Application of microbial inoculants in sustainable

soil management of contaminated or urban areas

Paula Castro

UCP-ESB

2nd PhytoSUDOE Stakeholder Workshop, 17th April 2018, Porto

International Seminar - Phytotechnologies to RecoverContaminated Sites

EROSION

COMPACTION

NUTRIENT MINING

LOSS OF ORGANICA MATTER

SALINIZATION

CONTAMINATION

BASIS FOR FOOD PRODUCTION

BASIS FOR FUEL AND FIBER PRODUCTION

CRUTIAL FOR WATER AVAILABILITY

NUTRIENT RECYCLING

CRUTIAL FOR ORGANIC CARBON STOCKS

REPRESENTS ¼ OF THE GLOBAL BIODIVERSITY

SOIL - amongst the planet most vital resource

Resource Threats

2.5 million of potentially contaminated sites in Europe

342,000 sites (14 %) are contaminated and in need of remediation measures

Area with high probability

Few data

No data

increase by 50% in 2025 (European Environment Agency, 2012).

van Liedekerke, M. et al. 2014. Progress in the Management of Contaminated Sites in Europe. JRC Reference Report of the European Commission

Following over 200 years of industrialization, soil contamination is a widespread problem in Europe

Smelting

Waste disposal Mining

Agriculture & Foresty

Fuel Fossil combustion

Industrial effluents

SOURCES OF CONTAMINATION

Phytoremediation - an alternative?

Belowground “invisible” interactions

required for a successful restoration

and a sustainable plantcover.

What kind of services can microorganisms provide?

- Acquisition of nutrients- Pest and disease resistance- Alleviating abiotic stress: drought, salinity, pollutants, …

Role of microbial inoculants in assisting phytoremediation?

Tools to enhance phytomanagement success

Well characterised

organisms (no

pathogens)

Mechanisms of action

Adapted to field

practices

Easy production

–reasonable

cost

Shelf-life

Formulations

Bioinoculants – ChallengesHow to deliver these microbial inoculants in fieldconditions?

Challenge: MECHANISMS

Improvement of plant

nutrition

Siderophores

P solubilization

Ammonia

Plant growth promotion

ACC-deaminase activity

Indole acetic acid (IAA)

Biocide production

Extracellular enzymes

HCN

PLANT GROWTH PROMOTING BACTERIA

Less competition for nutrients

Protection from high stress soilsBacterial endophytes

Plant Growth Promoting Bacteria (PGPB) Play a key role in the establishment of plants in stressed areas

Root protection against adverse conditions - water deficiency- extreme pH and temperatures- heavy metal or toxin stresses

Fungi Plant

Water and nutrients

Ectomycorrhiza+

Carbohydrates

Symbiosis between plant root and soil fungi

Mycorrhizal fungi

Inocula productionInoculation of

seedlings at nursery stage

Transplantation to forest

Fungi Isolation 40 Fungi Library Pure Culture

Hunting for production of mycorrhiza fungi

Contaminated streams

Industrial sitesIsolamento de bactérias

Bacterial dentification

Field trials

Bacterial collection

Hunting for bacterial bioinoculants - PGPB

• Phytoremediation

• Reforestation

• Agriculture

Evidencesand

challenges

Use of bioinoculants - examples

Challenge: size of inoculum and different PGPB

Selection of metal resistant PGPR for the growth and accumulation of energy maize in a mine soil

Panasqueira Mine

(extends over 2000 ha)

Economic exploitation:

mainly wolframite,

cassiterite and chalcopyrite.

Huge tailing piles and two

mud dams exposed to

atmospheric conditions.

Surface runoff and water percolation

leach the tailings to Casinhas

stream, which drains to Zêzere

river.

