Phytophthora cinnamomi

The phylum Oomycota comprises important tree pathogens like Phytophthora quercina, involved in central European oak decline, and Phytophthora cinnamomi shown to affect holm oaks among many other hosts. Despite the importance to study the distribution, dispersal and niche partitioning of this phylum, metabarcoding surveys, and studies considering environmental factors that could explain oomycete community patterns are still rare. We investigated oomycetes in the rhizosphere of evergreen oaks in a Spanish oak woodland using metabarcoding based on Illumina sequencing of the taxonomic marker cytochrome c oxidase subunit II (cox2). We developed an approach amplifying a 333 bp long fragment using the forward primer Hud-F (Mycologia, 2000) and a reverse primer found using DegePrime (Applied and Environmental Microbiology, 2014). Factors reflecting topo-edaphic conditions and tree health were linked to oomycete community patterns. The majority of detected OTUs belonged to the Peronosporales. Most taxa were relatives of the Pythiaceae, but relatives of the Peronosporaceae and members of the Saprolegniales were also found. The most abundant OTUs were related to Globisporangium irregulare and P. cinnamomi, both displaying strong site-specific patterns. Oomycete communities were strongly correlated with the environmental factors: altitude, crown foliation, slope and soil skeleton and soil nitrogen. Our findings illustrate the significance of small scale variation in habitat conditions for the distribution of oomycetes and highlight the importance to study oomycete communities in relation to such ecological patterns. Members of the phylum Oomycota are globally distributed and adapted to a large variety of ecosystems . Although oomycetes display a great variety of lifestyles, it is thought that they are largely adapted to a pathogenic lifestyle given the high number of known pathogens . The phylum contains many well-known plant pathogens including the causative agent of sudden oak death, Phytophthora ramorum (Grünwald, Garbelotto, Goss,

The genetic diversity of Phytophthora spp. was investigated in potted ornamental and fruit tree species. A metabarcoding approach was used, based on a semi-nested PCR with Phytophthora genus-specific primers targeting the ITS1 region of the rDNA. More than 50 ITS1 sequence types representing at least 15 distinct Phytophthora taxa were detected. Nine had ITS sequences that grouped them in defined taxonomic groups (P. nicotianae, P. citrophthora, P. meadii, P. taxon Pgchlamydo, P. cinnamomi, P. parvispora, P. cambivora, P. niederhauserii and P. lateralis) whereas three phylotypes were associated to two or more taxa (P. citricola taxon E or III; P. pseudosyringae, P. ilicis or P. nemorosa; and P. cryptogea, P. erythroseptica, P. himalayensis or P. sp. 'kelmania') that can be challenging to resolve with ITS1 sequences alone. Three additional phylotypes were considered as representatives of novel Phytophthora taxa and defined as P. meadii-like, P. cinnamomi-like and P. niederhauserii-like. Furthermore, the analyses highlighted a very complex assemblage of Phytophthora taxa in ornamental nurseries within a limited geographic area and provided some indications of structure amongst populations of P. nicotianae (the most prevalent taxon) and other taxa. Data revealed new host-pathogen combinations, evidence of new species previously unreported in Italy (P. lateralis) or Europe (P. meadii) and phylotypes representative of species that remain to be taxonomically defined. Furthermore, the results reinforced the primary role of plant nurseries in favouring the introduction, dissemination and evolution of Phytophthora species.

The efficacy of biofumigation with Brassica carinata pellets (BioFence) to control vegetative and reproductive structures of Phytophthora cinnamomi was investigated in vitro at different doses and temperatures. Biofumigation was effective in inhibiting mycelial growth (culture diameter) and chlamydospore and zoospore germination, and was lethal at 24 mg of pellet per plate (approximately 0.4 mg/liter). The 50% effective concentration values showed that efficacy of B. carinata pellets in inhibiting or killing the vegetative and reproductive structures of P. cinnamomi was maximum at 15°C and decreased as temperature rose to 25°C. However, the fungicide effect was independent of the temperature. In vivo biofumigation of Quercus cerris seedlings with Bio-Fence confirmed efficacy by reducing the inoculum density (CFU/g) of P. cinnamomi, thus protecting the host from root infection. The use of BioFence provides an alternative to synthetic pesticides to control P. cinnamomi within disease management programs in agroforestry systems. Phytophthora cinnamomi is among the most destructive pathogens associated with the decline of forest, ornamental, and fruit species worldwide . In Europe, it is known to be responsible for extensive mortality of Quercus ilex L. and Q. suber L. in the "dehesa", an important agroforestry ecosystem and cultural landscape used primarily for grazing and production of nontimber forest products such as wild game, mushroom, honey, cork, and firewood . The pathogen has been also associated with deteriorating crown status of Q. alba L. (Balci et al. 2007), stem cankers of Q. cerris L. (Oh et al. 2011) and Q. laurifolia Michx. (Wood and Tainter 2002), and ink disease of Q. rubra L. ). Furthermore, P. cinnamomi is recognized as a primary pathogen of Castanea sativa L. and Juglans spp. in Europe . Climatic variables, including winter and summer temperature, precipitation regime, and host susceptibility, can determine the aggressiveness of the pathogen . In a scenario of global warming, the increase of temperature is likely to increase the impact of P. cinnamomi due to more favorable conditions for overwintering and spread ). P. cinnamomi is disseminated through zoospore or chlamydospore production in soil and water and as mycelial growth by root-to-root contact . Furthermore, activities such as road building, timber harvesting, mining, bush walking, and movement of wild native and feral animals have also been implicated in its dispersal . Once established, it is unlikely that P. cinnamomi can be eradicated from natural ecosystems, because it can survive in the soil profile and in tolerant hosts. Currently, the use of chemicals together with quarantine and hygiene practices are the only control methods available. However, increasing restrictions on the utilization of synthetic pesticides in natural and naturalized ecosystems, owing to their adverse effects in the

The experiment compared productivity and vegetative growth of the Hass avocado on three avocado root rot resistant rootstocks and one susceptible rootstock. Hass trees on Duke 7 reported the largest number of fruit per tree and on G 755c the smallest five years after planting. Trees on Topa Topa and Duke 7 reported the highest average production four years after planting. Trees on G 755c were significantly lower in the amount of leaf N. Trees on Toro Canyon and G 755c showed significantly lower amounts of Na. Trees on Duke 7 showed a significantly higher level of Mn. Trees on G 755c were significantly smaller two years after planting. Trees on Topa Topa and Duke 7 showed a significantly larger canopy diameter than those on G 755c four years after planting. Trees on G 755c showed the smallest mean shoot growth four years after planting. Trees on G 755c had significantly larger trunk circumferences three and four years after planting. No statistical differences were found among rootst...

