Biostimulants for stress management

1. A iosti ula t app oa h
to edu i g st ess o you tu f
a d i p o i g tu f uality -
ith i depe de t t ial data

5. Product Foliar Rate/Ha Active Benefits Cost/Ha
Phylgreen 15 Ascophyllum nodosum Improve root growth/heat tolerance 290
Alexin 5 Salicylic acid Improve ability of plant to fight disease 76
Delfan 2 24% amino acids; 9% N; 23% carbon Improve plant recovery 35
EDTA Ca 5 14% w/w calcium oxide (CaO) Harden cell wall 148
Silica 1-2 100% silica Harden turf 80-160
Green Fer Energy 5-8 21% iron EDDHA; 17% Humic acid Improve colour and root growth 80-161Monthly
Frequency
Monthly
Monthly
Monthly
Monthly
Monthly

7. Why is this approach beneficial ?
STRESS Ma age e t
Suppl of o ple sugars
Suppl of a tio ida ts
Suppl i oligo-ele e ts
Suppl of Potassiu
Suppl i os oregula ts

8. MIX Ph lg ee WITH YOUR OTHER FOLIAR PRODUCT“ !
Appli atio :
s all doses ut egula
CONTACT EFFECT
pH EFFECT
SYNERGIES ith Catio s
Fe, Ca, Mg, M …

9. Effe ts:
MAIN BENEFITS.
REDUCE stress effe ts fro soil a d eather o ditio s
E ha e faster re o er after frost, draught or sali it stress
I pro e utrie t a sorptio espe iall ke ele e ts
Mai l atio s like K+, Ca +, Mg +, Fe +, Se+…
I rease i ro iologi al di ersit of the soils
Pseudomonassp., Bacillus subtilis
I pro e the root de elop e t i parti ular root le gth
Redu e da agi g effe ts of pathoge s a d pesti ide
treat e ts effe t a idi pH a d a tio ida ts

10. L A N D L A B
Drought Stress
La dla Resea h “tatio
Qui to Vi e to
Ital
D . Ad ia o Altissi o
Rossella Bo tolaso
T ials ea

11. - “oil: % sa d + % la dla
- epli ates / plot . – “tats ANOVA
- Lolium perenne & Festuca rubra
- 0 % (high stress), 25% (medium Stress) and 50% (low stress)
of Evapotranspiration (water restitution using sprinkler)
Drought Stress
E peri e tal Setup:
Sprinkler for irrigation
- Spraying of 0 (control), 30 and 60 litres AG200 every 3 weeks
Para eters Assessed:
- % Li i g G ou d Co e LGC usi g Digital I age A al sis / eek
- Visual Tu f Qualit TQ afte ea h ut
- Chlo oph ll o te t efo e e e ut
Chlorophyll Meter (CM)- Che k of the soil hu idit / eek

12. Drought Stress
d Mai Out o e: R eg ass Lolium perenne – Be efits of phylgree
Co t ol
L/Ha
-As the stress i rease,
the TQ is de reasi g
.
.
.
.TQ(TurfQuality)
Ryegrass -Turf Quality at high drought stress

13. Drought Stress
d Mai Out o e: R eg ass Lolium perenne – Be efits of the phylgree
Co t ol
L/Ha
As the st ess i ease,
the Chlo oph ll o te t
is de easi g
. . . . . . . . . . . . . . . . . .
CM
Ryegrass - Chlorophyll Measure (CM) at high drought
stress

14. Drought Stress
rd Mai Out o e: R eg ass Lolium perenne – Be efits of the phylgree
Co t ol
L/Ha.
.
.
.
.
.
.
.
.
-“ep -“ep -O t -O t -O t -O t -No -No
% Dr Pat hes o R egrass after phylgree treat e t
i high drought stress
Co t ol
15 L/ha

15. Drought Stress
Co trol 15 L/Ha
R eg ass Lolium perenne – % ate estitutio ediu st ess – e d of t ial De e e
.9 .
. .
. .
% Gree
Chlo. M.
% D

16. Meloidogyne minor
Nematodes
“ pto s o a g ee …
Pi tu e ou tes of D . Coli Fle i g

17. RESULTS RELATION TO NEMATODES i e peri e ts ith AFBI,
I ease of Ne atode ates i o t ol a d significant de ease i Ne ati ide
Mai Co lusio s:
AG pu e a d AG Roots a e li iti g de elop e t of e atodes at ost of appli atio s
Effe t of o petitio et ee the i o ial life a d Ne atodes
Nematodes

