Natural cropping system based on Brassicas biomasses for plant

management and defense

Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria, cerealicoltura e colture industriali

Luca LazzeriSenior Researcher

CREA CI Bologna Italy

The EU is requesting to produce more. with less chemistry (IPM techniques)

A growing interest for non chemical systems as totalor partial alternative to chemical in agriculture

totally widespread in the environment

in contact with operators during and after distribution

closest to food chain with all the problems of food health due to residue problem and sustainability

Chemicals in European agriculture are more and more controlled (Directive 2009/128/EC “ Sustainable use of pesticides”) in the framework of a wider

strategy of their registration (EC Regulation REACH 1907/2006). It encouragethe application of natural materials as non chemical strategies

Pesticides and fertilisers are:

Soil fertility concept

Soil fertility as the whole Physical, Chemical, and Biological characteristics of a soil is strongly linked to the organic matter amount, composition and

dynamics requesting a plan for increasing soil organic matter

It is not possible to produce high quality agriculture on lowfertility soils, without a wide application of Chemicals

In the last 50 years the wide application of chemistry in agriculture, linked to the climate change determined a progressive decreasing of soil fertility, moreover in coastal areas, where the soils have very low organic matter

amounts

4

The Brassicaceae Family

350 genera Around 3700 specie More than 200 glucosinolates Generally herbaceous Present in each continent

An ancient Sanskrit text dated 10 century describes the effects of organic amendments and green manures

(including crucifers)

Surapala: The Science of plant life

The glucosinolate- myrosinase system (1)

+ D-GlucoseHOHO OH

OHO

S CR

N-O3SO

R CNOSO3--

SH

Myrosinase

H2O

R C N + S + HSO4-R N C S + HSO4-NitrileIsothiocyanate

Glucosinolate

Federal Register of rule and regulations: May 20, 1996, Vol. 61,No 98, page 25152No 25152--25153

“Allyl-isothiocyanate (AITC), as component of mustard oil, is on the FDA Generally Recognized as a Safe (GRAS) Substance”

EPA - 40 CFR Part 18040 180

7

Glucosinolate Biodiversity

Glucosinolati con catena R alchilica Glucosinolati con catena R ossidrilica

Nome Struttura Nome Struttura

Glucocapparina x

Progoitrina /Epiprogoitrina OH

x

Sinigrina

x

Gluconapoleiferina OHx

Gluconapina

x

Glucosisimbrina x

HO

Glucobrassicanapina

x Glucoconringina

xHO

Glucosinolati con catena R

tiofunzionalizzata Glucosinolati con catena R aromatica

Glucoerucina S x Glucotropeolina

x

Glucoiberina xSO

Gluconasturtina x

Glucorafanina x

SO

Sinalbina

HO

x

Glucoalissina SO

x

Glucolimnantina

x

OMe

Glucorafasatina S x Glucosinolati indolici

Glucorafenina x

SO

Glucobrassicina N

x

H

Glucocheirolina SO O

x

4-Idrossi glucobrassicina N

x

H

OH

4-Metossi glucobrassicina

N

x

H

OMe

Neoglucobrassicina NOMe

x

Different R side chain

DifferentIsothiocyanates

Different biologicaleffect

Different Physico-chemical properties

The CREA CI collection of purified glucosinolates

“Pest and desease suppression by glucosinolatecontaining plants arising specifically from the

biocidal properties of the glucosinolate hydrolysisproducts, particularly the isothiocyanates,

released from incorporated tissues or rotationcrops, notably the Brassicaceae”.

