Forest tree associated bacteria for potential biological control of Fusarium solani and of Fusarium kuroshium, causal agent of Fusarium dieback

Journal Pre-proof Forest tree associated bacteria for potential biological control of Fusarium solani and of Fusarium kuroshium, causal agent of Fusarium dieback ´ Nailea Baez-Vallejo, David A. Camarena-Pozos, Juan L. ´ ´ Monribot-Villanueva, Monica Ram´ırez-Vazquez, Gloria L. ´ Carrion-Villarnovo, Jose´ A. Guerrero-Analco, Laila P. ´ erique ´ Partida-Mart´ınez, Fred Reverchon

PII:

S0944-5013(19)30921-8

DOI:

https://doi.org/10.1016/j.micres.2020.126440

Reference:

MICRES 126440

To appear in:

Microbiological Research

Received Date:

16 August 2019

Revised Date:

9 November 2019

Accepted Date:

15 February 2020

´ Please cite this article as: Baez-Vallejo N, Camarena-Pozos DA, Monribot-Villanueva JL, ´ ´ Ram´ırez-Vazquez M, Carrion-Villarnovo GL, Guerrero-Analco JA, Partida-Mart´ınez LP, Reverchon F, Forest tree associated bacteria for potential biological control of Fusarium solani and of Fusarium kuroshium, causal agent of Fusarium dieback, Microbiological Research (2020), doi: https://doi.org/10.1016/j.micres.2020.126440

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Forest tree associated bacteria for potential biological control of Fusarium solani and of Fusarium kuroshium, causal agent of Fusarium dieback

Nailea Báez-Vallejo1, David A. Camarena-Pozos2, Juan L. Monribot-Villanueva1, Mónica Ramírez-Vázquez1, Gloria L. Carrión-Villarnovo3, José A. Guerrero-Analco1, Laila P. Partida-

Red de Estudios Moleculares Avanzados, Instituto de Ecología, A.C. Carretera antigua a Coatepec 351,

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Martínez2, Frédérique Reverchon4*

Col. El Haya, 91070 Xalapa, Veracruz, México.

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Nailea Báez-Vallejo: [email protected]; Juan L. Monribot-Villanueva: [email protected]; Mónica Ramírez-Vázquez: [email protected]; José A. Guerrero-Analco:

Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados. Km. 9.6 Lib.

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[email protected]

Nte. Carr. Irapuato-León. 036824 Irapuato, Guanajuato, México.

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David A. Camarena-Pozos: [email protected]; Laila P. Partida-Martínez: [email protected]

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Red de Biodiversidad y Sistemática, Instituto de Ecología, A.C. Carretera antigua a Coatepec 351, Col.

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El Haya, 91070 Xalapa, Veracruz, México.

Gloria Luz L. Carrión-Villarnovo: [email protected]

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Red de Estudios Moleculares Avanzados, Instituto de Ecología, A.C. Avenida Lázaro Cárdenas 253,

61600 Pátzcuaro, Michoacán, México. Frédérique Reverchon: [email protected] *Corresponding author: Frédérique Reverchon: [email protected]

Abstract

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Although the use of crop-associated bacteria as biological control agents of fungal diseases has

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gained increasing interest, the biotechnological potential of forest tree-associated microbes and their natural products has scarcely been investigated. The objective of this study was to identify

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bacteria or bacterial products with antagonistic activity against Fusarium solani and Fusarium kuroshium, causal agent of Fusarium dieback, by screening the rhizosphere and phyllosphere of

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three Lauraceae species. From 195 bacterial isolates, we identified 32 isolates that significantly

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reduced the growth of F. solani in vitro, which mostly belonged to bacterial taxa Bacillus, Pseudomonas and Actinobacteria. The antifungal activity of their volatile organic compounds

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(VOCs) was also evaluated. Bacterial strain Bacillus sp. CCeRi1-002, recovered from the rhizosphere of Aiouea effusa, showed the highest percentage of direct inhibition (62.5%) of F. solani and produced diffusible compounds that significantly reduced its mycelial growth. HPLCMS analyses on this strain allowed to tentatively identify bioactive compounds from three

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lipopeptide groups (iturin, surfactin and fengycin). Bacillus sp. CCeRi1-002 and another strain identified as Pseudomonas sp. significantly inhibited F. solani mycelial growth through the emission of VOCs. Chemical analysis of their volatile profiles indicated the likely presence of 2nonanone, 2-undecanone, disulfide dimethyl and 1-butanol 3-methyl-, which had been previously 2

reported with antifungal activity. In antagonism assays against F. kuroshium, Bacillus sp. CCeRi1-002 and its diffusible compounds exhibited significant antifungal activity and induced hyphal deformations. Our findings highlight the importance of considering bacteria associated with forest species and the need to include bacterial products in the search for potential antagonists of Fusarium dieback.

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Keywords: Bacillus; Bacterial volatiles; Lauraceae; Lipopeptides; Phyllosphere; Rhizobacteria.

1. Introduction

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Plant-associated microorganisms fulfill important functions for plant growth and health

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(Barea et al., 2005; Berg, 2009). Rhizosphere microorganisms are particularly important for plant development as they participate in processes such as nitrogen (N) fixation, phosphorus (P)

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solubilization, plant growth promotion through the production of phytohormones, or plant protection against phytopathogens (Bais et al., 2006; Bakker et al., 2013). The aerial surface of

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plants, named phyllosphere, also hosts communities of microorganisms that are adapted to harsh environmental conditions and have been associated with important plant functional traits and growth (Kembel et al., 2014). Moreover, phyllosphere microbes form an important barrier for the protection of their host against pathogen infections (Vorholt, 2012).

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Recently, integrating biological control in pest and disease management strategies has been

promoted as a promising alternative to the use of chemical pesticides, due to its low environmental impact and lower economic cost (Ab Rahman et al., 2018). The biocontrol of diseases in plants aims at controlling the populations of pathogens using other organisms (Yang 3

et al., 2001). Moreover, the application of natural products and chemical compounds extracted from different sources, such as plant extracts, natural or modified organisms or gene products are other examples of biological control (McSpadden Gardener and Fravel, 2002; Pal and Gardener, 2006). One strategy to find potential biocontrol agents is to isolate bacteria that are present in the phyllosphere or rhizosphere of plants of interest. Such bacteria may produce diffusible antibiotic compounds or emit volatile organic compounds (VOCs) with antifungal activity (Cazorla et al.,

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2007; Kai et al., 2009). Bacteria from the genera Bacillus and Pseudomonas, for example, are

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considered as important biological control agents of fungal phytopathogens, as they produce compounds such as lipopeptides (Cawoy et al., 2015), 2,4-diacetylphloroglucinol (DAPG)

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(Bergsma-Vlami et al., 2005) and VOCs that may inhibit mycelial growth or fungal spore

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germination (Yuan et al., 2012; Ossowicki et al., 2017).

Tropical montane cloud forests, despite their limited distribution, are highly diverse

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ecosystems and critical providers of ecosystem services (Williams-Linera, 2002). One of the most representative plant family of tropical montane cloud forests are the Lauraceae (Lorea-

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Hernández, 2002). Members of the Lauraceae family have been reported to be threatened by several pathogenic fungi, vectored by invasive ambrosia beetles. The highly pathogenic fungus Raffaelea lauricola forms a symbiosis with the ambrosia beetle Xyleborus glabratus and causes laurel wilt (Harrington et al., 2008). On the other hand, the ambrosia beetles Euwallacea spp. nr.

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fornicatus, with their fungal symbionts Fusarium euwallaceae and Fusarium kuroshium, among others, are responsible for Fusarium dieback (Lynch et al., 2016; Na et al., 2018), which causes serious damage to more than 58 plant families, among them the Lauraceae (Eskalen et al., 2013). In 2015, the ambrosia beetle Euwallacea sp. nr. fornicatus, also known as Kuroshio Shot Hole 4

Borer (KSHB), was detected in Tijuana, Mexico, representing a serious threat to the agricultural and forestry sectors of the country (García-Ávila et al., 2016; Lira-Noriega et al., 2018). It is thus important to explore different alternatives for the treatment and control of these emerging plant diseases, and to consider biological control as an option (Dunlap et al., 2017). Although several studies have investigated the antifungal activity of rhizobacteria from avocado trees (Persea americana Mill.) against the pathogenic fungi vectored by the ambrosia beetles Euwallacea spp.

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nr. fornicatus (Guevara-Avendaño et al., 2018, 2019), there are no reports so far on the potential

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of bacteria associated with forest trees to control the causal agents of Fusarium dieback.

Furthermore, there is still limited information regarding the biotechnological potential of forest

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tree-associated microbes and their microbial products (Cazorla and Mercado-Blanco, 2016) to

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tackle forest tree diseases. The objectives of this study were therefore 1) to evaluate the antifungal activity of bacteria isolated from the rhizosphere and phyllosphere of three selected

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Lauraceae forest tree species and 2) to characterize the chemical compounds present in crude extracts and in volatiles obtained from the bacterial isolate showing the strongest antifungal

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activity.

2. Materials and methods

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2.1. Sampling and isolation of phyllosphere and rhizosphere bacteria from three Lauraceae species

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Samples of phyllosphere and rhizosphere soil of Lauraceae species Aiouea effusa (Meisn.) R. Rohde & Rohwer, Damburneya salicifolia (Kunth) Trofimov & Rohwer and Ocotea psychotrioides Kunth., were collected in April and August 2017 at the Ecological Centre “Las Cañadas” located in Huatusco, Veracruz State, Mexico. The study site is a private reserve constituted by 118 ha of lower tropical montane cloud forest growing on Andosols. Vegetation is dense and dominated by Juglans pyriformis Liebm., Quercus insignis M. Martens & Galeotti, Q.

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leiophylla A. DC., and Q. xalapensis Bonpl. (Mehltreter and García-Franco, 2008). Five different

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adult trees per Lauraceae species, without any visual disease symptoms, were randomly selected within different points of the reserve. Five leaves were collected per tree, from different points of

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the canopy. Four rhizosphere soil samples were collected per tree, at the four cardinal points,

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approximately 50 cm away from the trunk and at a depth of 5–10 cm. Leaf and soil samples from a same tree were then mixed to form one composite sample from the phyllosphere and one from

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the rhizosphere, for each tree. All samples were transported in sterile bags, at 4 ºC, and were immediately processed upon arrival at the laboratory.

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Phyllosphere solutions were prepared by cutting a 1 cm diameter disk from five different leaves of each tree and adding 5 mL of sterile 0.9% (w/v) NaCl solution. Rhizosphere samples were processed as follows: loose soil was removed from the roots and the soil that was strongly adhered to the roots (rhizosphere soil) was recovered using a small brush. Subsequently,

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rhizosphere solutions were prepared with 1 g of rhizosphere soil and 99 mL of sterile distilled water and homogenized by shaking vigorously. As in our previous study (Guevara-Avendaño et al., 2019), and due to quarantine restriction,

screening for antifungal activity was first implemented with Fusarium solani, which was selected 6

as a model organism for being phylogenetically related to F. euwallaceae (O’Donnell et al. 2015) and F. kuroshium (Na et al. 2018). The strain of F. solani used in this study, provided by Mauricio Luna-Rodríguez (Universidad Veracruzana), was isolated from chilli (Capsicum annum L.) and was the same as that used in our other studies (Guevara-Avendaño et al., 2019; Reverchon et al., 2019). To carry out the screening for bacterial isolates with antifungal activity, Petri dishes with potato dextrose agar (PDA, Sigma-Aldrich, Mexico) and tryptone soy agar

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(TSA, Sigma-Aldrich, Mexico) were inoculated with a 5 mm diameter disk of F. solani

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mycelium, placed on the left side of the plate. The Petri dishes were incubated at 28 ºC in the dark until mycelial growth reached 1 cm diameter. Dilutions of 1:10 (v/v) were prepared from

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phyllosphere and rhizosphere solutions and then streaked on the right side of the Petri dishes

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(opposite side from the F. solani mycelium disk), in triplicate. Plates were incubated at 30 ºC in the dark for 7 days. Bacteria that were able to grow in contact with the fungus and showed visual

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evidence of mycelial growth inhibition were taken from the plates as they grew and sub-cultured in Luria Bertani (LB, Sigma-Aldrich, Mexico) agar until pure cultures were obtained. Culture

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purity was verified using standard Gram staining procedure and microscopic observations. Pure bacterial isolates were preserved in LB medium and 20% (v/v) of glycerol and subsequently stored at -20 ºC.