18. Start of AutuStart of Spri g
Application:
Small doses but frequent
“t ess
e ha i al
“t ess
heat / ate
Possi le “t ess
Fu gi
“t ess
F ost
Possi le
“t ess A so ptio
N, P
Oligo elts
K
AG Hu i s
AG Pu e
Use as ase produ t for a foliar spra i g – i ith other produ ts i parti ular fu gi ides

19. A atu al i of Biosti ula ts
Biostimulant
Type/Familly
Active
compounds
Main
Origin
Action
Plant
Growth
Regulators
(PGR)
Plant hormones
Amino Acids
Humic acids
Sugars (Carbon)
Bacteria
Synthetic
Synthetic
Natural
Natural
Natural
Enhance development
of key phases:
Roots, leaf, flowers…
(direct or indirect)
Plant Defense
Stimulators
Auxines
Glutamic acid
Oligosaccharides
Synthetic
Both
Natural
Direct stimulation of
Plant defense system
Osmoregulants
Antioxidants
Glycin Betain
Mannitol
Salicylic acid
Vitamins, tannins
Natural
Natural
Both
Both
Help plant regulate
Water inside/outside
Neutralise free radicals
Biofungicides
Biopesticides
Trichoderma sp.
Garlic, Mustard…
Natural
Natural
Compete with pathos
Act on pest physiology
Seaweed
Cold Extracts -
Plant
Hormone like (+) ,
A.A. (+),
Sugars (+++)
Auxins (+),
Glutamic Acid (+)
Oligosaccharides (+++)
Mannitol (++),
Vitamins (+,
Tannins (++)
In addition:
Alginates (- charged) =
surfactant & water retention

21. Here are a few important effects of amino acids:
•Increase chlorophyll production
•Provide rich source of organic nitrogen
•Stimulate synthesis of vitamins
•Influence various enzymatic systems
•Higher brix level (quality increase)
•Increased pest and pathogen resistance
•Improve stress resistance

22. Plants are “autotrophic” or in other words, they make their
own food.
Plants take up nitrogen through the roots predominantly in
the form of nitrate ions. The plant cells then convert the
nitrogen into amino acids, usually in the form of glutamic
acid.
The glutamic acid is then transported throughout the plant
and rebuilt into all of the other amino acids.

23. Some amino acids can also be transported directly into
the plant through the plant roots.
For example, the amino acid “tryptophan” is absorbed
through the roots, transported upward to the leaves, then
changed into the natural growth hormone “auxin”.
The auxin (IAA) is then pumped to the growing tips to
stimulate the new growth of roots and shoots. On the
other hand, plants also produce their own tryptophan.
Some of the extra tryptophan is leaked from the roots to
feed beneficial microorganisms that live on the surface of
the roots. Amazingly, some to the microorganisms also
change the tryptophan into IAA, and directly stimulate root
growth.

24. L-amino acids are produced through a process called
“enzymatic hydrolysis”.
As bacteria grow and reproduce, they exude digestive
enzymes so large molecules such as proteins are broken
down into smaller molecules such as amino acids.
The amino acids are then used as a carbon and nitrogen
source for the microorganisms. The amino acids produced
through enzymatic hydrolysis are left-handed molecules
and they are biologically active.
On the other hand, synthetic amino acids, produced
through acid or alkaline hydrolysis, are right-handed
molecules and they are not biologically active. So when
purchasing amino acids for plant growth, make sure
that they are L-amino acids.

25. In the following example you can see that for example
Isoleucine is only 11% free, Glutamic Acid only 25% free
even though the label states total amino acid contents of
0.9 and 5.2 respectively. It’s the free amino acid content
which is important.