(Kirkegaard and Matthiessen “Developing and refining the biofumigation concept”Proceeding of the First International Symposium “Biofumigation: a possible alternative to methylbromide?” Firenze 29 marzo-1 aprile 2004 - Agroindustria, 2004, 3 (3),233-239)

__________________________________________________

Biofumigation definition

Green manure and cover crops could offer a fundamental help to

the farmer for:

(i) Improving soil fertility in nutrient (Nitrogen, soil structure

etc.) and the biodiversity in the system

(ii) Playing a role in the decrease of CO2 in the atmosphere thanks to

the SINK effect of organic matter in soil

(iii) Improving the resistence of the agro-system to disease and pest

for a multifunctional and more sustainable agriculture

Green manure and cover crop roles

Biofumigant green manure plants (1)

Plants selected for their capability of releasing volitile ITCs from theirepigean part. (B. juncea. B, rapa, Sinapis alba)

They are applied in soil for containing soil bornefungi, nematodes and wireworms

Correlations of glucosinolate content in plants and mortality of Globodera pallida eggs

R2 = 0,843 P‹ 0,0001

Biofumigant green manure: catch crops (2)

Nematodes attack biofumigant plant roots, penetrates the root tissues and

activates the enzymatic hydrolysis and isothiocyanates from glucoerucin or

glucoraphasatin are released.

The nematode feeds a poisoned media and does not end its growing cycle into

the root during the time of biocidal crop cultivation (8-10 weeks).

Plants selected for a high GL content in roots. (Eruca sativa sel. Nemat. Raphanus sativum cv. Karacter,

(Crotalaria juncea)

Catch crop effect of E. sativa green manureNematode: Meloidogyne incognita

Post J2 larvae

J4 youngfemales

Females

Necrosisonrootsafternematode attack

Defatted seed meals

Biofumigant pellets and mealsbased on B. carinata, B.juncea,

B. nigra et al. seeds

USA Patent N° US7,749,549B2 of 2010-06-06

0,0

25,0

50,0

75,0

100,0

0 100 200 300 400 500

Rate % Formulated defatted mealsPellet

Untreated defatted meals

In vials In soils

Grafico1

153060

3060120

4590180

60120300

75150360

9018012.7

105210

120240

135270

150330

165390

180

240

Formulated defatted meals

Pellet

Untreated defatted meals

Rate %

6.1307757289

2.3061885653

1.5892263026

20.8481501661

13.0369236209

4.8179901364

32.6102548322

21.7847834447

4.0764371596

41.7714239182

35.5393882981

6.3781769727

47.0503136484

43.9589544253

12.3485561119

61.9648152177

55.1052304148

63.4681055429

53.1773001693

64.4197255392

59.6142247626

68.7178389894

62.0571993387

66.4580354124

69.5232258687

71

87.2924240587

72

74

Foglio1

1-1,5mediad.s.

15152045152013145713149923.6666666673647

30406459435815452491431588.33333333323305

45588334706940674809656694.33333333361343

6075335589335584935983202371592

751048937818748931470933051.666666667115103

901136787117237913463011218489112110

1051198486134000912032831247259.3333333380359

1201281177132775011874881265471.6666666771438

135136156614154411266183134773075585

1501258147147536111799361304481.33333333153066

1651179649151878012011981299875.66666667189883

1801349560150939213124791390477104639

240111428113327321223506.5154468

1-1,5mediad.s.

15149923.6666666673647

30431588.33333333323305

45656694.33333333361343

6083202371592

75933051.666666667115103

901218489112110

1051247259.3333333380359

1201265471.6666666771438

135134773075585

1501304481.33333333153066

1651299875.66666667189883

1801390477104639

2401223506.5154468

minore 0,25mediaD.S

10297602304666382987328418.33333333347390

2070510466129075538370725947084

301159053113924411863061161534.3333333323629

401520839124392812131631325976.66666667169455

501623005168446514731591593543108690

601715839184744015727391712006137391

701852658180273916281351761177.33333333117891

801801920187383216928301789527.3333333391135

901726939184959618055501794028.3333333362135

1001722507189296516538001756424123137

11018033081697200175025475030

120177618516669701721577.577227

Formulated pelletsScaglie - 0,25

mediad.s.mediad.s.

