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Background – In the successful cultivation of crops for improved quality and high yield, the health status of the originating seed is of utmost importance as this can affect the performance and quality of the ensuring plant after germination. Seed sanitation and use of biological control agents (BCAs) have become a major method of reducing pathogen and insect infestation and improving crop yield. This applies to most crops including Quinoa whose cultivation in the more humid and colder regions of the world have resulted in the manifestation of new plant pathogens and diseases. Methods – A total of 4 growth experiments were conducted using the Puno and Vikinga quinoa variety with 3 replicates of 30 seeds each, comprising of 9 different treatments (1 control with just water, 2 with pathogens alone, 2 with BCAs alone. 4 with 1 pathogen plus 1 BCA) to evaluate the effect of pathogens on quinoa seedlings. The impact of beneficial fungi that show antagonistic activity towards pathogenic fungi was also investigated to determine their potential as biocontrol agents. Physical measurements and dry weight analyses on the harvested seedings showed increased biomass accumulation in seedlings with pathogen plus BCA as against those with the pathogen alone. In addition, their seed germination rates were also higher. Results – Statistical analysis showed significant difference between plant biomass accumulation (dry weight) and treatments. Supporting our hypothesis that BCAs do improve the growth of quinoa seedlings inoculated with certain fungal pathogens. Conclusions – The obtained results underlined the importance and role of biopriming in the prevention and control of certain seed-borne pathogens. It’s further study and adoption will have a significant positive impact in overall plant yield, biodiversity and sustainable agriculture.

Quinoa (Chenopodium quinoa Willd.) is a native American crop mainly grown in the Andes of Bolivia and Peru. During the last decades, the cultivation of quinoa has expanded to more than 125 countries. Since then, several diseases of quinoa have been characterized. A leaf disease was observed on quinoa plants growing in an experimental plot in Eastern Denmark in 2018. The symptoms produced by the associated fungi consisted of small yellow blotches on the upper surface of leaves with a pale chlorotic halo surrounding the lesion. These studies used a combination of morphology, molecular diagnostics, and pathogenicity test to identify two different Alternaria species belonging to Alternaria section Infectoriae and alternata as the causal agent of observed disease symptoms. To the best of our knowledge, this is the first report of Alternaria spp. as foliar pathogens of quinoa. Our findings indicate the need for additional studies to determine potential risks to quinoa production.

High-throughput profiling of key enzyme activities of carbon, nitrogen, and antioxidant metabolism is emerging as a valuable approach to integrate cell physiological phenotyping into a holistic functional phenomics approach. However, the analyses of the large datasets generated by this method represent a bottleneck, often keeping researchers from exploiting the full potential of their studies. We address these limitations through the exemplary application of a set of data evaluation and visualisation tools within a case study. This includes the introduction of multivariate statistical analyses which can easily be implemented in similar studies, allowing researchers to extract more valuable information to identify enzymatic biosignatures. Through a literature meta-analysis, we demonstrate how enzyme activity profiling has already provided functional information on the mechanisms regulating plant development and response mechanisms to abiotic stress and pathogen attack. The high robustness of the distinct enzymatic biosignatures observed during developmental processes and under stress conditions underpins the enormous potential of enzyme activity profiling for future applications both in basic and applied research. Enzyme activity profiling will complement molecular -omics approaches to contribute to the mechanistic understanding required to narrow the genotype-to-phenotype knowledge gap and to identify predictive biomarkers for plant breeding to develop climate-resilient crops.

Agriculture has always been the most susceptible sector to climate change, as environmental stresses severely impact plant development and final yield. The interest in the application of plant growth promoting rhizobacteria (PGPR) in agriculture is exponentially increasing, as it provides a sustainable solution against hazardous agrochemicals. A vast amount of research has indicated that PGPRs can have bio stimulatory activity and function as biocontrol agents, providing protection against biotic and abiotic stresses and therefore offering a less environmentally invasive approach to food supply. This study was conducted to enlighten the effects of two PGPRs, the Enterobacter spp., and the commercially applied Bacillus amyloliquefaciens FZB42, on root growth and architecture as well as on carbohydrate-metabolic and antioxidant enzyme activity in barley (Hordeum vulgare cv Guld). Preliminary experimental results have shown that the impact of Enterobacter spp. is very similar to the effect of cytokinins on root growth. The bacterial inoculation response of the plants was initially characterized by phenotypical and biochemical analysis. There was no distinct effect on the root system and plant metabolism with FZB42 treatment, therefore further focus was given only to the impact of Enterobacter spp. Enterobacter spp. caused a strong inhibitory impact on root growth and affected the carbohydrate and antioxidant enzyme activity of plants grown in vitro. This specific effect on roots did not appear under soil conditions, in greenhouse experiments. The study around Enterobacter spp. was complemented with analysis of the available RNA Sequencing data. Expression profiling of the roots of barley plants identified the number of differentially regulated genes. Functional annotation of these differentially expressed genes was performed using Gene Ontology (GO) and the Kyoto Encyclopedia