1Universidad Michoacana de San Nicolás de Hidalgo-Instituto de Investigaciones Agropecuarias y Forestales. Carretera Morelia-Zinapécuaro-Tarímbaro km 9.5, Michoacán, México. CP. 58880. 01 (443) 1940975. (alexsotoppv@gmail.com; gra.labpv@gmail.com; marle-dc@yahoo.com.mx). 2Colegio de Posgraduados-Programa de Fruticultura. Carretera México-Texcoco km 36.5, Montecillo, Texcoco, Estado de México. México. CP. 56230. Tel. 01 (55) 24905471.(fernandezpavia@hotmail.com; yletif@yahoo.com.mx; lexquilax@yahoo.com.mx). 3Facultad de Agrobiología-Universidad Michoacana de San Nicolás de Hidalgo. Paseo Lázaro Cárdenas esq. Berlín, Colonia Viveros, Uruapan, Michoacán, México. CP. 60090. 01 (452) 1126710. (marelpesa@yahoo.com.mx). §Autora para correspondencia: fernandezpavia@hotmail.com.

El origen del aguacate de acuerdo a Williams (1977b) tuvo lugar en las partes altas del centro y este de México, y partes altas de Guatemala. Esta misma región está incluida en lo que se conoce como Mesoamérica, y también es considerada como el área donde se llevó a ...

Ecosystems worldwide must simultaneously cope with several global change drivers with potential strong effects on ecosystem functioning. These drivers might interact in unexpected ways, but our still limited understanding of these interactive effects precludes us from predicting the impact of global change on ecosystem functioning. In this study we assessed the direct effects of pathogen-induced tree mortality and predicted warming and drought on C, N and P in Mediterranean forest soils affected by the decline of their dominant tree species (i.e. Quercus suber) due to the invasive pathogen Phytophthora cinnamomi. We also explored the potential indirect effects due to species replacement after Q. suber mortality. To achieve our goal, we conducted a soil incubation experiment using soils collected under Q. suber trees with different health status (i.e. healthy, defoliated and dead trees) and from coexistent shrubs (i.e. pioneer and late successional shrubs). These soils were incubated under controlled temperatures and soil moistures, mimicking various climate change scenarios predicted for 2050 and 2100 in the Mediterranean Basin. Our results showed that P. cinnamomi-induced mortality and future warming and drought may interact to simultaneously alter biogeochemical cycles in Q. suber forest soils. Resistance of studied variables to changes in temperature and moisture tended to be lower for dead trees than for healthy and defoliated trees. Moreover, we found that soil respiration and nutrient availability might be affected indirectly by P. cinnamomi-induced mortality due to species replacement. Overall, our results support a high potential of invasive pathogen species for modifying the response of soil functioning to climatic stressors.

Pyrosequencing analysis was performed on soils from Italian chestnut groves to evaluate the diversity of the resident Phytophthora community. Sequences analysed with a custom database discriminated 15 pathogenic Phytophthoras including species common to chestnut soils, while a total of nine species were detected with baiting. The two sites studied differed in Phytophthora diversity and the presence of specific taxa responded to specific ecological traits of the sites. Furthermore, some species not previously recorded were represented by a discrete number of reads; among these species, Phytophthora ramorum was detected at both sites. Pyrosequencing was demonstrated to be a very sensitive technique to describe the Phytophthora community in soil and was able to detect species not easy to be isolated from soil with standard baiting techniques. In particular, pyrosequencing is an highly efficient tool for investigating the colonization of new environments by alien species, and for ecological and adaptive studies coupled with biological detection methods. This study represents the first application of pyrosequencing for describing Phytophthoras in environmental soil samples.

An increasing decline and mortality of cork oak trees have been recently observed in central Italy and Sardinia Island. Following surveys conducted in three declining cork oak forests, a Phytophthora species was consistently isolated from soil samples collected from trees displaying different level of decline. Based on morphological features, growth rates at different temperatures and analysis of DNA sequences of the ITS region, all isolates were identified as Phytophthora cinnamomi Rands. This pathogen caused large brownish lesions on inoculated freshly cut branches of cork oak. It was re-isolated from all infected tissues. These findings represent the first report of P. cinnamomi on cork oak trees in Italy.

The genus Quercus holds significant ecological and economic value in the Northern Hemisphere Mediterranean-
type climate (MTC) regions. However, Quercus species and ecosystems are threatened by decline. Despite the
importance of this genus, a comprehensive analysis of the causes of oak decline across these regions is still
lacking. This study maps and analyzes research on oak decline in MTC regions, examining causal factors and
Quercus species studied, while identifying knowledge gaps and future research priorities. We systematically
reviewed 241 peer-reviewed articles on oak decline in MTC regions up to 2022, retrieved from the Web of
Science and Scopus databases.
Our findings show that scientific articles on oak decline began to be published in 1981 and peaked in 2021.
Most studies focused on the Mediterranean Basin, with disparities between Northern Africa and Southern Europe.
The most frequently studied species were Quercus suber L. and Quercus ilex L. in the Mediterranean Basin, and
Quercus agrifolia N´ee and Quercus kelloggii Newb. in California, comprising 53% of all species mentioned.
Pathogens (48%) and climate factors (17%) were identified as the most researched causes of oak decline.
Additionally, multiple interactions between factors were identified, confirming that these combinations poten­
tially exacerbate oak decline. Climate factors were the most frequently found in combination with others.
Conversely, pathogens were mostly studied in isolation, reflecting their widely recognized role in oak decline.
The study highlights the potential risk of pathogen and pest transference between MTC regions and the critical
role of management practices in influencing oak decline in interaction with biotic and abiotic factors.

