# HARNESSING USEFUL RHIZOSPHERE MICROORGANISMS FOR PATHOGEN AND PEST BIOCONTROL, VOLUME II

EDITED BY : Aurelio Ciancio, Corné M. J. Pieterse and Jesús Mercado-Blanco PUBLISHED IN : Frontiers in Microbiology and Frontiers in Plant Science

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# HARNESSING USEFUL RHIZOSPHERE MICROORGANISMS FOR PATHOGEN AND PEST BIOCONTROL, VOLUME II

Topic Editors:

Aurelio Ciancio, Istituto per la Protezione Sostenibile delle Piante, CNR, Italy Corné M. J. Pieterse, Urecht University, Netherlands Jesús Mercado-Blanco, Instituto de Agricultura Sostenible, CSIC, Spain

Image: Aurelio Ciancio.

The use of biocontrol agents and beneficial organisms for management of plant and pest diseases appears as an environment-friendly and economic procedure. However, this option is not always available, depending on the lack of knowledge on the mechanisms of natural regulation, locally effective. In this view, this eBook considers studies and experimental works illustrating a range of problems and solutions based on microbial resources, suitable for management of biotic stress factors. These examples show how detailed data and knowledge on the organisms involved are of paramount importance to achieve a sustainable and durable management capability.

Citation: Ciancio, A., Pieterse, C. M. J., Mercado-Blanco, J., eds. (2019). Harnessing Useful Rhizosphere Microorganisms for Pathogen and Pest Biocontrol, Volume II. Lausanne: Frontiers Media. doi: 10.3389/978-2-88963-200-8

# Table of Contents

*07 Editorial: Harnessing Useful Rhizosphere Microorganisms for Pathogen and Pest Biocontrol - Second Edition*

Aurelio Ciancio, Corné M. J. Pieterse and Jesús Mercado-Blanco

## SECTION 1

## PHYTOPHTHORA

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

Mout De Vrieze, Fanny Germanier, Nicolas Vuille and Laure Weisskopf

*25 Versatile Antagonistic Activities of Soil-Borne* Bacillus *spp. and*  Pseudomonas *spp. Against* Phytophthora infestans *and Other Potato Pathogens*

Simon Caulier, Annika Gillis, Gil Colau, Florent Licciardi, Maxime Liépin, Nicolas Desoignies, Pauline Modrie, Anne Legrève, Jacques Mahillon and Claude Bragard

## SECTION 2

## BIOCONTROL OF FUNGAL DISEASES

*40 Involvement of the Transcriptional Coactivator ThMBF1 in the Biocontrol Activity of* Trichoderma harzianum

M. Belén Rubio, Alonso J. Pardal, Rosa E. Cardoza, Santiago Gutiérrez, Enrique Monte and Rosa Hermosa

Nicola Imperiali, Francesca Dennert, Jana Schneider, Titouan Laessle, Christelle Velatta, Marie Fesselet, Michele Wyler, Fabio Mascher, Olga Mavrodi, Dmitri Mavrodi, Monika Maurhofer and Christoph Keel

*103 Potential of Finger Millet Indigenous Rhizobacterium* Pseudomonas *sp. MSSRFD41 in Blast Disease Management—Growth Promotion and Compatibility With the Resident Rhizomicrobiome* Jegan Sekar, Kathiravan Raju, Purushothaman Duraisamy and Prabavathy Ramalingam Vaiyapuri

*119 Indigenous* Pseudomonas *spp. Strains From the Olive (*Olea europaea *L.) Rhizosphere as Effective Biocontrol Agents Against* Verticillium dahliae*: From the Host Roots to the Bacterial Genomes*

Carmen Gómez-Lama Cabanás, Garikoitz Legarda, David Ruano-Rosa, Paloma Pizarro-Tobías, Antonio Valverde-Corredor, José L. Niqui, Juan C. Triviño, Amalia Roca and Jesús Mercado-Blanco

*138* Bacillus velezensis *FZB42 in 2018: The Gram-Positive Model Strain for Plant Growth Promotion and Biocontrol*

Ben Fan, Cong Wang, Xiaofeng Song, Xiaolei Ding, Liming Wu, Huijun Wu, Xuewen Gao and Rainer Borriss

Kihyun Kim, Yoonji Lee, Areum Ha, Ji-In Kim, Ae Ran Park, Nan Hee Yu, Hokyoung Son, Gyung Ja Choi, Hae Woong Park, Chul Won Lee, Theresa Lee, Yin-Won Lee and Jin-Cheol Kim

*169 Antifungal Activity of Lipopeptides From* Bacillus *XT1 CECT 8661 Against*  Botrytis cinerea

Laura Toral, Miguel Rodríguez, Victoria Béjar and Inmaculada Sampedro

Fatima H. Kamil, Esam E. Saeed, Khaled A. El-Tarabily and Synan F. AbuQamar

*230 How* Streptomyces anulatus *Primes Grapevine Defenses to Cope With Gray Mold: A Study of the Early Responses of Cell Suspensions*

Parul Vatsa-Portugal, Aziz Aziz, Marine Rondeau, Sandra Villaume, Hamid Morjani, Christophe Clément and Essaid Ait Barka

## SECTION 3

## BACTERIAL DISEASES

Sumera Yasmin, Fauzia Y. Hafeez, Muhammad S. Mirza, Maria Rasul, Hafiz M. I. Arshad, Muhammad Zubair and Mazhar Iqbal

## SECTION 4

## MANAGEMENT OF NEMATODE AND INSECT PESTS

*279 Rhizosphere Microbiomes Modulated by Pre-crops Assisted Plants in Defense Against Plant-Parasitic Nematodes*

Ahmed Elhady, Shimaa Adss, Johannes Hallmann and Holger Heuer

*288* Klebsiella pneumoniae *SnebYK Mediates Resistance Against* Heterodera glycines *and Promotes Soybean Growth*

Dan Liu, Le Chen, Xiaofeng Zhu, Yuanyuan Wang, Yuanhu Xuan, Xiaoyu Liu, Lijie Chen and Yuxi Duan

*301 Chitosan Increases Tomato Root Colonization by* Pochonia chlamydosporia *and Their Combination Reduces Root-Knot Nematode Damage*

Nuria Escudero, Federico Lopez-Moya, Zahra Ghahremani, Ernesto A. Zavala-Gonzalez, Aurora Alaguero-Cordovilla, Caridad Ros-Ibañez, Alfredo Lacasa, Francisco J. Sorribas and Luis V. Lopez-Llorca

*311 Combined Field Inoculations of* Pseudomonas *Bacteria, Arbuscular Mycorrhizal Fungi, and Entomopathogenic Nematodes and Their Effects on Wheat Performance*

Nicola Imperiali, Xavier Chiriboga, Klaus Schlaeppi, Marie Fesselet, Daniela Villacrés, Geoffrey Jaffuel, S. Franz Bender, Francesca Dennert, Ruben Blanco-Pérez, Marcel G. A. van der Heijden, Monika Maurhofer, Fabio Mascher, Ted C. J. Turlings, Christoph J. Keel and Raquel Campos-Herrera

## SECTION 5

## SUPPRESSIVE SOILS

*328 Screening and Characterization of Potentially Suppressive Soils Against*  Gaeumannomyces graminis *Under Extensive Wheat Cropping by Chilean Indigenous Communities*

Paola Durán, Milko Jorquera, Sharon Viscardi, Victor J. Carrion, María de la Luz Mora and María J. Pozo

Jun Chen, Linkun Wu, Zhigang Xiao, Yanhong Wu, Hongmiao Wu, Xianjin Qin, Juanying Wang, Xiaoya Wei, Muhammad U. Khan, Sheng Lin and Wenxiong Lin

## SECTION 6

## SOIL ENVIRONMENT

*379 Long-Term Irrigation Affects the Dynamics and Activity of the Wheat Rhizosphere Microbiome*

Dmitri V. Mavrodi, Olga V. Mavrodi, Liam D. H. Elbourne, Sasha Tetu, Robert F. Bonsall, James Parejko, Mingming Yang, Ian T. Paulsen, David M. Weller and Linda S. Thomashow

Hyun G. Kong, Teak S. Shin, Tae H. Kim and Choong-Min Ryu

## SECTION 7

## FUTURE CHALLENGES

*415 Challenges and Approaches in Microbiome Research: From Fundamental to Applied*

Chrysi Sergaki, Beatriz Lagunas, Ian Lidbury, Miriam L. Gifford and Patrick Schäfer

*427 Microbial Phosphorus Solubilization and its Potential for Use in Sustainable Agriculture*

Elizabeth T. Alori, Bernard R. Glick and Olubukola O. Babalola

*435 The Chemistry of Plant–Microbe Interactions in the Rhizosphere and the Potential for Metabolomics to Reveal Signaling Related to Defense Priming and Induced Systemic Resistance*

Msizi I. Mhlongo, Lizelle A. Piater, Ntakadzeni E. Madala, Nico Labuschagne and Ian A. Dubery

*452 Inner Plant Values: Diversity, Colonization and Benefits From Endophytic Bacteria*

Hongwei Liu, Lilia C. Carvalhais, Mark Crawford, Eugenie Singh, Paul G. Dennis, Corné M. J. Pieterse and Peer M. Schenk

# Editorial: Harnessing Useful Rhizosphere Microorganisms for Pathogen and Pest Biocontrol - Second Edition

#### Aurelio Ciancio1 \*, Corné M. J. Pieterse2 and Jesús Mercado-Blanco3

1 Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, Bari, Italy, 2 Department of Biology, Science4Life, Utrecht University, Utrecht, Netherlands, 3 Instituto de Agricultura Sostenible, Agencia Estatal Consejo Superior de Investigaciones Científicas, Córdoba, Spain

Keywords: biocontrol, induced resistance, plant growth promotion, rhizosphere microbiology, plant microbe interaction soil microbiology

### **Editorial on the Research Topic**

### **Harnessing Useful Rhizosphere Microorganisms for Pathogen and Pest Biocontrol - Second Edition**

#### Edited by:

Brigitte Mauch-Mani, Université de Neuchâtel, Switzerland

#### Reviewed by:

Felix Mauch, Université de Fribourg, Switzerland Estrella Luna Diez, University of Birmingham, United Kingdom

> \*Correspondence: Aurelio Ciancio<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Microbiology

Received: 11 June 2019 Accepted: 06 August 2019 Published: 28 August 2019

#### Citation:

Ciancio A, Pieterse CMJ and Mercado-Blanco J (2019) Editorial: Harnessing Useful Rhizosphere Microorganisms for Pathogen and Pest Biocontrol - Second Edition. Front. Microbiol. 10:1935. doi: 10.3389/fmicb.2019.01935 There is a worldwide interest in the exploitation of beneficial plant-associated microorganisms as an alternative to pesticides for pest and disease management. It is underpinned by practical and social reasons, including safety of consumers, farmers, and field workers, as well as the need for sustainable practices safeguarding the environment and protecting its biodiversity. Cost of conventional pesticides and the insurgence of resistance in pests also re-direct farmers' choice toward safer approaches. This trend is observed also in fast-growing population economies, propelling the global demand for eco-sustainable technologies.

Understanding the role of rhizosphere microorganisms in the control of pests and diseases appears as a growing research field, as shown by the sharp increase of studies carried out during the period 2000–2019. The number of records retrieved through a Google Scholar query with keywords "microorganisms," "control," "pest," and "diseases" increased from around 5000 (2000–2005) to ∼8500 and >20,000 (2006–2010 and 2011–2019, respectively), when the term "rhizosphere" was added. Without the latter the records instead lowered from around 17,000 to ∼15,000 in the last period (interrogation dated August 2, 2019). However, in spite of this increased interest in rhizosphere ecology managing and exploiting living organisms to regulate or control other noxious species still remains a complex task. Detailed data on interacting variables and processes are needed, as their final result often differs significantly from the simple sum of effects. Any information boosting our capacity to solve problems related to safer plant protection is, therefore, more than welcome.

## BIOCONTROL OF PHYTOPHTHORA

The Oomycete Phytophthora infestans, the most widespread and severe pathogen of potato, is a global threat due to its capacity to develop new genotypes overcoming the resistance present in many cultivars (Fry, 2008; Cooke et al., 2011). Rhizo/phyllosphere Pseudomonas strains have been tested against this pathogen. The potato cultivar to which bacteria were originally associated, and the combined use of strains were found to underpin beneficial effects, including a higher disease inhibition (De Vrieze et al.). These authors tested the rhizosphere complexity, with combinations of native bacteria yielding benefits, not previously inferred from in vitro data. This approach showed that multiple strains can be exploited in combination, using poorly competitive bacteria that do not

interfere each other. Results stressed the potential of communities based on native microorganisms rather than single isolates, inhibiting P. infestans or halting its progression.

In a similar approach, Caulier et al. screened Bacillus and Pseudomonas spp. strains against potato pathogens including P. infestans. The most significant antagonistic activity appeared to be related to production of metabolites such as bacilysin, biosurfactants, and siderophores, characterizing some Bacillus spp. strains. Data indicated that indigenous rhizosphere microflora may yield effective bio-management tools.

## BIOCONTROL OF FUNGAL DISEASES

The exploitation of rhizosphere microorganisms for management of fungal diseases is a fertile research field (Toyota and Shirai, 2018). Species mostly include antagonistic fungi such as Trichoderma spp. and bacteria of genera Pseudomonas, Bacillus, and Streptomyces. As assays showed biocontrol efficacy for many isolates, there is a need for characterizing metabolic and molecular mechanisms underpinning these activities.

