Coastal Ecosystems as Sources of Biofertilizers in Agriculture: From

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ORIGINAL RESEARCH

published: 19 August 2021

doi: 10.3389/fmars.2021.685076

Edited by:

Bernardo Duarte,

Center for Marine and Environmental

Sciences (MARE), Portugal

Reviewed by:

Ricardo Cruz de Carvalho,

University of Lisbon, Portugal

Isabel Caçador,

Center for Marine and Environmental

Sciences (MARE), Portugal

*Correspondence:

Salvadora Navarro-Torre

[email protected]

Specialty section:

This article was submitted to

Coastal Ocean Processes,

a section of the journal

Frontiers in Marine Science

Received: 24 March 2021

Accepted: 28 July 2021

Published: 19 August 2021

Citation:

Pajuelo E, Arjona S,

Rodríguez-Llorente ID,

Mateos-Naranjo E,

Redondo-Gómez S, Merchán F and

Navarro-Torre S (2021) Coastal

Ecosystems as Sources

of Biofertilizers in Agriculture: From

Genomics to Application in an Urban

Orchard. Front. Mar. Sci. 8:685076.

doi: 10.3389/fmars.2021.685076

Coastal Ecosystems as Sources of

Biofertilizers in Agriculture: From

Genomics to Application in an Urban

Orchard

Eloísa Pajuelo

, Sandra Arjona

, Ignacio D. Rodríguez-Llorente

Enrique Mateos-Naranjo

, Susana Redondo-Gómez

, Francisco Merchán

and

Salvadora Navarro-Torre

Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain,

Department

of Plant Biology and Ecology, Faculty of Biology, University of Seville, Seville, Spain

Pantoea agglomerans RSO7, a rhizobacterium previously isolated from Spartina

maritima grown on metal polluted saltmarshes, had demonstrated good plant growth

promoting activity for its host halophyte, but was never tested in crops. The aims of this

study were: (1) testing PGP activity on a model plant (alfalfa) in vitro; (2) testing a bacterial

consortium including RSO7 as biofertilizer in a pilot experiment in urban orchard; and

(3) identifying the traits related to PGP activities. RSO7 was able to enhance alfalfa

growth in vitro, particularly the root system, besides improving plant survival and

protecting plants against fungal contamination. In addition, in a pilot experiment in urban

orchard, a consortium of three bacteria including RSO7 was able to foster the growth

and yield of several winter crops between 1.5 and 10 fold, depending on species.

Moreover, the analysis of chlorophyll ﬂuorescence revealed that photosynthesis was

highly ameliorated. Genome analysis of RSO7 depicted the robustness of this bacterial

strain which showed resilience to multiple stresses (heat, cold, UV radiation, several

xenobiotics). Together with wide metabolic versatility, genes conferring resistance to

oxidative stress were identiﬁed. Many genes involved in metal resistance (As, Cu, Ni,

Co, Zn, Se, Te) and in tolerance toward high osmolality (production of a battery of

osmoprotectans) were also found. Regarding plant growth promoting properties, traits

for phosphate solubilization, synthesis of a battery of siderophores and production of

IAA were detected. In addition, the bacterium has genes related to key processes in the

rhizosphere including ﬂagellar motility, chemotaxis, quorum sensing, bioﬁlm formation,

plant-bacteria dialog, and high competitiveness in the rhizosphere. Our results suggest

the high potential of this bacterium as bioinoculant for an array of crops. However,

the classiﬁcation in biosecurity group 2 prevents its use according to current European

regulation. Alternative formulations for the application of the bioinoculant are discussed.

Keywords: Pantoea agglomerans RSO7, saltmarshes, PGPR, bioinoculant, genome analysis, PGP traits,

metabolic versatility, resilience

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Pajuelo et al. Pilot Experiments Using Coastal Biofertilizers

INTRODUCTION

Interest in the environment is increasingly present in people’s

lives. This concern has evolved so much that climate risks

are at the forefront of the planet’s concerns today (World

Economic Forum, 2020). Among these attempts, searching

for less polluting fertilization methods than the commonly

used chemicals is prioritized in order to evolve toward a more

sustainable agriculture

. One of the most challenging politics in

this new green revolution is the use of biofertilizers, included

in the recent regulation of the European Union on fertilizers

(2019/1009). Biostimulants were deﬁned by du Jardin (2015)

as “any substance or microorganism applied to plants with the

aim of improving nutritional eﬃciency, tolerance to abiotic

stress and/or quality traits of the crop, regardless of its nutrient

content.”

Of particular interest are plant growth promoting

rhizobacteria (PGPR), which are soil bacteria able to establish

beneﬁcial relationships with plants (Backer et al., 2018).

These bacteria are a real alternative to agrochemicals and

are proven to improve plant resilience toward an array of

stress situations (drought, salinity, high temperature, poor

and degraded soils) together with defense against plant

pathogens (Backer et al., 2018; Enebe and Babalola, 2018). In

this particular, coastal ecosystems are a source of rhizosphere

bacteria with particular properties, since they are able to

tolerate an array of stress situations such as high salinity,

extreme temperature, irradiation, xenobiotic pollutants, heavy

metals, etc. (Mesa-Marín et al., 2019; Rodríguez-Llorente et al.,

2019).

There are multiple mechanisms by which PGPRs can

promote plant growth, which are classiﬁed into direct and

indirect mechanisms. Direct mechanisms are those that provide

nutrients or regulate plant growth, including phosphate

solubilization, nitrogen ﬁxation, iron acquisition or the

secretion of growth stimulating phytohormones. Indirect

mechanisms are those that protect plants from acquiring

infections (biotic stress) or help the plant to grow healthy

during a period of environmental stress (abiotic stress) for

instance through regulation of ethylene production due to

the enzyme aminocyclopropane (ACC) deaminase (Goswami

et al., 2016; Singh and Jha, 2017). Some of the most frequent

bacterial genera used as PGPR are Pseudomonas, Bacillus,

Azospirillum, Azotobacter, Rhizobium, Enterobacter, etc.

(Ferreira et al., 2019).

