Transcriptome analysis of the fungal pathogen Rosellinia necatrix during infection of a susceptible avocado rootstock identifies potential mechanisms of pathogenesis.

BACKGROUND: White root rot disease caused by Rosellinia necatrix is one of the most important threats affecting avocado productivity in tropical and subtropical climates. Control of this disease is complex and nowadays, lies in the use of physical and chemical methods, although none have proven to be fully effective. Detailed understanding of the molecular mechanisms underlying white root rot disease has the potential of aiding future developments in disease resistance and management. In this regard, this study used RNA-Seq technology to compare the transcriptomic profiles of R. necatrix during infection of susceptible avocado 'Dusa' roots with that obtained from the fungus cultured in rich medium. RESULTS: The transcriptomes from three biological replicates of R. necatrix colonizing avocado roots (RGA) and R. necatrix growing on potato dextrose agar media (RGPDA) were analyzed using Illumina sequencing. A total of 12,104 transcripts were obtained, among which 1937 were differentially expressed genes (DEG), 137 exclusively expressed in RGA and 160 in RGPDA. During the root infection process, genes involved in the production of fungal toxins, detoxification and transport of toxic compounds, hormone biosynthesis, gene silencing and plant cell wall degradation were overexpressed. Interestingly, 24 out of the 137 contigs expressed only during R. necatrix growth on avocado roots, were predicted as candidate effector proteins (CEP) with a probability above 60%. The PHI (Pathogen Host Interaction) database revealed that three of the R. necatrix CEP showed homology with previously annotated effectors, already proven experimentally via pathogen-host interaction. CONCLUSIONS: The analysis of the full-length transcriptome of R. necatrix during the infection process is suggesting that the success of this fungus to infect roots of diverse crops might be attributed to the production of different compounds which, singly or in combination, interfere with defense or signaling mechanisms shared among distinct plant families. The transcriptome analysis of R. necatrix during the infection process provides useful information and facilitates further research to a more in -depth understanding of the biology and virulence of this emergent pathogen. In turn, this will make possible to evolve novel strategies for white root rot management in avocado.

Observations on Texas hypoxylons, including two new Hypoxylon species and widespread environmental isolates of the H. croceum complex identified by a polyphasic approach.

Two new species and a new combination of Hypoxylon from Texas were identified and described based on morphological, multigene phylogenetic (ITS [nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2], 28S [5' 1200 bp of nuc 28S rDNA], RPB2 [partial second largest subunit of the DNA-directed RNA polymerase II], TUB2 [partial ß-tubulin]), and chemotaxonomic data. Hypoxylon olivaceopigmentum is characterized by its pulvinate to glomerate stromata, olivaceous KOH-extractable pigments, equilateral ascospores, and indehiscent perispore. Hypoxylon texense can be distinguished from morphologically similar species by its rust to dark brick KOH-extractable pigments and the high-performance liquid chromatography (HPLC) profile of its stromatal secondary metabolites. Hypoxylon hinnuleum is proposed as the sexual morph of Nodulisporium hinnuleum, featuring dark vinaceous glomerate stromata with dark brick KOH-extractable pigments composed of cohaerin-type azaphilones and smooth equilateral ascospores with indehiscent perispore. Based on these diagnostic characters, H. hinnuleum forms a complex with H. croceum and H. minicroceum. More than 50 ITS sequences with high identity originating from North American and East Asian environmental isolates formed a well-supported clade with the type of N. hinnuleum, demonstrating the widespread distribution of the species complex. In addition, updated descriptions and comprehensive illustrations with detailed information on the diagnostic features of H. fendleri and H. perforatum are provided. The multilocus phylogenetic reconstruction of Hypoxylon supported the status of the new species and broadened the knowledge about intergeneric relationships.

Weed Roots Facilitate the Spread of Rosellinia necatrix, the Causal Agent of White Root Rot.

Rosellinia necatrix causes white root rot in various plants, including the Japanese pear. PCR assays using specific primers for R. necatrix detected the fungus on the roots of nine weed species from infested pear orchards. The soil inoculation experiment revealed that the spread of R. necatrix was similar between weed-mowed and non-weed-mowed treatments under field conditions. The spread of R. necatrix was also observed when rescue grass (Bromus catharticus) was grown in planter boxes under greenhouse conditions, but was limited without the grass, suggesting that some weeds facilitate the spread of R. necatrix in soil.