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2.2. In vitro evaluation of antifungal activity exhibited by the obtained bacterial isolates

Bacterial isolates that were obtained in the first screening were evaluated in vitro for

antagonistic activity against F. solani. Bacterial isolates were re-streaked onto LB agar plates and 7

incubated at 30 ºC during 48 h. An isolate of F. solani was incubated onto PDA at 28 ºC for 7 days before setting up the dual culture assays. One plug of 5 mm of diameter was taken from the border of the F. solani mycelium and placed on the center of a PDA plate. Bacterial isolates were taken from a single colony with a toothpick and inoculated at a 2 cm distance from the mycelial growth, following the procedure described in Guevara-Avendaño et al. (2018). Three different bacterial isolates were tested per plate and a sterilized toothpick mark was used as a control on

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each plate. The combination of isolates with the control was carried out in triplicate. The direct

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antagonism assays were incubated at 30 ºC in the dark and the growth of the fungus towards the bacterial and control treatments was measured on the seventh day. Subsequently, the percentage

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of inhibition of mycelial growth was calculated using the following formula:

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% Inhibition = [(R-r)/R] * 100

where R is the radius of fungal growth from the center of the plate towards the control treatment,

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and r is the radius of fungal growth towards the bacterial treatment.

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2.3. Molecular identification of bacterial isolates with antifungal activity

Bacterial isolates that showed a significant inhibition of mycelial growth of F. solani were identified through 16S rRNA gene sequencing. DNA was extracted from each bacterial isolate

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using the DNeasy® Blood and Tissue kit (Qiagen, Germany), following the instructions of the included protocol. The 16S rRNA region was amplified by PCR using universal primers 27F (5´AGAGTTTGATCMTGGCTCAG-3´) and 1492R (5´-TACGGYTACCTTGTTACGACTT-3´). Each PCR reaction was carried out in a total volume of 50 μl, containing 25-150 ng of template 8

DNA, 1X of Taq buffer, 0.2 mM of each dNTP, 2.5 mM of MgCl2, 0.4 μM of each primer, and 1.75U of Taq DNA polymerase (Invitrogen, USA). Amplification was performed in a SureCycler 8800 (Agilent Technologies, California), under the following conditions: initial denaturation for 4.5 min at 95 ºC, followed by 40 cycles of denaturation for 1 min at 95 ºC, annealing for 1 min adapted at 53 ºC, extension for 2 min at 72 ºC and final extension step of 5 min at 72 ºC (Hogg and Lehane, 1999). To evaluate the success of the amplification, electrophoresis was performed

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on a 1% agarose gel and the PCR products were purified using the Wizard® SV Gel and PCR

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Clean-Up System kit (Promega, USA) following the instructions of the included protocol. The DNA integrity was verified by electrophoresis on a 1% agarose gel and the final concentration of

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pure DNA was quantified in the NanoDrop™ 2000 (Thermo Fisher, USA). Purified DNA

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amplicons were then sent to Macrogen Inc. (Seoul, Korea) for sequencing, using primer 27F. Sequences were manually edited in BioEdit 7.2.5 program (Hall, 1999). Edited sequences

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were compared with the GenBank nucleotide database (www.ncbi.nlm.nih.gov), from which their best matches were retrieved and used to construct an alignment with the multiple alignment

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program MUSCLE integrated in MEGA 7 (Kumar et al., 2016). Three Maximum-Likelihood trees (one per phylum) were constructed using a Kimura two parameter model with Gamma distribution and a Bootstrap method with 1000 replicates (Wang et al., 2007). Sequences were

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deposited in GenBank (accession numbers MH538904 to MH538932).

2.4. In vitro evaluation of the antifungal activity of bacterial VOCs against F. solani

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We evaluated the antifungal activity of bacterial VOCs emitted by the 29 bacterial isolates obtained from the phyllosphere and rhizosphere of A. effusa, D. salicifolia and O. psychotrioides that exhibited significant antifungal activity in the direct antagonism assays and could be successfully sequenced. The bacterial isolates were grown in TSB (tryptic soy broth, Bioxon) in constant agitation at 150 rpm, at 28 ºC in the dark during 19 h. After that time, 1 mL of each bacterial culture was taken and placed in a microtube, and centrifuged during 3 min at 12,000

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rpm. The supernatant was discarded and the remaining pellet was washed twice with 1 mL of

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PBS (phosphate buffered saline). The concentration of the obtained bacterial culture was adjusted to 1×108 CFU·mL-1.

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The evaluation of the antifungal activity of bacterial VOCs was carried out by the sealed

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double plate method described in Giorgio et al. (2015). One plug of 5 mm of diameter was taken from the border of the F. solani mycelium and placed on the center of a PDA plate. In the center

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of another plate with PDA or TSA medium, 50 μl of bacterial culture, prepared as aforementioned, were inoculated. The treatments and the control (PDA or TSA plate without

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bacterial culture) were carried out in triplicate. The assays were incubated at 28 ºC in the dark and the growth of the fungus towards the bacterial and control treatments was measured on the seventh day. Subsequently, the percentage of inhibition of mycelial growth was calculated using the following formula:

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% Inhibition = [(D-d)/D] * 100,

where D is the diameter of fungal growth in the control, and d is the diameter of fungal growth when exposed to bacterial volatiles.

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2.5. Analysis of bacterial VOCs by SPME-GC-MS

The emitted bacterial VOCs with potential antifungal activity against F. solani were trapped by solid phase micro-extraction (SPME) and further analyzed by gas chromatography coupled to mass spectrometry (GC–MS), following the procedure described in Camarena-Pozos et al. (2019). Briefly, volatiles were collected employing a gray SPME fiber, coated with 50/30 μm of

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Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) (Supelco 57348-U, placed

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on a SPME holder, 57330-U, Sigma-Aldrich, Mexico). The fiber was inserted into the headspace of the Petri plate through a hole previously obtained by piercing the Petri plate wall. Volatile

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trapping was carried out during 20 h. The fibers were then placed in the injector port of a gas

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chromatograph (Agilent 5890 Series II; Agilent, Foster City, CA) equipped with a MS detector (HP 5972) and the Chemstation Software (Hewlett-Packard Co.). Volatile compounds were

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desorbed at 230 ºC for 30 s. Helium was used as the carrier gas (1 mL min-1) and the detector temperature was 260 ºC. A free fatty acid-phase capillary column (HP-FFAP) of 25 m length ×

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0.52 mm inner diameter × 0.32 μm film thickness was used. The GC–MS was operated following the conditions described previously (Camarena-Pozos et al., 2019). The mass fragments were analyzed using electron impact ionization at 70 eV and a scan rate of 1.9 scan s-1. The mass spectrum data of volatile compounds were compared with those from NIST 08 and AMDIS. Only

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compounds with match values superior to 700 were taken and compared to the mass spectra of the NIST / EPA / NIH Mass Spectral Library version NIST 08. The VOCs profiles of bacterial isolate were compared with those of the control (PDA and TSA medium, without bacteria). Only those compounds that were not detected in the control chromatograms were reported. 11

2.6. Preparation of bacterial crude extracts

The characterization of the chemical composition of bacterial crude extracts was only carried out on the bacterial isolate that showed the highest percentage of inhibition in the direct antagonism assays. Bacterial isolate CCeRi1-002, obtained from the rhizosphere of A. effusa, was

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incubated on LB agar at 30 °C for 24 h. Subsequently, a bacterial dilution was prepared in sterile

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distilled water at a concentration of 1.5 × 108 CFU (Colony Forming Units) mL-1, using the McFarland scale. For each 500 mL of LB broth, 1 mL of bacterial dilution was added and

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incubated at 30 °C for 7 days and constant stir at 180 rpm, in dark conditions (Lavermicocca et

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al., 1997). The obtained bacterial culture was centrifuged at 20,000 × g for 15 min at 25 °C to separate the supernatant from the cell biomass. Crude extracts were obtained by subjecting the

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supernatant (5 L) to a partitioning procedure (1:1, v/v), first with ethyl acetate (EtOAc) to extract the non-polar and medium polar compounds, and subsequently with n-butanol (n-BuOH) for

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extraction of the compounds of greater polarity. Each extraction was conducted at least three times with each solvent. The same procedure was carried out for the control, which was prepared in LB broth, and 1 mL of sterile distilled water was added per 500 mL of medium. The resulting organic phases were filtered through a layer of anhydrous sodium sulfate

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placed in a funnel and plugged with paper filter to remove the remaining moisture, and the solvent excess was removed by evaporation under reduced pressure in a Rotavapor® (BUCHI, RII, Switzerland) thus obtaining the crude extracts of EtOAc and n-BuOH, respectively. Subsequently, 50 mg of each crude extract were dissolved in 1 mL of methanol with formic acid 12

(0.1%, v/v). Each sample was filtered through 13 mm syringe filters with 0.2 µM nylon membrane (Pall Life Sciences, East Hills, NY) to remove impurities and used for the tentative identification of antifungal compounds using ultra-high performance liquid chromatography (UPLC) and high resolution mass spectrometry (HRMS).

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2.7. Chemical profiling of antifungal compounds using UPLC -HRMS

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The chromatographic system was an UPLC Class I of Waters coupled to a Synapt G2-Si HDMi mass spectrometer. The chromatography was carried out on an Acquity BEH column (1.7 m,

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2.1 × 50 mm) with a column and sample temperatures of 40 °C and 15 °C. The mobile phase

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consisted of (A) water and (B) acetonitrile, both with 0.1% (v/v) of formic acid (Sigma). The gradient conditions of the mobile phases were 0-13 min linear gradient 1 - 80% B, 13-14 min

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80% B isocratic, 14-15 min linear gradient 80 - 1% B (total run time 20 min). The flow rate was 0.3 mL min-1 and 1 μL of each extract was injected. The mass spectrometric analysis was

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performed with an electrospray ionization source in positive mode with a capillary, sampling cone and source offset voltages of 3,000, 40 and 80 V, respectively. The source temperature was 100 °C and the desolvation temperature was 20 °C. The desolvation gas flow was 600 L h-1, and the nebulizer pressure was 6.5 Bar. Leucine-enkephalin was used as the lock mass (556.2771,

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[M+H] +). The conditions used for MS analysis were: mass range 100-2000 Da, function 1 CE, 6 V, function 2 CER 10-30 V, scan time 0.5 sec. The data were acquired and processed with MassLynx (version 4.1, Waters™) software packages. Accurate mass values (m/z ratios) of the major peaks exclusively detected in the extracts obtained from inoculated media were compared 13

with those previous studies that reported m/z of bacterial compounds with antifungal activity (Sun et al., 2006; Li et al., 2012b; Liao et al., 2016; Torres et al., 2016, 2017).

