# HARNESSING USEFUL RHIZOSPHERE MICROORGANISMS FOR PATHOGEN AND PEST BIOCONTROL

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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ISSN 1664-8714 ISBN 978-2-88945-059-6 DOI 10.3389/978-2-88945-059-6

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# **HARNESSING USEFUL RHIZOSPHERE MICROORGANISMS FOR PATHOGEN AND PEST BIOCONTROL**

Topic Editors:

**Aurelio Ciancio,** Consiglio Nazionale delle Ricerche, Italy **Corné M. J. Pieterse,** Utrecht University, Netherlands **Jesús Mercado-Blanco,** Agencia Estatal Consejo Superior de Investigaciones Científicas, Spain

Root-knot nematode eggs parasitized by hyphae of an entomopathogenic Lecanicillium sp. Image by Aurelio Ciancio

Growing demographic trends require sustainable technologies to improve quality and yield of future food productions. However, there is uncertainty about plant protection strategies in many agro-ecosystems. Pests, diseases and weeds are overwhelmingly controlled by chemicals which pose health risks and cause other undesirable effects.Therefore, an increasing concern on control measures emerged in recent years. Many chemicals became questioned with regard to their sustainability and are (or will be) banned. Alternative management tools are studied, relying on biological, and low impact solutions. This ResearchTopic concerns microbial biocontrol agents, root-associated microbiomes, and rhizosphere networks. Understanding how they interact or respond to (a)biotic environmental cues is instrumental for an effective and sustainable impact. The rhizosphere is in this regard a fundamental object of study, because of its role in plant productivity.

This e-book provides a polyhedral perspective on many issues in which beneficial microorganisms are involved. Data indeed demonstrate that they represent an as yet poorly-explored resource, whose exploitation may actively sustain plant protection and crop production. Given the huge number of microbial species present on the planet, the microorganisms studied represent just the tip of an iceberg. Data produced are, however, informative enough about their genetic and functional biodiversity, as well as about the ecosystem services they provide to underpin crop production. Challenges for future research work concern not only the biology of these species, but also the practices required to protect their biodiversity and to extend their application in the wide range of agricultural soils and systems present in the world. Agriculture cannot remain successfully and sustainable unless plant germplasm and useful microbial species are integrated, a goal for which new knowledge and information-based approaches are urgently needed.

**Citation:** Ciancio, A., Pieterse, C. M. J., Mercado-Blanco, J., eds. (2016). Harnessing Useful Rhizosphere Microorganisms for Pathogen and Pest Biocontrol. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-059-6

# Table of Contents

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

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

# **Section 1. Microbial Volatiles and other Compounds**

*12 Impact of a Bacterial Volatile 2,3-Butanediol on* **Bacillus subtilis** *Rhizosphere Robustness*

Hwe-Su Yi, Yeo-Rim Ahn, Geun C. Song, Sa-Youl Ghim, Soohyun Lee, Gahyung Lee and Choong-Min Ryu

*23 Augmenting Sulfur Metabolism and Herbivore Defense in* **Arabidopsis** *by Bacterial Volatile Signaling*

Mina Aziz, Ranjith K. Nadipalli, Xitao Xie, Yan Sun, Kazimierz Surowiec, Jin-Lin Zhang and Paul W. Paré

# **Section 2. Soil Amendments**

*62 Chitin Mixed in Potting Soil Alters Lettuce Growth, the Survival of Zoonotic Bacteria on the Leaves and Associated Rhizosphere Microbiology*

Jane Debode, Caroline De Tender, Saman Soltaninejad, Cinzia Van Malderghem, Annelies Haegeman, Inge Van der Linden, Bart Cottyn, Marc Heyndrickx and Martine Maes

*77 Microbial Profiling of a Suppressiveness-Induced Agricultural Soil Amended with Composted Almond Shells*

Carmen Vida, Nuria Bonilla, Antonio de Vicente and Francisco M. Cazorla

# **Section 3. Biocontrol and Suppression**

*91 Characterization of Soil Suppressiveness to Root-Knot Nematodes in Organic Horticulture in Plastic Greenhouse* Ariadna Giné, Marc Carrasquilla, Maira Martínez-Alonso, Núria Gaju

and Francisco J. Sorribas

*106 Concilience in Entomopathogenic Nematode Responses to Water Potential and Their Geospatial Patterns in Florida*

Fahiem El-Borai, Nabil Killiny and Larry W. Duncan

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

Anouk Guyer, Mout De Vrieze, Denise Bönisch, Ramona Gloor, Tomke Musa, Natacha Bodenhausen, Aurélien Bailly and Laure Weisskopf

# **Section 4. Mycorrhizae, Endophytes, Symbioses**

*174 Arbuscular Mycorrhizal Fungi as Natural Biofertilizers: Let's Benefit from Past Successes*

Andrea Berruti, Erica Lumini, Raffaella Balestrini and Valeria Bianciotto

*187 Arbuscular Mycorrhizal Fungi for the Biocontrol of Plant-Parasitic Nematodes: A Review of the Mechanisms Involved*

Nele Schouteden, Dirk De Waele, Bart Panis and Christine M. Vos

# **Section 5. Virulence and Induced Resistance**

*235 Crucial Roles of Abscisic Acid Biogenesis in Virulence of Rice Blast Fungus*  **Magnaporthe oryzae**

Carla A. Spence, Venkatachalam Lakshmanan, Nicole Donofrio and Harsh P. Bais

Pilar Martínez-Hidalgo, Juan M. García and María J. Pozo

*267 The importance of chorismate mutase in the biocontrol potential of*  **Trichoderma parareesei** Esclaudys Pérez, M. Belén Rubio, Rosa E. Cardoza, Santiago Gutiérrez,

Wagner Bettiol, Enrique Monte and Rosa Hermosa

# **Section 6. -OMICS**

*281 Biocontrol agent* **Bacillus amyloliquefaciens** *LJ02 induces systemic resistance against cucurbits powdery mildew*

Yunlong Li, Yilin Gu, Juan Li, Mingzhu Xu, Qing Wei and Yuanhong Wang

*296 Genome mining and metabolic profiling of the rhizosphere bacterium*  **Pseudomonas** *sp. SH-C52 for antimicrobial compounds*

Menno Van Der Voort, Harold J. G. Meijer, Yvonne Schmidt, Jeramie Watrous, Ester Dekkers, Rodrigo Mendes, Pieter C. Dorrestein, Harald Gross and Jos M. Raaijmakers

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

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

1 Dipartimento di Scienze Agroalimentari, Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, Bari, Italy, 2 Department of Biology, Faculty of Science, Institute of Environmental Biology, Utrecht University, Utrecht, Netherlands, 3 Instituto 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

**The Editorial on the Research Topic**

### **Harnessing Useful Rhizosphere Microorganisms for Pathogen and Pest Biocontrol**

### Edited by:

Laure Weisskopf, University of Applied Sciences Western Switzerland, Switzerland

### Reviewed by:

Paolina Garbeva, Nederlands Instituut voor Ecologie (Koninklijke Nederlandse Akademie van Wetenschappen), Netherlands

\*Correspondence:

Aurelio Ciancio<EMAIL_ADDRESS>
### Specialty section:

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

Received: 16 July 2016 Accepted: 28 September 2016 Published: 19 October 2016

### Citation:

Ciancio A, Pieterse CMJ and Mercado-Blanco J (2016) Editorial: Harnessing Useful Rhizosphere Microorganisms for Pathogen and Pest Biocontrol. Front. Microbiol. 7:1620. doi: 10.3389/fmicb.2016.01620 Growing demographic trends require sustainable technologies to improve quality and yield of future food productions. However, there is uncertainty about plant protection strategies in many agro-ecosystems. Pests, diseases, and weeds are overwhelmingly controlled by chemicals which pose health risks and cause other undesirable effects. Therefore, an increasing concern on control measures emerged in recent years. Many chemicals became questioned with regard to their sustainability and are (or will be) banned. Alternative management tools are studied, relying on biological, and low impact solutions.

This Research Topic concerns microbial biocontrol agents, root-associated microbiomes, and rhizosphere networks. Understanding how they interact or respond to (a) biotic environmental cues is instrumental for an effective and sustainable impact. The rhizosphere is in this regard a fundamental object of study, because of its role in plant productivity.

# MICROBIAL VOLATILES AND OTHER COMPOUNDS

Roots are surrounded by a flow of molecules released by microorganisms. Some volatiles may affect growth through different mechanisms, such as biochemical signals eliciting local defense reactions or systemic resistance (Kai et al., 2007; Chung et al., 2016). Yi et al. studied the effects of 2,3-butanediol produced by a Bacillus subtilis isolate overexpressing the bud operon synthesis encoding genes. They showed that the isolate persisted on pepper roots more than the wild type, thereby interfering with colonization by fungi. Exudates from isolate pre-treated roots inhibited colonization by Trichoderma sp., by another B. subtilis strain, and by the soil-borne pathogen Ralstonia solanacearum. Application of 2,3-butanediol to roots followed by R. solanacearum exposure enhanced the expression of pathogenesis-related (PR) genes. The volatile triggered the secretion of root exudates modulating fitness of soil fungi and bacteria, thus acting as a plant defense inducer.

Bacteria metabolism affects nutrient assimilation. Aziz et al. reported a new mechanism in the plant growth-promoting (PGP) Bacillus amyloliquefaciens GB03, which activates genes involved in sulfur assimilation and uptake by Arabidopsis. Other transcripts encoding for proteins involved in the biosynthesis of sulfur-rich aliphatic and indolic glucosinolates were also expressed. The enhanced sulfur assimilation increased plant glucosinolate biosynthesis, and conferred protection against the beet armyworm Spodoptera exigua.

Bacterial metabolites may also protect plants by inhibiting herbivores (Mithöfer and Boland, 2012). Ganassi et al. reported that also fungal metabolites may have this effect, as shown by a Trichoderma citrinoviride isolate, interfering with feeding of the cereal aphid Rhopalosiphum padi. Different long-chain primary alcohols (LCOHs) showed a phagodeterrent effect restraining aphids from settling on treated leaves. The LCOHs, perceived through taste receptor neurons and effective at low concentrations, hold potential for insect control in synergy with other compounds.

Lysobacter spp. may affect soil-borne pathogens through extracellular enzymes and other metabolites (Folman et al., 2003). Members of this genus appeared abundant in soil suppressive to the root pathogen Rhizoctonia solani. Some strains showed in vitro activity against R. solani and other phytopathogens such as Pythium ultimum, Aspergillus niger, Fusarium oxysporum, and Xanthomonas campestris. In soil, however, suppression of R. solani damping-off on sugar beet and cauliflower was low, and no PGP effect was found on sugar beet, cauliflower, onion and Arabidopsis thaliana, likely due to poor rhizosphere colonization. Antagonistic Lysobacter spp. are an important source of new enzymes and antimicrobial compounds, although their role in disease suppressiveness needs to be confirmed (Gómez Expósito et al.).

# SOIL AMENDMENTS

The characterization of organic matter amendments enhancing biocontrol and influencing resident soil communities is a growing research field (Bailey and Lazarovits, 2003; Bonilla et al., 2012). Debode et al. studied the effect of chitin on lettuce growth and survival of pathogenic Escherichia coli O157:H7 and Salmonella enterica colonizing leaves. Chitin addition increased yields and reduced phyllosphere survival of both bacteria, increasing fungal and bacterial biomass in the rhizosphere. An increase was observed for bacterial genera Cellvibrio, Pedobacter, Dyadobacter, and Streptomyces and the fungi Lecanicillium and Mortierella. These taxa include species involved in biocontrol, PGP, nitrogen cycle, and chitin degradation, showing a potential for chitin-based amendments.

A positive effect of amendments on soil microbial communities was reported by Vida et al. Composted almond shells elicited suppression of Rosellinia necatrix, causal agent of white root rot on avocado, increasing Proteobacteria and Ascomycota, and reducing Acidobacteria and Mortierellales. Beneficial Pseudomonas, Burkholderia spp., or Actinobacteria were found only in the amended soil.

# BIOCONTROL AND SUPPRESSION

Soil suppressiveness is an unfrequent condition, regulating noxious organisms, including root-knot nematodes (Meloidogyne spp.; Bent et al., 2008). In two organic horticulture greenhouses in Spain, nematodes decreased progressively in a crop rotation, a decline associated to eggs parasitism by the hyphomycete Pochonia chlamydosporia. Controlled assays confirmed suppressiveness, as indicated by lower egg densities and reduced Meloidogyne reproduction, observed in nonsterilized soil. A higher microbial diversity was also reported from suppressive soils (Giné et al.).

Entomopathogenic nematodes rely on phoretically associated, insect-killing bacteria (Lewis et al., 2015). In Florida, Steinernema sp. and S. diaprepesi occupy habitats with different soil properties and water potential. They have opposite migration behaviors in relation to soil water potential, the latter being attracted toward drier soil. After three migration cycles through soil to infect insect larvae, S. diaprepesi dominated the nematode community when soil was maintained at 6% moisture, whereas Steinernema sp. was dominant at 18% moisture. The nematode responses to water potential and osmotic gradients explained their geospatial patterns. Steinernema sp. was recovered only from shallow water tables and moist soils, whereas S. diaprepesi inhabited ecoregions with well-drained soils and deeper water tables. Differential expression of proteins involved in thermo or mechano-sensation and movement was associated to soil moisture. Proteins involved in metabolism, lectin detoxification, gene regulation, and cell division also differed between the two conditions. Modifying soil moisture in orchards may favor effective entomopathogens, useful in different environments (El-Borai et al.).

Late blight caused by the oomycete Phytophthora infestans is the most severe disease of potato (Nowicki et al., 2012). To find alternative solutions to copper-based products, three Pseudomonas isolates originating from potato phyllo—and rhizosphere were studied for their protective effect against late blight. When sprayed on potato leaves they survived 15 days in the greenhouse and 8 days in the field. Pseudomonas chlororaphis R47 was the most active in vitro, and the best protectant in the greenhouse. Its beneficial effects against P. infestans, high rates of phyllosphere survival and rhizosphere colonization suggested a potential for application as tuber treatment or leaf spray (Guyer et al.).

