Biostimulants for Putting Greens - PowerPoint Presentation

Slide1

Biostimulants for Putting Greens

Erik H. Ervin, Ph.D.Professor, Turfgrass Culture & PhysiologyCrop and Soil Environmental Sciences Department; eervin@vt.eduVIRGINIA TECH

November 29, 2017,

MoGICSlide2

Schmidt and Ervin definition

A biostimulant is an organic material that, when applied in small quantities, enhances plant growth and development such that the response cannot be attributed to application of traditional plant nutrients, GCM (2002)Slide3

European Biostimulants Industry Council

www.Biostimulants.eu: 2014“Agricultural biostimulants include diverse formulations of compounds, substances and micro-organisms that are applied to plants or soils to improve crop vigour, yields, quality, and tolerance of abiotic stresses.”

“Biostimulants operate through different mechanisms than fertilisers, regardless of the presence of nutrients in the products; they do not have any direct action against pests.”Slide4

Agricultural Uses of Plant Biostimulants

Calvo et al., 2014. Plant and Soil 383:3-41.Microbial inoculantsHumic & fulvic

acidsProtein hydrolysates & amino acidsSeaweed extractsWe’ll review research data from 3 categories with a focus on improved efficiency and tolerance to abiotic stressSlide5

Microbial inoculants

Products containing living organisms that promote growth by several mechanisms such as increasing supply of nutrients, increasing root biomass or area, and increasing nutrient uptake capacity of the plantFree living bacteria, fungi, and arbuscular mycorrhizal fungi (AMF) that were isolated from environments such as soil, plants, plant residues, water, and processed manuresSlide6

Microbial inoculants, considerations

Microbes must survive in the commercial formulation; shelf life, temperature effects, UVAre microbes compatible with fertilizers and crop protectants?Slide7

From AAPFCO talk, OR Dept Ag

Serial dilution plating on growth agar of various microbial inoculant products, 2014Bacillus (9 M cfu/ml) on label; no detectionBacillus (35 M cfu/ml) on label;

3000Bacillus (787 K; 5000 detected), Pseudomonas (24 K; no detect), Trichoderma (2.4 K, no detect)Summary: 2 out of 21 formulated products met label guarantee; all numbers dropped while on shelf for 3 months; dry slightly better than liquidSlide8

Microbial inoculants, mechanisms

Nitrogen fixationNutrient solubilizationFe-sequestration via siderophore productionProduction of volatile organic compounds

Production of plant growth hormonesSlide9

5. Microbial inoculants, phytohormones

Production of plant growth regulators (hormones) by bacteria has been reported for over 30 years (Barea et al., 1976).Auxins, cytokinins, gibberellins, and ethylene can be synthesized by rhizosphere bacteria effecting many physiological processes such as root initiation, root elongation, and root hair formationSlide10

Cytokinins

Promote cell division at meristemsIn combination with auxin they regulate ratio of tillering to root initiationRetard senescence and chlorophyll degradation = “Stay-Green” effectBingru Huang’s group at Rutgers has transformed bentgrass (ipt gene) to activate cytokinin synthesis when exposed to heat stress (> 90 F). From: Proc. PGRSA Annual Meeting, 2006.Slide11

Stay-green mutant of corn due to maintenance of high leaf cytokinin levels

Loss of ACC oxidase expression stops ethylene’s effects on cytokinin degradation and senescence is delayed under drought (above) and light deprivation (below)

From: D. R. Gallie, Biochemistry, UC-RiversideSlide12

Root-injection of synthetic cytokinin improved bentgrass heat stress tolerance

Adapted from Liu et al., 2002. Cytokinin effects on creeping bentgrass responses to heat stress. Crop Sci. 42:457-472.

Means are from 56 d of heat treatments and 56 d after Zeatin Riboside (ZR) injection into the root zone; All means are sig diff. at P=0.05Slide13

Adapted from Liu et al., 2002. Cytokinin effects on creeping bentgrass responses to heat stress. Crop Sci. 42:457-472.