## Trichoderma

The role of gene regulatory elements may reveal key clues in biocontrol agents. In Trichoderma harzianum the transcription factor THCTF1 is involved in the production of 6-pentyl-pyrone, a volatile secondary metabolite active in interspecies cross-talk. THCTF1A is a homolog of the multiprotein bridging factor 1 (mbf1). Expression and regulation of both genes appeared to be important in modulating the activity of the fungus. Transformants overexpressing Thmbf1 were used to investigate the effect of this gene in biocontrol of Fusarium oxysporum f. sp. lycopersici race 2 (FO) and Botrytis cinerea. Thmbf1 affected production of volatile organic compounds (VOC) and low molecular weight molecules by T. harzianum with antifungal activity against FO. Thmbf1 overexpression negatively affected T. harzianum biocontrol efficacy against both pathogens on susceptible tomato (Rubio et al.).

Manganiello et al. investigated transcriptomic and metabolomic changes induced by a T. harzianum strain, or its secondary metabolite harzianic acid, in Micro-Tom tomato infected by the soil-borne pathogen Rhizoctonia solani. Genes involved in resistance to biotic stress were differentially expressed, confirming T. harzianum ability to activate a defense response in plants. Genes of the ethylene/jasmonate and salicylic acid signaling pathways were up-regulated, as well as genes involved in reactive oxygen species (ROS) detoxification. Harzianic acid induced expression of defense genes such as protease inhibitors, CC-NBS-LRR resistance proteins, and hormone interplay. Both treatments increased steroidal glycoalkaloids, confirming activation of metabolic chemical defenses.

## Pseudomonas

Rhizosphere competence is a key factor for a successful biocontrol agent, as plant species, soil type, and pathogen types affect the microbial community composition in the rhizosphere (Schreiter et al., 2014). The biocontrol efficacy of a Pseudomonas sp. isolate against R. solani on potato and lettuce was tested in three soil types. The antagonist colonized the rhizosphere of both crops and reduced the severity of black scurf on potato and bottom rot on lettuce in all soils. The Pseudomonas sp. isolate differently affected the bacterial community composition, depending on the host plant, as its effect was more evident in lettuce than potato (Schreiter et al.).

Pseudomonas spp. and antimicrobial metabolites such as 2,4-diacetylphloroglucinol (DAPG) or phenazines (PHZ), are likely involved in the insurgence of disease-suppressive soils, inhibiting soilborne plant diseases. However, antimicrobialproducing species can be found also in disease-conducive soils. Imperiali et al. compared soils from Swiss cereal farms, considering their resistance against two soilborne pathogens, the abundance of Pseudomonas spp. the biosynthesis DAPG, PHZ, and pyrrolnitrin, and the ability to support their production on roots. All these traits did not correlate to soil disease resistance. Data showed that this capacity depended on the pathogen species, as shown by soils exclusively not conducive for Gaeumannomyces tritici or Pythium ultimum. Factors related to soil structure and availability of nutrients were found to affect abundance and production of antimicrobial compounds.

A Pseudomonas spp. strain was tested for management of blast disease, caused by Pyricularia grisea, on the finger millet Eleusine coracona "Ragi," a cereal used in semi-arid regions of Asia and Africa. It inhibited P. grisea, solubilized phosphate and produced antifungal metabolites, siderophores, hydrolytic enzymes, and indolacetic acid. The strain had a direct effect on the pathogen, causing hyphal deformation, with a minimum impact on root microbial biomass and other rhizobacteria. It also reduced blast infestation and enhanced plant vigor and growth, showing a prolonged shelf-life when stored as a liquid formulation (Sekar et al.).

Similarly, several isolates collected by Gómez-Lama Cabanás et al. from olive rhizosphere showed potential against Verticillium wilt of olive, caused by Verticillium dahliae. Three Pseudomonas spp. strains were selected (PIC25, PIC105, and PICF141) with high in vitro inhibition against the pathogen. Effectiveness against the defoliating pathotype of V. dahliae was demonstrated, in particular for PICF141. Molecular analyses showed PICF141 as closest to P. lini ("Pseudomonas mandelii subgroup," within the "Pseudomonas fluorescens group"). Strains PIC25 and PIC105 were affiliated to the "Pseudomonas aeruginosa group," with P. indica as the closest relative. The isolates showed genotypic and phenotypic traits associated with plant growth promotion and/or biocontrol, including production of phytase, xylanase, catalase, cellulase, chitinase, glucanase activities, siderophores, and HCN. Pseudomonas indica PIC105 was identified for the first time as a biocontrol agent of fungi. Root colonization was useful for their application in biocontrol formulations.

## Bacillales

Several Bacillus species induce beneficial effects on plants through the production of antimicrobial compounds, as shown by species with growth-promotion and/or biocontrol activities such as the model strain Bacillus velezensis FZB42. Genes involved in synthesis of secondary metabolites, suppressing the growth of pathogens and inducing systemic resistance, were found together with VOC involved in biocontrol (Fan et al.).

Bacillus amyloliquefaciens strain JCK-12, isolated from soil, showed a significant antifungal activity vs. F. graminearum, the causal agent of Fusarium head blight. The fungus dramatically affects yields, with high risks to human and animal health due to its mycotoxins. JCK-12 fermentation broth and formulations reduced the disease incidence in wheat, likely related to production of cyclic lipopeptides including iturin A, fengycin, and surfactin. Iturin A inhibited F. graminearum spore germination, whereas the other lipopeptides showed, when mixed with iturin A, only a synergistic inhibitory effect on germination. Strain JCK-12 also affected production of trichothecenes. Moreover, it showed synergistic antifungal effects with synthetic fungicides in vitro, controlling the disease in greenhouse and field conditions (Kim et al.).

Lipopeptides production is a beneficial trait displayed by some biocontrol bacteria (Velho et al., 2011). Surfactin, bacillomycin, and fengycin, detected in cultures of B. methylotrophicus (syn. of B. velezensis) by mass spectrometry, inhibited in vitro growth of the necrotrophic plant pathogen B. cinerea. Electron microscopy observations confirmed this effect showing an alteration of the fungus morphology upon the interaction with lipopeptides, with degeneration of cell organelles and production of early stage resistance structures. Assays with tomatoes, grapes, and strawberries confirmed the lipopeptides efficacy against B. cinerea, with the induction of an antioxidant activity in fruit (Toral et al.).

Paenibacillus polymyxa (former B. polymyxa) strain HY96-2 is a plant-growth-promoting rhizobacterium and a biocontrol agent, commercialized in China as a microbial biopesticide. Comparison of complete genome sequence data with other P. polymyxa strains indicated the potential to antagonize plant diseases by biofilm formation, antibiotic production, and induced systemic resistance (Luo et al.).

## Streptomyces

Several Streptomyces spp. are effective biocontrol agents in different climatic conditions and agroecosystems. A S. globosus isolate, originating from healthy date palm rhizosphere in the United Arab Emirates, showed antagonism vs. Thielaviopsis punctulata, the causal agent of black scorch disease. Its activity was associated in vitro with production of diffusible antifungal metabolites that inhibited T. punctulata mycelial growth. Greenhouse and pathogenicity tests showed that the isolate suppressed black scorch disease and prevented its spread at a level comparable to that of a synthetic fungicide (Saeed et al.). In the same country, a selective in vitro screening showed two Streptomyces spp. and one Micromonospora sp. isolates with a strong inhibitory effect against mango dieback, caused by the fungus Lasiodiplodia theobromae. In particular, activity of S. samsunensis was related to antibiosis and production of cell-walldegrading enzymes (CWDE, i.e., chitinase) and siderophores, whereas S. cavourensis and M. tulbaghiae were associated with antibiotic and CWDE production, respectively. In greenhouse experiments the isolates showed high levels of disease protection in pre-inoculated mango seedlings, with a reduced number of defoliated leaves and lower counts of L. theobromae conidia (Kamil et al.).

Isolate S37 of S. anulatus from the rhizosphere of healthy Vitis vinifera promoted grapevine growth and induced resistance against B. cinerea, one of its most severe diseases. Interactions with S37, before and after the challenge with B. cinerea, induced local defense events including early responses such as oxidative burst, extracellular alkalinization, and activation of protein kinases, with expression of defense genes and phytoalexin accumulation. Moreover, Ca2+ also appeared to contribute upstream to the induced reactions. S37 primed grapevine cells for enhanced defense, with a decline in cell death rates. Grapevine cells also showed a distinct perception of beneficial and pathogenic microbes, as shown by desensitization assays based on alkalinization of extracellular pH. In fact, once the pH was increased by S. anulatus S37, the cells became refractory to further stimulation by B. cinerea (Vatsa-Portugal et al.).

## BACTERIAL DISEASES

Takishita et al. tested a Pseudomonas sp. strain against Clavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker. The strain activated a systemic resistance reaction in treated plants, as shown by marker genes such as PR1a and ACO. The observed reduction of C. michiganensis subsp. michiganensis cells was also linked to a better plant performance. The antagonist showed ability to solubilize inorganic phosphorus, and to produce siderophores, indole acetic acid, and hydrogen cyanide. These metabolic profiles, but hydrogen cyanide production, were also found in a strain of Pseudomonas aeruginosa active against Xanthomonas oryzae pv. oryzae, the causal agent of the bacterial leaf blight of Basmati rice, a severe disease in Pakistan. Yasmin et al. showed a significant pathogen suppression by the tested strain, likely mediated by secondary metabolites such as siderophores, rhamnolipids, and hydroxy-quinolines, underpinning an antibacterial activity. Moreover, the bacterium was capable of colonizing the rice rhizosphere, inducing the production of defense-related enzymes in treated plants, eventually increasing grain and straw yields by >50%.

## MANAGEMENT OF NEMATODE AND INSECT PESTS

Being one of the most severe crop limiting factors present in soil, the interaction of plant-parasitic nematodes with the rhizosphere microbiome has been the object of many studies. Several microorganisms both affect and are influenced by soil and rhizosphere food webs, of which nematodes represent a key component. Rhizosphere microorganisms may indeed counterbalance the reproduction of many nematodes, and in some cases they are determinant in soil suppressiveness (Westphal and Xing, 2011; Giné et al., 2016). However, cropping practices, and in particular monoculture, may dramatically shift this balance toward nematodes, due to their high reproductive rates in the presence of a suitable host. Elhady et al. investigated how the soil microbiome affects invasion and reproduction of plant-parasitic nematodes, studying the root knot nematode Meloidogyne incognita and the root lesion nematode Pratylenchus penetrans. They tested the effect of a transplanted rhizosphere microbiome from other crops (i.e., soybean, maize, or tomato), and the impact of a plant's own microbiome, in comparison to that from a fallow soil. The microbiome from maize or soybean significantly reduced root invasion by P. penetrans. Similarly, maize and tomato rhizosphere microbiomes affected the invasion of tomato roots by both P. penetrans and M. incognita, compared to soybean or bulk soil. Moreover, M. incognita was best suppressed on tomato by the own tomato rhizosphere microbiome. Data showed that the microbiome species composition, as selected by the previous cropping cycle, affect the subsequent development of a parasitic nematode population in soil.

In root-nematode interactions, the effect of soil bacteria may be deployed at multiple levels, depending on their metabolism and rhizosphere colonizing capability. Liu et al. studied the regulation of the soybean cyst nematode Heterodera glycines by an isolate of Klebsiella pneumoniae, a bacterium otherwise known for its clinical importance. One isolate, however, acts as plant growth promoter and biocontrol agent of sheath and seedling rice blights. The isolate tested showed an effect on nematode parasitism and density, when used as a seed coating or in field assays. Pot assays with split roots showed systemic resistance induced by K. pneumoniae in treated plants. Increased expression for some defense genes such as PR1, PR2, and PR5 (pathogenesis-related genes), as well as PDF1.2 (plant defensin), was observed. Moreover, the isolate improved the development of roots through the combined action of nitrogen fixation, phosphate solubilization, and siderophore production.

The links between plants and soil microorganism likely arise as the result of functional, coevolutive adaptations beneficial for the organisms involved. In this perspective it is useful to examine factors such as nutrients or substrates, present in the rhizosphere. Escudero et al. investigated the interaction of the hyphomycete Pochonia chlamydosporia with chitosan, a chitinderived product. The fungus has multiple behaviors in soil, as a specialized parasite of nematode eggs, a saprotroph, or a root endophyte (Manzanilla-López et al., 2013). Chitosan enhances P. chlamydosporia sporulation, production of extracellular enzymes and egg parasitism (Escudero et al., 2016). At low concentrations (up to 0.1 mg ml−1 ) it improved the fungal growth and did not affect chlamydospores viability and germination. Treatments increased tomato root and plant development in presence of both P. chlamydosporia and Meloidogyne javanica, but did not affect eggs parasitism, unless a highly suppressive (>70% prevalence in eggs) sterilized soil was used.

