C-coordinated O-carboxymethyl chitosan Cu(II) complex exerts antifungal activity by disrupting the cell membrane integrity of Phytophthora capsici Leonian.

Damage to the cell membrane is an effective method to prevent drug resistance in plant fungal diseases. Here, we proposed a negative remodeling model of the cell membrane structure induced by the C-coordinated O-carboxymethyl chitosan Cu (II) complex (O-CSLn-Cu). FITC-labeled O-CSLn-Cu (FITC-O-CSLn-Cu) was first synthesized via a nucleophilic substitution reaction and confirmed by FT-IR. FITC-labeled O-CSLn-Cu could pass through the fungal cell membrane, as detected by confocal laser scanning microscopy (CLSM) coupled with fluorescein isothiocyanate (FITC)-fluorescence. O-CSLn-Cu treatment led to apparent morphological changes in the membranes of P. capsici Leonian and giant unilamellar vesicles (GUVs) by transmission electron microscopy (TEM). Then, we performed component analysis of the cell membrane from the P. capsici Leonian affected by O-CSLn-Cu with a particular interest in membrane physicochemical properties. Many unsaturated fatty acids (UFAs) and key enzymes promoting UFA synthesis of the cell membrane were downregulated. Similarly, a large number of membrane proteins responsible for substance transport and biochemical reactions were downregulated. Furthermore, O-CSLn-Cu treatments increased plasma membrane permeability with significant leakage of intercellular electrolytes, soluble proteins and sugars, and lipid peroxidation with decreasing membrane fluidity. Finally, aquaporin 10 was proven to be a potential molecular target sensitive to antimicrobial agents according to composition analysis of membrane structure and immunohistochemistry.

Benzothiazole inhibits the growth of Phytophthora capsici through inducing apoptosis and suppressing stress responses and metabolic detoxification.

Benzothiazole (BZO) is an antimicrobial secondary metabolite volatilized by many plants and microbes. However, the mechanism of BZO against phytopathogens is still unclear. Here, we found that BZO has antimicrobial activity against the oomycete pathogen Phytophthora capsici. Transcriptome and proteome analyses demonstrated that BZO significantly suppressed the expression of genes and proteins involved in morphology, abiotic stress defense and detoxification, but induced the activity of apoptosis. Annexin V-FITC/PI staining confirmed that the process of apoptosis was significantly induced by BZO at concentration of 150 mg L-1. FITC-phalloidin actin-cytoskeleton staining combined with hyphal cell wall staining and hyphal ultrastructure studies further confirmed that BZO disrupted the cell membrane and hyphal morphology through disrupting the cytoskeleton, eventually inhibiting the growth of hyphae. These data demonstrated that BZO has multiple modes of action and may act as potential leading compound for the development of new oomycete fungicides. These results also showed that the combination of transcriptomic and proteomic approaches was a useful method for exploring the novel antifungal mechanisms of natural compounds.

Phytophthora palmivora establishes tissue-specific intracellular infection structures in the earliest divergent land plant lineage.

The expansion of plants onto land was a formative event that brought forth profound changes to the earth's geochemistry and biota. Filamentous eukaryotic microbes developed the ability to colonize plant tissues early during the evolution of land plants, as demonstrated by intimate, symbiosis-like associations in >400 million-year-old fossils. However, the degree to which filamentous microbes establish pathogenic interactions with early divergent land plants is unclear. Here, we demonstrate that the broad host-range oomycete pathogen Phytophthora palmivora colonizes liverworts, the earliest divergent land plant lineage. We show that P. palmivora establishes a complex tissue-specific interaction with Marchantia polymorpha, where it completes a full infection cycle within air chambers of the dorsal photosynthetic layer. Remarkably, P. palmivora invaginates M. polymorpha cells with haustoria-like structures that accumulate host cellular trafficking machinery and the membrane syntaxin MpSYP13B, but not the related MpSYP13A. Our results indicate that the intracellular accommodation of filamentous microbes is an ancient plant trait that is successfully exploited by pathogens like P. palmivora.

Antifungal Activity of Natural Volatile Organic Compounds against Litchi Downy Blight Pathogen Peronophythora litchii.