Functional Fungal Endophytes in Coleus forskohlii Regulate Labdane Diterpene Biosynthesis for Elevated Forskolin Accumulation in Roots.

Coleus forskohlii is a perennial medicinal shrub cultivated mainly for its forskolin content. The plant has been used since ancient times in ayurvedic traditional medicines for the treatment of hypertension, glaucoma, asthma, congestive heart failures, obesity, and cancer. Use of endophytic microorganisms presents a special interest for the development of value-added bioactive compounds through agriculture. Limited investigations have been undertaken on in planta enhancement of forskolin content using endophytic fungus in sustainable agriculture. Here we report specific roles of three fungal endophytes, Fusarium redolens (RF1), Phialemoniopsis cornearis (SF1), and Macrophomina pseudophaseolina (SF2), functionally acting as plant probiotic fungus, regulating secondary metabolite (forskolin) biosynthesis in C. forskohlii. The root endophyte, RF1, and shoot endophytes, SF1 and SF2, were found to enhance forskolin content by 52 to 88% in pot and 60 to 84% in field experiments as compared to uninoculated control plants. The three endophytes also enhanced total biomass owing to plant growth promoting properties. The expression of diterpene synthases (CfTPSs) like CfTPS1, CfTPS2, CfTPS3, and CfTPS4 were significantly upregulated in endophyte-treated C. forskohlii plants. Elevated expression of key diterpene synthases (CfTPS2) in the forskolin biosynthesis pathway, exclusively present in the root cork of C. forskohlii, was observed following SF2 endophyte treatment. Furthermore, endophyte treatments conferred a variety of antagonistic activity against nematode galls (80%) and plant pathogens like Fusarium oxysporum, Colletotricum gloeosporioides, and Sclerotium rolfsii. RF1 and SF1 fungal endophytes showed positive for IAA production; however, SF1 also indicated phosphate solubilization activity. Overall, the qualitative and quantitative improvement of in planta forskolin enhancement represents an area of high commercial interest, and hence, our work focused on novel insights for the application of three fungal endophytes for in planta enhancement of forskolin content for C. forskohlii cultivation by a sustainable approach.

Detection of White Root Rot in Avocado Trees by Remote Sensing.

White root rot, caused by the soilborne fungus Rosellinia necatrix, is an important constraint to production for a wide range of woody crop plants such as avocado trees. The current methods of detection of white root rot are based on microbial and molecular techniques, and their application at orchard scale is limited. In this study, physiological parameters provided by imaging techniques were analyzed by machine learning methods. Normalized difference vegetation index (NDVI) and normalized canopy temperature (canopy temperature - air temperature) were tested as predictors of disease by several algorithms. Among them, logistic regression analysis (LRA) trained on NDVI data showed the highest sensitivity and lowest rate of false negatives. This algorithm based on NDVI could be a quick and feasible method to detect trees potentially affected by white root rot in avocado orchards.

Rosellinia necatrix infection induces differential gene expression between tolerant and susceptible avocado rootstocks.

Rosellinia necatrix is the causal agent of avocado white root rot (WRR). Control of this soil-borne disease is difficult, and the use of tolerant rootstocks may present an effective method to lessen its impact. To date, no studies on the molecular mechanisms regulating the avocado plant response towards this pathogen have been undertaken. To shed light on the mechanisms underpinning disease susceptibility and tolerance, molecular analysis of the gene's response in two avocado rootstocks with a contrasting disease reaction was assessed. Gene expression profiles against R. necatrix were carried out in the susceptible 'Dusa' and the tolerant selection BG83 avocado genotypes by micro-array analysis. In 'Dusa', the early response was mainly related to redox processes and cell-wall degradation activities, all becoming enhanced after disease progression affected photosynthetic capacity, whereas tolerance to R. necatrix in BG83 relied on the induction of protease inhibitors and their negative regulators, as well as genes related to tolerance to salt and osmotic stress such as aspartic peptidase domain-containing proteins and gdsl esterase lipase proteins. In addition, three protease inhibitors were identified, glu protease, trypsin and endopeptidase inhibitors, which were highly overexpressed in the tolerant genotype when compared to susceptible 'Dusa', after infection with R. necatrix, reaching fold change values of 52, 19 and 38, respectively. The contrasting results between 'Dusa' and BG83 provide new insights into the different mechanisms involved in avocado tolerance to Phytophthora cinnamomi and R. necatrix, which are consistent with their biotrophic and necrotrophic lifestyles, respectively. The differential induction of genes involved in salt and osmotic stress in BG83 could indicate that R. necatrix penetration into the roots is associated with osmotic effects, suggesting that BG83's tolerance to R. necatrix is related to the ability to withstand osmotic imbalance. In addition, the high expression of protease inhibitors in tolerant BG83 compared to susceptible 'Dusa' after infection with the pathogen suggests the important role that these proteins may play in the defence of avocado rootstocks against R. necatrix.