In our case, the study involves the bacterium Pantoea

agglomerans RSO7, a Gram negative belonging to the

Enterobacteriaceae family. The genus Pantoea comprises

20 species with high diversity including PGPR, xenobiotic

degraders, antibiotic producers, biocontrol agents, plant

pathogens and opportunistic human pathogens (Walterson

and Stavrinides, 2015; Dutkiewicz et al., 2016). Concerning

the strain RSO7, it had been previously isolated from the

rhizosphere of Spartina maritima, a species present in

the marshes of southwestern Spain (Castillo et al., 2010)

https://ec.europa.eu/environment/archives/eussd/food.htm

and other parts of Europe. The bacterium was shown to

tolerate high levels of arsenic and heavy metals (Zn, Pb,

Cu), it shows high salt tolerance and display very good

PGPR properties (Paredes-Páliz et al., 2016a), which are

listed in Supplementary Table 1. Furthermore, inoculation

of Spartina densiﬂora plants with a bacterial consortium

containing RSO7 improved seed germination, enhanced plant

growth and ameliorated the physiological state of the host

halophyte in metal polluted soils (Paredes-Páliz et al., 2016b,

2017).

However, the PGP properties of this bacterium were never

tested in crops. In this particular, alfalfa (Medicago sativa) was

selected for a preliminary study in vitro for several reasons.

In the ﬁrst place, it is a legume or Fabaceae, which is a

family of plants with high importance for humans. Legumes are

considered as the second most important food for humanity,

second only to cereals. Their extensive consumption is justiﬁed

by their high nutritional value, since they provide essential

amino acids, complex carbohydrates, ﬁber, unsaturated fats,

vitamins and minerals (Kouris-Blazos and Belski, 2016; Maphosa

and Jideani, 2017). Besides alfalfa, RSO7 has been tested as

bioinoculant for an array of ﬁve winter crops (lettuce, spinach,

winter onion, radish and beet) in a pilot experiment in

an urban orchard.

In recent times, studies of the complete genomes of

PGPR are elucidating the genes that explain the plant growth

promoting activities and the plant-bacteria dialog in the

rhizosphere (Song et al., 2012; Usha et al., 2015; Bhattacharyya

et al., 2017). Accordingly, the draft genomes of several

Pantoea strains with PGPR activities have been compared

(Bruto et al., 2014; Palmer et al., 2018; Song et al., 2020).

In general these microorganisms show wide versatility and

adaptability, together with multiple PGP activities such as

phosphate solubilization, nitrogen ﬁxation, ACC deaminase

activity, auxins production and secretion of siderophores

(Shariati et al., 2017; Chen and Liu, 2019; Luziatelli et al.,

2020a).

Taking into account the precedent information, the

objectives of this work have been: (1) testing PGP

activity on a model plant in vitro (in this case, the

legume Medicago sativa, alfalfa); (2) Testing a bacterial

consortium including RSO7 as biofertilizer in a pilot

experiment in an urban orchard; and (3) identifying the

genes underlying the PGP activity as well as metal and salt

tolerance in this strain.

MATERIALS AND METHODS

Cultivation of the Bacterial Strain

The strain P. agglomerans RSO7 (Paredes-Páliz et al., 2016a)

was routinely maintained on TSA plates. For the preparation of

inocula, a single colony was cultivated in TSB liquid medium

and incubated at 28

◦

C for 48 h. Optical density of the cultures

at 600 nm was determined and adjusted to 1.0 with sterile TSB

medium in order to use always the same bacterial density in

all the procedures.

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Cultivation of Medicago sativa in Agar

Plates

Cultivation of alfalfa in vitro was performed in sterile

20 cm × 20 cm square plates ﬁlled with BNM (Buﬀered

Nodulation Medium, Ehrhardt et al., 1992) containing 0.9%

agar. This medium is commonly used for studies of rhizobium-

legume nodulation and lacks nitrogen. Since, in this case, the

alfalfa plants were not inoculated with rhizobia, the medium was

supplemented with 2.5 mM ammonium nitrate. The medium

was used at normal concentration (1 × BNM) and at half

concentration of all nutrients (1/2 × BNM) in order to simulate

poor or degraded soils.

Commercial M. sativa seeds were superﬁcially disinfected

in 90% alcohol for 5 min, after which they were thoroughly

washed with sterile distilled water. Subsequently, they were

treated with a sodium hypochlorite solution (commercial bleach

diluted 1: 5) for 5 min. Finally, they were exhaustively washed

with sterile distilled water to remove traces of bleach that could

inhibit germination. Seeds were pre-germinated for 24 h on

water-agar plates at 22

◦

C in the darkness. Twelve seeds at

the same developmental stage were transferred to each of the

agar plates, in duplicate. For each concentration (1 × BNM

or 1/2 × BNM) two treatments were performed: inoculated

and non-inoculated plates. For inoculation, 100 µL of a culture

of RSO7 was deposited on every seed. In the non-inoculated

plates 100 µL of TSB sterile medium was applied on every

seed. Plates were allowed to stand until the inoculum was

embedded in the agar and then sealed with ﬁlm-tape. The lower

part of the plates was covered with black paper to protect

roots from light and plates were incubated in a plant growth

incubator at 22

◦

C/18

◦

C and 16 h light/8 h darkness (120–

130 µE m

−2

−1

). The experiment was conducted for 16 days

(at longer periods, plant roots and stems reached the bottom

and the top of the plate, respectively, so length measurements

were not accurate). At day 8th, seeds were inoculated again

with 5 µL of an RSO7 culture as before, and the same

volume of sterile TSB was applied to each seed of the non-

inoculated plates.

Determination of the Effect of

Inoculation With RSO7 on Medicago

sativa Plant Survival and Growth in vitro

To ﬁnd out whether inoculation with P. agglomerans RSO7

bacteria aﬀected plant survival, the percentage of viable plants

was assessed at day 16

. Percentages per plate were calculated

and the mean of the duplicate plates was made. The eﬀect

of inoculation on root growth was evaluated every day for

the ﬁrst 8 days, and then at day 12

at ﬁnal time (day

). Roots were measured and the number of lateral roots

was consigned. The daily average of the size of the roots

and the average number of lateral roots per plate was

calculated and plotted against time to establish the kinetics

of root growth and development of inoculated and non-

inoculated plants in the two media considered. Finally, on

day 16

, the stem size was measured and the number of

trefoils was recorded.

