Minimal impacts on the wheat microbiome when Trichoderma gamsii T6085 is applied as a biocontrol agent to manage fusarium head blight disease.

Fusarium head blight (FHB) is a major fungal disease that causes severe yield and quality loss in wheat. Biological control can be integrated with other management strategies to control FHB. For this purpose, Trichoderma gamsii strain T6085 is a potential biocontrol agent to limit the infection of F. graminearum and F. culmorum in wheat. However, the possible impacts of T. gamsii T6085 on the broader microbiome associated with the wheat plant are not currently understood. Therefore, we identified bacteria and fungi associated with different wheat tissues, including assessment of their relative abundances and dynamics in response to the application of T6085 and over time, using amplicon sequencing. Residues of the prior year's wheat crop and the current year's wheat spikes were collected at multiple time points, and kernel samples were collected at harvest. DNA was extracted from the collected wheat tissues, and amplicon sequencing was performed to profile microbiomes using 16S v4 rRNA amplicons for bacteria and ITS2 amplicons for fungi. Quantitative PCR was performed to evaluate the absolute abundances of F. graminearum and T. gamsii in different wheat tissues. Disease progression was tracked visually during the growing season, revealing that FHB severity and incidence were significantly reduced when T6085 was applied to wheat spikes at anthesis. However, treatment with T6085 did not lessen the F. graminearum abundance in wheat spikes or kernels. There were substantial changes in F. graminearum abundance over time; in crop residue, pathogen abundance was highest at the initial time point and declined over time, while in wheat spikes, pathogen abundance increased significantly over time. The predominant bacterial taxa in wheat spikes and kernels were Pseudomonas, Enterobacter, and Pantoea, while Alternaria and Fusarium were the dominant fungal groups. Although the microbiome structure changed substantially over time, there were no community-scale rearrangements due to the T6085 treatment. The work suggests several other taxa that could be explored as potential biocontrol agents to integrate with T6085 treatment. However, the timing and the type of T6085 application need to be improved to give more advantages for T6085 to colonize and reduce the F. graminearum inoculum in the field.

Insights on KP4 Killer Toxin-like Proteins of Fusarium Species in Interspecific Interactions.

KP4 killer toxins are secreted proteins that inhibit cell growth and induce cell death in target organisms. In Fusarium graminearum, KP4-like (KP4L) proteins contribute to fungal virulence in wheat seedling rot and are expressed during Fusarium head blight development. However, fungal KP4L proteins are also hypothesized to support fungal antagonism by permeabilizing cell walls of competing fungi to enable penetration of toxic compounds. Here, we report the differential expression patterns of F. graminearum KP4L genes (Fgkp4l-1, -2, -3 and -4) in a competitive interaction, using Trichoderma gamsii as the antagonist. The results from dual cultures indicate that Fgkp4l-3 and Fgkp4l-4 could participate in the recognition at the distance of the antagonist, while all Fgkp4l genes were highly activated in the pathogen during the physical interaction of both fungi. Only Fgkp4l-4 was up-regulated during the interaction with T. gamsii in wheat spikes. This suggests the KP4L proteins could participate in supporting F. graminearum interspecific interactions, even in living plant tissues. The distribution of KP4L orthologous within the genus Fusarium revealed they are more represented in species with broad host-plant range than in host-specific species. Phylogeny inferred provides evidence that KP4L genes evolved through gene duplications, gene loss and sequence diversification in the genus Fusarium.

Fungal cross-talk: an integrated approach to study distance communication.

Despite the interest on fungi as eukaryotic model systems, the molecular mechanisms regulating the fungal non-self-recognition at a distance have not been studied so far. This paper investigates the molecular mechanisms regulating the cross-talk at a distance between two filamentous fungi, Trichoderma gamsii and Fusarium graminearum which establish a mycoparasitic interaction where T. gamsii and F. graminearum play the roles of mycoparasite and prey, respectively. In the present work, we use an integrated approach involving dual culture tests, comparative genomics and transcriptomics to investigate the fungal interaction before contact ('sensing phase'). Dual culture tests demonstrate that growth rate of F. graminearum accelerates in presence of T. gamsii at the sensing phase. T. gamsii up-regulates the expression of a ferric reductase involved in iron acquisition, while F. graminearum up-regulates the expression of genes coding for transmembrane transporters and killer toxins. At the same time, T. gamsii decreases the level of extracellular interaction by down-regulating genes coding for hydrolytic enzymes acting on fungal cell wall (chitinases). Given the importance of fungi as eukaryotic model systems and the ever-increasing genomic resources available, the integrated approach hereby presented can be applied to other interactions to deepen the knowledge on fungal communication at a distance.

