NUTRITION THERAPY IN CITRUS

Tripti Vashisth

Assistant Professor (Horticultural Sciences)

Plant Nutrition• Seventeen elements are essential

• Carbon (C), hydrogen (H) and oxygen (O), make up to 95% of tree biomass

Photosynthesis

Carbohydrates

Proteins Fatty acidsOrganic compounds

Minerals elements

• There are 14 essential mineral nutrients

Macronutrients Micronutrients

Nitrogen (N)Phosphorus (P)Potassium (K)

Calcium (Ca)Magnesium (Mg)Sulphur (S)

Iron (Fe)Zinc (Zn)Manganese (Mn)Boron (B)Copper (Cu)Molybdenum (Mo)Chlorine (Cl)Nickel (Ni)

Liebig's law of the minimumMineral Nutrition

Secondary Nutrients

Mineral Nutrition

Good Nutrition Management is Critical

Apple Bitter PitPhysiological DisorderDirectly related to CalciumAffected by Nitrogen, Potassium, and Boron

Good Nutrition Management is Complex

Relationship between fruit yields and N rates during 1993–1998 of >20-year-old ‘Hamlin’ orange trees on ‘Cleopatra mandarin’ rootstock planted in a Tavares fine sand. Alva et al. (2006). Relationship between SSC/TA ratio and N rates and

sources (A) and leaf N concentration (B), 1997– 2000 (SSC = soluble solid concentration and TA = titratable acid). Adapted from He et al. (2003)

Mineral Nutrition and Plant Defense

The effect of potassium nutrition on pest and disease resistance in plantsAnna Amtmann, Stephanie Troufflard, Patrick Armengaudhttps://doi.org/10.1111/j.1399-3054.2008.01075.x

HLB and Nutrition• HLB-affected leaves show blotchy mottle/chlorosis, often

show nutrient deficiencies

• Confused with nutrient deficiency and vice versa

Boron Zinc Potassium

Pen Test

Transmits bacteria

Root loss Water

+minerals uptake reduces

Phloem pluggingAsian citrus psyllid

feeds on leaf

Carbohydrates

To roots

To fruits, flower

Tree growth arrests and declineCLas thrives in

phloem

Carbohydrates

To roots

To fruits, flower

X

X

Model for HLB

Fruit quality lowers and fruit drop

HLB and Nutrition• Growers started nutritional therapy to address nutrient

deficiency symptoms

• Foliar spraysProc. Fla. State Hort. Soc. 125:61–64. 2012.

Fruit Production in a Southwest Florida Citrus Grove Using the Boyd Nutrient/SAR Foliar Spray

HLB and Nutrition

• The results were not consistent resulting in confusion

HLB and NutritionNutrition is a vast field:

• numerous products

• many chemical form, various analysis

• mature or young tree and often observational

• efficacy is dependent on various factors such as:

• method of application

• soil type and pH

• irrigation

• time of year

Citrus Grower Survey• June 2017

• Eight growers, spread throughout the state were surveyed-Growers who have claimed to be making profit

Every grower agreed that their grove management programs are more intensive

and better now then pre-HLB especially nutrition management.

Growers who had better nutrition management program pre-HLB and early on

adopted good psyllid control did not see as much decline as some other growers.

There is no fixed recipe for a management, programs are often site specific,

requires good observation and analyses and then responding to tree needs.

So what is happening?• Constant supply of nutrients is critical

• Focus on soil-applied nutrition

• HLB-affected plant have small root system, limiting nutrient uptake

• Reports suggest higher rate of nutrients is beneficial for HLB-affected trees

• The plant uptakes nutrients when they are in a solution

• During the water uptake by the plant, the dissolved mineral nutrients get

taken up by the plant and distributed throughout the canopy

• Mobile and immobile nutrients have equal and uniform distribution to all parts

of plant

Soil-Applied Nutrition Program

• Thick leaf cuticle limits the nutrient uptake

• Significant amount of foliar spray washes

away in soil:

• Pre HLB, trees had massive feeder root systems; therefore, could

easily take up washed up nutrients

• HLB-affected trees have few feeder roots therefore, may not be

effective in nutrient uptake

• With foliar sprays immobile nutrients can get locked in leaves

Foliar Nutrition Program

Current Research • Overdose-higher than recommended rate• Some concerns as the experimental design

was not robust• Nonetheless, there are increasing number of

evidence that HLB-affected trees benefits from higher rate of micronutrients

Study 1

Current Research• Micronutrient Field Trial

• Two locations: Fort Meade and Arcadia

• Valencia/Swingle; 10 to 15 year

• Completely Randomized Block Design

• Trial was initiated in February 2016 and will

end with 2019 harvest

• All the fertilizer treatments are applied 3

times a year by hand in the wetted zone

Study 2

Vashisth et al., Manuscript in preparation

Treatments1. Conventional granular fertilizer +

foliar

2. Conventional granular fertilizer + Tiger Micronutrient Mix

3. CRF + foliar

4. CRF + Tiger Micronutrient Mix

5. CRF + Tiger Micronutrient Mix + Tiger Mn elevated by 20%

6. CRF + Tiger Micronutrient Mix + Tiger Zn elevated by 20%

7. CRF + Tiger Micronutrient Mix + Tiger Fe elevated by 20%

8. CRF + Tiger Micronutrient Mix + Tiger B elevated by 20%

9. CRF + Tiger Micronutrient Mix + Tiger Mn and B elevated by 20%

10. CRF + Tiger Micronutrient Mix + Tiger Mn and B elevated by 50%

Study 2

Vashisth et al., Manuscript in preparation

Yield- 2019 (Boxes per acre)

444489 510 484 497

460 441470

551490

0

100

200

300

400

500

600

1 2 3 4 5 6 7 8 9 10

Boxe

s pe

r acr

e

Treatment #

Fort Meade

321

391442

497455 454 478

438385

483

0

100

200

300

400

500

600

1 2 3 4 5 6 7 8 9 10Bo

xes

per a

cre

Treatment #

Arcadia

bab

ab abab ab

abab a

ab

b

ab

aba ab

a a

ab

ab

a

Study 2

Vashisth et al., Manuscript in preparation

9081095 1088

12631136 1118

1310 1259

10481233

0

200

400

600

800

1000

1200

1400

1600

1800

1 2 3 4 5 6 7 8 9 10

Boxe

s pe

r acr

eTreatment #

Arcadia

893

1063 1047 1076 1039 1027913

9811130

1034

0

200

400

600

800

1000

1200

1400

1 2 3 4 5 6 7 8 9 10

Boxe

s pe

r acr

e

Treatment #

Fort Meade

3 Year Cumulative Yield (Boxes per acre)

b

ab ab abab ab

abab

a

ab

b

abab

a

aba

a

ab

ab

a

Study 2

Vashisth et al., Manuscript in preparation

Ranking based on cumulative yield of 3 yearsArcadia Site Fort Meade Site

Treatment # Treatment Total 3 Yr Yield

(boxes per acre) Treatment # Treatment Total 3 Yr Yield (boxes per acre)

7 CRF+Tiger MM + Fe 20% 1310 9 CRF+Tiger MM + Mn+B 20% 1130

4 CRF+Tiger MM 1263 4 CRF+Tiger MM 1076

8 CRF+Tiger MM +B 20% 1259 2 Conventional+ Tiger MM 1063

10 CRF+Tiger MM + Mn+ B 50% 1233 3 CRF+ foliar 1047

5 CRF+Tiger MM + Mn 20% 1136 5 CRF+Tiger MM + Mn 20% 1039

6 CRF+Tiger MM + Zn 20% 1118 10 CRF+Tiger MM + Mn+ B 50% 1034

2 Conventional+ Tiger MM 1095 6 CRF+Tiger MM + Zn 20% 1027

3 CRF+ foliar 1088 8 CRF+Tiger MM +B 20% 981

9 CRF+Tiger MM + Mn+B 20% 1048 7 CRF+Tiger MM + Fe 20% 913

1 Control 908 1 Control 893

Study 2

Vashisth et al., Manuscript in preparation

Current Research

Nutrient uptake in HLB-affected plants

• Investigate qualitative and quantitative difference in nutrient uptake of HLB

affected plants

• To evaluate efficiency of nutrient uptake in HLB affected plants verses healthy