2.8. In vitro evaluation of the antifungal activity of bacterial extracts

The antifungal activity of the bacterial extracts was evaluated by the agar well diffusion

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method (Balouiri et al., 2016). Prior to implementing the assays, F. solani was incubated in PDA

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for 7 days at 28 ºC in the dark. Subsequently, a spore solution was made by immersing the mycelium in 15 mL of sterile distilled water and homogenized by shaking vigorously. The

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number of spores was quantified using a Neubauer chamber (Celoromics, France), until reaching

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a concentration of 1.0 × 106 spores mL-1. Antagonism assays were carried out by inoculating 1 mL of spore solution per 100 mL of PDA, which were added to Petri dishes. Once the PDA

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medium with the spore solution was solidified, a well of approximately 9 mm of diameter was made in the center of each Petri dish, and inoculated with 100 μL of the following treatments: a)

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bacterial extract: 10 mg of extract dissolved in 1 mL of vehicle (H2O-Methanol-Dimethyl sulfoxide (DMSO) [2:1:1]); b) control extract: 10 mg of non-inoculated solvent (EtOAc or nBuOH) dissolved in 1 mL of vehicle (H2O-Methanol-Dimethyl sulfoxide (DMSO) [2:1:1]); c) positive control: 10 mg of Thiabendazole (Sigma-Aldrich) dissolved in 1 mL of sterile distilled

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water; d) negative control: vehicle only (H2O-Methanol-Dimethyl sulfoxide (DMSO) [2:1:1]); e) blank control (fungus only). Each treatment was carried out in triplicate. Assays were incubated at 27 ºC for 5 days in the dark after which the zone of inhibition was measured.

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2.9. In vitro evaluation of the antifungal activity of bacterial strain CCeRi1-002 and its crude extracts against F. kuroshium

Bacterial strain CCeRi1-002, which showed the highest percentage of inhibition in the first antagonism assays against F. solani, was selected for additional evaluation of its antifungal

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activity against the fungal pathogen F. kuroshium, one of the causal agents of Fusarium dieback.

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The same procedures as described before for antagonism assays via diffusible and volatile

compounds against F. solani were implemented, using three replicates. Antagonism assays

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against F. kuroshium were incubated at 27 ºC for seven days in dark conditions and were carried

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out at the quarantine facilities of SENASICA (Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria), through the CNRF (Centro Nacional de Referencia Fitosanitaria, Tecámac,

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México), where strain HFEW-16-IV-019 of F. kuroshium, isolated from the ambrosia beetle Euwallacea sp. nr. fornicatus (KSHB), was provided (Sánchez-Rangel et al., 2018). Moreover,

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the antifungal activity of crude extracts obtained from bacterial strain CCeRi1-002 was also evaluated against F. kuroshium, following the procedure described previously for assessment against F. solani.

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2.10. Microscopic analysis

The hyphal deformations of F. solani induced by the tested bacterial isolates were observed

by collecting mycelium from the antagonism assays described in section 2.2. The effect of each 15

bacterial isolate on F. solani was observed in triplicate; mycelium was collected from each assay after seven days, with sterile scalpel and tweezers, and control samples were collected from hyphae of F. solani growing without bacterial isolate. Hyphae were fixed and stained with a drop of Lactophenol Cotton Blue solution (Sigma Aldrich, USA) and in some cases with a 1% (w/v) aqueous solution of Congo red (Sigma Aldrich, USA), and immediately observed with an optical microscope LEICA DM500 (100X) (Largent et al., 1980).

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The mycelium of Fusarium kuroshium, which was subjected to bacterial diffusible

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compounds, bacterial VOCs and crude extracts of isolate CCeRi1-002 (prepared as described in section 2.6), was observed with Scanning Electron Microscopy (SEM). Samples for SEM were

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collected with sterile scalpel and tweezers from the antagonism assays (treatments and controls)

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described in section 2.9, in triplicate. The collected samples were then fixed in 4% paraformaldehyde + 0.2% cacodylate in PBS buffer, rinsed twice in the same buffer for 5 min,

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dehydrated in a graded ethanol series (30-100%) during 30 min for each concentration, dried in a Quorum K850 critical point dryer with CO2 and attached to aluminum stubs using a carbon

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adhesive prior to coating with gold in a sputter coater Quorum Q150. Samples were observed and photographed with a JEOL-IT300LV scanning electron microscope and software JSM-IT300 v. 1.020.

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2.11. Statistical analysis

To analyze mycelial growth data obtained on the seventh day from antagonism assays,

normality was checked by the Kolmogorov-Smirnov test. A one-way analysis of variance 16

(ANOVA) followed by a Tukey´s post-hoc test was implemented to evaluate differences in mycelial growth between treatments. For data that did not meet a normal distribution, we used the nonparametric Kruskal-Wallis test. These tests were performed using the SigmaStat 3.5.1 software. All the statistical tests were considered significant at P≤0.05.

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3. Results

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3.1. Antifungal activity of bacterial isolates against F. solani

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A total of 195 bacterial isolates were obtained from the phyllosphere and the rhizosphere of

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A. effusa (76 isolates), D. salicifolia (78 isolates) and O. psychotrioides (41 isolates). The 195 obtained isolates were tested in direct antagonism assays against F. solani, to determine if they

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could inhibit mycelial growth (Supplementary Material 1). The mycelial radial growth of F. solani was significantly reduced by 32 bacterial isolates, with inhibition percentages ranging

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from 6 to 62.5% (Figure 1). These 32 isolates were identified through 16S rRNA gene sequencing. DNA from three bacterial isolates could not be amplified. The closest sequence matches based on BLAST similarity analysis are presented in Table 1 for the 29 bacterial isolates that could be successfully sequenced. Most sequences belonged to the bacterial genera Bacillus

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and Pseudomonas, although the phylum Actinobacteria was also represented with genera such as Curtobacterium, Microbacterium, and Arthrobacter (Figure 2). Maximum likehood phylogenetic trees are available in Supplementary Materials 2 to 4.

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Bacterial strain CCeRi1-002, identified as Bacillus sp. (phylogenetically related to B. amyloliquefaciens) and obtained from the rhizosphere of A. effusa, displayed the greatest inhibition against F. solani. Morphological alterations induced by Bacillus sp. CCeRi1-002 in F. solani hyphae included the production of chlamydospores and vacuoles, extensive swelling, and in occasions a granulated cytoplasm, assessed visually seven days post-inoculation in comparison

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with the control (Figure 3a and 3b).

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3.2. Antifungal activity of bacterial VOCs against F. solani

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The 29 bacterial isolates that significantly inhibited F. solani mycelial growth in direct

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antagonism assays and could be successfully sequenced, were further tested to assess the antifungal activity of their emitted VOCs against F. solani. No fungal inhibition by bacterial

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VOCs was observed when bacterial isolates were grown on PDA medium. Only two bacterial strains, Bacillus sp. CCeRi1-002 and Pseudomonas sp. CCeRi5-020, significantly inhibited F.

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solani mycelial growth, by 50.6% and 64.7%, when grown in TSA (Supplementary Material 5). Both strains were obtained from the rhizosphere of A. effusa. The analysis of the bacterial volatile profiles by SPME-GC-MS tentatively identified 16 and 17 volatile compounds emitted by Bacillus sp. CCeRi1-002 and Pseudomonas sp. CCeRi5-020

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respectively, when inoculated on TSA medium (Table 2). The emitted VOCs on TSA medium belonged to different chemical classes, the most abundant compounds being aliphatic ketones and alcohols. Four compounds were emitted by both strains when growing on TSA medium (tentatively identified as 2-nonanone, 2-undecanone, 2-tridecanone and 2-undecanol), from the 18

ketone and alcohol class. When bacterial isolates were grown on PDA medium, 11 and 12 VOCs were tentatively identified as emitted by Bacillus sp. CCeRi1-002 and Pseudomonas sp. CCeRi5020 respectively (Supplementary Material 6). Sulfur-containing compounds were only emitted by Pseudomonas sp. CCeRi5-020, regardless of the culture medium. Compounds 2-nonanone, 2-undecanone, disulfide dimethyl, 1-butanol 3-methyl- and 1undecene, tentatively identified on both PDA and TSA culture media, have previously been

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reported for their antifungal or anti-oomycete activity as per the mVOC database (Table 2,

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Supplementary Material 6; Lemfack et al., 2018). The high similarity of their mass spectra with those of reference compounds reported in the NIST database is shown in Supplementary

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Materials 7 to 11.

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3.3. Antifungal activity of Bacillus sp. CCeRi1-002 against F. kuroshium

Bacillus sp. CCeRi1-002, which showed the highest percentage of inhibition (62.5%) in the

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first antagonism assays against F. solani, also showed significant antifungal activity against F. kuroshium, causal agent of Fusarium dieback. Bacillus sp. CCeRi1-002 reduced mycelial radial growth by 73.6% in direct antagonism assays. Microscopic observations evidenced a shriveling of hyphae and the occasional production of chlamydospores in the mycelium of F. kuroshium in

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contact with Bacillus sp. CCeRi1-002 (Figure 3d), as compared with the control (Figure 3c). On the other hand, no significant growth inhibition was detected when the mycelium of F. kuroshium was exposed to the VOCs emitted by Bacillus sp. CCeRi1-002, although microscopic

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observations showed that hyphae of F. kuroshium presented morphological alterations with apparent symptoms of lysis.

3.4. Antifungal activity of bacterial extracts from Bacillus sp. CCeRi1-002

The agar well diffusion method was used to determine the antifungal activity of the two crude

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extracts (EtOAc and n-BuOH) obtained by liquid-liquid extraction of strain Bacillus sp. CCeRi1-

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002 growing in the absence of F. solani. Both crude extracts significantly inhibited F. solani

mycelial growth when compared to their respective control (Figure 4a – 4d). Similar findings

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were obtained when the antifungal activity of the two crude extracts was assessed against F.

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kuroshium (Figure 4d). Alterations caused by both crude extracts on the hyphal morphology of F. kuroshium were analyzed with SEM. Microscopic observations showed that the mycelium that

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was exposed to both bacterial crude extracts presented similar evidences of substantial distortion, such as shriveling of hyphae, conglobated apical tips and expanded widths along hyphae (Figure

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4e – 4g).

3.5. Chemical profiling of bacterial diffusible compounds by UPLC-HRMS

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Crude bacterial extracts that were obtained from strain Bacillus sp. CCeRi1-002 were

analyzed by UPLC-HRMS. This analysis allowed to tentatively identify some bioactive compounds from three cyclo-lipopeptide groups (Table 3). In the EtOAc crude extract, ten compounds from the iturin and surfactin groups were tentatively identified (Figure 5a, Table 3). 20

In the n-BuOH extract, nine compounds from the iturin, surfactin and fengycin groups were detected (Figure 5b, Table 3). The presence of C-13 Iturin A1 in EtOAc and n-BuOH extracts was corroborated by the co-elution with an authentic commercial standard (Sigma-Aldrich, St. Louis) at the same analytical conditions. In both crude extracts, some compounds could not be assigned to a specific group (Table 3).