HM reach hazardous levels in the

surrounding soilse.g.: Cd and Zn

Soil: Mine soil (Barroca Grande, Panasqueira Mine (Castelo Branco district))

Improving mine land valorization by the application of PGPR and AMF for growth of energy maize

PGPR

R. eutropha

1C2

(B1)

C. humiECP37

(B2)

P. fluorescens

S3X

(B3)R.

radiobacter

(B4)

P. reactansEDP 28

(B5)

mg kg-1 AGRICULTURAL RESIDENTIAL COMMERCIAL INDUSTRIAL

Zn 200 200 360 360

Cd 1.4 10 22 22

Canadian guidelines

Selection of PGPB based on site characteristics

B5 increased root biomass Doubling inocula size: NO EFFECT

0.0

0.5

1.0

1.5

2.0

2.5

10 mL 20 mL

Bio

mas

s (g

po

t-1)

B0

B1

B2

B3

B4

B5

(a)

NSFI=0.578***FB=7.807**FIxB=3.788

b,c*

*

Root

a

b,cb,c

b,c,d

a

b,c,d

b,c

a,b

c d

b

0.0

0.5

1.0

1.5

2.0

2.5

3.0

10 mL 20 mL

Bio

mas

s (g

po

t-1)

B0

B1

B2

B3

B4

B5

(b)

***FI=53.483***FB=49.923

***FIxB=14.083a

*

* *

* Shoot

dc

b,c

c

a

cd

c,db

b

c

0

10

20

30

40

50

60

70

80

10 mL 20 mL

Sho

ot

elo

nga

tio

n (

cm)

B0

B1

B2

B3

B4

B5

(c)

**FI=9.687***FB=19.849**FIxB=4.466a

*

*

b c

b

c

ab

bb b

b b

+37%

+25%

Doubling bacterialinoculum size hadgenerally no significanteffect in plants’ biometricparameters.

Soil: Mine soil - Maize biomass in the root

0.0

0.5

1.0

1.5

2.0

2.5

10 mL 20 mL

Bio

mas

s (g

po

t-1)

B0

B1

B2

B3

B4

B5

(a)

NSFI=0.578***FB=7.807**FIxB=3.788

b,c*

*

Root

a

b,cb,c

b,c,d

a

b,c,d

b,c

a,b

c d

b

0.0

0.5

1.0

1.5

2.0

2.5

3.0

10 mL 20 mL

Bio

mas

s (g

po

t-1)

B0

B1

B2

B3

B4

B5

(b)

***FI=53.483***FB=49.923

***FIxB=14.083a

*

* *

* Shoot

dc

b,c

c

a

cd

c,db

b

c

0

10

20

30

40

50

60

70

80

10 mL 20 mL

Sho

ot

elo

nga

tio

n (

cm)

B0

B1

B2

B3

B4

B5

(c)

**FI=9.687***FB=19.849**FIxB=4.466a

*

*

b c

b

c

ab

bb b

b b

B2, B3 and B5 increased shoot biomass Doubling inocula size: GENERAL DECREASE

+7%

+17%

+59%

+18%+14%

Soil: Mine soil - Maize biomass in the shoot

0

20

40

60

80

10 mL 20 mL

Cd

acc

um

ula

tio

n (

mg

kg-1

)B0

B1

B2

B3

B4

B5

***FI=238.066***FB=18.054

***FIxB=11.655

(a)

*

*

*

*

*

*

*

*

*

*

Root

bbaa,b

aa

a

a

c c

b

a

0

10

20

30

40

50

60

10 mL 20 mL

Cd

acc

um

ula

tio

n (

mg

kg-1

)

B0

B1

B2

B3

B4

B5

***FI=232.748***FB=141.264***FIxB=20.161

(b)

*

*

*

*

*

*

*

*

*

*

Shoot

c

ba

a

b

a

a

d

c

bb

a

Cd

Doubling inocula size: INCREASE

+17%

Soil: Mine soil - Plant metal accumulation in the roots

Soil: Mine soil - Plant metal accumulation in the shoot0

20

40

60

80

10 mL 20 mL

Cd

acc

um

ula

tio

n (

mg

kg-1

)

B0

B1

B2

B3

B4

B5

***FI=238.066***FB=18.054

***FIxB=11.655

(a)