Design of a Pilot Experiment in Urban

Orchard

The pilot experiment was performed in the urban orchard of the

College I.E.S. Pablo Neruda, located in Castilleja de la Cuesta

(Seville, Spain: 37

◦

16.7

N; 6

◦

27.5

W). The experiment

was done in the frame of a collaborative research project called

“Doing research together” between the University of Seville and

the College I.E.S. Pablo Neruda in an attempt to disseminate the

knowledge generated in the university to society and also to foster

the interest of students for scientiﬁc research.

The orchard had four permaculture terraces for planting

(area 2 × 1 m) delimited by rails. Two of them were used for

inoculation and another two were kept without inoculation as

controls. For inoculation, a consortium of three bacteria was

used including Pantoea agglomerans RSO7, Pantoea agglomerans

RSO6 and Bacillus aryabathai RSO25 (Paredes-Páliz et al., 2016a).

The three bacteria were cultivated individually in 1 L of TSB

for 48 h at 28

◦

C and 150 rpm. The three cultures were mixed

before inoculation.

The experiment was done in the winter season of 2019. In

January, plants of ﬁve species were planted in the orchard, namely

winter onions, spinach, lettuce, radishes and beets. Commercial

local varieties of onions bulbs and seeds of the rest of the

plants were submerged in the culture of the three bacteria for

30 min and then planted in the terraces. The rest of the bacterial

culture was mixed with 50 L tap water and used to water

the inoculated plants. At the same time, analogous number of

onion bulbs and seeds was planted in the control terraces (non-

inoculated) and they were watered with 50 L tap water. Three

inoculations were done, the ﬁrst one at sowing and then once

every month (at the end of February and at the end of March).

The experiment was conducted for four months. During all this

time, the teachers and the students of the college watered the

plants twice a week and removed the weeds for keeping the

orchard in adequate conditions. Plants were ﬁnally harvested at

the end of April 2019.

Determination of the Effect of

Inoculation on the Photosynthesis and

Yield of Winter Crops

The saturation pulse method was used to determine leaf

light and dark-adapted ﬂuorescence parameters at midday

(1600 µmol m

−2

−1

) using a portable modulated ﬂuorimeter

(FMS-2; Hansatech Instruments Ltd., United Kindom) (Maxwell

and Johnson, 2000; Baker and Oxborough, 2004). Plants

were dark-adapted for 30 min using leaf–clips designed for

this purpose. The minimal ﬂuorescence level in the dark-

adapted state (F0) was measured using a modulated pulse

(< 0.05 µmol m

−2

−1

for 1.8 µs) too small to induce signiﬁcant

physiological changes in the plant. Maximal ﬂuorescence level

in this state (Fm) was measured after applying a saturating

actinic light pulse of 10000 µmol m

−2

−1

for 0.8 s.

Maximum quantum eﬃciency of PSII photochemistry (Fv/Fm)

were calculated from F0 and Fm. The same leaf section of

each plant was used to measure light-adapted parameters.

For this purpose, steady state ﬂuorescence yield (Fs) was

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Pajuelo et al. Pilot Experiments Using Coastal Biofertilizers

recorded at ambient light conditions and saturating actinic

light pulse of 10000 µmol m

−2

−1

for 0.8 s was then

used to produce the maximum ﬂuorescence yield (Fm

) by

temporarily inhibiting PSII photochemistry. Finally, using both

parameters the quantum eﬃciency of PSII (8PSII = (Fm

–

Fs)/Fm

) was calculated.

At the end of the experiment plants were harvested and the

aerial parts were measured and weighed. The bulbs of onions and

tubers of radishes and beets were unearthed and weighed.

Isolation of DNA, Whole Genome

Sequencing and Annotation of the RSO7

Genome

Pantoea agglomerans RSO7 was cultivated in 3 mL of liquid

TSB medium at 28

◦

C and 150 rpm for 24 h. 1.5 mL of the

culture were centrifuged and genomic DNA was extracted

using the G-spin

Genomic DNA Extraction Kit for

Bacteria (iNtRON Biotechnology Ltd, Korea) according to

the speciﬁcations of the supplier. Whole genome sequencing

was carried out through the company Sistemas Genómicos,

S.A. (Valencia) and by the MicrobesNG company (Birmingham,

United Kingdom) using Illumina technology. Kraken was

used to identify the closest available reference (Wood and

Salzberg, 2014). Quality of data was studied mapping the reads

with BWA mem (Li, 2013), de novo assembly of the genome

was performed using SPAdes (Bankevich et al., 2012) and,

then, more quality parameters were checked with BWA mem.

The whole genome was deposited in GenBank/EMBL/DDBJ

under the accession number CAJOSF01000000. Genome

annotation was performed with PROKKA (Seemann, 2014)

and basics statistics about the genome were extracted

using RAST server v2.0 (Aziz et al., 2008), QUAST v.4.6.3

software (Gurevich et al., 2013), SignalP 4.1 server (Petersen

et al., 2011), TMHMM server v.2.0 (Krogh et al., 2001),

and CRISPRFinder (Grissa et al., 2007) and PlasFlow

(Krawczyk et al., 2018).

Once annotated, genes have been classiﬁed in four categories:

genes related to adaptability and resilience under several stress

situations; genes related to plant growth promoting activities;

genes related to rhizosphere processes and genes involved in

toxicity and pathogenesis.

Statistical Analysis

For the in vitro experiment with alfalfa, the results obtained

were expressed as the mean ± standard error of 2 independent

experiments (2 plates for each condition with n = 10–12 plants

each depending on plant survival). For the orchard experiment,

the results are the mean ± standard error of 2 independent

experiments (2 independent terraces with variable number of

plants depending on the crop, n = 10–30). The means were

compared using the Student test and signiﬁcant diﬀerences at

p < 0.05 between inoculated and non-inoculated plants are

indicated in ﬁgures and tables.

Statistical analysis of chlorophyll ﬂuorescence was carried

out using Statistica v. 10.0 (Statsoft Inc.). Data diﬀerences

between inoculation treatments for the same vegetable species

TABLE 1 | Percentage of survival of inoculated and non-inoculated plants at day

in complete medium (1 × BNM) and half nutrient medium (1/2 × BNM).