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4. Discussion

Based on the adverse effects of conventional agrochemicals on human health, on the

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environment and on living organisms, research efforts are currently focusing on microorganisms

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and their natural products as viable alternatives for the management of pest and plant pathogens (Ab Rahman et al., 2018). Bacteria that can grow in the rhizosphere and the phyllosphere of

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target plants are considered as suitable candidates in the search for biological control agents since they can deliver their pathogen-antagonizing metabolites into the root and leaf systems where

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they directly suppress pathogenic microbial growth (Shoda, 2000; Vacher et al., 2016). In the present work, bacterial isolates showing significant inhibition of the mycelial growth of F. solani, obtained from the phyllosphere and the rhizosphere of three Lauraceae species, mostly belonged to the Bacillus and Pseudomonas genera. These genera have been frequently reported for their

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antimicrobial activity (Ligon et al., 2000; Schisler et al., 2004) and for their plant growthpromoting properties (Santoyo et al., 2012; Singh et al., 2015; Ortiz-Castro et al., 2017). Members of the Actinobacteria phylum were also detected, such as Curtobacterium, Microbacterium and Arthrobacter. Curtobacterium species have been shown to interact with 21

their host plant by controlling phytopathogens (Garrido et al., 2016), such as bacteria Pseudomonas syringae pv. lachrymans and Acidovorax citrulli, or fungus Colletotrichum orbiculare (Raupach and Kloepper, 1998; Horuz and Aysan, 2018). Although some species of Curtobacterium have been reported to be phytopathogenic, their potential to control plant pathogens cannot be discarded, and using their produced metabolites may constitute an alternative in biocontrol strategies (Magalhães et al., 2017). Microbacterium spp. have been

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isolated from the phyllosphere and the rhizosphere of various plant species and used as biocontrol

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agents against pathogens such as Rhizoctonia solani (Barnett et al., 2006), Fusarium

verticillioides (Pereira et al., 2007; Sartori et al., 2012) or Aspergillus flavus (Mannaa et al.,

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2017). On the other hand, Arthrobacter spp. have displayed antagonistic activities against fungal

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pathogens such as Botrytis cinerea, F. verticillioides and the oomycete Phytophthora cinnamomi, through the emission of the volatile dimethylhexadecylamine, among other mechanisms

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(Cavaglieri et al., 2004; Velázquez-Becerra et al., 2013).

Bacillus sp. CCeRi1-002, which was obtained from the rhizosphere of A. effusa and is related

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to Bacillus amyloliquefaciens, produced diffusible compounds that reduced F. solani mycelial growth by 62.5% and that of F. kuroshium by 73.6%. Bacillus amyloliquefaciens has been considered very important for the control of phytopathogens due to its ability to produce various antifungal lipopeptides (Stein, 2005; Arguelles-Arias et al., 2009), mainly surfactin, iturin

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(mycosubtilin, iturin A, and bacillomycin), and fengycin (Kim et al., 2010; Malfanova et al., 2012). In this study, we tentatively identified bioactive compounds from the same three cyclolipopeptide families, whose molecular weights are in the range of 1000-1600 Da, according to a previous report (Liao et al., 2016). Fengycin-like molecules were only detected in the n-BuOH 22

extract, and we tentatively identified some analogs of fengycin previously reported in Pathak et al. (2012) for B. subtilis. Other reports showed that lipopeptides from Bacillus spp. can induce hyphal damage, vacuolization and the production of resistance structures such as hyphal and conidial chlamydospores in phytopathogenic fungi (Souto et al., 2004; Alvarez et al., 2012; Li et al., 2012a), which is consistent with the chlamydospores and conglobations induced in F. kuroshium and observed by SEM in our study. The antifungal activity exhibited by Bacillus sp.

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CCeRi1-002 may also be due to the secretion of hydrolytic enzymes such as such as chitinase, β-

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glucanase and protease, which were not present in the bacterial extracts (Li et al., 2016; Khan et al., 2018). Altogether, this suggests the need for future studies to test the antifungal activity of

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antifungal enzymes by Bacillus sp. CCeRi1-002.

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pure compounds or enriched fractions with lipopeptides and to analyze the putative secretion of

The antifungal activity of bacteria can also occur through the production of VOCs, which can

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inhibit fungal growth (Guevara-Avendaño et al., 2019), induce systemic resistance in plants (Ryu et al., 2004; Tahir et al., 2017), promote plant growth (Raza et al., 2016; Camarena-Pozos et al.,

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2019) and prevent the colonization of plant tissues by phytopathogens (Farré-Armengol et al., 2016). Bacillus sp. CCeRi1-002 also exhibited significant antifungal activity through VOCs emission, against F. solani. An additional strain (CCeRi5-020), identified as Pseudomonas sp. and also obtained from the rhizosphere of A. effusa, inhibited F. solani mycelial growth through

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VOCs production. Rhizobacteria from the Bacillus genus have been previously isolated from avocado (Persea americana Mill.), a Lauraceae species, and shown to emit VOCs with antimicrobial properties (Méndez-Bravo et al., 2018; Guevara-Avendaño et al., 2019). Furthermore, Bacillus amyloliquefaciens has been reported to emit VOCs that inhibit mycelial 23

growth and spore germination of Fusarium oxysporum f. sp. cubense, mainly through the production of aromatic compounds and aliphatic ketones such as 2-nonanone and 2-decanone (Yuan et al., 2012). These compounds, as well as several other ketones, were tentatively found in the volatile profile of strain Bacillus sp. CCeRi1-002. The antifungal activity of 2-nonanone, 2decanone and 2-dodecanone was confirmed against F. solani using pure synthetic compounds by Guevara-Avendaño et al. (2019). These authors also confirmed the antifungal activity of

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dimethyl-disulfide against F. solani, which was tentatively detected in the volatile profile of

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Pseudomonas sp. in our study. Dimethyl disulfide, as well as 2-nonanone, 2-undecanone and 1undecene, have also been identified in common bean rhizobacteria Pseudomonas brassicacearum

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and P. putida, and were shown to reduce mycelial growth of Sclerotinia sclerotiorum, a

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necrotrophic pathogenic fungus causing white mold in a wide host range of plants (Giorgio et al., 2015). Volatile compound 1-undecene, which was tentatively identified as the most abundant

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compound emitted by strain Pseudomonas sp. CCeRi5-020, was also shown to reduce mycelial growth, sporangium formation, germination, and zoospore release in Phytophthora infestans, the

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causal agent of late blight in tomato and potato (Hunzinker et al., 2015). Altogether, these results support the biotechnological potential of bacterial VOCs for their application in biological control strategies.

The use of plant-associated bacteria, or the chemical compounds they produce, to tackle

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fungal diseases is gaining increasing interest, especially in agroecosystems (Beneduzi et al., 2012; Ciancio et al., 2016). However, the use of forest tree-associated microbes as biocontrol agents of fungal pathogens has been scarcely investigated, despite the economic and ecological importance of forest ecosystems (Terhonen et al., 2018). Our findings emphasize the importance 24

of considering bacterial isolates associated with forest species, and not only agricultural crops, for the screening of potential antagonists of phytopathogenic fungi. Naturally occurring bacteria associated with P. americana, a Lauraceae species of agricultural importance, have been recently described as promising for the biological control of Fusarium dieback (Guevara-Avendaño et al., 2018, 2019). Recent research from our group evidenced the potential of rhizobacteria from tropical montane cloud forest species to act as antagonists of F. solani and F. oxysporum

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(Reverchon et al., 2019). Although the implementation of field-based assays was not possible due

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to quarantine restrictions, the present study highlights the potential of bacterial natural products, especially bacterial crude extracts, for the control of Fusarium dieback. Further studies should

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aim at determining the bacterial compounds produced during the interaction with F. kuroshium.

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Recent findings have shown that some bacterial VOCs and lipopeptides are only produced in the presence of a fungal pathogen (Zihalirwa Kulimushi et al., 2017; Avalos et al., 2018), which

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suggests the existence of a triggering mechanism emitted by the fungus and perceived by the bacteria (Li et al., 2014). Consequently, bacterial metabolism may be modified upon interaction

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with the fungal pathogen, leading to differences in the profiles of emitted bacterial compounds (Chaves-López et al., 2015) and to the possible secretion of a different set of antifungal factors.

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5. Conclusions

We identified 32 bacterial isolates, retrieved from the phyllosphere and rhizosphere of three

Lauraceae species, which significantly reduced the mycelial growth of F. solani. These isolates belonged to the genera Bacillus, Pseudomonas, Curtobacterium, Microbacterium, Arthrobacter, 25

Methylobacterium, Erwinia and Hafnia. Bacillus sp. CCeRi1-002, obtained from the rhizosphere of A. effusa, exhibited the strongest antifungal activity against F. solani and produced diffusible compounds that significantly reduced F. solani mycelial growth. Analysis of crude extracts obtained from Bacillus sp. CCeRi1-002 by UPLC-HRMS allowed to putatively identify compounds belonging to three antifungal cyclo-lipopeptide groups: iturin, surfactin and fengycin. Volatiles emitted by Bacillus sp. CCeRi1-002, among which the presence of ketones, alcohols

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and alkenes was detected, inhibited the growth of F. solani by 50.6%. Another bacterial strain,

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identified as Pseudomonas sp., also exhibited significant antifungal activity against F. solani by VOCs emission. Bacillus sp. CCeRi1-002and its crude extracts also presented significant

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antagonistic activity against F. kuroshium, one of the causal agents of Fusarium dieback. Our

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findings highlight the importance of including natural products from bacterial isolates in the search for potential biocontrol agents, and the need to identify compounds which could not be

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phytopathogens.

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assigned to any category, in order to detect new bioactive compounds against fungal

Acknowledgements

This research was funded by the National Forestry Commission of Mexico (Comisión Nacional

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Forestal or CONAFOR) grant number 265677 and by the National Fund for Scientific and Technological Development (Fondo Nacional de Desarrollo Científico y Tecnológico or FORDECYT) grant number 292399. We thank CONACyT for the postgraduate fellowship awarded to the first author. We thank Ricardo Romero González for giving us access to the 26

sampling site, and Francisco Lorea and María de Jesús Peralta for their help with species identification. We also thank Clemente García-Ávila, Abel López Buenfil and Magnolia Moreno Velázquez at CNRF-SENASICA for providing the facilities to assess the antifungal activity of isolate CCeRi1-002 against F. kuroshium. We are grateful to Daniel García-Toscano and Karla Bravo for their help with the antagonism assays, and to Martín Camas for his help with taking the microscopy images. We finally thank Damaris Desgarennes, Ofelia Ferrera, Zelene Durán, María

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Vázquez, Mercedes G. López and Edgar Guevara for their support with some laboratory

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procedures.

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Compliance with Ethical Standards

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Funding

This research was funded by the National Forestry Commission of Mexico (Comisión Nacional

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Forestal or CONAFOR) grant number 265677 and by the National Fund for Scientific and Technological Development (Fondo Nacional de Desarrollo Científico y Tecnológico or

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FORDECYT) grant number 292399.

Conflict of Interest

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The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors. 27

References Ab Rahman, S.F.S., Singh, E., Pieterse, C.M., Schenk, P.M., 2018. Emerging microbial biocontrol strategies for plant pathogens. Plant Sci. 267, 102-111. https://doi.org/10.1016/j.plantsci.2017.11.012 Alvarez, F., Castro, M., Príncipe, A., Borioli, G., Fischer, S., Mori, G., Jofre, E., 2012. The plant‐

of

associated Bacillus amyloliquefaciens strains MEP218 and ARP23 capable of producing

ro

the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease. J. Appl. Microbiol. 112, 159-174.

