Moshe Lapidot

Volcani Center, ARO

Pelargonium zonate spot virus is transmitted via seed and pollen

in tomato

TBRT 2013, Chiang Mai, Thailand

Shlomo Cohen 1935 - 2012

In autumn 2007, a new disease with unknown etiology was observed in open-field tomato in the Lachish region of Israel

Symptoms included mild mosaic, leaf malformation and severe stunting of the plants

The causal agent was readily transmitted mechanically from sap of infected plants to indicator plants

Crude extracts from symptomatic tomato plants were negatively stained and analyzed by transmission electron microscope, revealing spherical particles, 25 - 30 nm in diameter

cDNA was synthesized from RNA isolated from the viral particles

Alignment of sequences from the unknown virus - 95% homology to RNA 3 of Pelargonium zonate spot virus from Italy (PZSV-It)

Year Identified in Disease of

1980 Italy Tomato

1996 Spain Tomato

2000 France Tomato

2006 California, USA Tomato

2006 Spain Pepper

2007 Israel Tomato

Distribution of PZSV

Pelargonium zonate spot virus – PZSV Family – Bromoviridae

Genus – Anulavirus

3383 nt

2435 nt

2659 nt

ICR-Internal Control Region

PZSV-Israel PZSV-Italy ORF Start Stop NT (%) AA (%) Start Stop RNA 1a 79 2,943 90 93 79 2,967 RNA 2a 82 2,145 95 97 82 2,145 RNA 3a 335 1,264 97 98 335 1,264 RNA 3b 1,618 2,244 92 96 1,619 2,245

Nucleotide and amino acid comparison of predicted ORFs between PZSV from Israel and PZSV from Italy

Partial Host Range

Pelargonium

Tomato M-82

Tomato R-13

Pepper

N. Glutinosa

Cucumber

Lettuce

Eggplant

Common name

Taxon Cultivar Inoculated leaf

Systemic leaves

PCR

Bean Phaseolus vulgaris

Wax 216 - - -

Cotton Gossypium hirsutum

Aqala - - -

Cucumber Cucumis sativus Shimshon - - -

Beit Alfa + + +

Eggplant Solanum melongena

HA1726 - + +

Geranium Pelargoniun zonale

Perlenkate Orange

- + +

Jimsonweed Datura stramonium

- + +

Lettuce Lactuca sativa Noga + - -

Melon Cucumis melo Arava + - -

Pepper Capsicum annuum

Maor - + +

Quinoa Chenopodium quinoa

- - -

Common name

Taxon Cultivar Inoculated leaf

Systemic leaves

PCR

Squash Cucurbita pepo Ma’ayan + - -

Sweet corn Zea mays Saccharata - - -

Tobacco N. benthamiana - + +

N. glutinosa + + +

N. tabacum Samsun - + +

Tomatillo Physalis floridana - - -

Tomato S. lycopersicum R-13 - + +

M-82 - + +

Watermelon Cirulus lanatus Malali - - +

Transmission

Proceedings 4th Plant Virus Epidemiology Workshop, Montpellier, France, 1989:302-305 Vovlas et al. (1989) found pollen collected from naturally infected tomato plants to be contaminated with PZSV, but the virus was not found to be transmitted via tomato seed. While looking for alternative hosts they found that Diplotaxis erucoides, an endemic weed in southern Italy, serves as a symptomless host for the virus. They also found that PZSV is seed transmitted in D. erucoides with an efficiency of 5%. When pollen from infected D. erucoides was rubbed gently onto leaves of tomato plants, 8 of 10 plants became infected within 10 days.

They also presented preliminary results showing that a thrips species (Melanothrips fuscus) feeding on flowers of D. erucoides carries pollen grains on its body. When these thrips were transferred to tomato seedlings, 1 of 10 plants showed PZSV symptoms 25 days later. Hence, it was suggested that PZSV might be transmitted by thrips in an unusual manner.

Proceedings 4th Plant Virus Epidemiology Workshop, Montpellier, France, 1989:302-305

Transmission by insect vectors

Insect Transmission

Whitefly (Bemisia tabaci) 0/5

Greenhouse whitefly (Trialeurodes vaporariorum) 2/5

Peach aphid (Myzus persicae) 0/5

Western flower thrips (Frankliniella occidentalis)

0/4

Onion thrips (Thrips tabaci) 0/3

Soil transmission

To test whether PZSV can be transmitted through contaminated soil, PZSV-infected N. glutinosa plants were grown for nearly 2 months and then removed while leaving their infected roots in the soil. 14-day-old tomato seedlings were transplanted into the contaminated soil, and allowed to grow for another 2 months.

Soil transmission

A total of 50 tomato seedlings were transplanted into the contaminated soil, none of which produced PZSV symptoms, and all of which tested negative for the virus by RT-PCR.

Seed transmission Infected tomato plants (cvs. M-82 and R-13) were grown until fruiting and the seeds were harvested, washed with water and dried. The seeds were sown in Todd planter flats, grown for 1 month in the greenhouse and then evaluated for the development of PZSV symptoms.

