TECHNICAL WORKSHOPS 2022

Paolo Battistel
CERES SRL

3rd November 2022

“The Prevention of Contact

Diseases in GreenHouses:
ToBRFV (PepMV, Clavibacter)”

ceres.srl@iol.it
CERES 1
 TOBAMOVIRUS GENUS
- 37 Species (+ 5 Candidates)
- Mechanical Transmission (Contact Diseases)
- Long Time preserved infectious in Soil and Tools
Tobacco Mosaic Virus (TMV)
Tomato Mosaic Virus (ToMV)
Tomato Mottle Mosaic Virus (ToMMV) Closely Related
Tomato Brown Rugose Fruit Virus (ToBRFV) to TMV at 82.4%
Pepino Mosaic Virus (PepMV) à Genus PotexVirus Genetic Identity
Cucumber Green Mottle Mosaic Virus (CGMMV) (RNA-Sequence
maybe RECOMBINANT)
Pepper Mild Mottle Virus (PMMoV)
Paprika Mild Mottle Virus (PaMMV)
ToBRFV is reproduced in the MERISTEMS
TOBRFV goes to STRONG SINKS à at 1st to Roots à then to Inflorescences
 ToBRFV MORPHOLOGY
Capsid (Proteins) RNA

300-310nm (55%)

Rod shape
ToBRFV CGMMV PepMV
Jordan/Israel October 2014

Breaking Tm-22 resistance in tomato (TMV, ToMV)

First report in October 2014 (8 years with us)
CERES 4
 1st Article
Nov 2015

• Department of Plant Protection, University of Jordan, Amman, Jordan

• Istituto per la Protezione Sostenibile delle Piante, CNR, Turin, Italy
• Department of Plant Pathology, University of California, Davis, USA

CERES
SYMPTOMS
Immature Fruits:
«Brown Rugosus»

CERES 6
SYPTOMS
Mature Fruits
Heavy Marbling

Light Marbling

Chlorotic Spotting

Ready for the Market

CERES 7
 SYMPTOMS
Truss Stalk & Sepals

CERES 8
 SYMPTOMS
Sepals, Young Fruits, Stem

Heavy Symptoms
on the Fruits

No Symptoms
on the Fruits CERES 9
 SYMPTOMS
Leaf Blistering

Chlorosis + Dark Green «Bubbles»

CERES 10
 SYMPTOMS
Leaf: Mosaic, Narrowing, Chlorosis

Mosaic Narrowing Chlorosis

(Needle-like Symptoms)
 SYMPTOMS
Old Leaves: Deformation, Rugosis, Yellowing

CERES 12
 SYMPTOMS
Head, Plant, Crop

The heads stop to grow after 6th truss Heads wilting Plant Death
CERES 13
Early Symptoms

Early Detection
Early Symptoms
Early Detection

CERES 15
Early Symptoms
Early Detection

CERES 16
ToBRFV & TOBAMOVIRUSES

TMV, ToMV, ToMMV: resistance genes Tm-1, Tm-2/Tm-22

ToBRFV vs PepMV

CERES 18
 PepMV: LEAVES

Blistering «Nettle Head» Leaf Browning Yellow Spots

CERES 19
 PepMV: FRUITS
PepMV
in Cherry Tomato

Exposed Seeds Dry Calyx Discolored Fruits

CERES 20
 ToBRFV + PepMV

Leaf Mosaic CERES

Leaf Narrowing 21
 ToBRFV vs TYLCV

TYLCV CERES
TYLCV+ToBRFV 22
 Pepper:

TSWV (Tospovirus)

TSWV
Tospovirus

TSWV

TSWV TSWV
ToBRFV
BRONZAGE!

PMMoV
ToBRFV CERES Tobamovirus
 ToBRFV & WEEDS!

Solanum nigrum
Chenopodium quinoa Nicotiana tabacum

Datura stramonium Portulaca oleracea Physalis pubescens

 ToBRFV & WEEDS!
Chenopodiastrum murale

Symptomatic Asymptomatic
CERES 25
ToBRFV Host Range

CERES 26
ToBRFV PEPPER

CERES 27
ToBRFV & L3/L4 PEPPERS

HR symptoms developed following root inoculation

in pepper plants (harboring gene L1,3,4) demonstrating
dried spots on stems leading to plant growth inhibition
ToBRFV & L3/L4 PEPPERS