-p

https://doi.org/10.1111/j.1365-2672.2011.05182.x

re

Arguelles-Arias, A., Ongena, M., Halimi, B., Lara, Y., Brans, A., Joris, B., Fickers, P., 2009. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary

lP

metabolites for biocontrol of plant pathogens. Microb. Cell Fact. 8, 63. https://doi.org/10.1186/1475-2859-8-63

ur na

Avalos, M., van Wezel, G.P., Raaijmakers, J.M. and Garbeva, P., 2018. Healthy scents: microbial volatiles as new frontier in antibiotic research?. Curr. Opin. Microbiol. 45, 84-91. https://doi.org/10.1016/j.mib.2018.02.011 Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S., Vivanco, J.M., 2006. The role of root exudates in

Jo

rhizosphere interactions with plants and other organisms. Ann. Rev. Plant Biol. 57, 233266. https://doi.org/10.1146/annurev.arplant.57.032905.105159

28

Bakker, P., Berendsen, R., Doornbos, R., Wintermans, P., Pieterse, C., 2013. The rhizosphere revisited: root microbiomics. Front. Plant Sci. 4, 165. https://doi.org/10.3389/fpls.2013.00165 Balouiri, M., Sadiki, M., Ibnsouda, S.K., 2016. Methods for in vitro evaluating antimicrobial activity: A review. J. Pharm. Anal. 6, 71-79. https://doi.org/10.1016/j.jpha.2015.11.005 Barea, J.M., Pozo, M.J., Azcón, R., Azcón-Aguilar, C., 2005. Microbial cooperation in the

of

rhizosphere. J. Exp. Bot. 56, 1761-1776. https://doi.org/10.1093/jxb/eri197

ro

Barnett, S.J., Roget, D.K., Ryder, M.H., 2006. Suppression of Rhizoctonia solani AG-8 induced disease on wheat by the interaction between Pantoea, Exiguobacterium, and

-p

Microbacteria. Soil Res. 44, 331-342. https://doi.org/10.1071/SR05113

re

Beneduzi, A., Ambrosini, A., Passaglia, L.M., 2012. Plant growth-promoting rhizobacteria (PGPR): their potential as antagonists and biocontrol agents. Genet. Mol. Biol. 35, 1044-

lP

1051. http://dx.doi.org/10.1590/S1415-47572012000600020 Berg, G., 2009. Plant–microbe interactions promoting plant growth and health: perspectives for

ur na

controlled use of microorganisms in agriculture. Appl. Microbiol. Biotechnol. 84, 11-18. https://doi.org/10.1007/s00253-009-2092-7 Bergsma-Vlami, M., Prins, M.E., Staats, M., Raaijmakers, J.M., 2005. Assessment of genotypic diversity of antibiotic-producing Pseudomonas species in the rhizosphere by denaturing

Jo

gradient gel electrophoresis. Appl. Environ. Microbiol. 71, 993-1003. https://doi.org/10.1128/AEM.71.2.993-1003.2005

Camarena‐ Pozos, D.A., Flores‐ Núñez, V.M., López, M.G., López‐ Bucio, J., Partida‐ Martínez, L.P., 2019. Smells from the desert: Microbial volatiles that affect plant growth 29

and development of native and non‐ native plant species. Plant Cell Environ. 42, 13681380. https://doi.org/10.1111/pce.13476 Cavaglieri, L., Passone, A., Etcheverry, M., 2004. Screening procedures for selecting rhizobacteria with biocontrol effects upon Fusarium verticillioides growth and fumonisin B1 production. Res. Microbiol. 155, 747-754. https://doi.org/10.1016/j.resmic.2004.06.001

of

Cawoy, H., Debois, D., Franzil, L., De Pauw, E., Thonart, P., Ongena, M., 2015. Lipopeptides as

ro

main ingredients for inhibition of fungal phytopathogens by Bacillus

subtilis/amyloliquefaciens. Microb. Biotechnol. 8, 281-295. https://doi.org/10.1111/1751-

-p

7915.12238

re

Cazorla, F.M., Mercado-Blanco, J., 2016. Biological control of tree and woody plant diseases: an impossible task? BioControl 61, 233-242. https://doi.org/10.1007/s10526-016-9737-0

lP

Cazorla, F.M., Romero, D., Pérez-García, A., Lugtenberg, B.J.J., Vicente, A.D., Bloemberg, G., 2007. Isolation and characterization of antagonistic Bacillus subtilis strains from the

ur na

avocado rhizoplane displaying biocontrol activity. J. Appl. Microbiol. 103, 1950-1959. https://doi.org/10.1111/j.1365-2672.2007.03433.x Chaves-López, C., Serio, A., Gianotti, A., Sacchetti, G., Ndagijimana, M., Ciccarone, C., Stellarini, A., Corsetti, A., Paparella, A., 2015. Diversity of food-borne Bacillus volatile

Jo

compounds and influence on fungal growth. J. Appl. Microbiol. 119, 487–499. https://doi.org/10.1111/jam.12847

30

Ciancio, A., Pieterse, C.M., Mercado-Blanco, J., 2016. Harnessing useful rhizosphere microorganisms for pathogen and pest biocontrol. Front Microbiol. 7, 1620. https://doi.org/10.3389/fmicb.2016.01620 Dunlap, C.A., Lueschow, S., Carrillo, D., Rooney, A.P., 2017. Screening of bacteria for antagonistic activity against phytopathogens of avocados. Plant Gene 11, 17-22. https://doi.org/10.1016/j.plgene.2016.11.004

of

Eskalen, A., Stouthamer, R., Lynch, S.C., Rugman-Jones, P.F., Twizeyimana, M., Gonzalez, A.,

ro

Thibault, T., 2013. Host range of Fusarium dieback and its ambrosia beetle (Coleoptera:

https://doi.org/10.1094/PDIS-11-12-1026-RE

-p

Scolytinae) vector in southern California. Plant Dis. 97, 938-951.

re

Farré-Armengol, G., Filella, I., Llusia, J., Peñuelas, J., 2016. Bidirectional interaction between phyllospheric microbiotas and plant volatile emissions. Trends Plant Sci. 21, 854-860.

lP

https://doi.org/10.1016/j.tplants.2016.06.005

García-Ávila, C.D.J., Trujillo-Arriaga, F.J., López-Buenfil, J.A., González-Gómez, R., Carrillo,

ur na

D., Cruz, L.F., Ruiz-Galván, I., Quezada-Salinas, A., Acevedo-Reyes, N., 2016. First report of Euwallacea nr. fornicatus (Coleoptera: Curculionidae) in Mexico. Fla. Entomol. 99, 555-556. https://doi.org/10.1653/024.099.0335 Garrido, L.M., Alves, J.M.P., Oliveira, L.S., Gruber, A., Padilla, G., Araújo, W.L., 2016. Draft

Jo

genome sequence of Curtobacterium sp. strain ER1/6, an endophytic strain isolated from Citrus sinensis with potential to be used as a biocontrol agent. Genome Announc. 4, e01264-16. https://doi.org/10.1128/genomeA.01264-16

31

Giorgio, A., De Stradis, A., Lo Cantore, P., Iacobellis, N.S., 2015. Biocide effects of volatile organic compounds produced by potential biocontrol rhizobacteria on Sclerotinia sclerotiorum. Front Microbiol. 6, 1056. https://doi.org/10.3389/fmicb.2015.01056 Guevara-Avendaño, E., Bejarano-Bolívar, A.A., Kiel-Martínez, A.L., Ramírez-Vázquez, M., Méndez-Bravo, A., von Wobeser, E.A., Sánchez-Rangel, D., Guerrero-Analco, J.A., Eskalen, A., Reverchon, F., 2019. Avocado rhizobacteria emit volatile organic

of

compounds with antifungal activity against Fusarium solani, Fusarium sp. associated

74-83. https://doi.org/10.1016/j.micres.2018.11.009

ro

with Kuroshio shot hole borer, and Colletotrichum gloeosporioides. Microbiol. Res. 219,

-p

Guevara-Avendaño, E., Carrillo, J.D., Ndinga-Muniania, C., Moreno, K., Méndez-Bravo, A.,

re

Guerrero-Analco, J.A., Eskalen, A., Reverchon, F., 2018. Antifungal activity of avocado rhizobacteria against Fusarium euwallaceae and Graphium spp., associated with

lP

Euwallacea spp. nr. fornicatus, and Phytophthora cinnamomi. Antonie van Leeuwenhoek 111, 563-572. https://doi.org/10.1007/s10482-017-0977-5

ur na

Hall, T.A., 1999. BioEdit: a friendly biological sequence alignment editor and analysis program for Window 95/98/NT. Nucleic Acids Symp. Ser. 41, 95-98. Harrington, T.C., Fraedrich, S.W., Aghayeva, D.N., 2008. Raffaelea lauricola, a new ambrosia beetle symbiont and pathogen on the Lauraceae. Mycotaxon 104, 399-404.

Jo

Hogg, J.C., Lehane, M.J., 1999. Identification of bacterial species associated with the sheep scab mite (Psoroptes ovis) by using amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 65, 4227-4229.

32

Horuz, S., Aysan, Y., 2018. Biological control of watermelon seedling blight caused by Acidovorax citrulli using antagonistic bacteria from the genera Curtobacterium, Microbacterium and Pseudomonas. Plant Protect. Sci. 54(3). https://doi.org/10.17221/168/2016-PPS Hunziker L, Bönisch D, Groenhagen U, Bailly A, Schulz S, Weisskopf L (2015) Pseudomonas strains naturally associated with potato plants produce volatiles with high potential for

of

inhibition of Phytophthora infestans. Appl. Environ. Microbiol. 81, 821-830.

ro

https://doi.org/10.1128/AEM.02999-14

Kai, M., Haustein, M., Molina, F., Petri, A., Scholz, B., Piechulla, B., 2009. Bacterial volatiles

re

https://doi.org/10.1007/s00253-008-1760-3

-p

and their action potential. Appl. Microbiol. Biotechnol. 81, 1001-1012.

Kembel, S.W., O´Connor, T.K., Arnold, H.K., Hubbell, S.P., Wright, J., Green, J.L., 2014.

lP

Relationships between phyllosphere bacterial communities and plant functional traits in a Neotropical forest. Proc. Natl. Acad. Sci. U. S. A. 111, 13715-13720.

ur na

https://doi.org/10.1073/pnas.1216057111

Khan, N., Martínez-Hidalgo, P., Ice, T.A., Maymon, M., Humm, E.A., Nejat, N., Sanders, E.R., Kaplan, D. Hirsch, A.M., 2018. Antifungal activity of Bacillus species against Fusarium and analysis of the potential mechanisms used in biocontrol. Front. Microbiol. 9, 2363.

