Oomycete Phytophthora Cinnamomi Research Articles

Identification of Phytophthora cinnamomi CRN effectors and their roles in manipulating cell death during Persea americana infection

The oomycete Phytophthora cinnamomi is a devastating plant pathogen with a notably broad host range. It is the causal agent of Phytophthora root rot (PRR), arguably the most economically important yield-limiting disease in Persea americana (avocado). Despite this, our understanding of the mechanisms P. cinnamomi employs to infect and successfully colonize avocado remains limited, particularly regarding the pathogen’s ability to maintain its biotrophic and necrotrophic lifestyles during infection. The pathogen utilises a large repertoire of effector proteins which function in facilitating and establishing disease in susceptible host plants. Crinkling and necrosis effectors (CRN/Crinklers) are suspected to manipulate cell death to aid in maintenance of the pathogens biotrophic and necrotrophic lifestyles during different stages of infection. The current study identified 25 P. cinnamomi CRN effectors from the GKB4 genome using an HMM profile and assigned putative function to them as either cell death inducers or suppressors. Function was assigned to 10 PcinCRNs by analysing their RNA-seq expression profiles, relatedness to other functionally characterised Phytophthora CRNs and tertiary protein predictions. The full-length coding sequences for these PcinCRNs were confirmed by Sanger sequencing, six of which were found to have two divergent alleles. The presence of alleles indicates that the proteins encoded may perform contradicting functions in cell death manipulation, or function in different host plant species. Overall, this study provides a foundation for future research on P. cinnamomi infection and cell death manipulation mechanisms.

Evidence for antagonistic effects of climate change and exotic pathogens on regeneration of Mediterranean forests

Abstract Understanding the interactive effects of global change drivers on tree demography is fundamental for realistic predictions of future forest dynamics. Multiple studies have shown increasing drought and exotic pathogens to severely threaten forest persistence by increasing mortality and decreasing growth of adult trees. However, much less is known about their effects on regeneration, and how they might affect seedling performance in additive and non‐additive (synergistic or antagonistic) ways. Here we aimed to fill this gap by experimentally exploring the effects of increasing drought and soil‐borne pathogens on tree regeneration in two types of mixed oak forests (Quercus suber‐Q. canariensis and Q. suber‐Olea europaea) invaded by the exotic soil‐borne oomycete Phytophthora cinnamomi, one of the most aggressive plant pathogens on earth. We conducted a seed sowing experiment with oomycete‐specific fungicide taking advantage of rainfall exclusion infrastructures that excluded 30% of the annual rainfall, simulating predictions of climate change models for Mediterranean systems. Seedling emergence, survival and growth of the three tree species were followed during 3 years. We found that neutral or positive drought effects on regeneration dominated over negative effects in the tree community. Moreover, most positive drought effects on the dominant species (Q. suber) were not direct, but indirectly mediated by soil‐borne pathogens. This was shown by the fact that positive drought effects disappeared with fungicide application. Synthesis: Overall, our results suggest that rainfall reductions predicted by climate change models for the Mediterranean region might have minor direct negative effects on early regeneration of tree species, but could play a major indirect role by limiting the negative effects of exotic pathogens on highly susceptible tree species. These findings highlight that antagonisms among global change drivers should be recognized as important forces that might slow down the current loss of tree health.

Tolerant Epitypes of Elicited Holm Oak Somatic Embryos Could Be Revealed by Challenges in Dual Culture with Phytophthora cinnamomi Rands.

Holm oaks (Quercus ilex L.) can suffer severe infection by the oomycete Phytophthora cinnamomi Rands; the production of more tolerant plants is, therefore, required. Embryo formation is a key period in the establishment of epigenetic memory. Somatic embryos from three holm oak genotypes were elicited, either over 3 days or 60 days, with methyl-jasmonate, salicylic acid (SA), β-aminobutyric acid (BABA), or benzothiadiazole (all at 50 μM and 100 μM), or 10% and 30% of a filtered oomycete extract (FILT10 and FILT30) to activate plant immune responses. The number of embryos produced and conversion rate under all conditions were recorded. Some elicited embryos were then exposed to P. cinnamomi in dual culture, and differential mycelial growth and the progression of necrosis were measured. The same was performed with the roots of germinated embryos. Within genotypes, significant differences were seen among the elicitation treatments in terms of both variables. Embryos and roots of 60-day BABA, SA, or FILT10 treatments inhibited mycelium growth. The 3-day BABA (either concentration) and 60-day FILT10 induced the greatest inhibition of necrosis. Mycelium and necrosis inhibition were compared with those of tolerant trees. Both inhibitions might be a defense response maintained after primed embryo germination, thus increasing the likelihood of tolerance to infection.