Straw Competition and Wheat Root Endophytism of Trichoderma gamsii T6085 as Useful Traits in the Biological Control of Fusarium Head Blight.

Trichoderma gamsii T6085 has been investigated for many years as a beneficial isolate for use in the biocontrol of Fusarium head blight (FHB) of wheat caused primarily by Fusarium graminearum. Previous work focused on application of T6085 to wheat spikes at anthesis, whereas application to soil before or at sowing has received limited attention. In the present study, the competitive ability of T6085 on plant residues against F. graminearum was investigated. Results showed a significant reduction of wheat straw colonization by the pathogen and of the development of perithecia, not only when T6085 was applied alone but also in the presence of a F. oxysporum isolate (7121), well known as a natural competitor on wheat plant residues. T6085 was able to endophytically colonize wheat roots, resulting in internal colonization of the radical cortex area, without reaching the vascular system, as confirmed by confocal microscopy. This intimate interaction with the plant resulted in a significant increase of the expression of the plant defense-related genes PAL1 and PR1. Taken together, competitive ability, endophytic behavior, and host resistance induction represent three important traits that can be of great use in the application of T6085 against FHB not only on spikes at anthesis but potentially also in soil before or at sowing.

Combined Comparative Genomics and Gene Expression Analyses Provide Insights into the Terpene Synthases Inventory in Trichoderma.

Trichoderma is a fungal genus comprising species used as biocontrol agents in crop plant protection and with high value for industry. The beneficial effects of these species are supported by the secondary metabolites they produce. Terpenoid compounds are key players in the interaction of Trichoderma spp. with the environment and with their fungal and plant hosts; however, most of the terpene synthase (TS) genes involved in their biosynthesis have yet not been characterized. Here, we combined comparative genomics of TSs of 21 strains belonging to 17 Trichoderma spp., and gene expression studies on TSs using T. gamsii T6085 as a model. An overview of the diversity within the TS-gene family and the regulation of TS genes is provided. We identified 15 groups of TSs, and the presence of clade-specific enzymes revealed a variety of terpenoid chemotypes evolved to cover different ecological demands. We propose that functional differentiation of gene family members is the driver for the high number of TS genes found in the genomes of Trichoderma. Expression studies provide a picture in which different TS genes are regulated in many ways, which is a strong indication of different biological functions.

Genome Resources for the Endophytic Fungus Paraphaeosphaeria sporulosa.

Paraphaeosphaeria genus includes plant pathogens or biocontrol agents as well as bioremediators and endophytic fungi. Paraphaeosphaeria sporulosa 10515 was isolated in 2013 as an endophyte of Festuca spp. collected on Mount Etna at 1,832 meters above sea level. Here, we present the first-draft whole-genome sequence of a P. sporulosa endophytic isolate. This data will be useful for future research on understanding the genetic bases of endophytism.

Carnauba wax enhances the insecticidal activity of entomopathogenic fungi against the blowfly Lucilia sericata (Diptera: Calliphoridae).

Blowfly, Lucilia sericata (Diptera: Calliphoridae), is a problematic synanthropic insect pest, a vector of microbial pathogens, and the causal agent of secondary myiasis. Fungal biopesticides are considered eco-friendly tools, alternative to synthetic pesticides, for the control of arthropod pests; however, to date, little is known about their bioactivity against blowflies. In this study, we assessed the insecticidal activity of three well-known entomopathogenic fungi, Beauveria bassiana, Beauveria pseudobassiana and Akanthomyces muscarius against L. sericata. In addition, we tested powdered carnauba wax as an electrically charged dust carrier in an attempt to enhance the virulence of fungal spores. Pathogenicity tests on adult flies, by adult immersion in conidial suspension (108 conidia mL-1), showed that the median lethal time (LT50) was 5.3, 5.9, and 6.2 days for B. bassiana, A. muscarius and B. pseudobassiana, respectively. In topical tests, when 108 dry conidia were mixed with or without carnauba wax, the LT50 was 7.7, 10.2, and 14 days without this carrier and 6.9, 8.6, and 13.8 days with it for B. bassiana, B. pseudobassiana and A. muscarius, respectively. Overall, our findings showed that, among the tested fungi, B. bassiana was the most virulent when formulated as a dry powder with carnauba wax, which greatly improved fungal efficacy against the blowfly. We discuss the utility of carnauba wax for electrostatic formulation powder of fungal spores in the integrated management of blowflies as an environmentally sustainable tool to reduce the over-reliance on chemical insecticides and their risk of resistance.

Secondary metabolites produced by Colletotrichum lupini, the causal agent of anthachnose of lupin (Lupinus spp.).