As many antagonists occur in soil at the same time, it is worth checking whether their combined application may yield synergic effects. Experimental data indicated, however, that this situation is difficult to observe in natural conditions. Imperiali et al. studied field application of beneficial rhizosphere organisms, inoculated alone or in combination at wheat seeding. Tested antagonists included Pseudomonas spp. arbuscular mycorrhizal fungi (AMF), and entomopathogenic nematodes (EPN). The beneficial organisms persisted in soil after their introduction, as confirmed by molecular identification and increased plant health and productivity. However, no difference was observed between combined and single applications. Best wheat survival was reported on plants exposed to a biotic stress such as infestation by the frit fly, Oscinella frit, an important pest of cereals, which was reduced in treatments with Pseudomonas and AMF. Inoculations with EPN displaced some other native EPN, but only on the short-term.

## SUPPRESSIVE SOILS

Soils showing presence of a pathogen with, however, a low disease insurgence on a susceptible crop, are known as suppressive. This trait is likely due to the beneficial, indigenous microorganisms (Schlatter et al., 2017). Suppressive soils may be found in extensive cropping systems or in areas with longterm cultivation. Durán et al. screened soils for suppressiveness against "take-all" disease caused by the fungus Gaeumannomyces graminis var. tritici, sampling extensive wheat fields managed by indigenous Mapuche communities in Southern Chile. In vitro growth inhibition tests allowed identification of putative soils suppressive for take-all disease. Suppressiveness was closely associated to the soil microbiome, being lost upon soil sterilization and then recovered by addition of a natural soil inoculum.

A review on Verticillium wilts, vascular diseases caused on annual crops and woody perennials by several species of the soil-borne fungus Verticillium, showed the potential of bacteria belonging to Bacillus and Pseudomonas and of non-pathogenic xylem-colonizing Verticillium and Fusarium isolates as biocontrol tools. Soils and composts suppressive to Verticillium wilt allowed isolation of biocontrol agents characterized by useful traits such as inhibition of primary inoculum germination, plant growth promotion, competition, and induced resistance. However, in vitro antibiosis and mycoparasitism did not correlate with in vivo or in planta activities. Most useful traits included activity against the pathogen microsclerotia, a significant xylem and/or cortex colonization with nutrients and/or space competition, the induction of resistance reactions, and growth promotion in treated plants. All these effects should be screened under field conditions for selection of biocontrol agents suitable for large scale production, stable formulation, and application (Deketelaere et al.).

However, the impact on soil microbial communities of external factors such as the farmers' technical decisions (i.e., selecting crop species with/without rotations) should also be evaluated. A long-term monoculture increased the diversity of F. oxysporum and had a negative effect on antagonistic Pseudomonas spp. as shown by a 3 years long cropping of the medicinal plant Radix pseudostellariae (Chen et al.).

## SOIL ENVIRONMENT

Several physical and climatic factors affect the microbiome diversity and the interactions of soil antagonists with plants, pests, and pathogens (Lareen et al., 2016). Mavrodi et al. characterized the establishment and activity of microbial communities in the rhizosphere of dryland wheat, and the microbiome response to irrigation. They also studied the population dynamics and activity of indigenous rhizobacteria producing antibiotics (phenazines), contributing to the natural suppression of wheat soilborne pathogens. Irrigation and soil moisture had a negative effect on plant colonization, density of bacteria and antibiotic levels. Although irrigation had a limited effect on the global microbiome diversity, differences were found for some groups such as the plant growth-promoting Bacteroidetes and Proteobacteria.

Atmospheric CO2 is a further physical factor related to climate change. Williams et al. studied its effect on strains of Pseudomonas simiae and P. putida in the rhizosphere of Arabidopsis. Differences in rhizosphere colonization were observed for the two bacteria, with an increase linked to CO2 levels (from 200 to 1,200 ppm), observed only for P. simiae in soil with relatively low C and N. Plant growth and systemic resistance to P. simiae varied in relation to the CO2 concentrations and soil type. Growth promotion and induced susceptibility were observed at sub-ambient CO2, whereas an opposite response was found at highest levels.

Stereoisomers of 2,3-butanediol, produced by some rootassociated bacteria, were reported as triggering immunity of pepper plants against Cucumber mosaic virus and Tobacco mosaic virus. In a field trial, treatments with an enantiomer (2R,3R-butanediol) and a meso compound (2R,3S-butanediol) significantly reduced the prevalence of naturally occurring viruses, compared with a further enantiomer (2S,3S-butanediol) and control. Moreover, 2R,3R-butanediol induced the expression of plant defense genes in the ethylene/jasmonate and salicylic acid pathways at levels similar to those of benzothiadiazole-treated control (Kong et al.).

## REFERENCES

## OUTLOOK AND FUTURE CHALLENGES

Soil microbial communities offer an unlimited potential to develop locally customized and innovative approaches, facing major threats for global food security (Sergaki et al.). Challenges mainly concern plant protection and soil fertility. Many microorganisms that mineralize insoluble phosphate hold potential to improve growth and yield of many crops, in a sustainable way. This technology appears ready for commercial exploitation in various regions of the world, but more detailed information is still needed to be considered as an effective replacement of conventional fertilizers (Alori et al.). Studies on induced resistance and chemical communication of roots with soil microorganisms open unlimited perspectives for development of sustainable management tools, as shown in the reviews by Mhlongo et al. and Liu et al. Due to the many links among below- and above-ground physiological processes, chemical communication has been only partially deciphered, thanks to advanced techniques such as mass spectrometric-based metabolomics. The complexity and stability of microbial communities also depend on the capacity of roots to screen and select rhizosphere/rhizoplane taxa, mostly dominated by Proteobacteria with favorable traits such as motility, plant cell-wall degradation and ROS scavenging, nutrients procurement, nitrogen fixation, and defense priming.

## AUTHOR CONTRIBUTIONS

AC wrote the manuscript. JM-B and CP revised the manuscript, helped structure and edit it, and approved its final version for publication.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Ciancio, Pieterse and Mercado-Blanco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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

#### Mout De Vrieze1,2, Fanny Germanier1† , Nicolas Vuille2† and Laure Weisskopf1 \*

1 Department of Biology, University of Fribourg, Fribourg, Switzerland, 2 Institute for Plant Production Sciences, Agroscope, Nyon, Switzerland

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.

Keywords: late blight, pseudomonads, Solanum tuberosum, consortium, biocontrol, rhizosphere, phyllosphere

## INTRODUCTION

Sustainable crop production faces the challenge of maintaining high yields to meet the food requirements of an increasing world population while limiting its own environmental impact. In potato production, the major yield-threatening disease is the oomycete Phytophthora infestans, causing late blight (Fry, 2008). In Europe, late blight's costs, resulting both from yield loss and

### Edited by:

Jesús Mercado-Blanco, Consejo Superior de Investigaciones Científicas (CSIC), Spain

#### Reviewed by:

David Ruano-Rosa, Instituto Tecnológico Agrario de Castilla y León, Spain Linda Thomashow, Agricultural Research Service (USDA), United States

\*Correspondence:

Laure Weisskopf<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Microbiology

Received: 02 July 2018 Accepted: 09 October 2018 Published: 29 October 2018

#### Citation:

De Vrieze M, Germanier F, Vuille N and Weisskopf L (2018) Combining Different Potato-Associated Pseudomonas Strains for Improved Biocontrol of Phytophthora infestans. Front. Microbiol. 9:2573. doi: 10.3389/fmicb.2018.02573

**12**

disease control, have been estimated to over 1 billion Euros per year (Haverkort et al., 2008). In conventional agricultural management, potato late blight, as many other diseases, is controlled by multiple applications of fungicides of varying modes of action during the growing season (Haverkort et al., 2008; Cooke et al., 2011). However, increasing awareness of the negative side effects of synthetic pesticides on environmental and human health has led to growing interest in organically produced food. In organic potato production, growers use copper-based products as alternative to synthetic fungicides to protect their crops from late blight, but since copper is not degradable, it accumulates in the soil and is toxic to the soil fauna (Du Plessis et al., 2005; Eijsackers et al., 2005). Therefore, there is a need for alternative measures to control late blight in an environmentally friendly manner. One such alternative is the use of natural enemies of the disease-causing agent, also termed "biological control" or "biocontrol." Microbial biocontrol agents such as the bacterium Bacillus thuringiensis or the entomopathogenic fungus Beauveria bassiana have been successfully applied to control insect pests (Arthurs and Dara, 2018), but only few examples (mostly based on bacteria of the genera Pseudomonas or Bacillus) exist where such biocontrol strategy was efficient enough against fungal pathogens to lead to product commercialization (reviewed in Velivelli et al., 2014). Beyond these few examples, controlling an aggressive pathogen such as P. infestans is a significant challenge, as evidenced by the number of biocompatible treatments that have been tested but did not, or only partially, meet the required level of reproducible efficacy (Dorn et al., 2007; Axel et al., 2012; Alaux et al., 2018).

One possible factor underlying this lack of success might be that P. infestans can infect its host plants by different means, e.g., through direct germination of sporangia or release of motile zoospores, both leading to host tissue penetration and mycelial development (Fry, 2008). An efficient biocontrol agent should therefore either induce plant resistance (Syed Ab Rahman et al., 2018) or, when acting directly on the pathogen, inhibit both types of infection routes. Alternatively, using different biocontrol agents each targeting one of the pathogen's modes of infection could improve biocontrol efficiency. Such approaches based on strain combinations rather than on single strains could not only bring functional complementary as exemplified above for sporangia vs. zoospore-mediated infection, but they could also be useful in providing functional redundancy. Indeed, a key element in the success of a biocontrol agent is the ability to colonize its host, which in turn is influenced by many factors including the resident microbiome (Sylla et al., 2013). Using strain consortia might therefore lead to enhanced protection robustness in the face of varying environmental and genetic (e.g., different crop varieties) conditions. To date, few studies have addressed this question and tested the impact of mixed strains rather than single ones on plant protection against diseases. Among these, Niu and co-workers could protect maize against Fusarium by applying a consortium of seven different bacterial species (Niu et al., 2017), while Hu and co-workers obtained significantly better protection of tomato against Ralstonia-induced wilt when using a mixture of 8 Pseudomonas strains than when applying each strain individually (Hu et al., 2016).

We have previously isolated and characterized the protective potential of single Pseudomonas strains isolated from the rhizosphere and phyllosphere of potato against P. infestans (Guyer et al., 2015; Hunziker et al., 2015). In the present study, we hypothesized that mixing different Pseudomonas strains, which differed in their phylogenetic identity, in their origin of isolation (rhizosphere vs. phyllosphere) and in their emission of volatile (De Vrieze et al., 2015) and non-volatile anti-Phytophthora metabolites could increase the disease-inhibiting potential of these strains. Since the discrepancy between in vitro activity and in planta protection is usually large (Dorn et al., 2007), and since we had recently established a high-throughput assay to screen for inhibition of late blight development on leaf material (Guyer et al., 2015), we started with a leaf diskbased screening rather than an in vitro screening procedure. To this end, all possible twofold and threefold combinations of nine selected Pseudomonas strains were tested for late blight inhibition on leaf disks of three potato cultivars of varying late blight susceptibility. This first experiment enabled to select five promising strains, which were further analyzed for better understanding of the mechanisms underlying the observed synergetic effects. This was done by characterizing their effect as single strains vs. in dual and triple combinations on the pathogen's development (both mycelial growth and zoospore release), as well as their growth behavior when inoculated alone or in dual and triple combinations, in order to identify putative synergetic or antagonistic effects.

## MATERIALS AND METHODS

## Bacterial Strains and Culture Media

Nine Pseudomonas strains were selected from a collection of strains isolated from the rhizosphere (R) and from shoots (S) of field grown potato plants, based on their in vitro activity against P. infestans (Guyer et al., 2015; Hunziker et al., 2015). The bacteria were routinely grown on Luria Bertani medium (LB), which was prepared by dissolving 20 g L−1 of Difco LB broth (Lennox, United States) in distilled water supplemented with 15 g L−1 of agar (Agar-agar, ERNE surface AG, Switzerland). For bacterial cell suspensions, cultures were prepared by suspending single colonies from 2-day-old plates in 0.9% NaCl and streaking the obtained suspensions on LB agar medium. After overnight incubation at 20◦C, bacterial cells were resuspended in 0.9% NaCl. For bacterial competition assays, rifampicinresistant derivative strains obtained as described in Guyer et al. (2015) were used. LB and Pseudomonas Isolation Agar (PIA), supplemented or not with rifampicin (50 µg mL−1 ) and nystatin (500,000 UL−1 ) were used for these experiments. PIA medium was prepared by dissolving 45 g L−1 of Pseudomonas Isolation Agar (Fluka) in distilled water, to which 20 mL L−1 of glycerol (Sigma–Aldrich) was added. Optical density (OD) measured at 570 nm was used to quantify and adjust bacterial density. The nine strains showed slightly different cell numbers/OD570 unit but these differences were within the same order of magnitude: most strains had between 1.4 × 108 and 2 × 108 cells per mL for an OD570 of 1, while slightly higher cell numbers were observed

for R32, S34 and S35 (between 4.5 × 108 and 5.5 × 108 cells per mL at OD570 of 1).

