Biological Disease Control by Beneficial (Micro)Organisms: Selected Breakthroughs in the Past 50 Years.

Biological control of plant disease by beneficial (micro)organisms is one of the main tools available to preserve plant health within the wider context of One Health and in line with the goals of the Agenda 2030 for Sustainable Development. The commercial development of biocontrol agents, together with a new perspective on the resident microbial community, all supported by innovative "omics" technologies, continues to gain in prominence in plant pathology, addressing the need to feed the increasing world population and to assure safe and secure access to food. The present review considers selected advances within the last 50 years, highlighting those that can be considered as breakthroughs for the biological control research field. Selected examples of successful biocontrol agents and strategies are reported, including the history of the progress in researching Trichoderma isolates as commercial biocontrol agents, the exploitation of mycoviruses to confer hypovirulence to plant pathogenic fungi, the role of microbial communities in the suppressiveness of soils, and evolving approaches including the establishment of synthetic microbial communities.

Genome Resources of Verticillium dahliae VdGL16: The Causal Agent of Vascular Wilt on the Invasive Species Ailanthus altissima.

Verticillium species are known as plant pathogens responsible for wilt diseases in a large variety of dicotyledon plants and crops in many parts of the world. Here we present the draft genome sequence of Verticillium dahliae Kleb. (strain VdGL16) isolated in Italy from the invasive alien species Ailanthus altissima (Mill.; commonly known as tree-of-heaven) showing Verticillium wilt symptoms. The comparison between the newly sequenced genome with those publicly available revealed candidate genes putatively involved in pathogenicity. The genome represents a new useful source for future research on Verticillium genetics and biology as well as research on novel approaches in the control of A. altissima.

Complete Genome Sequence of the Plant-Pathogenic Fungus Colletotrichum lupini.

Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long- and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into 11 nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41 Mb and 36.55 kb, respectively. In total, 18,324 protein-encoding genes have been predicted, of which only 39 are specific to C. lupini. This resource will provide insight into pathogenicity factors and will help provide a better understanding of the evolution and genome structure of this important plant pathogen.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

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.

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.

Whole-Genome Sequence of the Orchid Anthracnose Pathogen Colletotrichum orchidophilum.

Colletotrichum orchidophilum is a plant-pathogenic fungus infecting a wide range of plant species belonging to the family Orchidaceae. In addition to its economic impact, C. orchidophilum has been used in recent years in evolutionary studies because it represents the closest related species to the C. acutatum species complex. Here, we present the first-draft whole-genome sequence of C. orchidophilum IMI 309357, providing a resource for future research on anthracnose of Orchidaceae and other hosts.

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.

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.

Sustainable Strategies to Counteract Mycotoxins Contamination and Cowpea Weevil in Chickpea Seeds during Post-Harvest.

Mycotoxins contamination and pest infestation of foods and feeds represent a pivotal threat for food safety and security worldwide, with crucial implications for human and animal health. Controlled atmosphere could be a sustainable strategy to reduce mycotoxins content and counteract the vitality of deleterious organisms in foodstuff. Ozone treatment (O3, 500 ppb for 30, 60 or 90 min) and high nitrogen concentration (N2, 99% for 21 consecutive days) were tested in the post-harvest management of four batches of Cicer arietinum grains to control the presence of mycotoxigenic fungi and their secondary metabolites, as well as pest (i.e., Callosobruchus maculatus) infestation. At the end of the treatment, O3 significantly decreased the incidence of Penicillium spp. (by an average of -50%, independently to the time of exposure) and reduced the patulin and aflatoxins content after 30 min (-85 and -100%, respectively). High N2 concentrations remarkably reduced mycotoxins contamination (by an average of -94%) and induced pest mortality (at 100% after 5 days of exposure). These results confirm the promising potential of O3 and N2 in post-harvest conservation strategies, leading to further investigations to evaluate the effects on the qualitative characteristics of grains.

Isolation and characterization of Fusarium spp. From unhatched eggs of Caretta caretta in Tuscany (Italy).