Arbuscular mycorrhizal fungi presences in the rhizosphere of avocado trees with symptoms of root rot sadness caused by Phytophthora cinnamomi were determined. The investigation was done in the avocado orchard "Ojo de agua" in the town of Tancitaro, Michoacan, Mexico, in 21 previously selected trees. For the control of P. cinnamomi were applied three strains of Trichoderma (T. erinaceum, T. aggressivum and T. arundinaceum) before the application was made the first soil sampling, the second 6 months later, before the second application of Tricoderma strains and the last 12 months before the third application. To remove soil spores was used wet sieving and decantation protocol proposed by Gerdemann and Nicolson (1963), followed by centrifugation on sucrose (400 g L-1) at 2000 rpm. Taxonomic identification was based on the morphological characteristics of AMF spores, considering the shape, size and color, and thickness, ornamentations and number of the layers of the wall, coupling form and supporting hyphae, identifications were made by comparison with original descriptions available in the International Collection of Arbuscular Mycorrhizal Fungi and Glomeromycota species list. The first sampling were identified eleven species in seven genera: Glomus with two undetermined species, Glomus sp.1, Glomus sp.2, Glomus etunicatum and Glomus geosporum; genus Acaulospora, one undetermined species Acaulospora sp., A. spinosa, A. bireticulata and A. denticulate; genus Entrophospora, E. infrequens; genus Diversispora, D. aurantia; genus Scutellospora, S. pellucida; genus Racocetra, R. castanea and R. verrucosa and genus Gigaspora, Gi. decipiens. In the second and third sampling, the presence of new kinds of HMA there was not observed but the number of spores increased (average 38.09% and 30% respectively). The application of these species in the genus Trichoderma to control root pathogens of avocado encouraged the growth of HMA spores in the rhizosphere of the crop.

Phytophthora parasitica is the causal agent of the roselle's (Hibiscus sabdariffa) crown rot. In Ayutla, Tecoanapa and Tierra Colorada, Guerrero, Mexico, the attack of this disease has diminished crop production from 10 to 30%. The pathogenic variability of 30 P. parasitica strains and 25 monozoosporic isolates was determinated in this work. The most aggresive strains were TC1, TC8 and A5, which caused an initial necrosis in 11 days (Id) on roselle plants, and a final necrosis in 14 days (Fd); the least aggresive strains were TC2, A4 and TC7 because they produced the initial necrosis in 22 days and a final necrosis in 29 days. Monozoosporic isolates were obtained from strain TC1, which were inoculated onto roselle plants; the most pathogenic were 6 and 12, because they produced the initial necrosis in 8 days and caused plant death in 12 days; the least pathogenic were 9 and 14 (Id = 14, Fd = 23). These results demonstrated the pathogenic variability between strains and monozoos...

Phytophthora cinnamomi and P. cambivora are considered as the causal agents of Castanea sativa ink disease. These soil-borne plant pathogens invade and destroy the root system leading to the death of the trees. Most Phytophthora species secrete elicitins, a group of unique highly conserved proteins that are able to enhance plant defence responses in a systemic acquired resistance manner against infection by several pathogens. A cluster of four elicitin genes was identified in P. cinnamomi. In previous works one of these elicitins, αcinnamomin was shown to restrict the invasion of root cortical tissues by P. cinnamomi preventing vascular colonization in cork and holm oak. In the present work, roots of chestnut plantlets grown in vitro were allowed to absorb α-cinnamomin at 100 µg/ml for two days before being inoculated with P. cinnamomi. The effects of this elicitin on host-pathogen interaction were studied at histological and ultrastructural levels. P. cinnamomi was restricted to the outer cortex of 65% of the roots pre-treated with α-cinnamomin. In these roots, the vascular cylinders were free of pathogen. On the contrary, the pathogen reached the vascular cylinder, penetrating the phloem and xylem vessels in all non-treated assayed roots. The signs of pathogen degradation in the cortical parenchyma, mainly in the intercellular spaces, and the increase of a physical barrier in epidermal and sub-epidermal cell wall-media lamella and intercellular spaces by impregnation with phenol-like compounds strongly suggest that α-cinnamomin induced in chestnut defence reactions against P. cinnamomi.

La riboflavina (vitamina B2) se sintetiza por las plantas y muchos microorganismos y puede actuar como un activador de resistencia para contrarrestar el estrés biótico. En este trabajo se muestra que la aplicación exógena de riboflavina sobre la raíz de aguacate induce resistencia en contra del oomiceto Phytophthora cinnamomi. La riboflavina aplicada in vitro en una concentración de 5 mM a raíces de plántulas inhibió la colonización por P. cinnamomi, en comparación con las plántulas control, pero no tuvo efecto alguno en el crecimiento in vitro del oomiceto. Extractos de raíces tratadas con riboflavina mostraron actividad inhibitoria sobre el patógeno. Cuando diferentes concentraciones del extracto (50, 100 y 150 µl/ml) se mezclaron con zoosporas del patógeno (1x10 4 zoosporas/ml) y se analizaron al microscopio 6 días después, se observó una morfología alterada de las hifas o una disminución en la producción de clamidosporas, así como restos de hifas, sugiriendo un efecto tóxico sobre el patógeno. Estos resultados indican que los extractos de raíces de aguacate tratadas con riboflavina afectan la formación de clamidosporas y la estructura del micelio de P. cinnamomi.