Means are from 56 d of heat treatments and 56 d after Zeatin Riboside (ZR) injection into the root zone; All mean comparisons are diff. at P=0.05

Root-injection of synthetic

cytokinin

improved bentgrass heat stress toleranceSlide14

Auxin or IAA

Produced in new leavesCell division & Stem elongationInduction of rootingIn combination with cytokinins they regulate ratio of tillering to root initiation and growthBending of shoots toward light, roots towards gravityIAA overproducing mutants of Arabidopsis are known as “rooty” and “superroot”

Rogg & Bartel. 2001. Developmental Cell. 1(5):595Slide15

Microbial production of

Auxin in Biosolids

Organic matter

Tryptophan

Auxin

Microbial actionSlide16

Auxin Level in BiosolidsDetermined using

BioassayLC/MS/MSBiosolids* contains:

2.1-15.4 ppm

auxin

(average: 8.8 ppm)

*Zhang, Ervin,

Evanylo

, and

Haering

, 2009

Auxin-

13

C

AuxinSlide17

Picture from initial biosolids trial showing improved TF drought resistance due to biosolids-amendment relative to N controlsSlide18

Auxins (IBA drench or in boosted biosolids) improved tall fescue rooting

Drought

Control auxin-biosolids IBA-control Biosolids

A

A

B

B

b

ab

b

aSlide19

Total KBG Root Dry Weight (g/pot) Slide20

Univ of Wisconsin biostimulant research

Objective: determine how certain commercial biostimulants might alter the microbial community in a mature sand-based bentgrass greenTested: Flexx-Plus®

(ben. bacteria = Bacillus sp @ 11 billion cfu/lb)Colonize® T&O (ben. bacteria = Bacillus sp @ 45 billion cfu/lb)

Applied every 2 wk from late May through Aug.

Soil samples taken every 2 wk = 10 sample dates

Kussow, HortScience, 2006Slide21

Authors’ Conclusion: “The treatments did not effectively alter the putting green microbial community in terms of enzyme activities or substrate utilization.” Observed improvements in quality/wilting resistance may have been due to surfactant or SWE in products

Kussow, HortScience, 2006

NS

NS

NS

NS

*Slide22

Humic SubstancesSlide23

Humic Substances defined

Humic substances comprise 60-80% of soil OM and are the components of OM most resistant to microbial decompositionClassified into:Fulvic acid: smallest & soluble in acid & alkaliHumic acid: medium & soluble in alkali

Humin: largest, most persistent, dark, insolubleSourcesLeonardite (mined soft coal, lignite): 30-80% HA, 150 CECPeats: 5-25% HA, Biosolids/Compost: 3-15% HA Slide24

Liquid-applied Humic Substances

At 0.05 to 0.10% HA concentrations have been shown to mimic auxin in promoting root growth.* Are non-humic components (saccharides, organic acids) the biologically active fraction?

HA and FA also chelate Fe, Mg, Zn, Mn in exchangeable forms for more efficient root availabilityNo soil moisture or structural effects predictedVT Research rate is 15 g leonardite/M = ~1.5 lb/A = 0.4% concentration applied in our trials

*O’Donnell, 1973. Soil Sci. 116(2):106-112Slide25

Humic Acid has been shown to increase photosynthesis and root growth

In a greenhouse experiment, Crenshaw bent was grown in solution culture supplied with non-limiting nutrients (no stress) and 3 concentrations of HA.Photosynthesis and root growth was increased significantly from 1 to 4 weeks after treatment by HA at 400 ppm (0.0004%)

VT foliar spray rate ~5000 ppm

Liu, Cooper, Bowman. 1998. HortScience 33:1023-1025Slide26

KBG sod establishment on sand

Two humic sources compared against fertilizer only; fertilizer inputs equalizedPeat based versus leonardite, both applied at 3 oz/M every 2 wks for 6 applications

Table 4. Humic acid (HA) effects on monthly post-transplant root strength measurements of Kentucky bluegrass

Treatment

Root Strength

22 May 2002

21 June 2002

22 July 2002

------------------------------ kg m

-2

------------------------------

Control

367.4a

483.7a†

470.1a‡

Leonardite HA

401.1a

532.6a

587.6b

Peat HA

430.7a

600.5b

593.0b

Root mass

Treatment

--------

mg cm

-3

--------

Control

0.60a‡

Leonardite HA

0.85b

Peat HA

1.06b

34 and 73% transplant rooting increaseSlide27

KBG sod establishment on sand

Ervin et al., 2005. Acta HortSlide28

3. Seaweed ExtractsSlide29

Kelp or Seaweed Extracts (SWE)

