This work was supported by the Agricultural Science and Technology Innovation Program (ASTIP-TRIC07), Science Foundation for Young Scholars of Tobacco Research Institute of Chinese Academy of Agricultural Sciences (2016A01), Fundamental Research Funds for Central Non-profit Scientific Institution<PHONE_NUMBER>003), and Funded by Open Foundation of the State Key Laboratory of Bioactive Seaweed Substances (SKL-BASS1715).

# ACKNOWLEDGMENTS

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We would like to thank Editage (www.editage.cn) for English language editing.

## REFERENCES

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00048/ full#supplementary-material

FIGURE S1 | The MW of LNP, LNP-1, and LNP-2 measured by HPLC with dRI detector.

FIGURE S2 | Effects of LNP, LNP-1, and LNP-2 on growth of wheat seedlings.

AVP1 H+-pump. Proc. Natl. Acad. Sci. U.S.A. 98, 11444–11449. doi: 10.1073/ pnas.191389398

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

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

# Exposure in vitro to an Environmentally Isolated Strain TC09 of Cladosporium sphaerospermum Triggers Plant Growth Promotion, Early Flowering, and Fruit Yield Increase

Zhijian T. Li1 , Wojciech J. Janisiewicz1 , Zongrang Liu1 , Ann M. Callahan1 , Breyn E. Evans1 , Wayne M. Jurick II2 and Chris Dardick1 \*

1 Appalachian Fruit Research Station, United States Department of Agriculture – Agricultural Research Service, Kearneysville, WV, United States, 2 Food Quality Laboratory, Beltsville Agricultural Research Center, United States Department of Agriculture – Agricultural Research Service, Beltsville, MD, United States

#### Edited by:

Giuseppe Colla, Università degli Studi della Tuscia, Italy

#### Reviewed by:

Philipp Franken, Leibniz-Institut für Gemüse- und Zierpflanzenbau (IGZ), Germany Paolo Bonini, Ngalab, Spain Antonio Ferrante, University of Milan, Italy

> \*Correspondence: Chris Dardick

<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 26 September 2018 Accepted: 17 December 2018 Published: 01 February 2019

#### Citation:

Li ZT, Janisiewicz WJ, Liu Z, Callahan AM, Evans BE, Jurick WM II and Dardick C (2019) Exposure in vitro to an Environmentally Isolated Strain TC09 of Cladosporium sphaerospermum Triggers Plant Growth Promotion, Early Flowering, and Fruit Yield Increase. Front. Plant Sci. 9:1959. doi: 10.3389/fpls.2018.01959 A growing number of bacteria and fungi have been found to promote plant growth through mutualistic interactions involving elements such as volatile organic compounds (VOCs). Here, we report the identification of an environmentally isolated strain of Cladosporium sphaerospermum (herein named TC09), that substantially enhances plant growth after exposure in vitro beyond what has previously been reported. When cultured on Murashige and Skoog (MS) medium under in vitro conditions, tobacco seedlings (Nicotiana tabacum) exposed to TC09 cultures for 20 days increased stem height and whole plant biomass up to 25- and 15-fold, respectively, over controls without exposure. TC09-mediated growth promotion required >5 g/L sucrose in the plant culture medium and was influenced by the duration of exposure ranging from one to 10 days, beyond which no differences were detected. When transplanted to soil under greenhouse conditions, TC09-exposed tobacco plants retained higher rates of growth. Comparative transcriptome analyses using tobacco seedlings exposed to TC09 for 10 days uncovered differentially expressed genes (DEGs) associated with diverse biological processes including cell expansion and cell cycle, photosynthesis, phytohormone homeostasis and defense responses. To test the potential efficacy of TC09-mediated growth promotion on agricultural productivity, pepper plants (Capsicum annuum L.) of two different varieties, Cayenne and Minisweet, were pre-exposed to TC09 and planted in the greenhouse to monitor growth, flowering, and fruit production. Results showed that treated pepper plants flowered 20 days earlier and yielded up to 213% more fruit than untreated controls. Altogether the data suggest that exposure of young plants to C. sphaerospermum produced VOCs may provide a useful tool to improve crop productivity.

Keywords: microbial volatile organic compounds, Cladosporium sphaerospermum, plant growth promotion, biostimulant, increased productivity, Nicotiana tabacum L., Capsicum annuum L., expression profiling

# INTRODUCTION

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Innumerable bacterial and fungal microorganisms colonize various habitats and can influence the survival of plants. Many such microorganisms promote plant growth through mutualistic interactions which have been the subject of intensive studies for more than 150 years (Whipps, 2001; Berg, 2009). In general, beneficial microorganisms are categorized into a number of groups depending on their habitats and functional roles. They include organic matter decomposers, nitrogen fixing bacteria, mycorrhizal fungi, detoxifiers, mutualistic endophytes, and pathogen-antagonists (Barea et al., 2005). These microbes produce and release unique proteins, enzymes, antibiotics, phenolics, lipids, carbohydrates, and phytohormones improving the availability of essential nutrients and plant growth. In addition, they are capable of activating plant defenses and protecting against biotic and abiotic stresses (Wardle et al., 2004; Barea et al., 2005; Saharan and Nehra, 2011; De-la-Peña and Loyola-Vargas, 2014). Over the past several decades, concerted efforts have focused on the study of plant–microbe symbiotic relationships and endophytic interactions to elucidate plant growth promoting (PGP) activities of beneficial microorganisms (Barea et al., 2005; Berg and Smalla, 2009; Lambers et al., 2009; Saharan and Nehra, 2011; Ahemad and Kibret, 2014; De-la-Peña and Loyola-Vargas, 2014). Consequently, a wide range of microbial inoculants and biocontrol products were developed and marketed as biopesticides, plant strengtheners, phytostimulators, and biofertilizers to improve soil conditions, increase crop productivity and control soil borne pests while reducing reliance on agrochemicals. These products have the potential to encourage environmentally friendly and sustainable cultivation practices and protect biodiversity (Thakore, 2006; Berg, 2009; Baez-Rogelio et al., 2016).

The utilization of current microbial products designed to be applied to the rhizosphere or as inoculants for PGP commonly suffer from unpredictability and inconsistency (Berg, 2009; Ahemad and Kibret, 2014). Changes in soil conditions due to tillage and erosion, extreme weather events and capricious mutualistic interactions amongst microorganisms outside or within host plants can influence the biochemical, physiological and metabolic activities of microbial community and the survival of beneficial microorganisms (De Souza et al., 2015; Baez-Rogelio et al., 2016). A number of studies revealed that some beneficial microorganisms under certain growth and environmental conditions can also produce phytotoxic substances such as hydrogen cyanide (HCN) and other nitrogen and sulfur compounds that can adversely affect plant growth (Kremer and Souissi, 2001; Weise et al., 2013; Nadeem et al., 2014). Clearly, a better understanding of the responses of beneficial microorganisms to their environments will help the development of sustainable strategies that can maximize the effectiveness of microbial products and minimize the deleterious effects on plant growth.

Recently, research activities in the utilization of microorganisms that enhance plant growth via microbial volatile organic compounds (MVOCs) have been intensified based on the premise that they might provide an eco-friendly, cost-effective and sustainable strategy to benefit agriculture (Kanchiswamy et al., 2015a,b; Li et al., 2016). MVOCs are small (<300 Da) and vaporous/gaseous semiochemicals with relatively high vapor pressures, low boiling points and high levels of lipophilicity (Schulz and Dickschat, 2007; Lemfack et al., 2014). These compounds are mostly organic/hydrocarbon in nature (Audrain et al., 2015). Microorganisms from many bacterial and fungal species and genera are able to synthesize a plethora of MVOCs with various biological functions. According to a recent tally, 400 out of 10,000 microorganisms have been described for their ability to produce up to 1,000 MVOCs (Schulz and Dickschat, 2007; Lemfack et al., 2014; Peñuelas et al., 2014; Kanchiswamy et al., 2015b; Li et al., 2016). However, among the complex blends of MVOCs, only a handful have been demonstrated to function in PGP. For instance, 141 MVOCs were detected from 20 strains and 11 species of Trichoderma examined. Yet, only 18 compounds or 11% of the detectable pool were able to promote plant growth, whereas the remaining majority compounds were either neutral or inhibitory to plant growth (Lee et al., 2016). In some cases, phytopathogenic microorganisms are also capable of emitting PGP MVOCs but their positive activities are often subdued by strong phytotoxic substances and parasitic or pathogenic behaviors (Sánchez-López et al., 2016b; Cordovez et al., 2017). While specific correlation between microbe-derived volatiles and plant responses requires further examination and the mechanisms of action remain unknown in most cases, a growing number of studies have linked PGP MVOCs to diverse mechanisms.

Despite these studies, challenges remain in applying MVOC-mediated PGP technology to crop production. More research is needed to identify and fully exploit novel MVOC-emitting microorganisms and develop effective deployment strategies (Ahmad et al., 2008; Berg, 2009; Kanchiswamy et al., 2015a; Turner and Meadows-Smith, 2016). In this study, we report on the discovery of a non-pathogenic airborne fungus Cladosporium sphaerospermum, strain TC09 that strongly stimulates plant growth through exposure in vitro. By using tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) as models, conditions for fungal exposure and fungal cultivation to achieve optimal plant growth stimulation were investigated. We demonstrated growth stimulation, early flowering and fruit yield increases following a relatively short duration of in vitro MVOC exposure at seedling stage. Comparative transcriptome analysis was conducted to reveal differential gene expression associated with promoted plant growth and development. PGP activities following in vitro exposure using related fungal species and isolates were also compared.

# MATERIALS AND METHODS

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### Culture Media

Premixed medium powder containing basal salts and vitamins of MS medium (Murashige and Skoog, 1962) were purchased from PhytoTechnology Laboratories (Cat No. M519, Overland Park, KS, United States). For in vitro culture of plants, full strength MS medium supplemented with 30 g/L sucrose (Sigma-Aldrich, St. Louis, MO, United States, S5391) was prepared with pH adjusted to 5.8 using 1N KOH prior to addition of 7 g/L gelling agar (Sigma, A7921). The medium was then autoclaved at 121◦C for 20 min and cooled down to 45◦C prior to dispensing to Magenta GA7 vessels (100 ml per vessel) and 15 × 100 mm Petri dishes (30 ml/Plate).

#### Species Identification of TC09

TC09 was an environmentally isolated strain of unknown fungus and was subsequently deposited at the Agricultural Research Service Culture Collection (NRRL) with an assigned #: NRRL 67603. To determine the species identity, single spore cultures were transferred to MS medium in Petri plates and incubated at 25◦C. Fungal spores were collected from the cultures and kept in a 1.5 ml microcentrifuge tube at −20◦C. Genomic DNA was isolated using the DNEasy Plant Mini Kit (Qiagen, Hilden, Germany). Briefly, conidia were removed from the freezer and liquid nitrogen was added to the microcentrifuge tube. The conidia were ground to a fine powder using a motorized pestle mixer (VWR Pellet Mixer, VWR, Intl., Radnor, PA, United States). The DNA was isolated following the manufacturer's protocol with one exception; in the final step, DNA was eluted from the spin column using 100 µL hot (65◦C) nuclease-free water. DNA concentration was determined using a Qubit R 2.0 fluorometer and the dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, United States). Polymerase chain reaction (PCR) was performed with the genomic DNA as a template. The internal transcribed spacer 1 (ITS1) and ITS2 regions associated with the 5.8S ribosomal RNA (rRNA) gene in fungal organisms were targeted for species identification. Two reactions using primer pairs ITS1/2 and ITS3/4, respectively, were conducted in a Bio-Rad thermocycler using 30 reaction cycles each consisting of 94◦C for 1 min, 60◦C for 1 min, and 72◦C for 3 min. Sequences of the primer pairs are as described by White et al. (1990) (**Table 1**). Both amplicons were visualized on a 0.8% agarose gel stained with ethidium bromide (EtBr) following electrophoresis. DNA products in single bands were purified from isolated gel blocks using Qiagen PCR Clean Up kit and DNA concentrations were quantified using a Nanodrop spectrophotometer. Purified DNA Products were subject to Sanger sequencing by Eurofins MWG Operon (KY, United States) and sequence data were analyzed using Geneious software (Biomatters Inc., Newark, NJ, United States).

To determine phylogenetic relationships, DNA sequences of 145 related fungal species/isolates with over 95% homology to the consensus sequence of the ITS3/4 primer pair-amplified PCR products were retrieved via a BLAST search of GenBank database. The sequences were aligned and analyzed using default settings of the Phylogeny Module of CLC Genomics Workbench software program (Version 10, Qiagen, Redwood City, CA, United States) with Neighbor-Joining method with 1,000 bootstrap replicates.

For microscopic examination, TC09 was grown on PDA (Potato dextrose agar) or MS media for 7–10 days at 22◦C under continuous light. Squares of transparent adhesive tape (Scotch Magic tape, 3M, St. Paul, MN, United States) were gently placed along the edge of the colony with forceps. Tape squares were removed from the colony margin and stained for 20 min with 1% aqueous Calcofluor white M2R (Fluorescent brightener 28, Sigma, St. Louis, MO, United States). Tape squares were gently rinsed in sterile distilled water and mounted between drops of 50% glycerol under a glass cover slip. The cover slip was affixed in

TABLE 1 | Oligonucleotide primers and amplicons targeting the internal transcribed spacer 1 (ITS1) and ITS2 regions associated with the 5.8S ribosomal RNA (rRNA) gene in fungi.

place using clear nail polish. Mounted specimens were visualized through confocal microscopy (Zeiss LSM-800, Carl Zeiss AG, Oberkochen, Germany) and images were captured using the manufacturer software.

