MOSEL-w2, 15 compounds in E. cloacae MOSEL-w7 and 20 compounds found in Paenibacillus sp. MOSEL-w13. LC–MS/MS analyses showed different bioactive compounds in the bacterial extracts such as Cotinine (alkylpyrrolidine), l-tryptophan, l-lysine, l-Dopa, and l-ornithine. These results suggest that S. marcescens MOSEL-w2, E. cloacae MOSEL-w7, and Paenibacillus MOSEL-w13 are a source of bioactive metabolites and could be used in combination with other biocontrol agents, with other modes of action for controlling diseases caused by Phytophthora in crops. They could be a clue for the broad-spectrum biopesticides for agriculturally significant crops. [ABSTRACT FROM AUTHOR]

Giant viruses of amoebas, recently classified in the class Megaviricetes, are a group of viruses that can infect major eukaryotic lineages. We previously identified a set of giant virus sequences in the genome of Phytophthora parasitica, an oomycete and a devastating major plant pathogen. How viral insertions shape the structure and evolution of the invaded genomes is unclear, but it is known that the unprecedented functional potential of giant viruses is the result of an intense genetic interplay with their hosts. We previously identified a set of giant virus sequences in the genome of P. parasitica, an oomycete and a devastating major plant pathogen. Here, we show that viral pieces are found in a 550-kb locus and are organized in three main clusters. Viral sequences, namely RNA polymerases I and II and a major capsid protein, were identified, along with orphan sequences, as a hallmark of giant viruses insertions. Mining of public databases and phylogenetic reconstructions suggest an ancient association of oomycetes and giant viruses of amoeba, including faustoviruses, African swine fever virus (ASFV) and pandoraviruses, and that a single viral insertion occurred early in the evolutionary history of oomycetes prior to the Phytophthora–Pythium radiation, estimated at ∼80 million years ago. Functional annotation reveals that the viral insertions are located in a gene sparse region of the Phytophthora genome, characterized by a plethora of transposable elements (TEs), effectors and other genes potentially involved in virulence. Transcription of viral genes was investigated through analysis of RNA-Seq data and qPCR experiments. We show that most viral genes are not expressed, and that a variety of mechanisms, including deletions, TEs insertions and RNA interference may contribute to transcriptional repression. However, a gene coding a truncated copy of RNA polymerase II along a set of neighboring sequences have been shown to be expressed in a wide range of physiological conditions, including responses to stress. These results, which describe for the first time the endogenization of a giant virus in an oomycete, contribute to challenge our view of Phytophthora evolution.

Giant viruses of amoebas, recently classified in the class Megaviricetes, are a group of viruses that can infect major eukaryotic lineages. We previously identified a set of giant virus sequences in the genome of Phytophthora parasitica , an oomycete and a devastating major plant pathogen. How viral insertions shape the structure and evolution of the invaded genomes is unclear, but it is known that the unprecedented functional potential of giant viruses is the result of an intense genetic interplay with their hosts. We previously identified a set of giant virus sequences in the genome of P. parasitica , an oomycete and a devastating major plant pathogen. Here, we show that viral pieces are found in a 550-kb locus and are organized in three main clusters. Viral sequences, namely RNA polymerases I and II and a major capsid protein, were identified, along with orphan sequences, as a hallmark of giant viruses insertions. Mining of public databases and phylogenetic reconstructions suggest an ancient association of oomycetes and giant viruses of amoeba, including faustoviruses, African swine fever virus (ASFV) and pandoraviruses, and that a single viral insertion occurred early in the evolutionary history of oomycetes prior to the Phytophthora – Pythium radiation, estimated at ∼80 million years ago. Functional annotation reveals that the viral insertions are located in a gene sparse region of the Phytophthora genome, characterized by a plethora of transposable elements (TEs), effectors and other genes potentially involved in virulence. Transcription of viral genes was investigated through analysis of RNA-Seq data and qPCR experiments. We show that most viral genes are not expressed, and that a variety of mechanisms, including deletions, TEs insertions and RNA interference may contribute to transcriptional repression. However, a gene coding a truncated copy of RNA polymerase II along a set of neighboring sequences have been shown to be expressed in a wide range of physiological conditions, including responses to stress. These results, which describe for the first time the endogenization of a giant virus in an oomycete, contribute to challenge our view of Phytophthora evolution. [ABSTRACT FROM AUTHOR]