## Phytophthora infestans and Culture Media

Phytophthora infestans isolate Rec01 (originally isolated by H. Krebs, Agroscope) was used for all inhibition assays and grown on unclarified V8 (10%) medium for collection of sporangia and zoospores, or on Pea agar medium for mycelial growth assays. Unclarified V8 medium was prepared by diluting V8 juice (100 mL L−1 ) amended with CaCO3 (1 g L−1 ) and 15 g L−1 of agar according to Miller (1955). Pea agar medium was prepared by skimming 120 g of autoclaved frozen peas in water and adding 15 g of agar. The isolate was regularly inoculated on potato slices for host passage. Sporangia suspensions were prepared by scraping off the mycelium of 14-day-old plates and suspending it in demineralized water. After vigorous shaking, the suspension was filtered using cloth to discard the mycelium. Concentration of sporangia was determined using a Thoma chamber. Sporangia suspensions were maintained in the dark until use. To obtain zoospore suspensions, sporangia were subjected to a cold shock by adding ice-cold water to sporangia suspensions in Eppendorf tubes, which were subsequently incubated at 4◦C for 2 h and then left to rest at room temperature for 20 min to allow zoospore release.

## Effects of Single Strains vs. Strain Combinations on Disease Protection in a Leaf Disk Assay

The 3rd and 4th leaves of greenhouse grown potato plants of the cultivars Bintje, Lady Claire and Victoria were harvested 7 weeks after emergence. Using a cork borer, leaf disks (1.8 cm diameter) were cut and positioned abaxial face up on 1% water agar plates. Droplets of 10 µL of a mixture of bacterial and sporangial suspensions were pipetted in the center of each leaf disk, at final concentrations of 125,000 sporangia/mL for P. infestans and of OD570 = 0.9 for single bacterial strains (simple), OD570 = 0.45 for combinations of two strains (double), and OD570 = 0.3 for combinations of three strains (triple). For negative control plates, bacterial suspensions were replaced with a 0.9% NaCl solution. The plates were stored at 18◦C (the ideal growth temperature for P. infestans) and at high humidity in the dark. After seven days, the plates were photographed. Severity of infection was assessed through estimation of sporangiophore development using a macro-instruction in ImageJ as described previously (Guyer et al., 2015). Per experiment, each treatment was tested in five replicates consisting of five disks from five different plants in Lady Claire and Victoria, and in ten replicates consisting of ten leaf disks from ten different plants in Bintje (because we had more plants available for Bintje than for Lady Claire and Victoria). Each batch of five plants could be used to assess the efficiency of 27 different treatments by comparing infection severity in treated vs. untreated disks coming from the same plants. To enable comparison between different batches of plants, the infection severity quantified on untreated control disks was set to 100% and the infection severity of treated disks was expressed as percentage of the control (relative infection severity). Finally, treatment efficiency was calculated with the following formula: Treatment efficiency (%) = 100 – (relative infection severity of treatment). With this calculation, a treatment efficiency of 100% corresponds to no infection, an efficiency of 0% corresponds to the same infection as in untreated controls and a negative value indicates higher infection severity in treated disks compared to untreated controls.

## Effects of Strain Combinations on P. infestans Mycelial Growth

Bacterial strains and P. infestans were co-inoculated on Pea agar plates. Three drops of 10 µL of bacterial suspensions (prepared as described above) were pipetted on the medium equidistantly and 10 mm from the border of the plates. One 5 mm plug of a 14-day-old P. infestans culture was placed in the center. Negative control plates contained 10 µL of NaCl 0.9% instead of bacterial cell suspension. The plates were prepared in three replicates and were incubated in the dark for 6 days at 18◦C before being photographed. Mycelium growth was assessed by measuring the growth area of P. infestans using the ImageJ software. Relative mycelial growth was calculated by dividing the mycelial area quantified in treated plates by the mycelial area quantified in negative control plates (in the absence of bacteria). Treatment efficiency was then calculated as above (100 – relative mycelial growth).

## Effects of Strain Combinations on P. infestans Zoospore Release

Cold shock was applied to sporangia suspensions freshly mixed with bacterial suspensions. Thirty µL of the mixture were pipetted onto a 24-well plate (Costar), which contained one well per treatment. Per well, one picture was taken at 4 fold magnification using a Cytation5 plate reader (Biotek, United States) once the zoospores had settled and once more after shaking the plate to insure even distribution of the zoospores for counting. The experiment was repeated three times. An average of the three replicate experiments was calculated for each treatment. Relative zoospore release was calculated by dividing the number of zoospores released in treated samples by the average number of zoospores released in the untreated samples (negative controls non-exposed to bacteria). Treatment efficiency was calculated as above (100 – relative zoospore release).

## Growth/Survival of Bacteria as Single Strains vs. in Combinations With Other Strains

To find out whether the development of single bacterial strains was affected by the presence of other strains, the growth/survival of each bacterial strain was assessed alone and in combination. Rifampicin resistant and wild type bacteria were mixed in equal densities in NaCl (0.45%) and incubated at 18◦C without shaking, to mimic the conditions of the leaf disk experiment. We used 0.45% NaCl to have the same concentration as in leaf disk experiments, where bacterial cells suspended in 0.9% NaCl were mixed with equal volumes of P. infestans sporangia suspended in water. After 1 day and after 5 days, 10 µL of bacterial suspension were 10-fold serially diluted in NaCl (0.9%) and 15 µL of the diluted suspensions were plated onto differentially selective media to discriminate the two or three different strains, taking advantage of rifampicin resistance vs. sensitivity, of ability vs. inability to grow on PIA and of different colony morphology. Colony forming units (CFUs) were counted after two or three days depending on the medium.

## Statistical Analyses

fmicb-09-02573 October 25, 2018 Time: 15:1 # 4

All statistical analyses were performed using R software (Sasaki et al., 2005). When possible, one-way or two-way ANOVA was performed followed by Dunnett's test or Tukey's HSD test using agricolae and multcomp packages. If needed, boxcox transformation computed via the MASS package was used to meet normality and homogeneity of variances. For effects on zoospore release and mycelial growth data, Kruskal–Wallis test was used to discriminate between treatments.

## RESULTS

## Disease-Inhibiting Effects of Nine Pseudomonas Strains in Single, Dual and Triple Combinations

Nine Pseudomonas strains previously isolated from the rhizosphere (R) or phyllosphere (S) of field-grown potatoes and displaying various levels of Phytophthora-inhibiting activity in vitro were selected for this experiment. To determine whether these strains would confer higher protection when applied in combinations than when applied as single strains, we carried out a leaf disk infection experiment with 129 treatments (9 single strains, their 36 dual and 84 triple combination possibilities) using three potato cultivars differing in late blight sensitivity. We selected Bintje as highly sensitive, Lady Claire as sensitive and Victoria as moderately tolerant. This experiment revealed a strong cultivar effect, with higher overall protection efficacy in Bintje than in Lady Claire and Victoria (**Figure 1**). Among the nine treatments with single strains, only one strain (S35) significantly reduced disease progression in all three varieties, while 7 offered protection on some but not all varieties and one (R84) was inefficient in all varieties. Among the dual combinations, six (out of 36 possible combinations) provided protection on all three cultivars, i.e., R32/S34, R76/S49, R84/S35, R84/S49, S04/S49, and S19/S49. This latter dual combination offered best protection in terms of quantitative disease inhibition (**Figure 1**). Interestingly, four out of six of these efficient combinations contained the strain S49, while only one contained the strain S35, which showed consistent protection when applied as single strain. Among the 84 possible triple combinations, only seven were able to significantly reduce disease progression in Lady Claire, among which two were also efficient on the two other varieties. When considering less stringent conditions, e.g., triple combinations able to reduce disease progression in at least two of the three varieties, 16 combinations were found to be efficient, among which seven contained strain S35 and six contained strain S49, indicating putative synergistic effects of these two strains when applied in combinations with two additional other strains. In addition to the overrepresentation of S35 and S49 in the efficient combinations, we noticed that the duo R47/S35 combined with either S19, S34, or R76, yielded significant protection against P. infestans infection.

To gain a general view on the performance of the strains in single, dual and triple combinations, we calculated the percentage of efficient treatments, i.e., those significantly reducing disease symptoms, in the three varieties and for each strain in its respective modes of application (single or combinations) (**Figure 2A**). The relative efficiency of the strains in the different modes of application strongly depended on the variety, yet some strains showed consistent differences between the three application modes: R84 was consistently more efficient when applied with one or two other strains than when applied alone. The same was observed for S49 on Bintje and Lady Claire. In Bintje, the strains performed generally better in triple combinations than in dual combinations in terms of percentage of efficient treatments, while the opposite trend was observed in Lady Claire and Victoria (**Figure 2A**). Because the total bacterial cell density was not the same in single, dual and triple treatments (OD of 0.9 for single, 0.45 for dual and 0.3 for triple treatments), we wondered whether the low percentage of efficient treatments in triple combinations on Lady Claire and Victoria could be explained by these differences in inoculum density. We therefore tested the protective effect of selected single strains applied in the three different densities. This revealed that in Lady Claire and Victoria, cell density in the range of OD = 0.3 – 0.9 did generally not influence the extent of protection conferred by the strains (**Supplementary Figure S1**). However, in Bintje, a dose-dependent protective effect was observed, with applications at OD = 0.3 generally being less efficient than applications at OD = 0.9, although both concentrations were able to significantly reduce disease symptoms (with the exception of strains R32 and S19) (**Supplementary Figure S1**). Therefore, the high percentage of efficient treatments in triple combinations in Bintje that were observed despite overall lower cell density were likely due to synergetic effects between the strains, that were able to compensate the overall lower cell density.

To better understand the mechanisms underlying disease inhibition by strain combinations, we selected a subset of five strains to compare their effect as single strains and as double/triple combinations on two main developmental stages of P. infestans, mycelial growth and zoospore release. The selection of these five strains was carried out such as to maximize the chance to see synergetic effects and was based on the number of efficient treatments per strain (**Figure 2B**). We therefore selected R47, S19, S35 and S49 based on the results on Bintje, and included R32 for its efficient protection of Victoria (**Figure 2B**). R32 was remarkably efficient in protecting Victoria when applied in dual combinations, where it led to significant disease reduction when combined with six of the eight other strains (**Figure 1**). The last criterion for strain selection was to ensure some diversity, both in terms of phylogeny and in terms of origin of isolation (rhizosphere vs. phyllosphere). The main properties of these five selected strains are listed in **Table 1**.

## Mycelial Growth Inhibition by Five Selected Strains and Their Dual and Triple Combinations

When inoculated alone, three strains (R32, R47 and S49) were able to inhibit fully the mycelial growth of P. infestans, while the two others (S19 and S35) induced more moderate, but still significant growth inhibition. The dual combination of the two "weaker" strains did not result in stronger mycelial growth reduction, nor did the "stronger" strains lose their activity when mixed with other strong strains (**Figure 3**). Interestingly, when either S19 or S35 were mixed with R32, the same complete inhibition of mycelial growth was observed as when R32 was inoculated alone. However, this was not the case with either R47 or S49, whose effects were weakened when mixed with S19 or S35, especially in the S49/S19 and in the R47/S35 combinations, and to a lesser extent in the S35/S49 combination. Concerning the triple combinations, the beneficial impact of R32 was also clearly visible, since its addition conferred strong activity to any couple of strains, including the inactive S19/S35 but also the moderately active R47/S35 and S19/S49 (**Figure 3**). In contrast, adding the active R47 to the "inactive" couple S19/S35 did not improve mycelial inhibition efficiency. From this experiment, it appeared that (i) R32 was the best helper in dual and triple combinations, (ii) S49 only improved the efficiency of S19/S35 but had no positive effect on the other dual combinations, and (iii) R47 was not able to increase the efficiency of S19/S35 and had generally little positive influence in triple combinations (**Figure 3**). Here as well as in the leaf disk experiments, it should be noted that single strains were applied with an optical density of 0.9, against 0.45 for the double and 0.3 for the triple combinations. We therefore tested whether cell density changes at the start of the experiment would influence the extent of mycelial growth inhibition and this was not the case for any of the strains (**Supplementary Figure S2**).

## Zoospore Release Affected by Five Selected Strains and Their Dual and Triple Combinations

Preliminary experiments revealed that the total cell density of the strains in single or mixed applications influenced their effect on P. infestans zoospores (**Supplementary Figure S3**), therefore this assay was carried out with two different optical

densities, a high one (OD = 0.9) and a lower one (OD = 0.3). When bacteria were applied at an OD of 0.9, all treatments significantly and drastically inhibited zoospore release (**Figure 4**). Interestingly, the dual combination of S19/S49 caused lesser reduction in zoospore release than each of the strains applied individually, although it was still significant compared with the control. This decrease in activity was also observed in the more diluted applications (OD = 0.3) and was even more pronounced when S19 was mixed with R32, R47 or S35, where it led to complete loss of activity. Adding any other strain to the couples S19/R32, S19/R47 or S19/S35 could not restore the significant activity observed with the strains applied alone (**Figure 4**). In contrast to the observed antagonistic effects between S19 and other strains, some synergetic effects could also be seen in the experiment carried out with lower cell density: mixing S49 with R32 resulted in almost total inhibition of zoospore release, while the single strains still allowed ca. 20% of the sporangia to release the zoospores. Likewise, mixing S49 with S35 resulted in more consistent (less variable) inhibition of zoospore release than either of the strains applied alone. Remarkably, all combinations of strains containing this couple (S35/S49)

significantly reduced zoospore release, which was not the case for any other couple. When comparing the total number of efficient combinatory treatments for each strain (out of 10 possible dual and triple combinations), we observed that S49 scored best (7/10), followed by S35 and R32 (6/10), while combinations containing R47 (4/10) and especially S19 (2/10) were much less efficient. This suggests that in the experimental setup used to assess zoospore release, S49, S35 and R32 had a beneficial effect on other strains present in the respective mixtures, while R47 and especially S19 had deleterious effects on the same strains (**Figure 4**).