Plant culture medium Inoculation conditions Percentage

of survival

1 × BNM Non-inoculated 66.7%

(a)

Inoculated with RSO7 70.8%

(a)

1/2 × BNM Non- Inoculated 58.3%

(b)

Inoculated with RSO7 79.2%

(c)

Data are the mean of 24 plants (2 plates × 12 plants) and signiﬁcant differences at

p < 0.05 are indicated by different letters.

were recorded by using the Student test (t-test). Data were ﬁrst

tested for normality with the Kolmogorov-Smirnov test and for

homogeneity with the Brown-Forsythe test.

RESULTS AND DISCUSSION

Effect of Inoculation on Plant Survival

in vitro

The eﬀect of inoculation with P. agglomerans RSO7 on alfalfa

survival in vitro was studied. Data are shown in Table 1. In

the case of the complete medium (1 × BNM), the survival

index was 4.16% higher in the inoculated plates with regard

to non-inoculated; however, this diﬀerence was not statistically

signiﬁcant. In the case of the plates with half the nutrients,

the percentage of survival in non-inoculated plants decreased

to 58.3%. By contrast, in this case, inoculation had very

positive eﬀect and the survival of the inoculated plates increased

up to 79.2%. Since all the seeds were selected at the same

developmental stage (at emerging root) our results suggest

that the bacterium ameliorated plant survival in conditions

of nutrient limitation. This eﬀect could be a consequence of

biocontrol properties of the strain (for instance secretion of

siderophores) since much higher fungal contamination was

observed in the non-inoculated plates as compared to the

inoculated ones (not shown).

Effect of Inoculation With RSO7 on

Medicago sativa Growth in vitro

The eﬀect of inoculation with P. agglomerans RSO7 on root

growth was followed every day and, from day 8, every four

days. The results are shown in Figure 1. In complete medium

(1 × BNM) data showed a slight and late positive eﬀect

of inoculation that was observed from day 12

. Finally, on

day 16

, the mean of the root in non-inoculated plants was

6.6 cm, while in inoculated plants it was 8.5 cm. The results

in deﬁcit medium (1/2 × BNM) were very diﬀerent. In this

case, a marked positive eﬀect of inoculation was observed

from the 5

day. The roots became approximately twice as

long in inoculated plants as compared to non-inoculated ones.

Finally, on day 16

, the mean of the root in non-inoculated

plants was 5.48 cm, whereas in the inoculated plants it was

9.41 cm. These results indicated that the eﬀect of bacteria was

much greater when the plants were in a situation of lack of

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FIGURE 1 | Kinetics of root growth in non-inoculated and inoculated plants of Medicago sativa grown in vitro in square plates. Values represent mean ± SE, n = 24.

nutrients. In the presence of full nutrients all the plants grow

well, however, in the case of 1/2 × BNM where the availability

of nutrients is more limited, the beneﬁcial eﬀects of bacteria

can be better observed, since they can solubilize phosphate,

ﬁx nitrogen, synthesize siderophores, etc. (Paredes-Páliz et al.,

2016a) allowing better root growth with less supply of nutrients

(biofertilizer).

Concerning the number of secondary roots, the results are

shown in Figure 2. In this case, the data conﬁrmed that the

presence of the bacteria increased the number of secondary roots

of the plant, regardless of the medium considered (1 × BNM

or 1/2 × BNM); in the ﬁrst case, data showed a notable

diﬀerence between the number of secondary roots of the non-

inoculated plates, with an average of 0, and the number of

secondary roots present in the inoculated plates, with an average

of 3.54. The results in deﬁcit medium also showed mean

number of secondary roots of 0.5 for non-inoculated face to

average 2.42 lateral roots in the inoculated plants. These eﬀects

could be probably related to the bacterium ability to synthesize

auxins (Paredes-Páliz et al., 2016a), since these phytohormones

regulate, among other processes, the growth and branching of

the root and the development of a greater number of root hairs

(Weijers et al., 2018).

The eﬀect of inoculation has also been studied on shoot

growth by determining shoot length and the number of trefoils

(Table 2). There was an increase in the stem size of the inoculated

plants compared to those that were not inoculated in both media.

However, the diﬀerences were not signiﬁcant in 1 × BNM (20%

increase in shoot length) whereas it was a signiﬁcant diﬀerence

in the low-nutrients medium where the increase in shoot length

was 53%. When considering the number of trefoils, there were

signiﬁcant diﬀerences in both media, with increases of 55%.

Again, the results suggest that there is a greater growth promoting

eﬀect under conditions of nutrient limitation.

Determination of the Effect of

Inoculation on the Yield of Crop Plants in

the Pilot Experiment in Urban Orchard

The eﬀect of inoculation with a consortium of 3 bacterial

strains including RSO7 was analyzed on 5 winter crops in a

pilot experiment in urban orchad. This bacterial consortium

had been previously used for inoculation of the host plant

Spartina densiﬂora (Paredes-Páliz et al., 2017) having

demonstrated plant growth promotion and protection of

plant against stress by heavy metals (Paredes-Páliz et al., 2017,

2018).

In this opportunity, 5 winter crops were selected to test

the eﬀect of the inoculant, namely lettuce, spinach, winter

onion, beet and radish. As explained before, the experiment

was done within the frame of a collaborative teaching-service

project between our research group at the University of

Seville and the College I.E.S. Pablo Neruda with professors

of Biology and Applied Sciences and students aging 15-

16. Supplementary Figure 1 shows several moments of the

development of the project. Final data of yield after 4 months

are shown in Table 3. The results indicated an enhancement

of growth of all the plant species (also observed in Figure 3).

The highest eﬀects were found in lettuce and spinach which

increased their weights by 8 and 5 fold in average, respectively.

Concerning winter onions, the increase was about 15% and it was

remarkable that in the case of radish and beet, only inoculated

plants gave tubers.

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FIGURE 2 | Kinetics of appearance of secondary roots in non-inoculated and inoculated plants of Medicago sativa grown in vitro in square plates. Values represent

mean ± SE, n = 24.