plants

• Transcriptomic analysis of HLB affected plants in response to nutrient uptake

Vashisth et al., Manuscript in preparation

Study 3

Experimental Setup

• Same age ‘Midsweet’ grafted on Kuharske

rootstock: Healthy (HLY) and HLB-affected

• Plants were deprived from fertilizer for 6

months before experiment

• Hydroponic system with Hoagland solution

added at the beginning

• Two month study, sampling every 7 days

Vashisth et al., Manuscript in preparation

Study 3

Results

0

10

20

30

HLB HLY

Fres

h w

eigh

t (g)

roots shoots

leaves

*

HLB-affected plants were significantly low in root and shoot

biomass

*

*

0

0.2

0.4

0.6

0.8

Healthy HLB

Root

to S

hoot

Rat

io

*

Vashisth et al., Manuscript in preparation

Study 3

Chlorophyll content

0

10

20

30

40

50

12/8/2016 12/18/2016 12/28/2016 1/7/2017 1/17/2017 1/27/2017

Chl

orop

hyll

cont

ent

HLB Fertilized Helathy FertilizedHLB non-Fertilized Healthy non- Fertilized

(P < 0.05)

HLB plants were able to increase chlorophyll content as infertilized healthy plants.

Study 3

Leaf Nutrients Level- HLB affected plant metabolize nutrient at higher rate

0

50

100

150

Iron

(ppm

)

0

0.2

0.4

0.6M

agne

sium

(%)

0

0.2

0.4

0.6

Sulfu

r (%

)

0

1

2

3

4

Pota

ssiu

m (%

)

0

20

40

60

Man

gane

se (p

pm)

0

20

40

Zin

c (p

pm)

*

**

*

** *

*

Vashisth et al., Manuscript in preparation

Study 3

Efficiency- HLB affected plants had higher nutrient uptake efficiency for all nutrient

29

0

6

12

18

24

HLB-affected plants Healthy plants

Effic

iecn

y (%

)

Potassium

0

6

12

18

HLB-affected plants Healthy plants

Effic

iecn

y (%

)

Calcium

0

4

8

12

HLB-affected plants Healthy plants

Effic

iecn

y (%

)

Sulfur

0

4

8

12

16

HLB-affected plants Healthy plants

Effic

iecn

y (%

)

Nitrogen

HLB-affected fertilized plantsHealthy fertilized plants

(P < 0.034)(P < 0.022) (P < 0.015)

(P < 0.026)

*

* *

0

2

4

HLB-affected plants Healthy plants

Effic

iecn

y (%

)

Magnesium

0

2

4

HLB-affected plants Healthy plants

Effic

iecn

y (%

)

Phosphorus

(P < 0.046)

(P < 0.017)

*

*

Vashisth et al., Manuscript in preparation

Study 3

Transcriptomic AnalysisRNAseq for global gene expression study

Day 0, 7, and 14

Number of Differentially Expressed Genes

Day 0 HLB vs HLY 9

Day 7 HLB vs HLY 21

Day 14 HLB vs HLY 3246

Vashisth et al., Manuscript in preparation

Study 3

HLB vs. HLY D1 DEGs and functionOrange Gene ID Log FC Arabidopsis

homologsFunction

orange1.1g014036 2.07 AT3G51670.1 Phosphoglyceride transfer family protein

orange1.1g025435 2.91 AT5G62720.1 Integral membrane HPP family protein

orange1.1g031858 3.33 AT3G15680.1 Ran BP2/NZF zinc finger-like superfamily protein

orange1.1g030361 3.57 AT1G69700.1 HVA22 homologue C

orange1.1g033664 3.84 AT5G22580.1 Stress responsive A/B Barrel Domain

orange1.1g020451 4.76 AT1G49570.1 Peroxidase superfamily protein

orange1.1g018585 6.14 AT1G31260.1 zinc transporter 10 precursor

orange1.1g046055 6.44 AT1G75820.1 Leucine-rich receptor-like protein kinase family

protein

orange1.1g032726 7.76 Strictosidine synthase-related sub

family/biosynthesis of terpenoid indole alkaloids

present in meristematic tissue

Vashisth et al., Manuscript in preparation

Study 3

HLB vs. HLY D14 Enriched Categories Stress response

10%

Stimulus response16%

Cell death2%

Cell division/modi

fication1%

Developmental processes

9%

Nutrient metabolic process (N)

13%Nutrient metabolic process

(P)6%

Transport7%

Protein metabolic process

11%

Macromolecule metabolic process

23%

Other categories2%

Metabolic process,stress and stimulusresponse, growth anddevelopment, death,and transport weremostly enrichedcategories.