## Survival and Growth of Five Selected Strains Alone and in Their Dual and Triple Combinations

Some of the results described above hinted at possible direct stimulating or inhibiting effects of strains on each other. To assess whether co-incubation in conditions similar to those applied in the leaf disk assay would lead to preferential survival/growth of specific strains, we incubated the five selected strains alone,

Phylogenetic analysis was based on (De Vrieze et al., 2015; Hunziker et al., 2015). HCN and Phenazines are listed as the two major known determinants of anti-Phytophthora activity (Hunziker et al., 2015; Morrison et al., 2016).

FIGURE 3 | Relative mycelial growth of P. infestans when exposed to single, double and triple combinations of five Pseudomonas strains in a dual culture Petri dish assay. Untreated controls represent P. infestans grown without bacteria. Relative mycelial growth was calculated by dividing the mycelial area obtained in the respective treatments with that obtained in the untreated controls (not exposed to bacteria). Results are means of three replicates from the same experiment. They are expressed as treatment efficiency and calculated as above (100 – relative mycelial growth). Letters indicate significant differences between treatments according to Kruskal–Wallis test (p < 0.05, n = 3).

as well as in dual and triple combinations for five days in physiological solution and quantified their relative abundance after one day and at the end of the experiment. For this experiment, all treatments had the same global cell density at the start of the experiment (as estimated by optical density), meaning that each individual strain started with half the inoculum vs. 1/3 of the inoculum in dual vs. triple combinations compared with the treatment where it was inoculated alone. Despite these differences, the total CFU counts (taking all strains together) at the end of the experiment were much higher for dual and triple combinations compared to single inoculations (**Figure 5**), indicating that (i) strains were able to compensate the lower

initial cell density over the 5 days of growth, and (ii) strains generally did not grow at the expense of each other, although they were incubated in saline solution only (no nutrient supply). When inoculated alone, four of the five strains developed to a density of roughly a million CFU/mL after 5 days, while their abundance was slightly lower after 1 day, indicating mild growth from day 1 to day 5 even in these nutrient poor conditions. S19 was already less abundant than the others after 1 day, and this strain hardly grew or even decreased in abundance depending on the combinations during the four following days (**Figure 5** and **Table 2**). Overall, being incubated with different partners did not affect all strains in the same way (**Table 2**): S35 and, to a lesser extent, R32, were inhibited in their growth in many of the dual and triple combinations compared to when they were inoculated alone, while R47 grew less well mainly in triple combinations but was not affected by dual combinations. S19 was only growing less well in combinations than alone in three out of 10 possible combinations and S49 was only affected by the presence of R47 in dual combination, but otherwise grew as well with any partner as alone (**Table 2** and **Figure 5**). A striking fact was observed in the case of S35: beyond its general decrease in abundance when mixed with other strains, it appeared to be completely outcompeted or even killed when incubated with either of the two rhizosphere strains R32 and R47. However, these latter strains did not seem to profit from the presence of S35, since their abundance was not significantly higher in the presence of S35 than in its absence (**Figure 5** and **Table 2**). This inhibition of S35 in presence of either R32 or R47 was rescued when any additional strain was present, since S35 grew normally

in all tripartite combinations tested, even in the combination with R32 and R47.

## DISCUSSION

Efficient control of late blight by bacterial biocontrol agents has been observed in few cases in greenhouse or even field experiments (Puopolo et al., 2014; Caulier et al., 2018), but most of the studies reported lack of reproducibility in protection against this disease (reviewed in Dorn et al., 2007; Axel et al., 2012). Indeed, in contrast to a synthetic molecule acting directly on a specific target of the pathogen, biocontrol agents that are applied, e.g., on leaves need (i) to efficiently compete with the native microbiota to colonize this environment, and (ii) to survive there despite exposure to UV and to rapidly changing temperature and humidity. Once established, they can produce bioactive molecules that either trigger the host plant's immune defense or that directly inhibit the pathogen's development. Phytophthora infestans, as many other plant pathogens, undergoes different developmental stages during the infection season, such as producing/releasing spores (sporangia and zoospores) or growing mycelium to colonize the host tissues (Fry, 2008). Ideally, control measures should target as many of these stages as possible to maximize efficiency.

One possible way to increase the chances for biocontrol agents to overcome the above-mentioned hurdles consists in using mixtures of strains rather than single agents, to increase both functional polyvalence (targeting different stages of the pathogen

life cycle) and redundancy (maximizing the chances of successful host plant colonization in various environmental conditions). This "polymicrobial" approach has drawn considerable attention in recent years, although most studies have so-far focused on mixing well-known, commercially available microbial agents such as Trichoderma and Bacillus/Pseudomonas or mycorrhizal fungi and nitrogen-fixing bacteria (Xu et al., 2011; Reddy and Saravanan, 2013; Sarma et al., 2015; Parnell et al., 2016). Few

Treatments are ordered from highest (top) to lowest (bottom) abundance. For each strain (column), different letters indicate statistically different values of the treatments according to Kruskal–Wallis test, p < 0.05, n = 3. Bold font indicates significantly lower abundance of the combined treatments compared with the control (single strain).

studies compared the effect of such strain combinations with that of the respective strains applied alone, and they came to divergent conclusions: Pertot et al. (2017) performed a 4-year field study on biological control of Botrytis cinerea in grapevine using a combination of two fungi (Trichoderma, Aureobasidium) and a Bacillus. They observed good efficacy for each of the antagonist but no additive value of combining the three (Pertot et al., 2017). Using five commercially available biocontrol agents (two based on Bacillus, one on Streptomyces and two on Trichoderma strains) against Phytophthora ramorum in a detached leaf assay, Elliott et al. (2009) observed lower efficacy of the mixture compared to some of its individual components, suggesting antagonistic effects between the different strains composing the mixture (Xu et al., 2011). The performance of strain combinations compared to individual strains might also depend on the targeted disease, as observed in rice for a dual treatment of Trichoderma and Pseudomonas strains, which was more effective than its single constituents against blast (caused by a fungus) but not against blight (caused by a bacterium) (Jambhulkar et al., 2018). In contrast, protection against Ralstonia-induced wilt in tomato was much higher when a mixture of eight Pseudomonas strains was applied than when the strains were applied individually (Hu et al., 2016).

Most of the above-mentioned studies used strains available as commercial products or in strain collections; however, these strains might not be adapted to the plant host and its pathogens, depending on their origin of isolation. Moreover, most screening efforts leading to the discovery (and putative registration) of antagonist strains have been done in in vitro experiments, which does not necessarily reflect the true antagonistic potential in planta or even in field conditions. In the present study, we investigated whether protective effects of Pseudomonas strains would be higher when applied in combinations than as single strains. Using nine potato-associated Pseudomonas strains, we performed a leaf disk infection assay with all 129 possible dual and triple combinations to circumvent the bias of the in vitro selection procedure. We performed this leaf disk screening on three different potato cultivars since we expected that the strain performance would vary according to the host plant genotype and sensitivity to late blight. As expected, a strong cultivar effect was observed, but surprisingly, best overall protection occurred on Bintje, which is most sensitive to late blight, while the two other cultivars were less efficiently protected by the strains (**Figure 2B**). This, however, might be at least partially because the screening on Bintje was carried out on ten leaf disks, while only five leaf disks per treatment were analyzed for the other two cultivars (see Material and Methods for the underlying reason). Only one strain, P. fluorescens S35, conferred significant protection on all three cultivars when applied alone, but this strain was less represented among efficient dual combinations than, e.g., P. fluorescens S49. This might be due to inability of S35 to compete with other Pseudomonas strains, as evidenced by the fact that in most combinations tested, S35 grew less well than when incubated alone (**Table 2**). In contrast, S49 could grow to the same level when co-incubated with any other strain we tested, except with R47, where it was slightly inhibited in its growth. Interestingly, when S35 was co-incubated with either R32 or R47, it could not be recovered after five days, suggesting strong inhibition or even killing of S35 by R32 and R47. This observation could explain the loss of activity of S35 when mixed with R47 on Lady Claire and Victoria, on which R47 was not active on its own, while R47/S35 was still active on Bintje, where R47 was active on its own (**Figure 1**). Likewise, S49, which was offering significant protection on Victoria when applied alone, lost its activity when mixed with R47, while it kept it when mixed with other strains that did not interfere with its growth. These results indicate that the mutual influence of strains on each other, when incubated in very low nutrient conditions, might be a useful parameter to investigate when designing microbial consortia for protection against diseases.

The screening of the 129 different treatments did not lead to an overall "champion" combination, but it highlighted the consistent protective activity of some strains, either when applied alone (S35), or in dual combinations (S19/S49). This latter combination was particularly interesting since it was efficient on all three cultivars, but when applied alone, neither strain was efficient on Bintje, only S19 was efficient on Lady Claire and only S49 was efficient on Victoria, thereby suggesting a synergetic effect between the two strains. Likewise, the dual combination S35/S49 was efficient on Bintje although only S35 was efficient on this cultivar when applied alone (**Figures 1**, **6A**). We wondered whether such synergetic effects could be due to differential modes of action of the different strains, e.g., inhibiting specifically the mycelial or spore stage of the pathogen, and tested these three potato phyllosphere isolates, together with two rhizosphere isolates previously shown to display strong anti-Phytophthora activity in vitro (Guyer et al., 2015; Hunziker et al., 2015).

We observed that the dual combination of S19/S49, which was particularly efficient on leaf disks, was only moderately inhibiting the mycelial growth of P. infestans in the in vitro assay, much less than when S49 was applied alone. However, all triple combinations containing S19/S49 (including that with the otherwise moderately active S35) were highly efficient in inhibiting mycelial growth, in contrast to those containing R47/S35 (**Figure 3**). In the natural leaf infection cycle, as well as in our leaf disk assays, the infection starts with a sporangium that, depending on temperature, can either directly germinate or release motile zoospores (Fry, 2008). Originally we aimed at investigating the effects of the five selected strains on these two processes but due to unknown reasons and despite repeated trials in different conditions, the harvested P. infestans sporangia did not germinate (even in the control) as they did previously in our hands, but consistently released zoospores. We therefore focused on analyzing how the five selected strains would affect this important route of infection in single, dual and triple combinations. Interestingly, the strain inhibiting this stage of P. infestans development in the strongest and most consistent way was S19, while S35 was the least active one (**Figure 4**). However, mixing S19 with any other strain but S49 led to loss of the activity. Among triple combinations, all those containing both S35 and S49 led to significant inhibition of zoospore release, suggesting good tolerance of these strains toward additional members of the tripartite consortium.

In summary, our screening of 129 different treatments of single, dual and triple strain combinations against P. infestans on three potato cultivars led to the observation that despite strong cultivar specificity, some strains showed strong and consistent protective effects, either when applied alone (S35) or in combination (S19/S49). The effects of these three phyllosphere strains on disease development, mycelial growth and zoospore release are shown through representative picture of the respective assays in **Figure 6**. When investigating the effect of these strains on each other's growth, we observed that S35 was less able to compete with other strains than S49 or even S19 (**Figure 5** and **Table 2**), possible explaining its better leaf disk performance when applied alone than in combination. The successful combination of S19 and S49 could be explained by their different mode of action: while S49 had much stronger inhibiting effect on mycelial growth than S19, S19 was a very efficient inhibitor of zoospore release. Despite their difference, S19 and S49 were able to maintain sufficient population densities when grown together (**Figure 5**), which is a prerequisite for synergetic effects.

In previous studies, we had considered S19, S35 and even S49 as among the lesser active strains, because our activity screening was performed mostly on in vitro tests assessing mycelial growth inhibition in dual assays (Guyer et al., 2015; Hunziker et al., 2015). Interestingly, when screening for protection using leaf disks rather than in vitro assays, these three phyllosphere strains turned out to be the most promising ones, which might be due to a particular ability to survive on leaf tissues or to cope with plant defenses. In dual and triple combinations, mixing of either S19/S49 (leaf disks) or S35/S49 (zoospore release assay) proved efficient in inhibiting P. infestans development. This good compatibility of strains sharing the same – phyllosphere – origin was not observed when mixing strains from the phyllosphere with strains from the rhizosphere, which might indicate that these strains have different requirements with respect to environmental conditions. In addition to leaf blight, P. infestans also causes tuber blight and it would be interesting to see whether rhizosphere isolates would prove more efficient than phyllosphere isolates for this particular form of the disease. In contrast to foliar blight, tuber blight was shown in an earlier study to be efficiently controlled by a mixture of four strains, among which three were fluorescent pseudomonads (Slininger et al., 2007). These strains were originally isolated from suppressive soils supplemented with tuber slices (Schisler and Slininger, 1994) and their protection efficacy was much higher in the mixture than with either of the strains applied alone, which highlights the potential of such host plant- or even host-tissue derived consortia to fight oomycete diseases.

