The bacterial mechanosensitive channels MscS and MscL have been shown to protect cells from hypo-osmotic shock-induced lysis. Bacterial strains deficient for MscS and MscL are severely compromised and fail to survive a hypo-osmotic shock. Both channels exhibit redundant function such that re-introduction of either of these proteins is sufficient to restore cell survival. Several proteins paralogous to MscS have been identified in E. coli, but their function remains unknown. Mechanosensitive channel homologues are also being discovered in a variety of organisms including Archaea, plants and fungi and their function is starting to emerge.

Thesis (Ph. D.)--Ohio State University, 2005. / Document formatted into pages; contains 215 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 March 3.

Thesis (M.S.)--University of North Carolina at Wilmington, 2005. / Includes bibliographical references (leaves: [13]-17)

Les oomycètes phytopathogènes ont co-évolué avec les microbiotes des plantes hôtes. Il en résulte la formation de biofilms et des réseaux complexes d’interactions dont nous commençons juste à comprendre l’incidence sur la biologie et la virulence des oomycètes. Déterminer la nature de ces interactions et leur rôle dans le contexte d’une infection est aujourd’hui un enjeu cognitif qui concerne la caractérisation des mécanismes moléculaires et communautaires sous-jacents. C’est également une opportunité en termes d’innovation pour élaborer des méthodes de lutte alternative à l’usage de fongicides. Dans ce contexte, ce travail de thèse a consisté à étudier, d’une part, la formation de biofilm chez Phytophthora parasitica, un oomycète polyphage et tellurique, ainsi que, d’autre part, les interactions au sein de ce biofilm entre P. parasitica et le microbiote procaryote rhizosphérique de la plante hôte Solanum lycopersicum. L’analyse du génome de P. parasitica et du transcriptome du biofilm a conduit à la caractérisation d’une nouvelle famille de mucines restreinte à la lignée des oomycètes, les protéines MUCL. Ces 315 protéines sécrétées (25-30 kDa) sont réparties en 15 groupes et possèdent deux domaines : un domaine hautement conservé de fonction inconnue ; un domaine caractéristique des mucines, riche en résidus Sérine et Thréonine avec de très nombreux sites putatifs d’O-glycosylation. Chez P. parasitica, les 3 gènes PPMUCL1/2/3 sont exprimés et co-régulés spécifiquement au stade biofilm. / The interactions between a pathogen and the host surface resident microbiota are critical to disease outbreak. These interactions shape the distribution, the density and the genetic diversity of inoculum. However for most plant pathogens how each of these interactions acts on disease as a single one or as a member of a functional network remains to be specified. This issue is addressed here through the analysis of two types of interactions involving the polyphagous oomycete P.parasitica : (i) the intraspecific interaction that leads to monospecific biofilm formation by P. parasitica zoospores on plant surface; (ii) the interspecific interactions that occur between P. parasitica biofilm and the prokaryotic microbiota of Solanum lycopersicum rhizosphere. The biology of monospecific biofilm is investigated through the characterization of MUCL, a new oomycete-specific Mucin-like Protein family. Gene profiling, biochemical and immunohistological analyses define the extent of this family and lead to identify three members, PPMUCL1/2/3, as residing in P. parasitica biofilm. The Phytophthora parasitica-Microbiota interaction is explored using first a metagenomic approach. Two microbial metagenomes derived from a soil of a tomato greenhouse is defined and compared after 16S RNA gene sequencing: M1 which corresponds to the sub-rhizospheric microbiota able to colonize the roots of axenic tomato seedlings; M2, the sub-microbiota able to colonize the tomato seedling roots previously coated with P. parasitica monospecific biofilm. A representative collection of microorganisms from M2 were also obtained through in vitro selection on a medium prepared from P. parasitica extract.