Vashisth et al., Manuscript in preparation

Study 3

Biological Process HLB vs HLY Day 14

Vashisth et al., Manuscript in preparation

Study 3

Soil pH• Macronutrients (N, K,

S, Ca and Mg) highly available between soil pH 6.5 and 8

• Micronutrients (Mn, Zn B, and Fe) most available below soil pH 6.5

• Best soil pH range for most crops is 6 to 6.5

Study 4

Why soil acidification?

In Florida,

Optimum pH range: 5.5 to 6.5

Soil pH> 6.5

Irrigation water: Bicarbonates >100 ppm and pH> 7.5

No soil acidification in commercial citrus groves pre-HLB

HLB-symptoms intensify under high soil pH

Root density is related to bicarbonate stress thus reducing yield at high pH

High pH stress

Osmotic stress

Ion toxicity

Nutrient unavailability

Production of reactive oxygen

species (ROS)

Cell death

Stress due to high pH on citrus

(Hanumantharao et al., 2016)

Study 4

Why soil acidification? Higher soil pH reduces availability of Ca, Mg, Zn, Fe and Mn

Soil acidification increases availability of Ca, Mg, Zn, Fe and Mn to the plants

Soil acidification is helpful in managing existing and new planting groves

Biotic stressHLB reduces feeder root

density

Abiotic stressHigh pH restricts

nutrient availability

Water and nutrient stress

Poor performance by plants

Study 4

Current Research

Effect of irrigation water pH on HLB-affected plants

1.To investigate the effects of irrigation water pH on the performance of healthy (HLY) and HLB-affected citrus

2.To study the molecular regulation of pH-HLB interaction

Study 4

Vashisth et al., Manuscript in preparation

Experimental Details

• Experimental design: CRD (n=8), Factors:

pH : 5.8, 7.0 and 8.0

pH 5.8 pH 8.0

pH 7.0

Disease: HLY and HLB

• Planting material: ‘Midsweet’ on Kuharske

Study 4

Experimental details

Acclimatized and nutrient deprived for 2 weeks

Irrigated with phosphate buffer every 3 days

Fertilized at Day 0

Grown in autoclaved Candler fine sand (pH 7.2)

Final data collection: 60 days

Study 4

Vashisth et al., Manuscript in preparation

0 0

12.5

0

12.5

37.5

0

10

20

30

40

5.8 7.0 8.0

Dea

th (%

)

ResultsHLB-affected plants under pH 8.0 treatment had the highest death rate

Study 4

Vashisth et al., Manuscript in preparation

• At pH 5.8, HLB-affected plants showed significantly higher growth

0

2

4

6

8

5.8 7 8

Cha

nge

in h

eigh

t (%

)

HLY HLB

b

a

b

At pH 5.8, HLB-affected plants performed similar to HLY• Leaf chlorophyll content (SPAD) decreased significantly

with increase in pH

5.8 7.0 8.0

P = 0.0004

0

20

40

60

80

5.8 7 8SP

AD

HLY

HLB

5.8 7.0 8.0

aa

b

P < 0.0001

Study 4

Vashisth et al., Manuscript in preparation

pH 8.0 HLB treatment resulted in heavy leaf drop• Irrigation water pH had significant effect on reducing

leaf number

0

10

20

30

40

Day 0 Day 60

Leaf

num

ber

5.8 HLY 5.8 HLB 7.0 HLY7.0 HLB 8.0 HLY 8.0 HLB

}}

}

P < 0.0001

a

b

c

R² = 0.93y = 19.352x + 4.6805

y = 36.91x - 19.65R² = 0.99

0

20

40

60

80

100

5.8 7.0 8.0

Leaf

dro

p ra

te (%

)HLY HLB Linear (HLY) Linear (HLB)