# Assay of TC09 Promotion Activities on Tobacco Plant Growth

Tobacco seeds (Nicotiana tabacum cv. "Samsun") were surface-sterilized by rinsing briefly in 95% ethanol and then immersed in 20% (v/v) bleach containing 8.25% w/v sodium hypochlorite with constant agitation for 10 min. After 3× rinse with sterile water, seeds were then spread evenly on the surface of MS medium in Petri plates. The plates were sealed with plastic wrap and maintained at 25◦C under 16-h light (50 µmol m2 s 1 ) and 8-h dark photoperiods for 6 days. Seedlings with fully expanded cotyledons were selected based on a uniform plant size and an appearance free of abnormality (∼80–90% of sowed seeds).

TC09 inoculant in aqueous conidial suspension was prepared by first culturing the fungal conidia on MS plate for 1 week followed by collecting conidia in sterile 0.01% Triton X-100/water solution and adjusting density to 105 conidia per ml prior to use or storage at −80◦C. For initial experiments, fungal cultures grown in Eppendorf tubes were placed inside the plant tissue culture vessel but physically separated using sealed biological filters that restrict the movement of conidia but not volatiles. First, aliquots of 300 µl warm MS medium were poured into 1.5 ml Eppendorf microcentrifuge tubes. Tubes were positioned diagonally to form a slant surface while cooling in laminar flow hood. Ten microliters of an aqueous conidial suspension of TC09 was transferred onto the surface of the medium in each tube and thus inoculated tubes were plugged with a sterile aerosol substance- and liquid-resistant filter (Rainin #17001945, Mettler Toledo, Oakland, CA, United States). Two tubes were directly inserted at separate corners of a Magenta GA7 vessel containing three tobacco seedlings each. The vessels were then sealed with plastic wrap and maintained under light conditions with a 16-h photoperiod at 25◦C. Plant growth was monitored and compared to controls grown without fungal cultures.

In subsequent experiments open-end culture tube closures (Sigma C5791) were used to reduce premature build-up of condensation water on the surface of the fungal mycelium as found in above microcentrifuge tube setting. Each sterile closure contained 5 ml semi-solid MS medium followed by addition of 10 µl of TC09 suspension as inoculant. One inoculated closure was then placed in each Magenta GA7 vessel that contained three tobacco seedlings for fungal exposure treatment. For controls, a MS-containing closure without fungal inoculant was added. Vessels were placed under above-mentioned light conditions at 25◦C. To measure plant growth promotion under in vitro conditions, a 20-day exposure duration was employed which was comparable to previous studies using tobacco (Paul and Park, 2013; Lee et al., 2016).

Three plants were grown in each replicate vessel and three vessels were used per treatment. The experiment was conducted three times unless otherwise stated. Plant growth was determined by measurement of major growth parameters including total plant height, total plant fresh weight, stem length, number of traceable leaves, root system length, and length of largest leaf. Data were analyzed by using SAS statistics program with standard statistical approaches including standard error, t-test (two means), ANOVA and Tukey's HSD mean separation test (multiple means) with a statistical significance level P < 0.05.

# Effect of TC09 Exposure Duration and Sugar Concentration

For experiments to test the effect of exposure time under in vitro conditions, control tube closures with medium-only (CK) and those with fungal cultures were placed in vessels containing tobacco seedlings. Fungal cultures were then removed after an incrementally extended exposure duration from 1, 4, 10, or 20 days. Vessels containing medium only were treated as 0 day and served as a control. All plants were allowed to grow continuously in the vessels until 20 days after introduction of fungal cultures when growth was measured. The experiment was conducted two times.

A total of six sucrose concentrations including 0, 5, 10, 20, 30, and 40 g/L were incorporated in MS medium and used to culture tobacco seedlings. Introduction of TC09 cultures in tube closures to Magenta GA7 vessels was carried out as aforementioned. After a 20-day fungal exposure, plants were collected, measured and analyzed as described above. Experiments were conducted two times.

# Plant Growth Under ex vitro/Soil Conditions in the Greenhouse

In vitro grown plants following fungal exposure were transplanted to potting soil mix (Metro Mix 360, Sun Gro Horticulture, Canada) and maintained in a greenhouse to monitor growth. Soil mix was pre-sterilized by autoclaving at 121◦C for 90 min prior to use. Plants were maintained in a greenhouse. Six to ten individual tobacco plants from each treatment and untreated controls were transplanted for each test and experiments were conducted three times. Plant growth and development were evaluated via periodic measurement of plant height and total leaf numbers over the course of 70 days beginning from the time the seeds were sown. Experimental data were analyzed using standard statistical approaches as mentioned above.

# Effect of TC09 Growth Media on Plant Growth Promotion

A total of six media were examined for fungal cultivation including MS (Murashige and Skoog, 1962), PDA (potato dextrose agar), Czapek (CYA, Czapek-DOX Yeast agar), Malt (Malt extract agar), yeast (Yeast extract extract) and Hutner's medium. Formula and preparation procedures for the last five culture media were described previously (Sinclair and Dhingra, 1995). Organic potatoes were purchased locally and used to prepare potato infusion and PDA medium. For these experiments, tobacco and closure-mediated method for fungal exposure were employed. Final determination of plant

growth was made at the end of an exposure duration of 20 days. There were three plants per replicate vessel and three vessels per treatment. The experiment was repeated twice.

# Effect of TC09-Related Fungal Species/Isolates on Plant Growth Promotion

Comparative assays were performed to determine the effectiveness of exposure in vitro using various species and isolates belonging to Cladosporium that were kindly provided by Dr. Frank M. Dugan of USDA (59 Johnson Hall, WSU, Pullman, WA, United States). These include C. sphaerospermum NRRL8131, C. cladosporioides 113d, C. asperulatum 208db, C. subtilissimum WF99-209, C. cladosporioides W99-175a, and C. macrocarpum Clad ex Phyl 8. Tobacco plants were used for growth comparison using MS medium for fungal culture. Three replicate vessels with three plants each were used for each treatment. The experiment was repeated twice.

# Comparative Transcriptome Analysis of Tobacco Plants Following Exposure to TC09

Transcriptome analysis was carried out using total RNA isolated from tobacco seedlings grown under in vitro conditions with or without TC09 exposure for 10 days. For RNA isolation, shoot tips carrying the apical meristem and three to four terminal leaves were collected from each replicate plant, placed in 1.5 ml microcentrifuge tubes, and immediately immersed in liquid nitrogen. Samples were pulverized into a fine powder using Geno/Grinder 2010 (SPEX Sample Prep, Metuchen, NJ, United States). Total RNA was then extracted from 100 mg tissue powder using the RNeasy R Plant Mini Kit per manufacturer's instructions (Qiagen, Redwood City, CA, United States). There were four biological replicates from each treatment. RNA sequencing was performed by Genewiz via Illumina HiSeq platform (50 bp single-end reads) (South Plainfield, NJ, United States). Raw sequences were paired, trimmed and filtered to obtain quality reads. Reads were mapped to the Solanum lycopersicum reference genome with CLC Genomics Workbench (version 10, Qiagen, Redwood City, CA, United States) using default parameters (The Tomato Genome Consortium, 2012). Differentially expressed genes (DEGs) were identified using false discovery rate (FDR) ≤ 0.05. Genes were annotated per referenced tomato (S. lycopersicum) transcriptome dataset available on https:// solgenomics.net and associated gene ontology (GO) terms. The RNAseq data was deposited to NCBI GEO under reference ID GSE120288.

# Effect of TC09 Exposure in vitro on Pepper Plant Growth and Production

Two different varieties of pepper (Capsicum annuum), cayenne pepper 'Long Thin' and sweet bell pepper 'Minisweet Pepper Mixed,' were tested to determine growth response to TC09 exposure in vitro. Pepper seeds were acquired from commercial source (Plantation Product LLC., Norton, MA, United States) and sterilized by previously described method prior to in vitro cultivation or seeded directly in the soil as controls. Germinated pepper seedlings were exposed to closure-contained TC09 while culturing on MS medium in GA7 vessels for 20 days and then transplanted to soil in 8-inch pots. Plants were maintained in the greenhouse using standard management practice. Plant growth and fruit production were monitored continuously until fruit ripening. In these experiments each treatment consisted of four to five individual plants. Experiments were conducted two times each in a similar seasonal span from late winter to early spring in 2017 and 2018, respectively.

# RESULTS

# Isolation and Species Identification of TC09 Fungus

An airborne fungal contaminant on a tissue culture plate containing tobacco seedlings growing on MS medium was isolated and single-spore purified based on the preliminary visual observation that plants in the contaminated plate grew much larger than comparable plants on uncontaminated plates. To identify the species, DNA was extracted and the internal transcribed spacer was amplified by PCR and subsequently sequenced. The sequence of the two fragments generated from the primer pairs are specified in **Table 1**.

Upon comparison with existing sequences, the 138 bp ITS1/2 amplicon was 100% identical to a GenBank accession KU926349.1 belonging to Cladosporium sphaerospermum isolate UACH-124. The 249 bp ITS3/4 amplicon was 100% identical to a GenBank accession KX982238.1 of Cladosporium sphaerospermum strain 7. Phylogenetic analysis of 148 sequences homologous to the ITS3/4 amplicon that were available in GenBank showed that TC09 clusters with known isolates of C. sphaerospermum (**Supplementary Figure S1**).

To confirm the results of genetic identification, TC09 was cultured on PDA medium, which is commonly used as a standard condition for fungal species identification. TC09 colonies grew to 20 mm and 35 mm in diameter during 7 and 14 days, respectively, on PDA at 20◦C.=. Colonies were olivaceous-gray in the center and olivaceous to iron-gray in the outer region (**Supplementary Figure S3A**). On the reverse they were iron-gray and turned almost blackish at 14 days. Conidiophores were not much differentiated, occasionally branched with conidia produced in branching chains with variable shapes with smaller size toward the apex (**Supplementary Figures S3B,C**). Intercalary conidia were 1.7–3.2 × 2.4–8.0 µm and terminal conidia 1.9–2.5 × 2.1–2.9 µm. Hyphae was 2.6–3.2 µm wide, sparsely to profusely branched at 45–90◦ angles, distinctly septate with cell length averaging 17.5 µm and ranging from 12.4 to 27.3 µm (**Supplementary Figure S3D**). Collectively, the morphological results were consistent with TC09 being an isolate

of C. sphaerospermum as described by Dugan et al. (2008) and Ababutain (2013).

## Stimulation of Plant Growth Is due to in vitro Exposure to TC09

Since the fungal mycelium and tobacco plants appeared to be physically separated in the original contaminated culture plate, possible involvement of MVOCs in plant growth promotion was investigated. Tobacco plants were tested with and without the inclusion of biological filter-sealed Eppendorf tubes containing TC09 in Magenta culture vessels. In this manner, the plants were exposed only to volatiles produced by the fungus. Visual observation indicated that plants with fungal exposure for 10 days exhibited more vigorous growth and thicker stems, larger leaves, and a more robust root system relative to plants without fungal exposure (**Figure 1A**). No conidia from the filter-sealed Eppendorf tubes were visually detected in either the culture vessel headspace or plant culture medium.

Next, plant growth stimulation was quantified using a 20-day exposure duration and open-end culture tube closures to prevent excessive condensation in the fungal culture. Exposed plants grew larger and filled up the headspace of culture vessels, produced elongated stems with larger diameters and longer internodes as well as a larger root systems as compared to controls (**Figure 1B**). Plants were subsequently transplanted to soil and growth differences were maintained (**Figure 1C**).

As compared to controls, the individual plants following 20-day in vitro exposure treatment exhibited the following growth increase: 25-fold for stem length, 15-fold for shoot biomass, 15-fold for root biomass, 3-fold for base-to-top length of largest leaf, and 10-fold for largest leaf biomass (**Figure 2**). Results

Left and upper row: control plants without TC09 exposure in triplicates per vessel, Left and lower row: plants with exposure to fungal cultures in triplicates per vessel. Arrows indicate TC09 mycelium in closure. Right panel: representative plants of control (left) and TC09-exposed treatment (right). (C) Evaluation of plant growth in greenhouse following in vitro seedling treatment with or without TC09 exposure. Plants with (right three) or without (left two) exposure to TC09 for 20 days were transplanted to soil and maintained in greenhouse using standard management practices. Photograph was taken 70 days after seed sowing.

Frontiers in Plant Science | www.frontiersin.org

fpls-09-01959 January 30, 2019 Time: 17:57 # 7

from repeat experiments essentially gave rise to similar increased levels of plant growth with exposure to the fungal culture (data not shown). When planted in soil, the treated plants maintained a near twofold increase in height and in leaf number at 70 days post seed sowing relative to controls (**Figures 3A,B**).

#### Effect of TC09 Exposure Duration on Plant Growth

The potency of PGP activity of TC09 was ascertained using incrementally extended exposure durations. Visual inspection at the end of a 26-day culture period post seed sowing indicated that 1-day exposure of tobacco seedling to the freshly set-up TC09 culture was sufficient to double the plant size as compared to plants without exposure (0 day vs. 1 day) (**Figure 4A**). Plant growth was incrementally increased after 4-, 10-, and 20-day exposures with 10 days being maximal (**Figure 4A**). Based on measurements of major growth parameters, 10-day or 20-day exposure duration led to increase in stem length, stem caliper (diameter), and total plant biomass 16-, 3-, and 12-fold, respectively, over the control without TC09 exposure (**Figure 4B**). Repeated experiments yielded similar results (data not shown).

#### Effect of Sucrose Concentration and TC09 Exposure on Plant Growth

To investigate whether carbon source availability was a contributing factor for PGP activity, various concentrations of sucrose were tested in conjunction with TC09 exposure for 20 days. Without fungal exposure, tobacco plants did not show any

measurable increase in growth rate in response to increasing concentrations of sucrose in the medium (**Figure 5**). When plants were exposed to TC09, only minimal plant growth promotion was observed in media containing 0 and 5 g/L sucrose whereas significant growth promotion was achieved when plants were maintained on a medium containing > 10 g/L sucrose (**Figure 5**). These results were also confirmed in a repeat experiment (data not shown).

