Bacterial Volatiles Known to Inhibit Phytophthora infestans Are Emitted on Potato Leaves by Pseudomonas Strains.

Bacterial volatiles play important roles in mediating beneficial interactions between plants and their associated microbiota. Despite their relevance, bacterial volatiles are mostly studied under laboratory conditions, although these strongly differ from the natural environment bacteria encounter when colonizing plant roots or shoots. In this work, we ask the question whether plant-associated bacteria also emit bioactive volatiles when growing on plant leaves rather than on artificial media. Using four potato-associated Pseudomonas, we demonstrate that potato leaves offer sufficient nutrients for the four strains to grow and emit volatiles, among which 1-undecene and Sulfur compounds have previously demonstrated the ability to inhibit the development of the oomycete Phytophthora infestans, the causative agent of potato late blight. Our results bring the proof of concept that bacterial volatiles with known plant health-promoting properties can be emitted on the surface of leaves and warrant further studies to test the bacterial emission of bioactive volatiles in greenhouse and field-grown plants.

Evaluating the Antagonistic Potential of Actinomycete Strains Isolated From Sudan's Soils Against Phytophthora infestans.

Soil microorganisms play crucial roles in soil fertility, e.g., through decomposing organic matter, cycling nutrients or through beneficial interactions with plants. Actinomycetes are a major component of soil inhabitants; they are prolific producers of specialized metabolites, among which many antibiotics. Here we report the isolation and characterization of 175 Actinomycetes from rhizosphere and bulk soil samples collected in 18 locations in Sudan. We evaluated the strains' metabolic potential for plant protection by testing their ability to inhibit the mycelial growth of the oomycete Phytophthora infestans, which is one of the most devastating plant pathogens worldwide. Most strains significantly reduced the oomycete's growth in direct confrontational in vitro assays. A significant proportion of the tested strains (15%) were able to inhibit P. infestans to more than 80%, 23% to 50%-80%, while the remaining 62% had inhibition percentages lesser than 50%. Different morphologies of P. infestans mycelial growth and sporangia formation were observed upon co-inoculation with some of the Actinomycetes isolates, such as the production of fewer, thinner hyphae without sporangia leading to a faint growth morphology, or on the contrary, of clusters of thick-walled hyphae leading to a bushy, or "frozen" morphology. These morphologies were caused by strains differing in activity levels but phylogenetically closely related with each other. To evaluate whether the isolated Actinomycetes could also inhibit the pathogen's growth in planta, the most active strains were tested for their ability to restrict disease progress in leaf disc and full plant assays. Five of the active strains showed highly significant protection of potato leaves against the pathogen in leaf disc assays, as well as substantial reduction of disease progress in full plants assays. Using cell-free filtrates instead of the bacterial spores also led to full protection against disease on leaf discs, which highlights the strong crop protective potential of the secreted metabolites that could be applied as leaf spray. This study demonstrates the strong anti-oomycete activity of soil- and rhizosphere-borne Actinomycetes and highlights their significant potential for the development of sustainable solutions based on either cell suspensions or cell-free filtrates to safeguard potatoes from their most damaging pathogen.

S-methyl Methanethiosulfonate: Promising Late Blight Inhibitor or Broad Range Toxin?

(1) Background: S-methyl methanethiosulfonate (MMTS), a sulfur containing volatile organic compound produced by plants and bacterial species, has recently been described to be an efficient anti-oomycete agent with promising perspectives for the control of the devastating potato late blight disease caused by Phytophthora infestans. However, earlier work raised questions regarding the putative toxicity of this compound. To assess the suitability of MMTS for late blight control in the field, the present study thus aimed at evaluating the effect of MMTS on a wide range of non-target organisms in comparison to P. infestans. (2) Methods: To this end, we exposed P. infestans, as well as different pathogenic and non-pathogenic fungi, bacteria, the nematode Caenorhabditis elegans as well as the plant Arabidopsis thaliana to MMTS treatment and evaluated their response by means of in vitro assays. (3) Results: Our results showed that fungi (both mycelium and spores) tolerated MMTS better than the oomycete P. infestans, but that the compound nevertheless exhibited non-negligible toxic effects on bacteria, nematodes and plants. (4) Conclusions: We discuss the mode of action of MMTS and conclude that even though this compound might be too toxic for chemical application in the field, its strong anti-oomycete activity could still be exploited when naturally released at the site of infection by plant-associated microbes inoculated as biocontrol agents.

Linking Comparative Genomics of Nine Potato-Associated Pseudomonas Isolates With Their Differing Biocontrol Potential Against Late Blight.