We demonstrated the ability of salicylic acid (SA) to induce a compound in avocado roots that strengthens their defense against Phytophthora cinnamomi. The SA content of avocado roots, before and after the application of exogenous SA, was determined by High-Performance Liquid Chromatography (HPLC). After 4 h of SA feeding, the endogenous level in the roots increased to 223 g g -1 FW, which was 15 times the amount found in control roots. The methanolic extract obtained from SA-treated avocado roots inhibited the radial growth of P. cinnamomi. A thin layer chromatographic bioassay with the methanolic extract and spores of Aspergillus showed a distinct inhibition zone. The compound responsible for the inhibition was identified as phenol-2,4-bis (1,1-dimethylethyl) by gas chromatography and mass spectrometry. At a concentration of 100 g/mL, the substance reduced germinative tube length in Aspergillus and radial growth of P. cinnamomi. A commercial preparation of phenol-2,4-bis (1,1-dimethylethyl) caused the same effects on mycelium morphology and radial growth as our isolate, confirming the presence of this compound in the root extracts. This is the first report of the induction of this compound in plants by SA, and the results suggest that it plays an important role in the defense response of avocado.

RESUMEN El compostaje es un proceso bio-oxidativo en el que intervienen numerosos y variados microorganismos, cuya dinámica poblacional está influenciada por el tipo de sustrato orgánico empleado. Con el objetivo de cuantificar las poblaciones fúngica y actinomicetos, así como la identificación de hongos presentes durante el proceso de compostaje, se elaboraron tres compost con la inclusión de diferentes materiales orgánicos, diferenciándose por la inclusión de sustratos fibrosos de bora (Eichhornia crassipes), pergamino de café y residuos de jardinería. Se emplearon tres composteros de metal con dimensiones: 62,5 x 77,5 x 75,0 cm. El diseño estadístico fue de bloques al azar con medidas repetidas en el tiempo, con los tratamientos provenientes de un arreglo factorial 3x3, tres tipos de compost, tres tiempos de evaluación (20, 40 y 60 días) y tres repeticiones. Los valores obtenidos fueron transformados a log10 y realizado su respectivo análisis de varianza; las medias se compararon...

The genetic structure of a sample of isolates of the oomycete plant pathogen Phytophthora cinnamomi from natural and agricultural outbreaks and the long-distance movement of individual genotypes were studied using four microsatellite markers to genotype 159 isolates of Californian, Mexican, and worldwide origins. Allelic profiles identified 75 multilocus genotypes. A STRUCTURE analysis placed them in three groups characterized by different geographic and host ranges, different genic and genotypic diversity, and different reproductive modes. When relationships among genotypes were visualized on a minimum spanning network (MSN), genotypes belonging to the same STRUCTURE group were contiguous, with rare exceptions. A putatively ancestral group 1 had high genic diversity, included all A1 mating type isolates and all Papuan isolates in the sample, was rarely isolated from natural settings in California and Mexico, and was positioned at the center of the MSN. Putatively younger groups 2 a...

• Mediterranean oaks are endangered by infection with an invasive alien oomycete. • Forecasts based on SDM showed an expansion of the plant pathogen within Andalusia. • Our SDMs verified the known environmental suitability and provided new insights. • Phytosanitary management zones may be set from the current and future distribution.

Cover crops are used increasingly by growers to improve soil quality, prevent erosion, increase organic matter, and suppress root pathogens and pests. However, limited information is available on their use for suppressing pathogens (Rhizoctonia, Pythium, Fusarium, Thieloviopsis, Pratylenchus, and Meloidogyne) of vegetables grown in the Northeast. Thus, a collaborative project was initiated in 2009 to assess the efficacy of selected cover crops in suppressing root pathogens of vegetables and improving soil health in research and on-farm field trials in New York, Pennsylvania and Connecticut. In NY, strips (4.5 X 60 M) of 9 cover crops (rye grain + hairy vetch, oat, sudex, forage radish, red clover, rapeseed, buchwheat, wheat, and a fallow check) were randomized in 4 fields with 3 replications (3.2 ha total). The fields had different management histories resulting in varied levels of pathogen pressure and soil quality. In 2010, cover crop biomass was measured and collected soil samples were assessed for root health (greenhouse bean bioassay), nematode diversity and density, and selected soil health parameters (Cornell Soil Health Test). In general, root rot severity was lowest and yield of snap bean was highest in the field with the highest soil quality. After one year, the cover crops greatly affected root health and bean yield in this trial as well as the microplots and/or on-farm trials conducted in CT and PA. Another cycle of evaluations is in progress. biocontrol strains of non-aflatoxigenic Aspergillus flavus H. ABBAS (1), B. Horn (2), M. Weaver (3), X. Jin (3), C. Abel (4), W. T.

Invasive species threaten biodiversity in Australia. One of these species is the introduced root pathogen Phytophthora cinnamomi, which harms native and commercial plant species. Disease caused by this pathogen was listed as a key threatening process under the Commonwealth's Environment Protection and Biodiversity Conservation Act in 2000. There is considerable uncertainty regarding the best management strategies to address invasive species like Phytophthora cinnamomi; particularly where the efficacy of control methods is unknown. How should environmental managers decide whether management strategies are worthwhile or cost-effective? We demonstrate the use of a tool that can address these management challenges: the Investment Framework for Environmental Resources (INFFER). INFFER was designed to help environmental managers achieve the most valuable environmental outcomes with the limited resources they have available. The framework involves a systematic assessment of the trade-offs between variables such as asset value, likelihood that private individuals will adopt management or behavioural changes, delays in the realisation of benefits, and sources of uncertainty due to potential technical failure or socio-political risks. We present an application of INFFER to assess the cost-effectiveness of management strategies to address Phytophthora cinnamomi in the Fitzgerald River National Park, a national park in the South-West Botanical Province of Western Australia. The Fitzgerald River National Park is one of the largest parks in Australia, and has high conservation value because of its high floral diversity and numbers of endemic species. The model was developed in collaboration with park stakeholders, including park rangers, management and research officers, and members of a local natural resource management group. We found that despite uncertainty regarding the efficacy of existing containment and eradication methods, management strategies aimed at containing existing Phytophthora cinnamomi infestations and preventing future infestations, resulted in significantly greater benefits than costs (Benefit: Cost Index = 3.02). This result supports investment of public conservation funds to control Phytophthora cinnamomi in the Fitzgerald River National Park. A sensitivity analysis identified the need for better biophysical data to quantify the impact of management works, and demonstrated how uncertain funding environments prevent confidence in the accomplishment of project goals, through an inability to assure the future maintenance and upkeep of management works. Our analysis demonstrates the importance of using a rigorous decision support tool like INFFER to assess investment decisions when there is uncertainty regarding conservation benefits.