Pseudomonas aeruginosa is a model system due to endophytism and antagonism toward plant pathogens and pests. Nevertheless, it is also a human pathogen, a trait compromising its exploitation in agriculture (Deredjian et al., 2014). Thomas and Sekhar studied P. aeruginosa applications to control other phytopathogenic bacteria. Strain GNS.13.2a isolated from banana rhizosphere significantly reduced the density of native bacteria soon after inoculation. However, its density declined within a week, while a resilient response was observed for native soil bacteria. Assays under axenic conditions or with soil microbiota showed antagonism by the native microbial community, with varying interactive or antagonistic effects.

The use of bacteria, fungi and viruses for weed biocontrol received attention due to prevalence of herbicide-resistant weeds and pesticide bans. Benefits include low environmental impact, higher specificity, reduced costs, and identification of new herbicidal mechanisms. Harding and Raizada reviewed fungal bioherbicides from North America, based on genera Colletotrichum, Phoma, and Sclerotinia. Bioherbicides also include bacteria of genera Xanthomonas and Pseudomonas and some viruses. Weed suppression in field conditions is a challenge, as bioherbicides with cytotoxic mechanisms are more sensitive to environmental variables than conventional herbicides.

Bacterial formulations are increasingly produced for sustainable agriculture using biocontrol and PGP species, including endospore-forming Bacillus spp. as alternatives to pesticides. Wu et al. reviewed the use of B. amyloliquefaciens type strain FZB42, as an application example.

# MYCORRHIZAE, ENDOPHYTES, SYMBIOSES

Arbuscular Mycorrhizal Fungi (AMF) are primary soil components whose impairment affects the rhizosphere functioning. In their review, Berruti et al. highlighted AMF as valid alternatives to conventional fertilization in sustainable agriculture. Although soil inoculation appears a successful strategy, results may vary, depending on host plant, and fungi. Factors affecting AMF success and persistence include species compatibility with the soil environment, spatial competition and inoculation timing. Genomic and transcriptomic data advanced the knowledge on AMF interactions with the host-plant and other soil organisms, unraveling important factors. AMF can protect host plants against biotic stresses like plant-parasitic nematodes. Mechanisms include enhanced root tolerance, direct competition for nutrients and space, induced systemic resistance, and altered rhizosphere interactions. In a second AMF review, Schouteden et al. emphasized the importance of AMF-based systemic resistance for effective biocontrol of nematode pests.

Besides mycorrhizae, the root niche has been "discovered" also by other endophytic microorganisms contributing to plant health and disease containment (Mercado-Blanco and Lugtenberg, 2014; Hardoim et al., 2015). Bacterial root endophytes control the tomato resistance to R. solanacearum. Resistant cultivar Arka Abha showed a bacterial endophyte diversity higher than a susceptible cultivar, including species producing siderophores, HCN and antibiotics, of which three isolates (Pseudomonas oleovorans, Pantoea ananatis, and Enterobacter cloacae) were effective antagonists. Other resistant cultivars also showed higher prevalence for antagonistic than susceptible bacteria, confirming a role of root-associated endophytes in plant protection (Upreti and Thomas).

Colonization of lettuce roots and rhizosphere by five genetically modified Streptomyces spp. was studied by Bonaldi et al. The strains, transformed with a green fluorescent protein marker and apramycin resistance, inhibited in vitro the soil-borne pathogen Sclerotinia sclerotiorum. In a nonsterile substrate, a transformed strain colonized soil, roots and rhizosphere. When directly inoculated, the bacterium was reisolated from rhizosphere and roots at densities higher than after seed coating, showing that it is either rhizospheric, and endophytic.

Microbial symbioses also contribute to the competitiveness of invasive plant species. In this regard, the interactions of their microbiome with native species may provide indications for effective biocontrol. A research agenda aiming at developing novel microbial-based biocontrol strategies was proposed by Kowalski et al. based on the invasive plant Phragmites australis.

# VIRULENCE AND INDUCED RESISTANCE

Abscisic acid (ABA) is active in the rice interactions with the blast fungal pathogen Magnaporthe oryzae and the antagonistic bacterium P. chlororaphis EA105. Spence et al. reported that abscisic acid (ABA) affects plant defense by acting antagonistically on salicylic acid (SA), jasmonic acid (JA), and ethylene signaling. Magnaporthe oryzae-produced ABA enhanced plant susceptibility by accelerating pathogenesis through higher rates of spore germination and appressoria formation. Strain EA105 reduced the pathogen virulence by preventing its up-regulation of the ABA biosynthetic gene NCED3 in rice roots and of a β-glucosidase, activating ABA forms. EA105 counteracted the virulence-promoting effects of ABA on M. oryzae by inhibiting appressoria formation, preventing spores from increasing ABA biosynthesis and related signaling. ABA implication in plant protection by EA105 involved both direct mechanisms and plant signaling. The role of endogenous fungal ABA was confirmed through the inability of a knoc-kout mutant impaired in ABA synthesis to form lesions on rice, dramatically reducing fungal virulence. Studying the effect of virulence inducers (pH, temperature and acetosyringone concentration) on three homologous genes of Agrobacterium tumefaciens (VBP1, VBP2, and VBP3, involved in the T-DNA transfer), Yang et al. showed that vbp2 was affected by pH and by the deletion of vbp1. Results indicated that, in addition to Tcomplex recruitment, the three homologous genes were involved also in other biological processes.

Martínez-Hidalgo et al. reported how Micromonospora strains control fungal pathogens by stimulating plant immunity. This Gram-positive inhabits nitrogen fixing nodules of healthy leguminous plants, with PGP effects. Inoculation of tomato roots with antifungal Micromonospora isolates reduced leaf infection by Botrytis cinerea, with a durable induced systemic resistance. Gene expression analyses showed that Micromonospora stimulates plant defense, enhancing jasmonate-regulated defense pathways, an effects confirmed using defense-impaired tomato mutants. Nodule-isolated Micromonospora strains appear as excellent biocontrol agents, combining antifungal activity with the ability to elicit plant immunity.

Pérez et al. showed that the Trichoderma parareesei gene Tparo7 encodes a chorismate mutase (CM), an intermediate of aromatic amino acids, essential in protein synthesis and precursor of many secondary metabolites. Decreased levels of Tparo7 in silenced transformants showed reduced CM activity, lower growth rates and mycoparasite behavior against phytopathogenic fungi (R. solani, F. oxysporum, and B. cinerea) in dual cultures. Higher amounts of aromatic metabolites (tyrosol, 2-phenylethanol and SA) were produced from the silenced transformant, which inhibited growth of F. oxysporum and B. cinerea. The silenced transformants also showed reduced colonization of tomato root in vitro. In greenhouse assays the plants colonized by the silenced transformants were reduced, with a higher susceptibility to B. cinerea. The treated plants became yellowish and were defective in JA- and ethyleneregulated signaling pathways, as shown by expression analysis of of lipoxygenase 1, ethylene-insensitive protein 2, and PR protein 1 genes.

Greenhouse trials with fermentation broth of B. amyloliquefaciens LJ02 showed effective reduction of cucurbits powdery mildew in China. Li et al. reported that treated seedlings produced superoxide dismutase, peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase at levels higher than control. Free SA, with the PR-1 gene product, increased after leaf treatments, suggesting SA-mediated defense. Secretions from treated cucumber leaves also inhibited fungal spores germination in the rhizosphere. The bacterium and its fermented products showed SA induction, available for powdery mildew biocontrol through systemic resistance.

# -OMICS

Recent "-omics" research advances coupled to progressive cost reduction allow a better understanding on many trophic interactions in rhizosphere systems (Massart et al., 2015). The genomes of 12 Bacillus subtilis strains with PGP activity were sequenced and analyzed by Hossain et al. The strains exhibited high genomic diversity, except highly conserved B. amyloliquefaciens strains (B. subtilis group), with 32–90% gene family similarity among Bacillus genomes and 2839 core genome genes, similar to B. amyloliquefaciens subsp. plantarum. Comparative analyses identified genes linked to biocontrol and roots/leaf colonization, including 73 genes from subsp. plantarum with functions related to signaling, transport, secondary metabolites and carbon utilization. They encoded several secondary metabolites, with conserved polyketide biosynthetic clusters encoding difficidin and macrolactin. Deletion of secondary metabolite genes in B. amyloliquefaciens subsp. plantarum showed that expression of difficidin is critical to reduce damage by Xanthomonas axonopodis pv. vesicatoria on tomato.

Van der Voort et al. sequenced and analyzed the genome of Pseudomonas sp. SH-C52. This bacterium, from a R. solani suppressive soil, has antifungal activity attributed to the chlorinated 9-amino-acid lipopeptide thanamycin. Its 6.3 Mb genome showed 5579 predicted ORFs, matching Pseudomonas

# REFERENCES

corrugata as closest taxon. In silico, secondary metabolite analysis showed six non-ribosomal peptide synthetase gene clusters, including clusters for thanamycin and the 2-amino acid antibacterial lipopeptide brabantamide. Thanamycin is an antifungal whereas brabantamide has anti-oomycete activity, affecting phospholipases of Phytophthora infestans. A third lipopeptide, thanapeptin, was discovered, with structural variants active against P. infestans. Remaining clusters encode for another unknown lipopeptide. Results indicated a potential for SH-C52 lipopeptides with different antimicrobial activities.

Lichen microbioms were studied by Cernava et al. to analyze antagonism against bacteria and fungi. The Lobaria pulmonaria antagonistic community was dominated by Stenotrophomonas, Pseudomonas, and Burkholderia. 24.5% of isolates were antagonists, accounting for 7% of the metagenome, indicative of an overrepresentation in the culturable fraction. Stenotrophomonas bioactive components like spermidine showed PGP effects. Lichens represent important reservoirs for antagonistic bacteria, useful for biotechnological applications.

# OUTLOOK AND FUTURE CHALLENGES

Articles in this Research Topic provide a polyhedral perspective on many issues in which beneficial microorganisms are involved. Data indeed demonstrate that they represent an as yet poorlyexplored resource, whose exploitation may actively sustain plant protection and crop production. Given the huge number of microbial species present on the planet, the microorganisms studied represent just the tip of an iceberg. Data produced are, however, informative enough about their genetic and functional biodiversity, as well as about the ecosystem services they provide to underpin crop production. Challenges for future research work concern not only the biology of these species, but also the practices required to protect their biodiversity and to extend their application in the wide range of agricultural soils and systems present in the world. Agriculture cannot remain successfully and sustainable unless plant germplasm and useful microbial species are integrated, a goal for which new knowledge and informationbased approaches are urgently needed.

# AUTHOR CONTRIBUTIONS

AC wrote the paper, JM and CP have made direct contributions to the work, and approved it 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 © 2016 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) or licensor 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.

# Impact of a Bacterial Volatile 2,3-Butanediol on Bacillus subtilis Rhizosphere Robustness

Hwe-Su Yi1,2, Yeo-Rim Ahn1,3, Geun C. Song1 , Sa-Youl Ghim2 , Soohyun Lee1 , Gahyung Lee1 and Choong-Min Ryu1,4 \*

1 Molecular Phytobacteriology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea, 2 School of Life Science, Kyungpook National University, Daegu, South Korea, 3 Department of Biological Science, Korea Advanced Institute of Science and Technology, Daejeon, South Korea, 4 Biosystems and Bioengineering Program, School of Science, University of Science and Technology, Daejeon, South Korea

### Edited by:

Corné M. J. Pieterse, Utrecht University, Netherlands

### Reviewed by:

Jurriaan Ton, University of Sheffield, UK Harsh Bais, University of Delaware, USA

> \*Correspondence: Choong-Min Ryu<EMAIL_ADDRESS>
### Specialty section:

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

Received: 01 December 2015 Accepted: 09 June 2016 Published: 28 June 2016

### Citation:

Yi H-S, Ahn Y-R, Song GC, Ghim S-Y, Lee S, Lee G and Ryu C-M (2016) Impact of a Bacterial Volatile 2,3-Butanediol on Bacillus subtilis Rhizosphere Robustness. Front. Microbiol. 7:993. doi: 10.3389/fmicb.2016.00993 Volatile compounds, such as short chain alcohols, acetoin, and 2,3-butanediol, produced by certain strains of root-associated bacteria (rhizobacteria) elicit induced systemic resistance in plants. The effects of bacterial volatile compounds (BVCs) on plant and fungal growth have been extensively studied; however, the impact of bacterial BVCs on bacterial growth remains poorly understood. In this study the effects of a well-characterized bacterial volatile, 2,3-butanediol, produced by the rhizobacterium Bacillus subtilis, were examined in the rhizosphere. The nature of 2,3-butanediol on bacterial cells was assessed, and the effect of the molecule on root colonization was also determined. Pepper roots were inoculated with three B. subtilis strains: the wild type, a 2,3-butanediol overexpressor, and a 2,3-butanediol null mutant. The B. subtilis null strain was the first to be eliminated in the rhizosphere, followed by the wildtype strain. The overexpressor mutant was maintained at roots for the duration of the experiment. Rhizosphere colonization by a saprophytic fungus declined from 14 days post-inoculation in roots treated with the B. subtilis overexpressor strain. Next, exudates from roots exposed to 2,3-butanediol were assessed for their impact on fungal and bacterial growth in vitro. Exudates from plant roots pre-treated with the 2,3-butanediol overexpressor were used to challenge various microorganisms. Growth was inhibited in a saprophytic fungus (Trichoderma sp.), the 2,3-butanediol null B. subtilis strain, and a soil-borne pathogen, Ralstonia solanacearum. Direct application of 2,3-butanediol to pepper roots, followed by exposure to R. solanacearum, induced expression of Pathogenesis-Related (PR) genes such as CaPR2, CaSAR8.2, and CaPAL. These results indicate that 2,3-butanediol triggers the secretion of root exudates that modulate soil fungi and rhizosphere bacteria. These data broaden our knowledge regarding bacterial volatiles in the rhizosphere and their roles in bacterial fitness and as important inducers of plant defenses.