30690258497276998.51127660663037085668579.53219.4571747424

60301532265719283625.5253241201388411445491416954036.1655070128

9042088248326045207144108180116456133349124902.511945.1548545843

120751888681961716924.549446300201117152934177025.534070.5260379114

150935118822979879048.57929436027185335259731222557094.6299401266

18011055951081759109367716855

210104092310721841056553.522105

240113976012212411180500.557616Scaglie - 0,25

270128552011695631227541.581994mediad.s.

330148724312553661371304.51639626068579.53219.4571747424

390181747516094481713461.51470971201416954036.1655070128

180124902.511945.1548545843

300177025.534070.5260379114

36031222557094.6299401266

Untreated defatted meals

mediamg/lumoli/gresa

Formulated defatted mealsumoli/gresa6068579.50.01459261921.47162355621.6

mg/l%1201416950.04423982614.46145886634.8

15149923.6666666670.04757642354.79794508556.1180124902.50.03743072653.77478080984.1

30431588.3333333330.161787099216.3157623220.8300177025.50.05856579865.90619187686.4

45656694.3333333330.253064108325.520785431632.63603122250.113387109511.434762959612.3

608320230.32415717732.690316358441.812.7

75933051.6666666670.365122742336.821575461247.1

9012184890.480863176148.493664389462.0

1051247259.333333330.492529102349.670139397963.5

1201265471.666666670.499913922450.41487720764.4

13513477300.533268407153.778580793168.7

1501304481.333333330.515731740252.010058513766.5

1651299875.666666670.513864213551.821723826571.0

18013904770.55060165655.526588944672.0

2401223506.50.482897694948.698839744474.0

umoli/gresa

minore 0.25mg/l%

10328418.3333333330.119953256312.096939923314.7

207072590.273567300427.588473211233.6

301161534.333333330.457768937746.164677054656.3

401325976.666666670.524447766252.88904459264.4

5015935430.632941785363.830353496477.8

6017120060.680976703768.674536471883.7

701761177.333333330.700914920570.685248135586.1

801789527.333333330.71241040871.844534894787.5

901794028.333333330.714235494172.028589557987.8

10017564240.698987517270.490875070485.9

11017502540.696485677470.23857174485.6

1201721577.50.684857799269.065933763484.2

umoli/gresa

Pelletmediamg/l%

3076998.50.01800639381.81589287632.3

60283625.50.101790453610.265273659113.0

904520710.170092504417.153338484421.8

120716924.50.277486511527.98371434635.5

150879048.50.343225291634.613280714544.0

18010936770.430253836243.389858428655.1

2101056553.50.415200829841.871806153353.2

2401180500.50.465459425646.94024057859.6

2701227541.50.484533825148.863838759362.1

3301371304.50.542827503154.74258804969.5

3901713461.50.681566886468.734054703887.3

Foglio1

3646.57940175893646.579401758947389.656680897647389.656680897611276.231839581911276.2318395819

23305.252397974923305.252397974947083.502216806347083.502216806325323.615154633825323.6151546338

61342.902363137161342.902363137123628.917079149723628.917079149744107.906796854544107.9067968545

71591.917644382271591.9176443822169455.357012794169455.35701279449445.855888031749445.8558880317

115102.650631222115102.650631222108690.218014318108690.21801431879294.247335478379294.2473354783

112109.893069256112109.893069256137390.606545717137390.60654571716854.597236362516854.5972363625

80359.369723346580359.3697233465117890.514819189117890.51481918922104.865086672722104.8650866727

71437.737102553171437.737102553191135.144929567891135.144929567857615.767637861157615.7676378611

75584.812052422375584.812052422362134.905764258962134.905764258981993.981026048581993.9810260485

153065.788111954153065.788111954123137.104371509123137.104371509163961.799101193163961.799101193

189882.648534124189882.64853412475029.686338142275029.6863381422147097.302369894147097.302369894

104639.06086639104639.0608663977226.667107288877226.6671072888

154468.183456982154468.183456982

1-1,5

minore 0,25

Formulated pellets

Foglio2

Formulated defatted meals

Pellet

Untreated defatted meals

Rate %

Foglio3

minuti

Gall index and vigour after treatment with different DSM (2)