Response of the Biocontrol Agent Pseudomonas pseudoalcaligenes AVO110 to Rosellinia necatrix Exudate.

The rhizobacterium Pseudomonas pseudoalcaligenes AVO110, isolated by the enrichment of competitive avocado root tip colonizers, controls avocado white root rot disease caused by Rosellinia necatrix Here, we applied signature-tagged mutagenesis (STM) during the growth and survival of AVO110 in fungal exudate-containing medium with the goal of identifying the molecular mechanisms linked to the interaction of this bacterium with R. necatrix A total of 26 STM mutants outcompeted by the parental strain in fungal exudate, but not in rich medium, were selected and named growth-attenuated mutants (GAMs). Twenty-one genes were identified as being required for this bacterial-fungal interaction, including membrane transporters, transcriptional regulators, and genes related to the metabolism of hydrocarbons, amino acids, fatty acids, and aromatic compounds. The bacterial traits identified here that are involved in the colonization of fungal hyphae include proteins involved in membrane maintenance (a dynamin-like protein and ColS) or cyclic-di-GMP signaling and chemotaxis. In addition, genes encoding a DNA helicase (recB) and a regulator of alginate production (algQ) were identified as being required for efficient colonization of the avocado rhizosphere.IMPORTANCE Diseases associated with fungal root invasion cause a significant loss of fruit tree production worldwide. The bacterium Pseudomonas pseudoalcaligenes AVO110 controls avocado white root rot disease caused by Rosellinia necatrix by using mechanisms involving competition for nutrients and niches. Here, a functional genomics approach was conducted to identify the bacterial traits involved in the interaction with this fungal pathogen. Our results contribute to a better understanding of the multitrophic interactions established among bacterial biocontrol agents, the plant rhizosphere, and the mycelia of soilborne pathogens.

A novel experimental system using the liverwort Marchantia polymorpha and its fungal endophytes reveals diverse and context-dependent effects.

Fungal symbioses are ubiquitous in plants, but their effects have mostly been studied in seed plants. This study aimed to assess the diversity of fungal endophyte effects in a bryophyte and identify factors contributing to the variability of outcomes in these interactions. Fungal endophyte cultures and axenic liverwort clones were isolated from wild populations of the liverwort, Marchantia polymorpha. These collections were combined in a gnotobiotic system to test the effects of fungal isolates on the growth rates of hosts under laboratory conditions. Under the experimental conditions, fungi isolated from M. polymorpha ranged from aggressively pathogenic to strongly growth-promoting, but the majority of isolates caused no detectable change in host growth. Growth promotion by selected fungi depended on nutrient concentrations and was inhibited by coinoculation with multiple fungi. The M. polymorpha endophyte system expands the resources for this model liverwort. The experiments presented here demonstrate a wealth of diversity in fungal interactions even in a host reported to lack standard mycorrhizal symbiosis. In addition, they show that some known pathogens of vascular plants live in M. polymorpha and can confer benefits to this nonvascular host. This highlights the importance of studying endophyte effects across the plant tree of life.

Diversity of Neopestalotiopsis and Pestalotiopsis spp., Causal Agents of Guava Scab in Colombia.

Common guava (Psidium guajava L.) is a fruit tree of global economic importance. It is grown in Asia, South and Central America, and Hawaii for its exquisite aroma and flavor, and nutritional and medical properties. However, guava production is limited by guava scab, caused by fungi in the Pestalotiopsis genus. Characteristic symptoms of guava scab are corky, ovoid or round lesions on fruit surfaces. These lesions may thicken, affecting the flesh below and reducing fruit quality and commercial value. We characterized 81 isolates isolated from guava scab lesions on guava leaves and fruit in different regions of Colombia, and identified them as Pestalotiopsis and Neopestalotiopsis spp. We analyzed the morphology, pathogenicity, and genetic diversity of the isolates based on the sequences of the internal transcribed spacer, ß-tubulin, and elongation factor genes. Isolates were morphologically, pathogenically, and genetically diverse but the diversity did not correlate with geographical origin, or guava cultivar or tissue from which the isolates were recovered. Selected monosporic isolates included in the multiple-gene analysis were identified as belonging to two genera: Neopestalotiopsis (65 isolates with versicolorous conidia) and Pestalotiopsis (4 isolates with concolorous conidia).