Keywords: PGPR, ISR, volatile, 2,3-butanediol, bacteria robustness

# INTRODUCTION

fmicb-07-00993 June 24, 2016 Time: 13:58 # 2

The rhizosphere is defined as the narrow area surrounding the plant root system. Root exudates within the rhizosphere act as a food source for other organisms (Bowen and Rovira, 1999). As a result, the rhizosphere is an important habitat for many different microbes, and acts as a competitive arena for roots and soil-borne-pathogenic and rhizosphere bacteria (rhizobacteria; Vespermann et al., 2007). Among the rhizobacteria, plant growth-promoting rhizobacteria (PGPR) have been the subject of much research in recent decades. PGPR colonization of roots promotes plant growth and enhances crop yields (biostimulants), and can help protect against plant pathogens (bioprotectants; Kloepper and Metting, 1992; Kloepper et al., 2004; Ryu et al., 2004; Calvo et al., 2014; Chung et al., 2016). PGPRs act as biostimulants and bioprotectants by (1) acting antagonistically to target pathogens, (2) producing plant hormone mimics, and (3) inducing systemic resistance (Kloepper and Ryu, 2006).

Volatile compounds such as isoprene, terpenes, alkanes, alkenes, alcohols, esters, carbonyls, and acids can influence communication between organisms, including between bacteria and plants (Kesselmeier and Staudt, 1999; Ryu et al., 2005a; Kai et al., 2007). Previous research reported that PGPR bacilli emitted volatiles that triggered plant growth promotion and induced systemic resistance (ISR; Ryu et al., 2003, 2004; Chung et al., 2016). After this discovery, numerous studies identified further bacterial volatiles and determined their effects on plant responses. Some volatiles are now available for field applications (Cortes-Barco et al., 2010a,b; Farag et al., 2013; Chung et al., 2016). One well-characterized volatile is 2,3-butanediol, which was examined in multiple Gram-negative and Gram-positive bacterial species such as Bacillus spp., Aerobacter spp., Serratia spp., Enterobacter spp., and Klebsiella spp. (Barrett et al., 1983; Voloch et al., 1985; Ryu et al., 2004; Han et al., 2006). Acetoin is the last synthesis intermediate for 2,3-butanediol, and metabolic conversion of acetoin to 2,3-butanediol is reversible in most bacteria but irreversible in fungi such as yeast (Syu, 2001). Acetoin and 2,3 butanediol also mediate plant-beneficial effects such as growth promotion and ISR in model plants and crops under in vitro and in situ conditions (Ryu et al., 2003, 2004; Han et al., 2006; Hahm et al., 2012). Recent metabolic engineering approaches facilitated increased production of 2,3-butanediol in non-producer or lowproducer bacterial species such as Klebsiella oxytoca, Escherichia coli, and Paenibacillus polymyxa by introduction of new genes and modification of biosynthetic pathways (Ji et al., 2011, 2014; Yang et al., 2013; Bai et al., 2015). However, the benefit to bacteria of producing 2,3-butanediol in the anaerobic conditions of the rhizosphere remains unknown.

The role of 2,3-butanediol in bacterial fitness has not been intensively studied. Early research in a mouse model revealed that 2,3-butanediol synthesis in Vibrio cholerae conferred a survival advantage in vivo during infection of intestines (Yoon and Mekalanos, 2006; Xiao and Xu, 2007). It is thought that 2,3-butanediol acts as a neutralizer in the acidic conditions of the intestinal cells. A null mutant that was unable to produce 2,3-butanediol was unable to colonize or maintain the bacterial populations during infection (Xiao and Xu, 2007; Pradhan et al., 2010; Bari et al., 2011). We hypothesized that 2,3-butanediol might play a similar bacterial fitness role in the rhizosphere. In this study, the effects of 2,3-butanediol on rhizosphere colonization were examined using three strains: Bacillus subtilis 168, BSIP1174 [a 2,3-butanediol null mutant referred to as "2,3-B(−)"], and BSIP1171 [an overexpression strain referred to as "2,3-B(+++)"]. In addition, the indirect effects of 2,3 butanediol on secretion of root exudates were examined in pepper roots. Finally, the antimicrobial capacity of root exudates elicited by 2,3-butanediol treatment was assessed. Exudates exhibited selective antagonism against pathogenic bacteria such as Ralstonia solanacearum. To our knowledge, this is the first report to characterize a bacterial volatile under in situ conditions in plants and to validate in vitro.

# MATERIALS AND METHODS

# Plant Materials and Bacterial Preparation

Plants were grown were carried out as previously described (Kang et al., 2007). Briefly, seeds of Capsicum annuum were surface-sterilized with 6% sodium hypochlorite, washed four times with sterile distilled water (SDW), and then maintained at 25◦C for 3 days until germination on Murashige and Skoog medium (Duchefa, Haarlem, the Netherlands). Germinated seeds were then transplanted to soilless media (Punong Horticulture Nursery Media LOW, Punong, Co. Ltd., Gyeongju, South Korea). Plants were grown at 25 ± 2 ◦C under fluorescent light (12 h/12 h day/night cycle, 7000 lx light intensity) in a controlledenvironment growth room. After establishment of seedlings, plants were transferred to the KRIBB greenhouse facility in Daejeon, South Korea.

Three B. subtilis strains were used to assess the role of 2,3 butanediol on bacterial rhizosphere competence: 168, BSIP1174 [2,3-butanediol null mutant referred to as 2,3-B(−)], and BSIP1171 [2,3-butanediol overexpression mutant referred to as 2,3-B(++); Cruz et al., 2000]. Bacterial suspension (5 ml at 108 colony forming units/ml) was used to inoculate pepper roots, as described previously (Lee et al., 2012, 2013). A spontaneous rifampicin resistance mutant of wild-type B. subtilis 168 was isolated previously (Ryu et al., 2005b). Bacterial strains were isolated from plant roots using specific antibiotics in the tryptic soy broth agar growth medium (TSA, BactoTM, BD, Sparks, MD, USA): 50 µg/ml rifampicin for strain 168, 10 µg/ml spectinomycin for 2,3-B(−), and 10 µg/ml spectinomycin plus 5 µg/ml chloramphenicol for 2,3-B(++). The experiment was repeated three times with five replications (one plant per replication).

The naturally occurring soil fungus was isolated from dilution plating method of pepper root system when we attempted to assess B. subtilis population described above.

# Disease Assay of Ralstonia solanacearum

Spontaneous rifampicin resistant R. solanacearum, was grown on solid Casamino acid-Peptone-Glucose [CPG, 1 g casamino acid (casein hydrolysate), 10 g peptone, 5 g glucose, and 18 g

agar per 1 L water] medium containing 100 µg/ml rifampicin at 30◦C for 2 days, scraped off the plates, re-suspended in sterilized distilled water and adjusted to 108 cfu/ml concentration for further experiments (Lee et al., 2012). The plants pretreated with 1 mM BTH was used a positive control. The 10 ml suspension of R. solanacearum was drenched on 3 weeks-old pepper seedlings at 1 week after 1 mM and 10 µM 2,3-butanediol and BTH drenchapplication as describe previously (Lee et al., 2012). To assess pathogen multiplication, the root sample at 0 and 3 days after pathogen challenge collected, macerated with sterile mortar and pestle, and plating on CPG agar medium containing 100 µg/ml rifampicin. The number of CFU was counted at 2–3 days after incaution of the plates.

# Assessment of Bacterial Populations Isolated from Pepper Roots

Bacterial colonization on roots was determined at 0, 7, 14, 21, and 28 days after treatment, as described previously (Ryu et al., 2005b). Briefly, root samples were collected and, after removal of soil particles, roots were agitated in 20 ml of SDW in a flask. Samples of 10-fold serial dilutions were plated onto TSA containing appropriate antibiotics, as above. CFUs were counted after 1–3 days.

# Assessing the Antifungal Capacity of Root Exudates

An in vitro assay was developed to test the antifungal capacity of root exudates (**Figure 2A**). Pepper seeds (cv. Bukwang) were prepared as described above. After 7 days of germination at 25◦C, seeds were transferred to Petri dishes (diameter = 20 cm) and allowed to grow vertically. Plates were sealed with Saran wrap to retain moisture, and were half covered with aluminum foil (Daihan Eunpakgy Ind. Co., Ltd., Suwon, South Korea) to reduce exposure of roots to light from growth cabinets, which were set at 24 h light, 25◦C (Vision Bio Tech., Seoul, South Korea). Four days after transplanting, 5 ml of 2,3-butanediol (1 mM or 1 µM) was drench-applied to the root system. Drenches with 1 mM benzothiadizole (BTH), which was commercialized SAR trigger by Syngenta as Actigard in the USA and BION in Europe and water were used as positive and negative controls, respectively. After cultivation for 2 days on potato dextrose broth agar (PDA, Becton, Dickinson and Company, Sparks, MD, USA) at 30◦C, fungal spores were collected and their concentration was estimated using a hemocytometer. A sterilized cotton swab was used to inoculate the pepper root system with 105 CFU/ml fungus, avoiding direct contact with the root surface. To determine any inhibitory effects of the root exudate on fungal growth, growth of fungal mycelium was imaged daily for a week after spore inoculation using a digital camera (Nikon Coolpix 4500, Japan). Fungus-free zones around the pepper roots were measured, and the mean was calculated (n = 20). The experiments were repeated three times.

# ITS-Based Fungus Identification

Fungus was isolated from pepper rhizosphere during assessing population density of B. subtilis. The fungus was cultured on the Poate Dextrose Broth agar (TSA, BactoTM, BD, Sparks, MD, USA) Total genomic DNA was extracted from the purified isolates using AccuPrepr Genomic DNA Extraction Kit (Bioneer, Daejeon, South Korea). The nuclear ribosomal internal transcribed spacer (ITS) region of genomic DNA was amplified with ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 - TCCTCCGCTTATTGATATGC-30 ) primers using Quick PCR Premix containing Taq DNA polymerase, dNTPs, reaction buffer, and tracking dye (Genenmed, Daejeon, South Korea). PCR analyses were conducted in a PTC100 Thermal Cycler (MJ Research, Watertown, MA, USA) using an initial denaturation step of 95◦C for 5 min; followed by 29 cycles of denaturation for 1 min at 94◦C, primer annealing for 30 s at 52◦C, and extension for 30 s at 72◦C; with a final extension for 10 min at 72◦C. Amplified PCR products were detected by electrophoresis on a 0.75% agarose gel, and purified with AccuPrepr PCR Purification Kit (Bioneer, Daejeon, South Korea). The ITS region of the yeast isolates was sequenced using the same PCR primers and the ABI3700 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequence was submitted to NCBI1 for identification of the fungus.

# Collection of Root Exudates after Induction by 2,3-Butanediol Treatment

A new protocol was developed to collect root exudates from pepper seedlings. Seeds were germinated as described above, then positioned between two sterile filter papers (diameter = 120 mm) in a Petri dish (diameter = 150 mm and height = 20 mm). MS broth (10 ml) was applied to the Petri dish, which was then positioned vertically in an incubator at 25◦C. After 4 days of incubation, excess MS broth was removed. A further 15 ml of MS broth containing 1 mM 2,3-butanediol, 1 µM 2,3-butanediol, or 1 mM BTH was drench-applied to filter papers. Treated Petri dishes were sealed with Saran wrap and partially covered with foil, as described above. Petri dishes were incubated at 25◦C for a further 7 days before collection of root exudates. To collect root exudate in the hydroponic system, we modified our system previously described (**Figures 2D,F** inset; Song et al., 2015). Pepper seeds were surface-sterilized and germinated, as described above. Four-days-old seedlings were transferred to plates (60 mm × 15 mm, SPL) containing 26 ml of 0.5X MS liquid media. Plates were placed in the plastic container (phytohealth, 103 mm × 78.6 mm, SPL). 2 mM 2,3-butanediol, BTH and water control treatments were applied to plants as described above. The root exudates were collected at 7 and 14 days after treatments. For each replicate, containing 16 plants, 80 ml of root exudate was collected from plates. No media contamination was observed in the entire experiment.

# Assessment of Root Exudates on Bacterial Growth

A 96-well based assay was used to assess the effects of root exudates on bacterial growth. B. subtilis strains 168, 2,3-B(−), and 2,3-B(++) were cultured in TSB containing antibiotics

1http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE\_TYPE=BlastSearch&BLAST\_SPEC= TargLociBlast

as detailed above, then washed three times in 0.8% NaCl solution. Bacterial growth was monitored in 150 µl volumes containing TSB and root exudate (1:1 ratio). Optical density was determined every 15 min using a Bioscreen C system (Fluoroskan; Labsystems, Helsinki, Finland) at 30◦C with continuous shaking.

# Bacillus Growth on the Different pH

The phenotype analysis for different pH was carried out by using a new tool, Phenotype MicroArrays (PMs). The 2,3-butanediol over-producer [2,3-B (++)] and non-producer [2,3-B (−)] were assayed on PM (Biolog) lane A1 to A12 of microplates PM10, testing different pH range from 3.5 to 10. PM technology uses the irreversible reduction of tetrazolium violet to formazan as a reporter of active metabolism. All procedures were performed as indicated by the manufacturer and previous study (Zhang and Biswas, 2009). Strains were grown at 30◦C on BUG agar (Biolog), and then, each strain was picked with a sterile cotton swab from the agar surface and suspended in 15 ml of inoculation fluid (IF-0; Biolog) until a cell density of 85% transmittance was reached on a Biolog turbidimeter. In order to inoculate microplates PM10, 1% tetrazolium violet (vol/vol) was added to the suspension and the mixture was inoculated (100 µl per well). The photo was taken at 24 h after bacterial inoculation.