#### Effect of Fungal Culture Media on PGP Activities of TC09

replicated plants per treatment and two repeated experiments.

Initial PGP activity of TC09 was characterized using MS medium. Thus, to investigate the influence of culture conditions, several common fungal media were also tested. Variations in visual size and morphology of TC09 mycelium were discernable among various culture media used (**Figure 6**). Noticeable differences in the level of PGP activity of fungal cultures on various media were also observed (**Figure 6**). Twenty days post treatment, plants were measured to determine growth differences including total plant height, total plant biomass, stem length, and length of largest leaf. Results revealed that the order of growth stimulation level from highest to lowest among tested fungal culture media

was as follows: MS>PDA>Czapek>Yeast>Malt>Hutner's (**Figure 6B**). Hutner's medium contained rich vitamins and the same amount of sucrose as MS medium, but it showed the lowest levels of TC09 growth and PGP activity. On the other hand, Malt and Yeast media, both with reduced PGP activities as compared to MS, encouraged vigorous TC09 growth with comparable colony sizes to that of other media except for Hutner's medium. In addition, Malt medium seemed to increase hydrophilicity of the mycelium of TC09 and promote hyphal penetration into culture medium (**Figure 6A**). We conducted a repeat experiment and observed an identical trend of growth stimulation associated with various fungal media (data not shown).

# Comparison of PGP by TC09 and Other Related Fungi

To investigate whether the observed PGP activity was unique to TC09-or common among related fungal species, we compared a total of seven Cladosporium species/isolates for their ability to promote in vitro tobacco growth. All strains exhibited PGP activity as compared to control without fungal exposure, although there were measurable differences among the species/isolates (**Figure 7A**). After 20 days of exposure, TC09 gave the highest level or PGP activity in five out of seven growth parameters measured (**Table 2**). Exposure to C. sphaerospermum NRRL8131 produced the longest stems among all compared fungi (**Table 2**). Colony morphology and growth pattern of various Cladosporium fungi when cultured on MS medium were also noticeably varied (**Figure 7B**). Four of the tested fungi including C. sphaerospermum TC09 (1), C. sphaerospermum NRRL8131 (2), C. cladosporioides 113 db (3) and C. asperulatum 208 db (4) produced mycelium in a similarly large diameter and dark greenish brown color. The remaining fungi including C. subtilissimum WF99-209 (5), C. cladosporioides W99-175a (6) and C. macrocarpum Clad ex Phyl 8 (7) formed smaller, black colonies. C. cladosporioides W99-175a (6) is the only fungus which produced dark brownish compounds that penetrated into the culture medium, whereas all other fungi demonstrated hydrophobicity of the mycelium on MS medium (**Figure 7B**). Subsequently, we conducted a repeat experiment that yielded similar results (data not shown).

# RNAseq Analysis of Differential Gene Expression (DEG) Associated With TC09 Induced Growth Promotion

To assess host responses at the RNA level, tobacco seedlings treated with and without TC09 exposure for 10 days were subject to RNAseq analysis to identify DEGs. The sampling point was chosen based on the consistent levels of plant growth

stimulation achieved following 10 days of MOVC exposure (**Figure 4**). Experimental design included four replicate TC09 treated plants and four untreated control plants sampled after 10 days of exposure. Approximately 20 million quality reads per sample were obtained. A total of 3,562 DEGs were identified consisting of 1,594 up-regulated and 1,968 down-regulated genes (**Supplementary Table S1** and **Supplementary Figure S2**). Overall, the most highly repressed DEGs included reactive oxygen response genes such as those encoding catalases and superoxide dismutases involved in redox balance as well as photosystem functions including photosystem II D1 core genes along with constituents of the cytochrome and ATP synthase complexes. Among the over-represented DEGs were a number of genes involved in cell wall biosynthesis, cell expansion and multiplication. Numerous genes associated with the biosynthesis and response to phytohormones were differentially expressed with those related to auxin, gibberellin, and ethylene being the most abundant.

JA-inducible genes were notably up-regulated as the result of TC09 exposure. These included genes related to herbivore defense including proteinase inhibitors, Kunitz trypsin inhibitor, and O-methyltransferases – which catalyze multiple reactions in the biosynthesis of furanocoumarins (FCs) that act as insect repellants (**Table 3**).

Among cyclin-associated genes, cyclin A and B and two cyclin D genes increased twofold to threefold. Two cyclindependent kinases (CDKs) genes increased twofold to eightfold, respectively, in expression. In addition, one CDK regulator gene showed a 2.6-fold up-regulated expression. A number of genes encoding auxin regulators and sugar transporters were also upregulated. Expression of several gylcosyltransferases functioning in glycosylation was reduced 2- to 10-fold (**Table 3**).

### TC09 Exposure Increases Productivity in Peppers

Two varieties of pepper (Capsicum annuum) were used to investigate whether exposure to TC09 at the seedling stage could positively influence fruit production in a Solanaceous crop plant. Two independent experiments were carried out for each variety. The pepper seedlings were treated with and without TC09 for 20 days under in vitro conditions as described for tobacco. TC09–exposed cayenne and minisweet pepper plants responded to fungal stimulation in a growth pattern similar to tobacco

triplicate vessels per treatment. Standard errors of the means were labeled as vertical lines. Means sharing a letter in the group label are not significantly different (P > 0.05) according to Tukey's test.

and grew significantly larger than untreated controls that were either started in tissue culture or planted directly to soil. For cayenne pepper, following transplanting to soil, the first flowers among TC09-exposed plants were observed 20 days earlier than control plants along with a denser canopy and more branches (**Figure 8A**). By 116 days post seed sowing, visual inspection of the root system showed that TC09-exposed plants had more vigorous root growth than both direct soil seeding and tissue culture control plants without fungal exposure (**Figure 8B**).

Cayenne peppers longer than 1 cm in length were collected from all plants at 116 days post seed sowing. Results showed that TC09-exposed plants had the heaviest fruit load and the most vine-ripened fruits (reddish color) among all treatments (**Figure 8C**, left panel). Similar patterns of fruit production and development were also observed in minisweet pepper when all fruits were collected at 160 days post seed sowing (**Figure 8C**, right panel). For cayenne pepper, TC09-treated plants produced on average 49 fruit per plant whereas direct seeding and tissue

culture-treated plants only put out on average 10 and 18 fruit per plant, respectively. Likewise, for minisweet peppers, the number of fruits per plant from these three treatments were 28, 17, and 17, respectively (**Figure 9A**). Overall, the number of fruits per plant increased 174% and 62% for these two pepper varieties following exposure to TC09 as compared to tissue culture controls. When fruit weight was determined, cayenne pepper generated 173.1, 34.2, and 55.4 g of collected fruit per plant; and minisweet pepper produced 417.9, 247.8, and 272.0 g per plant for TC09, direct seeding and tissue culture treatments, respectively (**Figure 9B**). These results indicate that TC09 treatment gave rise to 213% and 54% yield increase over tissue culture control in cayenne and minisweet peppers, respectively. During the period from early 2017 to late 2018, we conducted a total of two experiments with minisweet pepper and three experiments with cayenne pepper. All results were highly consistent (data not shown).

# DISCUSSION

In recent years there have been an increasing number of reports on MVOC-mediated PGP by beneficial and, in some cases, phytopathogenic microorganisms (Ryu et al., 2003; Zou et al., 2010; Minerdi et al., 2011; Paul and Park, 2013; Kai and Piechulla, 2014; Kanchiswamy et al., 2015a; Sánchez-López et al., 2016b; Cordovez et al., 2017; Tahir et al., 2017). Reported MVOCs are able to stimulate photosynthesis, cell expansion and starch accumulation, early flowering and fruit development and enhance host defense and stress tolerance (Ryu et al., 2004; Zhang et al., 2007; Ahemad and Kibret, 2014; Pieterse et al., 2014; Liu and Zhang, 2015; Chung et al., 2016; Sánchez-López et al., 2016a; Cordovez et al., 2017). This work has highlighted potential benefits and opportunities to utilize MVOC-mediated technology to improve agriculture in an environmentally friendly and sustainable fashion (Berg, 2009; Kanchiswamy et al., 2015a; Baez-Rogelio et al., 2016). However, with the relatively low levels of growth enhancement demonstrated thus far and the few plant species studied, the practical application involving direct use of MVOC emitting microorganisms has remained limited (Song and Ryu, 2013; Li et al., 2016; Cordovez et al., 2017; Fincheira and Quiroz, 2018). Furthermore, all effective MVOC species, their interactions and direct correlation between MVOC and plant response need to be determined. In this study, we demonstrated that short term exposure in vitro to an environmentally isolated fungal strain TC09 identified as C. sphaerospermum dramatically promoted tobacco plant growth via MVOCs. We showed in pilot scale experiments under greenhouse conditions that crop productivity of pepper plants could be significantly enhanced by exposure in vitro to TC09 at the seedling stage.

The effects of TC09 exposure on plant growth were largely correlated with the duration of exposure. The stem length of treated tobacco plants doubled after 20 days relative to controls with as little as a 24-h exposure. Exposure duration of 10 days gave maximal PGP as durations beyond 10 days had no significant additive effect. These observed dynamic changes in growth stimulation could potentially be explained by the loss of MVOC emission by TC09 over longer culturing times, saturated plant signaling receptors, or alternatively as a consequence of the depletion of nutrients in the media by rapidly growing plants. To test the latter hypothesis, we performed experiments to test

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TABLE 2 | Fold increase over control of growth parameters from tobacco plants exposed to MVOCs emitted by various Cladosporium fungi.

Names of species and isolates are abbreviated and described in the section "Materials and Methods." Average values were derived from triplicate vessels with three plants each for each treatment. Highest values of fold increase over control (CK) in each category were in bold letters. 1 Significant difference label generated by Tukey's mean separation test. Average values sharing a letter within a growth parameter are not significantly different at P = 0.05. Data were averaged from three plants in each replicate vessel and three vessels per treatment. 2 The length of stem segment measuring from the base of the plant to the top of shoot apex. 3 The length of root system measuring from the base of the plant to the tip of longest root. 4 The vertical length of the blade of a largest leaf of each plant excluding petiole. 5 Fold over CK reflects average value fold increase of treatment over control as follows:

> Fold over CK = Average value of treatment − Average value of control Average value of control

the requirement for sucrose in the tissue culture media and found that sugar concentrations of >5 g/L were necessary for the observed PGP activity but did not enhance PGP beyond 10g/L. These data suggest that endogenous sugar production in young plants is not sufficient to support robust PGP by TC09 and that sustained PGP requires exogenous carbon sources. This finding may also imply that TC09 MVOCs function by activating some signaling pathway(s) in the plant that allows it to better utilize carbon resources available directly through root uptake.

Another contributing factor to PGP by TC09 was the fungal cultivation medium. MS medium supported the highest level of PGP while some common fungal growth media such as Hutner's medium not only suppressed fungal growth but also reduced PGP activity. MVOC-mediated PGP has been previously shown to vary according to microbial culture conditions. By using rhizobacteria Bacillus mojavensis RRC101, Rath et al. (2018) demonstrated such variability in microbial metabolism in conjunction with PGP activity. When cultured on 1/2 strength MS medium, the bacteria produced known PGP compounds acetoin and 2,3-butanediol, thus leading to improved growth of Arabidopsis seedlings. However, the use of nutrient agar for bacterial cultivation resulted in the production of relatively high levels of acetone and 2-propanone and correlated with phytotoxicity. In addition, the microbial volatile compound, e.g., β-caryophyllene which is commonly produced by fungi was shown to switch its mode of action from stimulatory to inhibitory depending on concentrations (Wang et al., 2009). Other studies also established that both the diversity and absolute quantity of MVOCs emitted by many microbes are dictated by growth conditions and external stimuli (Sunesson

TABLE 3 | DEGs, associated major biological functions and expression changes from tobacco seedlings with exposure to TC09 for 10 days.

(Continued)

#### TABLE 3 | Continued

fpls-09-01959 January 30, 2019 Time: 17:57 # 14

Fold change in expression levels was calculated based on means of four biological replicates for both control and MVOC-treated plants. Genes were annotated using GO terms and tomato (Solanum lycopersicum) reference genes available on https://solgenomics.net.

seeding, tissue culture and TC09 treatments at 50 days post seed sowing. (B) Representative belowground image of cayenne pepper plants from various treatments at 116 days post seed sowing to depict the density and development of fibrous root mass. (C) Lineup of fruits collected from cayenne pepper (left panel, at 116 days post seed sowing) and minisweet pepper (right panel, at 160 days post seed sowing) from all treatments. Fruits in reddish color indicate vine-ripening.

et al., 1995; Weikl et al., 2015; Lee et al., 2016; Nieto-Jacobo et al., 2017). Our findings of altered PGP activities derived from the use of different fungal culture media suggest that culture conditions for TC09 and other PGP microbes can be further exploited and optimized to achieve more effective PGP.

The observed levels of PGP upon exposure of tobacco seedlings to TC09 greatly exceeded those previously reported using other microorganisms (Ryu et al., 2003; Minerdi et al., 2011; Paul and Park, 2013; Bitas et al., 2015; Lee et al., 2016; Sánchez-López et al., 2016b; Cordovez et al., 2017; Tahir et al., 2017). We found that varied levels of PGP activity were also exhibited by diverse species of Cladosporium, however, this activity was largely strain specific. Paul and Park (2013) previously reported an approximately 10-fold increase in fresh weight of tobacco plants exposed to C. cladosporioides CL-1 cultured on PDA for 28 days (4 weeks). The results reported here suggest that differences between various studies could be due to several factors including the specific strain or isolate used, the culture media used for both the plant and fungus,

and the availability of sufficient carbon resources to support maximal plant growth rates. Nevertheless, the rapid response to growth stimulants should facilitate further investigation of the mechanisms underlying PGP activity as recently outlined by Fincheira and Quiroz (2018).

