Whitefly transmitted Ipomovirus, SqVYV-CA isolate in cucurbits:

An update on plant diseases from insect vectored virus in Imperial Valley, CA

E. Natwick, University of California ANR Cooperative Extension, Holtville CA; O. Batuman, Department of Plant Pathology, University of California-Davis, CA; W. M. Wintermantel, United States Department of Agriculture, Salinas CA; J. D. McCreight , United States Department of Agriculture, Salinas CA; T. Tian, California Department of Food and Agriculture, Sacramento CA and R. L. Gilbertson, Department of Plant Pathology, University of California-Davis, CA

2015 Vegetable Crops Program Team Meeting December 8, 2015

Insect Vectored Tomato Viruses & Phytoplasma Whitefly (Bemisia tabaci biotype B)

• Tomato yellow leaf curl virus (TYLCV)

• Tomato necrotic dwarf virus (ToNDV)

Aphid (various species) • Alfalfa mosaic virus (AMV); transmitted by at least 14 aphid species

• Cucumber mosaic virus (CMV); vectored by more than 60 aphid species

• Potato Virus Y (PVY); green peach aphid, Myzus persicae and other aphid species

Thrips (western flower thrips, Frankliniella occidentalis and onion thrips, Thrips tabaci) • Tomato spotted wilt virus (TSWV)

Leafhoppers • Beet curly top virus (BCTV), Beet severe curly top virus (BSCTV), Beet mild curly top

virus (BMCTV); transmitted by beet leafhopper, Circulifer tenellus

• Tomato big-bud mycoplasma (TBB); vector is brown leafhopper, Orosiun argentatus

• Aster yellows phytoplasma (AYP); vector Aster leafhopper, Macrosteles quadrilineatus

Insect Vectored Lettuce Viruses & Phytoplasma Sweetpotato Whitefly (Bemisia tabaci biotype B)

• Lettuce infectious yellows virus (LIYV); can no longer be found • Lettuce chlorosis virus (LCV)

Thrips (western flower thrips, Frankliniella occidentalis; tobacco thrips, F. fusca; onion thrips,Thrips tabaci) • Tomato spotted wilt virus (TSWV) • Impatiens necrotic spot virus (INSV) AMV

Aphid (various species) • Alfalfa mosaic virus (AMV); transmitted by at least 14 aphid species • Beet western yellows virus (BWYV); green peach aphid, Myzus persicae and 7 other

aphid species • Cucumber mosaic virus (CMV); vectored by more than 60 aphid species • Lettuce mosaic potyvirus (LMV) green peach aphid, Myzus persicae and many other

aphid species Leafhoppers

• Aster yellows phytoplasma (AYP) Aster leafhopper, Macrosteles quadrilineatus • Beet curly top virus (BCTV), Beet severe curly top virus (BSCTV), Beet mild curly

top virus (BMCTV) Beet leafhopper, Circulifer tenellus

Insect Vectored Beet Viruses & Phytoplasma Whitefly (Bemisia tabaci biotype B)

• Lettuce infectious yellows virus (LIYV); can no longer be found

• Lettuce chlorosis virus (LCV)

Aphid (various species) • Beet yellows virus (BYV); transmitted by at least 22 aphid species

• Beet yellow stunt virus (BYSV); Sow thistle aphid, Nasonovia lactucae

• Beet western yellows virus (BWYV); Green peach aphid, Myzus persicae and 7 other aphid species

• Cucumber mosaic virus (CMV); vectored by more than 60 aphid species

• Beet mosaic virus (BMV); transmitted by many aphid species

Leafhoppers • Aster yellows phytoplasma (AYP) Aster leafhopper, Macrosteles quadrilineatus

• Beet curly top virus (BCTV), Beet severe curly top virus (BSCTV), Beet mild curly top virus (BMCTV) Beet leafhopper, Circulifer tenellus

Low Desert U.S. history of Bemisia tabaci-transmitted and aphid-transmitted cucurbit infecting viruses

Cucurbit production in the desert southwest of the United States, including the Imperial Valley of California, is affected by a complex of insect-transmitted viruses, including Cucurbit yellow stunting disorder virus (CYSDV, crinivirus) and Cucurbit leaf crumple virus (CuLCrV) and Squash leaf curl virus (SLCV) (both are begomoviruses) all vectored by sweetpotato whitefly MEAM1 cryptic species of Bemisia tabaci also known as biotype B.