26. Free Total % Free
Alanine 2.94 5.4 54
Arginine < 0,05 0.5 1
Aspartic acid 0.87 1.6 54
Glutamic acid 1.30 5.2 25
Glycine 5.56 12 46
Hydroxylysine 2.1 0
Hydroxyproline 1.12 3.5 32
Histidine < 0,05 0.4 12.5
Isoleucine 0.11 0.9 11
Leucine 0.45 1.8 25
Lysine 0.53 1.7 31
Methionine 0.22 0.7 31
Omithine 2.5 0
Phenylalanine 0.37 1.2 30
Proline 2.08 6.9 30
Serine 0.18 0.3 59
Threonine 0.25 0.5 49
Tyrosine 0.38 1.2 32
Valine 0.29 1.6 18

27. We don’t compare Amino Acids contents individually as it
becomes a beauty content, I have more X than your Y
Amino Acid. I have more A than your B Amino Acid. As
stated previously it’s the free Amino Acids content is
what matters.
- The L-a free amino acids content is 40% Delfan (w/w
basis) with many competitors being less refined and less
plant available than Delfan. That’s what to focus on

28. Many products are misleadingly labelled in relation to
content on the label. So quoting Alanine as 5.4% and
Glycine 12% is irrelevant as they are in protein form (long
chain) and not plant available they will wash off the leaf in
first rain or irrigation and end up in the soil.

29.  Free amino acids – content of amino acids that can be
used fast by the plant
 Total amino acids – content of free and peptide amino
acids. Only part of the total amino acids can be used
 Peptides - short amino acid strains with two or more
amino acids
 Proteins - long chains of amino acids folded in a
special way. Proteins have important functions in plant
metabolism
Definitions

30. Delfan Plus is extremely high concentrated in free amino
acids, 24%. Products based on enzymatic hydrolysis
have a lower content of free amino acids
Delfan Plus – technical specifications
PHYSICAL-CHEMICAL CHARACTERISTICS
Aspect: Liquid
Colour: Brown
Density: 1,2 g/cc
pH: 7,2
CHEMICAL ANALYSIS
Parameter Guaranteed value (% w/w) Admitted deviation*
Free aminoacids** 24,00% -1,20
Total nitrogen (N) 9,00% -0,90
Organic matter 37,00% -3,00
Organic carbon 23,00% -2,00

31. The importance of the aminogram
Delfan Plus aminogram
Histidine
Threonine
Methionine
Tryptophane
Valine
Arginine
Serine
Lysine
Phenylalanine
Isoleucine
Leucine
Aspartic acid
Tyrosine
Hydroxyproline
Proline
Alanine
Glycine
Glutamic acid

32. 0
2
4
6
8
10
12
14
freeAAcontent[%w/w]
Vegetable aminogram vs. Delfan Plus
Typical vegetal AA from soybean and wheat Delfan Plus
No Hydroxiproline
and little proline
Differences in aminograms

33. The importance of Proline and Hydroxiproline
 Proline is an excellent anti stress agent and leads to tolerance to
a wide range of abiotic stresses
 Proline is especially important during osmotic stress (drought,
salinity and freezing)
Proline leads to:
 Osmotic adjustment (Compatible osmolyte)
 Stabilized cellular structures
 Prevention of cell damage due to free radicals (Radical
Scavenger)
 Provides energy needed for recovery

34.  Protein synthesis
 DNA synthesis
 Precursors of hormones
 Precursors of other functional molecules
 Stress metabolism
Functions of amino acids in plants
Amino acids are the building blocks of life
Without amino acids there is no life

35. Functions of amino acids
Amino acid Effect or function
All AA Protein synthesis
Glycine DNA synthesis, Alcaloid metabolism
Glutamic acid Chlorophyll synthesis
Tryptophane Auxin and phytoalexin precursor
Methionine Ethylen and polyamine precursor
Aspartate, glutamine and
glutamate
N and C storage amino acids, transport amino
acids
Proline stress metabolism, flowering
Serine Precursor glycine betaine, stress metabolism
Alanine
Precursor of certain antibiotics in some
species
Leucine, Lysine, tryptophane,
histidine, phenylalanine, tyrosine
and glycine
Alcaloid metabolism, plant protection against
pests and stress
Phenylalanine
Salicylic acid production, stress and disease
prevention
Tyrosine Glucosinolate precursors “Phytoanticipins”

36. Methionine
Arginine
Tryptophane
Rest
Delfan Plus content of hormone precursor amino acids

37. Serine
Proline
Rest
Osmolyte content of Delfan Plus

38. 34.5
35
35.5
36
36.5
37
37.5
Control Delfan
Yield[T/ha]
Yield of potatoes after Delfan applications of 1.5L/ha
compared to standard in a dry year
Delfan effect on potatoes during dry conditions

