Harnessing Chemical Ecology for Environment-Friendly Crop Protection.

Heavy reliance on synthetic pesticides for crop protection has become increasingly unsustainable, calling for robust alternative strategies that do not degrade the environment and vital ecosystem services. There are numerous reports of successful disease control by various microbes used in small-scale trials. However, inconsistent efficacy has hampered their large-scale application. A better understanding of how beneficial microbes interact with plants, other microbes, and the environment and which factors affect disease control efficacy is crucial to deploy microbial agents as effective and reliable pesticide alternatives. Diverse metabolites produced by plants and microbes participate in pathogenesis and defense, regulate the growth and development of themselves and neighboring organisms, help maintain cellular homeostasis under various environmental conditions, and affect the assembly and activity of plant and soil microbiomes. However, research on the metabolites associated with plant health-related processes, except antibiotics, has not received adequate attention. This review highlights several classes of metabolites known or suspected to affect plant health, focusing on those associated with biocontrol and belowground plant-microbe and microbe-microbe interactions. The review also describes how new insights from systematic explorations of the diversity and mechanism of action of bioactive metabolites can be harnessed to develop novel crop protection strategies.

Volatile Compounds Emitted by Diverse Verticillium Species Enhance Plant Growth by Manipulating Auxin Signaling.

Some volatile compounds (VC) play critical roles in intra- and interspecies interactions. To investigate roles of VC in fungal ecology, we characterized how VC produced by Verticillium spp., a group of broad-host-range soilborne fungal pathogens, affect plant growth and development. VC produced by 19 strains corresponding to 10 species significantly enhanced the growth of Arabidopsis thaliana and Nicotiana benthamiana. Analysis of VC produced by four species revealed the presence of diverse compounds, including those previously shown to affect plant growth. Using A. thaliana, we investigated the mechanism underpinning plant growth enhancement by Verticillium dahliae VC. Allometric analysis indicated that VC caused preferential resource allocation for root growth over shoot growth. Growth responses of A. thaliana mutants defective in auxin or ethylene signaling suggested the involvement of several components of auxin signaling, with TIR3 playing a key role. AUX1, TIR1, and AXR1 were also implicated but appeared to play lesser roles. Inhibition of auxin efflux using 1-naphthylphthalamic acid blocked VC-mediated growth enhancement. Spatial and temporal expression patterns of the auxin-responsive reporter DR5::GUS indicated that the activation of auxin signaling occurred before enhanced plant growth became visible. Results from this study suggest critical yet overlooked roles of VC in Verticillium ecology and pathology.

Phylogenomics reveals extensive misidentification of fungal strains from the genus Aspergillus.

Modern taxonomic classification is often based on phylogenetic analyses of a few molecular markers, although single-gene studies are still common. Here, we leverage genome-scale molecular phylogenetics (phylogenomics) of species and populations to reconstruct evolutionary relationships in a dense data set of 710 fungal genomes from the biomedically and technologically important genus Aspergillus. To do so, we generated a novel set of 1,362 high-quality molecular markers specific for Aspergillus and provided profile Hidden Markov Models for each, facilitating their use by others. Examining the resulting phylogeny helped resolve ongoing taxonomic controversies, identified new ones, and revealed extensive strain misidentification (7.59% of strains were previously misidentified), underscoring the importance of population-level sampling in species classification. These findings were corroborated using the current standard, taxonomically informative loci. These findings suggest that phylogenomics of species and populations can facilitate accurate taxonomic classifications and reconstructions of the Tree of Life.IMPORTANCEIdentification of fungal species relies on the use of molecular markers. Advances in genomic technologies have made it possible to sequence the genome of any fungal strain, making it possible to use genomic data for the accurate assignment of strains to fungal species (and for the discovery of new ones). We examined the usefulness and current limitations of genomic data using a large data set of 710 publicly available genomes from multiple strains and species of the biomedically, agriculturally, and industrially important genus Aspergillus. Our evolutionary genomic analyses revealed that nearly 8% of publicly available Aspergillus genomes are misidentified. Our work highlights the usefulness of genomic data for fungal systematic biology and suggests that systematic genome sequencing of multiple strains, including reference strains (e.g., type strains), of fungal species will be required to reduce misidentification errors in public databases.

Two bifunctional enzymes with ferric reduction ability play complementary roles during magnetosome synthesis in Magnetospirillum gryphiswaldense MSR-1.

