Amino acids: the scientific basis of the biostimulation David Cambri – Isagro SpA

Introduction Biostimulants are a wide category of products

based on various compounds of natural origin. Amino acids and peptides represent a relevant class of bio-molecules which enter in the composition of biostimulants

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… and they work

World wide penetration

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• to improve yield (quantity & quality) • to increase stress tolerance • as a physical and physiological adjuvant for agrochemicals

Good! But why do they work? to understand Æ to improve improve biostimulants biostimulants effectiveness effectiveness and and get get new targets

Amino acids: the scientific basis of the biostimulation

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Molecular biology biology tecniques tecniques allow allow to to investigate investigate on on the the effect effect of biostimulants on the plant physiology and the gene regulation Gene expression analysis allows to classify the biostimulants on the base of their molecular mode of action action..

Experimental conditions In order to demonstrate that the beneficial effects observed in plants treated with biostimulants was due to specific gene expression changes, a set of experiments was performed on the model plant Arabidopsis thaliana. Three week old Arabidopsis plants were sprayed with Aminoplant® formulation and analyzed for gene expression. A preliminary set of experiments was performed to set up the conditions and preliminarly verify that over expressed genes under stress (saline) condition are activated by the aminoplant formulation.

%[Aminoplant]

PR1 ►

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18S ►

0.00

0.01

0.10

Materials and Methods Arabidopsis ecotype Colombia were grown for 3 weeks under controlled conditions (24°C, 70% humidity and 16 hours of light). Aminoplant® 0.1% (and Proline 0.014%, Hydroxyproline 0.011%, Phenilalanine 0.002%, Glutammic acid 0.009%, Aspartic Acid 0.005%) was dissolved in 4 ml of MS 0.25% (Murashige and Skoog Basal Medium with Gamborg’s Vitamins) and sprayed on the leaves.

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4 plants were treated and 24 hour later total RNA was extracted from 3 different leaves. A semi-quantitative RT-PCR analysis was conducted to quantify the PR1, PDH, GSTF7, KIN2 and SAG12 gene expression since those genes are normally over-expressed in response to abiotic stress.

PD1 = salt PDH = drought GSTF7 = drought KIN2 = low temperature SAG12 = senescence

Results

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Semi-quantitative RT-PCR of the expression of PR1, PDH, GSTF7, KIN2 e SAG12 in Arabidopsis, after a treatment with, respectively, Aminoplant® 0.1% (S), Proline 0.014% (P), 4-hydroxyproline 0.011% (H), Phenylalanine 0.002% (F), Glutamic acid 0.009% (G), Aspartic acid 0.005% (A) and Buffer (B). The molecular analysis has shown that PR1, PDH, GSTF7, KIN2 are induced after Aminoplant® application. SAG12 was not affected by Aminoplant®; actually this last gene is related with senescence.

Results

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Therefore: 1. Aminoplant® is able to activate the transcription of genes related to abiotic stresses. Genes such as PDH and GSTF7, tipically activated by drought and osmotic stress, (Oono et al., 2003; Sappl et al., 2004) are expressed at higher levels if compared to control plants (buffer treated); KIN2, a gene whose expression is upregulated by low temperature (Kim et al., 2003) and that contributes to cold tollerance in plant, is also upregulated in Siapton treated plants.

Results

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Therefore: 2. the higher gene expression level is induced by Aminoplant®. It is higher if compared to that of the same genes analyzed in plant treated with the single aminoacid components of Aminoplant® itself; 3. considering that in all the experiments, the expression level of each of the genes tested is higher than the sum of the expression level induced by the sigle components of the Aminoplant® mixture, we can presume that the composition produces a sinergic effect. effect

What else?

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Roots of a tomato potted plant grown in high salinity conditions and treated with Aminoplant® as a root application (A), compared to roots of an untreated plant growth in the same challenging conditions (B) and roots of a plant grown in absence of salinity (C).

What else?

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Shooting length (cm) of potted lemon plants grown in a high salinity soil, untreated (A) and treated (B) with Aminoplant®, compared to data obtained from untreated plants grown in standard conditions (C).

