Knowledge of -plant interactions helps in understanding and managing the new invasive brown regose fruit (ToBRFV)

Robert L. Gilbertson Department of Plant Pathology University of California Davis

Photos: N. Salem What is a tobamovirus?

• It is a member of a genus (Tobamovirus) in the family

• The tobamovirus type species is Tobacco (TMV), a very famous plant virus!

• 37 species currently recognized

• have similar features -rigid rod-shaped virus particles (18 X 300 nm) -single positive-sense RNA genome of ~6.4 kb -similar genome -transmitted by contact and seed, no insect vector

• Many cause economically important diseases! Multiple tobamoviruses infect tomato • At least five tobamoviruses infect tomato and induce similar symptoms: - (TMV) - (ToMV) -Tobacco mild green mosaic virus (TMGMV) -Tomato mottle mosaic virus (ToMMV) -Tomato brown rugose fruit virus (ToBRFV)

Symptoms Symptoms Virus Emergent Leaves Fruit Tm-22 Distribution Importance

TMV No Mo, Di, SS Browning* Resistant WW Low ToMV No Mo. Di, SS Browning* Resistant WW High TGMMV No Mo, Di, SS Few or none Resistant WW Medium ToMMV Yes (2013) Mo, Di, SS Few or none Resistant* MX, USA, Medium ME, Spain ToBRFV Yes (2015) Mo, Di, SS Necrotic Susceptible ME, MX, High lesions* USA, Europe Type of tomato production and potential ToBRFV impact • Open field *Processing tomatoes in CA Minimal touching and 3 month tomato free period No findings in 2020 **Fresh market production (various) Overlapping crops and more touching of plants • Protected culture ***High technology greenhouses-temperate Overlapping of crops and lots of touching of plants Outbreaks eradicated (Europe) ***High technology greenhouses/screenhouses-other Same as for greenhouses but inoculum from outside **Low technology greenhouses/screenhouses-other Overlapping crops, lots of touching, more inoculum Plant Virus Movement and Spread • How does a virus move within a plant?

• Viral life cycle -inoculation -replication -movement -plant-to-plant spread

• Two types of movement: cell-to-cell and long distance

• Cell-to-cell via plasmodesmata

• Long distance via the vascular system, mostly the phloem Plant Virus Movement and Spread • Movement in the phloem is rapid and results in systemic infection and symptoms

• Once a virus accesses the phloem (form?) -rapid movement to roots (soil) -rapid movement to shoot apex -subsequently colonizes much of the plant -plant-to-plant spread

• The virus moves in the direction of sinks, e.g., root and shoot apices and fruits

• Disease symptoms appear in 7-14d and in newly emerged leaves

• Huge amounts of infectious virus exist in inclusion bodies Spread of a tobamovirus in a relatively young tomato plant

-~3 days to access phloem (latent period)

-Roots (4 d), then to shoot apex (5d)

-Unloading and spread (~12d)

-Symptoms at top (12-14d) and total colonization by 25d

-Very high titers of virus due to logarithmic replication

-What does it say about movement in the phloem? (Samuel, 1934) Sampling and Testing: Seed is a key target as it is the major primary inoculum and means of long-distance transmission

• Tobamoviruses are transmitted as contaminants on the surface of tomato seed and infection of seedlings is ‘inefficient’

• Worldwide spread of ToBRFV has occurred via seed

Comments:

• Seed tests for ToBRFV based on RT-qPCR and a tobacco bio-indicator are commonly required

• Seed disinfestation (e.g., 10% TSP for 30 min)

• Avoid trialing varieties or materials unless seeds were tested* Distribution of ToBRFV

www.cabi.org/dmpd Detection of Tomato brown rugose fruit virus (ToBRFV) in tomato and pepper seed (ISHI-Veg)

Seed extraction buffer (phosphate buffered saline [PBS], pH 7.2 – 7.4)

Fw: Forward Rv: Reverse Pr: Labeled Fluorescent Probes Sampling and Testing: Transplants

• Transplants -Symptoms often not visible -Testing of symptomless transplants (?)

• ToBRFV outbreak in an Arizona (September 2020) associated with tomato transplants from Mexico

Comments: • Use only certified ToBRFV-tested free seed (CA) • Testing of transplants is challenging • Regular monitoring of sites where transplants are grown

• Avoid obtaining transplants from distant locations Monitoring, Sampling and Testing of Tomato Plants in Production Systems • Monitoring plants (protected and open field production) -Regular monitoring (7-14d intervals) of new leaves -Visual symptoms and confirmatory tests (LFD) -Pattern of infection is informative -There must be a plan and a team in place for monitoring and hygiene for protected culture • Soil -Tobamoviruses can persist in soil with debris for years -Roots very good for infection -Soil sampling and testing difficult -Rotate (2-3 yr), soil treatments, resistant rootstocks, soil-less culture • Reservoir hosts -Weeds and volunteer tomato plants should be monitored and tested (more for open field production) Pattern of plant disease

Regular Extensive Contamination of Greenhourse

Random Seed borne

Clustered Soil borne (foci) Sampling and Testing: Commercial Tomato Fruits • Fruit symptoms include necrotic lesions, yellow blotches to no symptoms (e.g., resistant varieties)

• Tomato fruit can carry large quantities of tobamovirus

• Virus is not on the surface of intact fruits

• Infected commercial fruit were not linked to outbreaks in Europe

• What is the probability that virus from such infected fruit could reach commercial production?

Comments: • Fruit inspection and testing: Is it necessary? • Not a major inoculum source? • Impact of new varieties with intermediate resistance 1 2

3 4

5

Tomato fruits from Mexico: An avenue for establishment in California? Disease Resistance

• Pathogen-host interaction terms -Susceptible-typical symptoms -Tolerant-typical symptoms but still yields well -Resistant Strong-no symptoms and pathogen levels reduced Intermediate/moderate-reduced or no symptoms but may have high pathogen titer -Immunity-all members of a taxon (family) not infected

• Tobamovirus resistance: an important tool for management -Single dominant genes from wild species

-ToBRFV breaks the durable Tm22 R gene from S. peruvianum

-Resistance to ToBRFV has now been identified Deployment of Varieties with Resistant to ToBRFV

• Susceptible -Highly susceptible cvs replaced -Cherry tomatoes less susceptible -Switched to determinant cvs

• Resistant -Varieties with intermediate resistance (symptomless) available -These varieties has helped growers in Israel reduce losses due to ToBRFV from ~100% to ~20% -Growers in Mexico are growing cultivars with similar resistance

-Genotype with strong resistance, i.e., no symptoms or virus detection, have been identified (e.g., LA 3812)

• Comments: More extensive planting and development of resistant varieties and more fruits with symptomless infections? RT-PCR and LAMP tests for specific detection of tomato brown rugose fruit virus (ToBRFV) Where are we with ToBRFV? • Good detection tools and disinfectants

• Emphasis on seed is critical-is the RT-qPCR test sufficient?

• Planting of moderately resistant (symptomless) varieties: allowing protected culture to rebound (IS, MX) while serving as symptomless carriers? • Prioritize monitoring/sampling (depends on type of culture/cultivar): -Seed: test (+++) -Transplants: visual/test? (+) -Production: visual/test as needed (+++) -Commercial fruits (+) -Soil with history of ToBRFV (++)

• Implementation: Easier said than done! • Try to standardize approaches as much as possible Acknowledgements

UC Davis Dr. Tomas Melgarejo Margaret Cespedes