# Quantitative RT-PCR

Expression analysis of 2,3-butanediol-elicited defense genes was performed using quantitative real-time polymerase chain reaction (qRT-PCR), as described previously (Yang et al., 2011). Expression of C. annum basic β-1,3-glucanase (CaPR 2), 1-aminocyclopropane-1-carboxylic acid (CaACC), Systemic Acquired Resistance 8.2 (CaSAR8.2), phenylalanine ammonia (CaPAL), lipid transfer protein (CaLTP 1), glutathione S-transferases (CaGST), and basic class II chitinase (CaChi2) was reported previously during the defense response (Marrs, 1996; Garcia-Pineda and Lozoya-Gloria, 1999; Jung and Hwang, 2000; Lee et al., 2001; Hong and Hwang, 2002; Park et al., 2002; Mateos et al., 2009; Mazourek et al., 2009). The following primers were used: 50 -TAGTGAGACTAAGAAAGTTGGACG-30 (CaS AR8.2 Forward; GenBank accession no. AF327570.1), 50 -AA GAGTGCATGCAGTATCACAAAG-30 (CaSAR8.2 Reverse), 50 - ATTGGACGATGGAAGCCATCACCAG-30 (CaChi2 Forward; GenBank accession no. AF091235.1), 50 -ATATTCCGAATGT CTAAAGTGGTAC-30 (CaChi2 Reverse), 50 -TTTTAGCTATG CTGGTAATCCGCG-30 (CaPR2 Forward; GenBank accession no. AF227953.1), 50 -AAACCATGAGGACCAACAAAAGCG-3 0 (CaPR2 Reverse), 50 -CTCTAGGAAGGTGCTGTGGTGT C-30 (CaLTP1 Forward; GenBank accession no. AF118131.1), 5 0 -ACGGAAGGGCTGATTTCGGATG-30 (CaLTP1 Reverse), 50 -TCCACAAAGGGTCATGGTTT-30 (CaGST Forward; Gen-Bank accession no. HQ010689.1), 50 -GCCCTCTTCAATGA CAGGAA-30 (CaGST Reverse), 50 -ATTCGCGCTGCAACTAAG AT-30 (CaPAL Forward; GenBank accession no. EU61657- 5.1), 50 -CACCGTGTAAGGCCTTGTTT-30 (CaPAL Reverse), 5 0 -AGTGGCCTTCAACTCCTCAA-30 (CaACC Forward; Gen-Bank accession no. AJ011109.1), and 50 -TTCCGTTTGTGATCA CCTCA-30 (CaACC Reverse). Relative mRNA levels were calibrated and normalized to the level of CaActin mRNA (Gen-Bank accession no. AY572427). As a control, to ensure that equal amounts of RNA were used in each experiment, CaActin was analyzed using the primers 50 -CACTGAAGCACC CTTGAACCC-30 and 50 -GAGACAACACCGCCTGAATAGC-3 0 . Candidate priming genes were amplified from 100 ng of cDNA by PCR using an annealing temperature of 55◦C. A Chromo4 real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) was used to carry out qRT-PCR. Reaction mixtures (20 µl) contained 10 µl of 2× Bril-liant SYBR Green QPCR master mix (Bio-Rad Laboratories, Hercules, CA, USA), cDNA, and 10 pmol of each primer. The thermocycle parameters were as follows: 10 min at 95◦C, followed by 45 cycles of 30 s at 95◦C, 60 s at 55◦C, and 30 s at 72◦C. Conditions were determined by comparing threshold values in a series of dilutions of the RT product, followed by a non-RT template control and a non-template control for each primer pair. Relative RNA levels were calibrated and normalized to the level of CaACT1 mRNA (GenBank accession no. AY572427).

# Statistical Analysis

Data were subjected to ANOVA using JMP software version 4.0 (SAS Institute, Cary, NC, USA). Significance of biological or chemical treatment effects was determined by the magnitude of the F-value at P = 0.05. When a significant F-value was obtained for treatments, separation of means was accomplished using Fisher's protected LSD at P = 0.05. Results of repeated trials of each experiment outlined above were similar, and one representative trial of each experiment is reported.

# RESULTS

# Effect of 2,3-Butanediol Production on Rhizosphere Competence of Bacillus subtilis

The role of bacterial volatile 2,3-butanediol in situ was examined in wild-type B. subtilis 168 and wild-type-derived null and overexpression strains. Wild-type B. subtilis 168 was previously shown to produce 2,3-butanediol (Ryu et al., 2004). A pta-als double mutant, 2,3-B(−), was unable to produce 2,3-butanediol, and the pta mutant 2,3-B(++) was an overproducer of 2,3 butanediol. Rifampicin resistance was generated in the three strains to allow selection from the pepper roots (data not shown). We hypothesized that the population densities of the three strains in the pepper rhizosphere at different time points (7 days intervals from inoculation) would differ. Total bacterial populations on pepper roots at inoculation were 107–108 cfu/g root and did not significantly differ between treatments (**Figure 1C**). Initial populations of each strain were ∼107 cfu/g root [7.0, 7.2, and 7.0 log cfu/g root for 2,3-B(++), 2,3-B(−), and 168, respectively]. After 7 days, populations were 7.3, 6.1, and 5.3 log cfu/g root for 2,3-B(++), 2,3-B(−), and 168, respectively. After 14 days, the population densities of 2,3-B(++) and 168 were 2.8- and 3-fold higher than that of 2,3-B(−), respectively (**Figure 1A**). Populations of strains 168 and 2,3-B(++) remained at pepper

roots 21 days after inoculation, at 2.8 and 4.8 log cfu/g root, respectively (**Figure 1A**). Strain 2,3-B(−) was not found at pepper roots 21 days after inoculation. After 28 days, only strain 2,3-B(++) was present at pepper roots, at 102 cfu/g root (**Figure 1A**). These results indicated that 2,3-butanediol facilitated maintenance of bacterial populations in the pepper rhizosphere. Unexpectedly, fungal colonies developed on the TSA plates used for isolation of B. subtilis from roots. The fungus was identified 98% as Trichoderma sp. from morphological characteristics and sequencing of the 18S ribosomal RNA ITS (data not shown). No fungal colonies were observed on isolation plates for 2,3-B(++) at 14, 21, and 28 days after inoculation (**Figure 1B**). Larger fungal populations were isolated from roots treated with strains 168 and 2,3-B(−) (**Figure 1B**). Fungal populations were 5.2, 5.3, and 5.4 log cfu/g root in the 2,3-B(−) treatment at days 14, 21, and 28, respectively (**Figure 1B**). For treatment with strain 168, fungal populations gradually decreased with time, at 6.1, 5.8, and 4.2 log cfu/g root at days 14, 21, and 28 respectively (**Figure 1B**). The number of Trichoderma sp. showed similar pattern when the repeated experiment was conducted. These results suggested that fungal growth could be directly inhibited by 2,3-butanediol. To test this, the fungus was challenged by pharmacological applications of 2,3-butanediol at different concentrations (10 µM–1 mM); however, no inhibition was observed, indicating that 2,3-butanediol did not directly affect fungal growth (**Figure 1B**). These results suggested that exposure of pepper roots to 2,3-butanediol might trigger the production of root exudates antagonistic to fungal growth.

# Indirect Effect of 2,3-Butanediol on Inhibition of Soil Fungus

To determine whether exudates of pepper roots treated with 2,3-butanediol contained antifungal agents, a novel protocol was devised in which seedlings were cultivated on Petri dishes, drenched with 2,3-butanediol, and inoculated with fungus (**Figure 2A**). Fungal growth was inhibited with all treatments (1 mM 2,3-butanediol, 1 mM BTH, and water; **Figures 2B–D**), with clear root inhibition zones of 2.45, 1.41, and 0.875 mm, respectively (**Figure 2E**). These results indicated that root exudates elicited by treatment with 2,3-butanediol and BTH inhibited the growth of soil fungus. We therefore wished to test whether root exudates elicited by 2,3-butanediol could also inhibit the growth of other microorganisms such as saprophytic and pathogenic soil bacteria. To obtain the clear evidence of antifungal capacity in the root exudate from pepper plant treated with 2,3-butanediol, the hydroponic system was set-up and successfully obtained enough root exudates. The three time inoculation of root exudate from 2,3-butanediol pre-treated root only showed clear zone (No. 4 in **Figures 2E,F**) while one time root or chemical alone treatments did not show any inhibitory effect (**Figures 2E,F**).

# Growth Kinetics of B. subtilis after Exposure to 2,3-Butanediol and Root Exudates

To understand the role of root exudates (**Figure 3A**), and their effects on growth of B. subtilis strains 168, 2,3-B(++) (strain BSIP1171), and 2,3-B(−) (strain BSIP1174) and other soil-borne bacterial species, the growth kinetics of each treatment were assessed. B. subtilis strains 168, 2,3-B(++), and 2,3-B(−) had similar growth patterns on TSB medium, with maximum optical density of OD600 = 1 indicating that the mutation of the garget genes did not affect bacterial robustness under ideal growth condition (**Figure 3B**).

water treatments. Different letters above bars indicate significant differences between treatments as determined using LSD at P = 0.05.

When exposed to root exudate from pepper root elicited by 2,3-butanediol treatment, growth of strains 168 (the wild type) and 2,3-B(++) (overexpression mutant) was less inhibited than that of 2,3-B(−) (null mutant; **Figure 3C**). After 2 days (48 h), growth of overexpresser strain 2,3-B(++) was higher when treated directly with 1 mM 2,3-butanediol (control) than when treated with exudates of 2,3-butanedioltreated pepper root (**Figure 3C**). Until log phase, the growth curve of strain 168 was similar between bacteria exposed to control and exudates (**Figure 3B**); nevertheless, after log phase, growth of control-treated 168 exceeded that of exudate-treated 168 (**Figure 3B**). Conversely, growth of exudate-treated 2,3- B(++) slightly exceeded that of control-treated 2,3-B(++) (**Figure 3C**). Furthermore, 2,3-B(−) was more sensitive to 2,3 butanediol itself while 2,3-B(++) was more resistant compared to wild type indicating that 2,3-butanediol non-producer can be less fitness than 2,3-butanediol producer (**Figure 3C**). The data suggested that 2,3-butanediol played an important role in protecting B. subtilis cells against harmful plant root exudates.

We next examined the effect of 2,3-butanediol on other soil bacteria, namely, the non-pathogenic biological control

FIGURE 3 | Growth kinetics of B. subtilis wild-type and mutant strains after exposure to root exudates elicited by 2,3-butanediol. Initial cell culture concentrations were OD600 = 0.02 and data are shown as log-normal plots. Tryptic soy broth supplemented with root exudate at a 1:1 ratio was applied to pepper roots. Aliquots (150 µl) from each culture were transferred to 100 wells of a Bioscreen plate. Plates were incubated in a Bioscreen C (Fluoroskan; Labsystems, Helsinki, Finland) with shaking at 30◦C for ∼4 days. The OD600 of each well was measured every 15 min. 2.3B = 2,3-butanediol; 23BE = root exudate collected from 2,3-butanediol-treated root system; 168 = B. subtilis 168; BSIP 1174 = B. subtilis BSIP 1174 (non-producer); BSIP 1171 = B. subtilis BSIP 1174 (overproducer); Pf-5 = Pseudomonas protegens Pf-5; GMI1000 = Ralstonia solanacearum GMI1000; M = MS broth; ME = MS broth amended with root exudate without treatment. (A) Schematic of protocol to extract root exudates after 2,3-butanediol application. (B) Growth kinetics of the three strains in control TSB medium. The figure indicate background expression of three strains 168, 2,3-B(++), and 2,3-B(−). (C) Growth of bacterial strains 168, 2,3-B(++), and 2,3-B(−) after treatment with 2,3-butanedol alone (referred to as 2,3B) or 2,3-butanediol-elicited root exudate (referred to as 2,3BE). (D) Growth kinetics of P. protegens Pf-5, Ralstonia solanacearum GMI1000, and E. coli. P. protegens Pf-5 is a non-pathogenic saprophyte that inhabits soil, water, and plant surface environments. Growth of P. protegens Pf-5 was not inhibited by 2,3-butanediol-elicited exudate. Growth of GMI1000, a soil-borne bacterial wilt pathogen, was inhibited by root exudates. E. coli was included as a bacterial control. Data shown are mean ± SEM of triplicate experiments.

agent Pseudomonas protegens Pf-5, the bacterial wilt pathogen R. solanacearum GMI1000, and E. coli. Growth of the nonpathogenic saprophyte Pf-5 did not much affected by amendment of 2,3-butanediol-elicited root exudate (M + Pf-5, ME +Pf5, 2,3B +Pf5, and 2,3BE + Pf-5; **Figure 3D**). However, bacterial growth upon 2,3BE + Pf-5 gradually decreased after 60 h. In contrast growth of the pathogen GMI1000 was inhibited by exudates from pepper roots treated with 2,3-butanediol at 42 h after root exudate treatment (2,3B + GMI1000 vs. 2,3-BE + GMI1000). Although, the treatment ME (MS media solution plus root exudate without 2,3-butanediol treatment) also showed inhibitory effect on growth of strain GMI1000, the inhibition by root exudate collected from 2,3-butanediol treatment was greater (ME + GMI1000 vs. 2,3-BE + GMI1000). Growth of E. coli was totally inhibited by all treatment including control 2,3-butanediol treatments, TSB, MS, and root exudate mixtures (**Figure 3D**). These results indicate that 2,3-butanediol-elicited root exudate contains compounds that allow selective inhibition of bacterial growth depending on bacterial species. The bacterial growth upon different pH condition using by Phenotype Microarray system showed that the growth of 2,3-B(++) and 2,3-B(−) was indicated at pH 5 and pH 7 respectively (**Figure 4**). This results clearly showed that 2,3-butanediol production acts an important role on bacterial fitness under the acidic pH condition.

# Expression of Defense Genes in 2,3-Butanediol-Treated Pepper Roots

Induction of plant defense genes by 2,3-butanediol was assessed in pepper roots using qRT-PCR. At 3, 6, and 12 h after treatment, transcription of basic β-1,3-glucanase (CaPR 2) was higher in pepper roots treated with 1 mM 2,3-butanediol than in those treated with water (**Figure 5**). The effects of 2,3-butanediol on pathogen populations and gene expression in pathogenchallenged pepper roots were also assessed. Roots were exposed to the wilt pathogen R. solanacearum GMI1000 for 3 days, and bacterial populations were then determined. Fewer GMI1000 bacteria were recovered from roots treated with 1 mM and 10 µM 2,3-butanediol than from roots treated with water (**Table 1**). Root exudates of 2,3-butanediol-treated pepper were therefore able to inhibit growth of the bacterial pathogen GMI1000. Three days after pathogen challenge, expression levels of Basic pathogenesis systemic acquired resistance gene 8.2 (CaSAR8.2) and

phenylalanine ammonia (CaPAL) were higher in roots treated with 1 mM 2,3-butanediol than in positive control roots treated with 1 mM BTH (**Figure 5**). Conversely, expression levels of 1-aminocyclopropane-1-carboxylic acid (CaACC), lipid transfer protein (CaLTP 1), and basic class II chitinase (CaChi 2) were lower in roots treated with 1 mM and 10 µM 2,3-butanediol than in positive control roots, but higher than in negative control roots treated with water (**Figure 5**). Transcription of basic β-1,3-glucanase (CaPR 2) was similar in roots treated with 1 mM and 10 µM 2,3-butanediol and roots treated with 1 mM BTH (**Figure 5**).