• Effect of pH on leaf drop rate is linear in HLB

Study 4

Vashisth et al., Manuscript in preparation

Leaf biomass decreased with increase in pH

a

b

c

0

2

4

6

8

5.8 7.0 8.0

Leaf

dry

wei

ght (

g)

P < 0.0001

Study 4

Vashisth et al., Manuscript in preparation

Shoot system at Day60

pH: 5.8 7.0 8.0

HLY:

HLB:

Study 4

Vashisth et al., Manuscript in preparation

Root biomass decreased with HLB, regardless of the pH

a

b

0

10

20

30

40

HLY HLB

Roo

t dry

wei

ght (

g)

P = 0.001

Study 4

Vashisth et al., Manuscript in preparation

Root system at Day60

HLY:

pH: 5.8 7.0 8.0

HLB:

Study 4

Vashisth et al., Manuscript in preparation

EC increased with increase in pH and HLB at Day60

0

0.4

0.8

1.2

1.6

2

5.8 7 8

Soil

EC(m

S/cm

)

HLY

HLB

pH: P < 0.001Ds: P = 0.0018

5.8 7.0 8.0

a

b

a

0

2

4

6

8

10

5.8 7.0 8.0So

il pH

Day30Day60

pH of soil tends to rebound to its original pH

Study 4

Vashisth et al., Manuscript in preparation

520274

694

0

0

200

400

600

800

1000

1200

1400

pH 5.8 (HLY vs HLB) pH 8.0 (HLY vs HLB)

Num

ber o

f gen

es (#

)Up

Down

Greater numbers of genes were differentially regulated at pH 5.8 with HLB while fewer at pH 8.0

Study 4

Vashisth et al., Manuscript in preparation

response to red light

lipoprotein biosynthetic process

lipoprotein metabolic process

hydrogen peroxide metabolic process

post-embryonic root development

reactive oxygen species metabolic process

hormone metabolic process

regulation of hormone levels

secondary metabolite biosynthetic process

shoot system morphogenesis regulation of meristem

development

terpenoid metabolic processresponse to bacterium

isoprenoid metabolic process

immune response

meristem development

secondary metabolic process

photosynthesis

response to oxidative stress

developmental maturation

oxidation-reduction process

root system development

pH 5.8-HLB vs HLY: Enriched GO categories for upregulated genes• ROS detoxifying processes • Growth of root and shoot • Photosynthesis• Hormonal regulations • Secondary metabolic process

Root and shoot development

Secondary metabolite biosynthesis

ROS detoxifying processes

Hormonal regulations

Response to stresses

Study 4

Vashisth et al., Manuscript in preparation

oxylipin biosynthetic process

oxylipin metabolic process

glycine metabolic process

defense response to bacterium, incompatible

interaction

cellular lipid catabolic process

lipid oxidation

lipid modification

response to woundingpolyol metabolic process

serine family amino acid metabolic process

immune system process

immune response

single-organism catabolic process

aging

cellular polysaccharide metabolic process

defense response

Jasmonic acid related pathways

Defense or immune responses

Senescence

Aminoacid metabolic process

pH 5.8-HLB vs HLY: Enriched GO categories for down-regulated genes• Jasmonic acid related pathways • Defense and immune responses • Protein metabolic processes• Senescence

Study 4

Vashisth et al., Manuscript in preparation

lignin metabolic process

protein acylation

programmed cell death

response to light intensity

cell death

regulation of defense response

response to bacterium

secondary metabolic process

mRNA processing

protein ubiquitination

mRNA metabolic process

immune system process

root developmentroot system development

regulation of growth

immune response

response to abscisic acid

response to stress

cell division

Cell deathGrowth and development

Defense or immune responses

pH 8.0-HLB vs HLY: Enriched GO categories for upregulated genes• Cell death • Defense and immune responses • Growth and development

Study 4

Vashisth et al., Manuscript in preparation

Take Home Message

• HLB-affected trees do benefit from good nutrition program• Micronutrients are critical for HLB-affected plants, needed at slightly

elevated levels• Due to small root system in HLB-affected trees, nutrient uptake is

limited• HLB-affected trees are more efficient in nutrient uptake

• Fertilizer program should be site specific• Soil pH affects growth of HLB-affected trees• Effect of soil-pH on HLB-affected trees is not limited to nutrient

uptake