On soils with 30 J2 larvae in 100 cm-3 inoculum of Meloidogyne incognita

Oil N P K C C/N OM Gl

% on

DM9-12 5-6 0.7-1 1-1.5 40-45 7-8 80/85 4.1/4.7

That for a dose of 2.5 t ha-1 on dry matter means:

Kg ha-1

125/ 150

18/25

25/ 38

1000/ 1100

100/117

Biofumigant pellets composition

Pilot plant for post harvest biofumigation

Treatment cabin 0.1m3 Reactor:

DSM+water

NCS

AITC

Quality Maintaining,Extension of shelf-life

improvement of nutraceutical properties

Liquid biofumigant formulations

Liquid formulation are essentially vegetable oil/water emulsions where DSM are mixed

Oil in the emulsion plays a suffocatingeffect on pests or pathogens and

protecting plants from ITCs phytotoxicity

DSM in the emulsion play the wellknown «biofumigant effect»

The emulsion activates the hydrolysis reaction of DSM, releasing isothiocyanates (ITCs) that naturallyare solubilised in the oil phase

Different final preparation of the liquid formulations

Foliar formulations

Duolif Duofruit

The suspension is taken at the requested concentration in

water.DSM are micronized at a sizelower than 150 micron, a size

that permits the distribution of the emulsion still containing DSM

by mechanical pumps.

Root formulation

Biofence FL

The suspension is taken at the requested concentration of

water. DSM are applied at a sizeof 1000/2000 micron to make a simple remval the suspension by a simple filtration. In this way, no DSM remains in the emulsion to avoid problem to drip irrigation

system

Isothiocyanate release duringliquid formulation preparation

Time (min)

AIT

C Y

IELD

(%)

0

20

40

60

80

100

non-formulated DSMformulated DSM

Root treatment

Time (min)0 50 100 150 200 250 300 350

AIT

C Y

IELD

(%)

0

20

40

60

80

100

non-formulated DSMformulated DSM

Foliar treatment

The main target of foliar liquids is in epigeal apparatus defense, not only in horticulture, but even in floriculture, fruit, grape and several other crops

New open field application for foliar liquid

1) Pests and disease control2) 2) Biostimulant effect3) Earliness of ripening4) Leaf washing5) leaves functionality

TreatedUntreated

23

Pest and disease control (1)

The main target for foliar liquid formulation are pests and diseases, limited to

organisms of small dimension to permit a sufficient persistence of oil microfilm

enriched by ITCs

Scientific studies on their effect on :• Aphids. Cotton aphid (Aphis gossypii G.), (Aphis fabae S.), (Myzus persicae

S.)• Mites. Twospotted spider mites (Tetranychus urticae K.), Citrus red• mite (Panonycus citri McG.).• Scales. (Aonidiella aurantii, A.), Pyriform scale (Protopulvinaria

pyriformis C.), Japanese citrus scale (Unaspis yanonensis K.),Cottonycushion scale (Icerya purchasi M.).

• Whitefly. Woolly whitefly (Aleurothrixus floccosus M.).• Oidium (Podosphaera xanthii B.), (Golovinomyces cichoracearum DC),

sugar beet oidium (Erysiphe betae V), cucurbitacee oidium (Erysiphecichoracearum DC ).

Evaluation of Duofruit and DuolifUNITA’ DI RICERCA

PER L’ORTICOLTURAMonsampolo del Tronto (AP) Italy

Effect of the liquid foliar treatments on spiders on eggplants and Oidium on melon

Dr. V. FerrariDr. E. Piccinini

n° Acari / leaf

EGGPLANTCompared to the chemical fenazaquin (Magister)

in the control of Red spider(Tetranycus urticae)

ResultsDuofruit showed a higher containment effect in

comparison to Duolif with a persistence of around14 days

Defense strategy : a treatment every 15 days

Untreated

MELONCompared to the chemical product Penconazol(Topas) in the control of Oidium (Podosphaera

xanthii, Golovinomyces cichoracearum)