# DISCUSSION

The first examinations of bacterial volatile-mediated plant growth and ISR, which used B. subtilis and Arabidopsis thaliana (Ryu et al., 2003, 2004), were followed by numerous studies examining the effects of bacterial volatiles on plants. Of the many volatile compounds identified from bacteria, 2,3-butanediol generated particular interest due to its broad spectrum effects on bacterial cells and induction of host responses (Ryu et al., 2004; Xiao and Xu, 2007; Rudrappa et al., 2010; Moons et al., 2011; Hahm et al., 2012). However, the impact of 2,3-butanediol on bacterial cells is yet to be elucidated. This prompted us to ask why bacteria, and soil bacteria in particular, might secrete 2,3-butanediol. Our results suggest that 2,3-butanediol promotes bacterial cell robustness against the effects of harmful compounds, such as root exudates (**Figures 1A** and **3C**). Both 2,3-butanediol and its precursor acetoin were shown to trigger ISR in plants (Han et al., 2006; Cortes-Barco et al., 2010a,b; Rudrappa et al., 2010; Hahm et al., 2012). Our study provides new information regarding the roles of 2,3-butanediol in root-associated bacteria in situ.

The volatile compound 2,3-butanediol is produced by many bacterial species as a result of a synthetic cascade, termed butanediol fermentation (Xiao and Xu, 2007). The exact role that butanediol fermentation plays in bacterial fitness is largely unknown. Classic literature suggested that 2,3-butanediol was formed to divert the cellular metabolism away from production of acidic compounds (Johansen et al., 1975). It was later discovered that 2,3-butanediol provided an alkaline environment during cell multiplication and protected bacterial cells against unfavorable acidic conditions, such as are found in eukaryotic hosts (Yoon and Mekalanos, 2006; Pradhan et al., 2010; Bari et al., 2011). Our results showed that a 2,3-butanediol null B. subtilis mutant was eliminated from the rhizosphere by 21 days

TABLE 1 | Effect of 2,3-butanediol on elicitation of plant immunity against Ralstonia solanacearum.

The bacterial cell count was measured at 14 days after pathogen inoculation. Different letters indicate significant differences between treatments (P = 0.05 according to least significant difference). The experiment was repeated three times with similar results (sample size, n = 5 replications per treatment).

after inoculation; however, corresponding wild-type and 2,3 butanediol overexpressing strains persisted for 21 and 28 days, respectively (**Figure 1A**). This can be hypothesized that 2,3 butanediol production by B. subtilis increased robustness of the acidic rhizosphere environment similar with rhizosphere microorganism (Huang and Chen, 2003; Hinsinger et al., 2003). Root exudates, which include acidic root secretion products, contribute to lowering the rhizosphere pH by releasing H+ or OH− to compensate for unbalanced cation–anion uptake at the root surface (Hinsinger et al., 2003). In our system, the pH of root exudate was changed to 4.5 at 2 weeks after treatments compared to pH 5.8 at the beginning of experiment (data not shown). However, there are no difference upon pH between pretreatment of 2,3-butaendiol and water control. In addition to the proposed protective role of 2,3-butanediol in bacteria, 2,3 butanediol was recently shown to be critical for virulence of soft-rot plant pathogenic Pectobacterium spp. and Dicheya spp. Cell wall-degrading enzymes produced by the bacteria, such as protease, pectinase, and cellulose, require neutral pH for optimal function (Kwan et al., 2013). Bacterial robustness under acidic conditions and on normal artificial medium was compromised in a Serratia plymuthica budB mutant (Wevers et al., 2009).

In addition to the role of 2,3-butanediol as a bacterial protectant, 2,3-butanediol directly affects plant physiology and immunity (Han et al., 2006; Cortes-Barco et al., 2010a,b; Rudrappa et al., 2010; Hahm et al., 2012). In this study, the 2,3-butanediol null mutant could not stimulate plant defenses; however, wild-type B. subtilis successfully elicited a plant defense response against pathogens, indicating that bacterial 2,3-butanediol production played an important role in plant protection (Ryu et al., 2004; Rudrappa et al., 2010). No direct inhibition was observed when pathogenic bacteria and fungi were exposed to 2,3-butanediol, indicating that plant immunity rather than the direct effect of 2,3-butanediol provided inhibition (**Figure 1C**). Root application of 2,3 butanediol triggered root exudation and secretion of unknown compounds that differentially affected different species of bacteria in the rhizosphere. Exudate from roots treated with 2,3-butanediol suppressed growth of the soil-borne pathogen R. solanacearum, but enhanced growth of the saprophytic biocontrol bacterium P. protegens Pf-5 (**Figure 3D**). Our extra bioinformatics analyses revealed support the role of microbial production of 2,3-butanediol upon its robustness in rhizosphere: the P. protegens Pf-5 genome contains three major genes, acetolactate synthase (alsS), alpha-acetolactate decarboxylase (alsD), and 2,3-butanediol dehydrogenase (bdhA), needed for 2,3-butanediol production but R. solanacearum contains only the alsS gene with the result that less or no 2,3-butanediol can be produced (data not shown). Similarly, 2,3-butanediol null mutant of B. subtilis became more sensitive to pepper root exudates while the overexpressor of 2,3-butanediol was more resistance compared to wild type strain (**Figure 3C**). Moreover, 2,3-butanediol production help bacterial cells tolerate against acidic pH such as pH5 (**Figure 4**). Interestingly, the root exudate pH was stabilized as pH 4.5 in our hydroponic system indicating that B. subtilis may optimize the robustness using 2,3-butanediol production to acidification around root. Another possible explanation is that the two species may have different sensitivities to unknown compounds within the 2,3-butanediol-elicited root exudate. In our previous research, aboveground infestation of sucking insects like whitefly and aphids modulated the secretion of plant root exudates, leading to the recruitment of specific microbiota such as Gram-positive Bacillus spp. (Yang et al., 2011; Lee et al., 2012). Further research revealed that whitefly-infested tobacco plants secreted salicylic acid, which repressed Agrobacterium tumefaciens virulence genes and resulted in the suppression of crown gall formation (Song et al., 2015). We propose that plant defenses were induced by soil application of 2,3-butanediol, and that this induced the secretion of unknown compounds that targeted bacteria in a species-dependent manner. Detailed profiling of root exudates is required to characterize the compounds involved. The root exudate profiling was failed due to limitation to obtain large scale root exudates following 2,3-butanediol application.

Finally, one additional explanation is that different species may have different utilization capacities for root exudates. P. protegens Pf-5 is a saprophyte and encodes numerous enzymes that degrade organic materials, while R. solanacearum is a plant pathogen that primarily obtains nutrition from specific plant materials within xylem sap during the infection process (Salanoubat et al., 2002; Loper et al., 2007, 2012; Remenant et al., 2010).

The direct effect of 2,3-butanediol on pepper roots was demonstrated by changes in gene expression. Transcription of CaPAL, CaSAR8.2, CaACC, and CaPR2 was affected when roots were drenched with 2,3-butanediol (**Figure 5**). This suggested that bacterial secretion of 2,3-butanediol activated plant defenses in the roots, mainly via salicylic acid and ethylene signaling pathways. This is supported by recent research in which direct soil application of 2,3-butanediol stimulated defense responses against foliar pathogenic anthracnose fungus and Pseudomonas syringae (Han et al., 2006; Cortes-Barco et al., 2010a,b). Previous studies showed 2,3-butanediol to be a signaling molecule involved in activation of immune responses in animal hosts. In mammals, 2,3-butanediol produced by pathogenic bacteria was closely associated with lung infections, including those caused by Klebsiella pneumonia, Staphylococcus aureus, and Serratia marcescens. Under these conditions, 2,3 butanediol produced an anti-inflammatory effect via inhibition of NF-κB signaling (Hsieh et al., 2007). Taken together, these

In summary, this study demonstrates that the bacterial volatile 2,3-butanediol has two key roles in the rhizosphere. First, 2,3-butanediol-elicited root exudates selectively affect different bacterial species, and, secondly, 2,3-butanediol protects bacterial cells against putative harmful plant root exudates and low pH. To our knowledge, this study is the first to demonstrate the significance of 2,3-butanediol on bacterial robustness in planta.

# AUTHOR CONTRIBUTIONS

H-SY, Y-RA, S-YG, and C-MR conceived and designed research. H-SY, Y-RA, GCS, GL, and C-MR conducted all experiments. The

# REFERENCES

manuscript was written by H-SY, Y-RA, and C-MR and approved by all other authors. SL conducted the pH sensitivity and provide **Figure 4**. The **Figure 4** was input during revising the manuscript.

# ACKNOWLEDGMENTS

This research was supported by grants from grants from BioNano Health-Guard Research Center funded by the Ministry of Science, ICT, and Future Planning of Korea as a Global Frontier Project (Grant H-GUARD\_2013M3A6B2078953), the Next-Generation BioGreen 21 Program (SSAC grant #PJ009524), Rural Development Administration, South Korea, and the KRIBB initiative program, South Korea.

carotovora but against Pseudomonas syringae pv. tabaci in tobacco. Mol. Plant Microbe Interact. 19, 924–930. doi: 10.1094/mpmi-19-0924

fmicb-07-00993 June 24, 2016 Time: 13:58 # 10

**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 © 2016 Yi, Ahn, Song, Ghim, Lee, Lee and Ryu. 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) or licensor 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.

fmicb-07-00993 June 24, 2016 Time: 13:58 # 11

# Augmenting Sulfur Metabolism and Herbivore Defense in Arabidopsis by Bacterial Volatile Signaling

Mina Aziz1,2, Ranjith K. Nadipalli1 , Xitao Xie1 , Yan Sun1 , Kazimierz Surowiec1 , Jin-Lin Zhang3 and Paul W. Paré1 \*

1 Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA, 2 Center for Plant Lipid Research, University of North Texas, Denton, TX, USA, 3 College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, China

Sulfur is an element necessary for the life cycle of higher plants. Its assimilation and reduction into essential biomolecules are pivotal factors determining a plant's growth and vigor as well as resistance to environmental stress. While certain soil microbes can enhance ion solubility via chelating agents or oxidation, microbial regulation of plant-sulfur assimilation has not been reported. With an increasing understanding that soil microbes can activate growth and stress tolerance in plants via chemical signaling, the question arises as to whether such beneficial bacteria also regulate sulfur assimilation. Here we report a previously unidentified mechanism by which the growth-promoting rhizobacterium Bacillus amyloliquefaciens (GB03) transcriptionally activates genes responsible for sulfur assimilation, increasing sulfur uptake and accumulation in Arabidopsis. Transcripts encoding for sulfur-rich aliphatic and indolic glucosinolates are also GB03 induced. As a result, GB03-exposed plants with elevated glucosinolates exhibit greater protection against the generalist herbivore, Spodoptera exigua (beet armyworm, BAW). In contrast, a previously characterized glucosinolate mutant compromised in the production of both aliphatic and indolic glucosinolates is also compromised in terms of GB03-induced protection against insect herbivory. As with in vitro studies, soil-grown plants show enhanced glucosinolate accumulation and protection against BAW feeding with GB03 exposure. These results demonstrate the potential of microbes to enhance plant sulfur assimilation and emphasize the sophisticated integration of microbial signaling in plant defense.

Keywords: plant growth-promoting rhizobacteria (PGPR), Bacillus amyloliquefaciens GB03, bacterial volatile organic compounds (VOCs), glucosinolates (GSL), sulfur assimilation, plant-defense priming

### Edited by:

Jesú Mercado-Blanco, Consejo Superior de Investigaciones Científicas, Spain

### Reviewed by:

Stanislav Kopriva, University of Cologne, Germany Ana Pineda, Nederlands Instituut voor Ecologie – Koninklijke Nederlandse Akademie van Wetenschappen, Netherlands Aurélien Bailly, University of Zurich, Switzerland

> \*Correspondence: Paul W. Paré<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Plant Biotic Interactions, a section of the journal Frontiers in Plant Science

Received: 14 December 2015 Accepted: 24 March 2016 Published: 08 April 2016

### Citation:

Aziz M, Nadipalli RK, Xie X, Sun Y, Surowiec K, Zhang J-L and Paré PW (2016) Augmenting Sulfur Metabolism and Herbivore Defense in Arabidopsis by Bacterial Volatile Signaling. Front. Plant Sci. 7:458. doi: 10.3389/fpls.2016.00458

**Abbreviations:** 1MOI3M, 1-methoxyindol-3-ylmethyl glucosinolate; 4MOI3M, 4-methoxyindol-3-ylmethyl glucosinolate; 4-MSOB, 4-methylsulfinylbutyl glucosinolate; 4-MTB, 4-methylthiobutyl glucosinolate; 5-MSOP, 5-methylsulfinylpentyl glucosinolate; 6-MSOH, 6-methylsulfinylhexyl glucosinolate; 7-MSOH, 7-methylsulfinylheptyl glucosinolate; 7-MTH, 7 methylthioheptyl glucosinolate; 8-MSOO, 8-methylsulfinyloctyl glucosinolate; 8-MTO, 8-methylthiooctyl glucosinolate; APK, APS kinase; APR, APS reductase; APS, adenosine 50 -phosphosulfate; ATPS, ATP sulfurylase; BAW, beet armyworm; DMDS, dimethyl disulfide; BCAT, branched-chain amino acid amino transferase; CFU, colony forming unit; CYP, cytochromes P6450; DDW, double-distilled water; ESI-MS, electrospray ionization-mass spectrometry; FMOGS−OX, flavin monooxygenase; GST, glutathione-s-transferase; I3M, indol-3-ylmethyl glucosinolate; IGMT, indole glucosinolate methyl transferase; IPMDH, isopropyl malate dehydrogenase; IPMI, isopropyl malate isomerase; MAM, methylthioalkyl malate synthase; MSG, methylsulfinylalkyl glucosinolates; MTG, methylthioalkyl glucosinolates; PAPS, 30 -phosphoadenosine 5 0 -phosphosulfate; PDA, photodiode array; PGPR, plant growth-promoting rhizobacteria; RT-PCR, reverse-transcription PCR; SOT, sulfotransferase; SUR, super root; TSA, tryptic soy agar; UHPLC, ultra-HPLC; VOCs, volatile organic compounds.