TABLE 2 | Length of the primary root of alfalfa plants grown on 1 × BNM (normal concentration of nutrients) and 1/2 × BNM (half nutrients) and inoculated or

non-inoculated with P. agglomerans RSO7.

Plant growth medium Inoculation conditions Root length (cm)

at day 16

Number of lateral

roots at day 8

Shoot length (cm)

at day 16

Number of trefoils

at day 16

1 × BNM Non-inoculated 7.36 ± 0.77

(a)

5.79 ± 0.49

(a)

2.19 ± 0.85

(a)

Inoculated with RSO7 8.55 ± 0.75

(a)

3.50 ± 0.63

(b)

6.88 ± 0.63

(a)

3.41 ± 0.33

(b)

1/2 × BNM Non-inoculated 5.48 ± 0.66

(b)

0.50 ± 0.2

(c)

5.42 ± 0.66

(a)

2.28 ± 0.26

(a)

Inoculated with RSO7 9.41 ± 0.58

(c)

2.41 ± 0.52

(b)

8.31 ± 0.55

(c)

3.42 ± 0.24

(b)

Data correspond to day 16

and are the average ± standard error of 18–24 plants (2 plates with 8–12 plants, depending on survival). Signiﬁcant differences at p < 0.05

are indicated by different letters.

These results conﬁrm, in a pilot experiment, the usefulness of

the biofertilizer for enhancement of plant growth and yield. The

eﬀect is probably due to the plant growth promoting activities

of the strains which are able to solubilize phosphate, produce

siderophores, and secrete auxins (Paredes-Páliz et al., 2016a).

In addition, the survival of plants was much more eﬃcient

upon inoculation (not shown) being probably related to the

biocontrol properties of RSO7, protecting plants against fungal

contamination. Other Pantoea strains also are able to improve

the resilience of plants against several stressing environmental

conditions and display biocontrol activities (Chen and Liu, 2019;

Luziatelli et al., 2020a).

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TABLE 3 | Yield of winter crops grown in an urban orchard and inoculated or not with a consortium including the bacterial strains Pantoea agglomerans RSO7,

P. agglomerans RSO6, and Bacillus aryabatthai RSO25 (Paredes-Páliz et al., 2016a).

Crop Yield (g) Comment

Non- inoculated Inoculated

Lettuce 95 ± 12 (n = 9)

787 ± 176 (n = 10)

Spinach 20 ± 2,4 (n = 8)

113 ± 22 (n = 9)

Winter onion 60 ± 14 (n = 11)

87 ± 23 (n = 10)

Radish 0.9 ± 0.1 (n = 13)

25.4 ± 5.4 (n = 12)

Only inoculated plants produced

tubers and ﬂowers

Beet 0.6 ± 0.06 (n = 14)

1.9 ± 0.4 (n = 15) *

Only inoculated plants produced

tubers *Average weight of plants

without tubers **Average weight of

plants with tubers

n indicates the number of plants. Signiﬁcant differences at p < 0.05 are indicated by different letters.

FIGURE 3 | Aspect of lettuces, spinach and radish plants in the orchard one month after planting. Left: non-inoculated; right: inoculated. The red arrow points a key

used for size comparison. Values represent mean ± SE, n = 10 (for spinach and lettuces), and n = 50 (for radishes).

Determination of the Physiological State

of Crop Plants in the Pilot Experiment in

Urban Orchard

The ﬂuorescence parameters were determined in inoculated

and non-inoculated plants. Chlorophyll ﬂuorescence analysis

indicated that overall F0 and Fm values were higher in inoculated

plants compared with their non-inoculated counterparts (t-test,

P < 0.05; Figures 4A,B), which consequently led to greater

maximum quantum eﬃciency of PSII photochemistry (Fv/Fm)

for all species (t-test, P < 0.05; Figure 4C). In addition, a

very similar trend was recorded for the quantum eﬃciency

of PSII (8PSII) for all species except for spinach and beet,

which did not show any statistical diﬀerence between both

inoculation treatments (Figure 4D). These results suggested a

positive eﬀect of bacterial inoculation on PSII functionality, in

terms of optimization the antenna size to prevent photoinhibition

and increase photosynthetic eﬃciency (Adams and Demmig-

Adams, 2004; Ort et al., 2011) with a consequent increment in

plant biomass, as was aforementioned.

Analysis of the RSO7 Genome

The genome of P. agglomerans RSO7 is composed by a

chromosome of 4,829,129 bp and it does not contain plasmids

(Supplementary Table 2). According to other species of the

genus, the G + C content is 55.1%. The analysis depicted 4384

coding sequences from which 616 corresponded to proteins

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FIGURE 4 | Minimal ﬂuorescence level, F0 (A), maximal ﬂuorescence level, Fm (B); maximum quantum efﬁciency of PSII photochemistry, Fv/Fm (C) and quantum

efﬁciency of PSII, 8PSII (D) at midday in randomly selected, fully developed leaves of ﬁve winter vegetables in non-inoculated and inoculated plants. Asterisks

indicate means that are signiﬁcantly different between inoculation treatments (t-test, P < 0.05).

with signal peptides and 1051 to proteins with transmembrane

domains. Moreover, the sequence encoded 85 RNAs, from which

11 were rRNA, 73 were tRNA, and 1 tmRNA. Furthermore, 3

CRISPR sequences were identiﬁed.