10,0

15,0

20,0

25,0

30,0

35,0

T0 T+7 T+14

Testimone

Topas

Duolif

Duofruit

A.I.* / leaf

ResultsDuofruit showed a similar containment effect in

comparison to Topas

Defense strategy : a treatment every 20 days

∑ % superficie fogliare coperta da macchieI.A.*=

Log10 % foglie malate

Untreated

Grafico1

T0T0T0T0

T+7T+7T+7T+7

T+14T+14T+14T+14

T+21T+21T+21T+21

Testimone

Magister

Duolif

Duofruit

6

11

13

10

18

0.1

7

1

33

0.1

15

5

65

1

40

25

Foglio1

TestimoneMagisterDuolifDuofruit

T06.011.013.010.0

T+718.00.17.01.0

T+1433.00.115.05.0

T+2165.01.040.025.0

Per ridimensionare l'intervallo di dati del grafico, trascinare l'angolo inferiore destro dell'intervallo.

Grafico1

T0T0T0T0

T+7T+7T+7T+7

T+14T+14T+14T+14

Testimone

Topas

Duolif

Duofruit

14

13.4

14.5

13.9

21.1

13.7

16.5

14.2

33.4

15.5

18.7

15.8

Foglio1

TestimoneTopasDuolifDuofruit

T014.013.414.513.9

T+721.113.716.514.2

T+1433.415.518.715.8

Per ridimensionare l'intervallo di dati del grafico, trascinare l'angolo inferiore destro dell'intervallo.

I.A.*=∑ % superficie fogliare coperta da macchie

Log10 % foglie malate

Biofence liquid formulation efficacy on citrus in the control of Aonidiella (Erysiphe betae

0

10

2030

40

50

Veg

etab

leoi

l

Min

eral

oi

l

Biof

ence

form

ulat

ion

Biof

ence

with

min

eral

oil

Infe

sted

Lea

f Sur

face

(%

of t

he c

ontr

ol)

a a

bb

Grafico1

Vegetable oil

Mineral oil

Biofence formulation

Biofence with mineral oil

Infested Leaf Surface (% of the control)

42

42

12

5

Foglio1

Vegetable oil42b

Mineral oil42b

Biofence formulation12a

Biofence with mineral oil5a

Vegetable oil42.5

Mineral oil47

Biofence formulation97

Biofence with mineral oil100

control75.4a

chemical insechicide (MIRO)22b

biofence formulation1c

Foglio1

Infested Leaf Surface (% of the control)

Foglio2

Mortality (% of control)

OENIDIELLA

Foglio3

Mortality (% of control)

TETRANYCHUS URTICAE

Some trials on orange cultivation

26

020406080

100

Duofruit Mineraloil

Untreatedlar

val m

orta

lity

(%)

aa

b

Unaspis yanonensis Aphid gossypi

0

20

40

60

80

100

Livi

ng a

phid

per

leaf

(%)

b b a

c

Duolif + pirethrum

Duolif Imida-cloprid

Untreated

c

Protopulvinaria pyriformis

Larv

alm

orta

lity

(%)

Duolif Mineraloil

Untreated

b

aa

27

New application fields for root liquidformulates (1)

The main target for root liquid formulation are soil borne pests, even asrescue treatment after pre-plant with biofumigant green manure and defatted

seed meals.

Positive scientific results on:

• Nematodes: Southern root-knot nematode (Meloydogineincognita Chitw. Kofoid et White).

• Wireworms: (Agriotes sordidus, Agriotes litigiosus)

• Soilborn Fungi: (Armillaria mellea V.)