Litchi (Litchi chinensis Sonn.) is a commercially important fruit but its production and quality are restricted by litchi downy blight, caused by the oomycete pathogen Peronophythora litchii Chen. Volatile substances produced by a biocontrol antinomycetes Streptomyces fimicarius BWL-H1 could inhibited P. litchii growth and development both in vitro and in detached litchi leaf and fruit infection assay. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) analyses indicated that volatile organic compounds (VOCs) from BWL-H1 resulted in severe damage to the endomembrane system and cell wall of P. litchii cells in vitro and abnormal morphology of appressoria, as well as deformed new hyphae in infection process. VOCs could suppress mycelial growth, sporulation, while with no obvious effect on sporangia germination. Based on gas chromatography-mass spectrophotometric analyses, 32 VOCs were identified from S. fimicarius BWL-H1, the most abundant of which was phenylethyl alcohol. Eight VOCs, including phenylethyl alcohol, ethyl phenylacetate, methyl anthranilate, α-copaene, caryophyllene, humulene, methyl salicylate and 4-ethylphenol, that are commercially available, were purchased and their bioactivity was tested individually. Except for humulene, the other seven tested volatile compounds shown strong inhibitory activity against mycelial growth, sporulation, sporangia germination and germ-tube growth of P. litchii. Especially, 4-ethylphenol showed the highest inhibitory effect on sporulation at a very low concentration of 2 µL/L. Overall, our results provided a better understanding of the mode of action of volatiles from BWL-H1 on P. litchii, and showed that volatiles from BWL-H1 have the potential for control of postharvest litchi downy blight.

Morphological and Genetic Analyses of the Invasive Forest Pathogen Phytophthora austrocedri Reveal that Two Clonal Lineages Colonized Britain and Argentina from a Common Ancestral Population.

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.

Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology, mycelial growth and plant infection.

SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4',6-diamidino-2-phenylindole (DAPI) staining and PcSDA1-green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non-filamentous yeasts and human cells.

In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds against Phytophthora parasitica var. nicotianae.

AIM: To evaluate the antimicrobial effects of essential oils (EOs) from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components, against Phytophthora parasitica var. nicotianae; to investigate morphological changes in hyphae and sporangia in response to treatment with cinnamaldehyde; and to further evaluate potential biocontrol capacities against tobacco black shank under greenhouse conditions. METHODS AND RESULTS: The results revealed that the extent of mycelial growth inhibition was primarily dependent on the composition and concentration of the EOs and the structure of individual compounds. Cinnamaldehyde had a significantly higher inhibitory effect on mycelial growth, formation of sporangia, and production and germination of zoospores in P. parasitica var. nicotianae in vitro, achieving complete inhibition of these phenotypes at 72, 36, 36 and 18 mg l(-1), respectively. Scanning electron microscopic observations revealed that cinnamaldehyde can cause considerable morphological degenerations of hyphae and sporangia such as cytoplasmic coagulation, shrivelled mycelia and sporangia aggregates and swelling and lysis of mycelia and sporangia walls. In vivo assays with cinnamaldehyde demonstrated that this compound afforded protective effect against tobacco black shank under greenhouse conditions in susceptible tobacco plants. CONCLUSIONS: The results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a theoretical basis for the potential use of cinnamaldehyde as commercial agents or lead compounds that can be exploited as commercial biofungicides in the protection of tobacco plants from P. parasitica var. nicotianae infection.

Phytophthora borealis and Phytophthora riparia, new species in Phytophthora ITS Clade 6.

Phytophthora borealis and Phytophthora riparia, identified in recent Phytophthora surveys of forest streams in Oregon, California and Alaska, are described as new species in Phytophthora ITS Clade 6. They are similar in growth form and morphology to P. gonapodyides and are predominantly sterile. They present unique DNA sequences, however, and differ in temperature/growth relations and geographic distribution.

Phytophthora xserendipita sp. nov. and P. xpelgrandis, two destructive pathogens generated by natural hybridization.

The first natural hybrids in the genus Phytophthora were described in 1998, and they were the result of hybridization between P. nicotianae and P. cactorum. They were described formally as Phytophthora × pelgrandis in 2009. In 2007 a second type of P. cactorum hybrid species was described, generated by hybridization between P. hedraiandra and P. cactorum; it is described formally here as P. × serendipita sp. nov. The morphological description of P. ×pelgrandis was incomplete and here we also add several important diagnostic characters of P. × pelgrandis that were not in its original description. In addition, ITS-SSCP profiles are presented confirming the hybrid identity of both P. × pelgrandis and P. × serendipita.

Phytophthora aquimorbida sp. nov. and Phytophthora taxon 'aquatilis' recovered from irrigation reservoirs and a stream in Virginia, USA.