A global analysis of the genome revealed that P. agglomerans

RSO7 is a very versatile bacterium capable of adapting to a

large number of environmental situations. For example, operons

for the use of a large number of C sources (sorbitol, tartrate,

citrate, xylulose, galactofuranose, rhamnose, arabinose, galactose,

maltose, mannose, etc.) have been found. It can also use various

sources of N (nitrate, ammonium, amino acids, even some

ﬁxation genes have been detected) and S (sulfate, amino

acids like cysteine and methionine together with taurine). The

genus Pantoea is known by its robustness and by displaying

wide metabolic versatility (Shariati et al., 2017; Luziatelli et al.,

2020a). Along with its metabolic versatility, this bacterium

shows resistance toward many stresses, not only to heavy metals

(Shariati et al., 2017) and salinity as will be disclosed later, but also

to heat, cold, UV radiation, acidic conditions, etc. Besides, genes

are present for the degradation of diverse xenobiotics including

azo compounds (FMN-azoreductase), curcumin (curcumin

reductase), compounds with formyl groups (it has formylase

and formyl transferase), halogenated compounds (thanks to a

dehalogenase), compounds with nitro groups (nitroreductase),

etc. Some of these situations produce oxidative stress the

bacterium is able to deal with. In this particular, genes for

several antioxidant systems (catalase and various peroxidases, as

well as superoxide dismutase, glyoxylase, glutathione reductase,

glutathione transferase, etc.) were found. On the other hand, the

presence of several genes (Syx, crp) indicate that it is naturally

competent, being able to incorporate DNA and therefore acquire

new capabilities. It can acquire plasmid DNA by conjugation

having detected pilin precursor genes, an ATPase involved

in pilus retraction and ppdAB genes involved in conjugation.

The presence of genes encoding phage integrases (lysogeny)

suggests that it may also acquire genetic information by

transduction. All this indicates that it displays a high resilience

to many environmental variations and is very competitive in

the rhizosphere. In particular, among all the genes, three aspects

that are fundamental for its application as a bioinoculant will be

focused: (a) Resistance to heavy metals and salt (high osmolarity);

(b) PGP Properties, and (c) Important rhizosphere processes for

colonization and plant-microorganism interaction.

Resistance Toward Heavy Metals and Metalloids

The genome carries operons that encode resistance toward

metalloids As, Te and Se, and toward heavy metals such as Cu,

Zn, Co, and Ni (Table 4) (The complete list of genes for resistance

toward metals and metalloids is displayed in Supplementary

Table 3). One of the most frequent mechanisms of resistance of

prokaryotes against heavy metals consists in pumping the metal

out of the bacterial cell (Nies, 2003). In this particular, several

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TABLE 4 | Summary of mechanisms (and the corresponding traits) that justify the plant growth promoting properties and resilience of Pantoea agglomerans RSO7.

Traits Mechanisms Genes found in the genome

Resistance to

heavy metals

Resistance toward Cu, Zn, Co and Ni. Efﬂux pumps. CopA and YebZ (Grass and Rensing, 2001) ZntB and Zit (Grass

et al., 2001) RcnA for Co and Ni (Rodrigue et al., 2005)

Resistance to

metalloids

Resistance toward As. Resistance toward Se and Te. Methylation

and volatilization.

operon arrHBC (Fekih et al., 2018) tehB gene encoding a tellurite

and selenite methyltransferase (Chasteen and Bentley, 2003)

Resistance to

high osmolality

Synthesis of a battery of osmoprotectants. Permeases for uptake of

osmoregulatory substances. Recycling of carbon once the stress

disappears.

Ectoine: Doex (Schwibbert et al., 2011) Betaine: BetIABT Cánovas

et al., 2000) Trehalose: OtsAB (Kaasen et al., 1994) Permeases for

proline, glycine choline betaine and proline betaine, ectoine, and

pipecolic acid: ProPVWXY (Stirling et al., 1989) OsmVWXY

(Frossard et al., 2012) YehXYZ (Checroun and Gutierrez, 2004)

OusA (Gouesbet et al., 1996). Trehalases TreAF (Horlacher et al.,

1996); TreH (Carroll et al., 2007)

Iron acquisition Uptake of ferrous and ferric ions. Synthesis and transport of a

battery of siderophores.

Uptake of Fe

: EfeO, FeoAB, EfeU (Lau et al., 2016) Uptake of

: FbpBC (Wyckoff et al., 2006) heme HemH (Shepherd et al.,

2007); EfeB (Létoffé et al., 2015) hemin HmuSTUV (Hornung et al.,

1996) bacterial ferritines FntA (Yao et al., 2011) enterobactin

EntABCDEF (Reitz et al., 2017) achromobactin CrbD (Berti and

Thomas, 2009) ferrioxyamine FoxA and FhuE (Sauer et al., 1987)

ferri-bacillibactin BesA (Miethke et al., 2006) YfeBCD transport

multiple siderophores (Bearden et al., 1998).

PGP Phosphorous

acquisition

Uptake of inorganic phosphate and phosphite. Hydrolisis of C-P

bond (organic phosphorous). Accumulation of granules of

polyphosphate.

Uptake of phosphate: PstB and PitB (Willsky and Malamy, 1980)

Uptake of phosphite: PtxABC (Metcalf and Wolfe, 1998)

Exopolyphosphatases Ppx (Akiyama et al., 1993) Hydrolysis of

phosphonates PhnCDEF (Stasi et al., 2019) Phytase: Inositol-P

(Idriss et al., 2002) Polyphosphate kinase Ppk (Shiba et al., 2000)

Pyrophosphatase Ppa (Kajander et al., 2013).

Auxins Synthesis of the precursor tryptophan Several pathways of

synthesis of IAA. Degradation and conjugation of IAA. Auxin

transport permease.

Tryptophan monooxygenase: TM (Li et al., 2018) Indole-acetamide

hydrolase: IAH (Li et al., 2018) Nitrilases 1 and 2: N3 and N2 (Li

et al., 2018) Indole-3-pyruvate decarboxylase: IPAC (Li et al., 2018)

Indole-acetaldehyde dehydrogenase: IAD (Li et al., 2018)

Tryptophan transaminase: TT (Li et al., 2018) Monoamine oxidase:

AO (Li et al., 2018) Aromatic-L-amino-acid decarboxylase: AAD (Li

et al., 2018) Tryptophol oxidase: TO (Li et al., 2018)

Indole-3-acetate beta-glucosyltransferase: BoundA (Li et al., 2018)

Flavonol 3-sulfotransferase ATFS (Li et al., 2018)

Indole-acetaldehyde reductase IAR (Li et al., 2018) AUX1- like

permease: AUX1 (Li et al., 2018)

Rhizosphere

processes

Motility by ﬂagella. Chemotaxis to the root. Bioﬁlm formation.

Quorum sensing. Competition, antibiosis, lactonases. Synthesis of

amylovoran. Defense against plant antimicrobial peptides.

Pectinases.