A potential limitation for the use of MVOC-mediated PGP is plant damage resulting from the use of phytopathogens or microbes with parasitic behaviors. Phytopathogenic microorganisms can cause adverse effects on target plants in the case of escape and contamination during fungal exposure. In this study, TC09 did not induce any discernable pathogenic responses in all test plants, consistent with its non-pathogenic nature. The non-pathogenic behavior of TC09 may be attributed to its environmental origin and non-adaptive relationship with plants. C. sphaerospermum has long been known as one of the most ubiquitous airborne, saprobic fungal species and can be found in diverse environments, air and physical structures (Park et al., 2004; Zalar et al., 2007). In contrast, we observed leaf tissue necrosis in tobacco treated with C. sphaerospermum NRRL 8131 (data not shown). This particular isolate was originally referenced as Cladosporium lignicolum Corda based on its sylvan habitat and the ability to degrade and absorb nutrients from lignified woody materials (Dugan et al., 2008). Even though it promoted plant growth similar to TC09, its ability to induce phytotoxic reactions in target plants suggests pathogenic propensity. The ability of TC09 to significantly promote plant growth via MVOCs while lacking harmful phytotoxic effects makes it particularly attractive for further exploitation and the development of practical applications to improve crop productivity.

Transcriptome analyses previously revealed the correlation between differential gene expression and PGP as well as enhanced tolerance to biotic and abiotic stresses following treatment with

beneficial microorganisms and MVOCs (e.g., van Loon, 2007; Zhang et al., 2007; Sánchez-López et al., 2016a). Our RNAseq data from untreated and treated tobacco plants provided evidence that diverse metabolic and signaling pathways are altered as a result of exposure to TC09. The most striking observation was that TC09 treatment was accompanied by down-regulation of photosystem functions. This finding is somewhat inconsistent with several previous reports that MVOCs from various microbes caused substantial positive impacts on photosynthesis and chlorophyll production (see review by Fincheira and Quiroz, 2018). The transcriptome data also revealed a potential increase in cell wall metabolism including up-regulation of numerous cellulose synthase genes, endoglucanases, pectinesterases, fasciclins, and xyloglucan endotransglucosylases. A number of putative expansins were also induced which are known to induce slippage of cellulose microfibrils via a non-enzymatic cell wall loosening mechanism and are responsible for cell size increase (Sampedro and Cosgrove, 2005; Choi et al., 2008). In previous MVOC-related studies, Fusarium oxysporum reportedly was able to upregulate expression of an expansin A5 gene in lettuce (Minerdi et al., 2011). Volatiles from rhizobacteria B. subtilis (GB03) also reportedly increased expression of eight expansin genes ∼2-fold (Zhang et al., 2007). Our results indicate that up to 11 expansin genes in treated tobacco plants were up-regulated twofold to fivefold (five genes with more than threefold) over the control.

The rapid growth enhancement following MVOC or microbial exposure may also reflect promoted mitosis and cell cycle that give rise to increased cell numbers. We identified a number of cyclins and CDKs as DEGs in tobacco plants exposed to TC09. Mitotic-specific cyclins and CDKs are known to interact in partner-dependent fashion to form active complexes that function to modulate cell cycle during different phases of cell division (Mironov et al., 1999). This process is also regulated by mitogenic stimulation through protein docking, CDK phosphorylation, and proteolytic degradation of cell cycle proteins (Breyne et al., 2002). Homeostatic alterations in the expression of these proteins has been linked to either faster or slower cell multiplication (Komaki and Sugimoto, 2012). Noticeably, a previous study reported that ectopic over-expression of a cyclin B in Arabidopsis resulted in promoted cell proliferation in roots (Doerner et al., 1996). Our findings provide a direction for further study to address the influence of MVOCs on cell cycle regulation.

Phytohormones including cytokinin, GA3 and auxin can modulate plant growth and development and their activities are dictated by either biosynthesis or homeostasis of active forms. Interestingly, we observed a reduction (2- to 10-fold) in six UDP-glucose glycosyltransferases (UGGTs), which play an important role in the deactivation, homeostasis, storage and secretion of numerous macromolecules including phytohormones via glycosylation modification (Jones and Vogt, 2001; Ostrowski and Jakubowska, 2014). Kim et al. (2015) also reported the down-regulation of a glycosyltransferase in tobacco in response to MVOCs emitted by rhizobacteria B. subtilis although the functional involvement was not elucidated. We postulate that down-regulation of this class of genes may reduce UGGT activity. This consequently leads to minimized deactivation of phytohormones thus potentially increasing the quantity of active phytohormones galvanizing various aspects of plant growth. Early flowering induced by MVOCs was previously attributed to the enhancement of cytokinin action (Sánchez-López et al., 2016b).

Our transcriptome data indicated that the core transcriptional regulator JA-1 in jasmonic acid (JA) signaling pathway was up-regulated more than twofold in tobacco plants exposed to TC09. Also, many herbivore defense genes were upregulated as well, including proteinase inhibitor II (21 fold), seed storage type protease inhibitor (3.6-fold), Kunitz trypsin inhibitor (2.3-fold) and three cytochrome P450 proteins with sequences homologous to a previously reported protein associated with wound healing and pest resistance (Noordermeer et al., 2001; Dunaevsky et al., 2005). Increased expression levels (threefold to fourfold) of eight latex-like proteins were identified which have been shown to have insecticidal activities against herbivore pests (Konno, 2011). Likewise, seven O-methyltransferases belonging to a class of catalytic enzymes involved in biosynthesis pathway of FCs also showed elevated expression (2- to 15-fold) (Bourgaud et al., 2006, 2014). FCs are secondary metabolites that function mainly as insect repellants (Nitao et al., 2003). These findings prompt future studies to evaluate whether TC09-exposed plants exhibit enhanced resistance to insect pests. Indeed, previous studies have likewise identified MVOC-induced expression of genes associated with JA signaling pathway, broad-spectrum induced systemic resistance (ISR) and herbivore resistance (Song et al., 2013; Naznin et al., 2014; Kanchiswamy et al., 2015a; Chung et al., 2016).

In this study, we demonstrated significant stimulation of plant growth, early reproduction and fruit yield increase in pepper by exposing seedlings to TC09 for a relatively short duration. Although results were achieved under greenhouse conditions, the high level of productivity increase without the additional input of other resources warrants further investigation as to whether such technology could be practically applied at a production scale. Many considerations have to be taken into account including microbe cultivation and conditions, MVOC deployment and dosage control, plant treatment stage and duration, post treatment process and transplanting methodology. In addition, future investigations are needed to study the genome, transcriptome and proteome of TC09 to identify genes, pathways and regulatory mechanisms associated with VOC production and PGP activity. The TC09 strain described here provides a useful tool to carry out such studies and highlights the potential of microbe-based PGP technology to benefit agriculture in an environmentally friendly and sustainable fashion.

# AUTHOR CONTRIBUTIONS

ZTL, WJJ, and CD conceived the concept and designed the experiments. ZTL performed in vitro experiments, analyzed the data, and drafted the manuscript. CD carried out transcriptome assembly and analyses. WJJ, WMJ, and BE conducted fungal identification experiments. ZL and AC provided scientific advice and discussed results. ZTL, CD, WJJ, WMJ, ZL, and AC reviewed the final version of the paper.

### FUNDING

This research was supported in part by grants from AFRS, ARS, USDA and the Oak Ridge Institute for Science and Education (ORISE).

#### ACKNOWLEDGMENTS

fpls-09-01959 January 30, 2019 Time: 17:57 # 17

We thank Dr. Frank M. Dugan for kindly providing Cladosporium fungi and also gratefully acknowledged Dr. Margaret Pooler, Research Leader, FNPR, ARS-USDA, for providing financial support. We also thank Dr. Tracy C. Leskey, Director, AFRS, ARS-USDA, for excellent scientific advice and support for this research and Mark A. Demuth, Elizabeth Lutton, and Dennis R. Bennett for their excellent technical assistance. The handling and movement of all fungi described in the current study were carried using Standard Operation Procedures in accordance with the Animal and Plant Health Inspection Service of the United States Department of Agriculture. Acquisition and movement of mentioned fungal materials were authorized under a permit (P526P-17-02230) issued by the agency.

#### REFERENCES

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01959/ full#supplementary-material

FIGURE S1 | Neighbor-joining dendrogram of ITS sequences associated with TC09. The 249 bp ITS3/4 amplicon consensus sequence derived from PCR amplification using DNA samples of the fungal isolate was used to search GenBank database for homologous sequences. More than 148 sequence entries were retrieved and subject to phylogenetic analysis. Neighbor-joining method was used to reconstruct the phylogenetic tree with bootstrap values indicated for each branch. Sequence entries belonging to same species were labeled using identical colors. Asterisk indicates the caudal position of the fungal isolate within the monophyletic group Cladosporium sphaerospermum.

FIGURE S2 | Heat map depicting high (red) and low (blue) relative levels of differentially expressed genes (DEGs) in individual plants with and without exposure to TC09. Six-day-old tobacco seedlings were maintained under in vitro conditions with or without a 10-day exposure to TC09 and then subject to total RNA extraction using shoot tip tissues that include apical meristem and three to four young leaves. Heat map was generated by using CLC Genomics Workbench software program following transcriptome assembly using tomato transcriptome as a reference. Plant treatment type is marked by red (control) and blue (TC09-treated) line on top. There were four replicated individual plants for each treatment. Color-rendered scale of expression changes is marked on bottom.

FIGURE S3 | Colony of Cladosporium sphaerospermum TC09 on PDA medium (A); and confocal microscopy micrographs of branching chains of conidia (B); intercalary and terminal conidia (C); and mycelium (D).

TABLE S1 | List of all tobacco DEGs after exposure in vitro to TC09 for 10 days. Fold change in expression levels was calculated based on means of four biological replicates for both control and MVOC-treated plants. Genes were annotated using GO terms and tomato (Solanum lycopersicum) reference genes available on https://solgenomics.net.

compounds emitted by plant growth-promoting fungi in Arabidopsis thaliana. PLoS One 9:e86882. doi: 10.1371/journal.pone.0086882

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

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

fpls-09-01959 January 30, 2019 Time: 17:57 # 19

# Foliar Application of Vegetal-Derived Bioactive Compounds Stimulates the Growth of Beneficial Bacteria and Enhances Microbiome Biodiversity in Lettuce

Francesca Luziatelli1 , Anna Grazia Ficca1 , Giuseppe Colla2 , Eva Baldassarre Švecová2 and Maurizio Ruzzi1 \*

1 Department for Innovation in Biological, Agrofood and Forest Systems, University of Tuscia, Viterbo, Italy, 2 Department of Agriculture and Forest Sciences, University of Tuscia, Viterbo, Italy

#### Edited by:

Martine Dorais, Laval University, Canada

#### Reviewed by:

Antonio Ferrante, University of Milan, Italy Andrea Ertani, University of Padova, Italy

> \*Correspondence: Maurizio Ruzzi<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science

Received: 29 September 2018 Accepted: 16 January 2019 Published: 05 February 2019

#### Citation:

Luziatelli F, Ficca AG, Colla G, Baldassarre Švecová E and Ruzzi M (2019) Foliar Application of Vegetal-Derived Bioactive Compounds Stimulates the Growth of Beneficial Bacteria and Enhances Microbiome Biodiversity in Lettuce. Front. Plant Sci. 10:60. doi: 10.3389/fpls.2019.00060 Many studies on plant biostimulants and organic fertilizers have been focused on the ability of these products to increase crop productivity and ameliorate crop tolerance to abiotic stresses. However, little information is available on their effect on plant microbiota, whereas it is well known that microorganisms associated with plant play crucial roles on the health and productivity of their host. The aim of this study was to evaluate the effect of a vegetal-derived protein hydrolysate (PH), a vegetal-derived PH enriched with copper (Cu-PH), and a tropical plant extract enriched with micronutrients (PE) on shoot growth and the epiphytic bacterial population of lettuce plants and the ability of these products to enhance the growth of beneficial or harmful bacteria. The three plant-derived products enhanced shoot biomass of lettuce plants indicating a biostimulant effect of the products. Data obtained using culture-independent (Terminal Restriction Fragment Length Polymorphism and Next Generation Sequencing) and culture-dependent approaches indicated that foliar application of commercial products altered the composition of the microbial population and stimulated the growth of specific bacteria belonging to Pantoea, Pseudomonas, Acinetobacter, and Bacillus genus. Data presented in this work demonstrated that some of these strains exhibited potential plant growth-promoting properties and/or biocontrol activity against fungi and bacteria phytopathogens including Fusarium, Trichoderma, and Erwinia species. No indication of potential health risks associated to the enrichment of human or plant bacterial pathogens emerged by the analysis of the microbiota of treated and no-treated plants. Overall, the findings presented in this study indicate that the commercial organicbased products can enhance the growth of beneficial bacteria occurring in the plant microbiota and signals produced by these bacteria can act synergistically with the organic compounds to enhance plant growth and productivity.

Keywords: vegetal protein hydrolysates, Lactuca sativa L., plant microbiota, terminal restriction fragment length polymorphism, next generation sequencing, plant growth-promoting bacteria, biocontrol activity

# INTRODUCTION

fpls-10-00060 February 1, 2019 Time: 17:52 # 2

Plants provide a rich and diverse habitat which harbors a wide array of bacteria most of which contribute to the growth, and health of their plant hosts (Berendsen et al., 2012; Hirsch and Mauchline, 2012; Jackson et al., 2013; Leff and Fierer, 2013). Most microorganisms in the phyllosphere have ability to promote plant growth through different mechanisms that vary from changes in hormonal content, production of volatile compounds, increasing nutrient availability, or enhancing abiotic stress tolerance (Ruzzi and Aroca, 2015). In addition, some native plant epiphytic microbiota can be used for bio-control of foodborne pathogens (Lopez-Velasco et al., 2012).