The bacterial strain Magnetospirillum gryphiswaldense MSR-1 does not produce siderophores, but it absorbs a large amount of ferric iron and synthesizes magnetosomes. We demonstrated previously the presence of six types of ferric reductase isozymes (termed FeR1 through FeR6) in MSR-1. Of these isozymes, FeR5 was the most abundant and FeR6 showed the highest ferric reductase activity. In the present study, we cloned the fer5 and fer6 genes from MSR-1 and expressed them separately in Escherichia coli. FeR5 and FeR6 were shown to be bifunctional enzymes through analysis of amino acid sequence homologies, structural predictions (using data from GenBank), and detection of enzyme activities. FeR5 is a thioredoxin reductase and FeR6 is a flavin reductase, in addition to being ferric reductases. To elucidate the functions of the enzymes, we constructed two single-gene-deletion mutant strains (Δfer5 and Δfer6 mutants) and a double-gene-deletion mutant strain (Δfer5 Δfer6 [Δfer5+6] mutant) along with its complemented strains (C5 and C6). An evaluation of phenotypic and physiological properties did not reveal significant differences between the wild-type and single-gene-deletion strains, whereas the double-gene-deletion strain showed reduced iron absorption and no magnetosome synthesis. Complementation of the double-gene-deletion strain using either fer5 or fer6 resulted in the partial recovery of magnetosome synthesis. Quantitative real-time PCR analysis of fer5 and fer6 transcriptional levels in the wild-type and complemented strains demonstrated consistent transcription of the two genes and confirmed that FeR5 and FeR6 are bifunctional enzymes that play complementary roles during the process of magnetosome synthesis in MSR-1.

Multi-Pronged Investigation of Volatile Compound-Mediated Interactions of Fusarium oxysporum with Plants, Fungi, and Bacteria.

Proteins and many biogenic compounds require water as a medium for movement. However, because volatile compounds (VCs) can travel through the air and porous soils due to their ability to vaporize at ambient temperature, they can mediate diverse intra- and inter-kingdom interactions and perform ecologically functions even in the absence of water. Here, we describe several tools and approaches for investigating how Fusarium oxysporum interacts with plants and other microbes through VCs and how VC-mediated interactions affect its ecology and pathology. We also present a method for capturing F. oxysporum VCs for analysis via gas chromatography linked to mass spectrometry.

Fusarium oxysporum infection-induced formation of agarwood (FOIFA): A rapid and efficient method for inducing the production of high quality agarwood.

Agarwood, a non-wood product from the endangered Aquilaria and Gyrinops tress, is highly prized for its use in fragrances and medicines. The special formation process of agarwood is closely related to external injury and fungal infection. In this study, we demonstrate that infection of Aquilaria sinensis by Fusarium oxysporum, a soilborne fungus that causes vascular wilt diseases in diverse plants, induces agarwood formation. Based on these findings, an efficient method, termed F. oxysporum infection-induced formation of agarwood (FOIFA), was developed for the rapid production of quality agarwood. The agarwood formed in response to F. oxysporum infection was similar in structure and chemical composition to wild agarwood according to TLC (Thin-layer chromatography), HPLC (high performance liquid chromatography), and GC-MS (gas chromatography-mass spectrometry) analyses, except that the contents of alcohol-soluble extract, chromones, and essential oils (mainly sesquiterpenes) were higher in the formed agarwood.

Secreted metabolite-mediated interactions between rhizosphere bacteria and Trichoderma biocontrol agents.

Trichoderma has been used as an alternative to synthetic pesticides to control a variety of phytopathogenic fungi, oomycetes, and nematodes. Although its mechanism of pathogen suppression has been extensively studied, how Trichoderma interacts with non-target microbes is not well understood. Here, we investigated how two Trichoderma biological control agents (BCAs) interact with rhizosphere bacteria isolated from a tomato plant via secreted proteins, metabolites, and volatile compounds (VCs). Culture filtrates (CFs) of T. virens and T. harzianum, containing secreted proteins and metabolites, strongly inhibited (>75% reduction in growth) 39 and 19, respectively, out of 47 bacterial strains tested. Their CFs inhibited the remaining strains at lower degrees. Both metabolites and proteins are involved in inhibiting bacteria, but they seem to antagonize each other in inhibiting some strains. Trichoderma and bacteria suppressed the growth of each other using VCs. The secretion of antibacterial and antifungal molecules by T. virens and T. harzianum was significantly affected by VCs from some bacteria, suggesting that both Trichoderma BCAs and rhizosphere bacteria use VCs to influence each other in multiple ways. In light of these results, we discuss how metabolite-mediated interactions can potentially affect the effectiveness of biocontrol.

I Plate-based Assay for Studying How Fungal Volatile Compounds (VCs) Affect Plant Growth and Development and the Identification of VCs via SPME-GC-MS.