HR symptoms developed following root inoculation

in pepper plants (harboring gene L1,3,4) demonstrating
dried spots on stems leading to plant growth inhibition
ToBRFV & NON-L3/L4 PEPPERS

26 days after inoculation 2 months after mechanical inoculation

CERES 30
CLAVIBACTER

YOUNG
PLANTS:
Wilting of
Leaflets

NURSERY:
Wounds
on the
Cotyledons or
on New Shoots
31
CLAVIBACTER

CERES 32
 Wilting
CLAVIBACTER of the Heads

«peacock eyes»
CERES 33
 ToBRFV DETECTION TESTS Best is RT-qPCR:
- ISHI-Veg 2019
- Menzel&Winter 2021
GH+ buffer
Cross-Reaction (Guanidine Hydrochloride) ToBRFV
with Other Dead
Tobamoviruses or
Not Specific Alive?
Detection SENSITIVITY > 97%
but suitable for
ImmunoStrip (Field) Mass Screening RT-qPCR (Lab)
T
Leaf Seed o
Extract Extract b
SENSITIVITY
a
Tom 81%
Pep 43% c
c
o
DAS-ELISA (Lab) CERES BioAssay (Lab) 34
ToBRFV ImmunoStrip AGDIA

35
 Detection of ToBRFV in
Tomato & Pepper Seeds (ISHI-Veg)

Sampling for
Bio
Assay
(12x250=3000)

Sampling for PCR:

Tomato: 3 x 1000 = 3000 seeds
High Sensitivity: 1:1000 (0.1%)
Pepper: 6 x 500 = 3000 seeds
Medium Sensitivity: 1:500
CERES
 ToBRFV Seed Detection
Tomato Seeds Hairiness Seed Treatments:
ToBRFV should be located:
- on the seed coat NaClO 2.5% x 15’ (Tom)
- in the peri/endo-sperm ? TSP 10% x 180’ (Tom+Pep)
- NOT inside the embrio

ToBRFV

CERES possible moment 37

of contamination
 Seeds Treatments
Bioassay
T° time Germ ToBRFV Infected
14d by PCR Plants
100%
80C X 24h 100% at 7d 60% 0 Virus
75C X 48h 100% 80% 0 Detectable
Thermal but
70C X 96h 100% 100% 0 Not Infectious
65C X 120h 100% 100% 20%
Bioassay
Products % Time Germ
14d
Infection
Rate

TSP 10% 180’ 100% 3%

X
H2O2 4% X 30’ 100% 100%
Chemical HCl 2%
24h 0% NA Virus
NaClO 1.5% X
Detectable
NaClO 2.5% X 15’ 100% 0% but
100% at 7d Not Infectious
 14th Sep 2022

CERES 39
ToBRFV is Not-Infected Infected
Not Transmitted
to the Progenies
by Pollination

ToBRFV-infected Pollen
Grains don’t Germinate

Not-Infected Infected

Ovules are
ToBRFV-free
 WHY TOBRFV is DANGEROUS?
EASY TRANSMISSIBLE !

✋✋
2 h
Novel
Monitoring
System?

Grafting Temporary Workers

Crop
Residuals

Irrigation
or Drain soil
Water
CERES 41
ToBRFV is the new
Tomato’s Covid? ✋
SIMILAR CHARACTERISTICS:
- High Infectivity Rate (IR)
- Covid = Rt Wuhan 3 à Omicron5 17 à Cerberus 20
- PepMV (Pepino): IR ³ 1
- ToBRFV (Tobama): IR ³ 9
- High Persistency out of the Host:
- Covid = hours/days
- ToBRFV: weeks/months/years!!! PepMV: IR ³ 1 / Mixed ³ 4
- Long Incubation Time:
- Covid = Wuhan 21 days à Omicron 3 days
- ToBRFV: (2)3-4 weeks (Summer = hot)
6-8(9) weeks (Winter = cold)

Trichomes or
Glandular Hairs CERES
ToBRFV: IR ³ 9 / Mixed ³ 9
 High Spreading Capacity
(Experiment in Sicily Set 2018 - May 2019)
Month #1 #2 #3 #4 #5

Introduction of -------------------------------------------------------------> 61% Infected

2 Infected Plants on top of 480 (12 lines x 40 pts) Plants
High Spreading Capacity
(Experiment in Sicily: final result)
9th Month