Plant growth-promoting (PGP) activity of epiphytic microbes can be affected by the environmental conditions, including the exposure to biostimulants/fertilizers or to their degradation products (Timmusk et al., 2017; Thapa et al., 2018). Protein hydrolysates (PHs) and other plant extracts are widely used as plant biostimulants for their positive effects on plant growth and nutrition (Colla et al., 2014). In the last years, the use of biostimulants to promote plant growth has been widely studied (Parrado et al., 2007; Kowalczyk et al., 2008; Ertani et al., 2009; Gurav and Jadhav, 2013; Colla et al., 2015). Foliar and root applications have been shown to enhance the uptake of both macro and micronutrients (Ertani et al., 2009, 2012; Colla et al., 2015; Halpern et al., 2015) and to increase plant tolerance to environmental stress conditions. Biostimulant action of vegetal-derived products has been ascribed to the supply of bioactive compounds like amino acids, peptides, carbohydrates, humic substances, lignosulphonates, and phytohormones (Du Jardin, 2015). These bioactive compounds can be available in the plant-derived biostimulant or can be generated after foliar spray or substrate drench through the microbial activity.

Despite the use of biostimulants has been increasing and has become a common practice in the sustainable agriculture, little information is available on the effect of these products on the epiphytic bacterial microbiota. PHs and vegetal extracts-based products contain a wide range of compounds, as simple or complex carbohydrates and soluble organic nitrogen (Trouvelot et al., 2014), that can be utilized by both plants and bacteria as a source of carbon, nitrogen, and energy (Farrell et al., 2013). Therefore, foliar application of biostimulants can affect the epiphytic microbiota enhancing the development of bacteria that can be beneficial, neutral, or detrimental to plants (Colla et al., 2017a). Structural and functional modifications in the plant-associated microbiota have a crucial impact on the ecosystem, altering antagonistic and synergistic interactions among microorganisms and improving the fitness of the host by enhancing plant metabolic capacity, uptake of nutrients, and response of the plant to abiotic and biotic stresses (Kumar and Verma, 2018; Valencia et al., 2018). The exposure of plants to harsh environmental conditions can strongly influence the structure and composition of its microbiota, but this effect can be counteracted by enhancing the growth and survival of stresstolerant PGP bacteria (Gouda et al., 2018). Therefore, growth stimulation of beneficial epiphytic microbes might be one of the different modes of action of some plant biostimulants.

In the case of amino acids and small peptide-based products, several studies on plant growth have been reported (Mladenova et al., 1998; Qurartieri et al., 2002). Kauffman et al. (2005) showed a growth-regulator activity of Foliar, an array of free amino acids and other organic constituents, on turf. The assessment of effects of foliar-applied agricultural products on plant epiphytic microbes guarantees the consumers harmless final product, especially for leafy vegetables such as lettuce. In lettuce, the diversity and abundance of epiphytic microbial community depend on the cultivation environment, i.e., phyllosphere microbiota from laboratory-grown plants is distinct from that colonizing plants grown in the field (Williams and Marco, 2014), and can be affected by the climatic variations such as radiation, rainfall, wind, and temperature (Medina-Martínez et al., 2015). The alteration of the composition of epiphytic microbial community of lettuce may lead to the promotion of beneficial microbes which can compete for the nutrient sources with enteric pathogens (Cooley et al., 2006) and perhaps phytopathogens.

We hypothesized that foliar applications of commercial products containing vegetal-derived bioactive compounds can enhance crop growth not only directly through the activity of signaling molecule such as peptides (e.g., short peptides such as root-hair promoting peptides), amino acids (e.g., glutamate), and phytohormones (e.g., auxins, cytokinins) but also indirectly by changing the microbial community in the phyllosphere. Vegetal-derived organic compounds sprayed on the leaf surface may be used as carbon and energy sources by beneficial microorganisms to synthesize new organic compounds including plant growth stimulating molecules (e.g., hormones) and/or toxic substances for plant pathogens. Moreover, vegetalderived biostimulants are often enriched with copper or other micronutrients to improve plant nutrition and crop protection against pathogens. Several authors demonstrated that copper could alter the structure and function of the soil microbial communities, resulting in the selection of copper tolerant and copper resistant strains (Berg et al., 2012; Griffiths and Philippot, 2013; Nunes et al., 2016). Similarly, the addition of mineral elements to vegetal-derived biostimulants may also affect the microbial community in the phyllosphere and then the biostimulant activity of the product. To our knowledge, no information is available in the scientific literature on the influence of foliar applications of vegetal-derived bioactive compounds alone or enriched with micronutrients on bacterial community in the phyllosphere. Starting from the above considerations, a lettuce trial was carried out under greenhouse conditions to evaluate the influence of foliar sprays with three different commercial products containing vegetal-derived biostimulant compounds [tropical plant extract enriched with micronutrients (PE) "Auxym"; vegetal-derived PH "Trainer"; vegetal derived PH enriched with copper "Scudo"] on bacterial community from lettuce leaves in order to determine whether the productmediated growth stimulation of certain epiphytic microbes contributes to the plant growth promoting properties of these products.

# MATERIALS AND METHODS

fpls-10-00060 February 1, 2019 Time: 17:52 # 3

#### Growing Conditions and Treatments

The trial was conducted, 2012, in a 300-m2 polyethylene greenhouse situated at the Experimental Farm of Tuscia University, central Italy (latitude 42◦ 250 N, longitude 12◦ 080 , altitude 310 m). Plants were grown under natural light conditions. The mean values of day/night air temperature and relative humidity and their standard deviations were 15.6 ± 1.3/22.7 ± 4.5◦C, and 55.1 ± 8.2/80.0 ± 8.9%, respectively. Lettuce seeds (Lactuca sativa L. cv "Green Salad Bowl," SAIS S.p.A., Cesena, Italy) were sown on April 9 in polystyrene plug trays filled with vermiculite at a plant density of 720 plants m−2 . The floating raft system consisted of the polystyrene plug trays floating in plastic tanks with a constant volume of 60 L of aerated nutrient solution. An air compressor maintained the dissolved oxygen content above 6 mg/L. The composition of the nutrient solution in all treatments was: 10 mM NO3-N, 1.5 mM P, 4.5 mM K, 10 mM Ca, 5.0 mM S, 2 mM Mg, 20 µM Fe, 9 µM Mn, 0.3 µM Cu, 1.6 µM Zn, 20 µM B, and 0.3 µM Mo. The electrical conductivity and pH of the nutrient solutions in all treatments were 2.0 ± 0.2 dS m−1 and 6.0 ± 0.3, respectively. To prevent large fluctuation in the nutrient concentrations, electrical conductivity, and pH, the nutrient solutions in all treatments were renewed from all tanks weekly.

After 14 days from emergence, plants were sprayed with 2.5 ml L −1 of a vegetal-derived PH ("Trainer"), or 1.0 ml L−1 of a copper-based fertilizer ("Scudo"), or 1 ml L−1 of a plant extract ("Auxym").

The plant extract "Auxym" (PE) is a commercial vegetalderived biostimulant produced through water extraction and fermentation of tropical plant biomass. It contains mainly phytohormones with an auxin:cytokinin ratio 6:1, amino acids, vitamins, and microelements (**Table 1**; Colla et al., 2017b).

The legume-derived PH "Trainer" is a commercial biostimulant obtained through enzymatic hydrolysis of proteins derived from legume seeds. It contains mainly free amino acids, and soluble peptides with the following aminogram (g kg−1 ): Ala (12), Arg (18), Asp (34), Cys (3), Glu (54), Gly (12), His (8), Ile (13), Leu (22), Lys (18), Met (4), Phe (15), Pro (15), Thr (11), Trp (3), Tyr (11), Val (14). The total content of micronutrients being below 0.05 g kg−1 can be considered negligible (**Table 1**). It has been shown that small peptides and single amino acids present in this PH exhibit auxin-like and gibberellin-like activities (Colla et al., 2014).

"Scudo" is a copper-based fertilizer (Cu-PH) containing copper complexed with peptides, and amino acids (90 g kg−1 of copper). Scudo (Cu-PH) contains free amino acids, and soluble peptides with the same composition of Trainer (PH; **Table 1**). Trainer, Scudo, and Auxym were manufactured by Italpollina S.p.A., Rivoli Veronese, Italy.

Foliar applications were repeated three times, at weekly intervals and, in each application, control plants were sprayed with the same amount of water used for the three vegetal-derived products. The four treatments were arranged in a randomized block design with three replicates per treatment (total of 12 plots). The number of plants per experimental plot was 84. Each plot included a polystyrene plug trays floating in a plastic tank. One day and seven days after the last treatment, six leaves (two from three independent plants) were sampled in each plot providing a total of 18 leaves per treatment (=6 leaves per plot × 3 replicates) per time point. Before the end of the trial (May 14, 35 days after sowing), the Soil-Plant Analysis Development (SPAD) index was recorded on lettuce leaves. A portable chlorophyll meter (SPAD-502, Minolta corporation, Ltd., Osaka, Japan) was used to measure the relative leaf chlorophyll concentration as a rational unit. Measurements were made at the central point of the leaflet between the midrib and the leaf margin of the second leaf starting from the apical shoot. Twenty random readings per plot were taken for each replicate and averaged to a single SPAD value; therefore, there were a total of three averaged SPAD values for each treatment. At end of the trial (May 14, 35 days after sowing), 20 plants per plot (single replicate) were harvested and the mean fresh weight of shoot biomass was determined from the 60 plants harvested from each treatment.

#### Isolation of Culturable Bacteria From Lettuce Phyllosphere

Epiphytic bacterial populations from treated and no-treated plants were recovered incubating six leaves per replicate in 20 mL of saline solution (0.9% w/v NaCl) for 60 min, under shaking condition (180 r/min). There were three replicates for each treatment with a total of 18 leaves per treatment per time point. Cell suspensions were serially diluted onto LB agar plates (Sambrook and Russel, 2001) for counting of predominant culturable bacteria and individual colonies were then picked and streaked on fresh LB plates for further characterization.

#### DNA Extraction

At each time point, metagenomic DNA used for T-RFLP and NGS analysis was prepared from three biological replicates for each treatment and from each biological replicate, two replicate extractions were performed. DNA was extracted from cells collected by centrifugation (13,000 r/min for 10 min) from saline solution used for leaf washing (six leaves for each replicate, two leaves from three independent plants per plot). Total DNA of culturable isolates was obtained from cells grown overnight on LB medium. DNA was extracted using PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific, Italy) following the manufacturer protocol for Gram-positive bacteria1 . The quantity and quality of isolated DNA was measured using a Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Italy) and agarose gel electrophoresis, respectively.

### Molecular Identification of Culturable Bacteria

Two to four isolates for each morphotype were selected for the molecular analysis. The culturable isolates were characterized

1https://assets.thermofisher.com/TFS-Assets/LSG/manuals/purelink\_genomic\_ man.pdf

#### ND = not detected.

by amplifying, sequencing, and analyzing the 16S rRNA gene. Universal primer 63F (50 -CAGGCCTAACACATGCAAGTC-30 ) and 1389R (50 -ACGGGCGGTGTGTACAAG-30 ) were used to generate amplicons (about 1400 bp) that were cloned into the pGEM-Teasy vector (Promega, Madison, WI, United States) and sequenced using MACROGEN commercial service (Amsterdam, Holland).

All 16S rRNA sequences from isolates with the same morphotype were identical and only sequences that could be shown to be derived from independent templates were analyzed. The 16S rRNA sequences were compared with those of all known bacterial species available in the GeneBank database2 to identify potential phylogenetic relationships. All sequences were aligned using Clustal Omega (Sievers et al., 2011) and the unrooted phylogenetic tree was constructed using the neighbor-joining program contained in the PHYLIP phylogeny inference package (ver 3.6). The confidence values of the branches were determined by performing a bootstrap analysis based on 1000 replicates and the phylogenetic tree was displayed using iTOL (Letunic and Bork, 2006).

#### T-RFLP Analysis

To ensure that the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis was not biased by biological sample, DNA extraction, and amplicon library preparation, two DNA pools, each consisting of equimolar DNA samples from biological replicates of each treatment, were prepared. Thus, the profile of the epiphytic bacterial community was derived analyzing 18 leaves from nine plants for each treatment. PCR amplification targeting the 16S rRNA gene was carried out using primer 8F (50 -AGAGTTTGATCCTGGCTCAG-30 ), fluorescently labeled at the 50 end with 6-FAM (6-carboxyfluorescein), and 1387R (50 -GGGCGGWGTGTACAAGGC-30 ), with three replicates per DNA pool. PCR replicates were purified, using the Promega Wizard (Promega, Madison, WI, United States), quantified, using Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Italy), and pooled into a single tube to represent each amplicon library in equimolar amounts. Fluorescently labeled products were digested with 10 U of restriction enzyme SspI or AvrII (Thermo Fisher Scientific, Italy) by following the manufacturer's instructions. Digested products were purified and analyzed on an ABI3730 capillary sequencer in genotyping mode with the size standard ROX-labeled GS500. Only peaks that achieved a prevalence of more than 1% have been considered. Replicate T-RF profiles of each DNA pool and T-RF profiles of distinct DNA pools from samples of each treatment gave reproducible fingerprints. Consensus profiles were created as suggested by Dunbar et al. (2001), using the average values for peak heights. Total richness (S), Shannon's diversity index (H), and Simpson's evenness index (E) were calculated using PRIMER (v7, PRIMER-E Ltd., Plymouth, United Kingdom).