# INTRODUCTION

fpls-07-00458 April 6, 2016 Time: 17:36 # 2

Sulfur, a crucial element for plants, is ubiquitous in proteins, present in the antioxidant tripeptide glutathione, the Cysrich peptides phytochelatins that function in heavy metals detoxification and thioredoxins that are the major disulfide reductases responsible for maintaining the reduced state of proteins inside cells (Arnér and Holmgren, 2000; Cobbett, 2000). Sulfur can also be present in chloroplastic membrane lipids as well as certain coenzymes/vitamins (Falk et al., 2007). Sulfur is taken up by plants as inorganic sulfate via sulfate transporters and incorporated into APS by ATPS (Mugford et al., 2009). APS is then sequentially reduced by APR and sulfite reductase to sulfite and sulfide, and subsequently incorporated into O-acetylserine to form the sulfur containing amino acid cysteine. APS can also be phosphorylated to PAPS by the action of APK. PAPS is the sulfate donor for the formation of sulfated metabolites including glucosinolates, select flavonoids, phytosulfokines, and certain hormones.

From an ecological context, sulfur metabolites function in plant defense against pathogens and herbivores (Falk et al., 2007). Defensin and thionin peptides are sulfur-containing antimicrobial defenses with widespread plant distribution (Broekaert et al., 1995), whereas anti-feedant glucosinolates are limited to the Brassicale order (Falk et al., 2007). Brassica crops including cabbage, broccoli, cauliflower (Brassica oleracea) and rapeseed (B. napus) as well as Arabidopsis are rich in glucosinolates. In addition to these amino acid derivatives functioning in plant defense, glucosinolates are a nutritional source of sulfur and possess cancer-preventive properties (Sønderby et al., 2010). Glucosinolates are classified based on their amino acid precursor with aliphatic glucosinolates derived from Met, Ala, Leu, Ile, or Val; indolic glucosinolates derived from Trp; and aromatic glucosinolates derived from Phe or Tyr (Kliebenstein et al., 2001).

With plant damage, glucosinolates are rapidly converted into an array of toxic derivatives that can obfuscate phytochemical analysis. Enzymatically generated glucosinolate derivatives including isothiocyanates, epithionitriles, nitriles, and thiocyanates are produced in proportion to the amount of leaf damage as well as the reaction time (Halkier and Gershenzon, 2006; Wittstock and Burow, 2010; Winde and Wittstock, 2011). Therefore quantifying the pool of original glucosinolates requires deactivating the myrosinase enzyme before glucosinolates are enzymatically converted (Koroleva et al., 2000; Andréasson and Jørgensen, 2003; Zhao et al., 2008; Winde and Wittstock, 2011).

In addition to constitutive glucosinolate accumulation serving in chemical defense against herbivore damage, soil-borne microbes such as mycorrhizal fungi and PGPR can induce plant defense responses (van Loon, 2007; van Wees et al., 2008; Yang et al., 2009; Pineda et al., 2010, 2012). PGPR are naturally occurring soil microorganisms that colonize roots and stimulate plant growth. Such bacteria are applied to a wide range of agricultural crops for the purpose of growth enhancement, including increased seed germination, plant weight, harvest yields, and disease resistance (Kloepper et al., 1980, 1991, 1999). Bacillus subtilis (GB03), recently re-named

as B. amyloliquefaciens is a commercially available PGPR strain that can be introduced into the soil at the time of planting via seed coating since spores are stable over time (Choi et al., 2014). Unlike many plant-growth promoting rhizobacterial strains that activate plant growth by directly producing and releasing indole-3-acetic acid and/or gibberellins, GB03 emits a bouquet of volatile metabolites, devoid of classic phytohormones that are capable of triggering plant growth promotion (Ryu et al., 2003; Paré et al., 2005). These VOCs have been shown to activate differential expression of approximately 600 transcripts related to cell wall modifications, primary and secondary metabolism, stress responses, hormone regulation, and iron homeostasis (Ryu et al., 2003; Farag et al., 2006; Zhang et al., 2007). This Arabidopsis profiling of GB03-induced transcripts has resulted in a new paradigm for PGPR-mediated iron uptake. While some soil microbes are proposed to enhance iron mobility and uptake solely via production of bacterial siderophores (Neilands and Leong, 1986; Bar-Ness et al., 1992; Briat, 1992; Glick et al., 1999; Sharma et al., 2003), GB03 enhances Arabidopsis iron accumulation via activation of the plant's own iron acquisition machinery including the iron uptake-related genes FRO2 and IRT1 that encode for ferric reductase and iron transport enzymes, respectively (Zhang et al., 2009). GB03 also transcriptionally regulates the Fe-deficiency-induced transcription factor 1 (FIT1) that is necessary and sufficient for ferric reductase and iron transporter induction (Zhang et al., 2009). More recently, an upstream iron acquisition-related transcription factor MYB72 has been shown to be transcriptionally induced in Arabidopsis by bacterial VOCs with activation of the iron uptake-related genes FIT1, FRO2, and IRT1 (Zamioudis et al., 2015).

The current study reports a novel mechanism in which the growth-promoting rhizobacterium B. amyloliquefaciens strain GB03 induces Arabidopsis sulfur assimilation and accumulation by inducing the plant's own sulfur assimilation machinery. Moreover, the impact of GB03 in regulating primary and secondary sulfur metabolites to enhance plant defense against herbivory is examined.

# MATERIALS AND METHODS

# Plant Material and Treatments

Arabidopsis thaliana seeds were surface sterilized and stratified for 2 days at 4◦C in the absence of light. Seeds were planted in plastic Petri dishes (100 × 15 mm) containing a central partition (I-plates; Fisher Scientific), covered Magenta boxes (75 mm × 75 mm × 100 mm) or standard Petri dishes (150 mm × 15 mm), based on the specific experimental requirements. The bacterial culture is inoculated on the unplanted side of the partitioned plate, a glass vial (4 dr.) or a plastic plate (35 mm × 10 mm). All chambers contained halfstrength MS solid media prepared according to Murashige and Skoog (1962) with 1.5% (w/v) sucrose and 0.8% (w/v) agar (except where noted otherwise). Plants were grown under a 14-/10-h light/dark cycle with metal halide and high pressure sodium lamps for a total light intensity of 200 µmol photons m−2 s −1 ; temperature was 21 ± 4 ◦C and relative humidity 40 ± 10%. For

plant growth, the media surface was oriented horizontally for I-plates and Magenta boxes and vertically for the larger plates with media agar increased to 1.5% (w/v).

Bacillus amyloliquefaciens (GB03) was streaked onto TSA plates and incubated at 28◦C in the absence of light for 24 h. Cells were harvested in double distilled water (DDW) to yield 109 CFU mL−1 , as determined by optical density (OD600 = 0.7). Two days after seed germination, the bacterial suspension culture or DDW (25 µL for plates and 50 µL for Magenta boxes) was added to the non-plant portion of the chamber. Vials containing bacterial culture were replaced with fresh culture every 14 days.

For soil experiments, bacterial liquid cultures were mixed with sterile growing mix (Sunshine LC1 Mix; Sun Gro Horticulture, Canada) to a final PGPR concentration of 108 – 109 CFU/g soil. For water control, the bacterial suspension was replaced with sterile DDW. Seeds were sown in growing mix and fertilized weekly using 13:13:13 (N:P:K) fertilizer.

# Semi-quantitative RT-PCR

Plants were harvested 48- or 72-h after GB03 or water treatment. Total RNA was extracted using RNeasy plant mini kit (Qiagen, Valencia, CA, USA) with genomic DNA contamination excluded by DNase digestion. First strand cDNA was synthesized from 3- 5 µg total RNA using MuMLV-RT (Fisher Scientific, Houston, TX, USA); primer sequences are shown (**Table 1**). The PCR reaction included an initial 3 min denaturation at 94◦C, followed by 30 s at 94◦C, 30 s at 54◦C and 1 min at 72◦C with 24– 27 cycles (based on the optimized linear range for each pair of specific primers), a final 10 min extension at 72◦C (T100 Thermal Cycler, Bio-Rad, Hercules, CA, USA). No-reverse-transcription controls were included with the PCR runs to confirm the absence of DNA contamination. Agarose gel electrophoresis were imaged with a Kodak Gel Logic 100 Imaging System (Fisher Scientific, Houston, TX, USA) and quantified using Image J 1.33u1 (National Institute of Health, USA). TUB8 and UBQ10 were employed for normalization as they were uniformly expressed in all tissues examined.

# Total Sulfur Determination

Shoots and roots were separated, oven-dried, pulverized, and converted to dry-ash by heating at 550◦C for 3 h in the presence of Ag2O and NaHCO3 based on Kalra (1998). Dried tissue was then neutralized, diluted, and analysis via a barium chloride-gelatin turbidimetric assay (Tabatabai and Bremner, 1970). Standards were prepared as tissue material and diluted to a final concentration of 0–32 µg mL−1 . Total sulfur was quantified spectrophotometrically at 420 nm based on a sulfur standard curve.

### 35SO4 −2 Uptake Assay

For sulfate uptake measurements, plants were germinated on nylon mesh and grown vertically on media-containing plates with GB03 or water exposure for 11 days. Radio-labeling was initiated by submerging the roots into liquid media containing 37 MBq L−1 35SO4 −2 (Perkin–Elmer). After incubation for

1http://rsb.info.nih.gov/ij/

TABLE 1 | Sequence of primers employed in the semi-quantitative RT-PCR analysis.

30 min, roots were briefly rinsed with non-labeled medium to remove apoplastic radioactivity (modified protocol from Kataoka et al., 2004; Maruyama-Nakashita et al., 2004; Yoshimoto et al., 2007). After blotting, shoots and roots were weighed separately, transferred to scintillation vials and covered with 1 mL of 0.1 M HCl. Overnight-extracted samples were mixed with universal scintillation cocktail (4 mL; Fisher Scientific) and incorporated radioactivity measured by liquid scintillation counting.

# Cysteine Measurements

Whole plant tissue (0.1 g) was ground in liquid nitrogen and thiols were acid extracted using ice-chilled 0.1 N HCl (200 µL). The homogenate was centrifuged at 12,000 × g for 10 min at 4◦C. Supernatant aliquots were neutralized with 200 mM HEPES (pH 12.4), reduced with dithiothreitol and sulfhydryl groups derivatized with monobromobimane (VWR). Separation, detection and quantification of fluorescent adducts was based on Schupp and Rennenberg (1988).

# Glucosinolates Analysis

Plants were shoot and root separated, frozen in liquid nitrogen and lyophilized. Tissue (20–50 mg) was extracted for 15 min in boiling aqueous 7.5 mM Pb(OAc)2/Ba(OAc)2 (4 mL) with

0.57 µmol internal standard (sinigrin, Sigma–Aldrich) based on Reintanz et al. (2001). At room temperature, samples were gently shaken for 30 min, centrifuged at 4000 × g for 10 min and the supernatant was loaded on DEAE Sephadex A-25 column (120 mg, Sigma–Aldrich). Resin was rinsed with aqueous methanol (67%) and water and subsequently incubated with 50 µL sulfatase solution overnight (Graser et al., 2000). The resulting desulfoglucosinolates were eluted with 60% aqueous methanol (800 µL) and water (800 µL). The pooled extract was evaporated to dryness in vacuo and the residue was dissolved in HPLC-grade water (100 µL).

Desulfoglucosinolates were separated by HPLC on a Dionex Ultimate 3000 UHPLC system equipped with auto-sampler, column oven, and diode array detector. A C18 reversed phase column (Acclaim 120 mm × 3.0 mm, 150 mm × 3.0 mm i.d., 3-µm particle size) was run with a 400 µL/min flow rate at 25◦C; the injection volume was 10 µL. Elution was performed with a gradient (solvent A water; B acetonitrile) of 1.5 to 5% solvent B (6 min), 5 to 7% solvent B (2 min), 7 to 21% solvent B (10 min), 21 to 29% solvent B (5 min), and 29 to 57% solvent B (14 min), followed by a cleaning cycle (57 to 93% solvent B for 3 min, 6 min of hold, 93 to 1.5% solvent B for 3 min with a 5 min hold). Compounds were monitored at 229 nm.

Desulfoglucosinolates were identified by HPLC-PDA-MS based on method of Kusznierewicz et al. (2013). Samples were analyzed on a LCQ Fleet HPLC system equipped with PAL autosampler, Surveyor PDA detector, and Surveyor MS pump using an Alltima C18 reversed phase column (250 mm × 2.1 mm i.d., 5-µm particle size) with a 200 µL/min flow rate. The injection volume was 10 µL. Elution was performed with a gradient (solvent A water/0.1% formic acid; B acetonitrile/0.1% formic acid) of 1.5% solvent B (3 min) 1.5 to 13% solvent B (15 min), 13 to 33% solvent B (12 min), 33 to 57% solvent B (7 min), followed by a cleaning cycle (57 to 93% solvent B for 3 min, 6 min of hold, 93 to 1.5% solvent B for 3 min with a 5 min hold). Compounds were monitored by PDA at 229 nm, then subsequently by ESI-MS (LCQ Fleet Ion Trap MS) operated in positive ion mode, an acquisition time of 40 min with scanning from m/z 150 to 800 amu.

Previously reported desulfoglucosinolates were identified by MS via characteristic [M+H]+ and [M+Na]+ peaks except for 3-methylsulfinylpropyl glucosinolate (3MSOP) which could not be identified because of poor resolution. Positional isomers 4MOI3M and 1MOI3M with equivalent masses were differentiated based on retention time comparisons with literature values (Reintanz et al., 2001). Glucosinolates were quantified based on response factors established for individual desulfoglucosinolates relative to the internal standard at 229 nm (Brown et al., 2003).