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https://doi.org/10.3389/fmicb.2018.02363

Kim, P.I., Ryu, J., Kim, Y.H., Chi, Y.T., 2010. Production of biosurfactant lipopeptides Iturin A, fengycin and surfactin A from Bacillus subtilis CMB32 for control of Colletotrichum

33

gloeosporioides. J. Microbiol. Biotechnol. 20, 138-145. https://doi.org/10.4014/jmb.0905.05007 Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol., msw054. https://doi.org/10.1093/molbev/msw054 Largent, D.C., Johnson, D., Watling, R., 1980. How to Identify Mushrooms to genus III:

of

Microscopic Features. Mad. River Press, Eureka. 148 p.

ro

Lavermicocca, P., Iacobellis, N.S., Simmaco, M., Graniti, A., 1997. Biological properties and spectrum of activity of Pseudomonas syringae pv. syringae toxins. Physiol. Mol. Plant

-p

Pathol. 50, 129-140. https://doi.org/10.1006/pmpp.1996.0078

re

Lemfack MC, Gohlke BO, Toguem SMT, Preissner S, Piechulla B, Preissner R. 2018. mVOC 2.0: a database of microbial volatiles. Nucleic Acids Res. 46, D1261-D1265.

lP

https://doi.org/10.1093/nar/gkx1016

Li, B., Li, Q., Xu, Z., Zhang, N., Shen, Q., Zhang, R., 2014. Responses of beneficial Bacillus

ur na

amyloliquefaciens SQR9 to different soilborne fungal pathogens through the alteration of antifungal compounds production. Front. Microbiol. 5, 636. https://doi.org/10.3389/fmicb.2014.00636 Li, L., Ma, M., Huang, R., Qu, Q., Li, G., Zhou, J., Zhang, K., Lu, K., Niu, X., Luo, J., 2012a.

Jo

Induction of chlamydospore formation in Fusarium by cyclic lipopeptide antibiotics from Bacillus subtilis C2. J. Chem. Ecol. 38, 966-974. https://doi.org/10.1007/s10886-0120171-1

34

Li, X.Y., Mao, Z.C., Wang, Y.H., Wu, Y.X., He, Y.Q., Long, C.L., 2012b. ESI LC-MS and MS/MS characterization of antifungal cyclic lipopeptides produced by Bacillus subtilis XF-1. J. Mol. Microbiol. Biotechnol. 22, 83-93. https://doi.org/10.1159/000338530 Li, X., Zhang, Y., Wei, Z., Guan, Z., Cai, Y., Liao, X., 2016. Antifungal activity of isolated Bacillus amyloliquefaciens SYBC H47 for the biocontrol of peach gummosis. PloS One 11, e0162125. https://doi.org/10.1371/journal.pone.0162125

of

Liao, J.H., Chen, P.Y., Yang, Y.L., Kan, S.C., Hsieh, F.C., Liu, Y.C., 2016. Clarification of the

ro

antagonistic effect of the lipopeptides produced by Bacillus amyloliquefaciens BPD1

https://doi.org/10.3390/molecules21121670

-p

against Pyricularia oryzae via in situ MALDI-TOF IMS Analysis. Molecules 21, 1670.

re

Ligon, J.M., Hill, D.S., Hammer, P.E., Torkewitz, N.R., Hofmann, D., Kempf, H.J., van Pée, K.H., 2000. Natural products with antifungal activity from Pseudomonas biocontrol

lP

bacteria. Pest Manage. Sci. 56, 688-695. https://doi.org/10.1002/15264998(200008)56:8<688::AID-PS186>3.0.CO;2-V

ur na

Lira-Noriega, A., Soberón, J., Equihua, J., 2018. Potential invasion of exotic ambrosia beetles Xyleborus glabratus and Euwallacea sp. in Mexico: A major threat for native and cultivated forest ecosystems. Sci. Rep. 8, 10179. https://doi.org/10.1038/s41598-01828517-4

Jo

Lorea-Hernández, F.G., 2002. La familia Lauraceae en el sur de México: diversidad, distribución y estado de conservación. Bol. Soc. Bot. México 71, 59-70.

Lynch, S.C., Twizeyimana, M., Mayorquin, J.S., Wang, D.H., Na, F., Kayim, M., Kasson, M.T., Thu, P.Q., Bateman, C., Rugman-Jones, P., Hulcr, J., Stouthamer, R., Eskalen, A., 2016. 35

Identification, pathogenicity and abundance of Paracremonium pembeum sp. nov. and Graphium euwallaceae sp. nov.— two newly discovered mycangial associates of the polyphagous shot hole borer (Euwallacea sp.) in California. Mycologia 108, 313-329. https://doi.org/10.3852/15-063 Magalhães, V.C., de Oliveira Barbosa, L., Andrade, J.P., Soares, A.C.F., de Souza, J.T., Marbach, P.A.S., 2017. Burkholderia isolates from a sand dune leaf litter display

of

biocontrol activity against the bole rot disease of Agave sisalana. Biol. Control 112, 41-

ro

48. https://doi.org/10.1016/j.biocontrol.2017.06.005

Malfanova, N., Franzil, L., Lugtenberg, B., Chebotar, V., Ongena, M., 2012. Cyclic lipopeptide

-p

profile of the plant-beneficial endophytic bacterium Bacillus subtilis HC8. Arch.

re

Microbiol. 194, 893-899. https://doi.org/10.1007/s00203-012-0823-0 Mannaa, M., Oh, J.Y., Kim, K.D., 2017. Microbe‐mediated control of Aspergillus flavus in stored

lP

rice grains with a focus on aflatoxin inhibition and biodegradation. Ann. Appl. Biol. 171, 376-392. https://doi.org/10.1111/aab.12381

ur na

McSpadden Gardener, B.B., Fravel, D.R., 2002. Biological control of plant pathogens: Research, commercialization, and application in the USA. Online. Plant Health Progress. https://doi.org/10.1094/PHP-2002-0510-01-RV Mehltreter, K., García-Franco, J.G., 2008. Leaf phenology and trunk growth of the deciduous tree

Jo

fern Alsophila firma (Baker) DS Conant in a lower montane Mexican forest. Am. Fern J. 98, 1-13. https://doi.org/10.1640/0002-8444(2008)98[1:LPATGO]2.0.CO;2

Méndez-Bravo, A., Cortazar-Murillo, E.M., Guevara-Avendaño, E., Ceballos-Luna, O., Rodríguez-Haas, B., Kiel-Martínez, A.L., Hernández-Cristóbal, O,. Guerrero-Analco, 36

J.A., Reverchon, F., 2018. Plant growth-promoting rhizobacteria associated with avocado display antagonistic activity against Phytophthora cinnamomi through volatile emissions. PLoS ONE 13(3): e0194665. https://doi.org/10.1371/journal.pone.0194665 Minerdi, D., Bossi, S., Gullino, M.L., Garibaldi, A., 2009. Volatile organic compounds: a potential direct long‐ distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35. Environmental Microbiol. 11, 844-854. https://doi.org/10.1111/j.1462-

of

2920.2008.01805.x

ro

Na, F., Carrillo, J.D., Mayorquin, J.S., Ndinga-Muniania, C., Stajich, J.E., Stouthamer, R.,

Huang, Y.T., Lin, Y.T., Chen, C.Y., Eskalen, A., 2018. Two novel fungal symbionts

-p

Fusarium kuroshium sp. nov. and Graphium kuroshium sp. nov. of Kuroshio shot hole

re

borer (Euwallacea sp. nr. fornicatus) cause Fusarium dieback on woody host species in California. Plant Dis. 102, 1154-1164. https://doi.org/10.1094/PDIS-07-17-1042-RE

lP

O’Donnell, K., Sink, S., Libeskind-Hadas, R., Hulcr, J., Kasson, M.T., Ploetz, R.C., Carrillo, D., Campbell, A., Duncan, R.E., Liyanage, P.N.H., Eskalen, A., Na, F., Geiser, D.M.,

ur na

Bateman, C., Freeman, S., Mendel, Z., Sharon, M., Aoki, T., Cossé, A.A., Rooney, A.P., 2015. Discordant phylogenies suggest repeated host shifts in the Fusarium-Euwallacea ambrosia beetle mutualism. Fungal Genet. Biol. 82, 277-290. https://doi.org/10.1016/j.fgb.2014.10.014

Jo

Ortiz-Castro, R., López-Bucio, J.S., López-Bucio, J., 2017. Physiological and molecular mechanisms of bacterial phytostimulation, in: Singh, H.B., Sarma, B.K., Keswani, C. (Eds.), Advances in PGPR Research. CABI, p. 16-28. https://doi.org/10.1079/9781786390325.0016 37

Ossowicki, A., Jafra, S., Garbeva, P., 2017. The antimicrobial volatile power of the rhizospheric isolate Pseudomonas donghuensis P482. PloS one, 12(3), e0174362. https://doi.org/10.1371/journal.pone.0174362 Pal, K.K., Gardener, B.M., 2006. Biological control of plant pathogens. The Plant Health Instructor 2, 1117-1142. https://doi.org/10.1094/PHI-A-2006-1117-02 Pathak, K.V., Keharia, H., Gupta, K., Thakur, S.S., Balaram, P., 2012. Lipopeptides from the

of

banyan endophyte, Bacillus subtilis K1: mass spectrometric characterization of a library

ro

of fengycins. J. Am. Soc. Mass Spectrom. 23, 1716-1728. https://doi.org/10.1007/s13361012-0437-4

-p

Pereira, P., Nesci, A., Etcheverry, M., 2007. Effects of biocontrol agents on Fusarium

re

verticillioides count and fumonisin content in the maize agroecosystem: Impact on rhizospheric bacterial and fungal groups. Biol. Control 42, 281-287.

lP

https://doi.org/10.1016/j.biocontrol.2007.05.015

Raupach, G.S., Kloepper, J.W., 1998. Mixtures of plant growth-promoting rhizobacteria enhance

ur na

biological control of multiple cucumber pathogens. Phytopathology 88, 1158-1164. https://doi.org/10.1094/PHYTO.1998.88.11.1158 Raza, W., Ling, N., Liu, D., Wei, Z., Huang, Q., Shen, Q., 2016. Volatile organic compounds produced by Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of

Jo

Ralstonia solanacearum. Microbiol. Res. 192, 103-113. https://doi.org/10.1016/j.micres.2016.05.014

Reverchon, F., García-Quiroz, W., Guevara-Avendaño, E., Solís-García, I.A., FerreraRodríguez, O., Lorea-Hernández, F., 2019. Antifungal potential of Lauraceae 38

rhizobacteria from a tropical montane cloud forest against Fusarium spp. Braz. J. Microbiol. 50, 583-592. https://doi.org/10.1007/s42770-019-00094-2 Ryu, C.M., Farag, M.A., Hu, C.H., Reddy, M.S., Kloepper, J.W., Paré, P.W., 2004. Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiol. 134, 1017-1026. https://doi.org/10.1104/pp.103.026583 Sánchez-Rangel, D., Hernández-Domínguez, E.E., Pérez-Torres, C.A., Ortiz-Castro, R.,

of

Villafán, E., Rodríguez-Haas, B., Alonso-Sánchez, A., López-Buenfil, A., Carrillo-Ortiz,

ro

N., Hernández-Ramos, L., Ibarra-Laclette, E., 2018. Environmental pH modulates

transcriptomic responses in the fungus Fusarium sp. associated with KSHB Euwallacea

-p

sp. near fornicatus. BMC Genomics 19, 721. https://doi.org/10.1186/s12864-018-5083-1

re

Santoyo, G., Orozco-Mosqueda, M.D.C., Govindappa, M., 2012. Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas: a

lP

review. Biocontrol Sci. Technol. 22, 855-872.

https://doi.org/10.1080/09583157.2012.694413

ur na

Sartori, M., Nesci, A., Etcheverry, M., 2012. Production of Fusarium verticillioides biocontrol agents, Bacillus amyloliquefaciens and Microbacterium oleovorans, using different growth media: evaluation of biomass and viability after freeze-drying. Food Addit. Contam. Part A 29, 287-292. https://doi.org/10.1080/19440049.2011.563369