Phytophthora cinnamomi is an important plant pathogen responsible for dieback diseases in plant genera including Quercus, Fagus, Castanea, Eucalyptus, and Pinus, among others, all over the world. P. cinnamomi infection exerts tremendous ecological and economic losses. Several strategies have been developed to combat this pathogenic oomycete, including the search for novel anti-oomycete compounds. In this work, a Mediterranean vascular plant, Phlomis purpurea, has been screened for secondary bioactivity against this pathogen. The genus Phlomis includes a group of herbaceous plants and shrubs described as producers of many different bioactive compounds, including several triterpenoids. Triterpenoids are well-known molecules synthesized by plants and microorganisms with potent antioxidant, antitumoral, and antimicrobial activities. We have isolated by HPLC-DAD and characterized by HPLC-MS and NMR two nortriterpenoid compounds (phlomispentaol A and phlomispurtetraolone) from the root ex...

Climate change and exotic pests and pathogens are causing alarming forest declines worldwide. However, we still lack a comprehensive understanding of how damage caused by exotic pests and pathogens might vary under the different scenarios of water availability imposed by a changing climate, particularly in water-limited forests as those that occupy Mediterranean areas. In this paper we aimed to experimentally analyse the interactive effects of the aggressive exotic pathogen Phytophthora cinnamomi and climate change-related reductions in soil moisture on seedling performance of the Mediterranean host Quercus suber. We conducted a full-factorial greenhouse experiment where the physiology and growth of Q. suber seedlings was measured in soils with different combinations of P. cinnamomi inoculum density (0, 30, 60 and 120 colony forming units per gram of dry soil) and soil moisture (15%, 40%, 50% and 100% soil water holding capacity) simulating different invasion and climate change scenarios. We found additive effects of P. cinnamomi and drought on Q. suber performance aboveground, although these effects were not always negative. In fact, seedlings showed a compensatory physiological response to P. cinnamomi infection by increasing their net photosynthetic rates. Our results also supported important interactive effects of pathogens and soil moisture on belowground performance. Thus, the inoculum density in the soil required to cause significant root damage in experimental seedlings decreased as soil moisture increased. From a climate change perspective, these results suggest that an average drier climate might imply sub-optimal conditions for P. cinnamomi infections allowing for a slower advance of the disease in invaded areas. However, this effect will be modulated by the also predicted more frequent extreme climatic events. A higher frequency of extreme rain events that saturate the soil might be particularly 3 beneficial for P. cinnamomi, boosting its soil density beyond any possible response capacity of susceptible hosts.

Phytophthora cinnamomi is an important plant pathogen responsible for dieback diseases in plant genera including Quercus, Fagus, Castanea, Eucalyptus, and Pinus, among others, all over the world. P. cinnamomi infection exerts tremendous ecological and economic losses. Several strategies have been developed to combat this pathogenic oomycete, including the search for novel anti-oomycete compounds. In this work, a Mediterranean vascular plant, Phlomis purpurea, has been screened for secondary bioactivity against this pathogen. The genus Phlomis includes a group of herbaceous plants and shrubs described as producers of many different bioactive compounds, including several triterpenoids. Triterpenoids are well-known molecules synthesized by plants and microorganisms with potent antioxidant, antitumoral, and antimicrobial activities. We have isolated by HPLC-DAD and characterized by HPLC-MS and NMR two nortriterpenoid compounds (phlomispentaol A and phlomispurtetraolone) from the root ex...

The European chestnut tree (Castanea sativa Mill.) is widely cultivated throughout the world’s temperate regions. In the Mediterranean region, it has a significant economic role mainly because of the high quality of its edible nuts. The Oomycete Phytophthora cinnamomi is one of the most severe pathogens affecting European chestnuts, causing ink disease and significant losses in production. Ginkgobilobin-2 (Gnk2) in Ginkgo biloba is a secreted protein with a plant-specific cysteine-rich motif that functions as a lectin, and its carbohydrate-binding properties are closely related to its antifungal activity. The binding of lectins to mannose residues of the cell wall of Phytophthora species may disturb and disrup the cell wall structure. This work determined that the amino acid sequence has a signal peptide that directs the final protein peptide to the apoplast. The Cast_Gnk2-like expression was performed and optimized, and different in vitro antagonism tests were done against P. cinna...

The Japanese chestnut (Castanea crenata) carries resistance to Phytophthora cinnamomi, the destructive and widespread oomycete causing ink disease. The European chestnut (Castanea sativa), carrying little to no disease resistance, is currently threatened by the presence of the oomycete pathogen in forests, orchards and nurseries. Determining the genetic basis of P. cinnamomi resistance, for further selection of molecular markers and candidate genes, is a prominent issue for implementation of marker assisted selection in the breeding programs for resistance. In this study, the first interspecific genetic linkage map of C. sativa x C. crenataallowed the detection of QTLs for P. cinnamomi resistance. The genetic map was constructed using two independent, control-cross mapping populations. Chestnut populations were genotyped using 452 microsatellite and single nucleotide polymorphism molecular markers derived from the available chestnut transcriptomes. The consensus genetic map spans 49...