Flh, ﬂi operons (Nakamura and Minamino, 2019) Che, Tsr/Tar (Feng

et al., 2018) ariR, BssS (Zhang et al., 2015) BdlA bioﬁlm dispersion

protein (Morgan et al., 2006) Qse, Lux, Rhi (Altaf et al., 2017)

amsBCDFJKL (Koczan et al., 2009) MsgA, PagC (Pulkkinen and

Miller, 1991) Pectinases YesR and KdgF (Bhadrecha et al., 2020)

metal eﬄux pumps have been found, including CopA and YebZ

for Cu (Grass and Rensing, 2001); ZntB and ZitB for Zn (Grass

et al., 2001); RcnA for Co and Ni (Rodrigue et al., 2005) and the

arsenic resistance operon arrHBC (Fekih et al., 2018).

On this side, the resistance toward metalloids Se and Te (both

from group VIB of the periodic table) consists in methylation

and volatilization of volatile species such as dimethylselenium

and dimethyltellide through the membrane (Chasteen and

Bentley, 2003). The tehB gene encoding an enzyme with tellurite

methyltransferase and selenite methyltransferase activities has

been found in the RSO7 genome.

Resistance to High Osmolality

The RSO7 strain is equipped with a battery of genes that regulate

the resistance to salt (osmotic stress) (Table 4) (The complete list

of genes for resistance toward metals and metalloids is shown in

Supplementary Table 4). Analysis of the genome revealed the

presence of genes involved in the synthesis and degradation of

a large number of osmoprotectants, such as ectoine (Schwibbert

et al., 2011), betaine (Cánovas et al., 2000), trehalose (OtsAB)

(Kaasen et al., 1994). Besides, the bacterium has permeases for

uptake of osmoregulatory substances such as proline, glycine

choline betaine and proline betaine, ectoine and pipecolic acid

(Stirling et al., 1989; Gouesbet et al., 1996; Checroun and

Gutierrez, 2004; Frossard et al., 2012). Once the osmotic stress

conditions disappear, the osmotic metabolites are “recycled” as

carbon source by the activity of trehalases (Horlacher et al., 1996;

Carroll et al., 2007).

Plant Growth Promotion Traits

Genes involved in promoting plant growth were also carefully

extracted, highlighting those involved in iron transport and

metabolism, as well as those related to phosphate uptake and

solubilization and auxin production (Table 4).

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Genes Related to Iron Transport and Metabolism

Iron is a fundamental element for bacteria, being part of

many redox enzyme cofactors, electron transporters, etc. For

this reason, the bacteria make sure to achieve it and even

compete for this element, so production of siderophores is

besides a biocontrol trait (The complete list of genes involved in

control of osmolality is listed in Supplementary Table 5). The

bacterium has low and high aﬃnity transporters for free ion,

both in the form of ferrous (EfeO, FeoAB, EfeU, ESA_00329)

(Lau et al., 2016) and ferric ions (FbpBC) (Wyckoﬀ et al.,

2006). Besides, the bacterium produces and/or transports a great

diversity of siderophores including heme (Otto et al., 1992),

hemin (Hornung et al., 1996), several bacterial ferritines (Yao

et al., 2011), enterobactin (Reitz et al., 2017), achromobactin

(Berti and Thomas, 2009), ferrioxyamine (Sauer et al., 1987), and

ferri-bacillibactin (Miethke et al., 2006). Finally, several copies of

the YfeBCD complex able to transport multiple iron complexes

were found (Bearden et al., 1998).

Genes Related to Phosphorous Uptake and Solubilization

The complete list of genes involved in phosphorous uptake and

metabolism is shown in Supplementary Table 6. Phosphorous

can be captured both in inorganic and organic forms. In its

inorganic forms, it is most often found in the form of phosphate

(PO

−3

) which is captured by low and high aﬃnity transporters

of phosphate such as PstB and PitB (Willsky and Malamy, 1980).

Besides phosphite (P

) can be transported by the PtxABC

phosphite transporter (Metcalf and Wolfe, 1998).

Referring to organic forms of phosphorus, the bacterium

can hydrolyze C-P bonds by exopolyphosphatases (Akiyama

et al., 1993) and nucleases. Besides it can use phosphonates (the

PhnCDEF operon has been found, Stasi et al., 2019) and inositol-

P by the activity of phytase (Matsuhisa et al., 1995). The captured

phosphorus can accumulate in polyphosphate granules in the

cytoplasm thanks to the Ppk polyphosphate kinase (Shiba et al.,

2000) and be mobilized when necessary by means of the inorganic

pyrophosphatase Ppa (Kajander et al., 2013).

Genes Related to Auxin Production

Respecting auxins production, RSO7 genome showed genes

involved in the tryptophan biosynthesis and indole-3-acetic

acid (IAA) biosynthesis. The main precursor in the IAA

synthesis is tryptophan and ﬁve diﬀerent pathways to synthetize

IAA have been studied: indol-3-acetamide pathway, indole-3-

pyruvate pathway, tryptamine pathway, tryptophan side-chain

oxidase pathway and indole-3-acetonitrile pathway (Li et al.,

2018; Duca and Glick, 2020). Strain RSO7 has genes involved

in four of these pathways (indol-3-acetamide pathway, indole-3-

pyruvate pathway, tryptamine pathway and indole-3-acetonitrile

pathway) (Table 4) (The complete list of genes for auxin

biosynthesis is listed in Supplementary Table 7), and all of them

are completed from tryptophan to IAA, supporting the IAA

production for this strain in results obtained in a previous work

(Paredes-Páliz et al., 2016a). These genes have been detected

in other strain of P. agglomerans (Morris, 1995; Manulis et al.,

1998; Spaepen et al., 2007). Moreover, genes involved in the

IAA degradation or conjugation and a transporter have been

found (Table 4).

Genes Involved in Rhizosphere Processes Important

to Plant Colonization

The correct colonization of the root is an important trait

which depends on many factors, such as mobility, the ability to

form bioﬁlms and bacterial communication, among others. In

this sense, genes involved in rhizosphere processes important

for plant-bacterium interaction are displayed in Table 4 (the

complete list of genes involved in important rhizosphere

processes is displayed in Supplementary Table 8).