Theoretical and empirical studies have shown that species radiations are facilitated when a trait under divergent natural selection is also involved in sexual selection. It is yet unclear how quick and effective radiations are where assortative mating is unrelated to the ecological environment and primarily results from sexual selection. We address this question using sympatric grasshopper species of the genus Chorthippus, which have evolved strong behavioural isolation while lacking noticeable ecomorphological divergence. Mitochondrial genomes suggest that the radiation is relatively recent, dating to the mid-Pleistocene, which leads to extensive incomplete lineage sorting throughout the mitochondrial and nuclear genomes. Nuclear data shows that hybrids are absent in sympatric localities but that all species have experienced gene flow, confirming that reproductive isolation is strong but remains incomplete. Demographic modelling is most consistent with a long period of geographic isolation, followed by secondary contact and extensive introgression. Such initial periods of geographic isolation might facilitate the association between male signaling and female preference, permitting the coexistence of sympatric species that are genetically, morphologically, and ecologically similar, but otherwise behave mostly as good biological species.

Quinoa (Chenopodium quinoa) is an ancient crop from the Andes of Bolivia, Peru and Ecuador and is increasingly cultivated outside its native range on all continents. Cultivation in new environments may lead to incidence of diseases previously not described for quinoa that may interfere with a commercially successful production of this crop. To test this hypothesis, we identified foliar pathogens at different stages of quinoa development to understand their epidemiology, host interactions and sources of resistance. We grew three cultivars (Vikinga, Titicaca and Puno) in an experimental plot in Tåstrup, Eastern Denmark. A first set of symptomatic leaves was collected from cv. Puno at the end of the growing season in late September 2017 to identify pathogens. A diversity of symptoms were observed that included small yellowish anamorph blots on the upper leaf surface; a pale chlorotic halo surrounded the yellow lesion which occasionally had slight pink colouring in the centre; and light brown spots, concentric rings and apical necrosis on the lower leaf surface. The symptomatic sections of leaf tissue were surface sterilized and plated onto potato dextrose. After 10 days, three main fungal groups could be differentiated by their morphological characteristics such us conidia shape, colour and number/occurrence of septae, colony diameter, reverse colour and formation of rings. Monoconidial isolations allowed to establish pure cultures of each fungus. For molecular identification we PCR amplified the intergenic transcribed spacer (ITS) and conducted BLAST searches for identification. Using this protocol we identified the following species: Didymella chenopodii, Alternaria infectoria/methacromatica, Alternaria tenuissima and Epicocum nigrum. To validate their identity, Koch postulates and pathogenicity tests are in progress. To our knowledge this is the first demonstration of Alternaria infectoria

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.

Colletotrichum acutatum is a major fungal pathogen of fruit crops, which causes severe yield losses in strawberry productions. A potential key factor in plant-pathogen interactions is fungal sesquiterpenoids that have myco- and phytotoxic activities. The first committed step in sesquiterpenoids biosynthesised is performed by sesquiterpene synthases (TPS). Only a few TPS's have been functionally characterized from filamentous fungi and none from the Colletotrichum genus. Despite it as an important fungal pathogen to agriculture, it is poorly understood at the molecular and chemical level. The terpenoid biochemistry in C. acutatum strain SA 0-1 was studied and one C. acutatum TPS (CaTPS) was successfully cloned and characterized in yeast. CaTPS catalyses the biosynthesis of multiple sesquiterpenoids. The two major products are -caryophyllene and an unidentified sesquiterpenoid along with -humulene as one the minor sesquiterpenoid products. These products were also secreted by the fungus in strawberry fruit media along with several other sesquiterpenoids indicating other TPS's are active during in vitro growth. -Caryophyllene and -humulene are known cytotoxic products important for ecological interactions and are produced by SA 0-1. Interestingly, a gene expression analysis using qRT-PCR revealed a significant increase in expression of CaTPS during strawberry fruit infection thus indicating that it could be involved in fruit infection. This is the first characterization of TPS in Colletotrichum spp. and terpenoid profiles of C. acutatum, which could facilitate studies on the role of terpenoids in the ecology of C. acutatum.