Phytophthora cinnamomi is a hemibiotrophic that causes root rot, also known as ink disease. Little information has been acquired in chestnut on the molecular defense strategies against this pathogen. The expression of eight candidate genes potentially involved in the defense to P. cinnamomi was quantified by digital PCR in Castanea genotypes showing different susceptibility to the pathogen. Seven of the eight candidate genes displayed differentially expressed levels depending on genotype and time-point after inoculation. Cast_Gnk2-like revealed to be the most expressed gene across all experiments and the one that best discriminates between susceptible and resistant genotypes. Our data suggest that the pre-formed defenses are crucial for the resistance of Castanea crenata to P. cinnamomi. A lower and delayed expression of the eight studied genes was found in the susceptible Castanea sativa, which may be related with the establishment and spread of the disease in this species. A working model integrating the obtained results is presented.

Nowadays there is a great demand for chestnut rootstocks with improved resistance to Phytophthora cinnamomi, the causing agent of ink disease/root rot. The majority of genotypes that are being commercialized and used in the new plantations, were selected from the breeding programs of the XX century. The climate scenarios have been changing throughout the years, therefore the selection of new genotypes, better adapted to the current conditions of soil and climate, together with an effective and expedite production system, are essential to fulfill the needs of the market on better propagation materials. The INIAV’s chestnut breeding program, established in 2006, aims the improvement of resistance of chestnut to biotic stresses. By taking advantage of micropropagation, this program developed a pipeline to produce new hybrid varieties with improved resistance to P. cinnamomi. Micropropagation helps to overcome the recalcitrance that chestnut tissues present to traditional clonal propaga...

The Japanese chestnut (Castanea crenata) carries resistance to Phytophthora cinnamomi, the destructive and widespread oomycete causing ink disease. The European chestnut (Castanea sativa), carrying little to no disease resistance, is currently threatened by the presence of the oomycete pathogen in forests, orchards and nurseries. Determining the genetic basis of P. cinnamomi resistance, for further selection of molecular markers and candidate genes, is a prominent issue for implementation of marker assisted selection in the breeding programs for resistance. In this study, the first interspecific genetic linkage map of C. sativa x C. crenata allowed the detection of QTLs for P. cinnamomi resistance. The genetic map was constructed using two independent, control-cross mapping populations. Chestnut populations were genotyped using 452 microsatellite and single nucleotide polymorphism molecular markers derived from the available chestnut transcriptomes. The consensus genetic map spans 498,9 cM and contains 217 markers mapped with an average interval of 2.3 cM. For QTL analyses, the progression rate of P. cinnamomi lesions in excised shoots inoculated was used as the phenotypic metric. Using non-parametric and composite interval mapping approaches, two QTLs were identified for ink disease resistance, distributed in two linkage groups: E and K. The presence of QTLs located in linkage group E regarding P. cinnamomi resistance is consistent with a previous preliminary study developed in American x Chinese chestnut populations, suggesting the presence of common P. cinnamomi defense mechanisms across species. Results presented here extend

The most dangerous pathogen affecting the production of chestnuts is Phytophthora cinnamomi a hemibiotrophic that causes root rot, also known as ink disease. Little information has been acquired in chestnut on the molecular defense strategies against this pathogen. The expression of eight candidate genes potentially involved in the defense to P. cinnamomi was quantified by digital PCR in Castanea genotypes showing different susceptibility to the pathogen. Seven of the eight candidate genes displayed differentially expressed levels depending on genotype and time-point after inoculation. Cast_Gnk2-like revealed to be the most expressed gene across all experiments and the one that best discriminates between susceptible and resistant genotypes. Our data suggest that the pre-formed defenses are crucial for the resistance of C. crenata to P. cinnamomi. A lower and delayed expression of the eight studied genes was found in the susceptible Castanea sativa, which may be related with the esta...

Phytophthora cinnamomi (P. c) is a plant pathogenic oomycete that is capable of damaging plants in commercial production systems and natural ecosystems worldwide. The most common methods for the detection and diagnosis of P. c infection are expensive, elaborate and time consuming. This study was carried out to examine whether species specific and life cycle specific volatile organic compounds (VOCs) can be absorbed by solid-phase microextraction fibers and detected by gas chromatography that are produced by P. c and another oomycete Pythium dissotocum. A headspace solid-phase microextraction (HS-SPME) together with gas chromatography (GC) method was developed and optimized for the identification of the VOCs released by P. c. The optimized parameters included type of fiber, exposure time, desorption temperature and desorption time. Optimization was achieved with the analytes of P. c+V8A and V8A alone. To perform the HS-SPME, six types of fiber were assayed and compared: 7μm Polydimethylsiloxane (PDMS), 100μm Polydimethylsiloxane (PDMS), 50/30μm Divinylbenzene/Carboxen TM /Polydimethylsiloxane DVB/CAR/PDMS), 65μm Polydimethylsiloxane/Divinylbenzene (PDMS/DVB), 85μm Polyacrylate (PA) fibre and 85μm Carboxen TM / Polydimethylsiloxane (Carboxen™/PDMS). In a comparison of the efficacy of the fibers, the bipolar fiber DVB/CAR/PDMS had a higher extraction efficiency than the other fibers. An exposure time of 16h with DVB/CAR/PDMS fiber in the sample headspace was enough to reach the maximum extraction efficiency. A desorption time of 3min in the GC injector with the desorption temperature of 250°C was enough for the fiber to desorb the compounds of interest. The