39. Delfan and stress prevention
 Delfan contains osmoprotectants proline,
hydroxiproline and serine
 Better tolerance to saline conditions
 Protection against dry spells
 Enhanced cold hardiness
 Enhanced yield

40. Evaluation of Creeping Bentgrass (Agrostis stolonifera L.) Responses to
Foliarly Applied Branched-Chain Amino Acids
I.T. Mertz, N.E. Christians
Department of Horticulture, Iowa State University, USA
Introduction
The branched-chain amino acids (BCAA) leucine, isoleucine, and
valine are synthesized in plants and are essential to growth in most
organisms. Research has shown that when foliarly applied, these
compounds can be absorbed by the plant, however, plant catabolism
of BCAA is not completely understood. Since the BCAA compounds
contain nitrogen in their chemical structure, they could potentially
be used an organic nitrogen source in plants. In previous research,
the application of a BCAA containing solution to creeping bentgrass
(Agrostis stolonifera L.) putting greens resulted in increased plant
shoot density compared to applications of mineral nutrition only,
indicating a potential benefit to their use. However, the BCAA and
nitrogen (N) concentrations in that product led to in some cases,
excessive product application rates that did not make its use
economical. Following a metabolomics analysis of the product,
efforts were made to improve the viability of its use.
Primary Objective
Evaluate and compare creeping bentgrass responses to foliarly
applied BCAA versus applications of equivalent mineral nutrition
only.
Materials & Methods
 Completely randomized design with 15 replications
 Creeping bentgrass ‘007’ plugs taken from field and grown in
9.5-cm diameter pots in greenhouse
 Treatments were applied on a 14-d interval and were based on an
equal rate of N (3.4 kg N ha-1). All samples received an additional
application of urea at 3.4 kg N ha-1 halfway through each 14-d
treatment interval.
 All treatments and additional fertilizer applications were applied
foliarly, with irrigation being withheld until 24-h post application.
 Supplemental radiation was provided when day-time irradiance
dropped below 200 µmol m-2 s-1 to ensure a consistency of 16
hours of light per day, and ranged from 350 to 385 µmol m-2 s-1.
 Air temperature ranged from 22.3 to 23.6°C
 Relative Humidity ranged from 24.3 to 44.7%
 Samples were mist watered with 2 ml, 4 times daily, until rooting
occurred
 At the point samples had established roots, watering was
transitioned to 2.54-cm per week
 A total of 4 treatment applications occurred during the duration of
the study
 A light-box was used to record weekly digital images of each
sample in order to track growth and spreading rates.
 Samples were harvested 8 weeks after transplanting, or at 42-d
following initial treatment.
 Measurements included rooting biomass (g), above-ground shoot
weight (g), shoot density (# shoots 2.85-cm-2), and area of
coverage through digital image analysis (DIA)
Treatment List
ImageJ Calibration
ImageJ was used to track the area of coverage/growth rate of
each sample. Weekly pictures taken with a light-box were used
for DIA. While there is a plethora of literature over turfgrass and
DIA, the majority of that work involves the use of pictures taken
in the field. This study attempted to use DIA on samples grown in
a controlled environment. For this study, the color threshold of
each image was adjusted to include only green pixels. The
threshold ranges used for this study included Hue (0-97),
Brightness (92-255), and Saturation (110-255). While these
numbers differ from those in the literature, the calibration curve
below illustrates their capability for our application. After
calculation of the area covered in cm-2 by DIA software, this
number was entered into the calibration equation, and then
divided by the pot surface area (71.25 cm-2) in order to calculate
the percent cover of each pot.
Contact:
FIGURE-5: Calibration curve generated based on actual area of turfgrass plug
with known measurements vs amount of area calculated by ImageJ using the
parameters stated above.
Isaac Mertz
Iowa State University
Department of Horticulture
Ames, IA, USA 50011
imertz@iastate.edu
Dr. Nick Christians
Iowa State University
Department of Horticulture
Ames, IA, USA 50011