Aphid vectored potyviruses: Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus (PRSV) and Watermelon mosaic virus (WMV) Cucumber mosaic virus (CMV)

CuLCrV in melon & watermelon SLCV in squash CYSDV in melon WMV in watermelon

A new and potentially devastating whitefly-transmitted virus of cucurbits was found in fall 2014 in Imperial County, CA

In fall 2014, high levels of B. tabaci biotype B and unusual virus-like symptoms were observed on pumpkin (Cucurbita pepo L.) by Eric Natwick and sent to UC Davis for further analysis. Also melon (Cucumis melo L.) showing similar virus-like disease symptoms on were taken to USDA ARS Salinas, CA by James McCreight for analysis.

Infected plants were stunted and leaves showed mottling, yellowing, epinasty and crumpling.

RT-PCR and PCR tests of symptomatic plants revealed mixed infection with Cucurbit yellow stunting disorder virus and Squash leaf curl virus;

RT-PCR with degenerate potyvirus primers revealed a positive result with the cylindrical inclusion (CI) but not with helper component proteinase primer pair.

First report of an ipomovirus in California: identification of a divergent strain of Squash vein

yellowing virus in the Imperial Valley of California

A potentially new ipomovirus was first identified by Dr. Ozgur Batuman in the Dr. Robert Gilbertson’s laboratory at UC Davis.

The presence of this virus was verified by Dr. William Wintermantel’s USDA-ARS laboratory, Salinas, CA.

This was the first report of an ipomovirus infecting cucurbits in the Imperial Valley of California

The new virus appears to be related to but distinct from Squash vein yellowing virus (SqVYV) a Bemisia tabaci-transmitted ipomovirus (family Potyviridae) that occurs in Florida.

Identification of an Ipomovirus New to California RT-PCR on leaf tissue with degenerate potyvirus cylindrical inclusion (CI)

and helper component proteinase (HC-Pro) primers revealed only the expected size ~700 bp fragment with the CI primer pair for 17 of 21 Pumpkin plants tested.

Sequence analysis of the CI fragments from 9 plants revealed 82-84% identity with the CI gene of Squash vein yellowing virus (SqVYV) from Florida. SqVYV is a whitefly (B. tabaci)-transmitted virus in the genus Ipomovirus and the family Potyviridae. This is the first report of such a virus in California.

RT-PCR analysis with primers specific for the capsid protein (CP) gene of SqVYV directed the amplification of the expected size ~1,100 bp fragment from the 17 leaf samples in which the CI fragment was amplified with RT-PCR, but not from healthy pumpkin plants or the leaf samples in which the CI fragment was not amplified.

Sequence analysis of fragments amplified with the SqVYV CP primers revealed 89-99% identity with the SqVYV CP gene.

Further evidence for an ipomovirus was mechanical (sap) transmission of the virus from symptomatic pumpkins leaves to non-infected pumpkin and squash plants (CYSDV and SLCV are not mechanically-transmitted).

Pumpkin (C. pepo cv. Small Sugar) and squash (C. pepo cv. Pavo) plants inoculated with sap prepared from leaves of 3 pumpkin plants (RT-PCR-positive for the presence of the ipomovirus) where it induced mild mottling, vein clearing and yellowing symptoms typical of those induced by SqVYY (9/11 pumpkin and 6/6 squash plants).

These plants tested positive for ipomovirus infection based on RT-PCR with the CP primers and negative for infection by CYSDV and SLCV.

Watermelon plants, similarly inoculated, were initially symptomless, but wilted and collapsed ~1 month after inoculation; whereas non-inoculated control plants were symptomless. RT-PCR analysis with the SqVYV primers confirmed the presence of the ipomovirus in the plants that developed symptoms after sap-inoculation.

