02/07/2023

New generation of plant

biostimulants
Giuseppe Colla
giucolla@unitus.it

© 2023 Giuseppe Colla

.
Content
✓ Biostimulant types

✓ Biactive compounds

✓ Biostimulant effects

✓ Practical applications

© 2023 Giuseppe Colla

1
 02/07/2023

What is a plant biostimulant?

.

Definition

Legislation

© 2023 Giuseppe Colla

Agricultural Inputs
.

Pesticides Biostimulants Fertilizers

Weed, disease, insect Nutrient availability Macro and micro
mite, snail, rodent Nutrient use efficiency nutrients
control
Crop quality traits
Plant growth Soil conditioners
regulators Abiotic stress tolerance

Product claims and marketing intent are critical

Fertilizing Products

Regulation (EU)
© 2023 Giuseppe2019/1009
Colla

2
 02/07/2023

Plant biostimulants
. Non-microbial plant Microbial plant
biostimulants biostimulants

Humic
PGPR
substances
Protein
hydrolysates
Seaweed Beneficial fungi
extracts
Plant extracts

© 2023 Giuseppe Colla

Biostimulant development
Product looking for purpose
.

Purpose-built product

© 2023 Giuseppe Colla

6 (Bonini et al., 2020)

3
 02/07/2023

Biostimulant development

© 2023 Giuseppe Colla

Protein hydrolysates
A cocktail of bioactive peptides and amino acids with multiple
functions
.
Metabolism

Hormone-like Crop
activity resistance to
stress

Plant
nutrition
© 2023 Giuseppe Colla

4
 02/07/2023

Peptides as plant signaling molecules

• 2-50 chains of amino acids
.
• specific amino acid sequence
• biological activity at very low concentration (nM)

• Control plant growth & development

• Flowering and fertilization
• Adaptation to environment

© 2023 Giuseppe Colla

Root Hair Promoting Peptide (RHPP)

Peptide stimulating root growth and development identified in a
. vegetal-derived protein hydrolysate
Gly-Gly-Ile-Arg-Ala-Ala-Pro-Thy-Gly-Asn-Glu-Arg
(Matsumiya, 2011)

© 2023 Giuseppe Colla

10

5
 02/07/2023

Production of signaling peptides

Enzymatic hydrolysis for the release of bioactive peptides from proteins

Key factors: protein source, enzyme, time, pH, temperature

© 2023 Giuseppe Colla

11

Vegetal biomass as protein source

.
❑ High content of proteins

❑ Protein quality

❑ Availability

❑ Easy to handle and store

❑ Cheap

© 2023 Giuseppe Colla

12

6
 02/07/2023

Fractionation of protein hydrolysate

.
Whole product

Peptide separation
based on molecular size

Dialysis membranes

PH1 PH2 PH3 PH4

(0.5-1 kDa) (1-5 kDa) (5-8 kDa) (5-8 kDa)
Peptide range Peptide range Peptide range Peptide range

5AA→9AA 9AA→45AA 45AA→73AA

© 2023 Giuseppe Colla >73AA

13

Tomato rooting test

Foliar vs root applications

© 2023 Giuseppe Colla

14

7
 02/07/2023

Signaling peptides
.
Local Effect & Systemic Effect

© 2023 Giuseppe Colla

15

Crop response to biostimulant

.

© 2023 Giuseppe Colla

16

8
 02/07/2023

Bioassay
. A bioassay is a simple, inexpensive, accurate, and direct method of
determining the hormone-like activity and other biological activities on
seedlings or plant tissues in controlled environment

Tomato rooting test Auxin like activity

Auxin-like activity ❑ Root growth
❑ Flowering
❑ Fruit set

Gibberellin-like activity
Lettuce hypocotyl elongation test ❑ Seed germination
Gibberellin-like activity
❑ Fruit growth
❑ Stem elongation
❑ Bud break

Cucumber cotyledon expansion test Cytokinin-like activity

Cytokinin-like activity ❑ Shoot and fruit growth
❑ Senescence
❑ Resistance to stress
© 2023 Giuseppe Colla

17

Bioassay
Tomato rooting test →Auxin-like activity
.

PH3

Control

© 2023 Giuseppe Colla

18

9
 02/07/2023

Controlled environment trials

© 2023 Giuseppe Colla

19

Controlled environment trials

© 2023 Giuseppe Colla

20

10
 02/07/2023

Agronomic trials
.

The increases of agronomic traits induced

Plot trials by biostimulants are often 5-10%

Farm trials

© 2023 Giuseppe Colla

21

Biostimulants & plant nutrition

Vegetal protein hydrolysate vs Control
.
Crop Leaf nutrient concentration increase
N P K Ca Mg K/Na
Lettuce (Trial 1) = = +9% +10% +8%
Lettuce (Trial 2) +23%
Tomato (greenhouse trial) = +36% +13% = +43%
Tomato (field trial) +22% +8% = +22%
Spinach (trial 1) = +36% = +25%
Spinach (trial 2) +13% = +13% +10%

© 2023 Giuseppe Colla

22

11
 02/07/2023

Nitrogen use efficiency (NUE)

.
N-uptake N-utilization
NUE = Efficiency X Efficiency

Y NP Y
= X
NF NF NP

Y = Crop biomass
NF = Nitrogen application rate
NP = Nitrogen content in plant
© 2023 Giuseppe Colla

23

Biostimulants & NUE

. N-use efficiency N-uptake efficiency N-utilization efficiency

© 2023 Giuseppe Colla

24

12
 02/07/2023

© 2023 Giuseppe Colla

25

© 2023 Giuseppe Colla

26

13
 02/07/2023

Biostimulant & abiotic stress

.

Godoy, F.; Olivos-Hernández, K.; Stange, C.; Handford, M. Abiotic Stress in Crop Species: Improving
Tolerance by Applying Plant Metabolites. Plants 2021, 10, 186. https://doi.org/10.3390/plants10020186
Hasanuzzaman, M.; Parvin, K.; Bardhan, K.; Nahar, K.; Anee, T.I.; Masud, A.A.C.; Fotopoulos,
© 2023 V. Colla
Giuseppe
Biostimulants for the Regulation of Reactive Oxygen Species Metabolism in Plants under Abiotic Stress.
Cells 2021, 10, 2537. https://doi.org/10.3390/cells10102537

27

Biostimulants & oxidative stress

.

© 2023 Giuseppe Colla

28

14
 02/07/2023

Biostimulants & crop quality traits

. Fruit size

© 2023 Giuseppe Colla

29

© 2023 Giuseppe Colla

30

15
 02/07/2023

© 2023 Giuseppe Colla

31

© 2023 Giuseppe Colla

32

16
 02/07/2023

© 2023 Giuseppe Colla

33

Planning biostimulant application

.

© 2023 Giuseppe Colla

34

17
 02/07/2023

Which biostimulant should I use?

.

© 2023 Giuseppe Colla

35

When should I apply the biostimulant?

.

© 2023 Giuseppe Colla

36

18
 02/07/2023

Biostimulant application
.

© 2023 Giuseppe Colla

37

Biostimulant application
.

© 2023 Giuseppe Colla

38

19
 02/07/2023

Biostimulant application
.

© 2023 Giuseppe Colla

39

Biostimulant application
.

© 2023 Giuseppe Colla

40

20
 02/07/2023

Biostimulants & economic benefits

.

© 2023 Giuseppe Colla

41

© 2023 Giuseppe Colla

42

21