#### NGS Analysis

The V4 hypervariable region of the 16S rRNA gene was amplified using modified universal bacterial primer pairs 515F/806R designed for use with the Illumina platform (forward primer: 5 0 -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGC CAGCMGCCGCGGTAA-30 ; reverse primer: 50 -GTCTCGTGGG CTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTW TCTAAT-30 ), with three replicates per DNA pool. PCR replicates were purified, quantified, and pooled in equimolar amounts as described before. Subsequently, amplicons were indexed and sequenced according to the Illumina MiSeq 16S Metagenomic Sequencing Library Preparation protocol3 . Sequencing was performed on the Illumina MiSeq platform (Illumina, San Diego, CA, United States) at Molecular Digital Diagnostics S.r.L. (Viterbo, Italy).

The Quantitative Insights Into Microbial Ecology (QIIME, v1.9.0; Caporaso et al., 2010) software was used to analyze the 16S rRNA sequence generated from paired-end amplicon sequencing. Paired-end reads were merged with PEAR (Zhang et al., 2014), setting a p-value cutoff of 0.05. Chimeras were detected and

2https://www.ncbi.nlm.nih.gov/genbank/

3https://support.illumina.com/content/dam/illumina-marketing/documents/ products/other/16s-metagenomics-faq-1270-2014-003.pdf

filtered from the paired-end reads using USEARCH (v6.1; Edgar, 2010). Operational taxonomic units (OTUs) were assigned to the reads using an open reference approach with UCLUST algorithm (Edgar, 2010) against the SILVA database release 132 that was clustered at 97% identity.

Based on the genus-level classification, principal component analysis (PCA) was performed to evaluate the similarity among various metagenomic communities. The PCA plots were displayed using PAST 3 (Hammer et al., 2001). The relative microbial abundance of all samples was summarized in a taxa plot.

### Indole Acetic Acid Production

Estimation of extracellular indole acetic acid (IAA) was determined using the colorimetric method described by Patten and Glick (2002). Bacteria were grown for 16 h at 30◦C, in LB medium (50 mL) without or with L-tryptophan (0.1% wt/vol). After growth, spent medium was recovered by centrifugation at 8000 r/min for 10 min and directly used for IAA quantification using Salkowski's reagent (0.5 M FeCl3 in 35% perchloric acid). The mixture was incubated at room temperature for 30 min and absorbance of the developed pink color was read at 530 nm. IAA concentration in the culture was determined by using a calibration curve of pure IAA (Sigma–Aldrich, Italy), as a standard. All these experiments were carried out in triplicate.

#### Phosphate Solubilization

Phosphate solubilization activity was determined on NBRIP medium agar plates containing insoluble Pi. NBRIP medium contained (per liter of distilled water): glucose, 10 g; MgCl2 . 6H2O, 5 g MgSO4 . 7H2O, 0.25 g; KCl, 0.2 g; (NH4)2SO4, 0.1 g; Ca3(PO4)2, 5 g (Nautiyal, 1999). Plates were incubated for 2–5 days at 30◦C. Colonies with clear halos were considered as phosphate solubilizing colonies. Solubilization index (SI) was calculated as: SI = (colony diameter + halo zone diameter)/colony diameter (Premono et al., 1996). All these experiments were carried out in triplicate.

# Antimicrobial Activity

Evaluation of antimicrobial activity was performed on the following strains: Erwinia amylovora Ea273 strain (ATCC 49946); Trichoderma reseii DIBAF-10 an environmental strain isolated from poplar chips, Trichoderma viride T-67 strain (ATCC 28020), Phytophthora cinnamomi isolate 1, Fusarium culmorum isolate 485, F. culmorum isolate J1, Fusarium oxysporum isolate 2, and Fusarium graminearum isolate 3, were kindly supplied by Prof. G. Chilosi (DIBAF, University of Tuscia).

Production of diffusible compounds with antifungal activity was tested on Potato Dextrose Agar (PDA) using a dualculture in vitro assay. First, a 5-mm-diameter mycelium disk from a 5-day-old fungal culture was placed on the surface of the agar plate in the center of the petri dish. Then, a bacterial suspension from an overnight culture on LB medium was streaked, on three sides, at a distance of about 3 cm from the fungus plug. Zones of inhibition were measured after 5 days of incubation at 30◦C according to the method of Geels and Schippers (1983). Bacterial strains that caused an inhibition zone of at least 2 mm were judged as positive. All these experiments were repeated independently at least twice.

Production of bioactive volatile organic compounds (VOCs) with antimicrobial activity was assessed by the double plate technique. This analysis was carried out on Bacillus strains F13 and F14 which exhibited the best inhibition against phytopathogenic Fusarium and Phytophthora in dual-culture assay. One hundred microliters of a bacterial suspension from an overnight culture on LB medium was spread on a LB-agar petri dish and a 5-mm disk of a 5-days-old pure culture of the fungus was placed at the center of another Petri dish containing PDA. Both half plates were placed face to face, preventing any physical contact between the fungal pathogen and the bacterium, and sealed to isolate the inside atmosphere and prevent loss of volatiles formed during the growth. Each pair of plates was incubated at 30◦C for 48 h and the growth of the pathogen was measured and compared to a control prepared in the same manner but without bacteria.

For the determination of the inhibitory effect of the bacterial isolates on pathogenic fungi, the percentage of inhibition of radial growth (PIRG %) was calculated according to the following formula 100 × (R1 − R2)/R1, where R1 and R2 are the radial growth of the pathogen in the absence and in the presence of the antagonist, respectively.

Antibacterial activity was assessed on plates according to Homma et al. (1989). Bacterial isolates were grown as spots on LB agar plates for 18 h and exposed to chloroform vapors for 30 min. After aeration, plates were covered with a suspension of E. amylovora Ea273 strain, obtained by mixing 5 mL of diluted LB soft agar (0.6%) with 200 µL of a stationary phase culture. Plates were incubated at 30◦C until inhibition halos were detected.

Fungal and bacterial inhibition assays were repeated independently at least twice.

## Nucleotide Sequence Accession Numbers

Sequences from independent templates/clones and amplicon libraries were deposited in GenBank under accession number: MH329697; MH338201; MH341118; MH375452; MH375453; MH375462; MH375472- MH375478; MH3755548; MH375601; MH375635; MH375635; MH375636; MH375453; MH376404; MH376429; MH376688; MH3756691; MH376690; MH376691; MH379797.

### Statistical Analysis

Analysis of variance of the data was calculated using the software package, SPSS 10 for Windows, 2001. Tukey HSD test was performed at p = 0.05 on each of the significant variables measured. PCA was carried out compare NGS data obtained with samples collected 1 day after the last treatment. The PCA outputs include variable loading to each selected component and treatment component scores.

# RESULTS

## Effects on Plant Growth and Epiphytic Bacteria

fpls-10-00060 February 1, 2019 Time: 17:52 # 6

Effects of vegetal-derived products on plant growth and composition of bacterial communities associated with lettuce leaves were determined on 5-weeks-old plants treated for 21 days with commercial extracts or hydrolysates of vegetal proteins. Leaf chlorophyll content expressed by the SPAD index was significantly increased by PE and PH (avg. 24.9) in comparison with Cu-PH and untreated control (18.9; **Table 2**). Fresh shoot biomass was also significantly enhanced by foliar applications of plant extracts or hydrolysates of vegetal proteins (avg. 6.69 g/plant) in comparison with untreated control (6.05 g/plant). After 1 day from the foliar application, the highest culturable aerobic epiphytic bacteria were observed in PE treatment whereas Cu-PH gave the lowest value. However, after 7 days from the foliar application the culturable aerobic epiphytic bacteria showed a different trend with the highest value in Cu-PH treatment (**Table 2**). Similarly, to fresh shoot biomass, leaf fresh weight sampled for the determination of abundance of culturable aerobic epiphytic bacteria was highest in lettuce plants treated with commercial products (**Table 2**).

Differences in the culturable count at 1 day were less pronounced when no-treated samples were compared to those collected from leaves of PE- or PH-treated lettuce. As shown in **Table 2**, the number of culturable epiphytic bacteria increased twofold in PE-treated plants (from 2.48 ± 0.03∗103 to 4.92 ± 0.02∗103 CFU/g of biomass) and had a slight decrease (about 0.3-fold) in samples from PH-treated plants (from 2.48 ± 0.03∗103 to 1.76 ± 0.05∗103 CFU/g of biomass).

In no-treated and in PE- or PH-treated plants, the abundance of culturable bacteria significantly decreased over the time and, 7 days after the last treatment, reached the same final value (between 0.92 ± 0.01∗ 103 and 1.11 ± 0.01∗ 103 CFU/g of biomass; **Table 2**).

#### Molecular Characterization of Culturable Bacteria

Based on colony morphology, we identified 23 morphotypes which were differentially distributed in the epiphytic population collected from leaves of treated and no-treated lettuce (**Table 3**). Only seven morphotypes were present in the aerobic bacterial population from no-treated control plants, while the larger number of morphotypes occurred in PH-treated lettuce (15 out of 23; **Table 3**).

For each morphotype, at least two independent colonies were characterized to the genus/species level using 16S rRNA gene as DNA barcode (see section "Materials and Methods"). Sequence data of 16S fragments were used to generate a phylogenetic tree to evaluate the genetic relatedness among these bacteria and known species (**Figure 1**). All strains exhibiting the same morphotype had identical 16S rRNA gene sequences (not shown). The 23 morphotypes belonged to six family and eight different genera of which Pseudomonas, Bacillus, and Exiguobacterium were the most recurrent. As shown in **Figure 1**, all Exiguibacterium strains clustered with Exiguibacterium indicum; Bacillus strains clustered into different clades corresponding to Bacillus cereus group (strain 5a, 5b, and F14), Bacillus pumilus (strain F12), and Bacillus mojavensis (strain F13); Pseudomonas strains could be organized in four different groups clustering with Pseudomonas putida (strain C3, C7, and F9), Pseudomonas psychrotolerans (strain F1G and F16), Pseudomonas rhizosphaerae (strain F4), and Pseudomonas moraviensis (strain F5), respectively (**Figure 1**).

#### Molecular Characterization of Bacterial Epiphytic Community

The structure of the total epiphytic bacterial community was evaluated using a molecular approach based on the T-RFLP of 16S rRNA gene (Osborn et al., 2000). To identify T-RFs in the community profiles, AvrII and SspI fingerprints of target gene from culturable bacteria reported in **Table 3** were generated. Completed digestion of PCR products gave T-RF of: 107 (218) bp for AvrII and 351 (354) bp for SspI on Pseudomonas DNA; 351 bp (SspI) on Acinetobacter and Micrococcus DNA; 259 (AvrII) and 355 bp (SspI) on Enterobacter/Pantoea DNA; 361 bp (SspI) on Sphingobacterium 16S gene. PCR product from Bacillus C5a and C5b generated a single peak of 174 bp after SspI digestion. Both enzymes do not generate detectable T-RFs using, as a template, DNA from Exiguobacterium and other Bacillus isolates.

As shown in **Figure 2**, SspI and AvrII digestions of amplicons from epiphytic bacterial community gave a total of 10 and 17

PE = tropical plant extract; PH = protein hydrolysate; Cu-PH = copper-based PH. Values ( ± SD) with no letter in common significantly differ at p ≤ 0.05 (Tukey HSD test).a∗ , ∗∗ Significant at P ≤ 0.05 and 0.01, respectively.

(+) = present; (−) = absent/no-detectable.

peaks, respectively. Differences in the absolute number and in the relative height of discernible peaks could be seen comparing no-treated and treated samples, as well as samples collected 1 or 7 days after the last treatment. Differences in T-RF profiles of samples collected at 1 and 7 days were observed with both treated and no-treated plants. Three SspI T-RFs (351, 361, 364 bp) and one AvrII T-RF (107 bp) occurred in all samples, albeit the relative abundance (RA) of each of these phylotypes was significantly different in the single T-RFs pattern. In T-RF profiles of notreated samples (1 and 7 days), a lower number of peaks were observed and with both enzymes 80% of the total peak area was associated to a single peak (351 bp in SspI profiles, 107 bp in AvrII profiles). In T-RF profiles of treated samples (1 and 7 days), specific additional peaks were observed at: 106, 355, and 356 bp for SspI, 120, 255, 259, and 261 bp for AvrII.

To describe the changes in the dominance among the phylotypes, the ecological diversity indices were calculated combining the data of both enzymes (**Figure 3**). The maximal number of species determined by T-RFLP analysis (15) was found in 1-day PH-treated samples and 7 days after the last treatment in samples from PE-treated plants (**Figure 3**). As shown in **Figure 3**, for PE and Cu-PH we observed an increase in species richness from 1 to 7 days. In contrast, in no-treated and PH-treated samples, we observed an overall reduction in the species richness over the time. In samples collected 1 and 7 days after the last treatment from PE- and PH-treated plants, the diversity (H) and evenness (E) were much higher than in no-treated plants (**Figure 3**). Interestingly, in samples collected 1 day after the last treatment with Cu-PH, we observed a reduction in the species richness (S), as well as an increase in the evenness compared to no-treated plants (**Figure 3**). These observations indicated that, on leaves of Cu-PH-treated plants, a lower number of species were present but a more equitable distribution in species abundance occurred.