International trade in plants, especially with potting substrates, is recognised as the main pathway of plant pathogen dissemination on a global scale. In the last 20 years, the wide use of internet commerce has become common in the nursery sector and, due to the nature of online sales, may be aggravating this risk. Oomycetes in the genera Phytophthora, Pythium and Phytopythium, cause a range of important plant diseases, responsible for serious economic and biological losses. This research focused on the detection of Oomycetes in imported potted ornamental plants in the UK and The Netherlands, including internet sales and asymptomatic plants. Isolation techniques and molecular protocols were developed to quantify pathogen load in ornamental plants, using TaqMan PCR and Next Generation Sequencing (NGS) to assess Oomycete diversity using a multi-locus approach. Survival of Phytophthora cinnamomi and Fusarium verticillioides was estimated in two commercial potting mixes used in ornamental plant production. Oomycetes were detected in all samples analysed with the NGS approach, with 38 Phytophthora spp. and 48 Pythium/Phytopythium spp. identified. Phytophthora ramorum, P. alni subsp. alni and P. cryptogea were common. TaqMan PCR quantification showed high numbers of Oomycetes in all samples, especially in substrates, followed by roots and baiting waters. During sampling by isolation, Pythium kashmirense was recovered from Viburnum plicatum, the first record of this species in the UK. The survival experiment showed that Fusarium verticillioides remained viable after 17 months, whereas Phytophthora cinnamomi was viable up to 7 months after inoculation. This work clearly demonstrated the widespread presence of Oomycete pathogens in the plants for planting pathway. Moreover, the protocols developed and findings of this work contribute greatly to the understanding of the potential for pathogens to spread in the international horticultural trade and may help to improve plant biosecurity protocols in the UK and Europe.

Oomycetes are eukaryotic fungus-like microorganisms that are known to be pathogenic to plants and animals, causing both ecological and economic damage. Animal pathogenic oomycetes, such as Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Phytophthora species cause a number of plant diseases. Pythium are less understood as many of these species are saprotrophs which are not thought to be pathogenic. Oomycete research has focussed mainly towards the characterisation and control of pathogenic species with very little information on the ecology of Oomycetes. In this thesis, oomycete species were isolated from water samples collected from rivers in Scotland and Northern England. Baiting and water filtration were used to compare the reliability of both methods, and nested PCR was used to compare the number and types of species obtained in comparison to conventional culturing methodologies. The results suggest that water filtration provided a fast, reliable method for isolating abundant, hardier species such as Pythium undulatum, Saprolegnia diclina, Saprolegnia delica, and Saprolegnia ferax. Water chemistry, surrounding land use, seasonality and land elevation were found to be important factors in Oomycete diversity. Nested PCR of oomycetes directly from filters to obtain target DNA from organisms which are more difficult to culture or less numerous proved to be useful for some species, but will need more refinement of primer and methodologies to obtain species of interest. Sampling of the rivers resulted in the isolation of three new Pythium spp. isolated from the Rivers Spey and Dochart in Scotland. Phylogenetic analysis, infection studies, growth rates and microscopy were used to characterise these three species.

Tesis por compendio / [ES] Phytophthora es uno de los géneros fitopatógenos más relevantes y agresivos en la agricultura y silvicultura. Muestreos realizados en la última década han revelado una gran cantidad de interacciones entre especies de Phytophthora y plantas, desconocidas con anterioridad. La introducción de nuevos patógenos de suelo, como Phytophthora en los bosques de Fagaceae, modifica la comunidad microbiana presente en la rizosfera, con importantes consecuencias ambientales y económicas. El género Quercus es uno de los géneros de Fagaceae más extendidos en Europa, y Quercus ilex es la especie dominante en España. El vínculo entre la dispersión de Phytophthora en los ecosistemas naturales y las actividades del ser humano, ha sido previamente estudiado. Numerosos muestreos en viveros y espacios públicos, mostraron la presencia de gran diversidad de especies de Phytophthora que podían suponer una amenaza para la producción y los ecosistemas naturales. En este contexto, se realizó un muestreo de