The wider
The Round
The longer
The Infection

The First 2
Infected
Plants

( only 4 pts
Not Infected)
CERES 44
Evolution of the Infection

Normal
Growing
Practices

Presence
of
Bumble
Bees

CERES 45
 Sicily: Infected Seed Lots (Nov 2018 – Jan 2020)

Seeds
15 months

Today:
0.08%
CERES 46
 Sicily: Infected Plantlets (Nov 2018 – Jan 2020)

Plantlets

Empty
GH

CERES 47
 Sicily: Infected Greenhouses (Nov 2018 – Jan 2020)

GreenHouses

Empty
GH

CERES 48
Sicily: ToBRFV outbreak Set 2018

CERES
In Only 15 Months! 49
 ToBRFV Big Problem:
Transmission by Bumble Bees

Hives coming
INFECTED
PLANT
from an 4wks
Infected GH
after 2wks

INFECTION 22%
3wks
NetHouse
GlassHouse
NetHouse
Inside 3
Healthy
INFECTION 60%
GreenHouses
2wks

Plants were growing

for 41 days until INFECTION 11%
Maturation of 1st Truss
 Transmission by Bumble Bees

ABDOMEN = 50% of ToBRFV

Bumble Bees + TEMOINE

Hives Components No Hives
(cotton & comb) + +
Plant’s Organs Untouched Plants
were analyzed
CERES 51
for ToBRFV
 A Possible Solution
to work with Bumble Bees
0,27mm x 0,85mm
1,0-1,2
(20x10 wires/cm2)
ha
Shadow 20%
WF Ventilation 30%
net

BB 4-7mm x 2,3 mm
net Shadow 10%
Ventilation 90%

Hives Disinfection Mats

Small Disinfection Units

To Isolate Each Block of 1,0-1,2ha of the Farm with BB-Insect Nets

To Destroy and To Change 4-5 Hives/ha every 2wks, max 3wks
CERES 52
 POLLINATION:
Possible Alternative Methods

Don’t Touch
The Plants!

Manual or Electrical Heads Shaking by «Bee Robots»

Vibration Air Blowers
CERES 53
 3 PHASES of REACTION
1. BEFORE 2.DURING 3. AFTER
THE INFECTION

Planning (Crop Rotation)

Prevention / Hygiene

Team Work
Good Strategy
(Fighting! 💪)
Scouting / Early Detection CERES Science (Breeding)
 PREVENTING MEASURES
1. GENETIC RESISTANCE
- PEPPER: varieties with Gene L4 (resistant and available)
- TOMATO: first Tolerant/Resistant varieties in 2022-23
2. VACCINE
- PepMV: 2 vaccines already available
- ToBRFV: not available, maybe possible within 5-8 years
3. HYGIENE
- Preventing Measures BEFORE entering the grenhouse
- Preventing Measures INSIDE the grenhouse
4. SOIL DISINFECTION: some possibilities
5. CROP ROTATION: Cucurbits or other crops
6. GRAFING on EGGPLANT (not confirmed)
on Tolerant/Resistant Tomato RootStocks
CERES 55
 Hygiene Hygiene Hygiene!
Measures BEFORE entering the greenhouse:
- Install a hygiene station to disinfect hands and footwear.
- Clean hands with disinfecting soap and then disinfect the dry hands again.
- Place disinfection mats where personnel and transport have to pass before entering.
- Before re-entering the greenhouse, after a break, disinfect and clean the hands again.
- Let visitors wear gloves, overlays and hairnets everywhere.
- Provide daily a clean work-clothing for the staff.

Measures INSIDE the greenhouse:

- Disinfect hands while working.
- Disinfect the knives and scissors after each row of crops.
- Disinfect all moving equipment that comes into contact with the crop.
- Tobamoviruses can survive for a long time, both on organic and non-organic material.
- Clean and disinfect very well the entire greenhouse during the crop rotation.

CERES 56
 Hygiene Protocols

Sensor
Soap
Disinfectant
Mats Mobiles Inside
Sealed Bags !!!

Nederland
Mid 2019 à 01 June 2022
Virkon 2%
57 total nr of infected farms
H2O+Soap+Dry
20’’
41 still fighting with ToBRFV
30’’ 12 have eliminated the virus
2 farms got a 2nd infection
Shoes
Brush + Disinfectant CERES 57
Hygiene Protocols
Mobiles

Washi
ng+Di
sinf ection
+Drying
Foam Knives
Disinfection Over-Night

Mobile
Disinfection
Units
Inside the GH
CERES 58
Disinfection Mats
ENTRANCE of the GH

CERES
 CLOTHES DISINFECTION

Min 60C!