To gain more insights about the effect of vegetal PHs on the structure of bacterial community of lettuce phyllosphere, samples collected one day after the last treatment were also analyzed using a next generation sequencing (NGS) approach. This analysis was only carried out on PH-treated and notreated plants for two major reasons: the treatment with PH gave the highest species diversity (**Figure 3**); PH does not contain inorganic compounds, such as copper salts (Cu-PH) and micronutrients (PE), whose combined effect with vegetal PHs could not be easily uncoupled. NGS analysis based on the sequencing of V3–V4 region of 16S rDNA gene allowed us to generate a number of reads per sample comprised between 120,000 and 150,000 (not shown). Approximatively 90% of raw reads per sample passed merging, trimming, and chimera filtering steps and were analyzed for OTU search. In both samples, two phyla accounted for 90% of total sequence reads with the majority belonging to Firmicutes (74.7% of total bacteria in no-treated samples) or Proteobacteria (72.2% in samples from PH-treated plants:

**Figure 4**). In PH-treated (compared to no-treated) samples, we observed a reduction of Bacillales (from 74.5 to 27.3% of total bacteria) and an increase in Pseudomonadales (from 21.5 to 44.4%) and Enterobacteriales (from 3.6 to 26.7%; **Figure 4**). Other orders that occurred as minor forms in PH-treated (and were not detectable in no-treated) samples comprised Sphingomonadales, Flavobacteriales, Micrococcales, and Pasteurellales.

A deeper phylogenetic classification of the reads at genus level (**Figure 4**) revealed that the most over-represented genera in PH-treated samples were Bacillus (25.5% of total bacteria), Pantoea (24.9%), Pseudomonas (24.6%), and Acinetobacter (19.3%). In no-treated samples, Bacillus and related genera represented 73.1% of total bacteria, major genera under the order of Pseudomonadales were Acinetobacter (15.8% of total bacteria) and Pseudomonas (5.4%), whereas Enterobacteriales included bacteria belonging to Pantoea genus (3.3%; **Figure 4**).

Principal component analysis analysis suggested substantial differences in the epiphytic microbial community between PHtreated and no-treated plants, as shown from the distribution in different zones of the PCA graph of data sets obtained from different samples (**Figure 5**). The two axes were involved in 99.9% of the total variance and could explain most variations in bacterial community structure. The first principal component (PC1) explained 78.4% and the second (PC2) 21.5% of the variance at genus level. Along the first axis, variability was mainly explained by an increase in the population of bacteria belonging

FIGURE 2 | Percentage of relative abundance of T-RFs after SspI and AvrII digestion among no-treated and PE-, Cu-PH-, and PH-treated samples at 1 and 7 days after the last treatment. The numbers indicate the size (in bp) of the T-RF fragments.

to Pantoea and Pseudomonas genus (**Figure 5**). Variability along the second PCA axis corresponded to a decrease in the RA of some bacteria belonging to Bacillus and Acinetobacter genus (**Figure 5**).

# Phenotypic Characterization of Culturable Bacteria

Epiphytic isolates from lettuce plants were tested for features known to contribute to plant growth promotion, such IAA production and mineral phosphate solubilization, or plant growth protection against phytopathogens. Results reported in **Table 4** indicated that about 30% of all isolates (7 out of 23) were able to solubilize mineral phosphate, with a SI ranging between 2.5 and 4.8.

About 35% of isolates (8 out of 23) produced indoleacetic acid in tryptophan supplemented LB medium. The highest level of IAA was obtained with Pantoea strain C1 (>100 mg L−1 ), and good levels of production were obtained with either Pseudomonas and Micrococcus (5–36 mg L−1 ) or Acinetobacter strains (≤5 mg L−1 ).

The antagonistic activity against phytopathogens was assayed using a dual culture technique (Oldenburg et al., 1996). Results reported in the **Table 4** indicated that most Bacillus strains had a high antagonist activity against F. oxysporum and E. amylovora. Bacillus strains F13 and F14 also exhibited strong inhibitory activity against P. cinnamomi (**Table 4**). E. amylovora was also strongly inhibited by Pantoea sp. C1 and Micrococcus sp. F3 strains. Biocontrol activity against fungi belonging to Fusarium and Phytophthora genus although at lower inhibitory levels were also observed with Pseudomonas strains F1G and F16. The latter one also exhibited antagonistic activity against E. amylovora (**Table 4**).

# Antifungal Activity in vitro

The production of volatile metabolites active against fungi belonging to different Fusarium (F. graminearum, F. culmorum, and F. oxysporum) and Trichoderma (T. viride and Trichoderma reesei) species, was further investigated using Bacillus strains F13 and F14 which exhibited the best inhibition against phytopathogenic fungi in dual-culture assay.

Results from double plate assays indicated that both strains produce volatile compounds with strong inhibitory activity (higher than 65%) against these phytopathogens (**Figure 6**). With F13 strain, PIRG % against Fusarium strains varied between 72.2 ± 0.3 (vs F. graminearum) and 78.0 ± 0.9 (vs F. culmorum isolate J1), whereas no significant difference was observed between PIRG values against T. viride and T. reesei (about 75%; **Figure 6**).

Interestingly, the metabolites produced by Bacillus F13 strain were stable and remained biological active over a wide range of pH (between 2 and 10) and temperature (between 4 and 100◦C), showing no loss of activity even after autoclaving (data not shown).

# DISCUSSION

The primary object of this work was to evaluate the effect of foliar applications of commercial products containing vegetal-derived bioactive compounds on the structure of epiphytic bacterial community and unforeseen implications on useful/deleterious bacteria enrichment.

TABLE 4 | Plant growth-promoting (PGP) activity and inhibitory effect on the growth of plant pathogens of culturable epiphytic bacteria isolated from treated and no-treated lettuce plants.

∗Phosphate solubilization index; § indole acetic acid production.

Foliar applications of the three commercial products increased fresh weights of shoots and leaves in lettuce plants compared to control treatment; moreover, leaf chlorophyll content expressed by the SPAD index was enhanced by foliar applications of PE and PH in comparison with Cu-PH and control treatments. These results agree with the previous findings on the effect of PH and PE on corn, tomato and spinach (Colla et al., 2014, 2017b; Rouphael et al., 2018). Bioactive compounds such as amino acids, peptides, and phytohormones supplied by the commercial products may be responsible for the increase of foliar biomass (**Table 2**). To better understand the effect of plant-derived bioactive products on leaf-associated bacteria, data obtained using different approaches were combined to have insight into the distribution of specific taxa and on the potential ability of strains belonging to these taxa to promote plant growth and/or suppress diseases. PE was the unique tested product that determined a significant twofold increase in the number of cultivable epiphytic bacteria compared to samples from no-treated plants (**Table 2**). This positive effect of PE on plant growth could be due to the stimulation of cell proliferation induced by signaling compounds (e.g., phytohormones, amino acids), the better protection of cells from oxidative damage resulting from the supply of antioxidant compounds (vitamins), the enhancement of plant metabolism arising from the supply of micronutrients (**Table 1**; Colla et al., 2017b) as well as of its microbiota. Similar evidences were obtained analyzing differences in the bacterial communities between treated and no-treated plants 1 day after the last treatment. Ecological diversity measures indicated a significant increase (P < 0.05) in species richness, evenness, and diversity indices in PE-treated (as well as in PH-treated) compared to notreated plants (**Figure 3**). This microbial diversity persisted for at least a week (**Figure 3**), whereas no significant difference in the abundance of culturable bacteria was observed comparing treated (PE or PH) and no-treated plants 7 days after the last treatment (**Table 2**). In contrast, the use of copper-containing products,

such as Cu-PH, had a transient negative effect on the epiphytic bacteria, as demonstrated by the reduction, at 1 day, of total aerobic count (25-fold compared to no-treated plants **Table 2**) and total species richness (1.25-fold vs no-treated control; **Figure 3**). A similar effect on the reduction of the bacterial biodiversity was also observed by Almeida et al. (2017) studying the dynamic of the bacterial community in sediments exposed to copper and the use of these bacterial communities resistant to copper to improve phytoremediation of copper-contaminated sediments. A comparison of T-RFLP profiles (**Figure 2**) and indices of diversity and evenness (**Figure 3**) of samples from no-treated and Cu-PH-treated lettuce also demonstrated that Cu-PH strongly affected the structure of the bacterial epiphytic community. In fact, in samples collected 1 day after the last treatment, we observed a significant increase (P < 0.05) in both Shannon–Weaver diversity and evenness when plants were treated with Cu-PH (**Figure 3**). In particular, the RA (% of total peak height) of the 351-bp peak in SspI T-RFLP profile decreased from 83 (no-treated) to 58% (Cu-PH), while RA of SspI peak sized 174 bp, the representative peak of Bacillus morphotypes C5a and C5b (**Table 3**), increased ninefold (from 3 to about 27%; **Figure 2**). A similar stimulatory effect was also observed with other minor forms that were undetectable in the SspI T-RFLP profile of no-treated samples, such as 106-, 355-, and 356-bp peak. The latter SspI peaks were also detected in T-RFLP profiles of samples collected from PE- and PHtreated plants, indicating that plant-derived bioactive compounds have the ability to stimulate the growth of specific groups of bacteria related to morphotypes C1, C4, and C6 (SspI peak of 355 bp) belonging to Enterobacter/Pantoea group (**Figure 1**) and to other not yet defined species (106- and 356-bp peaks). Interestingly, SspI peak sized 355 bp and the corresponding 259 bp AvrII peak became the most abundant ones (RA of 61 and 71%, respectively) in T-RFLP profiles obtained with samples collected from lettuce leaves 7 days after the last treatment with

Cu-PH (**Figure 2**). Therefore, we can conclude that the use of vegetal-PHs and plant extracts can determine the enrichment of epiphytic bacteria related to Enterobacter/Pantoea group (**Figure 1**), and that this effect can be enhanced combining these products with copper. Interestingly, Enterobacter/Pantoea group includes beneficial bacteria with PGP traits and biological control against phytopathogens (Taghavi et al., 2009; Madhaiyan et al., 2010; Dutkiewicz et al., 2016; Singh et al., 2018), as well as strains having the ability to interfere with the quorum-sensing which control biofilm and EPS formation in some food-borne pathogens, such as Yersinia enterocolitica (Gopu et al., 2016). The ability of copper-tolerant bacteria to promote plant growth was also observed by Liu et al. (2014) analyzing bacteria isolated from mine tailings. Our observation is also in agreement with current literature on phytoremediation indicating that bacteria with PGP traits can facilitate the removal of inorganic contaminants stimulating either plant growth or phytoremediation activity (Glick, 2010; Glick and Stearns, 2011; Ullah et al., 2015).

As shown in **Figure 3**, effects on the structure of the epiphytic bacterial community were also observed treating lettuce with PE and PH. These products selectively stimulated the growth of Pseudomonas-related bacteria that were not detected in notreated samples at 1 day (SspI T-RF sized 354 bp; **Figure 2**). This conclusion was supported by the information that in T-RFLP profiles peaks corresponding to Pseudomonas morphotypes F1G and F4 (SspI T-RF of 354 bp and AvrII T-RF of 107 bp; **Table 2**) and morphotype F9 (SspI T-RF of 354 bp and AvrII T-RF of 218 bp; **Table 2**) were predominant (>50%) in PE- (1 day) and PH-treated (1 and 7 days) samples (**Figure 2**). Interestingly, strains F1G, F4, and F9 are related to members of different P. putida subclusters (**Figure 1**) that have been characterized for their ability to solubilize inorganic phosphate, such as P. rhizosphaerae (Kwak et al., 2015), enhance plant growth or antagonize fungal phytopathogens, such as Pseudomonas fulva/Pseudomonas parafulva/P. putida (Pena et al., 2016), fix nitrogen and enhance nutrient uptake, such as P. psychrotolerans (Liu et al., 2017).

Peaks corresponding to Pseudomonas morphotypes C3, C7, and F5 (SspI T-RF of 351 bp and AvrII T-RF of 107 bp) and morphotype F16 (SspI T-RF of 351 bp and AvrII T-RF of 218 bp; **Table 2**) were predominant in no-treated and Cu-PH-treated at 1 day (RA of 83 and 58%, respectively), remained the major peaks in no-treated samples at 7 days (RA of 90%) and became the most abundant ones in PE-treated samples at 7 days (RA of 54%; **Figure 2**). These peaks were also present in T-RFLP profiles of samples from PH-treated lettuce, whereas they were not the most predominant ones and their RA (16–18%) did not change over the time (**Figure 2**). In conclusion, treatments with hydrolysates of plant proteins lead to changing patterns of Pseudomonas populations which are quite complex to analyze and might lead to a specific enrichment of strains, such as morphotype F1G, showing plant promoting traits (**Table 4**).

As mentioned before, peaks corresponding to Enterobacter/ Pantoea morphotypes (SspI T-RF of 355 bp and AvrII T-RF of 259 bp; **Table 2**) were also present in PE- and PH-treated samples and their RA increased, over the time (1→7 day after the last treatment), from 6 to 27% in samples from PE-treated plants and from 9 to 16% in those from PH-treated plants (**Figure 2**).

Comparing differences in the relative fluorescence of AvrII peaks specific for Pseudomonas (107 and 218 bp) and 351 bp SspI peak belonging to Pseudomonas and other species, and considering that vegetal-derived PHs and plant extracts analyzed in this work also stimulated the growth of cultivable Acinetobacter and Micrococcus strains with a SspI T-RF peak of 351 bp (**Table 3**), we can postulate that, in samples from treated plants, the relative high values of fluorescence associated with the 351-bp SspI peak (compared to the combined fluorescens of 107- and 218-bp peak in AvrII profile) reflects the presence in the epiphytic community, in addition to Pseudomonas, of a more abundant population of bacteria belonging to Acinetobacter and Micrococcus genus. It is worth pointing out that Acinetobacter and Micrococcus strains isolated from treated plants (**Table 3**) have PGP traits, such as ability to produce plant-related hormones (IAA) or solubilize inorganic phosphate (**Table 4**) and, therefore, their enrichment can be valuable for the plant.

Strains with PGP traits were enriched with all commercial products examined in this work, albeit each product stimulated the growth of a specific group of microorganisms. For example, Pseudomonas and Pantoea strains able to produce high levels of IAA were specifically stimulated by PH or PE (morphotypes C1, F1G, and F5), whereas Micrococcus sp. strains (F3 and F10) able to produce IAA and solubilize inorganic phosphate were specifically enriched on plants treated with Cu-PH (**Tables 3**, **4**). Other strains able to solubilize inorganic phosphate such as Pseudomonas sp. F1G or F5 were enriched by treatment with PE/PH or PH/Cu-PH, respectively.