viveros ornamentales y forestales en cuatro comunidades autónomas españolas, centrándose en los posibles síntomas asociados a Phytophthora en diferentes hospedantes e incluyendo muestras de agua de los viveros. Los resultados mostraron 17 filotipos de Phytophthora que afectan a 22 especies vegetales incluidas en 19 géneros. Algunas de estas interacciones se citaron por primera vez en España. Entre los patógenos de suelo aislados en los viveros, se identificó una gran cantidad de formas asexuales tipo Cylindrocarpon en las raíces de hospedantes leñosos. Se caracterizó una colección de aislados mediante estudios morfológicos y moleculares. Se identificaron 12 especies pertenecientes a los géneros Cylindrodendrum, Dactylonectria e Ilyonectria en hospedantes pertenecientes a 15 géneros y otras cuatro nuevas especies se describieron. El estudio demostró la prevalencia de este grupo fúngico asociado con plántulas de diversos hospedantes que muestran síntomas de decaimiento en viveros forestales. Se evaluó la susceptibilidad de Q. ilex a la inoculación con ocho especies de Phytophthora obtenidas de muestreos en viveros. Las especies más agresivas fueron Phytophthora cinnamomi, Phytophthora cryptogea, Phytophthora gonapodyides, Phytophthora plurivora y Phytophthora psychrophila, seguidas de Phytophthora megasperma, mientras que Phytophthora quercina y Phytophthora nicotianae fueron las especies menos agresivas. Los resultados obtenidos en el ensayo de patogenicidad confirmaron que todas las especies de Phytophthora evaluadas podrían representar una amenaza para los encinares. En este contexto, se realizó un estudio para verificar la presencia y / o detección de especies de Phytophthora en dos áreas de España (dehesas del sudoeste y bosque del noreste) utilizando diferentes métodos de aislamiento y detección. El aislamiento directo y el método de trampeo vegetal en muestras obtenidas a partir de encinas con y sin decaimiento, identificaron Phytophthora cambivora, P. cinnamomi, P. gonapodyides, P. megasperma y Phytophthora pseudocryptogea en las dehesas, mientras que, en el bosque del noreste, no se aisló Phytophthora spp. Los análisis estadísticos indicaron que no había una relación significativa entre la frecuencia de aislamiento de las especies de Phytophthora y la expresión de los síntomas de la enfermedad en las encinas de las dehesas. Además, P. quercina se detectó con mayor frecuencia que P. cinnamomi en las dos áreas estudiadas utilizando sondas TaqMan de PCR a tiempo real. Se evaluaron seis masas de Q. ilex ubicadas en tres comunidades autónomas de España mediante “Next Generation Sequencing” (NGS) para tener un mayor conocimiento sobre la diversidad de Phytophthora spp. en los bosques de encinas. Se detectaron 37 filotipos de Phytophthora pertenecientes a los clados 1 al 12, excepto los clados 4, 5 y 11, lo que demuestra una gran diversidad de Phytophthora en los encinares estudiados. Los filotipos más abundantes fueron P. quercina, P. psychrophila, P. cinnamomi y P. plurivora. / [EN] Phytophthora is one of the most relevant and aggressive plant pathogenic genus in agriculture and forestry. Due to the increasing environmental threat of invasive plant pathogens, monitoring new areas in the last decade has revealed a large number of new Phytophthora species-plant host interactions. The introduction of soilborne pathogens, such as Phytophthora in Fagaceae forests modifies the microbial community present in the rhizosphere with relevant environmental and economic consequences. The genus Quercus is one of the most extended Fagaceae genera in Europe, and Q. ilex is the dominant tree in Spain. The link between Phytophthora dispersion in natural ecosystems and human derived activities has been previously studied. Numerous sampling in nurseries and public spaces revealed a great diversity of Phytophthora species that compromised production and threated natural ecosystems. In this context, sampling ornamental and forest nurseries in four Spanish regions was carried out focusing on possible symptoms associated to Phytophthora on different hosts and including water samples from the nurseries. The results showed 17 Phytophthora phylotypes affecting 22 plant species included in 19 plant genera and some of them reported for the first time in Spain. Among the soilborne pathogens isolated in the nurseries, a large number of Cylindrocarpon-like asexual morphs were identified from the roots of woody hosts. A collection of Cylindrocarpon-like isolates recovered from Spanish nurseries was characterised by morphological and molecular studies. Twelve species belonging to the genera Cylindrodendrum, Dactylonectria and Ilyonectria were identified from damaged roots of 15 different host genera and other four species were newly described. The study