Jo

Schisler, D.A., Slininger, P.J., Behle, R.W., Jackson, M.A., 2004. Formulation of Bacillus spp. for biological control of plant diseases. Phytopathology 94, 1267-1271. https://doi.org/10.1094/PHYTO.2004.94.11.1267

39

Shoda, M., 2000. Bacterial control of plant diseases. J. Biosci. Bioeng. 89, 515-521. https://doi.org/10.1016/S1389-1723(00)80049-3 Singh, M., Awasthi, A., Soni, S.K., Singh, R., Verma, R.K., Kalra, A., 2015. Complementarity among plant growth promoting traits in rhizospheric bacterial communities promotes plant growth. Sci. Rep. 5, 15500. https://doi.org/10.1038/srep15500 Souto, G.I., Correa, O.S., Montecchia, M.S., Kerber, N.L., Pucheu, N.L., Bachur, M., Garcia,

of

A.F., 2004. Genetic and functional characterization of a Bacillus sp. strain excreting

ro

surfactin and antifungal metabolites partially identified as iturin‐like compounds. J. Appl. Microbiol. 97, 1247-1256. https://doi.org/10.1111/j.1365-2672.2004.02408.x

-p

Stein, T., 2005. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol.

re

Microbiol. 56, 845-857. https://doi.org/10.1111/j.1365-2958.2005.04587.x Sun, L., Lu, Z., Bie, X., Lu, F., Yang, S., 2006. Isolation and characterization of a co-producer of

lP

fengycins and surfactins, endophytic Bacillus amyloliquefaciens ES-2, from Scutellaria baicalensis Georgi. World J. Microbiol. Biotechnol. 22, 1259-1266.

ur na

https://doi.org/10.1007/s11274-006-9170-0

Tahir, H.A.S., Gu, Q., Wu, H., Niu, Y., Huo, R. and Gao, X., 2017. Bacillus volatiles adversely affect the physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt. Sci. Rep. 7, 40481.

Jo

https://doi.org/10.1038/srep40481

Terhonen, E., Kovalchuk, A., Zarsav, A., Asiegbu, F.O., 2018. Biocontrol potential of forest tree endophytes, In: Pirttilä, A., Frank, A. (Eds.), Endophytes of Forest Trees. Springer, Cham, pp. 283-318. https://doi.org/10.1007/978-3-319-89833-9_13 40

Toffano, L., Fialho, M.B., Pascholati, S.F., 2017. Potential of fumigation of orange fruits with volatile organic compounds produced by Saccharomyces cerevisiae to control citrus black spot disease at postharvest. Biol. Control 108, 77-82. https://doi.org/10.1016/j.biocontrol.2017.02.009 Torres, M.J., Brandan, C.P., Petroselli, G., Erra-Balsells, R., Audisio, M.C., 2016. Antagonistic effects of Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina

of

phaseolina: SEM study of fungal changes and UV-MALDI-TOF MS analysis of their

ro

bioactive compounds. Microbiol Res. 182, 31-39. https://doi.org/10.1016/j.micres.2015.09.005

-p

Torres, M.J., Brandan, C.P., Sabaté, D.C., Petroselli, G., Erra-Balsells, R., Audisio, M.C., 2017.

re

Biological activity of the lipopeptide-producing Bacillus amyloliquefaciens PGPBacCA1 on common bean Phaseolus vulgaris L. pathogens. Biol. Control 105, 93-99.

lP

https://doi.org/10.1016/j.biocontrol.2016.12.001

Vacher, C., Hampe, A., Porté, A.J., Sauer, U., Compant, S., Morris, C.E., 2016. The

ur na

phyllosphere: microbial jungle at the plant–climate interface. Annu. Rev. Ecol. Syst. 47, 1-24. https://doi.org/10.1146/annurev-ecolsys-121415-032238 Velázquez-Becerra, C., Macías-Rodríguez, L.I., López-Bucio, J., Flores-Cortez, I., Santoyo, G., Hernández-Soberano, C., Valencia-Cantero, E., 2013. The rhizobacterium Arthrobacter

Jo

agilis produces dimethylhexadecylamine, a compound that inhibits growth of phytopathogenic fungi in vitro. Protoplasma 250, 1251–1262. https://doi.org/10.1007/s00709-013-0506-y

41

Vorholt, J.A., 2012. Microbial life in the phyllosphere. Nat. Rev. Microbiol. 10, 828-840. https://doi.org/10.1038/nrmicro2910 Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261-5267. https://doi.org/10.1128/AEM.00062-07 Williams-Linera, G., 2002. Tree species richness complementarity, disturbance and

of

fragmentation in a Mexican tropical montane cloud forest. Biodivers. Conserv. 11, 1825-

ro

1843. https://doi.org/10.1023/A:1020346519085

Yang, C.H., Crowley, D.E., Menge, J.A., 2001. 16S rDNA fingerprinting of rhizophere bacterial

-p

communities associated with healthy and Phytophthora infected avocado roots. FEMS

re

Microbiol. Ecol. 35, 129-136. https://doi.org/10.1111/j.1574-6941.2001.tb00796.x Yuan, J., Raza, W., Shen, Q., Huang, Q., 2012. Antifungal activity of Bacillus amyloliquefaciens

lP

NJN-6 volatile compounds against Fusarium oxysporum f. sp. cubense. Appl. Environ. Microbiol. 78, 5942-5944. https://doi.org/10.1128/AEM.01357-12

ur na

Zihalirwa Kulimushi, P., Argüelles Arias, A., Franzil, L., Steels, S., Ongena, M., 2017. Stimulation of fengycin-type antifungal lipopeptides in Bacillus amyloliquefaciens in the presence of the maize fungal pathogen Rhizomucor variabilis. Front. Microbiol. 8, 850.

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https://doi.org/10.3389/fmicb.2017.00850

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Figure captions

Figure 1. Inhibition percentage of mycelial radial growth of F. solani in dual cultures with antagonistic bacterial isolates obtained from A. effusa, D. salicifolia and O. psychotrioides.

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Values represent the average of three replicates ± standard deviation (SD). All isolates significantly inhibited mycelial radial growth in comparison with a control.

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Figure 2. Abundance of bacterial genera with antifungal activity against Fusarium solani in in vitro antagonism assays, per host species and habitat. Numbers between brackets represent the

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total number of bacterial isolates retrieved from each habitat.

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Figure 3. Antifungal activity of strain Bacillus sp. CCeRi1-002 against F. solani (above) and F. kuroshium (below) on PDA medium. a) Normal hyphal growth of F. solani and b) hyphal

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deformations of F. solani induced by Bacillus sp. CCeRi1-002, stained with Congo red and visually analyzed by microscope observations (100X) on the seventh day of the antagonism

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assays; c) normal hyphal growth of F. kuroshium and d) hyphal deformations of F. kuroshium induced by Bacillus sp. CCeRi1-002 on the seventh day of the antagonism assays, visually analyzed by scanning electron microscopy (scale bars represent 1μm). Representative images are

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shown (n=3).

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Figure 4. Antifungal activity of crude extracts from strain Bacillus sp. CCeRi1-002 against F.

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solani and F. kuroshium, five days post-inoculation. Above: inhibition assays of F. solani

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mycelial growth by crude extracts. a) Blank control (F. solani alone); b) bacterial extract (EtOAc); c) bacterial extract (n-BuOH). Table: data represent the mean ± standard deviation

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(SD), n=3 replicates. Below: scanning electron microscopy images of the morphological alterations in hyphae of F. kuroshium induced by crude extracts. d) Blank control (F. kuroshium

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alone); e) bacterial extract (EtOAc); f) bacterial extract (n-BuOH). Scale bars represent 1μm.

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Representative images are shown (n=3).

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Figure 5. Representative chromatogram from the UPLC-HRMS analysis in positive ionization mode of the crude extracts EtOAc (a) and n-BuOH (b) obtained from Bacillus sp. CCeRi1-002.

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Numbers in red represent the number assigned to each compound as reported in Table 3. The presence of C-13 Iturin A1 in both extracts was corroborated by the co-elution with a commercial

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standard (Sigma-Aldrich, St. Louis) at the same analytical conditions.

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Table 1 Molecular identification of the 29 bacterial isolates that showed significant antagonism against F. solani and their closest matches

Habitat

CCeH1-001

A. effusa

Phyllosphere

CCeH1-003

A. effusa

Phyllosphere

CCeH1-004

A. effusa

Phyllosphere

CCeRi1-002

A. effusa

CCeRi5-017

NCBI best match (accession number)

Identity %

Accession number

Bacillus sp.

B. subtilis (NR_104873.1)

99

MH538904

Bacillus sp.

B. subtilis (NR_104873.1)

99

MH538905

Bacillus sp.

B. subtilis (NR_104873.1)

99

MH538906

Rhizosphere

Bacillus sp.

B. amyloliquefaciens (NR_117946.1)

99

MH538907

A. effusa

Rhizosphere

Pseudomonas sp.

P. helleri (NR_148763.1)

99

MH538908

CCeRi5-019

A. effusa

Rhizosphere

Pseudomonas sp.

P. koreensis (NR_025228.1)

99

MH538909

CCeRi5-020

A. effusa

Rhizosphere

Pseudomonas sp.

P. koreensis (NR_025228.1)

100

MH538910

CCeRi5-022

A. effusa

Rhizosphere

Pseudomonas sp.

P. koreensis (NR_025228.1)

99

MH538911

CNsH2-013

D. salicifolia

Phyllosphere

Erwinia sp.

E. endophytica (NR_148650.1)

99

MH538912

CNsH2-014

D. salicifolia

Phyllosphere

Curtobacterium sp.

C. luteum (JQ660078.1)

99

MH538913

CNsH3-023

D. salicifolia

Phyllosphere

Microbacterium sp.

M. testaceum (NR_026163.1)

99

MH538914

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Taxonomic assignment

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Host species

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Bacterial isolate

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based on the NCBI databases “16S ribosomal RNA sequences (Bacteria and Archaea)” and “Nucleotide collection (nr/nt)”

CNsH4-029

D. salicifolia

Phyllosphere

Curtobacterium sp.

C. flaccumfaciens (NR_025467.1)

99

MH538915

CNsH4-030

D. salicifolia

Phyllosphere

Methylobacterium sp.

M. radiotolerans (NR_074244.1)

98

MH538916

CNsRi1-012

D. salicifolia

Rhizosphere

Arthrobacter sp.

A. kerguelensis (NR_029027.1)

99

MH538917

49

D. salicifolia

Rhizosphere

Pseudomonas sp.

P. monteilii (NR_114224.1)

MH538919

CNsRi2-015

D. salicifolia

Rhizosphere

Pseudomonas sp.

P. putida (NR_113651.1)

99

MH538920

CNsRi2-018

D. salicifolia

Rhizosphere

Pseudomonas sp.

P. baetica (NR_116899.1)

99

MH538921

CNsRi3-025

D. salicifolia

Rhizosphere

Bacillus sp.

B. toyonensis (NR_121761.1)

99

MH538922

CNsRi3-027

D. salicifolia

Rhizosphere

Pseudomonas sp.

P. koreensis (NR_025228.1)

98

MH538918

CNsRi4-031

D. salicifolia

Rhizosphere

Hafnia sp.

H. alvei (NR_112985.1)

99

MH538923

CNsRi5-041

D. salicifolia

Rhizosphere

Pseudomonas sp.