Total Inactivation:
90Cx 10’ or 2.5 < pH > 9

Professional Washing Machines The Working Clothes and

to provide Clean Clothes The Working Shoes
to All Personel EVERY DAY CERES Never Leave the Farm 60
CLOTHES DISINFECTION

* 10 min
** 4 hours
CERES 61
 DISINFECTANTS
1. HANDS / TOOLS:
Virkon-S 2% à hands before entering the GH + Tools
1% à hands in contact with plants (5’ or 50 pts)

2. TOOLS (Knives, Scissors, Boxes, Trolleys):

Sodium Hypochloride 0.5% - 1% à 20’ – 10’ min time
Peroxide + Peracetic Acid (Jet5) 2% à 15’ min time

3. MATS (Shoes, Wheels):

Potassium (or Sodium) Hydroxyde (KOH, NaOH) 2% (pH 14)

4. HANDS during crop management:

!
Formaline,
Virkon S 1% Alcohol
Skimmed Milk > 3.5% proteins Kill Bacteria
Lactoferrin 0.5% CERES Don’t Kill Viruses 62
DISINFECTANTS TEST
in Young Plants

ToBRFV!

CERES 63
DISINFECTANTS TEST
in Young Plants

CERES 64
 SCOUTING & MONITORING
Waste of
Old Crops

Tuta
White Fly
Mites
Fusarium etc.
START EVERY YEAR 5ha
in B1
WHY???

Block 1 Block 2 Block 3 Block 4

SCOUTING IPM GM
TEAM IPM Gerant B1 IPM Gerant B2 IPM Gerant B3 IPM Gerant B4
13p
Help2 Help1 Help2
20ha Help1 Help2 Help1 Help2 Help1

CERES 65
 Practical Example
Stock
of Diseases

Transplanting wk03 Transplanting wk03

1 st Plant wk10 1 st Plant wk10

Immediate Phase Out

of 0,6ha
Quarantine Area (0.6ha)

Visitors
2021 2022
60% loss of 5ha 0,6ha lost (12%) of 5ha
75.000 pt lost 15.000 pt lost 66
 Working Method

EVERY BLOCK
ONLY ONE TEAM

To Work
Always
on One Side
of the Corridor
Every
To Enter into
Line
To Work The Lines
a
Always in Always from
Different
One Direction The Right Side
CERES
Tool
 Working Method
NL Morocco
Control Board

Plastic Foil
Protection

Timer
5min
(50 pts)

Virkon 1%

Trolleys are disinfected

?
at the End of each line CERES 68
 POSSIBLE SOLUTIONS for
MOROCCO

- To provide new protecting clothes to each worker every day

- To keep the working shoes always inside the farm
- To disinfect hands before entering the greenhouse (Soap+Virkon 2%)
- To use gloves to touch the plants
- To change the gloves after every line
- To spray stilts and bags after each line with Virkon 2%
- To provide each worker with a spray bottle 250-300cc Virkon 1%
- To inform workers by a whistle to spray hands every 5-10 min
- To link every knife or scissors to only one line
- To work always in only one direction
- To arrange only one team for each block of the greenhouse

CERES 69
 VEHICLES & BOXES

A PACKSTATION

DISINFECTION OF BOXES:
- High Pressure Washing
- Low Pressure Hot Steam
- Sodium Hypochloride 1%
- Peroxide + Peracetic Acid 2%
- Virkon-S 2%
70
 CROP ROTATION
Crop Removal

Mexico: Tomato Crop Residuals

1 year old à ToBRFV Foam
Products
Still Alive + Still Infectious

High Pressure
Cleaning
Strong Chemicals
to Destroy the RNA (NVWA NL)
Menno Florades 4%
VirkonS 4% * 30’ - 1L * 40 m2
HyperClean-X (NaClO 5%Cl) 1% 71
 NURSERIES
Irrigation Systems
BASIC RULES anti-ToBRFV:
- GSPP Nurseres à only GSPP Seeds
- To keep separated the big lots of seeds
- To avoid wounds on the roots
- To disinfect blades at any cut for grafting
- To change the blades after each tray
- To change daily the clothes of the workers Ebb & Flood
- To disinfect continuosly all internal trays
- Frequent scouting and monitoring
- To pack the plants with carton boxes
- To deliver the plants only by own tracks
- Not any contact Drivers/Clients/Nursery
- To disinfect the trucks after each delivery
- Not any food with Tomato or Pepper
- Not any contact Pt/Growers - Pt/Visitors
Spray Systems
72
 To re-plant an infected GH
1. Intermediate Healthy Soil