Two distinct subgroups (L2 and A(-2)) were recovered from irrigation reservoirs and a stream in Virginia, USA. After molecular, morphological and physiological examinations, the L2 subgroup was named Phytophthora aquimorbida and the A(-2) designated as Phytophthora taxon 'aquatilis'. Both taxa are homothallic. P. aquimorbida is characterized by its noncaducous and nonpapillate sporangia, catenulate and radiating hyphal swellings and thick-walled plerotic oospores formed in globose oogonia mostly in the absence of an antheridium. P. taxon 'aquatilis' produces plerotic oospores in globose oogonia mostly with a paragynous antheridium. It has semi-papillate, caducous sporangia with variable pedicels, but it does not have hyphal swelling. Analyses of ITS, CO1, ß-tubulin and NADH1 sequences revealed that P. aquimorbida is closely related to P. hydropathica, P. irrigata and P. parsiana, and P. taxon 'aquatilis' is related to P. multivesiculata. The optimum temperature for culture growth is 30 and 20 C for P. aquimorbida and P. taxon 'aquatilis' respectively. Both taxa were pathogenic to rhododendron plants and caused root discoloration, pale leaves, wilting, tip necrosis and dieback. Their plant biosecurity risk also is discussed.

Study of inhibitory effects and action mechanism of the novel fungicide pyrimorph against Phytophthora capsici.

The antifungal activity of the novel fungicide pyrimorph, (E)-3-[(2-chloropyridine-4-y1)-3-(4-tert-butylpheny1)acryloyl]morpholin, against Phytophthora capsici was investigated in vitro. Pyrimorph inhibited different stages in the life cycle of P. capsici including mycelial growth, sporangium production, zoospore release, and cystospore germination with EC(50) values of 1.84, 0.17, 4.92, and 0.09 microg mL(-1), respectively. The effect of pyrimorph on mycelial growth was reduced by the addition of different concentrations of ATP, which suggested that the action mechanism of pyrimorph was connected with impairment of the energy generation system. Meanwhile, pyrimorph exhibited certain inhibition on metabolic approaches of Embden-Meyerhof-Parnas (EMP), tricarboxylic acid cycle (TCA), and hexosemonophosphate (HMP) by measuring the oxygen consumption of pyrimorph combining with three representative inhibitors to the metabolic approaches. The results indicated that pyrimorph could inhibit the approach of HMP significantly. Morphological and ultrastructural studies showed that pyrimorph caused excessive septation and swelling of hyphae, distortion and disruption of most vacuoles, thickening and development a multilayer cell wall, and accumulation of dense bodies. These results suggested pyrimorph exhibited multiple modes of action including impairment of the energy generation system and effect on cell wall biosynthesis directly or indirectly.

Two new Phytophthora species from South African Eucalyptus plantations.

A recent study to determine the cause of collar and root rot disease outbreaks of cold tolerant Eucalyptus species in South Africa resulted in the isolation of two putative new Phytophthora species. Based on phylogenetic comparisons using the ITS and beta-tubulin gene regions, these species were shown to be distinct from known species. These differences were also supported by robust morphological characteristics. The names, Phytophthora frigida sp. nov. and Phytophthora alticola sp. nov. are thus provided for these taxa, which are phylogenetically closely related to species within the ITS clade 2 (P. citricola, P. tropicali and P.multivesiculata) and 4 (P. arecae and P. megakarya), respectively. Phytophthora frigida is heterothallic, and produces stellate to rosaceous growth patterns on growth medium, corraloid hyphae, sporangia with a variety of distorted shapes and has the ability to grow at low temperatures. Phytophthora alticola is homothallic and has a slower growth rate in culture. Both P. frigida and P. alticola are pathogenic to Eucalyptus dunnii. In pathogenicity tests, they were, however, less pathogenic than P. cinnamomi, which is a well-known pathogen of Eucalyptus in South Africa.

[Heteroplasmosis in Phytophthora infestans].

APCR and monozoospore plating was used to demonstrate a simultaneous presence of the mitochondria with mitochondrial DNA of haplotypes ILa and IIa in the mycelium of several Phytophthora infestans strains.

Phytophthora siskiyouensis, a new species from soil, water, myrtlewood (Umbellularia californica) and tanoak (Lithocarpus densiflorus) in southwestern Oregon.