Phytophthora parasitica is one of the most widespread Phytophthora species, which is known to cause root rot, foot rot/gummosis and brown rot of fruits in citrus. In this study, we have analyzed the transcriptome of a commonly used citrus rootstock Carrizo citrange in response to P. parasitica infection using the RNA-seq technology. In total, we have identified 6692 differentially expressed transcripts (DETs) among P. parasitica-inoculated and mock-treated roots. Of these, 3960 genes were differentially expressed at 24 h post inoculation and 5521 genes were differentially expressed at 48 h post inoculation. Gene ontology analysis of DETs suggested substantial transcriptional reprogramming of diverse cellular processes particularly the biotic stress response pathways in Carrizo citrange roots. Many R genes, transcription factors, and several other genes putatively involved in plant immunity were differentially modulated in citrus roots in response to P. parasitica infection. Analysis reported here lays out a strong foundation for future studies aimed at improving resistance of citrus rootstocks to P. parasitica.

The purpose of the study was to investigate the antifungal activity of essential oils derived from the Zingiberaceae family, such as Zingiber officinale Roscoe, Alpinia officinarum. Hance, and Curcuma longa Linn., against Phytophthora parasitica Dastur, (the pathogen that causes root and stem rot diseases). In vitro antifungal activity was measured using poisoned food technologies at a final concentration of 1000 mg/L in a completely randomized design with triplications. The essential oil of A. officinarum demonstrated the highest antifungal activity against P. parasitica, according to findings (p < 0.05). Gas chromatography-mass spectrometry was also used to analyze the oil. Twenty constituents representing 99.1% of the total content were identified. Eucalyptol was the most abundant component in A. officinarum rhizome oil (52%). The oil’s half-inhibitory concentration (IC50) of 432.89 mg/L had higher antifungal activity than eucalyptol (>1000 mg/L). The results suggested that A. officinarum oil should be expanded further for a new generation of fungicides as an environmentally acceptable agent and to reduce the use of chemicals in crop protection.

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 species are destructive plant pathogens that cause significant crop losses worldwide. To understand plant susceptibility to oomycete pathogens and to explore novel disease resistance strategies, we employed the Arabidopsis thaliana–Phytophthora parasitica model pathosystem and screened for A. thaliana T‐DNA insertion mutant lines resistant to P. parasitica. This led to the identification of the resistant mutant 267‐31, which carries two T‐DNA insertion sites in the promoter region of the ethylene‐responsive factor 19 gene (ERF019). Quantitative reverse transcription PCR (RT‐qPCR) assays showed that the expression of ERF019 was induced during P. parasitica infection in the wild type, which was suppressed in the 267‐31 mutant. Additional erf019 mutants were generated using CRISPR/Cas9 technology and were confirmed to have increased resistance to P. parasitica. In contrast, ERF019 overexpression lines were more susceptible. Transient overexpression assays in Nicotiana benthamiana showed that the nuclear localization of ERF019 is crucial for its susceptible function. RT‐qPCR analyses showed that the expression of marker genes for multiple defence pathways was significantly up‐regulated in the mutant compared with the wild type during infection. Flg22‐induced hydrogen peroxide accumulation and reactive oxygen species burst were impaired in ERF019 overexpression lines, and flg22‐induced MAPK activation was enhanced in erf019 mutants. Moreover, transient overexpression of ERF019 strongly suppressed INF‐triggered cell death in N. benthamiana. These results reveal the importance of ERF019 in mediating plant susceptibility to P. parasitica through suppression of pathogen‐associated molecular pattern‐triggered immunity. [ABSTRACT FROM AUTHOR]