Biogenic volatile compounds (VCs) mediate various types of crucial intra- and inter-species interactions in plants, animals, and microorganisms owing to their ability to travel through air, liquid, and porous soils. To study how VCs produced by Verticillium dahliae, a soilborne fungal pathogen, affect plant growth and development, we slightly modified a method previously used to study the effect of bacterial VCs on plant growth. The method involves culturing microbial cells and plants in I plate to allow only VC-mediated interaction. The modified protocol is simple to set up and produces reproducible results, facilitating studies on this poorly explored form of plant-fungal interactions. We also optimized conditions for extracting and identifying fungal VCs using solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS).

Do volatile compounds produced by Fusarium oxysporum and Verticillium dahliae affect stress tolerance in plants?

Volatile compounds (VCs) produced by diverse microbes seem to affect plant growth, development and/or stress tolerance. We investigated how VCs released by soilborne fungi Fusarium oxysporum and Verticillium dahliae affect Arabidopsis thaliana responses to abiotic and biotic stresses. Under salt stress, VCs from both fungi helped its growth and increased chlorophyll content. However, in contrast to wild-type A. thaliana (Col-0), V. dahliae VCs failed to increase leaf surface area in auxin signalling mutants aux1-7, tir1-1 and axr1-3. Compared to wild-type Col-0, the degree of lateral root density enhanced by V. dahliae VCs in these mutants was also reduced. Consistent with the involvement of auxin signalling in fungal VC-mediated salt torelance, A. thaliana line carrying DR5::GUS displayed increased auxin accumulation in root apex upon exposure to V. dahliae VCs, and 1-naphthylphthalamic acid, an auxin transport inhibitor, adversely affected V. dahliae VC-mediated salt tolerance. F. oxysporum VCs induced the expression of PR1 but not PDF1.2 in A. thaliana lines containing PR1::GUS and PFD1.2::GUS. When challenged with Pseudomonas syringae after the exposure to F. oxysporum VCs, A. thaliana showed reduced disease symptoms. However, the number of bacterial cells in F. oxysporum VC-treated plants was not significantly different from that in control plants.

Volatile Compound-Mediated Recognition and Inhibition Between Trichoderma Biocontrol Agents and Fusarium oxysporum.

Certain Trichoderma strains protect plants from diverse pathogens using multiple mechanisms. We report a novel mechanism that may potentially play an important role in Trichoderma-based biocontrol. Trichoderma virens and T. viride significantly increased the amount/activity of secreted antifungal metabolites in response to volatile compounds (VCs) produced by 13 strains of Fusarium oxysporum, a soilborne fungus that infects diverse plants. This response suggests that both Trichoderma spp. recognize the presence of F. oxysporum by sensing pathogen VCs and prepare for attacking pathogens. However, T. asperellum did not respond to any, while T. harzianum responded to VCs from only a few strains. Gene expression analysis via qPCR showed up-regulation of several biocontrol-associated genes in T. virens in response to F. oxysporum VCs. Analysis of VCs from seven F. oxysporum strains tentatively identified a total of 28 compounds, including six that were produced by all of them. All four Trichoderma species produced VCs that inhibited F. oxysporum growth. Analysis of VCs produced by T. virens and T. harzianum revealed the production of compounds that had been reported to display antifungal activity. F. oxysporum also recognizes Trichoderma spp. by sensing their VCs and releases VCs that inhibit Trichoderma, suggesting that both types of VC-mediated interaction are common among fungi.

Fusarium Oxysporum Volatiles Enhance Plant Growth Via Affecting Auxin Transport and Signaling.

Volatile organic compounds (VOCs) have well-documented roles in plant-plant communication and directing animal behavior. In this study, we examine the less understood roles of VOCs in plant-fungal relationships. Phylogenetically and ecologically diverse strains of Fusarium oxysporum, a fungal species complex that often resides in the rhizosphere of assorted plants, produce volatile compounds that augment shoot and root growth of Arabidopsis thaliana and tobacco. Growth responses of A. thaliana hormone signaling mutants and expression patterns of a GUS reporter gene under the auxin-responsive DR5 promoter supported the involvement of auxin signaling in F. oxysporum volatile-mediated growth enhancement. In addition, 1-naphthylthalamic acid, an inhibitor of auxin efflux, negated F. oxysporum volatile-mediated growth enhancement in both plants. Comparison of the profiles of volatile compounds produced by F. oxysporum strains that differentially affected plant growth suggests that the relative compositions of both growth inhibitory and stimulatory compounds may determine the degree of plant growth enhancement. Volatile-mediated signaling between fungi and plants may represent a potentially conserved, yet mostly overlooked, mechanism underpinning plant-fungus interactions and fungal niche adaption.