Castanea crenata Ginkbilobin-2-like Recombinant Protein Reveals Potential as an Antimicrobial against Phytophthora cinnamomi, the Causal Agent of Ink Disease in European Chestnut

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. cinnamomi using different purified protein concentrations. As a result of one of these assays, Cast_Gnk2-like significantly reduced the mycelia growth of P. cinnamomi in liquid medium as shown by the mycelia weight (g) in control treatments was 377% higher than in the treatments. These insights reveal the potential of Cast_Gnk2-like for agricultural uses and biotechnological developments for the pathosystem chestnut/P. cinnamomi.

Effect of Methyl Jasmonate in Gene Expression, and in Hormonal and Phenolic Profiles of Holm Oak Embryogenic Lines Before and After Infection With Phytophthora cinnamomi.

The dieback syndrome affecting Quercus ilex and other oak species impels the search for tolerant plant genotypes, as well as methods of plant immunization against such infections. Elicitation treatments can be an effective strategy to activate plant defense response and embryogenic lines represent a promising tool to generate new tolerant genotypes and also to study early markers involved in defense response. The aim of the presented work was to investigate changes in gene expression, and in hormonal and phenolic profiles induced in three holm oak embryogenic lines (ELs) elicited with methyl jasmonate (MeJA) before and after infection with the oomycete Phytophthora cinnamomi, which is the main biotic agent involved in this pathogenic process. The three ELs, derived from three genotypes, showed different basal profiles in all tested parameters, noting that the VA5 naïve genotype from a scape tree was characterized by a basal higher expression in NADPH-dependent cinnamyl alcohol dehydrogenase (CAD) and chalcone synthase (CHS) genes and also by higher caffeic acid content. Our work also identifies changes triggered by MeJA elicitation in holm oak embryogenic lines, such as increases in ABA and JA contents, as well as in levels of most of the determined phenolic compounds, especially in caffeic acid in Q8 and E00 ELs, but not in their biosynthesis genes. Irrespective of the EL, the response to oomycete infection in holm oak elicited plant material was characterized by a further increase in JA. Since JA and phenols have been described as a part of the Q. ilex defense response against P. cinnamomi, we propose that MeJA may act as an induced resistance (IR) stimulus and that in our embryogenic material induced both direct (detected prior to any challenge) and primed (detected after subsequent challenge) defense responses.

Genome of the destructive oomycete Phytophthora cinnamomi provides insights into its pathogenicity and adaptive potential

BackgroundPhytophthora cinnamomi is an oomycete pathogen of global relevance. It is considered as one of the most invasive species, which has caused irreversible damage to natural ecosystems and horticultural crops. There is currently a lack of a high-quality reference genome for this species despite several attempts that have been made towards sequencing its genome. The lack of a good quality genome sequence has been a setback for various genetic and genomic research to be done on this species. As a consequence, little is known regarding its genome characteristics and how these contribute to its pathogenicity and invasiveness.ResultsIn this work we generated a high-quality genome sequence and annotation for P. cinnamomi using a combination of Oxford Nanopore and Illumina sequencing technologies. The annotation was done using RNA-Seq data as supporting gene evidence. The final assembly consisted of 133 scaffolds, with an estimated genome size of 109.7 Mb, N50 of 1.18 Mb, and BUSCO completeness score of 97.5%. Genome partitioning analysis revealed that P. cinnamomi has a two-speed genome characteristic, similar to that of other oomycetes and fungal plant pathogens. In planta gene expression analysis revealed up-regulation of pathogenicity-related genes, suggesting their important roles during infection and host degradation.ConclusionThis study has provided a high-quality reference genome and annotation for P. cinnamomi. This is among the best assembled genomes for any Phytophthora species assembled to date and thus resulted in improved identification and characterization of pathogenicity-related genes, some of which were undetected in previous versions of genome assemblies. Phytophthora cinnamomi harbours a large number of effector genes which are located in the gene-poor regions of the genome. This unique genomic partitioning provides P. cinnamomi with a high level of adaptability and could contribute to its success as a highly invasive species. Finally, the genome sequence, its annotation and the pathogenicity effectors identified in this study will serve as an important resource that will enable future studies to better understand and mitigate the impact of this important pathogen.