What else? (1) Target Crop Location Appl. method Appl. timing Treatments Dosage Assessments

yield increase Carrot A var. Zetor B var. Kiev Wolskie, Central Poland foliar T1 stage T1 – July 1 T2 – August 16 1 – UTC (untreated control) 2 – Aminoplant® (T1, T2) 5 l/ha yield root quality

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Application method

What else? (1) Weight of 100 roots (kg)

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16,5 16,0 15,5 15,0 14,5 14,0 13,5 13,0 12,5 12,0 11,5

UTC Aminoplant

A

Varieties

T

T

Root homogenity

B

T Leaf quality at harvest

What else? (2)

Location: Trzciana (southeast part of Poland) Assessment: yield

Date of sowing: 13.04 Date of harvesting: 13.09 HARVEST

SIAPTON 0,3% (3 l /ha) 4 weeks before harvesting (14/08) Cultivar: Kazan F1

+23%

90 80 70 t/ha

60 50 40 30 20 10 0

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control

Aminoplant®

What else? (2)

Location: Trzciana (southeast part of Poland) Assessment: yield

Date of sowing: 13.04 Date of harvesting: 13.09 NITRATE CONTENT

SIAPTON 0,3% (3 l /ha) 4 weeks before harvesting (14/08) Cultivar: Kazan F1 mg NO3/kg

90 80 70 60 50

- 62%

40 30 20 10 0

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control

Aminoplant®

What else? (3)

Location: Trzciana (south-east part of Poland) EFFECT OF SIAPTON ON CARROT YIELD SIAPTON 0,3% (3 l /ha) 4 & 8 weeks before harvesting SIAPTON 0,3% (3 l /ha) 11 weeks after sowing , 4 & 8 weeks before harvesting (27.06 26.07 14.08) Cultivar: Kazan F1

Date of sowing: 13.04 Date of harvesting: 13.09 MARKETABLE YIELD 90 85

87,3 85,5

83,4

80 75 70 65 60 55

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50

Check

AM 2x

AM 3x

What else? (3)

Location: Trzciana (south-east part of Poland)

Date of sowing: 13.04 Date of harvesting: 13.09 NITRATE CONTENT

mg NO3/kg

EFFECT OF SIAPTON ON CARROT YIELD SIAPTON 0,3% (3 l /ha) 4 & 8 weeks before harvesting SIAPTON 0,3% (3 l /ha) 11 weeks after sowing , 4 & 8 weeks before harvesting (27.06 26.07 14.08) Cultivar: Kazan F1

60 50

- 33%

40 30 20

- 91%

10 0

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control

AMINOPL. 2x

AMINOPL. 3x

What else? (3)

Location: Trzciana (south-east part of Poland)

Date of sowing: 13.04 Date of harvesting: 13.09 SOLUBLE SUGAR CONTENT

+5%

6000

4000 3000 2000 1000 0 control

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+7%

5000 mg / 100g

EFFECT OF SIAPTON ON CARROT YIELD SIAPTON 0,3% (3 l /ha) 4 & 8 weeks before harvesting SIAPTON 0,3% (3 l /ha) 11 weeks after sowing , 4 & 8 weeks before harvesting (27.06 26.07 14.08) Cultivar: Kazan F1

AMINOPL. 2x

AMINOPL. 3x

What else? (3)

Location: Trzciana (south-east part of Poland)

Date of sowing: 13.04 Date of harvesting: 13.09 CAROTENOIDS CONTENT

+14%

25

+9%

20 mg / 100g

EFFECT OF SIAPTON ON CARROT YIELD SIAPTON 0,3% (3 l /ha) 4 & 8 weeks before harvesting SIAPTON 0,3% (3 l /ha) 11 weeks after sowing , 4 & 8 weeks before harvesting (27.06 26.07 14.08) Cultivar: Kazan F1

15 10 5 0

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control

AMINOPL. 2x

AMINOPL. 3x

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What else?

What else? Chinese Chinese Cabbage Cabbage higher higher yield yield up up to to +58% +58%

Cauliflower Cauliflower

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Broccoli Broccoli

less less nitrates nitrates down down to to -35% -35%

What else? higher higher yield yield up up to to +22% +22%

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Lettuce Lettuce (& (& leafy leafy vegetables) vegetables)

less less nitrates nitrates down down to to -31% -31% (especially (especially during during summer summer & & autumn) autumn)

Conclusion salinity

that’s that’s why why ®® works Aminoplant Aminoplant works

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drought

low temperature

even even under under “normal” “normal” conditions conditions

Aknowledgments Fabio Apone1, Stefania Arciello1, Gabriella Colucci1, Lucio Filippini2, Domenico Portoso2

1.

Arterra Bioscience S.r.l., CEINGE, Via Comunale Margherita 482, 80145 Napoli

2. Isagro Ricerca S.r.l. , Via Fauser 4, 28100 Novara

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Thank Thank you you