Proteases secreted by pathogens have been shown to be important virulence factors modifying plant immunity, and cysteine proteases have been demonstrated to participate in different pathosystems. However, the virulence functions of the cysteine proteases secreted by Phytophthora parasitica are poorly understood. Using a publicly available genome database, we identified 80 cysteine proteases in P. parasitica, 21 of which were shown to be secreted. Most of the secreted cysteine proteases are conserved among different P. parasitica strains and are induced during infection. The secreted cysteine protease proteins PpCys44/45 (proteases with identical protein sequences) and PpCys69 triggered cell death on the leaves of different Nicotiana spp. A truncated mutant of PpCys44/45 lacking a signal peptide failed to trigger cell death, suggesting that PpCys44/45 functions in the apoplastic space. Analysis of three catalytic site mutants showed that the enzyme activity of PpCys44/45 is required for its ability to trigger cell death. A virus‐induced gene silencing assay showed that PpCys44/45 does not induce cell death on NPK1 (Nicotiana Protein Kinase 1)‐silenced Nicotiana benthamiana plants, indicating that the cell death phenotype triggered by PpCys44/45 is dependent on NPK1. PpCys44‐ and PpCys45‐deficient double mutants showed decreased virulence, suggesting that PpCys44 and PpCys45 positively promote pathogen virulence during infection. PpCys44 and PpCys45 are important virulence factors of P. parasitica and trigger NPK1‐dependent cell death in various Nicotiana spp. [ABSTRACT FROM AUTHOR]

Phytophthora parasitica is one of the most widespread Phytophthora species, which is known to cause root rot, foot rot/gummosis and brown rot of fruits in citrus. In this study, we have analyzed the transcriptome of a commonly used citrus rootstock Carrizo citrange in response to P. parasitica infection using the RNA-seq technology. In total, we have identified 6692 differentially expressed transcripts (DETs) among P. parasitica-inoculated and mock-treated roots. Of these, 3960 genes were differentially expressed at 24 h post inoculation and 5521 genes were differentially expressed at 48 h post inoculation. Gene ontology analysis of DETs suggested substantial transcriptional reprogramming of diverse cellular processes particularly the biotic stress response pathways in Carrizo citrange roots. Many R genes, transcription factors, and several other genes putatively involved in plant immunity were differentially modulated in citrus roots in response to P. parasitica infection. Analysis reported here lays out a strong foundation for future studies aimed at improving resistance of citrus rootstocks to P. parasitica. [ABSTRACT FROM AUTHOR]

Summary: RXLR effectors encoded by Phytophthora species play a central role in pathogen–plant interactions. An understanding of the biological functions of RXLR effectors is conducive to the illumination of the pathogenic mechanisms and the development of disease control strategies. However, the virulence function of Phytophthora parasitica RXLR effectors is poorly understood. Here, we describe the identification of a P. parasitica RXLR effector gene, PPTG00121 (PpE4), which is highly transcribed during the early stages of infection. Live cell imaging of P. parasitica transformants expressing a full‐length PpE4 (E4FL)‐mCherry protein indicated that PpE4 is secreted and accumulates around haustoria during plant infection. Silencing of PpE4 in P. parasitica resulted in significantly reduced virulence on Nicotiana benthamiana. Transient expression of PpE4 in N. benthamiana in turn restored the pathogenicity of the PpE4‐silenced lines. Furthermore, the expression of PpE4 in both N. benthamiana and Arabidopsis thaliana consistently enhanced plant susceptibility to P. parasitica. These results indicate that PpE4 contributes to pathogen infection. Finally, heterologous expression experiments showed that PpE4 triggers non‐specific cell death in a variety of plants, including tobacco, tomato, potato and A. thaliana. Virus‐induced gene silencing assays revealed that PpE4‐induced cell death is dependent on HSP90, NPK and SGT1, suggesting that PpE4 is recognized by the plant immune system. In conclusion, PpE4 is an important virulence RXLR effector of P. parasitica and recognized by a wide range of host plants. [ABSTRACT FROM AUTHOR]