demonstrated the prevalence of this fungal group associated with seedlings of diverse hosts showing decline symptoms in forest nurseries in Spain. The susceptibility of Quercus ilex to the inoculation with eight Phytophthora species obtained from nurseries was evaluated. The most aggressive species were Phytophthora cinnamomi, Phytophthora cryptogea, Phytophthora gonapodyides, Phytophthora plurivora and Phytophthora psychrophila followed by Phytophthora megasperma, while Phytophthora quercina and Phytophthora nicotianae were the least aggressive species. Results obtained in the pathogenicity test confirmed that all Phytophthora species tested could represent a threat to holm oak ecosystems. In this context, a study to verify the presence and/or detection of Phytophthora species was conducted in two holm oak areas of Spain (southwestern dehesas and northeastern woodland) using different isolation and detection methods. Direct isolation and baiting methods in declining and non-declining holm oak trees revealed Phytophthora cambivora, P. cinnamomi, P. gonapodyides, P. megasperma, and Phytophthora pseudocryptogea in the dehesas, while in the northeastern woodland, no Phytophthora spp. were recovered. Statistical analyses indicated that there was not a significant relationship between the Phytophthora spp. isolation frequency and the disease expression of the holm oak stands in the dehesas. Phytophthora quercina and P. cinnamomi TaqMan real-time PCR probes showed that P. quercina was detected in a higher frequency than P. cinnamomi in both studied areas. A better understanding of the Phytophthora spp. diversity in holm oak forests was assessed using Next Generation Sequencing (NGS) in six Q. ilex stands located in three regions in Spain. Thirty-seven Phytophthora phylotypes belonging to clades 1 to 12, except for clades 4, 5 and 11, were detected in this study, demonstrating a high diversity of Phytophthora species in holm oak Spanish forests. The most abundant phylotypes were P. quercina, P. psychrophila, P. cinnamomi and P. plurivora. In summary, this Thesis demonstrated a high diversity of Phytophthora and Cylindrocarpon-like species in Spanish nurseries, reporting new pathogen-plant host in / [CA] Phytophthora és un dels gèneres fitopatògens més rellevants i agressius en l'agricultura i la silvicultura. Mostrejos realitzats en l'última dècada han revelat una gran quantitat d'interaccions desconegudes fins ara entre espècies de Phytophthora i plantes. La introducció de nous patògens del sòl, com Phytophthora en els boscos de Fagaceae, modifica la comunitat microbiana present en la rizosfera, amb importants conseqüències ambientals i econòmiques. El gènere Quercus és un dels gèneres de Fagaceae més estesos a Europa, i Quercus ilex és l'espècie dominant a Espanya. El vincle entre la dispersió de Phytophthora en els ecosistemes naturals i les activitats de l'ésser humà, ja ha sigut estudiat prèviament. Nombrosos mostrejos en vivers i espais públics, van mostrar la presència d'una gran diversitat d'espècies de Phytophthora que podien suposar una amenaça per a la producció i els ecosistemes naturals. En aquest context, es va realitzar un mostreig de vivers ornamentals i forestals en quatre comunitats autònomes espanyoles, centrant-se en els possibles símptomes associats a Phytophthora en diferents hostes i incloent mostres d'aigua dels vivers. Els resultats van mostrar 17 filotipus de Phytophthora que afecten 22 espècies vegetals incloses en 19 gèneres. Algunes d'aquestes 'interaccions es van citar per primera vegada a Espanya. Entre els patògens del sòl aïllats en els vivers, es va identificar una gran quantitat de formes asexuals tipus Cylindrocarpon en les arrels de plantes llenyoses. Es va caracteritzar una col·lecció d'aïllats mitjançant estudis morfològics i moleculars. Es van identificar 12 espècies pertanyents als gèneres Cylindrodendrum, Dactylonectria i Ilyonectria en hostes pertanyents a 15 gèneres i altres quatre noves espècies es van descriure. L'estudi va demostrar la prevalença d'aquest grup fúngic associat amb plàntules de diversos hostes que mostren símptomes de decaïment en vivers forestals espanyols. Es va avaluar la susceptibilitat de Q. ilex a la inoculació amb huit espècies de Phytophthora obtingudes de mostrejos en vivers. Les espècies més agressives van ser Phytophthora cinnamomi, Phytophthora cryptogea, Phytophthora gonapodyides, Phytophthora plurivora i Phytophthora psychrophila, seguides de Phytophthora megasperma, mentre que Phytophthora quercina i Phytophthora nicotianae van ser les espècies menys agressives. Els resultats obtinguts en l'assaig de patogenicitat van confirmar que totes les espècies de Phytophthora avaluades podrien representar una amenaça per als ecosistemes d'alzines. En aquest context, es