Pestalotiopsis-Like Species Causing Gray Blight Disease on Camellia sinensis in China.

Gray blight of tea, caused by several Pestalotiopsis-like species, is one of the most destructive foliar diseases in tea cultivation yet the characteristics of these pathogens have not been confirmed until now. With morphological and multigene phylogenetic analyses, we have identified the gray blight fungi as Pseudopestalotiopsis camelliae-sinensis, Neopestalotiopsis clavispora, and Pestalotiopsis camelliae. Phylogenetic analyses derived from the combined internal transcribed spacer, ß-tubulin, and translation elongation factor 1-α gene regions successfully resolved most of the Pestalotiopsis-like species used in this study with high bootstrap supports and revealed three major clusters representing these three species. Differences in colony appearance and conidia morphology (shape, size, septation, color and length of median cells, and length and number of apical and basal appendages) were consistent with the phylogenetic grouping. Pathogenicity tests validated that all three species isolated from tea leaves were causal agents of gray blight disease on tea plant (Camellia sinensis). This is the first description of the characteristics of the three species Pseudopestalotiopsis camelliae-sinensis, N. clavispora, and Pestalotiopsis camelliae as causal agents of tea gray blight disease in China.

Diversity of Diatrypaceae Species Associated with Dieback of Grapevines in South Africa, with the Description of Eutypa cremea sp. nov.

Recent studies in grape-growing areas including Australia, California, and Spain have revealed an extensive diversity of Diatrypaceae species on grapevines showing dieback symptoms and cankers. However, in South Africa, little is known regarding the diversity of these species in vineyards. The aim of this study was, therefore, to identify and characterize Diatrypaceae species associated with dieback symptoms of grapevine in South Africa. Isolates were collected from dying spurs of grapevines aged 4 to 8 years old, grapevine wood showing wedge-shaped necrosis when cut in cross section as well as from perithecia on dead grapevine wood. The collected isolates were identified based on morphological characters and phylogenetic analyses of the internal transcribed spacer region (ITS) and ß-tubulin gene. Seven Diatrypaceae species were identified on grapevine, namely Cryptovalsa ampelina, C. rabenhorstii, Eutypa consobrina, E. lata, E. cremea sp. nov., Eutypella citricola, and E. microtheca. The dying spurs yielded the highest diversity of species when compared with the wedge-shaped necrosis and/or perithecia. C. ampelina was the dominant species in the dying spurs, followed by E. citricola, whereas E. lata was the dominant species isolated from the wedge-shaped necroses and perithecia. These results confirm E. lata as an important grapevine canker pathogen in South Africa, but the frequent association of C. ampelina with spur dieback suggests that this pathogen plays a more prominent role in dieback than previously assumed. In some cases, more than one species were isolated from a single symptom, which suggests that interactions may be occurring leading to decline of grapevines. C. rabenhorstii, E. consobrina, E. citricola, E. microtheca, and E. cremea are reported for the first time on grapevine in South Africa.

Nematicidal activity of grammicin produced by Xylaria grammica KCTC 13121BP against Meloidogyne incognita.

BACKGROUND: The endolichenic fungus Xylaria grammica KCTC 13121BP showed strong nematicidal activity against Meloidogyne incognita. This study aimed to identify the nematicidal metabolites and to evaluate the efficacy of the strain as a biocontrol agent under pot and field conditions. RESULTS: Bioassay-guided fractionation and instrumental analyses led to grammicin being identified as the nematicidal metabolite. Because patulin is a mycotoxic isomer of grammicin and is known to have strong antibacterial and cytotoxic activities, several biological activities of the two compounds were compared. Grammicin showed strong second-stage juvenile killing and egg-hatching inhibitory effects, with a 50% effective concentration at 72 h (EC50/72 h ) of 15.9 µg/mL and a 50% effective concentration at 14 days (EC50/14 days ) of 5.87 µg/mL, respectively, whereas patulin was virtually inactive in both respects. Patulin was strongly active toward various phytopathogenic bacteria in vitro, whereas grammicin was weakly so. Patulin at the concentration range of 0.1-10 µg/mL also showed dose-dependent cytotoxicity toward the human first-trimester trophoblast cell line SW.71, whereas grammicin was not toxic toward this cell line. In pot and field experiments, a wettable powder-type formulation and fermentation broth filtrate of X. grammica KCTC 13121BP effectively suppressed the development of root-knot nematode disease on tomato and melon plants. CONCLUSION: The results suggest that X. grammica and grammicin may have potential applications for control of root-knot nematode disease of various crops. © 2017 Society of Chemical Industry.