# Herbivore Feeding

Spodoptera exigua (BAW) eggs were purchased from Benzon research (Carlisle, PA, USA). After hatching, neonate larvae were transferred to feed on artificial media for 6 days with a transfer to fresh media every 2–3 days. Since an acclimation period is required whenever larvae are transferred from one diet to another, 1 day before the experiment, third-instar larvae were transferred to feed on non-experimental wild-type Arabidopsis plants (Mewis et al., 2005). After this pre-feeding, larvae of the same developmental stage were weighted and transferred to 29-day-old GB03- or water-treated plants (one larva/plant); the initial average weight of larvae was recorded for both GB03 and water treatments. Shoot biomass was recorded after 56 h of feeding. Additional GB03-treated and untreated plants were reserved to serve as undamaged controls. Plants were harvested, rinsed, and weighted. Milligrams eaten per plant were calculated based on the weight difference between BAW eaten and uneaten plants. The quadruple glucosinolate knock-out mutant (myb28 myb29 cyp79b2 cyp79b3) was treated the same as Col-0.

For soil experiments, herbivore weights were collected. Neonate larvae were transferred to 28-day-old GB03- or water-treated plants with a transfer to fresh plants every 2– 3 days. Larvae weight was measured at 7 and 9 days after feeding.

# Statistical Analysis

For herbivore feeding experiments, statistical analyses were performed using R software2 . First, a Levene's test was performed to check the homogeneity of variance (Levene, 1960); homogeneous variance was achieved after transforming the data into the corresponding square root. Then, twoway ANOVAs were performed separately for wild-type and knock-out mutant lines. Tukey's method was used to do pairwise comparisons of means and an "lsmeans" package was used for means' grouping. For all other experiments, pairwise comparison of means was performed using Excel 2007 with significant difference between treatments was based on Student's t-test at P-values ≤ 0.05. The number of biological replicates is shown in each figure legend with minimum of three replicates.

# RESULTS

# Elevated Sulfate Assimilation with GB03 Exposure

The sulfate assimilation pathway with previously identified genes is depicted in **Figure 1A**. Mining whole-plant microarray data of GB03-exposed Arabidopsis seedlings identified sulfateassimilation gene induction for ATPS and APR. Of the four ATPS and the three APR isozymes present, ATPS1 and ATPS3 as well as APR1 and APR2 were found to be induced at 72 h post GB03 exposure (Supplementary Figure 1). RT-PCR analysis confirmed GB03 induction for ATPS1, ATPS2, and ATPS3 and all APR genes (**Figure 1B**). Another branch of sulfur assimilation involves APS conversion to PAPS by APK. There are four functional APK isoforms in Arabidopsis, among them APK1 and APK2 are the most active isoforms (Mugford et al., 2009). From the microarray data, both APK1 and APK2 are GB03 up regulated relative to controls (Supplementary

2www.R-project.org

Figure 1). APK transcript induction confirmation via RT-PCR analysis showed GB03 induction only in shoots (APK1, 1.3 ± 0.08; APK2, 1.5 ± 0.05). In addition, the amino acid cysteine, a precursor of many organic sulfur metabolites increased 28 ± 11, 32 ± 8, 37 ± 10, and 93 ± 15 % with GB03 exposure at 5, 7, 9, and 14 days, respectively (**Figure 1C**).

# GB03 Enhances Sulfur Accumulation and Uptake

As sulfate assimilation and reduction genes were GB03 induced, sulfur accumulation was examined. While total sulfur accumulation per tissue weight decreased ca. twofold in shoots 11 days post GB03 exposure, shoot sulfur accumulation per plant increased ca. 75% (**Figure 2A**). In roots, increases of ca. 50-fold and ca. 100-fold on a dry-weight and perplant basis, respectively, were observed (**Figure 2B**). To better characterize the process of inducible sulfur metabolism, plant sulfur movement was monitored with radioactive sulfate ( 35SO4 −2 ) to examine sulfur uptake and translocation. Although there was a ca. 30% reduction in total sulfur uptake per tissue weight, GB03 exposure enhanced total sulfur uptake per plant by ca. twofold, relative to untreated controls, within 30 min of radio-labeling (**Figure 3A**). Shoot sulfur translocation per tissue weight was ca. twofold less with GB03 treatment; however, similar translocation rate per plant was observed for both GB03 and controls (**Figure 3B**). And in roots, sulfur uptake and retention was higher with GB03 exposure on both a tissue weight and per-plant basis (**Figure 3C**).

Since select bacterial volatiles such as 2,3-butandiol have been previously shown to induce growth promotion and induced systemic resistance in Arabidopsis (Ryu et al., 2003, 2004), an array of 2,3-butandiol concentrations were assayed to examine for enhanced sulfur accumulation albeit no sulfur-associated changes were detected (data not shown). Similarly, collected bacterial volatiles reintroduced to plants also did not enhance sulfur accumulation.

# GB03 Induces Glucosinolate Biosynthetic Transcripts

The aliphatic and indolic glucosinolate biosynthetic pathways with previously identified genes is depicted in **Figures 4A,B**, respectively. Mining microarray data for transcripts encoding glucosinolate biosynthesis revealed that the majority of aliphatic

pathway genes are GB03 induced (Supplementary Figure 2A). For indolic glucosinolate biosynthesis, microarray data showed transcript induction limited to GSTF9, SUR1, UGT74B1, and SOT16 (Supplementary Figure 2B). GSTF9 is a GST which is responsible for the conjugation of the activated aldoximes to the sulfur donor glutathione, where the resulting S-alkylthiohydroximates are converted to thiohydroximates by a carbon-sulfur lyase, SUR1. Thiohydroximates are in turn S-glucosylated by the glucosyltransferases UGT74B1 to form desulfoglucosinolates. Finally, desulfoglucosinolates are sulfated to the corresponding glucosinolates by the sulfotransferase SOT16. Monitoring select shoot and root transcripts separately by RT-PCR confirmed gene induction with CYP79F1 induction in shoots ca. threefold, while root induction was ca. 30% (**Figures 4C,D**). CYP79F1 catalyzes the first committed step in biosynthesis of the aliphatic glucosinolate core structure that involves conversion of amino acids to corresponding aldoximes (a rate-limiting step in glucosinolates biosynthesis; Mikkelsen and Halkier, 2003). FMOGS−OX3, a gene that encodes one of the five flavin monooxygenases responsible for S-oxygenation of aliphatic glucosinolates resulting in conversion of MTG to MSG (Sønderby et al., 2010) was induced in shoots

within 48 h while root induction was delayed to 72 h (**Figures 4C,D**). SUR1 gene expression was induced ca. threefold in shoots (**Figures 4C,D**). To link transcriptional regulation with downstream glucosinolate accumulation, qualitative and quantitative glucosinolate analysis was performed.

# GB03 Induces Glucosinolate Accumulation

Desulfoglucosinolates were separated by HPLC based on relative polarity, with MSG eluting first in increasing order of their side-chain length, followed by indolic glucosinolates; long chain MTGs eluted last off the column (**Figure 5A**). GB03 exposure resulted in ca. 33 and 70% greater glucosinolate accumulation in shoots and roots, respectively (**Figure 5B**). Specifically, GB03

increased indolic glucosinolates in shoots (55%) and roots (twofold) while MSGs were induced in shoots by 45%. MTG accumulation differences with regard to tissue or GB03 treatment was not observed. In shoots, I3M was the most GB03 induced indolic glucosinolate (68%; **Table 2**); while among MSG, there was a 35, 37, 69, 73, and 65% GB03 induction of 4-MSOB, 5-MSOP, 6-MSOH, 7-MSOH, and 8-MSOO, respectively. For roots, the most abundant glucosinolate, 1MOI3M, increased threefold with no other statistically significant accumulation changes. Glucosinolate accumulation was not induced with plant exposure to 2,3-butandiol or collected bacterial volatiles (data not shown).

FIGURE 5 | Glucosinolate accumulation in Arabidopsis by GB03. Representative UHPLC-PDA chromatogram showing shoot glucosinolate profile in both GB03- and water-treated 30-day-old plants (A); an asterisk (∗ ) indicates peak alignment between the lower and upper chromatograms. GB03 induces total glucosinolate (GLS) accumulation in shoots (dark blue-GB03 versus light blue bar-H2O control) and roots (dark green-GB03 versus light green bar-H2O control) in 30-day-old plants (B). The aliphatic MSG and MTG as well as indolic glucosinolates are shown; an asterisk (∗ ) indicates statistically significant difference between treatments (t-test, P-value ≤ 0.05, n = 6, mean ± SE). Internal standard, sinigrin; 4-MSOB, 4-methylsulfinylbutyl glucosinolate; 5-MSOP, 5-methylsulfinylpentyl glucosinolate; 6-MSOH, 6-methylsulfinylhexyl glucosinolate; 7-MSOH, 7-methylsulfinylheptyl glucosinolate; 4-MTB, 4-methylthiobutyl glucosinolate; 8-MSOO, 8-methylsulfinyloctyl glucosinolate; I3M, indol-3-ylmethyl glucosinolate; 4MOI3M, 4-methoxyindol-3-ylmethyl glucosinolate; 1MOI3M, 1-methoxyindol-3-ylmethyl glucosinolate; 7-MTH, 7-methylthioheptyl glucosinolate; and 8-MTO, 8-methylthiooctyl glucosinolate.

TABLE 2 | GB03-induced glucosinolate (nmol/mg DW) accumulation with 30-day-old plants.

Bold values indicate statistically significant difference between treatments (t-test, P-value ≤ 0.05, n = 6, mean ± SE).

# GB03 Induces Plant Biomass Protection with Herbivory

GB03-treated plants were approximately twice the weight of water controls (**Figure 6A**). For feeding experiments, third instar larvae were pre-fed for 1 day on non-exposed Arabidopsis seedlings and initial BAW weight was monitored for larvae that were to feed on GB03 (46.39 ± 2.48) or water (46.48 ± 1.68) treated plants to exclude the effect of larvae weight and developmental stage variation on larval feeding. In addition, vials containing GB03 were removed from plant chambers before introducing BAW to avoid any direct interactions between bacterial volatiles and larvae as well as to avoid unequal PGPR-mediated growth for plants without BAW. With larval feeding, GB03-treated plants lost 24% shoot weight while controls lost 62% weight within 56 h of BAW feeding; plant tissue eaten per plant was 469 ± 54 and 658 ± 20 mg for GB03 and water treatments, respectively (t-test, P = 0.004, n = 11).

# GB03 Induces Glucosinolate-Dependant Plant Biomass Protection with Herbivory

For plants without herbivory, similar GB03-induced growth promotion was observed for both the Col-0 and a glucosinolate knock-out line (**Figures 6A–D**) compromised in both aliphatic and indolic glucosinolate production, myb28 myb29 cyp79b2 cyp79b3 (Müller et al., 2010). With larval feeding on the knockout line, shoot weight loss of 55% was observed for both GB03 and water treated plants; tissue consumed per plant was 962 ± 13

and 364 ± 11 mg for GB03 and water treatments, respectively (t-test, P = 1.89E – 20, n ≥ 11).

# GB03 Induces Plant Biomass Protection with Herbivory In Vivo

GB03-treated soil-grown Col-0 plants accumulate ca. 25% higher levels of glucosinolates compared to water controls in shoots for 35-day-old plants (**Figure 7A**). In addition, larval weight was lower when fed on GB03-treated plants for 7 and 9 days compared with controls (**Figures 7B–D**). Moreover, in the presence BAW, plant tissue eaten per plant was less for GB03 treated plants versus water controls (**Figures 7E,F**). A model for GB03-conferred protection against herbivory is proposed (Supplementary Figure 3).

# DISCUSSION

Several responses are induced in Arabidopsis by the PGPR strain GB03 including enhanced photosynthetic efficiency (Zhang et al., 2008), increased iron assimilation (Zhang et al., 2009) and elevated reproductive success (Xie et al., 2009), however, the ability of PGPR to induce sulfur assimilation via established mechanisms operational in plants has not been previously reported. Here is described that sulfur assimilation and glucosinolate biosynthetic genes are transcriptionally up regulated with GB03 exposure in Arabidopsis, from literature extracted microarray data (Zhang et al., 2007) and RT-PCR analysis. At the metabolite level, enhanced sulfate uptake along with elevated total sulfur, cysteine and sulfated aliphatic/indolic glucosinolate accumulation is observed. GB03-exposed plants also exhibit greater protection against the generalist herbivore BAW, while enhanced protection is compromised in a glucosinolate quadruple knockout line. Consistent with in vitro studies, GB03 enhanced glucosinolate accumulation and protection against larval feeding with soil-grown plants.

The PGPR strain Bacillus sp. B55 has previously been shown to promote tobacco growth by enhancing sulfur nutrition via uptake of sulfur volatiles including the major bacterial volatile component, DMDS (Meldau et al., 2013). B55 DMDS sulfur uptake observed in tobacco is subsequently incorporated into plant proteins and accompanied by reduced gene expression involved in sulfur assimilation, Met biosynthesis and sulfur recycling. In contrast, GB03 VOCs are low in sulfur emissions (Farag et al., 2006) and up-regulate genes that mediate sulfur assimilation. Since the volatilome has only been chemically characterized for GB03 (Farag et al., 2006), a unified mechanism for chemical incorporation and/or signaling inducing sulfur metabolism by GB03 and B55a is not possible. Moreover, different sulfur demands between glucosinolate-rich cruciferous plants such as Arabidopsis and glucosinolate-deficient tobacco (Falk et al., 2007), also prevents direct comparisons between the two sulfur induction studies.

In Arabidopsis, sulfate is taken up by roots and although root plastids contain the enzymatic machinery for sulfate reduction, sulfate conversion to sulfide and subsequent incorporation into cysteine predominantly takes place in shoot chloroplasts (Davidian and Kopriva, 2010). GB03 induces several Arabidopsis sulfate reduction genes including the key drivers of sulfate assimilation ATPS1 and APR2 (Loudet et al., 2007; Koprivova et al., 2013). While low-level gene activation does not constitute transcriptional regulation, the comprehensive induction of sulfate assimilation genes observed with GB03 exposure is consistent with coordinated transcriptional control. GB03 induced sulfur assimilation correlates with enhanced sulfur uptake and accumulation in roots. In shoots, sulfur uptake and accumulation per tissue weight is lower with GB03 exposure (**Figures 2** and **3**) which may be in part due to a dilution of plant sulfur with enhanced growth induced by GB03. On a whole-plant basis, sulfur uptake and accumulation is uniformly higher with GB03 treatment: 286.37 ± 59 versus 151 ± 35 pmol/30 min sulfur uptake and 732.6 ± 20 versus 391.36 ± 10 nmol accumulation for GB03 versus water treatments, respectively. Sulfur content in for in vitro grown Arabidopsis with and without GB03 exposure are consistent with published ICP-MS sulfur quantification under the same experimental conditions (Kwon et al., 2010). In addition to enhancing sulfur accumulation, GB03 has been previously shown to enhance Arabidopsis iron and copper accumulation (Zhang et al., 2009; Kwon et al., 2010), suggesting that this may be a coordinated effort by bacteria to increase plant growth by effectively enhancing the accumulation of essential elements.