An unknown Phytophthora species was recovered in southwestern Oregon from rhododendron and tanoak leaf baits used for monitoring streams and soils for the presence of Phytophthora ramorum, from a blighted shoot of myrtlewood and from tanoak bark cankers. Isolates of this species yielded ITS-DNA sequences that differed substantially from other Phytophthora sequences in GenBank. Morphological features also differed from available descriptions of known Phytophthora species. Based on the combination of unique morphology and unique ITS sequences a new species is proposed. The new species, Phytophthora siskiyouensis, is homothallic with globose to subglobose oogonia, which may be terminal, sessile or laterally intercalary. Antheridia are capitate and mostly paragynous but sometimes amphigynous. Oospores are mostly aplerotic. Sporangia are variable but commonly ovoid to reniform, with apical, subapical or lateral semipapillae (occasionally more than one). Sporangia are terminal, subterminal or occasionally intercalary on unbranched sporangiophores, with basal, subbasal or lateral attachment. Sporangia are weakly deciduous, with variable length pedicels. This combination of characters clearly separates Phytophthora siskiyouensis from other known Phytophthora species.

Stromata, sporangiomata and chlamydosori of Phytophthora ramorum on inoculated Mediterranean woody plants.

Three types of multihyphal structures, stromata, sporangiomata and chlamydosori, are described for the plant pathogen Phytophthora ramorum. Their morphology, morphogenesis and position on the host organ were observed by dissecting, compound and scanning electron microscopy. Stromata were consistently formed one to two weeks after zoospore inoculation of detached leaves and fruits of an assortment of Mediterranean sclerophyll shrubs. Stroma initials appeared subcuticularly or subepidermally and developed as small hyphal aggregates by repeated branching, budding, swelling and interweaving, eventually forming a prosenchyma. They always emerged through the adaxial side of the leaf by rupture of the overlying host tissue. Occasionally sporangia and chlamydosori (packed clusters of chlamydospores) were formed on the stromata. Sporangiomata bore short sporangiophores and clusters of 20-100 sporangia and resembled sporodochia of the mitosporic fungi. The biological significance of these multihyphal structures is discussed. Some epidemiological aspects were also studied: several understorey species of the holm oak (Quercus ilex) woodland were susceptible to in vitro infection with three isolates of P. ramorum originally collected from different ornamental hosts. The risk of spread to this ecosystem is evaluated.

Phytophthora captiosa sp. nov. and P. fallax sp. nov. causing crown dieback of Eucalyptus in New Zealand.

A locally severe crown disease of exotic plantation Eucalyptus trees has been recorded periodically in New Zealand since 1986. Symptoms include leaf spots, petiole infection and twig and small branch lesions. Outbreaks of disease are episodic and individual trees may show marked variation in crown symptoms ranging from unaffected to total defoliation. Two previously unknown species of Phytophthora are associated with the disease. These are described and formally designated here as P. captiosa, from Eucalyptus botryoides and E. saligna; and P. fallax, from E. delegatensis, E. fastigata, E. nitens and E. regnans. Both P. captiosa and P. fallax have non-papillate, non-caducous sporangia and both are self-fertile. Phylogenetic analysis on the basis of ITS rDNA sequence data indicates they are closely related to each other but evolutionarily distant from the majority of described Phytophthora taxa. They share a common ancestor with another assemblage of Phytophthora lineages that includes P. insolita, P. macrochlamydospora and P. richardiae. Sporulation of P. captiosa and P. fallax has not been observed in the field. The mode of infection and spread of these non-caducous Phytophthora species in the eucalypt tree canopy remains unknown. This issue, and the possible geographic origins of these two Phytophthora species are discussed.

Phytophthora nicotianae PnPMA1 encodes an atypical plasma membrane H+ -ATPase that is functional in yeast and developmentally regulated.

PnPMA1, a gene encoding a putative P-type plasma membrane H(+)-ATPase, has been isolated by differential screening of a Phytophthora nicotianae germinated cyst cDNA library. PnPMA1 is differentially expressed during pathogen asexual development with a more than 10-fold increase in expression in germinated cysts, the stage at which plant infection is initiated, compared to vegetative or sporulating hyphae or motile zoospores. PnPMA1 proteins are encoded by two closely linked genes that have no introns and encode identical proteins having 1,068 amino acid residues and a molecular mass of 116.3kDa. PnPMA1 shows moderate identity (30-50%) to plant and fungal plasma membrane H(+)-ATPases and weak identity to other P-type cation-transporting ATPases. PnPMA1 contains all the catalytic domains characteristic of H(+)-ATPases but also has a distinct domain of approximately 155 amino acids that forms a putative cytoplasmic loop between transmembrane domains 8 and 9, a feature that is not present in PMA1 proteins from other organisms. Polyclonal antibodies raised against the 155 residue domain were shown by immunogold labelling to react with a protein in the plasma membrane of P. nicotianae germinated cysts but not with the plasma membrane of motile zoospores. Genetic complementation experiments demonstrated that the P. nicotianae PnPMA1 is functional in yeast, Saccharomyces cerevisiae.

