The transition from conventional to organic agriculture is often challenged by the adaptation of biological control agents to environments heavily exposed to agrochemical pollutants. We studied Trichoderma species isolated from living leaf tissues of wild Rubiacaeae (coffee family) plants to determine their fungicide tolerance and potential for bioremoval. First, we assessed the in vitro tolerance to fungicides of four Trichoderma isolates ( Trichoderma rifaii T1, T . aff. crassum T2, T . aff. atroviride T3, and T . aff. strigosellum T4) by placing mycelial plugs onto solid media supplemented with seven different systemic and non-systemic fungicides. After a week, most of the fungicides did not significantly inhibit the growth of the isolates, except in the case of cyproconazole, where the only isolate able to grow was T1; however, the colony morphology was affected by the presence of fungicides. Second, biological removal potential was established for selected isolates. For this experiment, the isolates T1, T2, and T4 were independently inoculated into liquid media with the fungicides azoxystrobin, chlorothalonil, cyproconazole, and trifloxystrobin. After 14 days of incubation, a removal of up to 89% was achieved for chlorothalonil, 46.4% for cyproconazole, and 33.1% for trifloxystrobin using viable biomass. In the case of azoxystrobin, the highest removal (82.2%) occurred by adsorption to fungal biomass. Ecotoxicological tests in Daphnia magna revealed that T1 has the highest removal potential, achieving significant elimination of every fungicide, while simultaneously detoxifying the aqueous matrix (except in the case of cyproconazole). Isolate T4 also exhibited an intermediate efficiency, while isolate T2 was unable to detoxify the matrix in most cases. The removal and detoxification of cyproconazole failed with all the isolates. These findings suggest that endosphere of wild plants could be an attractive guild to find new Trichoderma species with promising bioremediation capabilities. In addition, the results demonstrate that attention should be placed when combining certain types of agrochemicals with antagonistic fungi in Integrated Pest and Disease Management strategies or when transitioning to organic agriculture.
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The endophytobiome of wild Rubiaceae as a source of antagonistic fungi against the American Leaf Spot of coffee ( Mycena citricolor )
The American leaf spot, caused by Mycena citricolor, is an important disease of coffee (Coffea arabica), mostly in Central America. Currently, there are limited pathogen control alternatives that are environment friendly and economically accessible. The use of fungi isolated from the plant endomycobiota in their native habitats is on the rise because studies show their great potential for biological control. To begin to generate a green alternative to control M. citricolor, the objectives of the present study were to (i) collect, identify, screen (in vitro and in planta), and select endophytic fungi from wild Rubiaceae collected in old-growth forests of Costa Rica; (ii) confirm endophytic colonization in coffee plantlets; (iii) evaluate the effects of the endophytes on plantlet development; and (iv) corroborate the antagonistic ability in planta.
Through in vitro and in planta antagonism assays, we found that out of the selected isolates (i.e. Daldinia eschscholzii GU11N, Nectria pseudotrichia GUHN1, Purpureocillium aff. lilacinum CT24, Sarocladium aff. kiliense CT25, Trichoderma rifaii CT5, T. aff. crassum G1C, T. aff. atroviride G7T, T. aff. strigosellum GU12, and Xylaria multiplex GU14T), Trichoderma spp. produced the highest growth inhibition percentages in vitro. Trichoderma isolates CT5 and G1C were then tested in planta using Coffea arabica cv. caturra plantlets. Endophytic colonization was verified, followed by in planta growth promotion and antagonism assays.
Results show that Trichoderma isolates CT5 and G1C have potential for plant growth promotion and antagonism against Mycena citricolor, reducing incidence and severity, and preventing plant mortality.