Picturing pathogen infection in plants.

Several imaging techniques have provided valuable tools to evaluate the impact of biotic stress on host plants. The use of these techniques enables the study of plant-pathogen interactions by analysing the spatial and temporal heterogeneity of foliar metabolism during pathogenesis. In this work we review the use of imaging techniques based on chlorophyll fluorescence, multicolour fluorescence and thermography for the study of virus, bacteria and fungi-infected plants. These studies have revealed the impact of pathogen challenge on photosynthetic performance, secondary metabolism, as well as leaf transpiration as a promising tool for field and greenhouse management of diseases. Images of standard chlorophyll fluorescence (Chl-F) parameters obtained during Chl-F induction kinetics related to photochemical processes and those involved in energy dissipation, could be good stress indicators to monitor pathogenesis. Changes on UV-induced blue (F440) and green fluorescence (F520) measured by multicolour fluorescence imaging in pathogen-challenged plants seem to be related with the up-regulation of the plant secondary metabolism and with an increase in phenolic compounds involved in plant defence, such as scopoletin, chlorogenic or ferulic acids. Thermal imaging visualizes the leaf transpiration map during pathogenesis and emphasizes the key role of stomata on innate plant immunity. Using several imaging techniques in parallel could allow obtaining disease signatures for a specific pathogen. These techniques have also turned out to be very useful for presymptomatic pathogen detection, and powerful non-destructive tools for precision agriculture. Their applicability at lab-scale, in the field by remote sensing, and in high-throughput plant phenotyping, makes them particularly useful. Thermal sensors are widely used in crop fields to detect early changes in leaf transpiration induced by both air-borne and soil-borne pathogens. The limitations of measuring photosynthesis by Chl-F at the canopy level are being solved, while the use of multispectral fluorescence imaging is very challenging due to the type of light excitation that is used.

Contributions of North American endophytes to the phylogeny, ecology, and taxonomy of Xylariaceae (Sordariomycetes, Ascomycota).

The Xylariaceae (Sordariomycetes) comprise one of the largest and most diverse families of Ascomycota, with at least 85 accepted genera and ca. 1343 accepted species. In addition to their frequent occurrence as saprotrophs, members of the family often are found as endophytes in living tissues of phylogenetically diverse plants and lichens. Many of these endophytes remain sterile in culture, precluding identification based on morphological characters. Previous studies indicate that endophytes are highly diverse and represent many xylariaceous genera; however, phylogenetic analyses at the family level generally have not included endophytes, such that their contributions to understanding phylogenetic relationships of Xylariaceae are not well known. Here we use a multi-locus, cumulative supermatrix approach to integrate 92 putative species of fungi isolated from plants and lichens into a phylogenetic framework for Xylariaceae. Our collection spans 1933 isolates from living and senescent tissues in five biomes across the continental United States, and here is analyzed in the context of previously published sequence data from described species and additional taxon sampling of type specimens from culture collections. We found that the majority of strains obtained in our surveys can be classified in the hypoxyloid and xylaroid subfamilies, although many also were found outside of these lineages (as currently circumscribed). Many endophytes were placed in lineages previously not known for endophytism. Most endophytes appear to represent novel species, but inferences are limited by potential gaps in public databases. By linking our data, publicly available sequence data, and records of ascomata, we identify many geographically widespread, host-generalist clades capable of symbiotic associations with diverse photosynthetic partners. Concomitant with such cosmopolitan host use and distributions, many xylariaceous endophytes appear to inhabit both living and non-living plant tissues, with potentially important roles as saprotrophs. Overall, our study reveals major gaps in the availability of multi-locus datasets and metadata for this iconic family, and provides new hypotheses regarding the ecology and evolution of endophytism and other trophic modes across the family Xylariaceae.