Overall, our study clearly shows the potential added value of combining different, but compatible strains, with the example of a dual combination that led to stronger and more consistent protection than that obtained with the single strains. This study also highlights the complexity of interactions taking place even in such limited tripartite consortia. When increasing the number of partners, much higher complexity shall be expected, opening a wide range of fascinating questions related to the role of each strain in the consortium and the broader community (Lindemann et al., 2016), as recently exemplified by Niu and coworkers, with the identification of one "keystone" species in a 8-member consortium (Niu et al., 2017). Beyond the traditional way of systematically isolating strains to test them later in single or combined applications, future endeavors might rely on the plant's ability to specifically recruit beneficial microbes when facing a particular pathogen attack, as recently demonstrated in the model plant Arabidopsis thaliana (Berendsen et al., 2018). Such recruited microbes might then be assembled in synthetic communities and investigated for protective potential against the original pathogen, as well as for other desired features to be conferred to the plant. This new and booming field of microbiome management is likely to provide innovative alternatives to our current ways of protecting plants against diseases (Herrera Paredes et al., 2018; Syed Ab Rahman et al., 2018), which would ideally be combined with more traditional strategies such as adapted crop management or selection of resistant varieties to achieve more durable and sustainable crop protection.

## AUTHOR CONTRIBUTIONS

LW and MDV designed the research. NV, FG, and MDV performed the experiments. MDV analyzed the data. LW and MDV wrote the manuscript with help from FG and NV.

## FUNDING

The support of the Swiss National Science Foundation (Grants 149271, 177093, and 179310 to LW) is gratefully acknowledged. MDV received financial support from the Agroscope Research Program MicBioDiv.

## ACKNOWLEDGMENTS

The authors are grateful to Dr. Floriane L'Haridon, Dr. Brice Dupuis, and Dr. Katia Gindro for access and management of laboratories, and to Dr. Mónica Zufferey and Fanny Louviot for technical help.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018. 02573/full#supplementary-material

FIGURE S1 | Phytophthora infestans relative infection severity in leaf disks of three potato cultivars treated with five Pseudomonas applied as single strains at low, medium and high cell density (OD570 = 0.3, OD570 = 0.45, OD570 = 0.9). Results are expressed as treatment efficiency (100 – relative infection severity in each treatment compared with the untreated leaf disks), with means and standard errors of 15 replicates for Bintje, Lady Claire and Victoria. Within each variety, different letters indicate significant differences between the treatments according to Tukey's HSD test (P < 0.05, n = 15).

FIGURE S2 | Relative mycelial growth of P. infestans when exposed to five Pseudomonas applied as single strains at three different bacterial cell densities in a dual culture Petri dish assay. Untreated controls represent P. infestans grown without bacteria (see Materials and Methods for details). Relative mycelial growth was calculated by dividing the mycelial area obtained in the respective treatments with that obtained in the untreated controls (not exposed to bacteria). Results are means of four replicates from the same experiment. They are expressed as treatment efficiency and calculated as above (100 – relative mycelial growth). Letters indicate significant differences between treatments according to Kruskal–Wallis test (p < 0.05, n = 4).

## REFERENCES

FIGURE S3 | Relative zoospore release from P. infestans sporangia exposed to five Pseudomonas strains applied at three different cell densities. Sporangia suspensions pre-mixed with saline instead of bacterial cell solution were used as untreated controls. Released zoospores were counted and the relative release rate was calculated by dividing values obtained for treatments by those obtained for the untreated controls (see Materials and Methods for details). Results are means of three experiments with one sample per treatment. They are expressed as treatment efficiency and calculated as above (100 – relative zoospore release). Letters indicate significant differences between treatments according to Kruskal–Wallis test (p < 0.05, n = 3).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 De Vrieze, Germanier, Vuille and Weisskopf. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Versatile Antagonistic Activities of Soil-Borne Bacillus spp. and Pseudomonas spp. against Phytophthora infestans and Other Potato Pathogens

Simon Caulier 1,2†, Annika Gillis 2†, Gil Colau1 , Florent Licciardi 2 , Maxime Liépin1 , Nicolas Desoignies 1 , Pauline Modrie2 , Anne Legrève1 , Jacques Mahillon2 \* and Claude Bragard1 \*

1 Phytopathology-Applied Microbiology, Earth and Life Institute, Université Catholique de Louvain, Louvain-la-Neuve, Belgium, 2 Laboratory of Food and Environmental Microbiology, Earth and Life Institute, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

#### Edited by:

Aurelio Ciancio, Consiglio Nazionale Delle Ricerche (CNR), Italy

Reviewed by:

Bhim Pratap Singh, Mizoram University, India Michelina Ruocco, Istituto per la Protezione Sostenibile delle Piante (CNR), Italy

#### \*Correspondence:

Jacques Mahillon<EMAIL_ADDRESS>Claude Bragard<EMAIL_ADDRESS>
† These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Microbiology

Received: 21 August 2017 Accepted: 23 January 2018 Published: 13 February 2018

#### Citation:

Caulier S, Gillis A, Colau G, Licciardi F, Liépin M, Desoignies N, Modrie P, Legrève A, Mahillon J and Bragard C (2018) Versatile Antagonistic Activities of Soil-Borne Bacillus spp. and Pseudomonas spp. against Phytophthora infestans and Other Potato Pathogens. Front. Microbiol. 9:143. doi: 10.3389/fmicb.2018.00143 The world potato is facing major economic losses due to disease pressure and environmental concerns regarding pesticides use. This work aims at addressing these two issues by isolating indigenous bacteria that can be integrated into pest management strategies. More than 2,800 strains of Bacillus-like and Pseudomonas-like were isolated from several soils and substrates associated with potato agro-systems in Belgium. Screenings for antagonistic activities against the potato pathogens Alternaria solani, Fusarium solani (BCCM-MUCL 5492), Pectobacterium carotovorum (ATCC 15713), Phytophthora infestans (CRA-W10022) and Rhizoctonia solani (BCCM-MUCL 51929) were performed, allowing the selection of 52 Bacillus spp. and eight Pseudomonas spp. displaying growth inhibition of at least 50% under in vitro conditions, particularly against P. infestans. All 60 bacterial isolates were identified based on 16S rRNA gene sequencing and further characterized for the production of potential bio-active secondary metabolites. The antagonistic activities displayed by the selected strains indicated that versatile metabolites can be produced by the strains. For instance, the detection of genes involved bacilysin biosynthesis was correlated with the strong antagonism of Bacillus pumilus strains toward P. infestans, whereas the production of both bio-surfactants and siderophores might explain the high antagonistic activities against late blight. Greenhouse assays with potato plants were performed with the most effective strains (seven Bacillus spp. and four Pseudomonas spp.) in order to evaluate their in vivo antagonistic effect against P. infestans. Based on these results, four strains (Bacillus amyloliquefaciens 17A-B3, Bacillus subtilis 30B-B6, Pseudomonas brenneri 43R-P1 and Pseudomonas protegens 44R-P8) were retained for further evaluation of their protection index against P. infestans in a pilot field trial. Interestingly, B. subtilis 30B-B6 was shown to significantly decrease late blight severity throughout the crop season. Overall, this study showed that antagonistic indigenous soil bacteria can offer an alternative to the indiscriminate use of pesticide in potato agro-systems.

Keywords: Bacillus spp., bacilysin, bio-control, lipopeptides, potato diseases, Phytophthora infestans, Pseudomonas spp., siderophores

## INTRODUCTION

The increasing production of potato (Solanum tuberosum L.) is still facing important losses due to bacteria, fungi and fungus-like microorganisms, insects and viruses (Locke, 2002). Among these pathogens, the oomycete Phytophthora infestans (Mont.) de Bary, is responsible for causing late blight disease. The management of potato diseases, particularly late blight, is based on a massive use of chemical pesticides (i.e., fungicides such as mancozeb), causing tremendous costs, both direct and environmental (Cooke et al., 2011). The search for an environmental-friendlier pest management approach has led to study microbial agents with antagonistic capacities. Bacteria from both Bacillus and Pseudomonas genera are known to be appropriate candidates to be used in a bio-control approach due to their predominance in various environments, resilience and survival ability, but also for the number of bio-active molecules they are potentaially able to produce (Kloepper et al., 2004; Haas and Defago, 2005; Raaijmakers et al., 2010). Most of these bio-active compounds are secondary metabolites that exhibit direct and/or indirect antagonistic effects mediated through mechanisms such as antibiosis, competition, stimulation of plant growth and/or defenses (Emmert and Handelsman, 1999; Ongena and Jacques, 2008; Mavrodi et al., 2010).

Bacteria belonging to the genus Bacillus, particularly Bacillus thuringiensis strains with insecticidal properties, have been used in bio-control strategies since the mid-1930s (Bravo et al., 2011). Since then, the interest in Bacillus spp. has grown and numerous remarkable agricultural applications have been found. Some of them are currently exploited by the phytopharmaceutical industry which is commercializing some Bacillus-derived products: EcoGuard R by Novozymes (Gladsaxe, Hovedstaden, Denmark), Serenade R by Bayer (Leverkusen, North Rhine-Westphalia, Germany) or Kodiak R by Gustafson (Plano, TX, USA) as plant growth promoters and/or antagonists of phytopathogens with variable efficacy (Brannen and Kenney, 1997; Jacobsen et al., 2004; Kong et al., 2010; Lahlali et al., 2012). These claimed activities mostly rely on the enormous diversity of secondary metabolites produced by these bacteria, which can be divided into ribosomally synthesized peptides (e.g., bacteriocins), non-ribosomally synthesized peptides (e.g., lipopeptides, siderophores), polyketides (macrolides, polyenes) and volatile in-/organic compounds (hereinafter referred to as VIC and VOC). These compounds produced by Bacillus spp. are accompanied by various modes of action. Lipopeptides and VOCs, for instance, have direct antifungal activities as it is the case for the lipopeptide iturin A on Rhizoctonia solani (Yu et al., 2002) or volatiles pyrazine (2,5-dimethyl), benzothiazole and phenol-(4-chloro-3-methyl) against Alternaria solani and Botrytis cinerea on tomato plants (Gao et al., 2017). But other VOCs (2,3-butanediol, methyl jasmonate or methyl salicylate), as well as other lipopeptides (surfactin, fengycin) are also able to induce systemic resistance in plants through an ethylenedependent pathway (Ryu et al., 2004; Ongena et al., 2007; Jourdan et al., 2009). Besides these activities, plant growth promotion was also reported using volatiles (2,3-butanediol, acetoin) on Arabidopsis thaliana (Ryu et al., 2003). Other compounds produced by Bacillus spp. like phytohormones (cytokinin, auxin) (Hussain and Hasnain, 2009; Lim and Kim, 2009) or siderophores (Yu et al., 2011) showed similar growth promoting effects on cucumber or red-pepper. In this last study, for example, the catecholic siderophore bacillibactin was proved to be involved in reducing the incidence of Fusarium wilt on pepper through induced systemic resistance, in addition to a direct bio-control effect.

Pseudomonas spp. are also known to produce a broad range of bio-active metabolites including lipopeptides, siderophores, polyketides, and volatile compounds with interesting activities against phytopathogens. It has been shown that these metabolites enable pseudomonads to directly compete with plant pathogens, promote plant growth or induce systemic plant resistance. The best example among these Pseudomonas spp. metabolites is the 2,4-diacetylphloroglucinol (DAPG). The antifungal activity of this polyketide has been extensively reviewed on dampingoff, take-all or root rot diseases in various crops (Keel et al., 1992; Cronin et al., 1997; Huang et al., 2004; Ahmadzadeh and Sharifi Tehrani, 2009). Moreover, a clear link has been established between the disease-suppressiveness of some soils and the amount of DAPG producers (Weller et al., 2002) that include a large set of pseudomonads. But besides its direct antagonistic activity, DAPG is also involved in inducing systemic resistance through jasmonic acid and ethylene-dependent pathways (Iavicoli et al., 2003), highlighting its versatile mode of action. Lipopeptides are another example of the multiple antagonistic mechanisms used by pseudomonads. Some are known to lyse zoospores of P. infestans (de Souza et al., 2003; de Bruijn et al., 2007) and to induce resistance in tomato plants infected by this pathogen (Bakker et al., 2007; Tran et al., 2007). Pseudomonads siderophores (e.g. pseudobactin) are also able to induce systemic resistance in plants (Leeman et al., 1996; Meziane et al., 2005) and participate in direct competition with soil-borne plant pathogens for bio-available iron (Loper and Buyer, 1991). Siderophores produced by Pseudomonas putida or Pseudomonas fluorescens, for instance, are also able to promote plant growth and improve nutritional quality of major crops, such as rice, by enhancing the grain iron content (Sharma et al., 2013). Besides these soluble compounds, some VOCs produced by Pseudomonas spp. are also able to directly inhibit the development of P. infestans (de Vrieze et al., 2015) or R. solani (Kai et al., 2007).

In this study, we show the wealth of naturally occurring indigenous Bacillus spp. and Pseudomonas spp. strains associated with potato agro-systems that have the potential to be used at implementing biological control of potato diseases. For this purpose, a large collection of strains was isolated. Those displaying the highest in vitro antagonistic activity toward P. infestans were retained. An extensive characterization of these strains, including the detection of the genetic determinants of known antifungal metabolites, the in vitro assessment of lytic enzymes, the production of lipopeptides and siderophores and the in vitro activities against other potato pathogens, was assessed. The most interesting strains were tested on potato plants under greenhouse conditions. The implemented methodology allowed the selection of Bacillus spp. and Pseudomonas spp. bacteria with versatile mode of actions and very effective against P. infestans. Because in vitro antagonistic activities have been shown to be rarely reproducible in field conditions (Ravensberg, 2011; Glare et al., 2012), a pilot field trial was also conducted with the most promising strains selected in this study. The results pointed out the attractive potential of B. subtilis 30B-B6 to be used as a bio-control agent against late blight in field conditions.