- Award ID(s):
- 1638976
- NSF-PAR ID:
- 10414138
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Journal of Applied Microbiology
- Volume:
- 134
- Issue:
- 5
- ISSN:
- 1365-2672
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Fungal endophytes are pivotal components of a plant's microbiome, profoundly impacting its health and fitness. Yet, myriad questions remain concerning the intricate interactions between these microorganisms and their hosts, particularly in the context of agriculturally important plants such as
Coffea arabica . To bridge this knowledge gap and provide a comprehensive framework, this study investigated how farming practices shape the taxonomic and functional diversity of phylloplane endophytes in coffee. Coffee plant leaves from two distinct producing regions in Costa Rica were sampled, ensuring the representation of various coffee varieties (Obatá, Catuaí, and Caturra), agricultural management methods (organic vs. conventional), sun exposure regimes (full sunlight/monoculture vs. natural shade/agroforestry), and leaf developmental stages (newly emerged asymptomatic vs. mature leaves). Fungal communities were characterized by employing both culture‐dependent and independent techniques (internal transcribed spacer 2 nuclear ribosomal DNA metabarcoding). The results showed a greater diversity of endophytes in mature leaves and conventionally managed plants, with coffee variety exerting an unclear influence. The effect of sun exposure was surprisingly negligible. However, data emphasize the benefits of agroforestry and organic farming, which are linked to reduced putative pathogens and heightened levels of potentially mutualistic fungi, fostering functionally diverse communities. Despite the role that plant microbiomes might play in agricultural production, the knowledge to shape endophytic communities through breeding or management is lacking. The results from this study provide a framework to understand how both plant and agricultural practices influence endophyte diversity within coffee crops. These insights hold promise for guiding future efforts to manipulate coffee microbial communities effectively. -
Wilson, Richard A. (Ed.)more » « less
Trichoderma is a cosmopolitan genus with diverse lifestyles and nutritional modes, including mycotrophy, saprophytism, and endophytism. Previous research has reported greater metabolic gene repertoires in endophytic fungal species compared to closely-related non-endophytes. However, the extent of this ecological trend and its underlying mechanisms are unclear. Some endophytic fungi may also be mycotrophs and have one or more mycoparasitism mechanisms. Mycotrophic endophytes are prominent in certain genera likeTrichoderma , therefore, the mechanisms that enable these fungi to colonize both living plants and fungi may be the result of expanded metabolic gene repertoires. Our objective was to determine what, if any, genomic features are overrepresented in endophytic fungi genomes in order to undercover the genomic underpinning of the fungal endophytic lifestyle. Here we compared metabolic gene cluster and mycoparasitism gene diversity across a dataset of thirty-eightTrichoderma genomes representing the full breadth of environmentalTrichoderma ’s diverse lifestyles and nutritional modes. We generated four newTrichoderma endophyticum genomes to improve the sampling of endophytic isolates from this genus. As predicted, endophyticTrichoderma genomes contained, on average, more total biosynthetic and degradative gene clusters than non-endophytic isolates, suggesting that the ability to create/modify a diversity of metabolites potential is beneficial or necessary to the endophytic fungi. Still, once the phylogenetic signal was taken in consideration, no particular class of metabolic gene cluster was independently associated with theTrichoderma endophytic lifestyle. Several mycoparasitism genes, but no chitinase genes, were associated with endophyticTrichoderma genomes. Most genomic differences betweenTrichoderma lifestyles and nutritional modes are difficult to disentangle from phylogenetic divergences among species, suggesting thatTrichoderma genomes maybe particularly well-equipped for lifestyle plasticity. We also consider the role of endophytism in diversifying secondary metabolism after identifying the horizontal transfer of the ergot alkaloid gene cluster toTrichoderma . -
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Background Fungal endophytes inhabit symptomless, living tissues of all major plant lineages to form one of earth’s most prevalent groups of symbionts. Many reproduce from senesced and/or decomposing leaves and can produce extracellular leaf-degrading enzymes, blurring the line between symbiotrophy and saprotrophy. To better understand the endophyte–saprotroph continuum we compared fungal communities and functional traits of focal strains isolated from living leaves to those isolated from leaves after senescence and decomposition, with a focus on foliage of woody plants in five biogeographic provinces ranging from tundra to subtropical scrub forest.