GB03-induced sulfur assimilation enhances accumulation of cysteine, the precursor of methionine, GSH and subsequently select glucosinolates. Methionine is the main substrate for aliphatic glucosinolates (Ravanel et al., 1998) while the active sulfate donor for glucosinolate biosynthesis is PAPS, a phosphorylated derivative of APS produced by APK (Mugford et al., 2009; Sønderby et al., 2010). The crucial role of APK1 and APK2 in glucosinolate biosynthesis has previously been established using an apk1 apk2 double mutant which resulted in an 80% glucosinolate reduction and a concomitant increase in desulfoglucosinolates (Mugford et al., 2009). The transfer of the sulfate group from PAPS to the free hydroxyl group of desulfoglucosinolates is catalyzed by SOTs (Sønderby et al., 2010). The parallel transcriptional up-regulation of APKs and SOTs suggest a coordinated regulation of the sulfate donor formation and the sulfate transfer reaction by GB03.

The induction of glucosinolate accumulation in response to herbivore attack has been extensively studied (Mewis et al., 2006; Hopkins et al., 2009), however, much less is known with regard to microbial glucosinolate induction (van de Mortel et al., 2012). With the root-colonizing Pseudomonas fluorescens strain SS101 (Pf.SS101), the phytoalexin camalexin and glucosinolates were correlated with induced systemic resistance in Arabidopsis against several bacterial pathogens, including Pseudomonas syringae pv tomato (Pst). In addition, herbivore mortality rate was greater with BAW feeding on Pf.SS101-root-colonized Arabidopsis and mortality-rate differences with SS101 root inoculation were lost when an indolic glucosinolate deficient line was assayed (van de Mortel et al., 2012). In this current report, inducible sulfur assimilation and/or partitioning is/are linked with elevated endogenous glucosinolates, foliar plant biomass with herbivory

and larval weight. By monitoring enhanced plant protection against BAW feeding by bacterial volatiles albeit devoid of direct plant–bacteria contact, induced plant defense responses independent of potential confounding bacterial anti-feedant effects can be identified. Without BAW larvae present, GB03 induced plant growth in both the wild-type and glucosinolate mutant line (**Figure 6**), indicating that glucosinolates play no role in GB03-triggered growth promotion. However, greater GB03 induced growth promotion in the mutant line compared to Col-0 (**Figure 6C**) may be in part due to additional energy available for growth promotion without glucosinolate biosynthesis operative. With BAW herbivory, GB03-treated Col-0 plants lost less shoot weight than water controls (**Figures 6A,B**), indicating GB03 induction of plant defense(s). With such GB03 plant protection against larval feeding compromised in the glucosinolate mutant line (**Figures 6C,D**), a causal relationship is established between GB03-enhanced glucosinolate accumulation and conferred plant protection. Interestingly, in the mutant line without glucosinolate defenses present, larval-consumed plant tissue per plant was greater with versus without GB03 exposure; tissue consumed per plant was 962 ± 13 and 364 ± 11 mg for GB03 versus water treatments, respectively. Future experiments will examine if GB03-induced plants contain greater amounts of young leaves that have yet to accumulate non-glucosinolate based chemical defenses or if such plants dilute non-inducible chemical defenses making the GB03-induced glucosinolate mutant line more palatable for feeding larvae.

Soil-grown GB03-treated plants exhibited enhanced glucosinolate accumulation and plant protection against BAW is consistent with I-plate experiments; however, elicitation differences limit direct comparisons between in vitro and in vivo systems. For example, chemical signaling is confined to bacterial VOCs in vitro while non-volatile metabolites can also serve as potential signaling molecules in the in vivo soil system. Moreover, although the soil is sterilized before planting and bacterial inoculation, the non-sterile environment in which soil-grown plants are exposed is conducive to bacterial proliferate of leaves and roots by other bacterial strains besides GB03. Down-stream signaling pathways can also be differentially regulated in media and soil systems. For example, ethylene signaling is operative with in vivo PGPR signaling but not with in vitro growth promotion (Ryu et al., 2005). Future studies will examine several mutant lines to elucidate which of the different plant signaling pathways are involved in eliciting enhanced sulfur metabolism and protection against herbivores by GB03 both in vitro and in vivo. Moreover, since it has been widely recognized that the plant hormone jasmonic acid (JA) plays a crucial role in plant defense against pathogens and herbivores as well as in glucosinolate accumulation (van Dam et al., 2004; van Dam and Oomen, 2008), JA mutant lines will be assayed.

Since the growth promotion signal 2,3-butandiol (Ryu et al., 2003, 2004) as well as collected GB03 VOCs re-introduced to plants do not exhibit enhanced sulfur assimilation or glucosinolate accumulation a more effective absorbent may be necessary to trap biologically active bacterial volatiles. Alternatively, as the genome sequence of GB03 has been recently identified (Choi et al., 2014), testing different GB03 mutant lines

could help deciphering the effect of different VOCs products on inducing sulfur metabolism.

Glucosinolate accumulation differs between shoots and roots. Without GB03 exposure, total glucosinolates are higher in shoots with aliphatic glucosinolates being the most abundant compared with roots. The subclass of indolic glucosinolates accumulates predominately in roots as has been reported previously in Arabidopsis (Brown et al., 2003) as well as in other Brassica species (Rosa, 1997; Kirkegaard and Sarwar, 1998). An absence of detectable short chain aliphatic glucosinolates in roots (**Table 2**) is consistent with recent findings where rosette leaves are the major source and storage site for short chain aliphatic glucosinolates (Andersen et al., 2013). GB03 induces a 33 and 70% increase in total glucosinolate content in shoots and roots, respectively. Although tissue perception of bacterial VOCs is unknown, GB03 induced increase in glucosinolates is higher in roots, suggesting that GB03 may have initially been recognized as a pathogen with glucosinolates potentially induced as a defense mechanism. In fact, Arabidopsis indolic glucosinolates are pathogen-induced by Erwinia carotovora (Brader et al., 2001).

In agriculture, in addition to generating defense-rich plants, sulfur-rich cruciferous crops such as canola (Brassica napus L. cv) require uniform sulfur uptake independent of soil sulfur content (Scherer, 2001). Other plant specific soil bacteria, active in triggering canola growth promotion have been examined, although their role in regulating sulfur assimilation has yet

to be characterized (Kloepper et al., 1988; Bertrand et al., 2001). Commercial canola inoculants have been developed that oxidize elemental sulfur to the sulfate form that is more readily taken up by plants. Such bacterial inoculants are agriculturally relevant since elemental sulfur, an industrial byproduct, is economically viable for regenerating sulfur deficient soils. Here, GB03 transcriptionally induces sulfate assimilation and coordinates this process with enhanced sulfate uptake as well as elevated sulfur, cysteine, and glucosinolate accumulation. In addition to the role of glucosinolates in plant defense, select sulfur metabolites possess cancer-preventive properties. For humans, isothiocyanates derived from the hydrolysis of MSG are potent cancer-preventive agents (Hansen et al., 2007; Li et al., 2008). The cancer-preventive properties of MSG have been targeted for elevated production by plant breeders of cruciferous crops (Li et al., 2008) and here they are shown to be selectively induced by GB03, relative to other aliphatic glucosinolates.

# AUTHOR CONTRIBUTIONS

MA designed the project, performed experiments, collected data, analyzed results, and wrote up the study; RN performed experiments, collected data, and analyzed results; XX designed

# REFERENCES

the project, performed experiments, collected data, and analyzed data; YS collected data; KS collected data; J-LZ designed the project and analyzed the results; and PP designed the project, analyzed results, and wrote up the study.

# FUNDING

Financial support was provided in part by the Robert Welch Foundation (D-1478).

# ACKNOWLEDGMENTS

We would like to thank Prof. Barbara Ann Halkier at the University of Copenhagen for the generous gift of the glucosinolates mutant lines, Dr. Mohamed Ali Farag at Cairo University for the technical assistance with MS analysis, and Gamage Pemantha Lakraj at Texas Tech University for assistance with the statistical analyses.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.00458

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component of low-affinity sulfate transport system in the root vasculature. Plant Physiol. 136, 4198–4204. doi: 10.1104/pp.104.045625

fpls-07-00458 April 6, 2016 Time: 17:36 # 13

Brassica species. New Phytol. 161, 801–810. doi: 10.1111/j.1469-8137.2004. 00984.x

coordinate MYB72 expression in Arabidopsis roots during onset of induced systemic resistance and iron-deficiency responses. Plant J. 84, 309–322. doi: 10.1111/tpj.12995

**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 © 2016 Aziz, Nadipalli, Xie, Sun, Surowiec, Zhang and Paré. 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) or licensor 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.

# Long Chain Alcohols Produced by *Trichoderma citrinoviride* Have Phagodeterrent Activity against the Bird Cherry-Oat Aphid *Rhopalosiphum padi*

Sonia Ganassi 1 , Pasqualina Grazioso1 , Antonio De Cristofaro2 , Fabio Fiorentini 3 , Maria Agnese Sabatini 1 , Antonio Evidente4 and Claudio Altomare5 \*

1 Department of Life Science, University of Modena and Reggio Emilia, Modena, Italy, 2 Department of Agricultural, Environmental and Food Sciences, University of Molise, Campobasso, Italy, 3 CBC (Europe) S.R.L.-Biogard Division, Cesena, Italy, 4 Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy, 5 Institute of Sciences of Food Production, National Research Council, Bari, Italy

### *Edited by:*

Jesús Mercado-Blanco, Consejo Superior de Investigaciones Científicas, Spain

### *Reviewed by:*

Enrique Quesada Moraga, University of Cordoba, Spain Regiane Cristina Oliveira De Freitas Bueno, Universidade Estadual Paulista "Júlio de Mesquita Filho," Brazil

> *\*Correspondence:* Claudio Altomare<EMAIL_ADDRESS>
### *Specialty section:*

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

*Received:* 17 December 2015 *Accepted:* 23 February 2016 *Published:* 10 March 2016

### *Citation:*

Ganassi S, Grazioso P, De Cristofaro A, Fiorentini F, Sabatini MA, Evidente A and Altomare C (2016) Long Chain Alcohols Produced by Trichoderma citrinoviride Have Phagodeterrent Activity against the Bird Cherry-Oat Aphid Rhopalosiphum padi. Front. Microbiol. 7:297. doi: 10.3389/fmicb.2016.00297 In this study we report the effects of fungal metabolites isolated from cultures of the fungus Trichoderma citrinoviride ITEM 4484 on the feeding preference of the aphid Rhopalosiphum padi, a major pest of cereal crops. Different phagodeterrent metabolites were purified by a combination of direct and reverse phase column chromatography and thin-layer chromatography. Chemical investigations, by spectroscopic and chemical methods, led to the identification of different long chain primary alcohols (LCOHs) of the general formula R-OH, wherein R is a long, unbranched, unsubstituted, linear aliphatic group. LCOHs have been reported as components of lepidopteran pheromone blends, but their phagodeterrent effect to aphids is herein reported for the first time. The effects of LCOHs on R. padi were studied by behavioral and electrophysiological bioassays. Feeding preference tests that were carried out with winged and wingless morphs of R. padi showed that LCOHs had high phagodeterrent activity and restrained aphids from settling on treated leaves at a concentration as low as 0.15 mM (0.036 g/l). The results of different electrophysiological analyses indicated that taste receptor neurons located on the aphid tarsomeres were involved in the LCOHs perception. Behavioral assays carried out with some commercial agrochemicals, including azadirachtin A, pyrethrum and a mineral oil-based product, in combination with 1-hexadecanol, the LCOH most abundantly produced by T. citrinoviride ITEM 4484, showed that these different active principles could be applied together, resulting in a useful increase of the phagodeterrent effect. The data shown indicate that these compounds can be profitably utilized for novel applications in biotechnical control of aphid pests. Furthermore, the tested LCOHs have no chiral centers and therefore can be obtained with good yield and at low cost through chemical synthesis, as well as from natural sources.

Keywords: *Trichoderma*, long-chain alcohols, biocontrol, aphids, phagodeterrence

# INTRODUCTION

Aphids are insect pests of great economic importance for agriculture and represent one major cause of damage and loss of both quantity and quality of produce in horticultural, cereal and tree crops (Blackman and Eastop, 2007; Dedryver et al., 2010). Aphids use their piercing sucking mouthparts to feed on host plants causing damage by subtraction of sap, injection of saliva which has phytotoxic effects, and spread of insect-transmitted virus diseases (Hull, 2002; Ng and Perry, 2004; Katis et al., 2007). Moreover, aphids produce large amounts of honeydew, a sugary liquid waste on which sooty molds often grow hindering the plant photosynthetic capability (Dedryver et al., 2010). Various insecticides can be used to control aphids, however the intensive use of chemical insecticides has led to environmental pollution and onset of resistant populations of various aphid species (Vanlerberghe-Masutti and Guillemaud, 2007). In addition, the phasing out of some active principles and the more and more strict registration procedures for new agrochemicals have in recent times led to a progressive reduction of the number of insecticides available for aphid control (Dedryver et al., 2010; Roubos et al., 2014). Therefore, there is a growing interest in the development of innovative control strategies that may lead to a progressive reduction of chemicals, with the aim to minimize the environmental impact of pest management and improve the safety of the agro-food chain.

Signaling chemicals produced by plants (Reddy and Guerrero, 2004; Honda et al., 2010) and microorganisms (Davis et al., 2013) may evoke different behavioral responses in insects, such as attraction, repellence or aggregation stimulation. The concept of using nontoxic compounds as insect antifeedants in crop protection has grown with the demonstration of the potent feeding deterrent effect of azadirachtin and neem seed extracts to a large number of pest species (Isman, 2006). The identification of new compounds with phagodeterrent activity, which possess the capability to interfere with aphid host plant selection and host acceptance, is currently becoming of great interest for the design of innovative biotechnical strategies in control of phytophagous insects.