COLLETOTRICHUM LUPINI: is the causal agent of lupin (Lupinus albus L.) anthracnose, a destructive seed-borne disease affecting stems and pods. Despite that several biological studies have been carried out on this pathogen, the production of secondary metabolites has not yet been investigated. Thus, a strain of C. lupini, obtained from symptomatic stems of L. albus, has been grown in vitro to evaluate its ability to produce bioactive compounds. From its culture filtrates, a 3-substituted indolinone, named lupindolinone, and a 5,6-disubstituted tetrahydro-α-pyrone, named lupinlactone, were isolated together with the known (3R)-mevalonolactone and tyrosol. Lupindolinone and lupinlactone were characterized as 3-ethylindolin-2-one and 5-hydroxy-6-methyltetrahydropyran-2-one by spectroscopic methods (essentially nuclear magnetic resonance [NMR] and high-resolution electrospray ionization mass spectrometry [HR ESI-MS]). The R absolute configuration (AC) at C-5 of lupinlactone was determined by applying the modified Mosher's method. Thus, considering its relative stereochemistry assigned by NMR spectroscopy, the AC of lupinlactone could be formulated as 5R,6S. Lupindolinone was isolated as racemic mixture as shown by investigation using chiroptical methods. The metabolites were assayed in different biological tests and proved to have some activities at the used concentration.

Evolution and comparative genomics of the most common Trichoderma species.

BACKGROUND: The growing importance of the ubiquitous fungal genus Trichoderma (Hypocreales, Ascomycota) requires understanding of its biology and evolution. Many Trichoderma species are used as biofertilizers and biofungicides and T. reesei is the model organism for industrial production of cellulolytic enzymes. In addition, some highly opportunistic species devastate mushroom farms and can become pathogens of humans. A comparative analysis of the first three whole genomes revealed mycoparasitism as the innate feature of Trichoderma. However, the evolution of these traits is not yet understood. RESULTS: We selected 12 most commonly occurring Trichoderma species and studied the evolution of their genome sequences. Trichoderma evolved in the time of the Cretaceous-Palaeogene extinction event 66 (±15) mya, but the formation of extant sections (Longibrachiatum, Trichoderma) or clades (Harzianum/Virens) happened in Oligocene. The evolution of the Harzianum clade and section Trichoderma was accompanied by significant gene gain, but the ancestor of section Longibrachiatum experienced rapid gene loss. The highest number of genes gained encoded ankyrins, HET domain proteins and transcription factors. We also identified the Trichoderma core genome, completely curated its annotation, investigated several gene families in detail and compared the results to those of other fungi. Eighty percent of those genes for which a function could be predicted were also found in other fungi, but only 67% of those without a predictable function. CONCLUSIONS: Our study presents a time scaled pattern of genome evolution in 12 Trichoderma species from three phylogenetically distant clades/sections and a comprehensive analysis of their genes. The data offer insights in the evolution of a mycoparasite towards a generalist.

Is Exploitation Competition Involved in a Multitrophic Strategy for the Biocontrol of Fusarium Head Blight?

Trichoderma gamsii T6085 was used in combination with a Fusarium oxysporum isolate (7121) in order to evaluate, in a multitrophic approach, their competitive ability against F. graminearum, one of the main causal agents of Fusarium head blight (FHB) on wheat. The two antagonists and the pathogen were coinoculated on two different natural substrates, wheat and rice kernels. Both T6085 and 7121, alone and coinoculated, significantly reduced the substrate colonization and mycotoxin production by the pathogen. The two antagonists did not affect each other. Using a metabolic approach (Biolog), we investigated whether exploitation competition could explain this antagonistic activity. The aim was to define whether the three fungi coexist or if one isolate nutritionally dominates another. Results obtained from Biolog suggest that no exploitative competition occurs between the antagonists and the pathogen during the colonization of the natural substrates. Interference competition was then preliminarily evaluated to justify the reduction in the pathogen's growth and to better explain mechanisms. A significant reduction of F. graminearum growth was observed when the pathogen grew in the cultural filtrates of T. gamsii T6085, both alone and cocultured with F. oxysporum 7121, thus suggesting the involvement of secondary metabolites. As far as we know, this is the first time that an ecological study has been performed to explain how and which kind of competition could be involved in a multitrophic biocontrol of FHB.

Corrigendum: UV-B Pre-treatment Alters Phenolics Response to Monilinia fructicola Infection in a Structure-Dependent Way in Peach Skin.

[This corrects the article DOI: 10.3389/fpls.2018.01598.].

UV-B Pre-treatment Alters Phenolics Response to Monilinia fructicola Infection in a Structure-Dependent Way in Peach Skin.