The ability of vegetal-derived bioactive compounds to promote shifts in the composition of epiphytic bacterial communities, stimulating the growth of specific strains and increasing bacterial diversity, was confirmed by comparison of NGS data from no-treated and PH-treated plants (**Figure 4**). In fact, in samples collected 1 day after the last treatment, we observed that PH treatment altered the structure of the leafassociated microbiome from phylum to genus level determining a reduction in the RA of bacteria belonging to Bacillales and, at the same time, an increase in the population of Pseudomonas, Pantoea, and other minor forms (**Figure 4**). Interestingly, the analysis at the level of individual OTUs demonstrated that PH specifically stimulates the growth of specific members of the epiphytic microbiota (**Figure 5**). This effect explains the increase in bacteria biodiversity, the alteration of the population of specific taxa, such as Bacillus, and the enrichment of rare species and specific strains which can play an important role in plant growth and protection (Shade et al., 2014; Jousset et al., 2017).

In agreement with data reviewed by Shafi et al. (2017), we demonstrated that microorganism belonging to Bacillus genus play an important role in lettuce microbiota as biocontrol agents (BCAs) against fungal and bacterial pathogens. Interestingly, the biocontrol activity is associated with strains related to B. mojavensis (F13) and B. cereus group (C5a, C5b, and F14; **Figure 1**), whose growth was stimulated by vegetal PHs and plant extracts (**Table 3**).

Among Bacillus strains isolated from treated plants, strain F13 exhibited the highest inhibitory activity (>71%) on the radial growth of all fungal pathogens tested in this work (**Figure 6**). The latter result is in agreement with data demonstrating that endophytic and epiphytic B. mojavensis strains have broadspectrum antibacterial properties related to their ability to produce lipopeptides, surfactin and fengycin (Vágvölgyi et al., 2013; Kalai-Grami et al., 2014; Kim et al., 2015; Blacutt et al., 2016; Jasim et al., 2016). This wide range of metabolites can reduce pathogen attack by suppressing fungal growth or inducing the plant immune system (Khan et al., 2017). The ability of these bacteria to produce spore facilitates the use of Bacillus as bioinoculant.

#### CONCLUSION

The present study revealed that vegetal PHs and extracts containing vegetal-derived bioactive compounds can stimulate the growth of epiphytic bacteria with PGP and/or biological control activity against pathogens. Metagenomic analysis also demonstrated that these products can stimulate the growth of rare members of the microbial community that can promote

## REFERENCES

the biostimulant effects of vegetal PHs and have direct and indirect effects on the ecosystem functioning and the plant health.

This is the first report indicating that the use of this class of biostimulants can enrich autochthonous bacterial strains able to enhance plant growth.

# AUTHOR CONTRIBUTIONS

MR, GC, and FL conceived the study and wrote the manuscript. AF and EBŠ contributed to corrections and suggestions. GC and EBŠ performed experiments on plants. FL isolated the culturable bacteria and extracted the metagenomic DNA. AF, MR, and FL performed the analysis of T-RFLP and NGS data and performed the phenotypic and molecular characterization of culturable bacteria. All authors read and approved the final manuscript.

# FUNDING

This research was partially supported by Italpollina S.p.A. (Rivoli Veronese, Italy).

degrade ethylenethiourea. Curr. Microbiol. 66, 243–250. doi: 10.1007/s00284- 012-0263-8

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

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

# Understanding the Biostimulant Action of Vegetal-Derived Protein Hydrolysates by High-Throughput Plant Phenotyping and Metabolomics: A Case Study on Tomato

, Luigi Lucini2

#### Edited by:

Kenny Paul1†

Martin Trtílek1

Mariateresa Cardarelli4

, Mirella Sorrentino1†

, Klára Panzarová1

, Paolo Bonini5

Jorge Almiro P. Paiva, Institute of Plant Genetics (PAN), Poland

#### Reviewed by:

Andrea Ertani, University of Padova, Italy Giacomo Cocetta, University of Milan, Italy

#### \*Correspondence:

Klára Panzarová<EMAIL_ADDRESS>Giuseppe Colla<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

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

Received: 02 November 2018 Accepted: 14 January 2019 Published: 08 February 2019

#### Citation:

Paul K, Sorrentino M, Lucini L, Rouphael Y, Cardarelli M, Bonini P, Reynaud H, Canaguier R, Trtílek M, Panzarová K and Colla G (2019) Understanding the Biostimulant Action of Vegetal-Derived Protein Hydrolysates by High-Throughput Plant Phenotyping and Metabolomics: A Case Study on Tomato. Front. Plant Sci. 10:47. doi: 10.3389/fpls.2019.00047 1 Photon Systems Instruments (PSI, spol.sr.o.), Drásov, Czechia, 2 Department for Sustainable Food Process, Research Centre for Nutrigenomics and Proteomics, Università Cattolica del Sacro Cuore, Piacenza, Italy, 3 Department of Agricultural Sciences, University of Naples Federico II, Naples, Italy, 4 Centro di Ricerca Orticoltura e Florovivaismo, Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria, Pontecagnano Faiano, Italy, 5 NGAlab, Tarragona, Spain, 6 Italpollina USA, Inc., Anderson, IN, United States, 7 Nixe, Valbonne, France, 8 Department of Agriculture and Forest Sciences, University of Tuscia, Viterbo, Italy, 9 Arcadia Srl, Rivoli Veronese, Italy

\* and Giuseppe Colla8,9

, Hélène Reynaud6

, Youssef Rouphael3

\*

,

,

, Renaud Canaguier7

Designing and developing new biostimulants is a crucial process which requires an accurate testing of the product effects on the morpho-physiological traits of plants and a deep understanding of the mechanism of action of selected products. Product screening approaches using omics technologies have been found to be more efficient and cost effective in finding new biostimulant substances. A screening protocol based on the use of high-throughput phenotyping platform for screening new vegetal-derived protein hydrolysates (PHs) for biostimulant activity followed by a metabolomic analysis to elucidate the mechanism of the most active PHs has been applied on tomato crop. Eight PHs (A–G, I) derived from enzymatic hydrolysis of seed proteins of Leguminosae and Brassicaceae species were foliarly sprayed twice during the trial. A non-ionic surfactant Triton X-100 at 0.1% was also added to the solutions before spraying. A control treatment foliarly sprayed with distilled water containing 0.1% Triton X-100 was also included. Untreated and PH-treated tomato plants were monitored regularly using high-throughput non-invasive imaging technologies. The phenotyping approach we used is based on automated integrative analysis of photosynthetic performance, growth analysis, and color index analysis. The digital biomass of the plants sprayed with PH was generally increased. In particular, the relative growth rate and the growth performance were significantly improved by PHs A and I, respectively, compared to the untreated control plants. Kinetic chlorophyll fluorescence imaging did not allow to differentiate the photosynthetic performance of treated and untreated plants. Finally, MS-based untargeted metabolomics analysis was performed in order to characterize the functional mechanisms of selected PHs. The treatment modulated the multi-layer

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regulation process that involved the ethylene precursor and polyamines and affected the ROS-mediated signaling pathways. Although further investigation is needed to strengthen our findings, metabolomic data suggest that treated plants experienced a metabolic reprogramming following the application of the tested biostimulants. Nonetheless, our experimental data highlight the potential for combined use of highthroughput phenotyping and metabolomics to facilitate the screening of new substances with biostimulant properties and to provide a morpho-physiological and metabolomic gateway to the mechanisms underlying PHs action on plants.

Keywords: protein hydrolysates, integrative image-based high-throughput phenotyping, metabolomics, morphophysiological traits, functional biostimulant characterization, ROS signaling

# INTRODUCTION

Over the past decade, interest in plant biostimulants (PBs) has been on the rise, compelled by the growing interest of researchers, private industry and farmers in integrating these products in the array of environmentally friendly tools that secure improved crop productivity and yield stability under environmental stressors (Ertani et al., 2012, 2013; Haplern et al., 2015; Colla et al., 2017a; Yakhin et al., 2017; Rouphael et al., 2017a,c, 2018). Based on the new EU regulation, PBs are defined as 'CE marked products which stimulate plant physiological processes independently of the their nutrient content by improving one or more of the following characteristics of the plant rhizosphere or phyllosphere: nutrient use efficiency, tolerance to abiotic stress, crop quality, availability of confined nutrients in the soil and rhizosphere, humification and degradation of organic compounds in the soil' (European Commission, 2016). Protein hydrolysates (PHs) are an important category of PBs which are produced by chemical, enzymatic or by combining chemical and enzymatic hydrolysis of proteins from animal or plant source (Ertani et al., 2009, 2017; Niculescu et al., 2009; Calvo et al., 2014; Colla et al. 2015, 2016, 2017a,b). Over the past 10 years, plant-derived PHs produced through enzymatic hydrolysis have received huge interest from farmers due to their high agronomic value and the lack of limitation in their application on organically produced crops (Colla et al., 2014; Nardi et al., 2016). PH-based biostimulants can be applied to plants through foliar application or soil/substrate drenching. PHs sprayed in foliar way reach mesophyll cells by absorption through cuticle, epidermal cells and stomata (Fernández and Eichert, 2009) while in drench application, the absorption occurs through root epidermal cells and gets redistributed through xylem (Subbarao et al., 2015). PHs can also be applied as seed treatments especially for field crops such as wheat, corn, and soybean (Rouphael et al., 2018). PH application stimulates plant uptake of macro and micronutrients and helps in rapid plant growth and biomass accumulation, interfering with the carbon and nitrogen metabolic activities (Ertani et al., 2009, 2016; Colla et al., 2017a). PHs can also improve crop tolerance to abiotic stresses such as drought, salinity, and thermal stress (Ertani et al., 2013; Lucini et al., 2015; Colla et al., 2017a). Therefore, improving metabolic and physiological traits by PH-based biostimulant treatments provides novel strategies for maximizing biomass yield (Dudits et al., 2016). Development of highly effective PH-based biostimulants requires an accurate evaluation of the effects of candidate products on morpho-physiological traits of selected crops during different developmental stages and environmental conditions. As conventional screening methods are time consuming, destructive (e.g., fresh and dry weight estimation), labor intensive and expensive, high-throughput plant phenotyping procedures were recently proposed as effective and high-precision tools for product screening in order to increase the probability of finding new bioactive substances in a more cost- and time-effective manner (Povero et al., 2016; Rouphael et al., 2018; Ugena et al., 2018). 'Phenomics' as a technological tool considers systematic management of complex traits in genome (G) × environment (E) interactions (Houle et al., 2010). Plant phenotyping systems are fully automated robotic systems usually installed in a controlled environment or in semi-controlled greenhouse conditions. The phenotyping platforms are designed to ensure not only noninvasive monitoring of plants in throughput of few up to several hundreds of plants, but also provide means for automated cultivation and handling of the plants such as automated watering/weighing or nutrient delivery units (Fahlgren et al., 2015; Großkinsky et al., 2015). High-throughput phenotyping systems, which can capture plant growth, morphology, color and photosynthetic performance using RGB and chlorophyll fluorescence (ChlF) imaging tools, are highly promising and essential tools for dissecting physiological components in product screening and for dynamic quantitative analysis of plant growth and physiological performance (Rahaman et al., 2015; Awlia et al., 2016; Rouphael et al., 2018). RGB imaging is used to estimate the true color of each pixel and by using image processing algorithms for identification of plant-derived pixels. For identified plant objects, morphological and geometrical features are quantified including color properties (Rahaman et al., 2015). The pixel number-based assessment of plant volume or total leaf area correlates with fresh and dry weight of above ground plant biomass and can be thus used to evaluate green/fresh weight of the plants without cutting and measuring them (Fehér-Juhász et al., 2014; Fahlgren et al., 2015). Further image-based automated phenotyping permits time-series measurements that are necessary to follow the progression of growth performance and stress responses on individual plants.

Chlorophyll fluorescence is a popular technique in plant physiology used for rapid non-invasive measurement of

photosystem II (PSII) activity. PSII activity is very sensitive to a range of biotic and abiotic factors and therefore the chlorophyll fluorescence technique is used as a rapid indicator of photosynthetic performance of plants in different developmental stages and/or in response to changing environment (Baker, 2008). The advantage of chlorophyll fluorescence measurements over other methods for monitoring stresses is that changes in chlorophyll fluorescence kinetic parameters often occur before other effects of stress are apparent (Murchie and Lawson, 2013). Chlorophyll fluorescence imagers integrated in highthroughput phenotyping platforms are becoming important tools for rapid screening for better photosynthetic performance and characterization of a plant's ability to harvest light energy, which is directly related to plant biomass formation and plant architecture (Tschiersch et al., 2017).

Nonetheless, the comprehension of biochemical processes and physiological functions underlying the changes observed at phenotype level is of primary relevance to scientifically demonstrate and support the use of plant biostimulants, likely providing some clues on the best scenarios where these products can be used. It is expected that in the near future, provided that a regulatory framework will be implemented at least in the EU and United States, the information on mechanism/mode(s) of action will support biostimulants authorization and implementation. In this regard, metabolomics is being proposed as a close link between an organism's genotype and phenotype (Lamichhane et al., 2018), including plant-environment interactions (Feussner and Polle, 2015). In fact, recent advances in metabolomics, data treatment and multi-variate statistics offer the possibility to achieve a rather inclusive phytochemical profile in biological systems, including plants, thus opening new opportunities (Meier et al., 2017; Tsugawa, 2018). This makes metabolomics a promising tool to elucidate, among others, the mode of action rather than the physiological processes involved in plant response to biostimulants.