Identification of an Ipomovirus New to California

Greenhouse Inoculation Research at UC Davis In sap inoculation studies, pumpkin and squash plants (Cucurbita pepo L.)

inoculated with sap prepared from leaves of pumpkin plants in which the California ipomovirus was detected by RT-PCR, developed mild mottling, vein clearing and yellowing symptoms whereas watermelon (Citrullus lanatus) plants were initially stunted and yellowed and eventually collapsed, similar to those reported for SqVYV.

California ipomovirus infected watermelon plants showing vine collapse symptoms (right) and mock inoculated healthy watermelon (left) at 30 days-post-inoculation.

Leaves of squash plant infected with California ipomovirus showing mild mottling, vein clearing and yellowing symptoms at 30 days-post-inoculation.

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Identification of a New Ipomovirus in California Transmission electron microscope examination of leaf-dip and

mini-purification preparations from squash and pumpkin plants infected by sap-inoculation revealed potyvirus virions, i.e., long flexuous rods ~722 X13 nm.

ELISA performed with an antiserum raised against a Florida isolate of SqVYV was positive (readings of 1.25) for leaves of all cucurbit plants infected with the ipomovirus after sap-inoculation, and negative for healthy control plants.

The origin of the California ipomovirus is not clear, but our results indicate that an ipomovirus, most likely a divergent strain of SqVYV from Florida was infecting pumpkin and melon plants in California.

Potential Threat to California Agriculture This is the first report of an ipomovirus in California and it represents a

potential threat to cucurbit production in the state, especially watermelons, which can develop the devastating vine decline disease found in Florida. Warmer, drier winters have allowed B. tabaci biotype B populations to overwinter and increase further north in CA and Tomato necrotic dwarf virus (ToNDV) only previously found in Imperial County has now been found in Kern county.

Significantly, RT-PCR tests of melon plants with yellowing symptoms from five commercial melon fields outside of the DREC were also positive for the SqVYV during the fall of 2014. Cucurbit plants from research plots at DREC (watermelon, squash, melon and cucumber), and commercial melon fields tested positive for SqVYV during the spring/summer of 2015.

Together with the fact that this virus has not been previously detected in the Imperial Valley, despite annual surveys and virus testing over the past 8 years (since the introduction of CYSDV), these results suggest that this maybe the initial introduction of this ipomovirus to the Imperial Valley.

Field infection of watermelon fruit with SqVYV SqVYV-CA strain symptoms of discoloration in mature

watermelon fruit at DREC in 2015.

California Ipomovirus Delimiting Survey A delimiting survey of the California ipomovirus was initiated by the California Department of Agriculture and the Imperial County Agricultural Commissioners Office.

California Ipomovirus Delimiting Survey

Field (UC DREC) and greenhouse (UC ANR) studies are underway to further investigate the biology of the California ipomovirus (SqVYV) and to ascertain the potential for the virus to impact cucurbit production in southern California (R. Gilbertson, T. Tian, O. Batuman, J. McCreight W. Wintermantel and E. Natwick).

Field surveys tracking the progression of the spread of the California ipomovirus in the desert southwest U.S. will continue in Imperial County and Riverside County in California and in Yuma County in Arizona (J. Palumbo, R. Gilbertson, T. Tian, O. Batuman, J. McCreight W. Wintermantel and E. Natwick)

Insecticide efficacy studies are being conducted at UC DREC for management of sweetpotato whitefly, B. tabaci biotype B and the California ipomovirus (E. Natwick & M. Lopez).

Host plant resistance in melons to the vector, B. tabaci and to viruses it transmits are ongoing at the University of California Desert Research & Extension Center (J. McCreight, W. Wintermantel and E. Natwick).

The epidemiology of viruses transmitted by leafhoppers, aphids and whiteflies viruses are ongoing in the Imperial Valley, CA (R. Gilbertson, O. Batuman, W. Wintermantel and E. Natwick).

Continuing Research at UC DREC and UC Davis

Differences in B. tabaci-transmitted virus disease symptom expression among melon accessions in plots at UC DREC

Acknowledgements

We wish to thank:

Connie Valenzuela, Imperial County Agricultural Commissioner and her staff for their kind assistance with our research

California Department of Agriculture for their assistance and cooperation with our research