PZSV is seed-transmitted in tomato

No. germinated seedlings

Tomato cv. Treatment Infected Total Infection rate

M-82 Water 121 646 18.7 %

Effect of seed treatment on PZSV transmission

No. germinated seedlings

Tomato cv. Treatment Infected Total Infection rate

R-13 Water 19 136 13.9 %

HCL+TSP 17 154 11.0 %

TSP = trisodium phosphate

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M

Water HCL HCL+TSP HCL+TSP+Bleach

Identification of PZSV RNA in tomato seed by RT-PCR

RNA extracted from: 20 seeds of control non-infected plants (lanes 1, 5, 9, 13) 5 seeds of PZSV-infected plants (lanes 2, 6, 10, 14) 10 seeds of PZSV-infected plants (lanes 3, 7, 11, 15) 20 seeds of PZSV-infected plants (lanes 4, 8, 12, 16)

Does PZSV reach tomato pollen?

RT-PCR identification of PZSV-RNA in pollen collected from flowers of

PZSV-infected tomato plants

1 2 3 M

Lanes 1 & 2: pollen from infected plants Lane 3: pollen from a control non-infected plant

Is PZSV pollen- transmitted ?

Plant pollination

Twenty clean tomato “mother” plants (cv. M-82) were grown in isolation in a greenhouse. The plants were tested by RT-PCR to verify no infection with PZSV. Once the plants started to flower, flowers were manually emasculated and pollinated with pollen collected from PZSV-infected plants. Once the mother plants gave fruit, seeds were extracted from each individual fruit, treated with HCl and TSP, and sown. The mother plants were also tested for PZSV by RT-PCR. The germinated seedlings were monitored for PZSV symptoms. The symptomatic seedlings were also tested by RT-PCR.

Seed transmission of PZSV following hand pollination of healthy mother plants with pollen collected from infected tomato plants

No. germinated seedlings Plant No. No. of fruit Infected Total Infection rate (%)

1 1 3 9 33.3 2 7 30 75 40.0 3 4 15 57 26.3 4 9 83 280 29.6 5 10 35 120 29.2 6 4 37 131 28.2 7 4 25 98 25.5 8 4 14 56 25.0 9 7 74 270 27.4 10 2 12 28 42.9 11 4 22 86 25.6 12 5 41 165 24.8 13 2 13 79 16.5

Total 404 1,454 Average 28.8

Variability in seed transmission among fruit of the same plant following hand pollination

No. germinated seedlings

Plant No. Fruit No. Infected Total Infection rate (%)

5 1 7 23 30

5 1 10 10

6 3 17 29

8 7 8 88

9 1 7 35 20

2 15 44 34

3 19 55 35

5 11 51 22

6 3 6 50

12 1 27 89 30

2 3 12 25

3 7 32 22

4 0 18 0

5 4 14 29

Summary

Summary

• The causal agent of a new tomato disease in Israel was identified as a new isolate of PZSV.

• The Israeli isolate has a very high homology to the

Italian isolate, app. 93% NT, 96% AA.

• The virus was readily transmitted mechanically and

induced symptoms resembling those described before.

Summary • The host range was essentially similar between the

different PZSV isolates, with the major differences being:

• (i) Isolates from Israel, France and US infect N. Benthamiana, the Italian isolate does not.

• (ii) ) Isolates from Israel, Spain, France and US infect

Datura stramonium, the Italian isolate does not.

• (iii) Reaction of cucumber to PZSV was not clear – Italian isolate induced local lesions + systemic infection; Spain & France only local lesions; We found the reaction of cucumber plants to be cultivar- dependent: Shimshon was a nonhost while Beit Alpha was a systemic host.

Summary

• PZSV-IL was not soil-transmitted. • PZSV-IL was not transmitted by an insect vector.

• PZSV-IL was found to be seed transmitted in tomato,

with transmission rate of 11-29%.

• Attempts to disinfect seeds using HCL and TSP failed to eliminate PZSV seed transmission.

• The virus was detected by RT-PCR in total RNA extracted from as as few as 5 seeds.

Summary • The virus was also detected by RT-PCR in total RNA

extracted from pollen grains collected from flowers of infected plants.

• Pollen grains collected from infected plants were used

to hand-pollinate healthy mother tomato plants. None of pollinated mother plants became infected, 29% of the seedlings produced from seeds harvested from these plants were infected. • This is the first demonstration of transmission of

PZSV via both seed and pollen in tomato.

Does PZSV poses a threat to

tomato production?

Plant Sciences Moshe Lapidot

Dana Guenoune-Gelbart Tzion Machbash Shlomo Cohen

Plant Pathology Amit Gal-On

Diana Leibman Vered Holdengreber

Entomology Michael Davidovitz Hazera Genetics

Smadar Klieman-Shoval

Volcani Center, ARO