## MATERIALS AND METHODS

## Sampling Procedure and Bacteria Isolation

The sampling campaign was performed during the potato crop season from March to September 2012, mainly in loamy, shalesandstone and sandy soils in Belgium. A total of 48 field soils from different locations and five soils from gardens were sampled from two soil horizons (A: 0–12 cm; B: 13–24 cm). Twentyfive other samples were also collected as follows: four from compost, eight from manure and 13 from potato plants divided in three parts (leaf, root and tuber) (Table S1). Plant samples were directly homogenized in 30 mL sterile Ringer's solution. Soil, compost and manure samples (25 g) were placed in blender bags with filters (pore size 330µm; VWR) and mixed with 30 mL of sterile Ringer's solution in a Stomacher R machine (Seward) for 10 s. Samples were then collected through the blender bag filter and decanted overnight at 4◦C. Each sample was then divided in aliquots supplemented with glycerol (3:1 vol/vol) and cryopreserved at −80◦C for further bacterial selective isolation. Bacillus-like strains were isolated as follows: 1 mL of each sample was subjected to heat treatment (75◦C, 20 min), 10-fold diluted with sterile Ringer's solution, plated on Lysogeny Broth (LB; also known as Luria-Bertani) (NaCl, 5 g L−1 ; yeast extract, 5 g L −1 ; tryptone, 10 g L−1 ) solidified with 1.4% (wt/vol) agar and incubated at 30◦C for 24 h. Pseudomonas-like selective isolation relied on growth (30◦C, 24 h) on Cephalothin-Sodium Fusidate-Cetrimide (CFC) agar medium (Biokar Diagnostics R ), combined with oxidase test for presumptive Pseudomonas strains. After selective isolation, nine strains of each genus were retained for each sample. The final collection of more than 2,800 isolates was then conserved in LB-glycerol (70:30 vol/vol) at −80◦C.

## Microorganisms, Culture Conditions and Potato Plant Varieties Used in this Study

Five potato pathogens were used in this work (**Table 1**): A. solani and P. infestans were cultivated on V8-agar (CaCO3, 1 g L−1 ; agar, 15 g L−1 ; vegetable juice V8, 200 mL L−1 ) in the dark, at room temperature and 18◦C, respectively. F. solani and R. solani were cultivated on potato dextrose-agar (PDA, Oxoid) in the dark at 22◦ and 20◦C, respectively. Pectobacterium carotovorum was grown overnight at 28◦C on LB-agar. Bacteria belonging to the Bacillus and Pseudomonas genera were routinely grown on LB-agar (Bacillus spp. and Pseudomonas spp.) or PDA (Pseudomonas spp.). Certified tubers of S. tuberosum "Bintje" and "Challenger" varieties from CONDIPLANT R (Gembloux, Belgium) were used in greenhouse experiments and field trials. For greenhouse experiments, tubers were grown in pots containing twice-sterilized compost, daily watered and, TABLE 1 | Potato pathogens used in this study.

aATCC, American Type Culture Collection, Manassas, VA, United States.

bCRA-W, Walloon Agronomical Research Center, Libramont, Belgium.

cBBCM-MUCL, Microbial Collection of the Université catholique de Louvain, Louvain-la-Neuve, Belgium.

maintained under controlled conditions with a photoperiod of 16:8 h at 25/15◦C (day/night). For field trials, 10 acres of soil were supplemented with a mix of Nitrogen/Phosphorus/Potassium (NPK: 12-9-22, 1000 Kg ha−1 ) and NH4 (27%, 300 kg ha−1 ). Prior to tuber plantation, an herbicide treatment was performed (2 Kg ha−1 Artist R , 1 L ha− 1 Linuron R , 2 L ha−1 Challenge R ; water 250 L ha−1 ). No chemical weed control was performed before harvest and weather conditions were daily registered throughout the growing season.

## Direct Antagonism Assays

Direct antagonism activities were assessed through confrontation on solid media in Petri dishes. For this purpose, bacterial cultures were prepared in 10 mL LB medium inoculated by a single colony and incubated 24 h at 30◦C and 120 rpm. Antagonistic activities against P. carotovorum were evaluated by streaking the bacterial culture of interest in one third of the Petri dish containing LBagar. Then, an overnight (O/N) broth culture of P. carotovorum (28◦C, 120 rpm) was streaked three times perpendicularly to the bacterial inoculum (**Figure 1A**). LB medium was used as negative control. Petri dishes were scanned (HP Scanjet G4010) after 48 h of incubation. To evaluate the potential antagonistic activity against the other pathogens, two streaks of two cm of the bacterial culture of interest were made at each side of a piece of agar (Ø 5 mm) covered with the pathogen mycelium aged of 7–10 days (**Figures 1B–E**). As negative control, LB medium was streaked at each side of the piece of agar covered with the pathogen mycelium. Petri dishes were scanned (HP Scanjet G4010) after 7–10 days of incubation. Image analysis (Image J R software) was used to quantitatively determine the Growth Inhibition Percentage (GIP) by comparing the surface covered by the pathogen in the presence of bacterial isolate and the negative controls. Antagonistic activity was categorized in four different classes: (i) GIP < 30%, (ii) GIP ≥ 30%, (iii) GIP ≥ 50% and GIP ≥ 70% (Figure S1). Each test was repeated twice with three technical replicas and statistically treated. At the end of those

tests, the 60 most active strains (GIP ≥ 50%) were selected for further characterization.

## Bacterial Isolates Identification

Bacterial identification was performed through 16S rRNA gene sequencing. For this, bacterial isolates were grown overnight on LB-agar and one single colony was picked-up for "colony-PCR" using primers pair 27F (AGAGTTTGAT CCTGGCTCAG) and 1492F (GGTTACCTTGTTACGACTT). PCR reactions were performed using the GoTaq R G2 Flexi DNA Polymerase (Promega) with colorless buffer following the manufacturer's recommendations. Thermal cycling parameters were as follows: a denaturation step at 95◦C for 5 min followed by 30 cycles at 95◦C for 1 min, 55◦C for 30 s and 72◦C for 90 s. Finally, an elongation step at 72◦C for 10 min. PCR amplicons were verified by gel electrophoresis, purified using the GenElute PCR cleanup kit (Sigma) and sequenced in both orientations at Macrogen Europe (Amsterdam, The Netherlands). For each bacterial isolate, nucleotide sequences were trimmed, aligned and compared with the BLASTn search available in GenBank database. Sequences were deposited in the NCBI database under GenBank accession numbers MF062580 to MF062639. Phylogenetic relationships based on partial 16S rRNA gene sequences were determined with MEGA 6.0 software (Tamura et al., 2013) using maximum likelihood (ML) method with the General Time-Reversible plus gamma model of nucleotide substitution and bootstrap values of 1,000 iterations.

## PCR Screening for Genes Related to Known Antagonistic Metabolites and Virulence Factors

Total DNA from bacterial isolates were extracted by using the Wizard R Genomic DNA Purification Kit (Promega) following manufacturer's instructions. DNA extractions were PCR-screened for the presence of the genetic determinants of previously reported antagonistic metabolites and virulence factors (Table S2). All PCR reactions were performed using the One Taq R Quick-Load 2X Master Mix with standard buffer (New England BioLabs) following the manufacturer's recommendations. Thermal cycling parameters were 5 min at 94◦C, then 35 cycles of 1 min at 94◦C, 1 min at the annealing temperature, followed by 1 min/kb of the PCR amplicon (Table S2) at 68◦C, and finally, 10 min at 68◦C. Reference strains used as positive controls for each PCR are indicated in Table S2. PCR amplicons were analyzed by agarose gel electrophoresis.

## Production of Enzymes, Siderophores and Bio-Surfactants by the Bacterial Isolates

The production of enzymes (Ariffin et al., 2006; Youcef-Ali et al., 2014), siderophores (Neilands, 1981) and bio-surfactants (Siegmund and Wagner, 1991; Youssef et al., 2004) were assessed for each selected bacterial isolate. Briefly, the production of proteolytic, cellulolytic and chitinolytic enzymes, along with the siderophore production, were respectively assessed throughout halo formation on specific solid media: Skimmed Milk (SM) agar [Skimmed milk, 28g L− 1 ; casein hydrolysate, 5 g L−1 ; yeast extract, 2 g L−1 ; dextrose, 1 g L−1 ; agar, 15 g L−1 at pH 7.0 ± 0.2]; Carboxy-methyl Cellulose (CMC)-agar [KH2PO4, 1 g L−1 ; MgSO4.7H2O, 0.5 g L−1 ; NaCl, 0.5 g L−1 ; FeSO4.7H2O, 0.01 g L−1 ; MnSO4.H2O, 0.01 g L−1 ; NH4NO3, 0.3 g L−1 ; CMC, 10 g L− 1 ; agar, 12 g L−1 at pH 7.0 ± 0.2]; Colloidal Chitin (CC)-agar [NH4H2PO4, 1 g L−1 ; KCl, 0.2 g L−1 ; MgSO4.7H2O, 0.2 g L−1 ; CC, 10 g L−1 ; agar, 20 g L −1 at pH 6.0 ± 0.2]; and modified Chrome Azurol S (CAS) agar [10 mL of FeCl3.6H2O, 27 mg/100 mL of HCl 10 mM; 50 mL of CAS, 1.2 g L−1 ; 40 mL of HDTMA, 1.82 g L− 1 ; 900 mL of LB-agar for Bacillus spp. and King Broth agar for Pseudomonas spp. at pH 6.8 ± 0.2]. A 10 µL drop of a bacterial culture in LB (24 h, 30◦C, 120 rpm) was spotted on the above-mentioned media and activity was observed after an incubation of 24 h at 30◦C (proteolytic enzymes), 120 h at 37◦C (cellulolytic enzymes), 120 h at 30◦C (chitinolytic enzymes and siderophores). Each test was repeated twice with

three technical replicas. Bio-surfactant production was assessed via a "drop collapse test" based on amphiphilic properties of compounds like lipopeptides. Twenty-five µL drops of filtered supernatant (0.22µm Minisart R , Sartorius Stedim) from a 48 h bacterial culture (Bacillus spp.: LB, 30◦C, 120 rpm; Pseudomonas spp.: KB, 25◦C, 120 rpm) were spotted on a hydrophobic surface (Petri dish Sarstedt R ). Drops of sterile media (LB; KB) were used as negative control. After 5 min of incubation at room temperature, plates were scanned (HP Scanjet G4010) and the area covered by the drop was measured with an image analysis software (Image J R ). The percentage of "spreading" was then calculated by comparison with the negative control and normalized with the optical density at 600 nm (OD600nm) of the initial culture (Multiskan FC, Thermo Scientific). Each test was performed four times with three technical replicas. Bacillus amyloliquefaciens S499, B. subtilis GA1 and Pseudomonas aeruginosa PAO1 were used as positive controls, since they are known as bio-surfactant producers (de Souza et al., 2003; Touré et al., 2004; Arguelles-Arias et al., 2009; Nihorimbere et al., 2012). Pseudomonas spp. biosurfactant production was also assessed via a culture of 5 days on Siegmund and Wagner (SW) medium [glycerol, 20 g L−1 ; KH2PO4, 0.7 g L−1 ; Na2HPO4, 0.9 g L−1 ; NaNO3, 2 g L−1 ; MgSO4.7H2O, 0.4 g L−1 ; CaCl2.2H2O, 0.1 g L−1 ; 2 mL L−1 of trace elements containing FeSO4.7H2O, 2 g L−1 ; MnSO4.H2O, 1.5 g L−1 ; (NH4)6Mo7O24.4H2O, 0.6 g L−1 ; at pH 6.7 ± 0.2] supplemented with cetyltrimethylammoniumbromide (CTAB) methylene blue agar [CTAB-methylene, 0.2 g L−1 ; methylene blue, 0.005 g L−1 ; agar, 15 g L−1 ] where bio-surfactant producers are able to form a dark blue halo around the colony (Siegmund and Wagner, 1991).

## Bio-Control Assays Against Late Blight in Greenhouse

Greenhouse experiments were performed in order to evaluate the antagonistic effect of selected bacteria against potato late blight. Assays were conducted with seven Bacillus spp. and four Pseudomonas spp. showing high in vitro antagonistic activities (GIP ≥ 70%) against P. infestans. Potato plants were obtained by planting potato tubers from sensitive "Bintje" variety and growing them for a month in a G2 greenhouse facility with cycles at 25/15◦C (day/night) with 70% relative humidity (RH) and 16:8 photoperiod. Potato plants were arranged every 30 centimeters and were daily irrigated. Bacteria were grown in LB for 48 h at 30◦C and 120 rpm. Bacterial cultures adjusted to an OD600nm of 1 with LB were sprayed on potato leaves using 6 plants by treatment (2 mL/leaf and 3 leaves/potato plant) 4 h before pathogen inoculation. Sterile LB was used as negative control. Prior to the pathogen inoculation, P. infestans strain CRA-W10022 pathogenicity was restored in vitro on potato leaves as described by Clinckemaillie et al. (2016). Suspensions of P. infestans (ca. 15,000 sporangia mL−1 ) were applied through pulverization (2 mL/leaf). Plants were placed in a humid chamber for 24 h at 90% RH to enable infection and then moved back to the G2 greenhouse. Quantitative scoring of the symptoms was done daily as described by Clinckemaillie et al. (2016) and the disease progression was followed for 7–10 days after inoculation. At the end of the experiment, the area under the disease progression curve (AUDPC) was calculated for each replica (Campbell and Madden, 1990) in order to determine a normalized protection index (PI) calculated as follows:

$$\text{PI} = \left[ \left( 1 - \frac{AUDPC \,\, treatment}{AUDPC \,\,control} \right) \* 100 \right]$$

The greenhouse assays were repeated four times using 18 technical replicas (6 plants, 3 leaves/potato plant) each and were statistically treated.