For plants, the advantages of associating with beneficial bacteria include plant growth promotion, reduction of abiotic and biotic stresses and enhanced protection against various pests and diseases. Beneficial bacteria rightly equipped for successful plant colonization and showing antagonistic activity toward plant pathogens seem to be actively recruited by plants. To gain more insights into the genetic determinants responsible for plant colonization and antagonistic activities, we first sequenced and de novo assembled the complete genomes of nine Pseudomonas strains that had exhibited varying antagonistic potential against the notorious oomycete Phytophthora infestans, placed them into the phylogenomic context of known Pseudomonas biocontrol strains and carried out a comparative genomic analysis to define core, accessory (i.e., genes found in two or more, but not all strains) and unique genes. Next, we assessed the colonizing abilities of these strains and used bioassays to characterize their inhibitory effects against different stages of P. infestans' lifecycle. The phenotype data were then correlated with genotype information, assessing over three hundred genes encoding known factors for plant colonization and antimicrobial activity as well as secondary metabolite biosynthesis clusters predicted by antiSMASH. All strains harbored genes required for successful plant colonization but also distinct arsenals of antimicrobial compounds. We identified genes coding for phenazine, hydrogen cyanide, 2-hexyl, 5-propyl resorcinol and pyrrolnitrin synthesis, as well as various siderophores, pyocins and type VI secretion systems. Additionally, the comparative genomic analysis revealed about a hundred accessory genes putatively involved in anti-Phytophthora activity, including a type II secretion system (T2SS), several peptidases and a toxin. Transcriptomic studies and mutagenesis are needed to further investigate the putative involvement of the novel candidate genes and to identify the various mechanisms involved in the inhibition of P. infestans by different Pseudomonas strains.

Endophytes and Epiphytes From the Grapevine Leaf Microbiome as Potential Biocontrol Agents Against Phytopathogens.

Plants harbor diverse microbial communities that colonize both below-ground and above-ground organs. Some bacterial members of these rhizosphere and phyllosphere microbial communities have been shown to contribute to plant defenses against pathogens. In this study, we characterize the pathogen-inhibiting potential of 78 bacterial isolates retrieved from endophytic and epiphytic communities living in the leaves of three grapevine cultivars. We selected two economically relevant pathogens, the fungus Botrytis cinerea causing gray mold and the oomycete Phytophthora infestans, which we used as a surrogate for Plasmopara viticola causing downy mildew. Our results showed that epiphytic isolates were phylogenetically more diverse than endophytic isolates, the latter mostly consisting of Bacillus and Staphylococcus strains, but that mycelial inhibition of both pathogens through bacterial diffusible metabolites was more widespread among endophytes than among epiphytes. Six closely related Bacillus strains induced strong inhibition (>60%) of Botrytis cinerea mycelial growth. Among these, five led to significant perturbation in spore germination, ranging from full inhibition to reduction in germination rate and germ tube length. Different types of spore developmental anomalies were observed for different strains, suggesting multiple active compounds with different modes of action on this pathogen. Compared with B. cinerea, the oomycete P. infestans was inhibited in its mycelial growth by a higher number and more diverse group of isolates, including many Bacillus but also Variovorax, Pantoea, Staphylococcus, Herbaspirillum, or Sphingomonas strains. Beyond mycelial growth, both zoospore and sporangia germination were strongly perturbed upon exposure to cells or cell-free filtrates of selected isolates. Moreover, three strains (all epiphytes) inhibited the pathogen's growth via the emission of volatile compounds. The comparison of the volatile profiles of two of these active strains with those of two phylogenetically closely related, inactive strains led to the identification of molecules possibly involved in the observed volatile-mediated pathogen growth inhibition, including trimethylpyrazine, dihydrochalcone, and L-dihydroxanthurenic acid. This work demonstrates that grapevine leaves are a rich source of bacterial antagonists with strong inhibition potential against two pathogens of high economical relevance. It further suggests that combining diffusible metabolite-secreting endophytes with volatile-emitting epiphytes might be a promising multi-layer strategy for biological control of above-ground pathogens.

Biocontrol Activity of Three Pseudomonas in a Newly Assembled Collection of Phytophthora infestans Isolates.

Late blight caused by the oomycete Phytophthora infestans constitutes the greatest threat to potato production worldwide. Considering the increasing concerns regarding the emergence of novel fungicide-resistant genotypes and the general demand for reducing inputs of synthetic and copper-based fungicides, the need for alternative control methods is acute. Several bacterial antagonists have shown anti-Phytophthora effects during in vitro and greenhouse experiments. We report the effects of three Pseudomonas strains recovered from field-grown potatoes against a collection of P. infestans isolates assembled for this study. The collection comprised 19 P. infestans isolates of mating types A1 and A2 greatly varying in fungicide resistance and virulence profiles as deduced from leaf disc experiments on Black's differential set. The mycelial growth of all P. infestans isolates was fully inhibited when co-cultivated with the most active Pseudomonas strain (R47). Moreover, the isolates reacted differently to exposure to the less active Pseudomonas strains (S19 and R76). Leaf disc infection experiments with six selected P. infestans isolates showed that four of them, including highly virulent and fungicide-resistant ones, could be efficiently controlled by different potato-associated Pseudomonas strains.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Combining Different Potato-Associated Pseudomonas Strains for Improved Biocontrol of Phytophthora infestans.