28

Unsatisfactory effects of foliar liquid formulations

Diseases: Plasmopara viticola B., Aspergillus and fungiproducing mycotoxins

Pests: Ceratitis capitata W., Red weevil (Rhynchophorusferrugineus O.), Spotted Wing Drosophila (Drosophilasuzukii M. and all the insect of “bigger” size (includingbees) and with rapid movement as Thrips (Frankliniellaoccidentalis)

At moment unsatisfactory effect on:

This root liquid was studied as a support of plant during cultivation, but itpermitted to open new application fields not only in horticulture for

nematode control, but even on fruit replant (apple, peaches, grape) and othercrops

New field of application for root liquidformulations

1) Containment of root infestation

2) Improvement of soil biological and chemical fertility

3) Biostimulation of root system 4) Lengthening of harvesting period

Untreated TreatedUntreatedTreated

Biofumigant green manureB. juncea sel ISCI20 – ISCI99

Green manure catch cropEruca sativa sel Nemat

Biofumigant pelletsEU Patent N° 03 792 616.9 -

1219

Plants and materials for biofumigation

Liquid root treatmentPCT EP 2009/050143

Liquid foliar treatmentPCT W 2006/136933 A2

The biofumigant purposal, even considering all the potential sinergies with other non chemical

alternatives (viz. Solarization, antagonists etc. ) hasto be considered as a real cropping system that has

to be applied with a year after year strategy for a total or partial alternative to chemicals.

The production line can be considered as a biorefinery approach in the ambit of circular

economy

Brassica cropping system

Brassica cropping system

The biofumigant purposal, even considering all the potential sinergies with other non chemicalalternatives (viz. Solarization, antagonists,

biostimulant etc. ) has to be considered as a realcropping system that has to be applied with a year

after year strategy for a total or partial alternative to chemicals.

The production line follows a biorefinery approach in the ambit of circular economy

It becomes crucial to evaluate not only the economic balance (€/kg), but even the environmental one (kg CO2eq/kg)

Seeds Residues

Inputs

Outputs

Luca Lazzeri, Lorenzo D’Avino “Green Chemistry, a first step towards” - S. Lucia, Alma Mater Studiorum-Università di Bologna, Italy. November 27th , 2013

Environmental balance

GHG emission by Biofumigation Kg CO2e ha-1

ec from cultivation phase 1523±707ep from processing 159ef for pellet production 41

ef for epigeal formulation 166

ef for hypogeal formulation 449Eb Emissions of biorefinery =

ec + ep + Σ (ef+ epk+ etd)2338

GHG emission by Conventionaleai from Metham sodium production 2127eai from paraffinic oil production 943eai from Oxamyl production 746Ec Emission of conventional proposal

= Σ (eai+epk+etd)

3816

Biofumigation material evaluation by the Biorefinery approach of carbon foot print

Biofumigant plants and materials…

• Could change the farmer role from user of chemicals to producer of its own bio-based materials for plant management and defense in the framework of a new multifunctional agriculture

• Have not to be considered a simple product innovation but a systemone, that considers soil fertility management and plant health as a

fundamental starting point for a real sustainable production.

The GL-MYR system management plays a fundamental role of bioactivemolecules• products from a biorefinery chain linked to the territory with

positive environmental balances

• Can be surely improved and diversified for new materials and applications. The topic request more and more research for defining

the system limits.

Diapositiva numero 1Diapositiva numero 2Diapositiva numero 3Diapositiva numero 4Diapositiva numero 5 The glucosinolate- myrosinase system (1)Diapositiva numero 7Diapositiva numero 8Diapositiva numero 9Diapositiva numero 10Diapositiva numero 11Diapositiva numero 12Diapositiva numero 13Diapositiva numero 14Diapositiva numero 15Diapositiva numero 16Diapositiva numero 17Diapositiva numero 18Diapositiva numero 19Different final preparation of the liquid formulationsDiapositiva numero 21Diapositiva numero 22Diapositiva numero 23 Diapositiva numero 25 Some trials on orange cultivationDiapositiva numero 27Diapositiva numero 28Diapositiva numero 29Diapositiva numero 30Diapositiva numero 31Diapositiva numero 32Diapositiva numero 33Diapositiva numero 34Diapositiva numero 35