A robust technique was developed to identify Phytophthora cinnamomi using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography (GC) coupled to a flame ionization detector (FID) for analyzing volatile organic compounds (VOCs). Six fiber types were evaluated and results indicated that the three-phase fiber 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/ PDMS) had the highest extraction efficiency for both polar and nonpolar GC columns. The maximum extraction efficiency (equilibrium absorption) was achieved 16 h after fiber exposure in the HS. Absorbed compounds on the fiber were completely desorbed in the GC injector after 5 min at 250°C. Compared with the nonpolar column, the polar column showed optimum separation of VOCs released from P. cinnamomi. Under the optimized HS-SPME and GC/FID conditions, lower detection limits for the four external standards was found to be between 1.57 to 27.36 ng/liter. Relative standard deviations <9.010% showed that the method is precise and reliable. The method also

Volatile organic compounds (VOCs) from Phytophthora cinnamomiinfected lupin seedlings were collected by headspace solid-phase microextraction (HS-SPME). The sampling was done 28 to 44, 52 to 68, and 76 to 92 h after inoculation (HAI). The HS-SPME samples were analyzed by gas chromatography-flame ionization detector (GC-FID) to assess the differences in volatile compounds between the P. cinnamomi-infected lupin seedlings and the control. Three specific peaks were identified after 52 to 68 h with the infected lupin seedlings, at which time there were no visible aboveground symptoms of infection. Subsequently, the VOCs of five different substrates (V8A, PDA, lupin seedlings, soil, and soil + lupin seedlings) infected with P. cinnamomi and the corresponding controls were analyzed by gas chromatography-mass spectrometry (GC/MS). A total of 87 VOCs were identified. Of these, the five most abundant that were unique to all five inoculated substrates included: 4-ethyl-2-methoxyphenol, 4-ethylphenol, butyrolactone, phenylethyl alcohol, and 3-hydroxy-2-butanone. Therefore, these metabolites can be used as markers for the identification of P. cinnamomi in different growing environments. Some VOCs were specific to a particular substrate; for example, 2,4,6-rrimethylheptanes, dl-6-methyl-5-hepten-2-ol, dimethyl trisulfide, 6,10-dimethyl-5,9-undecadien-2-ol, and 2-methoxy-4-vinylphenol were specific to P. cinnamomi + V8A; heptanes and 5-methyl-3-heptaneone were specific to P. cinnamomi + PDA; 3-methyl-1-butanol, ethyl acetate, 2methyl-propanoic acid, ethyl ester, and ethyl ester 2-methyl-butanoic acid were specific to P. cinnamomi-inoculated lupin seedlings; and benzyl alcohol and 4-ethyl-1, 2-dimethoxybenzene were only detected in the headspace of inoculated soil + lupin seedlings. Results from this investigation have multiple impacts as the volatile organic profiles produced by the pathogen can be utilized as an early warning system to detect the pathogen from contaminated field soil samples. Data from this investigation have also illuminated potential metabolic pathways utilized by the oomycete during infection which may serve as potential targets for the development of specific control strategies.

Unlike the USA and Europe, Australasia has not in the last two years faced any unexpected developments in new Phytophthora diseases in forests and natural ecosystems. Consequently, we will focus on the way forward for disease management and examine the challenges that will need to be faced over the next few years to control the impact and rate of spread of this disease.

Cork oak (Quercus suber) is one of the most important forest species in Portugal and in the Mediterranean basin, but in recent decades a progressive decline has been registered in the montado ecosystem. The main phytopathogenic agent involved in this process, Phytophthora cinnamomi, has a high infection potential and survival ability, and became a serious threat to this ecosystem. Extensive areas of cork oak are infected in Portugal, being imperative to find new ways to control this disease. The complex lifecycle of this pathogen and the small number of effective agrochemicals currently authorized against Phytophthora spp., with phosphonates taking advantage, justifies this work where new products with antagonism against Oomycetes or biostimulant properties were tested. This study aimed to evaluate the efficacy of BLAD (a polypeptide with antifungal, anti-oomycete and biostimulant properties), potassium phosphonate and salicylic acid in Q. suber infected with P. cinnamomi, quantifying the severity of root lesions. BLAD, potassium phosphonate and salicylic acid treatments showed lower root lesion severities than the inoculated control, and proved to be effective as they provided a slower disease evolution. The lowest lesion severity in cork oak roots observed with BLAD treatments allowed us to consider BLAD as the most promising product among the three we tested.

Soil borne Phytophthora cinnamomi and Phytophthora cambivora are considered the most pathogenic species associated with chestnut (Castanea sativa) decline in Europe. Mapping their incidence and distribution from nursery and plantation soils may offer valuable information for limiting spread. As conventional biological baiting and taxonomic confirmation is generally time consuming, labour, logistically and space intensive, we have focused on the development of a specific touchdown nested multiplex Polymerase Chain Reaction (PCR) approach for the simultaneous detection of both species direct from soil. Pre-existing and novel primers, based on Internal Transcribed Spacer (ITS) sequences, have been evaluated for their specificity and use in a multiplex capacity in various combinations. Coupled to this we have modified a mechanical lysis procedure for DNA extraction from up to 10 g of chestnut under storey soils (ranging from 0.5 to 25 mg DNA g À1 fresh soil). Using serial dilutions and/or polyvinylpolypyrrolidone chromatography purification, both species have been successfully detected, in artificially and naturally infected soils. Levels of assay detection are comparable to other Phytophthora species where PCR based diagnostic systems have been reported. A qualitative evaluation of this approach against conventional baiting is presented.