Phytophthora parasitica infection of Choisya ternata can cause important economical loses due to root rot disease. This research focused on testing the potential benefit of chemical treatment (Mefenoxam) and Biological Control Agents (Glomus intraradices, Gliocladium catenulatum, Trichoderma atroviridae and Bacillus amyloliquefaciens) in protecting C. ternata against P. parasitica. BCAs were applied as individual and/or combined treatments. The effect of the treatment was observed by monitoring C. ternata symptoms. A real-time PCR targeting the ypt1 gene was also adapted to evaluate P. parasitica development in the substrate. The use of Mefenoxam provided the higher level of plant protection. However, a significant reduction in plant symptoms and P. parasitica development was also observed with the combined treatment of G. intraradices with G. catenulatum and G. intraradices with T. atroviridae. Another combined treatment with G. catenulatum and B. amyloliquefaciens increased the pathogen density and severity. No individual treatment had a significant effect on the pathogen. Our results highlight the potential of biological control in protecting C. ternata against P. parasitica and the advantage of combined strategies. [ABSTRACT FROM AUTHOR]

The objective of this research was to conduct a spatial analysis of the incidence of Phytophthora nicotianae Breda de Haan and Phytophthora infestans (Mont.) De Bary, during five seasons of potato in Fields Company Miscellaneous Crops in Horquita, Cienfuegos (2003-2004 to 2007-2008). Information about pathogens, collected by the Plant Protection Station of Yaguaramas, was used in order to do so. An alpha numerical database was made, oriented to a group of areas under center pivot irrigation machines (Kubans and Bayamón) and its quadrants. Parallel to this, mapping of center pivot machines and quadrants was generated in MapInfo GIS 8.5. Several automatic geo codifications were made in order to relate the alphanumeric database and the mapping, and thematic maps were generated in the presence or absence of pathogens. The two study agents have presented high spatial variability during the five planting seasons. Both have influenced the same geographical area with similar appearance date. P. infestans has not expressed a defined dispersion pattern and it has spread at random from the primary source of incidence, while Phytophthora nicotianae has become an endemic agent which shows a dispersion pattern towards neighboring areas or areas connected by the road network from the primary sources, which have been associated with poor land leveling and late or intermediate planting seasons.

A study was carried out for identification and detection of Phytophthora spp. from soil and plant samples, collected from Solanaceous crops in Egypt and Italy. The samples were screened with specific and universal primers of Phytophthora 18S, ITS1-ITS2 and 28S regions, followed by PCR product sequencing. The Phytophthora spp. detected were P. infestans (Egypt) and P. parasitica (Italy). A molecular beacon probe was also developed based on the avr3a gene of P. infestans to detect a variant associated with virulence traits. The probe was suitable for avr3a allele identification from P. infestans and also from P. parasitica PCR products. [ABSTRACT FROM AUTHOR]

In the field, eggplants are frequently attacked by Alternaria porri f. sp. solani, Botrytis cinerea and Phytophthora parasitica pathogens, which diminishes yield in quantitative terms and depreciates qualitatively. For the control of these pathogens three variants of fungicide treatments with different active ingredients were tested: chlorotalonil 500 g/l, pyraclostrobin 5% + metiram 55%, metiram 80%, iprovalicarb 8.4% + Cu oxychloride 40.6%, azoxystrobin 200 g/l + diphenoconazole 125 g/l. The best efficacy was obtained at variant 2 with the following schedule of treatments: treatment 1 - pyraclostrobin 5% + metiram 55% - 0.2%; treatment 2 - iprovalicarb 8.4% + Cu oxychloride 40.6% - 0.2%; treatment 3 - pyraclostrobin 5% + metiram 55% - 0.2%; treatment 4 - iprovalicarb 8.4% + Cu oxychloride 40.6% - 0.2%; treatment 5 - azoxystrobin 200 g/l + diphenoconazole 125 g/l - 0.1%; treatment 6 - pyraclostrobin 5% + metiram 55% - 0.2%. In this variant, the average efficacy was 85.5% and the yield 39.7 t/ha (123.17%) compared to 31.8 t/ha in the untreated control variant. [ABSTRACT FROM AUTHOR]