Phenolic compounds represent a large class of secondary metabolites, involved in multiple functions not only in plant life cycle, but also in fruit during post-harvest. phenolics play a key role in the response to biotic and abiotic stresses, thus their accumulation is regulated by the presence of environmental stimuli. The present work aimed to investigate how different pre-UV-B-exposures can modulate the phenolic response of peach fruit infected with Monilinia fructicola. Through HPLC-DAD-MSn, several procyanidins, phenolic acids, flavonols, and anthocyanins were detected. Both UV-B radiation and fungal infection were able to stimulate the accumulation of phenolics, dependent on the chemical structure. Regarding UV-B exposure, inoculated with sterile water, 3 h of UV-B radiation highest concentration of phenolics was found, especially flavonols and cyanidin-3-glucoside far from the wound. However, wounding decreased the phenolics in the region nearby. When peaches were pre-treated with 1 h of UV-B radiation, the fungus had an additive effect in phenolic accumulation far from the infection, while it had a subtractive effect with 3 h of UV-B radiation, especially for flavonols. Canonical discriminant analysis and Pearson correlation revealed that all phenolic compounds, except procyanidin dimer, were highly regulated by UV-B radiation, with particularly strong correlation for quercetin and kaempferol glycosides, while phenolics correlated with the fungus infection were quercetin-3-galactoside, quercetin-3-glucoside, kaempferol-3-galactoside and isorhamnetin-3-glucoside. Modulation of pathogen-induced phenolics also far from inoculation site might suggest a migration of signaling molecules from the infected area to healthy tissues.

Polyketide synthases of Diaporthe helianthi and involvement of DhPKS1 in virulence on sunflower.

BACKGROUND: The early phases of Diaporthe helianthi pathogenesis on sunflower are characterized by the production of phytotoxins that may play a role in host colonisation. In previous studies, phytotoxins of a polyketidic nature were isolated and purified from culture filtrates of virulent strains of D. helianthi isolated from sunflower. A highly aggressive isolate (7/96) from France contained a gene fragment of a putative nonaketide synthase (lovB) which was conserved in a virulent D. helianthi population. RESULTS: In order to investigate the role of polyketide synthases in D. helianthi 7/96, a draft genome of this isolate was examined. We were able to find and phylogenetically analyse 40 genes putatively coding for polyketide synthases (PKSs). Analysis of their domains revealed that most PKS genes of D. helianthi are reducing PKSs, whereas only eight lacked reducing domains. Most of the identified PKSs have orthologs shown to be virulence factors or genetic determinants for toxin production in other pathogenic fungi. One of the genes (DhPKS1) corresponded to the previously cloned D. helianthi lovB gene fragment and clustered with a nonribosomal peptide synthetase (NRPS) -PKS hybrid/lovastatin nonaketide like A. nidulans LovB. We used DhPKS1 as a case study and carried out its disruption through Agrobacterium-mediated transformation in the isolate 7/96. D. helianthi DhPKS1 deleted mutants were less virulent to sunflower compared to the wild type, indicating a role for this gene in the pathogenesis of the fungus. CONCLUSION: The PKS sequences analysed and reported here constitute a new genomic resource that will be useful for further research on the biology, ecology and evolution of D. helianthi and generally of fungal plant pathogens.

Geosmithia-Ophiostoma: a New Fungus-Fungus Association.

In Europe as in North America, elms are devastated by Dutch elm disease (DED), caused by the alien ascomycete Ophiostoma novo-ulmi. Pathogen dispersal and transmission are ensured by local species of bark beetles, which established a novel association with the fungus. Elm bark beetles also transport the Geosmithia fungi genus that is found in scolytids' galleries colonized by O. novo-ulmi. Widespread horizontal gene transfer between O. novo-ulmi and Geosmithia was recently observed. In order to define the relation between these two fungi in the DED pathosystem, O. novo-ulmi and Geosmithia species from elm, including a GFP-tagged strain, were grown in dual culture and mycelial interactions were observed by light and fluorescence microscopy. Growth and sporulation of O. novo-ulmi in the absence or presence of Geosmithia were compared. The impact of Geosmithia on DED severity was tested in vivo by co-inoculating Geosmithia and O. novo-ulmi in elms. A close and stable relation was observed between the two fungi, which may be classified as mycoparasitism by Geosmithia on O. novo-ulmi. These results prove the existence of a new component in the complex of organisms involved in DED, which might be capable of reducing the disease impact.

Correction to: Geosmithia-Ophiostoma: a New Fungus-Fungus Association.