The mobility of the bacteria is determined by the presence of

ﬂagella (genes for synthesis, rotation and regulation were found;

Nakamura and Minamino, 2019). The mobility of the bacteria in

the rhizosphere is determined by chemotaxis toward the root,

which secretes bacteria-attracting compounds (root exudates).

In this particular, genes involved in chemotaxis such as che

and Tsr/Tar were identiﬁed (Feng et al., 2018). Genes involved

in bioﬁlm formation such as ariR and BssS are also present

(Zhang et al., 2015). Besides, the BdlA gene encoding a bioﬁlm

dispersion protein (Morgan et al., 2006) was identiﬁed. For all

these rhizosphere processes to occur there must be a minimum

cell density, detected by quorum sensing systems such as Qse, Lux,

and Rhi (Altaf et al., 2017).

Another group of genes present in RSO7 have to do with

plant-bacteria interaction. In order to avoid the initial plant

defense (Bordiec et al., 2011), RSO7 synthetizes amilovoran, an

extracellular polysaccharide that functions as a virulence factor

in the formation of bioﬂims (Koczan et al., 2009). Once the

bacterium attaches and multiplies on the root, it has pectinases

that degrade the plant cell wall (Bhadrecha et al., 2020) and allow

it to survive in the host (even as an endophyte) and defend

against antibacterial peptides produced by the host (Pulkkinen

and Miller, 1991; Tu et al., 2016).

Finally, genes were found that ensure the competition of

the bacterium in the rhizosphere, for example, it has lactonase

able to inhibiting quorum sensing signals of other bacteria

(Zhang et al., 2007). It also produces toxins (Shidore and

Triplett, 2017) and antibiotics such as colicin V and carbepenem

(Kenawy et al., 2019).

Genes Involved in Toxicity and

Pathogenicity

From the point of view of biosecurity, Pantoea agglomerans

is included in group 2. Only group 1 microorganisms are

authorized by European legislation to be used as inoculants

(GRAS microorganisms, which stand for Generally Recognized

as Safe). The species Pantoea agglomerans causes opportunistic

infections, particularly in nosocomial patients with previous

pathologies such as cystic ﬁbrosis, cancer, etc. (Cruz et al., 2007).

Some plant endophytic PGPR strains have also caused infections

in workers such as gardeners who, after being pricked by plants,

became infected (Jain et al., 2012). In this particular, it has to

be noticed that in our experiment, protocols for working with a

microorganism of the group 2 have been followed: students and

teachers were previously instructed; no immunocompromised

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people has participated in the experiment; the microorganism

does not transmit via inhalation, but only by punching with

branches of plants; all people participating in the experiment

wore gloves; the organism was isolated from soil and it has

been not modiﬁed; the orchard perimeter was delimited by

lateral “walls” 25 cm high in order to avoid interference between

inoculated plants and not inoculated controls.

In this sense, a search has been made for genes that

are related to pathogenicity. The RSO7 strain has several

determinants of resistance to multiple antibiotics including

polymyxin B, phosphinothricin, chloramphenicol, fumonisin,

novobiocin, bicyclomycin, nitroimidazole, and bacitracin. Along

with these activities, it has several beta-lactamases that break

the beta-lactam ring. On the other hand, the presence of a

large number of pumps for eﬄux of toxic substances increases

its resistance to antibiotics including erythomycin, tetracycline,

ampicillin and norﬂoxacin. It is also capable of synthesizing

toxins (hemolysins, RNAase). It possesses the YejABEF operon

for resistance to host antibacterial defense peptides. Finally, the

production of siderophores is also a mechanism that ensures

the competitiveness of this bacterium. All these factors are

considered as virulence factors.

For these reasons it cannot be used as an inoculant. Strategies

have been designed to take advantage of the PGP characteristics

in cell-free extracts. In this way, the bacteria are grown and

only the culture supernatant is used to inoculate the plants. In

this supernatant are the molecules and proteins secreted by the

bacteria which exert their eﬀect on the plant without the risk of

using a microorganism of the group 2 (Luziatelli et al., 2020b).

CONCLUSION

The use of biofertilizers rises up as a real alternative for

a more sustainable and aﬀordable agriculture, particularly in

poor countries. In this context, some strains isolated from

the rhizosphere of coastal plants, such as Pantoea agglomerans

RSO7 have excellent PGP properties and high resilience toward

multiple stresses, together with high competitiveness in the

rhizosphere. These properties have been demonstrated when

using this bacterium as inoculant both in vitro experiments

and pilot experiments in an urban orchard with summer and

winter crops. Full genome analysis has allowed the identiﬁcation

of the traits behind this important biofertilization capability.

With the previous results, the great potential of this bacterium

as a promoter of plant growth can be concluded. Finally,

restrictions for the use of microorganisms of the biosafety

group 2 as bioinoculants could limit its use, and therefore

possible bio-sure alternatives, such as using the supernatant of

cultures, are proposed.

DATA AVAILABILITY STATEMENT

The datasets presented in this study can be found in online

repositories. The names of the repository/repositories and

accession number(s) can be found below: https://www.ebi.ac.uk/

ena, CAJOSF01000000.

AUTHOR CONTRIBUTIONS

EP: design of the work, supervision, funding, pilot experiment,

and writing the manuscript. SA: in vitro experiments and

genome analysis. IR-L: design of the experiments and pilot

experiment. EM-N and SR-G: photosynthesis measurements,

pilot experiment, and draft writing. FM: supervision of the

work and funding. SN-T: genome analysis, supervision of the

work, and writing the manuscript. All authors contributed to the

article and approved the submitted version.

FUNDING

This work was ﬁnanced by Project P11-RNM-7274-MO (Junta

de Andalucía), Project FIUS19/0065 (FIUS, University of Seville),

Project US-1262036 (Program I + D + i FEDER 2014-2020, Junta

de Andalucía), and AE2-18 of the University of Seville.

ACKNOWLEDGMENTS

The professors and students of the College I.E.S. Pablo Neruda

(Castilleja de la Cuesta, Seville) are heartily acknowledged, in

particular José Juan Pastor Milán.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found

online at: https://www.frontiersin.org/articles/10.3389/fmars.

2021.685076/full#supplementary-material

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