*

*

*

*

*

*

*

*

*

*

Root

bbaa,b

aa

a

a

c c

b

a

0

10

20

30

40

50

60

10 mL 20 mL

Cd

acc

um

ula

tio

n (

mg

kg-1

)

B0

B1

B2

B3

B4

B5

***FI=232.748***FB=141.264***FIxB=20.161

(b)

*

*

*

*

*

*

*

*

*

*

Shoot

c

ba

a

b

a

a

d

c

bb

a

Cd

Doubling inocula size: INCREASE

+31%

Inocula size and bacteria species have different

outcomes

Na, K, P, N accumulation

PlantBiomass

C- ControlB2 - Chryseobacterium humiECP37E - Ochrobacterium haematophilum ZR3-5F – Rhizophagus irregularisMix – B + E+ F

Saline water0, 1 e 2 g NaCl l1

60 days

SoilEnzymes

Challenge: mixed formulations

Promotion of sunflower growth under saline water irrigation by the inoculation of PGPB

C B MIXFE

After 4 weeks, significant growth differences were observed between inoculated and non-inoculated plants

After 4 weeks

Plant Biomass

Plants irrigated with saline water showed reductions in shoot and root biomass

Bioinocula application increased biomass production

SHOOT ROOT

increases of 21, 8 and 41% for 0, 1 and 2 g NaCl l-1, respectively - all treatmentscontributed similarly

increases of 85, 82 and 62% for 0, 1 and 2 g NaCl l-1, respectively – rhizobacteria (B)was the best inoculation scheme

ROOT

SHOOT

Plant Biomass

Maize inoculated withPseudomonas fluorescens atincreasing doses

Maize inoculated withPseudomonas reactans atincreasing doses

Growth of maize under saline water irrigation –the effects of inoculum size

Inocula size affects yieldparameters and that can bedeterminant for economicfeasibility of application

Challenge: Bioinoculants must be able to rapidly colonize and persist in the rhizosphere of plants?

Phosphate-solubilizing rhizobacteria enhance Zea mays growth in P-deficient soils

Effect of PGPB - PSB inoculation in plant biomass

PSB inoculation enhanced Z. mays biomass production in all P-treatments

Bioinocula application increased P availability in soils

• Inoculated strains were detectable in the maize rhizosphere soils after 2 days of inoculation and after 45days in all treatments

• At the end of experiment (90 days) DGGE profiles did not allow to confirm the presence of rhizobacteriain soils

2 days 45 days

BORRALHA MINE

PHYTOSUDOE – The project in Borralha Mine

Sunflower was able to establish and grow in spite

of the high HM concentrations in the soil

Inoculation at Sowing

Harvesting

Plant Growth MF

PGPR

Sunflower sowedand inoculatedwith:• AMF• PGPR

Establishment of field trial – 1st year

S3: BORRALHA MINE

0

1

2

3

4

5

6

7

C B F Mix C B F Mix C B F Mix C B F Mix

0 2.5 5 10

Sh

oo

t b

iom

ass

(g

po

t-1)

Shoot

C0 B0 F0 Mix0

C2.5 B2.5 F2.5 Mix2.5 C10 B10 F10 Mix10C5 B5 F5 Mix5

S3: Borralha mine soil – mixed formulations

Ectomycorrhizal fungi - bioinoculants for tree crops in stress conditions

Importance of reforestation for ecossystems resilience… (e.g. control of erosion and water retention)

10.000

Pinus pinaster

3.000

Quercus rubra

Viveiro do Cruzeiro (Miranda), Concelho de Arcos de Valdevez

Two and five years after

transplantation to a post-fire

site

S. bovinus, S. granulatus,L. laccata, L. deliciosus, H.

crustuliniforme,P. tinctorius, R.

vulgaris

Tree height Root colar diameterMycorrhization status Ectomycorrhizal fungal

community establishedDiversity and Equitability

indexes

T. terrestris, R. vulgaris,

P. tinctorius, S. bovinus, L.

laccata,L. determinus

Impact of nursery inoculation on plant performance after transplantation to fire impacted soils