va realitzar un estudi per a verificar la presència i / o detecció d'espècies de Phytophthora en dues àrees d'Espanya (deveses del sud-oest i bosc del nord-est) utilitzant diferents mètodes d'aïllament i detecció. L'aïllament directe i el mètode de parany vegetal en mostres obtingudes a partir d'alzines, amb i sense decaïment, van identificar Phytophthora cambivora, P. cinnamomi, P. gonapodyides, P. megasperma i Phytophthora pseudocryptogea en les deveses, mentre que, en el bosc del nord-est, no es va aïllar Phytophthora spp. Les anàlisis estadístiques van indicar que no hi havia una relació significativa entre la freqüència d'aïllament de les espècies de Phytophthora i l'expressió dels símptomes de la malaltia en les alzines en les deveses. A més, P. quercina es va detectar amb major freqüència que P. cinnamomi en les dues àrees estudiades utilitzant sondes TaqMan de PCR a temps real. Es van avaluar sis masses de Q. ilex situades en tres comunitats autònomes d'Espanya mitjançant "Next Generation Sequencing" (NGS) per a tindre un major coneixement sobre la diversitat de Phytophthora spp. en els boscos d'alzines. Es van detectar 37 filotipus de Phytophthora pertanyents als clades 1 al 12, excepte els clades 4, 5 i 11, la qual cosa demostra una gran diversitat d'espècies de Phytophthora en els alzinars estudiats. Els filotipus més abundants van ser P. quercina, P. psychrophila, P. cinnamomi i P. plurivora. / Mora Sala, B. (2020). Detection and identification of Phytophthora spp. in woody plant nurseries and holm oak forests [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153808 / TESIS / Compendio

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Oomycetes are fungi-like eukaryotic microorganisms, which are actually phylogenetic relatives of diatoms and brown algae, within the kingdom Stramenopila. Many oomycete species, mainly in the genera Phytophthora, Pythium and downy mildews, are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Some oomycetes are also animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. Phytophthora species, represented by the Irish Potato Famine pathogen P. infestans and the soybean pathogen P. sojae, are arguably the most destructive pathogens of dicotyledonous plants among the oomycete species and thus have been extensively studied. This dissertation focuses on the model oomycete pathogen P. sojae to investigate specific aspects of its molecular biology and establish an efficient genetic manipulation tool. Specifically, in Chapter 1, I briefly introduce the basic concepts of oomycete biology and pathology, and summarize the experimental techniques used for studies of oomycete genetics over the past two decades. Because the approach to studying fungi and oomycetes are similar (indeed they were incorrectly placed in the same taxonomic group until recently), a special section reviews the emerging genome editing technology CRISPR/Cas system in these organisms together. Chapter 2 and Chapter 3 focus on one of the most important intracellular activities, nuclear localization of proteins, and describe the characterization of nuclear localization signals (NLSs) in P. sojae. This focus stemmed from my early work on genome editing in P. sojae, when I discovered that conventional NLS signals from SV40 used to target the TAL effector nuclease (TALEN) to the nucleus worked poorly in P. sojae. In the first part of this work (Chapter 2), I used confocal microscopy to identify features of nuclear localization in oomycetes that differ from animals, plants and fungi, based on characterization of two classes of nuclear localization signals, cNLS and PY-NLS, and on characterization of several conserved nuclear proteins. In the second part (Chapter 3), I determined that the nuclear localization of the P. sojae bZIP1 transcription factor is mediated by multiple weak nuclear targeting motifs acting together. In Chapter 4 and Chapter 5, I describe my implementation of nuclease-based technology for genetic modification and control of P. sojae. In Chapter 4, I describe the first use of the CRISPR system in an oomycete, including its use to validate the function of a host specificity gene. This is of particular importance because molecular techniques such as gene knockouts and gene replacements, widely used in other organisms, were not previously possible in oomycetes. The successful implementation of CRISPR provides a major new research capability to the oomycete community. Following up on the studies described in Chapter 4, in Chapter 5, I describe the generalization and simplification of the CRISPR/Cas9 expression strategy in P. sojae as well as methods for mutant screening. I also describe several optimized methodologies for P. sojae manipulation based on my 5 years of experience with P. sojae. / Ph. D.