Healthy Soil

Infected Soil
ToBRFV
Healthy
is entering the Soil
Plant Cells through
Wounded Roots Planting Pit
During Transplanting CERES 73
 To re-plant an infected GH
2. Intermediate Substrate

Possibility
to wrap the Plugs PERLITE
with Degradable (Coco Peat)
Plastic Foils CERES 74
 To re-plant an infected GH
with Intermediate Media
CAREFUL PLANTING REGULAR PLANTING
Plants directly from the Tray To walk in only one CAREFUL PLANTING
to the Planting Pit Direction inside the GH To Use Protecting
Clothes, Gloves and
Over-Shoes

CERES 75
 To re-plant an infected GH
3. Soil Disinfectants
Experiments 2016-2019

1. ToBRFV Inoculum 2. Disinfectant 3. Careful Transplant

 To re-plant an infected GH
3. Soil Disinfectants
Field Application Stabilized
Chlorine
2000 ppm

1. Planting Pit 2. Disinfectant 3. Careful

Preparation Application Transplanting
 To re-plant an infected GH
3. Soil Disinfectants
EXP. #1 TREAT Rep. 1 Rep. 2 Rep. 3 Total IR%
Temoine 31/50 20/100 46/100 97/250 38.8
3 Products KlorBac 2/50 6/50 8/50 16/150 10.7
with
Stabilized ChloRun 2/50 5/50 2/50 9/150 6.0
Chlorine Taharn 4/50 3/50 1/50 8/150 5.3

EXP. #2 TREAT Rep. 1 Rep. 2 Rep. 3 IR%

Wounded or Temoine 6/52 5/57 17/51 17.5

Wounded R
Not Wounded
Roots Temoine 3/51 4/51 --- 6.9
Not Wound
Stabilized 4/50 2/55 6/46 7.9
H2O2
Stabilized H2O2
vs GreeUp-D 0/52 0/48 0/49 0
Stabilized Chlorine
Taharan 0/53 0/52 0/51 0
78
 4. Grafting Tomato:
A. Eggplant
B. ToBRFV-resistant Tomato RS

TOLERANT
RESISTANT
Variety

Infected
Crop

Tomato

EggPlant
TRIAL

TOLERANT
RESISTANT
RootStock
CERES 79
New «Survival Strategies»
Loose Beef Tomato Crop
100% ToBRFV-infected
o Market
tt
Strigh
- Density: 3 heads/m2
- Grafting (strong rootstocks)
- «Careful Transplanting»
- Plants not touched for 3 wks
- Not any de-leafing
- Pollination by Bumble Bees
- Crop pinched at 8-9 Trusses
- 2 Crops/year
Light Marbling
 Genetic Tolerance
Susceptible (S) Tolerant (T)
The Tolerance Trait is Recessive
and Segregated as a
X
Single Recessive Gene

F1 F2

CERES
 Genetic Resistance
Susceptible (S) Resistant (T)
The Tolerance Trait is Polygenic.
It is controlled by at least 2
X Interacting Loci: 1 is Recessive and
1 is Semi-dominant

F1 F2

CERES
ToBRFV-Vaccine?
5-8 years
To find out a mild strain
To validate the treatment
To register the vaccine
To distribute the Product
Worldwide

25 Partners x 12 Countries
are investigating the ToBRFV
V
Cross Protection
83
 SOME POINTS of DISCUSSION
1. To choose Seeds and Nurseries GSPP certificated
2. To train all the staff to recognize the earliest symptoms
3. Frequent scouting by a specialized group of workers
4. To identify a group of specialized labs to do fast analysis
5. To take fast and strong decisions in case of detection
6. To keep always high levels of hygiene
7. To provide clean working clothes every day + shoes
8. To isolate each block of 1-1.2ha à new type of doors
9. To replace bumblee bees with air blowers in the infected blocks
10. Only one team for each block & To work in only one direction
11. To disinfect the hands while working every 5-10min
12. Only one knife or only one scissors for each growing line
13. To choose tolerant or resistant varieties when available
14. To share information along all the production chain
THANK YOU FOR YOUR ATTENTION!

CERES STATION – Agadir - Research & Training

You are All invited

to Contact or to Visit us!

ceres.srl@iol.it 85