Expression of Castanea crenata Allene Oxide Synthase in Arabidopsis Improves the Defense to Phytophthora cinnamomi.

Allene oxide synthase (AOS) is a key enzyme of the jasmonic acid (JA) signaling pathway. The AOS gene was previously found to be upregulated in an Asian chestnut species resistant to infection by the oomycete Phytophthora cinnamomi (Castanea crenata), while lower expression values were detected in the susceptible European chestnut (Castanea sativa). Here, we report a genetic and functional characterization of the C. crenata AOS (CcAOS) upon its heterologous gene expression in a susceptible ecotype of Arabidopsis thaliana, which contains a single AOS gene. It was found that Arabidopsis plants expressing CcAOS delay pathogen progression and exhibit more vigorous growth in its presence. They also show upregulation of jasmonic acid and salicylic acid-related genes. As in its native species, heterologous CcAOS localized to plastids, as revealed by confocal imaging of the CcAOS-eGFP fusion protein in transgenic Arabidopsis roots. This observation was confirmed upon transient expression in Nicotiana benthamiana leaf epidermal cells. To further confirm a specific role of CcAOS in the defense mechanism against the pathogen, we performed crosses between transgenic CcAOS plants and an infertile Arabidopsis AOS knockout mutant line. It was found that plants expressing CcAOS exhibit normal growth, remain infertile but are significantly more tolerant to the pathogen than wild type plants. Together, our results indicate that CcAOS is an important player in plant defense responses against oomycete infection and that its expression in susceptible varieties may be a valuable tool to mitigate biotic stress responses.

A Histopathological Study Reveals New Insights Into Responses of Chestnut (Castanea spp.) to Root Infection by Phytophthora cinnamomi.

The European chestnut (Castanea sativa) is threatened by the hemibiotrophic oomycete Phytophthora cinnamomi, the causal agent of ink disease. Chestnut species have different susceptibility levels to P. cinnamomi, with the Asian species (C. crenata; C. mollissima) exhibiting the highest level of resistance. A histological approach was used to study the responses exhibited by susceptible and resistant chestnut genotypes by characterizing the early stages of P. cinnamomi infection and the cellular responses it induces in roots. C. sativa (susceptible) and C. crenata (resistant) plantlets were inoculated with a P. cinnamomi virulent isolate with a zoospore suspension or by direct contact with mycelia agar pieces. Root samples were collected at 0.5, 3.5, 24, 48, and 72 h after inoculation (hai) for microscopic observations. Penetration was observed in both species at 0.5 and 3.5 hai with mycelium and zoospore inoculations, respectively. In both inoculation methods, following penetration into the rhizodermis, P. cinnamomi hyphae grew inter- and intracellularly through the cortex and into the vascular cylinder. C. crenata cells displayed a delay in the pattern of infection by having fewer cell layers colonized compared with C. sativa. At 72 hai, the collapse of the first layers of C. sativa cortical cells was observed, indicating the beginning of necrotrophy. C. crenata was able to respond more efficiently to P. cinnamomi than C. sativa by restricting the pathogen's growth area through the early activation of resistance responses such as callose deposition around some intracellular hyphae, hypersensitive response-like cell death, cell wall thickening, and accumulation of phenolic-like compounds.

Methods of supporting the development of Phytophthora cinnamomi rands on representatives of the Ericaceae DC family. In the orangeries of the Botanical garden of Peter the Great

In recent years, in the orangeries of the Peter the Great Botanical Garden, the affection of representatives of the Ericaceae family, the genus rhododendron (Rhododendron (L.) oomycete Phytophthora cinnamomi, has been growing. In the study of the rhizospheric soil of sick and healthy plants, a wide distribution of Phytophthora was found. representatives of this family. The dynamics of the spread of the disease from 2012 to 2019 is presented. The population of Phytophthora cinnamomi in the soils is unevenly distributed, changing its structure (abundance, survival, seasonality, life cycle) in accordance with various micro -phytocenoses. The population structure is governed by coenotic relationships with soil microorganisms. The Phytophthora cinnamomi population is “pulsating,” with a maximum population (in May-June) and a minimum in NovemberDecember. The creation of suppressive soils and suppression factors may serve as a preliminary strategy when creating measures to protect plants of the Peter the Great Botanical Garden. The results of studies of the effects of biological products (Vitaplan, Sternifag, Gliokladin) and fertilizers (Potassium humate, Ecofus) on the pathogen are presented. To increase immunity, resistance inducers (immunocytophyte, sillplant, chitosan) were used.