Why do they tend to be in every biostimulant?Where do they come from and what’s in them?N. Atlantic rockweed=brown algae=Ascophyllum nodosumSlide30

Composition of Ascophyllum nodosum

alkaline extract

ItemValuepH~10

Carbohydrates

~50%

Amino Acids

~5%

Nitrogen

0.8 to 1.5%

Phosphorus

0.5

to

1.0%

Potassium

14

to 18%

Calcium

0.3 to 0.6%

Sodium

3.0 to 5.0%

Micronutrients

1 to 250 ppm (Fe)

Source: Acadian

Seaplants

Limited, Dartmouth, Nova Scotia, 2012 Slide31

Reported Cytokinins and Auxin Contents in Seaweed Extracts (SWE)

Author, year

Cytokinins level, DWMethod

Tay

,

1985

7 µg/L

GC/MS

Sanderson, 1986

1.3 µg/L

GC/MS

Tay

, 1987

13 µg/L

GC/MS

Stirk

, 2004

50 µg/L

Plant bioassay

Ervin, 2004, 2008

70 & 45 µg/L

ELISA

Ervin, 2009

12 µg/L

LC/MS/MS

Wally, 2013

10

µg/L

LC/MS/MS

Auxin (IAA) level

Ervin, 2009

12

µg/L

LC/MS/MS

Wally, 2013

3 to 47 µg/L

LC/MS/MS

Cytokinins

level found in

bentgrass

leaf tissue 5 to 60 ng/g FW*

*Zhang and Ervin. 2004, 2008, 2010.

C

rop Science Slide32

Scientific Consensus on SWE

“The chemical compositions of several seaweed extracts are known, and because they can maintain plant-promoting bioactivity at relatively low concentrations (<0.01% w/v) (Crouch van Staden, 1993), it is unlikely that the growth-promoting ability is due to nutrient composition alone (Wally, 2013).”Similar statements in Blunden 1972, 1991 and Khan et al., 2009.

Blunden, G. 1972. Proc Int Seaweed Symp 7:584-589.Blunden, G. 1991. Seaweed Resources in Europe: uses and potential, pp. 65-81.

Crouch, I.J. and J. van

Staden

. 1993. Plant Growth Regulation. 13:21-29.

Khan W., et al., 2009. J Plant Growth Regulation 28:386-399.

Wally, O.S.D., et al., 2013.

J Plant Growth

Regulation 32:324-339.Slide33

Ervin SWE Research

Products used primarily on putting greensEqualized nutrient inputs, HoaglandsFew commercial products used, just generic alkaline seaweed extracts at 0.15% w/vChallenged bentgrass with drought or heat stressSlide34

Drought in Greenhouse

0.15% w/v SWE solution (estimated 3.5 µg/L cytokinins) or ashed SWE applied 7 d prior to 28-d dry down; complete fertilizer solution supplied evenlyLeaf Cytokinin Relative Levels at Soil Moisture Wilting Point (5%):

Exp. 1 then Exp. 2Control = 100% 100%Ashed SWE = 101% nsSWE = 123% sig 158% sigZhang and Ervin, 2004. Crop Science, 44:1737-1745Slide35

Drought in Greenhouse

We also tested Humic Acid (HA at 0.43% w/v) from leonardite, alone and in combination with SWE

Treatment

PE*

Vit

. E

Shoot

Wt

Root

Wt

CK

HA

0.33b

17.3b

1.28ab

0.80a

19.3b

SWE

0.30b

14.3b

1.46a

0.69b

28.9a

HA + SWE

0.40a

23.7a

1.49a

0.83a

26.6a

Control

0.23c

10.7c

0.96b

0.59b

18.3b

*PE = Photochemical Efficiency (0.7 = healthy); Vitamin E; CK =

Zeatin

riboside (

cytokinin

) levelSlide36

Roots left after 28-d dry downSlide37

Ascophyllum nodosum extracts

testedAcadian Seaplants (Dartmouth, Nova Scotia)ASL liquid concentrate: KOH extracted, 14.4% solids

Applied to foliage at 3.5 mg ZR/L = 28 mL SWE/L = 10 µmol ZR = 1.3 lb solids/acre =

3.3

oz

/M

Ocean Organics (Waldoboro, Maine)

Liquid concentrate:

KOH extracted, 8% solids

Applied to foliage at 3.5 mg ZR/L = 28.2 mL SWE/L

= 10 µ

mol

ZR

=

0.75

lb

solids/acre =

3.3

oz

/M

Zhang and Ervin, 2008.