Methods We cultured fungi from the interior of surface-sterilized leaves that were living at the time of sampling (i.e., endophytes), leaves that were dead and were retained in plant canopies (dead leaf fungi, DLF), and fallen leaves (leaf litter fungi, LLF) from 3–4 species of woody plants in each of five sites in North America. Our sampling encompassed 18 plant species representing two families of Pinophyta and five families of Angiospermae. Diversity and composition of fungal communities within and among leaf life stages, hosts, and sites were compared using ITS-partial LSU rDNA data. We evaluated substrate use and enzyme activity by a subset of fungi isolated only from living tissues vs. fungi isolated only from non-living leaves.
Results Across the diverse biomes and plant taxa surveyed here, culturable fungi from living leaves were isolated less frequently and were less diverse than those isolated from non-living leaves. Fungal communities in living leaves also differed detectably in composition from communities in dead leaves and leaf litter within focal sites and host taxa, regardless of differential weighting of rare and abundant fungi. All focal isolates grew on cellulose, lignin, and pectin as sole carbon sources, but none displayed ligninolytic or pectinolytic activity
in vitro . Cellulolytic activity differed among fungal classes. Within Dothideomycetes, activity differed significantly between fungi from living vs. non-living leaves, but such differences were not observed in Sordariomycetes.Discussion Although some fungi with endophytic life stages clearly persist for periods of time in leaves after senescence and incorporation into leaf litter, our sampling across diverse biomes and host lineages detected consistent differences between fungal assemblages in living vs. non-living leaves, reflecting incursion by fungi from the leaf exterior after leaf death and as leaves begin to decompose. However, fungi found only in living leaves do not differ consistently in cellulolytic activity from those fungi detected thus far only in dead leaves. Future analyses should consider Basidiomycota in addition to the Ascomycota fungi evaluated here, and should explore more dimensions of functional traits and persistence to further define the endophytism-to-saprotrophy continuum.
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Hom, Erik F. (Ed.)more » « less
ABSTRACT Terpenes are among the oldest and largest class of plant-specialized bioproducts that are known to affect plant development, adaptation, and biological interactions. While their biosynthesis, evolution, and function in aboveground interactions with insects and individual microbial species are well studied, how different terpenes impact plant microbiomes belowground is much less understood. Here we designed an experiment to assess how belowground exogenous applications of monoterpenes (1,8-cineole and linalool) and a sesquiterpene (nerolidol) delivered through an artificial root system impacted its belowground bacterial and fungal microbiome. We found that the terpene applications had significant and variable impacts on bacterial and fungal communities, depending on terpene class and concentration; however, these impacts were localized to the artificial root system and the fungal rhizosphere. We complemented this experiment with pure culture bioassays on responsive bacteria and fungi isolated from the sorghum rhizobiome. Overall, higher concentrations (200 µM) of nerolidol were inhibitory to
Ferrovibrium and tested Firmicutes. While fungal isolates ofPenicillium andPericonia were also more inhibited by higher concentrations (200 µM) of nerolidol,Clonostachys was enhanced at this higher level and together withHumicola was inhibited by the lower concentration tested (100 µM). On the other hand, 1,8-cineole had an inhibitory effect onOrbilia at both tested concentrations but had a promotive effect at 100 µM onPenicillium andPericonia . Similarly, linalool at 100 µM had significant growth promotion inMortierella , but an inhibitory effect forOrbilia . Together, these results highlight the variable direct effects of terpenes on single microbial isolates and demonstrate the complexity of microbe-terpene interactions in the rhizobiome.Importance Terpenes represent one of the largest and oldest classes of plant-specialized metabolism, but their role in the belowground microbiome is poorly understood. Here, we used a “rhizobox” mesocosm experimental set-up to supply different concentrations and classes of terpenes into the soil compartment with growing sorghum for 1 month to assess how these terpenes affect sorghum bacterial and fungal rhizobiome communities. Changes in bacterial and fungal communities between treatments belowground were characterized, followed by bioassays screening on bacterial and fungal isolates from the sorghum rhizosphere against terpenes to validate direct microbial responses. We found that microbial growth stimulatory and inhibitory effects were localized, terpene specific, dose dependent, and transient in time. This work paves the way for engineering terpene metabolisms in plant microbiomes for improved sustainable agriculture and bioenergy crop production.