nchris@iastate.edu
Results/Conclusions:
At trial end (42-days), plants that received applications of leucine, isoleucine, and valine in a 4:1:1 ratio exhibited a
37% and 27% increase in rooting and shoot density respectively, compared to those receiving urea only. When
applied in a 2:1:1 ratio, those increases were less pronounced (18% and 13.5% increase in rooting and shoot
density respectively, compared to urea only). This indicates that in terms of plant performance, BCAA may be
suitable for the substitution of urea when fertilizing creeping bentgrass. Shoot density of creeping bentgrass is of
the upmost importance, and can be directly related to playing surface quality. Due to the increases in shoot
density observed, these results also show the potential increased benefits of including an organic source of
nitrogen in a fertilizer program, however, further research is needed in order to fully understand plant catabolism
of BCAA.
While the potential benefits are indicated in this study, further research needs to be done. Future studies will
focus on the use of isotopic forms of the BCAA, as well as their effect on growth under less optimal conditions.
This work will hopefully lead us to a better understanding of BCAA catabolism by plants, furthering our knowledge
of the turfgrass plant
Treatment Product
Rate
(kg Product/ha)
Rate
(kg N/ha)
1 Control - -
2 Urea 7.4 3.4
3 BCAA (2 : 1 : 1)* 15 : 7.5 : 7.5 3.4
4 BCAA (4 : 1 : 1)* 20 : 5 : 5 3.4
Masks Generated During ImageJ Calibration
0% Coverage 20% Coverage 40% Coverage
60% Coverage 80% Coverage 100% Coverage
Weekly pictures of each sample were taken with a light-box in order to track the
growth/spreading rate in each pot, these pictures were used for DIA in ImageJ.
These pictures illustrate the process each image had to undergo in order for the
ImageJ software to quantify the number of green pixels of each sample.
FIGURE-3: Creeping bentgrass shoot density (shoots-1 2.85-cm-2) responses to treatment
application. Data next to treatment labels are statistically similar at α = 0.05.
FIGURE-4: Creeping bentgrass average growth responses of each treatment overtime, in
terms of the percent area being covered by green vegetation in each pot. Dates with * or **
indicates a significant difference among treatments at p ≤ 0.05 and ≤0.0001, respectively.
FIGURE-2: Root biomass responses of creeping bentgrass to treatment
application. Bars sharing the same letter are statistically similar at α = 0.05.
FIGURE-1: Above-ground shoot weight responses of creeping bentgrass to treatment
application. Bars sharing the same letter are statistically similar at α = 0.05.
Treatment
Control Urea BCAA (2:1:1) BCAA (4:1:1)
Above-GroundShootWeight(g)
0.0
0.1
0.2
0.3
0.4
0.5
0.365b
0.396ab
0.404ab
0.440a
LSD = 0.049
* BCAA Treatments consisted of L-valine:L-leucine:L-isoleucine
Treatment
Control Urea BCAA (2:1:1) BCAA (4:1:1)
RootingBiomass(g)
0.0
0.1
0.2
0.3
0.4
0.5
0.277b 0.276b
0.325ab
0.378a
LSD = 0.087
Calculated Area (cm2
)
0 20 40 60 80
AreaCalculatedbyImageJ(cm
2
)
0
10
20
30
40
50
60
70
y = 0.8032x + 2.7481
R2
= 0.9644
Days After Initial Treatment
0 7 14 21 28* 35* 42* 49* 56*
PotPercentCover(%)
10
15
20
25
30
35
40
Control
Urea
BCAA (2:1:1)
BCAA (4:1:1)

42. A liquid organic nutrient complex containing
salicylate derivates to boost the immune
response of the plant.
Alexin can safely be included in a disease
management programme and is highly tank
compatible.

43. Salycilic acid
Plants manufacture salicylic acid to trigger
natural defences against disease.
The addition of Salycilic acid acts as
an activator of ‘Systemic Acquired Resistance’
(SAR). However, plants often don’t produce
the acid quickly enough to prevent injury when
attacked by a pathogen and so spraying
Alexin enables the ability of the plant to fight
against infection.

44. Plants have never been totally defenceless
against disease attack. The issue has been
that often plants do not recognize their microbial
attackers in time.
Spraying salicylic acid in the form of Alexin
puts their defences on high-alert against future
attacks.

45. Research has found:
• Salt stress (salty water) (Borsani et al., 2001).
 improved plant tolerance to heat on
creeping bentgrass
(Larkindale and Huang, 2004)
• Improved heat tolerance of Kentucky bluegrass