Anna Rosa Sprocati Department for Sustainability, ENEA-Rome

Enabling barley production in arid soils by only exploiting the indigenous microbial biodiversity

Co-authors: P.Casale1, F. Tasso1, G. Falasca3, T. El- Hasan4, K. Khleifat4, P. Paganin2, G. Migliore1, C. Alisi1, G.B. De Giudici2

1 ENEA- Italian National Agency for New Technologies, Energy and Sustainable EconomicDevelopment, Rome 2 University of Cagliari, (Italy) 3 University of Rome, Sapienza (Italy) , 4 Mutah University –Jordan ERANETMED2-PROJECT SUPREME Developing tools for SUstainable food PRoduction in mEditerranean area using MicrobEs Tailoring a microbial formula to be used as a bioaugmentation agent for supporting the cultivation of barley (Hordeum vulgare L.) in the semi-arid soil of the Al-Ghweir agricultural station in Jordan. WORK-PLAN RESULTS

• Microbial load 8 ∙ 10⁶ UFC ∙ g -1 • 42 colony-forming morphotypes Functional biodiversity: 94%

Phylogenetic Classes Percentage distribution of the different PGP functions JOR31 Serratia odorifera Selection of the microbial formula JOR21 Serratia odorifera JOR32 Serratia odorifera JOR36 Serratia odorifera JOR17 Serratia odorifera JOR22 Pantoea sp. JOR33 Pantoea agglomerans JOR2 Pantoea sp. JOR29 Pseudomonas frederiksbergensis JN11 Pseudomonas sp. JOR6 Pseudomonas helmanticensis JN7 Pseudomonas sp. JOR25 Pseudomonas sp. JOR14 Pseudomonas sp. JOR11 Pseudomonas sp. JOR9 Stenotrophomonas rhizophila

JOR19 Stenotrophomonas rhizophila PROTEOBACTERIA -

JOR20 Stenotrophomonas rhizophila γ JN8 Variovorax paradoxus JOR18 Rhizobium sp. PROTEOBACTERIA JN2 Rhizobium nepotum JOR4 Bacillus simplex JOR1 Bacillus simplex FIRMICUTES JOR7 Bacillus simplex JOR3 Bacillus pumilus JOR24 Streptomyces spiroverticillatus JN1 Nocardioides albus JOR37 Nocardioides albus JN9 Rhodococcus erythtopolis JOR26 Microbacterium sp. JOR16 Microbacterium murale JN5 Microbacterium hydrocarbonoxydans JN12 Curtobacterium flaccumfaciens JOR5 Glutamicibacter arilaitensis JOR27 Arthrobacter globiformis JN10 Arthrobacter defluvii JN14 Curtobacterium sp. JOR35 Paenarthrobacter nitroguajacolicus

JN3 Pseudarthrobacter siccitolerans ACTINOBACTERIA JOR8 Paenarthrobacter nitrguajacolicus JN15 Curtobacterium sp. SIGLA IdentificazioneJOR (microbial formula)Classe filogenetica N-FIX O-CAS P-SOL AUX JOR5 Arthrobacter sp. Actinobacteria + +++ + / - - JN12 Curtobacterium flaccumfaciens Actinobacteria + - + - JN14 Curtobacterium sp. Actinobacteria + - + / - + JN15 Curtobacterium sp. Actinobacteria + - - + JOR16 Microbacterium murale Actinobacteria + / - ++ + - JOR8 Paenarthrobacter nitroguajacolicus Actinobacteria + + + / - - JOR35 Paenarthrobacter nitroguajacolicus Actinobacteria + - - ++ JOR7 Bacillus simplex Bacilli + ++ + / - - JOR2 Pantoea sp. Gammaproteobacteria + - + / - + JOR22 Pantoea sp. Gammaproteobacteria + - ++ + JOR33 Pantoea agglomerans Gammaproteobacteria + ++ ++ + / - JOR11 Pseudomonas sp. Gammaproteobacteria + +++ + - JOR29 Pseudomonas frederiksbergensis Gammaproteobacteria + - ++ - JOR17 Serratia odorifera Gammaproteobacteria + +++ + - JN2 Rhizobium nepotum Proteobacteria + - - ++ JOR18 Rhizobium sp. Proteobacteria + - - ++ 16 7 12 8 Pot experiments tillering phase 1 2 Italy Jordan ENEA Mutah University Bioaugmentation inoculum: SUP-JOR 108CFU mL-1 3 Treatments Control (W) Chemical fertilizers (DAP) Bacteria (B) 2 Water levels 3 Water levels

OPTIMAL* =100 OPTIMAL* =100 STRESS=25 MID=50 STRESS=25 *calculated on the water retention capacity of the soil Pot experiment n.1 With the kind collaboration with the University of Rome “Sapienza”

Barley (Hordeum vulgare L)

• 20 days • Room temperature (up to 25°C) • 70% H • Light/dark cycle 14/10 hours

WHC (Wernitznig et al., 2014). 100W 100W+B 100W+DAP

25W 25W+B 25W+DAP CLPP-AWCD 1200

1000

800

600

100W OD*1000 100W+B 400 100W+DAP 25W 200 25W+B 25W+DAP 0 0 1 2 3 4 5 6 7 incubation days

25W 25W 25W +B +DAP Functional 87 94 87 diversity(%) Plant physiological parameters PFresesho fre wsceoight ANOVA test 1.5

Student's t-test α α,β aerial parts

)

g 1.0 root system

(

i β

m

m ε

a r 0.5 g ζ δ

0.0

W B P W B P 0 + A 5 + A 0 W D 2 W D 1 0 + 5 + 0 W 2 W 1 0 5 0 2 1 trattamenti Organi aerei Apparato radicale

Test ANOVA- Test Student’s t

AverageNuNumbermero m eofd i oA dRootsi RA Average Number of LR 20

α α 15 α β LR AR

n 10 PR

5

0

W B P W B P 0 + A 5 + A AR Average length 0 W D 2 W D 1 0 + 5 + 0 W 2 W 1 0 5 (cm) 0 2 1 trattamenti

Pot experiment n.2 at Mutah University –Jordan

Barley-(Hordeum vulgare L) (Nov16 –Jan14) DAP B W OPTIMAL =100

3 Water levels MID=50

STRESS=25

Control (W)

3 Treatments Chemical fertilizers (DAP)

Bacteria (B)

• 8 weeks • Room temperature (up to 25°C) • 65% H • Light/dark cycle 12/12 hours Average values

SUMMARY CONCLUSIONS • A tailor-made microbial formula (JOR) has • When optimal water was supplied, no major been established and used as an alternative to differences are observed, thus bacteria can chemical fertilizers (DAP) for supporting the replace chemical fertilizers growth of barley crop (Hordeum vulgare L) in the semi-arid soil of the Al-Ghweir agronomic • Under water stress, the bacterial formula station in Jordan. proved to be the only treatment enabling • The formula is composed by 16 survival and healthy of barley plants . autochthonous performing PGPB strains • A “knowledge-based” bioaugmentation • The composition was designed to reflect as technique will be the key to develop new closely as possible the structure of the native approaches towards the recovery of bacterial community. degraded soils , saving water and replacing • The formula JOR was tested as agent of chemicals. functional bioaugmentation to enhance the PGP functions of soil under water stress, in • On these basis a two-year field trial is two pot experiments . underway at the Al-Gweir station. Positive results obtained in the first cycle have to be • Differences between the addition of bacteria confirmed in the next crop cycle. and chemical fertilizers were monitored over the tillering phase. THANK You for Your ATTENTION by