Crop Sci. 48(1):364-370.Slide38

Procedures

L-93 creeping bentgrass grown from seed to maturity in conetainers in a 75/68 F greenhouse.1st SWE treatment foliar-applied 7 d prior to moving to 95/77 F growth chamber

Conetainers suspended in ¼-strength Hoagland’s solution (aerated and changed weekly)2nd SWE treatment applied 14 d after heat stress beganWhole conetainer subsamples were taken destructively every 7 days (for 49 days) to quantify responses over timeSlide39

2

nd

app

NS

b

a

a

a

ab

b

a

a

b

b

b

b

b

a

a

Bars with the same letter are not diff at P=0.05Slide40

NS

NS

a

b

a

ab

b

b

Bars with the same letter are not diff at P=0.05

b

b

b

abSlide41

End of 2006 trial, 49 days of heat stress

Check Aca SWE OO SWE CK-check ashed-SWESlide42

Bentgrass Heat Stress Study:

We investigated 5 rates of SWE against 5 equivalent rates of pure cytokinin (zeatin riboside, ZR) against 1 True Foliar program at 95-100 F day/ 77-82 F night and 70-80% RHTreatments (4 apps @ 2

wk frequency)1: Fert Control = 0.05 lb N/M solution of 20-20-202-6: SWE at 0.1 1, 10, 100, 200 uM ZR (or 0.028, 0.28, 2.8, 28, and 56% solutions)

7-11: Pure ZR at 0.1, 1, 10, 100, 1000

uM

ZR

Treatments 2-11 also mixed with 0.05

lb

N/M 20-20-20

12: CPR (3

oz

) + True Foliar K (1

oz

), T-F NK (4

oz

) + T-F Ca (1

oz

) =

same SWE and

fert

inputs as 2-4

Slide43
Slide44

RESULTS (Zhang and Ervin. 2010. Crop Sci. 50:316-320)

The 2.8% (10 uM=3.5 ppm CK) SWE and ZR solutions worked better than the weaker ones but the same as the 28% solutionsThe 200 and 1000 uM solutions caused phytotoxicity

The True Foliar program mix (= 2.1% SWE solution) worked the best Slide45

Photochemical EfficiencySlide46

8 weeks at 95 F day/77 F night, treated every 2

wk

with 0.05

lb

N + SWE or CK

Fert Control 10 uM ZR 10 uM ZR from SWE True Foliar mixSlide47

Effects of Commercial Products Containing

Bacillus

spp

.

plus

Biostimulants

on an

Agrostis

stolonifera

Putting Green

Erik Ervin* and X. Zhang, Virginia Tech

M. Accorsi, D.

DiGioa

, G.

Dinelli

, University of Bologna

Sponsored by Lebanon Turf Products, USASlide48

Background, Turf Biostimulants

A 3-0-20 liquid fertilizer, with micronutrients, plus

22 million

cfu

/g of

Bacillus

spp.

Humic

acid, 3%

Seaweed extract, 4.1%

7.2%

maltodextrinSlide49

Background, Turf Biostimulants

Contains no

humate

, seaweed extract, or

maltodextrinSlide50

Background, Turf Biostimulants

Contains humate as fulvic acid, seaweed extract, but NO

maltodextrin or BacillusSlide51

Bacillus as a Plant Growth Promoting

RhizobacteriaNo direct turfgrass research found, but many reports for other crops that Bacillus produce auxin and cytokinins in the rhizosphere and increase plant fitness

Our objective was to compare Bacillus turf fertility products, containing seaweed extract + humate or not, for improved summer putting green healthSlide52

5 treatments, 4 Reps, RCBD, Spray applications (400 L/ha) every 14 d, 4 total over 60 d trial (June-July); Primarily ‘Penncross’ creeping bent/ Poa on USGA sand; N equalized at 0.1 lb/M/14d