P. helmanticensis (NR_126220.1)

99

MH538924

CNsRi5-043

D salicifolia

Rhizosphere

Pseudomonas sp.

P. koreensis (NR_025228.1)

100

MH538925

CNsRi5-044

D. salicifolia

Rhizosphere

Pseudomonas sp.

P. putida (NR_113651.1)

99

MH538926

COpH1-002

O. psychotrioides

Phyllosphere

Pseudomonas sp.

P. koreensis (NR_025228.1)

99

MH538927

COpH5-008

O. psychotrioides

Phyllosphere

Methylobacterium sp.

M. radiotolerans (NR_074244.1)

99

MH538928

COpRi1-001

O. psychotrioides

Rhizosphere

Pseudomonas sp.

P. koreensis (NR_025228.1)

99

MH538929

COpRi1-003

O. psychotrioides

Rhizosphere

Pseudomonas sp.

P. koreensis (NR_025228.1)

100

MH538930

COpRi4-022

O. psychotrioides

Rhizosphere

Pseudomonas sp.

P. koreensis (NR_025228.1)

100

MH538931

COpRi5-028

O. psychotrioides

Pseudomonas sp.

P. baetica (NR_116899.1)

99

MH538932

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Rhizosphere

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98

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CNsRi2-013

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Table 2 Chemical composition of VOC produced by bacterial strains with antifungal activity against F. solani on TSA medium at 28 °C, as

RT (min)

Bacillus sp. CCeRi1-002 7.01 8.72 9.31 10.29

2-Nonanone

13187

13.73

2-Decanone

12741

15.94

8163

22556 15448 61031 42265 11622

2-Undecanone

0.01 0.01 0.00 0.01

±

S (%)

Chemical class

Chemical formula

Already in mVOCs DB?*

Antifungal activity?

3.78 3.32 3.31 1.79

± ± ± ±

1.97 2.72 1.07 1.64

89.00 88.67 85.67 46.33

± ± ± ±

1.43 2.15 3.54 0.71

Ketone Ketone Ketone Ketone

C7H14O C8H16O C8H16O C8H16O

Yes Yes Yes Yes

0.00

6.71

±

1.94

92.00

±

2.83

Ketone

C9H18O

Yes

±

0.00

3.49

±

0.92

59.33

±

0.71

Ketone

C10H20O

Yes

20.36

±

0.00

6.81

±

0.60

89.33

±

2.08

Ketone

C11H22O

Yes

21.98 23.07 23.76 24.03 24.63

± ± ± ± ±

0.00 0.00 0.00 0.00 0.00

17.34 1.80 3.46 2.24 14.33

± ± ± ± ±

4.35 0.29 0.73 0.48 4.76

90.00 82.67 51.67 78.67 96.00

± ± ± ± ±

1.39 14.85 2.12 6.26 2.96

Ketone Alcohol Aldehyde Alcohol Ketone

C12H24O C11H24O C12H24O C13H28O C13H26O

Yes Yes Yes Yes Yes

Unknown Unknown Unknown Unknown Yes (Giorgio et al., 2015) Unknown Yes (Giorgio et al., 2015) Unknown Unknown Unknown Unknown Unknown

7654

24.84

±

0.02

2.49

±

1.95

67.00

±

4.24

Ester

C10H12O2

Yes

Unknown

75364

25.53

±

0.00

8.29

±

3.83

72.67

±

12.08

Ketone

C14H28O

Yes

Unknown

Jo

2-Dodecanone 2-Undecanol Undecanal, 2-methyl1-Dodecanol, 2-methyl2-Tridecanone Acetic acid, 2phenylethyl ester 2-Tetradecanone

± ± ± ±

RA (%)

lP

8051 13572 28965 8093

ur na

2-Heptanone 2-Heptanone, 6-methyl2-Heptanone, 5-methyl2-Octanone

-p

Compounds

re

PubChem ID

ro

measured by SPME-GC–MS

51

6421508

26.39

±

0.00

11.37

±

0.62

13.00

±

Ester

C21H42O3

No

Unknown

of

Carbonic acid, ethyl octadecyl ester 2-Hexadecanol

28.57

±

0.01

9.47

±

0.93

48.33

3.52

±

0.00

0.48

±

0.18

28.33

Disulfide, dimethyl

12232

3.97

±

0.00

1.58

±

0.33

1-Undecene

13190

5.53

±

0.02

31.53

±

1-Butanol, 3-methyl-

31260

7.84

±

0.00

2-Nonanone

13187

13.72

±

0.00

2-Undecanone

8163

20.35

546377

20.62

15448 13246 11622 15166 5364483

23.06 23.46 24.62 24.99 25.11

543425

87.33

Alcohol

C16H34O

No

Unknown

±

2.43

S-containing

C3H6OS

Yes

Unknown Yes (Giorgio et al., 2015) No (Giorgio et al., 2015), but activity vs. P. infestans (Hunziker et al., 2015) Yes (Toffano et al., 2017) Yes (Giorgio et al., 2015) Yes (Giorgio et al., 2015)

±

1.08

S-containing

C2H6S2

Yes

82.00

±

1.77

Aliphatic

C11H22

Yes

-p

re

14.50

±

0.04

68.00

±

5.66

Alcohol

C5H12O

Yes

2.07

±

0.19

92.33

±

3.54

Ketone

C9H18O

Yes

lP

0.54

0.00

6.59

±

2.38

91.33

±

0.35

Ketone

C11H22O

Yes

±

0.01

5.55

±

3.26

71.00

±

9.90

S-containing

C9H18OS

Yes

Unknown

± ± ± ± ±

0.00 0.01 0.00 0.00 0.01

0.77 14.65 11.22 1.90 2.83

± ± ± ± ±

0.27 2.35 3.51 0.14 0.05

76.00 40.67 93.33 66.00 49.33

± ± ± ± ±

5.66 3.54 4.60 2.83 5.30

Alcohol Ketone Ketone Alcohol Alcohol

C11H24O C12H22O C13H26O C10H20O C15H30O

Yes No Yes No No

Unknown Unknown Unknown Unknown Unknown

26.69

±

0.00

10.91

±

0.88

70.00

±

11.31

Alcohol

C14H26O

No

Unknown

5367672 23741 10441

26.87 28.21 29.46

± ± ±

0.00 0.00 0.01

1.84 3.95 3.07

± ± ±

0.46 0.67 0.63

43.67 56.67 70.67

± ± ±

7.78 4.95 1.77

Aldehyde Ether Ketone

C18H34O C16H32O C19H38O

No No No

Unknown Unknown Unknown

17629

30.21

±

0.00

0.50

±

0.08

34.33

±

5.30

Aromatic

C18H30

No

Unknown

ur na

±

Jo

Octanethioic acid, Smethyl ester 2-Undecanol Cyclododecanone 2-Tridecanone Cyclodecanol Z-10-Pentadecen-1-ol Cyclododecanol, 1ethenyl15-Octadecenal Oxirane, tetradecyl10-Nonadecanone Benzene, (1pentylheptyl)-

7.51

±

ro

85779

Pseudomonas sp. CCeRi5-020 Methyl thiolacetate 73750

2.83

52

lP

re

-p

ro

of

RT: Retention Time. RA: Relative peak Area (relative area concentration) of the different compounds detected for each bacterial isolate. Data are presented as means of three replicates ± standard deviation (s.d.). S: Similarity percentage. The tentative names of compounds were annotated according to the information provided by the NIST library. Only compounds with match values superior to 700 were considered. *Reported in the mVOC database (http://bioinformatics.charite.de/mvoc) as of October, 2019 (Lemfack et al., 2018).

Table 3

Analysis by UPLC-HRMS of the bacterial diffusible compounds present in the EtOAc and n-BuOH extracts obtained from bacterial

ur na

strain Bacillus sp. CCeRi1-002. An inoculum of 1 mL of Bacillus sp. CCeRi1-002 (1.5 × 108 CFU mL-1) was cultured in 500 mL of LB

Jo

broth at 30 °C for 7 days and constant stir at 180 rpm, in dark conditions, in the absence of F. solani

53

Compounds Calculated m/z [M+H]+

Observed m/z

Error (ppm)

C-13 Iturin A1

C48H74N12O14+

1043.5526

7.62

1043.5499

-2.6

2

C-14 Iturin A2

C49H76N12O14+

1057.5682

8.13

1057.5658

-2.3

3

C-16 Iturin A6

C50H78N12O14+

1071.5839

8.86

1071.5818

-2.0

4

C-17 Iturin A8

C51H80N12O14+

1085.5995

9.28

1086.5997

-4.3

5

C-12 Surfactin

C49H85N7O13+

980.6284

13.51

980.6247

-3.8

6

C-13 Surfactin

C50H87N7O13+

994.6440

14.36

994.6398

-4.2

7

C-12 Surfactin

C49H85N7O13+

980.6284

14.56

980.6259

-2.5

8

Not assigned

C47H65N18O9+

1026.5260

7.93

1026.5249

-1.1

9

Not assigned

C49H80N5O19+

1043.5526

7.96

1043.5511

-1.4

10

Not assigned

C48H70N19O9+

1057.5682

8.24

1057.5668

-2.3

11

Not assigned

C46H75N10O17+

1040.5389

8.44

1040.5391

0.1

12

Not assigned

C47H58NO3+

685.4494

9.02

685.4483

-1.8

13

Not assigned

C54H79N11O10+

1042.609

12.07

1042.6090

0.0

Not assigned

C49H89N10O13+

1026.6689

12.88

1026.6687

-0.2

Not assigned

C49H95N6O17+

1040.6832

13.63

1040.6827

-0.5

C-13 Iturin A1

C48H74N12O14+

1043.5526

7.62

1043.5493

-3.2

C-14 Iturin A2

C49H76N12O14+

1057.5682

8.13

1057.5656

-2.5

C-16 Iturin A6

C50H78N12O14+

1071.5839

8.86

1071.5786

-4.9

C-15 Surfactin

C53H95N7O13+

1036.6911

13.50

1036.6838

-6.9

20

C-14 Fengycin A

C70H106N12O20+

1435.7725

9.46

1435.7496

-0.3

21

C-15 Fengycin A

C71H108N12O20+

1449.7881

9.94

1449.7765

-0.3

14 15 16 17 18 n-BuOH

Jo

19

re

-p

ro

1

of

Molecular formula

lP

EtOAc

Number compound

ur na

Bacterial extract

Rt (min)

54

C-16 Fengycin A

C72H108N11O21+

1463.7799

10.10

23

C-17 Fengycin B

C75H116N12O20+

1505.8507

11.26

24

Not assigned

C39H76N16O18+

1057.5602

8.04

25

Not assigned

C49H75N11O15+

1058.5522

26

Not assigned

C30H62N5O12+

27

Not assigned

C50H95N3O18+

28

Not assigned

C70H102N19O17+

29

Not assigned

C71H110N14O21+

30

Not assigned

C73H105N16O17+

1505.8236

-3.12

1057.5604

0.2

8.26

1058.5525

0.3

685.4573

9.02

685.4471

-0.3

1026.6689

12.88

1026.6653

-3.5

1481.7779

9.65

1481.7881

4.0

1495.8048

10.05

1495.8044

-0.3

1478.7922

10.20

1478.7954

2.2

lP

re

-p

ro

-0.3

Jo

ur na

m/z: mass/charge ratio Rt: Retention time

1463.7795

of

22

55