Clonostachys rosea is a mycoparasitic fungus that can control several important plant diseases. Here, we report on the genome sequencing of C. rosea and a comparative genome analysis, in order to resolve the phylogenetic placement of C. rosea and to study the evolution of mycoparasitism as a fungal lifestyle. The genome of C. rosea is estimated to 58.3 Mb, and contains 14,268 predicted genes. A phylogenomic analysis shows that C. Tosco clusters as sister taxon to plant pathogenic Fusarium species, with mycoparasitic/saprotrophic Tfichoderma species in an ancestral position. A comparative analysis of gene family evolution reveals several distinct differences between the included mycoparasites. Clonostachys rosea contains significantly more ATP-binding cassette (ABC) transporters, polyketide synthases, cytochrome P450 monooxygenases, pectin lyases, glucose-methanol-choline oxidoreductases, and lytic polysaccharide monooxygenases compared with other fungi in the Hypocreales. Interestingly, the increase of ABC transporter gene number in C. rosea is associated with phylogenetic subgroups B (multidrug resistance proteins) and G (pleiotropic drug resistance transporters), whereas an increase in subgroup C (multidrug resistance-associated proteins) is evident in Tfichoderma virens. In contrast with mycoparasitic Tfichoderma species, C. rosea contains very few chitinases. Expression of six group B and group G ABC transporter genes was induced in C. rosea during exposure to the Fusafium mycotoxin zearalenone, the fungicide Boscalid or metabolites from the biocontrol bacterium Pseudomonas chiororaphis. The data suggest that tolerance toward secondary metabolites is a prominent feature in the biology of C. rosea.

Herbaria collections containing plants with disease symptoms are highly valuable, and they are often the only way to investigate outbreaks and epidemics from the past as the number of viable isolates in culture collections is often limited. Species belonging to the Colletotrichum acutatum complex infect a range of important crops. As members of the C. acutatum complex are easily confused with other Colletotrichum species, molecular methods are central for the correct identification. We performed molecular analyses on 21 herbaria specimens, displaying anthracnose symptoms, collected in Norway and Denmark before the first confirmed findings of C. acutatum complex members in this region. Sequencing parts of the fungal ITS regions showed that members of the species complex were present in 13 of the 21 specimens collected in different parts of Norway and Denmark between 1948 and 1991, representing seven plant hosts (three cherry species, apple, raspberry and rhododendron). This is the first time herbarium specimens have been used to study these pathogens under Nordic conditions. Differences in the ITS sequences suggest the presence of different genotypes within the complex, indicating a well-established population.

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing, together with associated bioinformatic tools have identified vast numbers of putative carbohydrate degrading and modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high-throughput and versatile semi-quantitative enzyme-screening technique which requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme cocktails and crude culture broths against single substrates, substrate mixtures and biomass samples. Moreover, we show that the technique can be used to analyse both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified un-characterised enzymes and by screening enzyme activities from fungal culture broths.

Colletotrichum acutatum is an economically important fruit rot pathogen in strawberry. At the molecular level, little is known about the infection. In my PhD study, I therefore set to comprehensively understand the interaction between C. acutatum and strawberry fruits by applying multiple omic/high-throughput methods. As a novel approach, I linked transcriptomic, metabolomics and proteomic data and suggest new fungal pathogenicity related pathways to be involved in plant - fungal interactions. I also found that sesquiterpenoids produced by C. acutatum are important for the interaction.However, these bioactive compounds are also of interest to industry and when cultured in vitro, various sesquiterpenoids are secreted by fungus, including unknowns. In addition, I have functionally characterized the first sesquiterpene synthase (STS) of Colletotrichum sp. using yeast expression systems and multiple sesquiterpenoid products are formed by STS.Furthermore, molecular diagnostics and molecular phylogenetic analyses of herbaria specimens with Colletotrichum sp. symptoms and of isolates collected from multiple plant species, revise the history of C. acutatum in the Nordic countries.

Fungal endophytes live naturally inside plants without causing symptoms. On the contrary, they can promote plant growth and increase tolerance to abiotic and biotic stress. These beneficial effects have increased the agricultural interest for exploitation of fungal isolates with an endophytic life-style. Clonostachys rosea occurs naturally world-wide and is capable of colonizing many different habitats. The fungus is primarily known as a versatile biological control agent. However, it has also been reported as a plant endophyte in, e.g., soybean, red clover and cacao. The C. rosea isolate IK726 efficiently controls seed- and soil-borne diseases and can furthermore promote plant growth. However, it is not known whether IK726 can colonize plants internally and therefore, the objective of the present study was to examine the possibility of an endophytic life-style of IK726 in tomato. Tomato seeds were sown in vermiculite or soil and grown at 24°C in a growth chamber. After two weeks, conidial suspensions of IK726 were applied to the roots either by root dipping or soil drenching around the stem and plants were subsequently incubated for at least two weeks before sampling for re-isolation of C. rosea. Stems were surface disinfected by dipping in ethanol followed by flaming and sections (0.5-14 cm from the soil surface) were placed on potato dextrose agar plates with Novobiocin and incubated at 25°C. Fungi growing from the stem pieces were identified by microscopy. Isolation of C. rosea was successful from two to five weeks after IK726 inoculation, from plants grown in both soil and vermiculite and from both root dip and drench inoculated plants. Verticillium, Penicillium, Cladosporium, Fusarium and Trichoderma were furthermore isolated from stems of soil grown plants. Surface disinfection of roots eradicated all culturable fungi. Therefore, root parts were washed only in water before incubation on the isolation medium and this revealed >50% colonization of t