Fert

control

Fert

control + MD

Complete

Bacillus-only

SWE+FA

Temperature was normal: 80 F/60 F

2x normal rainfall: 8 inches

Fungicides applied:

chlorothalonil

(3x);

trifloxystrobin

(1x)Slide53
Slide54

At trial end, Photosynthesis increased due to Complete and Bacteria-onlySlide55

Summary and Conclusions

Small, but significant, increases in summer putting green health (rooting, PS, NDVI) were measured due to SWE+humate and Complete treatmentsNo consistent correlation to Bacillus products, though 3 Bacillus species found in rhizosphere at trial endMust recruit microbiologists into more turf research for progress to be made:

$Slide56

4. Amino AcidsSlide57

20 essential amino acids

Nitrogen is stored and transported in amino formsProteins are chains of 100’s of amino acids, usually containing all 20 in their chains Slide58

What about foliar amino acid effects?

Why might a benefit be expected?They most likely can be absorbed by leaves = smaller than sucroseThey may conserve carbohydrate during summer stress as carbohydrates are broken down in respiration to produce chemical energy (ATP and NADPH) to make amino acids…then proteins Good candidates for formulations?

Glutamine: initial product in AA production cycle; its level controls need for more or less N-assimilationTryptophan: auxin precursor = root initiationProline

:

primary

osmoregulator

for drought protectionSlide59

What are the barriers to nutrient absorption through the leaves ?

Primary ports of nutrient entry are:Stomates (20,000/cm2

leaf) Tiny pores in cuticular wax (10 billion/cm2 leaf)

Nutrient absorption occurs by:

Diffusion

Active transportSlide60

Absorption is at its peak when stomates are fully open

Factors such as salinity, heat, and water stress will cause stomates to close

Stomate openings are about 10 µm wide

Cuticle pores are 1 nm or less = 1000 times smaller than stomate openings, but there are 10

6

more

Urea (COH

4

N

2

) is 0.44 nm in size; small enough to move through culticle pores

Slide61

Sizes of molecules to go through pores

Sucrose = 1 nm, mol. wt. = 342Glucose = 0.6 nmVarious inorganic ions 0.5 to 1 nm in their hydrated formsAmino acids have mol. wt. of 75 to 204; they should pass through cuticular pores Slide62

Bentgrass foliar uptake of N sources

Stiegler

& Richardson,

UArk

, 2008Slide63

Amino acid dosing trial

L93 bentgrass with complete nutrient solution: 0.25 lb N/M/month75 F on mist bench, 3 month trialTryptophan (3 mM/month)

Glutamine (3 mM/month)ControlGlutamine: initial product in AA production cycle; its level controls need for more or less N-assimilation = less root energy needed for nitrate uptake

Tryptophan:

auxin

precursor = root initiationSlide64

Results: Leaf ColorSlide65

Photochemical EfficiencySlide66

Leaf Protein content (mg/g FW)Slide67

Testing of glutamate-based foliar fertilizer product, end of drought stress responses

Treatment

ColorChl

Shoot wt

Root wt

Tillers

SOD

Exp1,

Glu

7.1 a

3.8 a

820 a

1.32 a

6.7 a

133 a

AS

6.0 b

3.3 b

923 a

1.08 a

6.1 a

128 a

No N check

3.5 c

1.6 c

428 b

1.06 a

3.8 b

120 b

Exp1,

Glu

and AS (Ammonium-sulfate) were applied to provide 0.06 lb N/M every 10 days for 50 days. L93 creeping

bentgrass

grown in USGA sand in pots, re-watered to 12% soil moisture every 2 days to maintain continuous moderate drought stress.

Sponsor: Ajinomoto USA, AmesSlide68

Salt stress: Aquaplex, 4 WAT

Watered twice weekly with 16

dS

/m salt-water; bent tolerance 3-6

dS

/m in soil

Aquaplex

applied at 4.5

oz

/M/14 d;

it is a mix of 16 amino acids and CaNO3

ACA 2786 + salt salt-alone water-control

=Aquaplex amino

Aquatrols

-sponsoredSlide69

Salt stress: Aquaplex, 8 WAT

ACA 2786 + salt salt-alone water-control

=Aquaplex amino

Watered twice weekly with 16 dS/m salt-water; bent tolerance 3-6 dS/m in soil

Aquaplex amino applied at 4.5 oz/M/14 d

Aquatrols

-sponsoredSlide70

Questions and Discussion