The soil biotic community protects Rhododendron spp. across multiple clades from the oomycete Phytophthora cinnamomi at a cost to plant growth.

The effects of whole soil biotic communities on plants is a result of positive and negative interactions from a complex suite of mutualists and pathogens. However, few experiments have evaluated the composite effects of whole soil biotic communities on plant growth and disease resistance. We conducted a factorial greenhouse experiment with 14 Rhododendron species grown with and without live conspecific soil biota and with and without the disease, Phytophthora cinnamomi. We tested the prediction that the presence of whole soil biotic communities influences survival in the presence of disease. We also explored functional trait correlations with disease susceptibility across the phylogeny. The presence of live soil biota led to higher survival in the presence of disease compared with sterilized soils, and the direction of this effect was consistent for seven species across four clades. The presence of live soil biota also significantly reduced plant growth rate and decreased shoot biomass, relative to plants grown in sterilized soil, indicating that live soil biota might influence plant allocation strategies. We found that Rhododendron species with higher Root Shoot Ratios were less susceptible to Phytophthora, suggesting that water relations influence disease susceptibility. Our findings that disease resistance and susceptibility occur independently across multiple clades and that whole soil biotic communities consistently enhance disease resistance across clades, suggest that soil biota may play an important role in disease resistance and can moderate disease-induced mortality.

Endophytic Trichoderma Species Isolated from Persea americana and Cinnamomum verum Roots Reduce Symptoms Caused by Phytophthora cinnamomi in Avocado.

Avocado root rot caused by the oomycete Phytophthora cinnamomi is a severe disease that affects avocado production in Mexico and worldwide. The use of biological control agents such as Trichoderma species isolated from places where the disease is always present, represents an efficient alternative to reduce losses. Thus, the objective of this research was to evaluate the biocontrol ability of 10 endophytic Trichoderma spp. strains against P. cinnamomi tested both in vitro and in the greenhouse. The endophytic Trichoderma spp. were recovered from Persea americana and Cinnamomum verum roots, isolated and purified on potato–dextrose–agar medium. Ten strains were identified by phylogenetic reconstruction of the internal transcribed spacer region of rDNA sequences as T. asperellum (T-AS1, T-AS2, T-AS6, and T-AS7), T. harzianum (T-H3, T-H4, and T-H5), T. hamatum (T-A12), T. koningiopsis (T-K8 and T-K11), and P. cinnamomi (CPO-PCU). In vitro dual-culture assay, the percentage of inhibition of radial growth (PIRG) between Trichoderma spp. and P. cinnamomi strains was measured according to the Bell’s scale. PIRG results indicated that T-AS2 reached the highest value of 78.32%, and T-H5 reached the lowest value of 38.66%. In the greenhouse, the infection was evaluated according to the percentage of disease incidence. Plants with the lowest incidence of dead by avocado root rot were those whose seedlings were inoculated with T-AS2 and T-AS7, resulting in only 5% death by root rot caused by P. cinnamomi. The disease incidence of seedlings with wilt symptoms and death decreased more than 50% in the presence of Trichoderma spp. Relying on the results, we conclude that T. asperellum and T. harzianum contribute to the biocontrol of soil-borne pathogenic oomycete P. cinnamomi.

An Overview of Phytophthora Species Inhabiting Declining Quercus suber Stands in Sardinia (Italy)