This study deals with in vitro-induced mutagenesis and selection of Phytophthora tolerant lines of Citrus jambhiri and their regeneration. For in vitro-induced mutagenesis, cotyledons were treated with ethyl methane sulfonate ( EMS) 100, 200 and 300 m m for different time durations viz. 1, 3, 6 and 9 hr. Callus cultures were established from EMS treated cotyledon explants on MS medium supplemented with 2.0 mg/L of 2,4-D and 500 mg/L of malt extract. Calli derived from cotyledon were challenged in vitro on selective MS medium containing 5%-100% of culture filtrate ( CF) of the Phytophthora parasitica. Selected mutagen-treated calli showed resistance in vitro on media containing CF. Calli treated with 100 m m EMS for 6-hr duration showed tolerance (24%) up to 75% CF after 4th selection cycle. While, calli treated with 200 m m for 6-hr duration showed maximum tolerance (76%) up to 75% CF. Resistant calli were then transferred to MS regeneration medium for shoot bud regeneration. A dose-dependent decrease in the regeneration capacity of the selected calli was observed with the increasing concentration of the CF. In randomly amplified polymorphic DNA analysis, plantlets showed different banding pattern in comparison with the control plant, which confirms the presence of variations at genetic level. [ABSTRACT FROM AUTHOR]

Small RNAs (sRNAs) are important non-coding RNA regulators, playing key roles in developmental regulation, transposon suppression, environmental response, host–pathogen interaction and other diverse biological processes. However, their roles in oomycetes are poorly understood. Here, we performed sRNA sequencing and RNA sequencing of Phytophthora parasitica at stages of vegetative growth and infection of Arabidopsis roots to examine diversity and function of sRNAs in P. parasitica, a model hemibiotrophic oomycete plant pathogen. Our results indicate that there are two distinct types of sRNA-generating loci in P. parasitica genome, giving rise to clusters of 25–26 nt and 21 nt sRNAs, respectively, with no significant strand-biases. The 25–26 nt sRNA loci lie predominantly in gene-sparse and repeat-rich regions, and overlap with over 7000 endogenous gene loci. These overlapped genes are typically P. parasitica species-specific, with no homologies to the sister species P. infestans. They include approximately 40% RXLR effector genes, 50% CRN effector genes and some elicitor genes. The transcripts of most of these genes could not be detected at both the vegetative mycelium and infection stages as revealed by RNA sequencing, indicating that the 25–26 nt sRNAs are associated with efficient silencing of these genes. The 21 nt sRNA loci typically overlap with the exon regions of highly expressed genes, suggesting that the biogenesis of the 21 nt sRNAs may be dependent on the level of gene transcription and that these sRNAs do not mediate efficient silencing of homologous genes. Analyses of the published P. infestans sRNA and mRNA sequencing data consistently show that the 25–26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in Phytophthora.