Genome analysis of Daldinia eschscholtzii strains UM 1400 and UM 1020, wood-decaying fungi isolated from human hosts.

BACKGROUND: Daldinia eschscholtzii is a wood-inhabiting fungus that causes wood decay under certain conditions. It has a broad host range and produces a large repertoire of potentially bioactive compounds. However, there is no extensive genome analysis on this fungal species. RESULTS: Two fungal isolates (UM 1400 and UM 1020) from human specimens were identified as Daldinia eschscholtzii by morphological features and ITS-based phylogenetic analysis. Both genomes were similar in size with 10,822 predicted genes in UM 1400 (35.8 Mb) and 11,120 predicted genes in UM 1020 (35.5 Mb). A total of 751 gene families were shared among both UM isolates, including gene families associated with fungus-host interactions. In the CAZyme comparative analysis, both genomes were found to contain arrays of CAZyme related to plant cell wall degradation. Genes encoding secreted peptidases were found in the genomes, which encode for the peptidases involved in the degradation of structural proteins in plant cell wall. In addition, arrays of secondary metabolite backbone genes were identified in both genomes, indicating of their potential to produce bioactive secondary metabolites. Both genomes also contained an abundance of gene encoding signaling components, with three proposed MAPK cascades involved in cell wall integrity, osmoregulation, and mating/filamentation. Besides genomic evidence for degrading capability, both isolates also harbored an array of genes encoding stress response proteins that are potentially significant for adaptation to living in the hostile environments. CONCLUSIONS: Our genomic studies provide further information for the biological understanding of the D. eschscholtzii and suggest that these wood-decaying fungi are also equipped for adaptation to adverse environments in the human host.

Melanin is required for the formation of the multi-cellular conidia in the endophytic fungus Pestalotiopsis microspora.

Melanin plays an important role in regulating various biological processes in many fungi. However, its biological role in conidiation remains largely elusive. We report here that conidia production, morphogenesis, integrity, germination and their viability in Pestalotiopsis microspora require the polyketide-derived melanin. A polyketide synthase gene, pks1, was identified and demonstrated responsible for melanin biosynthesis in this fungus. A targeted deletion mutant strain Δpks1 displayed a defect in pigmentation of conidia and had an albino colonial phenotype. Interestingly, Δpks1 produced approximately 6-fold as many conidia as the wild type did, suggesting a negative modulation of melanin on conidia production in this fungus. Moreover, the conidia failed to develop into the normal five-cell morphology, rather the three main-body cells separated via constriction at the original septum position to generate three independent mutant conidia. This result suggests a novel role of melanin in the formation of the multi-cellular conidia. Germ tubes could develop from the three different types of mutant conidia and kept elongating, despite a significantly lower germination rate was observed for them. Still more, the unpigmented conidia became permeable to Calcofluor White and DAPI, suggesting the integrity of the conidia was impaired. Deliberate inhibition of melanin biosynthesis by a specific inhibitor, tricyclazole, led to a similar phenotypes. This work demonstrates a new function of fungal melanin in conidial development.

Mutual Exclusion between Fungal Species of the Fusarium Head Blight Complex in a Wheat Spike.

Fusarium head blight (FHB) is one of the most damaging diseases of wheat. FHB is caused by a species complex that includes two genera of Ascomycetes: Microdochium and Fusarium. Fusarium graminearum, Fusarium culmorum, Fusarium poae, and Microdochium nivale are among the most common FHB species in Europe and were chosen for these experiments. Field studies and surveys show that two or more species often coexist within the same field or grain sample. In this study, we investigated the competitiveness of isolates of different species against isolates of F. graminearum at the scale of a single spike. By performing point inoculations of a single floret, we ensured that each species was able to establish independent infections and competed for spike colonization only. The fungal colonization was assessed in each spike by quantitative PCR. After establishing that the spike colonization was mainly downwards, we compared the relative colonization of each species in coinoculations. Classical analysis of variance suggested a competitive interaction but remained partly inconclusive because of a large between-spike variance. Further data exploration revealed a clear exclusion of one of the competing species and the complete absence of coexistence at the spike level.

The bZIP transcription factor PfZipA regulates secondary metabolism and oxidative stress response in the plant endophytic fungus Pestalotiopsis fici.