Cork oak forests are of immense importance in terms of economic, cultural, and ecological value in the Mediterranean regions. Since the beginning of the 20th century, these forests ecosystems have been threatened by several factors, including human intervention, climate change, wildfires, pathogens, and pests. Several studies have demonstrated the primary role of the oomycete Phytophthora cinnamomi Ronds in the widespread decline of cork oaks in Portugal, Spain, southern France, and Italy, although other congeneric species have also been occasionally associated. Between 2015 and 2019, independent surveys were undertaken to determine the diversity of Phytophthora species in declining cork oak stands in Sardinia (Italy). Rhizosphere soil samples were collected from 39 declining cork oak stands and baited in the laboratory with oak leaflets. In addition, the occurrence of Phytophthora was assayed using an in-situ baiting technique in rivers and streams located throughout ten of the surveyed oak stands. Isolates were identified by means of both morphological characters and sequence analysis of internal transcribed spacer (ITS) regions of ribosomal DNA. In total, 14 different Phytophthora species were detected. Phytophthora cinnamomi was the most frequently isolated species from rhizosphere soil, followed by P. quercina, P. pseudocryptogea, and P. tyrrhenica. In contrast, P. gonapodyides turned out to be the most dominant species in stream water, followed by P. bilorbang, P. pseudocryptogea, P. lacustris, and P. plurivora. Pathogenicity of the most common Phytophthora species detected was tested using both soil infestation and log inoculation methods. This study showed the high diversity of Phytophthora species inhabiting soil and watercourses, including several previously unrecorded species potentially involved in the decline of cork oak forests.

Soil-borne pathogens as determinants of regeneration patterns at community level in Mediterranean forests.

Emergent diseases are an increasing problem in forests worldwide. Exotic pathogens are now threatening forests where pathogens have not traditionally been considered to be major ecological drivers of tree demography, such as water-limited Mediterranean forests. However, how pathogens might limit regeneration in invaded forests is largely unknown. Here we used fungicide to analyse the impact of soil-borne oomycete pathogens on seedling establishment at community level in Mediterranean forests invaded by the exotic oomycete Phytophthora cinnamomi. Fungicide effects were modelled as a function of the tree neighbourhood composition, the seed mass of the target species, and the abiotic environment. Fungicide application had positive effects on seedling performance that varied in magnitude and spatial structure among coexisting species. Seed mass predicted fungicide effects on seedling emergence, but not on survival or growth. Positive fungicide effects were modulated by levels of abiotic resources, mainly water, increasing with soil moisture. Our results support a novel role for soil-borne oomycete pathogens as one more axis of the regeneration niche of woody species in water-limited forests. Given the increasing numbers of exotic oomycete pathogens worldwide, more research is needed to understand the role of this relevant microbial group as a factor shaping seedling establishment.

Directed energy system technology for the control of soilborne fungal pathogens and plant-parasitic nematodes.

It is challenging to manage soilborne pathogens and plant-parasitic nematodes using sustainable practices. Here, we evaluated a novel energy application system, Directed Energy System (DES). This system generates pulses of energy capable of impacting selected biological organisms. The oomycete Phytophthora cinnamomi, the fungus Verticillium dahliae, and the plant-parasitic nematodes Meloidogyne hapla and Globodera ellingtonae were added to soil. Then DES-generated energy was applied to soil and impacts on target organisms were determined. DES applied at 20, 40 and 70 J cm-3 to P. cinnamomi and V. dahliae resulted in ≥50% and 92% reductions (respectively) of propagules per gram of soil in comparison to the untreated control. There was a significant reduction of M. hapla eggs per gram of host tomato root between the untreated control, and 2.2, 13 and 25 J cm-3 DES dosages applied pre- or post-planting. Additionally, an 84% reduction in hatch from G. ellingtonae encysted eggs after treatment with 70 J cm-3 DES was observed. The dosages ranged from 40 or 80V mm-1 for nematodes to 200 V mm-1 for fungi. DES-generated energy reduced survival of the soilborne pathogens P. cinnamomi and V. dahlia, and the plant-parasitic nematodes M. hapla and G. ellingtonae. The application of this technology to a field setting remains to be considered. Published 2020. This article is a U.S. Government work and is in the public domain in the USA. Pest Management Science published by Wiley Periodicals, Inc. on behalf of © 2020 Society of Chemical Industry.

Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi.

The soil-borne oomycete Phytophthoracinnamomi is a highly destructive Phytophthora species associated with the decline offorest.This pathogen secretes a novel class of necrosis-inducing proteins known as Nep1-like proteins (NLPs). In this work, we report the sequencing and molecular characterization of one of these proteins, more specifically the necrosis-inducing Phytophthora protein 1 (NPP1). The ORF of the npp1 gene (EMBL database AM403130) has 768bp encoding a putative peptide of 256 amino acids with a molecular weight of approximately 25 kD. In order to understand its function, in vitro gene expression was studied during growth in different carbon sources (glucose, cellulose, and sawdust), and at different times of infection, in vivo by RT-qPCR. The highest expression of the npp1 gene occurred in glucose medium followed by sawdust. In vivo infection of Castanea sativa roots with P. cinnamomi revealed a decrease in npp1 expression from 12 to 24h; at 36h its expression increased suggesting the existence of a complex mechanism of defense/attack interaction between the pathogen and the host. Expression of recombinant npp1 gene was achieved in Pichiapastorisand assessed by SDS-PAGE analysis of the protein secreted into the culture supernatant, revealing the presence of the NPP1 protein.