## Pilot Field Trial against Late Blight

A pilot field assay aiming at assessing selected bacteria effectiveness as bio-control agents of P. infestans was conducted at the Alphonse de Marbaix Research Center in Louvain-la-Neuve (Belgium) from April to September 2016. A randomized complete block assay was designed with 7 treatments (4 replicas/treatment, 4 lines/replica, 48 plants/replica) and interstices of sensitive "Bintje" potato variety distributed on a 10,000 m² field (15 m²/replica). 'Bintje' potato plants were used as reservoir to improve natural P. infestans infection in field. Bacterial strains of Bacillus spp. and Pseudomonas spp. were selected on the basis of in vitro and in vivo assays performed against P. infestans. Treatments applied were divided as follows: (i) plants treated with antagonistic strain B. amyloliquefaciens 17A-B3, (ii) plants treated with antagonistic strain B. subtilis 30B-B6, (iii) plants treated with antagonistic strain Pseudomonas brenneri 43R-P1, (iv) plants treated with antagonistic strain Pseudomonas protegens 44R-P8, (v) plants treated with chemical fungicide Revus R (250 g L−1 mandipropamid; 300 L ha−1 ), (vi) plants treated with Cuprex R (300 L ha−1 ), an authorized fungicide for organic production and (vii) untreated potato plants from late blight semi-resistant "Challenger" variety. Bacterial cultures suspensions were produced as described for greenhouse experiments and 2.5 L/replica were foliar-sprayed once a week from 8th June to 17th August 2016 (12 applications). Fungicides, supplemented with Trend 90 R (1 mL L−1 ), were applied six times in the potato crop growing season following Belgian warning prediction system (CARAH, PCA). Late blight disease progression was monitored as described for greenhouse assays, three times a week throughout the growing potato crop season, with a quantitative scoring of all leaves of eight plants/replica. AUDPC and normalized PI were calculated as described above.

## Statistical Analysis

Data from antagonistic activity assays in vitro, greenhouse assays and pilot field trial were statistically treated using the oneway analysis of variance (ANOVA) using open source software R R (Ihaka and Gentleman, 1996). The Tukey HSD multiplecomparison test was used for comparison of the means, with confidence interval specified through p-value. The Pearson Correlation Index (PCI) was calculated with corrplot package in R R (Wei and Simko, 2016).

## RESULTS

## Isolation and in Vitro Screening of Bacteria Displaying Antagonistic Activities

A collection of 2,826 bacterial strains was isolated from a total of 157 collected samples (Table S1). Based on their in vitro antagonistic activities toward at least one of the five pathogens tested (A. solani, F. solani, R. solani, P. carotovorum, P. infestans) with a GIP ≥ 50%, 52 Bacillus spp. and eight Pseudomonas spp. strains were selected for further characterization (**Tables 2**, **3**). For this subset of 60 strains, 12 Bacillusspp. strains were shown to be active against F. solani (GIP ≥ 50%), whereas no Pseudomonas spp. showed a strong activity against this fungus. It is worth to note that due to the rhizoidal growth displayed by nine of the active Bacillus strains, their activity against F. solani might also be due to competition for surface colonization (Figure S2). Concerning the antagonism against late blight (P. infestans), 18 Bacillus spp. and seven Pseudomonas spp. strains displayed a strong inhibitory activity (GIP ≥ 70%). Also, 25 Bacillus spp. strains showed a good inhibition activity against A. solani (GIP ≥ 50%) and four against P. carotovorum, while none of the Pseudomonas strains were active against these two microbes. The assays against R. solani also revealed effective strains of B. amyloliquefaciens (one), B. pumilus (three) and P. putida (one). Taken together, a strong in vitro antagonistic activity (GIP ≥ 70%) against at least one of the pathogens tested was observed for 26 Bacillus spp. and five Pseudomonas spp. strains.

## Identification of the Antagonistic Bacteria

BLAST and phylogenetic analyses based on 16S rRNA gene sequences revealed that the majority of the selected Bacillus spp. strains belong to the groups of B. subtilis (i.e., B. amyloliquefaciens, B. licheniformis, and B. sonorensis) and B. cereus sensu lato (s.l.) (i.e., B. cereus and B. mycoides) (**Table 2**, Figure S3). It is worth noting that the B. mycoides strains were further identified through their characteristic rhizoid growth on LB plates. Strains belonging to the B. pumilus complex, along with B. megaterium and B. simplex, were also identified among the selected strains as displaying remarkable antagonistic activities against the pathogens tested. It is interesting to note that the strains that showed strong competitive abilities, which are not necessary antagonistic activities, are all B. mycoides except for one B. cereus strain. Selected isolates from the genus Pseudomonas were identified through 16S rRNA gene sequences as P. alkylphenolia, P. brenneri, P. chlororaphis, P. fluorescens, P. helmanticensis, P. protegens, P. putida, and P. salomonii (**Table 3**, Figure S4).

## PCR Screening for Known Antagonistic Metabolites

Using a PCR approach, the genetic determinants of potential antagonistic molecules screened for the collection of Bacillus strains were (Table S2): β-1,3-glucanase, AHL-lactonases, bacilysin, bacillomycin D, chitinases, difficidin, fengycin, iturin, macrolactin, plipastatin, surfactin and Zwittermicin A. As shown in **Table 2**, it seems evident that each species of Bacillus spp. present a specific pattern of detection associated with potential antagonistic metabolites. Indeed, B. mycoides strains were mostly positive to AHL-lactonases (PCI: 0.77), and exochitinases (PCI: 0.77), B. licheniformis strains to bacillomycin D (PCI: 0.47), B. megaterium strains to bacillomycin D (PCI: 0.48) and surfactin (PCI: 0.33), and B. pumilus strains to bacilysin (PCI: 0.61). Spectra of potential antagonistic metabolites were broader for B. subtilis strains (15A-B91, 15A-B92, 15A-B93, 27A-B4, 30B-B6) and even reached seven bio-active compounds for B. amyloliquefaciens strain (17A-B3). Bacilysin-related genes were the most commonly detected, in particular in all the strains displaying a strong inhibitory activity against P. infestans (GIP ≥ 70 %). These strains belong to the B. amyloliquefaciens, B. pumilus, and B. subtilis species (10 strains). Remarkably, five of these strains displayed positive PCR results only for bacilysin, which might indicate that this molecule was responsible for the strong antagonism observed toward P. infestans.

The genetic determinants associated with the production of phenazin, pyrrolnitrin, orfamid, syringopeptin, viscosin, 2,4 diacetylphloroglucinol, and pyoluteorin were detected by PCR in four strains of Pseudomonas spp. (**Table 3**) suggesting their potential implication in the in vitro antagonistic activities against the pathogens. In particular, P. protegens 44R-P8, which was positive for 5/10 molecules tested by PCR, showed an important in vitro antagonistic potential. Interestingly, although the P. brenneri 43R-P1 strain was negative to all tested genes, it displayed a strong antagonism toward P. infestans (GIP ≥ 70%; **Table 3**).

## PCR Screening for Virulence Factors

The detection by PCR of the genetic determinants related to potential virulence factors of B. cereus s.l. group and Pseudomonas aeruginosa species was performed on all strains of the collection since some members of these groups may be potential human pathogens. Among the collection of Bacillus spp., the screening indicated that strains belonging to B. mycoides, B. cereus and one strain of B. pumilus were positive for at least one gene coding for a potential virulence factor, mainly the non-hemolytic enterotoxin (Nhe), Hemolysin BL (HBL) or Cerolysin O (CerO) (**Table 2**). However, none of the Bacillus spp. were positive to the genetic determinants coding for the lethal toxins cereulide and cytotoxin K1 (CytK1), both associated with severe foodborne toxi-infections in humans, including fatalities (Lund et al., 2000; Naranjo et al., 2011). Among the selected Pseudomonas spp. strains, none were positive to any tested genes related to the potential virulence factors (Finnan et al., 2004; Lanotte et al., 2004; Khalifa et al., 2011; Holban et al., 2013).

## Evaluation of the Production of Enzymes, Siderophores, and Bio-Surfactants

The assays performed to evaluate enzymatic activities (i.e., proteolytic, cellulolytic and chitinolytic) of the selected strains indicated that the majority of the Bacillus spp. and Pseudomonas spp. were able to produce proteolytic and cellulolytic enzymes. On the contrary, very few Bacillus spp. and no Pseudomonas strains were able to display chitinolytic activity on colloidal chitin medium (**Tables 2**, **3**). No relation was found between any specific bacterial species, their enzymatic activities and TABLE 2 | Bacillus spp. strains isolated in this study and selected for their statistically relevant in vitro antagonistic activities, evaluated through observed growth inhibition percentage (GIP), against P. carotovorum, P. infestans, A. solani, F. solani, and R. solani.

Genbank accession number, isolate code, 16S identification, antagonism exhibited toward pathogens tested (scaled as described in color legend at the top of the table), PCR detection of related genes to known bio-active metabolites and virulence factors, and bio-active compound production (enzymes, siderophores, bio-surfactants) are indicated.

and

TABLE 3 |

Pseudomonas spp. strains isolated in this study and selected for their statistically

 relevant antagonistic

 activities, evaluated through observed growth inhibition percentage

 (GIP), against P. carotovorum,

their antagonistic behavior (data not shown). Regarding the siderophore production, while all Pseudomonas spp. strains produced siderophores under the condition tested, only 19 Bacillus spp. strains did, mostly strains of B. licheniformis, B. pumilus, and B. subtilis (**Tables 2**, **3**). Drop collapse tests also showed that all the Pseudomonas spp. strains were able to produce bio-surfactants (**Table 3**), while only four strains of the B. subtilis group did (**Table 2**).

## Greenhouse Assays against Late Blight

In vivo assays conducted under controlled greenhouse conditions were designed and tested beforehand to ensure a well-established and reliable pathosystem allowing the pathogen to grow under good conditions and perform a complete infection of inoculated leaves under 8 days. Results from statistical analysis (ANOVA) performed on 72 replicas for each tested strain, distributed among four assays, confirmed the strong antagonistic activities of seven Bacillusspp. and four Pseudomonasspp. against P. infestans on the sensitive potato variety "Bintje." As shown in **Figures 2**, **3**, three Bacillus spp. (30B-B6, PI: 67%; 15A-B2, PI: 65%; 17A-B3, PI: 62%) and two Pseudomonasspp. (44R-P8, PI: 83%; 43R-P1, PI: 64%) gave significant protection (PI > 60 %) against P. infestans after disease developmental level normalization.

## Pilot Field Trial against Late Blight

Based on greenhouse assays, B. amyloliquefaciens 17A-B3 and B. subtilis 30B-B6, along with P. brenneri 43R-P1 and P. protegens 44R-P8 were evaluated in a pilot field trial to assess their PI against P. infestans. Environmental conditions during field trial (April, 20 to September, 22, 2016) were appropriate for late blight infection and development. Potato plants of semi-resistant "Challenger" variety reached 99.8% of late blight infection at the end of evaluations, while the sensitive "Bintje" potato plants presented already 100% of late blight infection from the sixteenth day. During the assay, significant rainy events occurred and matched with temperatures favorable for the growth and transmission of P. infestans in the field (Figure S5). Disease progression was followed through quantitative evaluations from June, 27 (day 0) to August, 22 (day 56) (**Figure 4A**), allowing calculation of AUDPC and determination of a normalized PI (**Figures 4B–G**). The protection conferred by bacterial treatments was less effective than fungicide treatments at any time in the assay. As expected, the fungicide Cuprex and Revus treatments allowed a disease severity reduction of 83 and 98%, respectively. However, the bacterial treatments with P. protegens 44R-P8 strain provided a significant protection for 16 days after the first signs of symptoms (**Figures 4B–D**) and up to 45 days with B. subtilis 30B-B6 (**Figures 4B–F**). Moreover, treatment with 30B-B6 strain allowed shifts in time of 5 days to reach 25 and 50% of infection compared to controls. Interestingly, protection conferred by the treatment with B. subtilis 30B-B6 stabilized around 20% for nearly 30 days from day 14 to day 42 (**Figure 4G**). At the end of the trial, the treatment with B. subtilis 30B-B6 allowed a statistically significant reduction of late blight severity (PI: 22%; p-value < 0.001). Remarkably, monitoring of late blight progress showed that each application of B. subtilis 30B-B6 allowed a decrease in the disease development rate until an

FIGURE 2 | Mean of normalized protection index (PI) against late blight observed after foliar spray on sensitive "Bintje" variety of potato plants with Pseudomonas spp. (gray) or Bacillus spp. strains (white), and standard deviation based on four greenhouse assays.

FIGURE 3 | Late blight progression observed in greenhouse assay after foliar spray of P. infestants (15,000 sporangia mL-1) on: (a) sensitive "Bintje" variety of potato plant and (b) potato plant previously treated with P. protegens 44R-P8 suspension.