The bZIP transcription factors are conserved in all eukaryotes and play critical roles in organismal responses to environmental challenges. In filamentous fungi, several lines of evidence indicate that secondary metabolism (SM) is associated with oxidative stress mediated by bZIP proteins. Here we uncover a connection with a bZIP protein and oxidative stress induction of SM in the plant endophytic fungus Pestalotiopsis fici. A homology search of the P. fici genome with the bZIP protein RsmA, involved in SM and the oxidative stress response in Aspergillus nidulans, identified PfZipA. Deletion of PfzipA resulted in a strain that displayed resistant to the oxidative reagents tert-butylhydroperoxide (tBOOH), diamide, and menadione sodium bisulfite (MSB), but increased sensitivity to H2O2 as compared to wild type (WT). Secondary metabolite production presented a complex pattern dependent on PfzipA and oxidative reagents. Without oxidative treatment, the ΔPfzipA strain produced less isosulochrin and ficipyroneA than WT; addition of tBOOH further decreased production of iso-A82775C and pestaloficiol M in ΔPfzipA; diamide treatment resulted in equivalent production of isosulochrin and ficipyroneA in the two strains; MSB treatment further decreased production of RES1214-1 and iso-A82775C but increased pestaloficiol M production in the mutant; and H2O2 treatment resulted in enhanced production of isosulochrin, RES1214-1 and pestheic acid but decreased ficipyroneA and pestaloficiol M in ΔPfzipA compared to WT. Our results suggest that PfZipA regulation of SM is modified by oxidative stress pathways and provide insights into a possible role of PfZipA in mediating SM synthesis in the endophytic lifestyle of P. fici.

Organic amendments to avocado crops induce suppressiveness and influence the composition and activity of soil microbial communities.

One of the main avocado diseases in southern Spain is white root rot caused by the fungus Rosellinia necatrix Prill. The use of organic soil amendments to enhance the suppressiveness of natural soil is an inviting approach that has successfully controlled other soilborne pathogens. This study tested the suppressive capacity of different organic amendments against R. necatrix and analyzed their effects on soil microbial communities and enzymatic activities. Two-year-old avocado trees were grown in soil treated with composted organic amendments and then used for inoculation assays. All of the organic treatments reduced disease development in comparison to unamended control soil, especially yard waste (YW) and almond shells (AS). The YW had a strong effect on microbial communities in bulk soil and produced larger population levels and diversity, higher hydrolytic activity and strong changes in the bacterial community composition of bulk soil, suggesting a mechanism of general suppression. Amendment with AS induced more subtle changes in bacterial community composition and specific enzymatic activities, with the strongest effects observed in the rhizosphere. Even if the effect was not strong, the changes caused by AS in bulk soil microbiota were related to the direct inhibition of R. necatrix by this amendment, most likely being connected to specific populations able to recolonize conducive soil after pasteurization. All of the organic amendments assayed in this study were able to suppress white root rot, although their suppressiveness appears to be mediated differentially.

Strong Induction of Minor Terpenes in Italian Cypress, Cupressus sempervirens, in Response to Infection by the Fungus Seiridium cardinale.

Seiridium cardinale, the main fungal pathogen responsible for cypress bark canker, is the largest threat to cypresses worldwide. The terpene response of canker-resistant clones of Italian cypress, Cupressus sempervirens, to two differently aggressive isolates of S. cardinale was studied. Phloem terpene concentrations, foliar terpene concentrations, as well as foliar terpene emission rates were analyzed 1, 10, 30, and 90 days after artificial inoculation with fungal isolates. The phloem surrounding the inoculation point exhibited de novo production of four oxygenated monoterpenes and two unidentified terpenes. The concentrations of several constitutive mono- and diterpenes increased strongly (especially α-thujene, sabinene, terpinolene, terpinen-4-ol, oxygenated monoterpenes, manool, and two unidentified diterpenes) as the infection progressed. The proportion of minor terpenes in the infected cypresses increased markedly from the first day after inoculation (from 10% in the control to 30-50% in the infected treatments). Foliar concentrations showed no clear trend, but emission rates peaked at day 10 in infected trees, with higher δ-3-carene (15-fold) and total monoterpene (10-fold) emissions than the control. No substantial differences were found among cypresses infected by the two fungal isolates. These results suggest that cypresses activate several direct and indirect chemical defense mechanisms after infection by S. cardinale.