Dinámica del crecimiento in vitro de Phytophthora cinnamomi en medios de cultivo alternativos

México es el principal país productor, consumidor y exportador de aguacate el en mundo. El estudio de la macrobiótica del suelo cobra mayor importancia en el complejo marchitez del aguacate debido que en esta patología pueden estar implicados agentes causales de tipo biótico y abiótico, siendo el Oomycete Phytophthora cinnamomi Rands la principal enfermedad radical del aguacate. El objetivo de la presente investigación fue evaluar la dinámica del crecimiento in vitro de Phytophthora cinnamomi en medios de cultivo alternativos. En los meses de octubre y noviembre de 2016, se recolectaron muestras de raíces en arboles de aguacate (Persea americana Mill. var. Hass) en la huerta experimental del INIFAP ubicada en San Juan Nuevo Parangaricutiro, Michoacán. Los aislados se identificaron morfológica y molecularmente. Se registró las medidas de diámetros de crecimiento de cada cepa in vitro en función al tiempo. Se efectúo una regresión lineal mediante modelos lineales generalizados (GLM) para obtener los parámetros de intercepto y pendiente, significancia de la regresión y el valor de R2 ajustada. Los análisis se realizaron con el programa estadístico R 3.4. De acuerdo con los resultados obtenidos, el medio de cultivo Centeno-agar fue el más eficiente para la proliferación de P. cinnamomi.

Dissecting Resistance to Phytophthora cinnamomi in Interspecific Hybrid Chestnut Crosses Using Sequence-Based Genotyping and QTL Mapping

The soilborne oomycete Phytophthora cinnamomi-which causes root rot, trunk cankers, and stem lesions on an estimated 5,000 plant species worldwide-is a lethal pathogen of American chestnut (Castanea dentata) as well as many other woody plant species. P. cinnamomi is particularly damaging to chestnut and chinquapin trees (Castanea spp.) in the southern portion of its native range in the United States due to relatively mild climatic conditions that are conductive to disease development. Introduction of resistant genotypes is the most practical solution for disease management in forests because treatment with fungicides and eradication of the pathogen are neither practical nor economically feasible in natural ecosystems. Using backcross families derived from crosses of American chestnuts with two resistant Chinese chestnut cultivars Mahogany and Nanking, we constructed linkage maps and identified quantitative trait loci (QTLs) for resistance to P. cinnamomi that had been introgressed from these Chinese chestnut cultivars. In total, 957 plants representing five cohorts of three hybrid crosses were genotyped by sequencing and phenotyped by standardized inoculation and visual examination over a 6-year period from 2011 to 2016. Eight parental linkage maps comprising 7,715 markers were constructed, and 17 QTLs were identified on four linkage groups (LGs): LG_A, LG_C, LG_E, and LG_K. The most consistent QTLs were detected on LG_E in seedlings from crosses with both 'Mahogany' and 'Nanking' and LG_K in seedlings from 'Mahogany' crosses. Two consistent large and medium effect QTLs located ∼10 cM apart were present in the middle and at the lower end of LG_E; other QTLs were considered to have small effects. These results imply that the genetic architecture of resistance to P. cinnamomi in Chinese chestnut × American chestnut hybrid progeny may resemble the P. sojae-soybean pathosystem, with a few dominant QTLs along with quantitatively inherited partial resistance conferred by multiple small-effect QTLs.

The Influence of Contrasting Microbial Lifestyles on the Pre-symbiotic Metabolite Responses of Eucalyptus grandis Roots