The European chestnut (Castanea sativa Mill.) is a specie with great economic importance in Europe that have been present for thousands of years. In Portugal, the chestnut helps to maintain a positive trade balance, by contributing to the gross national product (GDP). One of the biggest threats for the chestnut is the ink disease caused by Phytophthora cinnamomi, this disease is problematic to chestnut crop with a damaging impact. Silicon (Si) is classified as a beneficial nutrient, having the ability to make plants more resistant to attacks by pathogens. Studies on the effect of silicon on chestnut are practically non-existent, so the aim of this study was to evaluate the impact of silicon in the resistance of chestnut plants to P. cinnamomi. The plants were treated by 0 mM, 5 mM, 7.5mM and 10 mM SiK ® with the analyzed mad at 0, 15 and 30 days after inoculation by P. cinnamomi. These findings showed that the Si-treated plants had higher survival rate resulted from the presence of phytoliths in root tissues, that acted as a mechanical barrier reducing the development of pathogenic structures and they arealso associated with the improvement on antioxidant activity through the increase of CAT and SOD, higher values of total phenols compounds and less oxidative damage. The presence of Si in PDA medium reduced the growth of P. cinnamomi all over the time, presenting high PI. This work shows that the Si fertilization in chestnut plants contributes to increase the resistance against P. cinnamomi infection.

The Japanese chestnut (Castanea crenata) carries resistance to Phytophthora cinnamomi, the destructive and widespread oomycete causing ink disease. The European chestnut (Castanea sativa), carrying little to no disease resistance, is currently threatened by the presence of the oomycete pathogen in forests, orchards and nurseries. Determining the genetic basis of P. cinnamomi resistance, for further selection of molecular markers and candidate genes, is a prominent issue for implementation of marker assisted selection in the breeding programs for resistance. In this study, the first interspecific genetic linkage map of C. sativa x C. crenata allowed the detection of QTLs for P. cinnamomi resistance. The genetic map was constructed using two independent, control-cross mapping populations. Chestnut populations were genotyped using 452 microsatellite and single nucleotide polymorphism molecular markers derived from the available chestnut transcriptomes. The consensus genetic map spans 498,9 cM and contains 217 markers mapped with an average interval of 2.3 cM. For QTL analyses, the progression rate of P. cinnamomi lesions in excised shoots inoculated was used as the phenotypic metric. Using non-parametric and composite interval mapping approaches, two QTLs were identified for ink disease resistance, distributed in two linkage groups: E and K. The presence of QTLs located in linkage group E regarding P. cinnamomi resistance is consistent with a previous preliminary study developed in American x Chinese chestnut populations, suggesting the presence of common P. cinnamomi defense mechanisms across species. Results presented here extend PLOS ONE |

Ink disease (caused by the pathogen Phytophthora cinnamomi) and chestnut blight (caused by the fungus Cryphonectria parasitica) are the two most destructive diseases affecting European chestnut (Castanea sativa) and American chestnut (C. dentata). Therefore, breeding for resistance to both pathogens is essential for the chestnut sustainability in Europe and the United States of America. Several genomic approaches have been implemented in order to map the resistance first to ink disease and then to blight chestnut using a hybrid population: C. sativa crossed with resistant Asian species (C. crenata and C. mollissima). The transcriptome of C. sativa and C. crenata inoculated and not with P. cinnamomi was recently sequenced and allowed the identification of genes putatively involved in ink disease resistance. Taking advantage of molecular markers (microsatellite and SNPs), developed from the referred tanscriptomes and from C. mollissima transcriptome, the first C. sativa x C. crenata genetic map was constructed. Additionally, a reliable phenotyping method was established to evaluate the level of P. cinnamomi resistance of each hybrid allowing the identification of two Quantitative Trait Loci (QTLs). These results are the first step for understanding the resistance to ink disease in chestnut.

Castanea sativa is susceptible to Phytophthora spp., a serious root pathogen causing ink disease, while C. crenata and C. mollissima show resistance to infection. Interspecific controlled crosses were established for introgression of resistance genes from the resistant species into the susceptible C. sativa, and two mapping populations were created. Phytophthora cinnamomi resistance of each progeny was evaluated by root and excised shoot inoculation tests. The number of days of survival after root inoculation was the best discriminator of resistance to P. cinnamomi while the percentage of shoots with internal lesions was the symptom most associated with survival. The lesion progression rate in the excised shoot inoculation test was strongly and negatively correlated with survival in the root inoculation test. The excised shoot inoculation test appears to be a reliable approach for screening the resistance of chestnut genotypes to P. cinnamomi. Strong genetic correlations were obtained between survival and ink disease symptoms and among symptoms, indicating that common or linked genes might influence resistance to P. cinnamomi. The most resistant genotypes selected from this study will be tested for other commercial variables, such as ease of vegetative propagation and stock-scion compatibility.

Exotic pests and pathogens are causing widespread decline and possible extirpation of several of our foundational forest tree species. The decline of such species has large-scale effects on their associated ecosystems and services they provide. A steady barrage of new threats to important forest species is anticipated to continue as a by-product of increasing global trade. The Forest Health Initiative (FHI) was conceived in recognition of these issues and the challenges they present to biodiversity and ecosystem sustainability. The FHI concept is a new paradigm that engages social, regulatory, and biological science experts and various stakeholders in developing and evaluating options for solving our most critical forest health problems. Blight resistance in American chestnut was selected as a test case for FHI, and although not completely solved, significant progress is being made and the promise of effective resistance and chestnut restoration has been advanced. Clearly additional work remains for American chestnut as well as the many other severely threatened forest tree species. To address these additional species, we are building on the FHI experience and developing two complementary initiatives designed to improve the ability of the research community to identify, produce, and deploy effective tree resistance. A projectoriented initiative, Forest.Health, will prioritize the most seriously threatened species, bring researchers and stakeholders together to reach consensus on a science-based solution, and seek collaborators and funding to carry out the work. At the same time, a network-based participatory tree breeding consortium is proposed to ensure long-term development of publically available, genetically improved forest trees.

development of bacteria (160x10 ufc g soil), mainly from Bacillus and Pseudomonas. In four of the non-sterile soils, the pH was always significantly (P <= 0.05) lower than the control and ranged dynamically between 7.33 and 8.24; the content of assimilable nitrogen, potassium, and the bacteria -1 g in soil were increased; Bacillus and Pseudomonas were incorporated; and the development of Penicillium was promoted.