Inoculation of plant seedlings

Production of mycorrhizal plants

Inoculum productionPlant production

Baldio de Vilar do Chão (Vieira do Minho)

Pinus Pinaster

Field Trial

- Inoculated plants with higher performance

Pinus pinaster

Quercus rubra

Nursery stage

- Inoculated plants were significantly higher than control plants

- Inoculation with ectomycorrhizal fungi favored plant growth under stress conditions

CO INO

Ro

ot

co

lla

r d

iam

ete

r (m

m)

0

2

4

6

8

10P=NS

A

CO INO

Heig

ht

(cm

)

0

10

20

30

40 P<0.005

B

Inoculation at nursery stage improved plant growth when transplanted to a post-fire site

ECMF inoculation at nursery stage promoted plant field establishment

Differences between the inoculated and non-inoculated P. Pinaster saplings persisted 5 yr after transplantation

0

50

100

150

200

250

300

350

CO INO CO INO

Stem

hei

ght (

cm)

a

b

c

d

June June 2011

0

20

40

60

80

100

CO INO CO INO

Ro

ot

colla

r d

iam

ete

r (m

m)

ab

c

d

June 2008 June 2011

0

40

80

120

160

CO INO CO INO

No

ECM

ro

ot

tip

s

a

abab

c

June June 2011

Pine Growth and ECMF root tips

After field transplantation…

- Inoculated strains persisted after 5 years

- Different microbial communities in inoculated and non inoculated plants

- Fungi promote higher root system, helping plant establishment

0 20 40 60 80 100

HV1

CG1

RH1

Lla

FN

HO1

HS2

FNII

HME

PAX

SC

Frequency of ECM fungal spp. (%)

INO CO

*

The majority of the inoculated fungi persisted in the root system several years after transplanting.

ControloS.gran + B2

Recolha de cogumelos micorrízicos

Isolamento de fungos em laboratório

Inoculação de plantas

Planta Micorrizada

Bioinoculationof Quercus suber

– mixed inoculation

,0

1,0

2,0

3,0

4,0

5,0

6,0

7,0

8,0

Control SgM Mix

Root dry weight (g) - Buçaco

,0

2,0

4,0

6,0

8,0

Control SgM Mix

Root dry weight (g) - Amarante

Control S. gran + B2 MIX

Bioinoculation of Quercus suber – mixed inoculation

2015 2016 2017

Bioinoculation of Quercus suber – water stress field trial

a

bab

,0

5,0

10,0

15,0

20,0

25,0

30,0

Control Sgran Mix

Diameter (mm)

CTR Mixed Comerc

Higher trunkdiameter

Same tree hight

Mortality less 10%

Bioinoculation of Quercus suber – 24 months in the field

UrbanmycoserveUnderstanding and Managing Urban Mycorrhizal Communities

Tilia in urban context - Porto

9 sites (3 for each treatment)

67 trees

Escola Básica dos Correios

Urban stress scnerarios

High salinity

Metal contamination

Water scarcity

Paxillus involutus

– Boletus edulis

– Boletus pinophilus

– Boletus reticulatus

– Lactarius deliciosus

– Lactarius sanguifluus

Ediblemushrooms

Ectomycorrhizal fungi – Fungal Collection

Russula virescens Russula parazurea

Ediblemushrooms

Urban forest park – Parque do AviosoMaia

Tilia ectomycorrhizal fungi – Isolation – May 2017

Fungus

Solid Liquid

Polymers Agar

Challenge: how to deliver to the field? Carriers

Challenge:

- Easy production - Reasonable cost

- Shelf-life

Field application of inoculants can contribute to sustainable environmental solutions

Alberto Vega, Albina Franco, Ana Marques, Cindy Serafim

Helena Moreira, Miguel Ramos, Nadine Sousa, Rui Oliveira , Sofia Pereira

Thanks for your attention