Phytophthora parasitica is one of the most widespread Phytophthora species, which is known to cause multiple diseases in tomato and is capable of infecting almost all plant parts. Our current understanding of tomato-Phytophthora parasitica interaction is very limited and currently nothing is known at the whole genome or transcriptome level. In this study, we have analyzed and compared the transcriptome of a resistant and a susceptible wild tomato accession in response to P. parasitica infection using the RNA-seq technology. We have identified 2657 and 3079 differentially expressed genes (DEGs) in treatment vs control comparison of resistant (Sp-R) and susceptible (Sp-S) samples respectively. Functional annotation of DEGs revealed substantial transcriptional reprogramming of diverse physiological and cellular processes, particularly the biotic stress responses in both Sp-R and Sp-S upon P. parasitica treatment. However, subtle expression differences among some core plant defense related genes were identified and their possible role in resistance development against P. parasitica is discussed. Our results revealed 1173 genes that were differentially expressed only in Sp-R accession upon P. parasitica inoculation. These exclusively found DEGs in Sp-R accession included some core plant defense genes, for example, several protease inhibitors, chitinases, defensin, PR-1, a downy mildew susceptibility factor, and so on, were all highly induced. Whereas, several R genes, WRKY transcriptions factors and a powdery mildew susceptibility gene (Mlo) were highly repressed during the resistance outcome. Analysis reported here lays out a strong foundation for future studies aimed at improving genetic resistance of tomato cultivars against to Phytopphthora species.

ROP is a subfamily of small GTP-binding proteins that uniquely exist in plants. It acts as versatile molecular switches that regulate various developmental processes. Some ROP proteins are also reported to affect defense responses, although their exact mechanism is not fully understood. Herein, ROP members in wheat were mined; the functions of three wheat ROP proteins were studied. RT-PCR results showed that the expression of TaRac1 was rapidly and strongly induced after leaf rust infection. TaRac1 interacted with TaCCR in yeast-hybridization assay. The overexpression of TaRac1 in tobacco promoted CCR and CAD gene expression, increased the total lignin content and sinapyl lignin proportion, and then enhanced resistance to tobacco black shank and bacterial wilt diseases. In contrast, TaRac3 and TaRac4 did not show to interact with TaCCR. Furthermore, the overexpression of TaRac3 and TaRac4 did not increase lignin gene expression and lignin accumulation either. Unlike TaRac1, the overexpression of TaRac3 increased susceptibility to both black shank and bacterial wilt pathogens, while overexpression of TaRac4 showed no effect on disease resistance but promoted the root growth in tobacco seedling. These data collectively suggest that TaRac1 in Group II is mainly involved in regulating lignin metabolism which, in turn, responsible for the observed roles in pathogen resistance. TaRac3 and TaRac4 have the minor roles in defense response but may act on regulation in plant developmental processes. These results shed light on the complexity and diverse function of ROP in plant defense pathway. [ABSTRACT FROM AUTHOR]

Betelvine (Piper betle L.) is a perennial dioecious creeper-belonging to the family Piperaceae usually grown under artificially erected structurals known as baroj that provides high moist and humid conditions favouring several diseases that in turn are major deterrents to good harvest. Betelvine suffers from many root and aerial diseases of which leaf rot caused by Phytophthora parasitica (Dastur) and leaf spot caused by Colletotrichum capsici Syd. (Butler and Bisby) are most important and are included in these studies for assessment. The extent of losses may vary from 20-40% for leaf rot and 10-20% for leaf spot, leading to almost total crop failure (Dasgupta and Sen, 1999). As the betel leaf is directly chewed immediately after harvest, it is not feasible to apply any pesticides that may cause toxic hazards to human being. An experiment was conducted for two consecutive years to study the effect of different crop canopy by maintaining three different plant to plant spacing (viz., 11.1, 9.53 and 8.3 cm.) on leaf yield, disease incidence and keeping quality (days to 50% rotting) of betelvine. With the above plant to plant spacing and standard row to row spacing (60cm), the plant population was maintained as 1.50, 1.75 and 2.00 lakh ha-1. The results revealed that when crop canopy was increased by reducing the plant to plant spacing from 11.1 cm to 8.3 cm there were significant increases in both the diseases under investigation (rot disease 19.76 to 22.30%; leaf spot disease 22.68 to 25.42%). Significant increase in yield (26.29 to 33.63 lakh ha-1year -1), decrease in fresh weight of 100 leaves (460.85 to 432.35g) and decrease in keeping quality of leaves (13.14 days to 10.28 days) were recorded when crop canopy was increased by reduction in plant to plant spacing from 11.1 cm to 8.3 cm. From these results it can be concluded that shifts in microclimate resulting from increase in canopy by reducing the plant spacing aggravated the spread and infection of disease. [ABSTRACT FROM AUTHOR]