Invasive hyphal growth: an F-actin depleted zone is associated with invasive hyphae of the oomycetes Achlya bisexualis and Phytophthora cinnamomi.

We have compared F-actin patterns in invasive and non-invasive oomycete hyphae. In Achlya bisexualis an F-actin depleted zone is present in 70% of invasive but only 9% of non-invasive hyphae. In Phytophthora cinnamomi these figures are 74 and 20%, respectively. Thus, the F-actin depleted zone appears to be associated with invasive growth. TEM images indicate that it is unlikely to represent areas of vesicle accumulation. Measurements of turgor indicate no significant increase under invasive conditions (0.65 MPa (invasive) and 0.63 MPa (non-invasive)). Similarly we found no difference in burst pressures (1.04 MPa (invasive) and 1.06 MPa (non-invasive)), although surrounding agarose may lead to overestimates of invasive tip strength. An F-actin depleted zone has the potential, along with wall softening, to increase protrusive force in the absence of turgor increases. Staining of F-actin in hyphae under hyperosmotic conditions suggests that decreases in F-actin at growing tips may also enable non-invasive growth at very low turgor.

Antimicrobial activities of the essential oils of various plants against tomato late blight disease agent Phytophthora infestans.

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of devastating oomycete pathogen Phytophthora infestans, causal agent of late blight disease of tomato. Antifungal activities of essential oils obtained from aerial parts of aromatic plants such as oregano (Origanum syriacum var. bevanii), thyme (Thymbra spicata subsp. spicata), lavender (Lavandula stoechas subsp. stoechas), rosemary (Rosmarinus officinalis), fennel (Foeniculum vulgare), and laurel (Laurus nobilis), were investigated against P. infestans. Both contact and volatile phase effects of different concentrations of the essential oils used were determined by using two in vitro methods. Chemical compositions of the essential oils were also determined by GC-MS analysis. Major compounds found in essential oils of thyme, oregano, rosemary, lavender, fennel and laurel were carvacrol (37.9%), carvacrol (79.8), borneol (20.4%), camphor (20.2%), anethole (82.8%) and 1,8-cineole (35.5%), respectively. All essential oils were found to inhibit the growth of P. infestans in a dose-dependent manner. Volatile phase effect of oregano and thyme oils at 0.3 microg/ml air was found to completely inhibit the growth of P. infestans. Complete growth inhibition of pathogen by essential oil of fennel, rosemary, lavender and laurel was, however, observed at 0.4-2.0 microg/ml air concentrations. For the determination of the contact phase effects of the tested essential oils, oregano, thyme and fennel oils at 6.4 microg/ml were found to inhibit the growth of P. infestans completely. Essential oils of rosemary, lavender and laurel were inhibitory at relatively higher concentrations (12.8, 25.6, 51.2 microg/ml respectively). Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. Sporangial production was also inhibited by the essential oil tested. Light and scanning electron microscopic (SEM) observation on pathogen hyphae, exposed to both volatile and contact phase of oil, revealed considerable morphological alterations in hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage.

Plant-induced cell death in the oomycete pathogen Phytophthora parasitica.

The activation of programmed cell death in the host during plant-pathogen interactions is an important component of the plant disease resistance mechanism. In this study we show that activation of programmed cell death in microorganisms also regulates plant-pathogen interactions. We found that a form of vacuolar cell death is induced in the oomycete Phytophthora parasitica--the agent that causes black shank disease in Nicotiana tabacum--by extracellular stimuli from resistant tobacco. The single-celled zoospores underwent cell death characterized by dynamic membrane rearrangements, cell shrinkage, formation of numerous large vacuoles in the cytoplasm and degradation of cytoplasmic components before plasma membrane disruption. Phytophthora cell death required protein synthesis but not caspase activation, and was associated with the production of intracellular reactive oxygen species. This characterization of plant-mediated cell death signalling in pathogens will enhance our understanding of the biological processes regulating plant-pathogen interactions, and improve our ability to control crop diseases.