Sea Turtle Egg Fusariosis (STEF) is a worldwide emergent fungal disease affecting eggs and causing embryos mortality in turtle's nests such as those of Caretta caretta. It is caused by a complex of species belonging to Fusarium genus, particularly those included in the Fusarium Solani Species Complex (FSSC). During the samplings carried out in summer 2020 along the Tuscany coastlines (Italy), C. caretta eggs showed clinical signs resembling those caused by STEF. A total of 32 fungal isolates were obtained from lesioned eggs whose molecular characterization allowing identifying as belonging to FSSC / Neocosmospora spp., Fusarium oxysporum Species Complex (FOSC) / F. oxysporum and Fusarium nodosum, i.e., fungal genera and speciesincluding also well-known plant pathogens. Isolates inoculated on several plant hosts did not result in any pathogenic activity but F. nodosum causing, on wheat spikes, disease symptoms.This is the first time F. nodosum has been isolated from portions of eggs showing evident signs of fungal infection. This work represents the first report of Fusarium spp. isolated from C. caretta eggs showing lesions resembling those caused by STEF on Tuscan coast thus posing a significant concern to loggerhead sea turtle conservation also in this region.

Mushroom-Based Supplements in Italy: Let's Open Pandora's Box.

Mushrooms and derivates are well known to the scientific community for having different health benefits and exhibit a wide range of pharmacological activities, including lipid-lowering, antihypertensive, antidiabetic, antimicrobic, antiallergic, anti-inflammatory, anticancer, immunomodulating, neuroprotective and osteoprotective actions. In Europe, medical mushrooms are mainly marketed in the form of food supplements as single components or combined with other nutraceuticals. In this context, the first peculiarity that distinguishes it is the safety established through the "history of consumption" that characterizes that mushroom. However, the cultivation of medicinal mushrooms on a large scale is performed mainly in China, where most of the production facilities do not have internationally recognized good manufacturing practices, despite that many European companies that sell myotherapies are supplied by Chinese manufacturers. This is particularly evident in Italy, where an arsenal of mushroom products is marketed in the form of powders and extracts not always of ascertained origin and sometimes of doubtful taxonomic identification, and thus not meeting the quality criteria required. The growing interest in mycotherapy involves a strong commitment from the scientific community to propose supplements of safe origin and genetic purity as well as to promote clinical trials to evaluate its real effects on humans. The purpose of this research is to analyze different mushroom-based dietary supplements used in medicine as monotherapy on the Italian market and to evaluate their composition and quality. The molecular identification of the sequences with those deposited in GenBank allowed for identifying 6 out of 19 samples, matching with those deposited belonging to the species indicated in the label, i.e., Lentinula edodes (samples 1, 4, 12 and 18) and Ganoderma lucidum (samples 5 and 10). Samples containing Ganoderma, labeled in the commercial product as G. lucidum, showed sequences that showed homology of 100% and 99% with G. resinaceum and G. sichuanense. An additional investigation was carried out in order to determine the active fungal ingredients, such as ergosterol, aflatoxins, heavy metals, nicotine and total glucan. The results obtained and shown in the manuscript highlight how the data were not only in line with what is expected with respect to what is indicated in the labels.

Characterization of Trichoderma species from forest ecosystems by high-throughput phenotypic microarray.

The use of beneficial organisms for the biocontrol of soil-borne pathogens in forestry is still poor explored. In this work, the nutritional demands of 10 previously selected isolates of Trichoderma for the biocontrol of forest soil-borne pathogens have been tested by Phenotype Microarray technology, to investigate about their C-source utilization and exploring the possibility to obtain a microbial consortia (SynCom), an innovative strategy for the biocontrol of plant disease. All Trichoderma isolates tested in this study showed a high spore germination percentage within 3 d and evidenced nutritional preference regardless of the species they belong to, and unrelated to their soil of origin. Results of growth curve analysis and MANOVA test revealed that all isolates assimilate a broad range of substrates, generally preferring complex compounds such as monosaccharides related compounds, nitrogen compounds, carboxylic acids and esters. No evidence of competition for nutritional resources have been observed among isolates of this study. As a result, a combination of different isolates could be proposed to obtain a SynCom useful for the practice of phytopathogen biocontrol in forestry. The addition of i-erythritol, adenosine and turanose to a growth substrate could be suggested as stimulating compounds for the growth of the selected Trichoderma isolates.

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.