The article Geosmithia-Ophiostoma: a New Fungus-Fungus Association, written by Alessia L. Pepori, Priscilla P. Bettini, Cecilia Comparini, Sabrina Sarrocco, Anna Bonini, Arcangela Frascella, Luisa Ghelardini, & Aniello Scala, Giovanni Vannacci, Alberto Santini.

Genome Sequence of Fusarium graminearum ITEM 124 (ATCC 56091), a Mycotoxigenic Plant Pathogen.

Fusarium graminearum is among the main causal agents of Fusarium head blight (FHB), or scab, of wheat and other cereals, caused by a complex of Fusarium species, worldwide. Besides causing economic losses in terms of crop yield and quality, F. graminearum poses a severe threat to animal and human health. Here, we present the first draft whole-genome sequence of the mycotoxigenic Fusarium graminearum strain ITEM 124, also providing useful information for comparative genomics studies.

The Constitutive Endopolygalacturonase TvPG2 Regulates the Induction of Plant Systemic Resistance by Trichoderma virens.

Trichoderma spp. are opportunistic fungi some of which are commonly present in the rhizosphere. Several species, such as T. virens, are also efficient biocontrol agents against phytopathogenic fungi and exert beneficial effects on plants. These effects are the consequence of interactions between Trichoderma and plant roots, which trigger enhanced plant growth and induce plant resistance. We have previously shown that T. virens I10 expresses two endopolygalacturonase genes, tvpg1 and tvpg2, during the interaction with plant roots; tvpg1 is inducible while tvpg2 is constitutively transcribed. Using the same system, the tomato polygalacturonase-inhibitor gene Lepgip1 was induced at the same time as tvpg1. Here we show by gene disruption that TvPG2 performs a regulatory role on the inducible tvpg1 gene and in triggering the plant immune response. A tvpg2-knockout strain fails to transcribe the inducible tvpg1 gene in neither in vitro in inducing media containing pectin or plant cell walls, nor during the in vivo interaction with tomato roots. Likewise, the in vivo induction of Lepgip1 does not occur, and its defense against the pathogen Botrytis cinerea is significantly reduced. Our data prove the importance of a T. virens constitutively produced endopolygalacturonase in eliciting plant induced systemic resistance against pathogenic fungi.

Gene family expansions and contractions are associated with host range in plant pathogens of the genus Colletotrichum.

BACKGROUND: Many species belonging to the genus Colletotrichum cause anthracnose disease on a wide range of plant species. In addition to their economic impact, the genus Colletotrichum is a useful model for the study of the evolution of host specificity, speciation and reproductive behaviors. Genome projects of Colletotrichum species have already opened a new era for studying the evolution of pathogenesis in fungi. RESULTS: We sequenced and annotated the genomes of four strains in the Colletotrichum acutatum species complex (CAsc), a clade of broad host range pathogens within the genus. The four CAsc proteomes and secretomes along with those representing an additional 13 species (six Colletotrichum spp. and seven other Sordariomycetes) were classified into protein families using a variety of tools. Hierarchical clustering of gene family and functional domain assignments, and phylogenetic analyses revealed lineage specific losses of carbohydrate-active enzymes (CAZymes) and proteases encoding genes in Colletotrichum species that have narrow host range as well as duplications of these families in the CAsc. We also found a lineage specific expansion of necrosis and ethylene-inducing peptide 1 (Nep1)-like protein (NLPs) families within the CAsc. CONCLUSIONS: This study illustrates the plasticity of Colletotrichum genomes, and shows that major changes in host range are associated with relatively recent changes in gene content.

Fusaproliferin, Terpestacin and Their Derivatives Display Variable Allelopathic Activity Against Some Ascomycetous Fungi.

Herbivorous mammal dung supports a large variety of fimicolous fungi able to produce different bioactive secondary metabolites to compete with other organisms. Recently, the organic extracts of the Solid State Fermentation (SSF) cultures of Cleistothelebolus nipigonensis and Neogymnomyces virgineus, showing strong antifungal activity, were preliminarily investigated. This manuscript reports the isolation of the main metabolites identified, using spectroscopic and optical methods, as fusaproliferin (1) and terpestacin (2). Furthermore, some key hemisynthetic derivatives were prepared and their antifungal activity was tested against the same fungi previously reported to be affected by the organic extracts obtained from SSF. These metabolites and their derivatives resulted able to reduce the growth of Alternaria brassicicola, Botrytis cinerea and Fusarium graminearum in a variable extent strongly dependent from chemical modifications and test fungi. The hydroxy enolic group at C(17) appeared to be a structural feature important to impart activity. This study represents the first report of these secondary metabolites produced by C. nipigonensis and N. virgineus.