Plant roots co-inhabit the soil with a diverse consortium of microbes of which a number attempt to enter symbiosis with the plant. These microbes may be pathogenic, mutualistic, or commensal. Hence, the health and survival of plants is heavily reliant on their ability to perceive different microbial lifestyles and respond appropriately. Emerging research suggests that there is a pivotal role for plant root secondary metabolites in responding to microbial colonization. However, it is largely unknown if plants are able to differentiate between microbes of different lifestyles and respond differently during the earliest stages of pre-symbiosis (i.e., prior to physical contact). In studying plant responses to a range of microbial isolates, we questioned: (1) if individual microbes of different lifestyles and species caused alterations to the plant root metabolome during pre-symbiosis, and (2) if these early metabolite responses correlate with the outcome of the symbiotic interaction in later phases of colonization. We compared the changes of the root tip metabolite profile of the model tree Eucalyptus grandis during pre-symbiosis with two isolates of a pathogenic fungus (Armillaria luteobubalina), one isolate of a pathogenic oomycete (Phytophthora cinnamomi), two isolates of an incompatible mutualistic fungus (Suillus granulatus), and six isolates of a compatible mutualistic fungus (Pisolithus microcarpus). Untargeted metabolite profiling revealed predominantly positive root metabolite responses at the pre-symbiosis stage, prior to any observable phenotypical changes of the root tips. Metabolite responses in the host tissue that were specific to each microbial species were identified. A deeper analysis of the root metabolomic profiles during pre-symbiotic contact with six strains of P. microcarpus showed a connection between these early metabolite responses in the root with later colonization success. Further investigation using isotopic tracing revealed a portion of metabolites found in root tips originated from the fungus. RNA-sequencing also showed that the plant roots undergo complementary transcriptomic reprogramming in response to the fungal stimuli. Taken together, our results demonstrate that the early metabolite responses of plant roots are partially selective toward the lifestyle of the interacting microbe, and that these responses can be crucial in determining the outcome of the interaction.

Biological activity of 7beta-acetoxywithanolide D isolated from Acnistus arborescens

Many oomycete species are plant pathogens and are responsible for causing significant losses in agriculture. Currently, plant pathogen control is carried out by chemical, biological and physical methods. However, due to the development of resistance to these methods by some pathogens, it is imperative that alternative methods are developed. Brazilian biodiversity is well-known for its species richness and is considered a promising source of natural products. Among the vascular plants, the family Solanaceae A. Juss. (Solanaceae) is considered one of the largest, with distributions across all tropical and temperate regions of the world. The Solanaceae family presents a high diversity of species of economic importance as sources of food, medicinal and ornamental properties. Plants of this family are sources of secondary metabolites of various chemical classes that possess potential diverse applications. Therefore, chemical and biological investigations of these compounds are extremely important as they present alternatives for their potential use in the control of plant pathogens. Here, we report for the first time, the biological activity of 7beta-acetoxywithanolide D, a compound isolated from Acnistus arborescens, against the oomycete Phytophthora cinnamomi. With these results, we emphasize the importance of such studies on plant secondary metabolites, which may present coadjuvant options in the control of plant pathogens.

Regulation by biotic stress of tannins biosynthesis in Quercus ilex: Crosstalk between defoliation and Phytophthora cinnamomi infection.

Sustainability of the Mediterranean forest is threatened by oak decline, a disease of holm oak and other Quercus species that is initiated by infection with the oomycete Phytophthora cinnamomi. Focusing on the role of tannins in the chemical defense of plants, this work investigated whether tannins content in Quercus ilex is regulated by biotic stress. Screening of published genomes allowed the identification of Quercus sequences encoding enzymes for early steps of the biosynthesis of phenolic compounds, like hydrolysable tannins and condensed tannins (CT) among others, plus genes involved in the late steps of CT biosynthesis. Four days after treatment of Q. ilex seedlings by mechanical defoliation, P. cinnamomi infection and both stressors simultaneously, mRNA concentrations for tannins biosynthesis enzymes were measured in leaves. Among the transcript amount for shikimate dehydrogenase (SDH, EC 1.1.1.25), anthocyanidin reductase (EC 1.3.1.77), anthocyanidin synthase (EC 1.14.11.19) and leucoanthocyanidine reductase (EC 1.17.1.3), defoliation induced gene expression for SDH2 isoenzyme. About 4 days after infection of roots by P. cinnamomi, this up-regulation was canceled and SDH enzyme activity decreased. Furthermore, during this late stage of biotrophic interaction the pathogen switched off the correlation engaged by defoliation between the expression of SDH1 and SDH2 encoding genes and chemical defenses corresponding to total tannins, which were down-regulated. Thus, tannins biosynthesis in seedlings of Q. ilex is induced after mechanical defoliation whereas infection by the pathogen interferes with this regulation, potentially increasing the susceptibility of plants to herbivory and aggravating the impact of biotic stress.