Late blight caused by Phytophthora infestans is considered as the most devastating disease of potato and is a re-emerging problem worldwide. Current late blight control practices rely mostly on synthetic fungicides or copper-based products, but growing awareness of the negative impact of these compounds on the environment has led to the search for alternative control measures. A collection of Pseudomonas strains isolated from both the rhizosphere and the phyllosphere of potato was recently characterized for in vitro protective effects against P. infestans. In the present study, we used a leaf disk assay with three different potato cultivars to compare the disease inhibition capacity of nine selected Pseudomonas strains when applied alone or in all possible dual and triple combinations. Results showed a strong cultivar effect and identified strains previously thought to be inactive based on in vitro assays as the best biocontrol candidates. One strain was much more active alone than in combination with other strains, while two other strains provided significantly better protection in dual combination than when applied alone. A subset of five strains was then further selected to determine their mutual influence on each other's survival and growth, as well as to characterize their activity against P. infestans in more details. This revealed that the two strains whose dual combination was particularly efficient were only weakly interfering with each other's growth and had complementary modes of action. Our results highlight the potential to harness the crop's native rhizosphere and phyllosphere microbiome through re-assembling strains with differing modes of action into small communities, thereby providing more consistent protection than with the application of single strains. We consider this as a first step toward more elaborate microbiome management efforts, which shall be integrated into global strategies for sustainable control of potato late blight.

The Anti-Phytophthora Effect of Selected Potato-Associated Pseudomonas Strains: From the Laboratory to the Field.

Late blight, caused by the oomycete Phytophthora infestans, is the most devastating disease of potato. In organic farming, late blight is controlled by repeated applications of copper-based products, which negatively impact the environment. To find alternative solutions for late blight management, we have previously isolated a large collection of bacteria from the phyllosphere and the rhizosphere of potatoes. Here we report the antagonistic potential of these strains when co-cultivated with P. infestans as well as with other potato pathogens. We then focused on three Pseudomonas strains and compared their protective impact against late blight to that of well-known biocontrol strains in planta using a high-throughput leaf disk assay with automated picture analysis. When sprayed on the leaves of potatoes in the greenhouse, the strains were able to survive for at least 15 days. Under field conditions, populations decreased faster but all tested strains could still be retrieved after 8 days. The most active strain in vitro, P. chlororaphis R47, was also the best protectant on leaf disks from plants grown in the greenhouse experiment, but its protection potential could not be verified in the field due to unfavorable infection conditions. However, its protective effect against P. infestans in planta, its survival in the phyllosphere as well as its ability to colonize the potato rhizosphere in very high population densities, suggest a potential for field application, e.g., in the form of tuber treatment or leaf spray.

Volatile Organic Compounds from Native Potato-associated Pseudomonas as Potential Anti-oomycete Agents.

The plant kingdom represents a prominent biodiversity island for microbes that associate with the below- or aboveground organs of vegetal species. Both the root and the leaf represent interfaces where dynamic biological interactions influence plant life. Beside well-studied communication strategies based on soluble compounds and protein effectors, bacteria were recently shown to interact both with host plants and other microbial species through the emissions of volatile organic compounds (VOCs). Focusing on the potato late blight-causing agent Phytophthora infestans, this work addresses the potential role of the bacterial volatilome in suppressing plant diseases. In a previous study, we isolated and identified a large collection of strains with anti-Phytophthora potential from both the phyllosphere and the rhizosphere of potato. Here we report the characterization and quantification of their emissions of biogenic volatiles, comparing 16 Pseudomonas strains differing in (i) origin of isolation (phyllosphere vs. rhizosphere), (ii) in vitro inhibition of P. infestans growth and sporulation behavior, and (iii) protective effects against late blight on potato leaf disks. We systematically tested the pharmacological inhibitory activity of core and strain-specific single compounds against P. infestans mycelial growth and sporangial behavior in order to identify key effective candidate molecules present in the complex natural VOCs blends. We envisage the plant bacterial microbiome as a reservoir for functional VOCs and establish the basis for finding the primary enzymatic toolset that enables the production of active components of the volatile bouquet in plant-associated bacteria. Comprehension of these functional interspecies interactions will open perspectives for the sustainable control of plant diseases in forthcoming agriculture.