Small RNAs (sRNAs) are important non-coding RNA regulators, playing key roles in developmental regulation, transposon suppression, environmental response, host-pathogen interaction and other diverse biological processes. However, their roles in oomycetes are poorly understood. Here, we performed sRNA sequencing and RNA sequencing of Phytophthora parasitica at stages of vegetative growth and infection of Arabidopsis roots to examine diversity and function of sRNAs in P. parasitica, a model hemibiotrophic oomycete plant pathogen. Our results indicate that there are two distinct types of sRNA-generating loci in P. parasitica genome, giving rise to clusters of 25-26 nt and 21 nt sRNAs, respectively, with no significant strand-biases. The 25-26 nt sRNA loci lie predominantly in gene-sparse and repeat-rich regions, and overlap with over 7000 endogenous gene loci. These overlapped genes are typically P. parasitica species-specific, with no homologies to the sister species P. infestans. They include approximately 40% RXLR effector genes, 50% CRN effector genes and some elicitor genes. The transcripts of most of these genes could not be detected at both the vegetative mycelium and infection stages as revealed by RNA sequencing, indicating that the 25-26 nt sRNAs are associated with efficient silencing of these genes. The 21 nt sRNA loci typically overlap with the exon regions of highly expressed genes, suggesting that the biogenesis of the 21 nt sRNAs may be dependent on the level of gene transcription and that these sRNAs do not mediate efficient silencing of homologous genes. Analyses of the published P. infestans sRNA and mRNA sequencing data consistently show that the 25-26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in Phytophthora. [ABSTRACT FROM AUTHOR]

The genus Phytophthora causes great damages to agricultural production, especially to potatoes and tomatoes cultures. To face these losses, it is of interest to reduce or inhibit the activity of this aggressive pathogen. Some species of Trichoderma have great potential for the biological control of several plant pathogens, including diseases caused by Phytophthora parasitica, Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum, Sclerotinia sclerotiorum etc. The purpose of this study was to assess the biocontrol efficacy of three Trichoderma strains (T. asperellum T36, T. asperellum T50, T. harzianum T78) against Phytophthora parasitica. In vitro tests were carried out using dual culture technique. In vivo tests were carried out with pepper seedlings (Capsicum annuum cv. Lamuyo) and conidial standard suspension of Trichoderma isolates as biocontrol agents. Of the three Trichoderma isolates tested for their effectiveness against mycelial growth of pathogen, T. asperellum T36 (81.2%) exhibited maximum inhibition of P. parasitica, compared with the control, followed by T. asperellum T50 (79.6%) and T. harzianum T78 (77.7%). Likewise for the in vivo test, the inoculation of the Trichoderma biocontrol agent showed that the percentage of dead plants seedlings was significant reduced. T. asperellum T36 is a useful biological alternative to pesticides for the control of P. parasitica in pepper seedlings. [ABSTRACT FROM AUTHOR]

Black shank, caused by Phytophthora parasitica var. nicotianae, is a widespread and destructive disease of tobacco. Crop rotation is essential in controlling black shank. Here, we confirmed that rotating black shank-infested fields with rapeseed (Brassica napus) suppressed the incidence this disease. Further study demonstrated that rapeseed roots have a strong ability to attract zoospores and subsequently stop the swimming of zoospores into cystospores. Then, rapeseed roots secrete a series of antimicrobial compounds, including 2-butenoic acid, benzothiazole, 2-(methylthio)benzothiazole, 1-(4-ethylphenyl)-ethanone, and 4-methoxyindole, to inhibit the cystospore germination and mycelial growth of P. parasitica var. nicotianae. Thus, rapeseed rotated with tobacco suppresses tobacco black shank disease through the chemical weapons secreted by rapeseed roots.