Identification of Additional Contributing to Lettuce Dieback Disease (and characterization of Lettuce dieback associated , LDaV)

William M Wintermantel USDA-ARS, Salinas, CA, USA

California Leafy Greens Research Program Annual Reseach Meeting St. Patty’s Day, 2020

What factors lead to high incidence of lettuce dieback?

• Flooding or poorly drained soil • Secondary factors that may be related to drainage issues and disease: – Elevated salinity and/or nitrates – Plant stress – Soil saturation Until recently it was believed that lettuce dieback was caused: exclusively by two tombusvirus (TBSV or MPV) resulting in infection of lettuce under specific environmental conditions and leading to disease development Two tombusviruses are known to infect lettuce and believed to cause lettuce dieback disease in CA and AZ production areas*

Moroccan pepper virus (MPV) Tomato bushy stunt virus (TBSV) • However, since 2014, many plants clearly symptomatic for lettuce dieback disease have been free of either MPV or TBSV • We have never had a 100% correlation between MPV and TBSV incidence with lettuce dieback, although in some years correlations were high (70% or greater). • This suggested another virus, perhaps a related virus in the , but more distantly related to TBSV or MPV, might be causing the more recent outbreaks, and is undetectable with current primers an antisera.

* Obermeier et al., 2001, Phytopathology 91: 797-806 The search for new pathogens associated with lettuce dieback disease • Requested lettuce dieback samples from UCCE and growers with dieback like symptoms. • Tested lettuce samples for the presence of TBSV and MPV, as well as using ‘degenerate primers’ to identify related tombusviruses and determine if other known viruses may be present. Selected those testing negative. • Passaged virus by grinding sap and manually inoculating leaves of test plants (mechanical transmission) to confirm presence of virus in tombusvirus-negative lettuce samples showing dieback symptoms. • Extracted total RNA from both lettuce and test plants and sequenced the transcriptomes (all RNA) of several symptomatic lettuces and test plants. – Compared results to all known viruses – Identified new viruses consistently associated with infection. Step 1: Test plant symptoms • Lettuce testing negative for MPV or TBSV by RT- PCR was passaged to test plants to confirm that a virus was present. • Passages from lettuce to Nicotiana benthamiana, N. clevelandii, Chenopodium quinoa, Datura stramonium, spinach and pepper often yielded infections. • Patterns were identified with certain test plants developing unique symptoms differing from those caused by TBSV and MPV. Step 2: Identifying new viruses associated with lettuce dieback • RNA sequencing (RNA-seq) was conducted by a commercial company on extracts from six selected diseased romaine plants from 2017-18. Prior to testing samples were vetted by passages to test hosts and RT-PCR to eliminate those less likely to identify causative agents. • Results yielded consistent identification of two new viruses across nearly all samples, as well as some of the same viruses seen previously. – A Soymovirus –likely aphid transmitted, but subsequent PCR did not confirm a close association with lettuce dieback disease – A virus distantly related to an obscure member of the , consistently associated with lettuce dieback disease and present in older achived lab samples Lettuce Dieback associated Virus (LDaV) 5/6 samples from the Salinas Valley, and one from Yuma were infected with a virus related to those in the family Phenuiviridae, Order . • Sequences of 6416-8347 nucleotides (large percentage) shared 31-36% identity with Watermelon crinkle leaf associated virus from China (distantly related), which was recently identified as well, but for which no biological information is available. • This means it is a distinct virus, unlike known viruses, but the genetics and consistent association with dieback suggest it may be associated with lettuce dieback disease. • The virus is mechanically transmissible based on pre- sequencing studies conducted by the Salinas lab, but vector relationships are unknown for this virus and the relative to which it shows homology. • This is the prime suspect: Nicknamed Lettuce dieback associated virus (LDaV) Detection of LDaV, the tombusviruses MPV & TBSV, and a soymovirus in field lettuce showing dieback symptoms

Number of Samples without any symptoms of LDB testing positive: LDaV 0/20 MPV 0/20 TBSV 0/20 RT-PCR evaluation of a 957 nt section of LDaV from frozen archived lettuce dieback samples in the USDA freezer

# PLANTS % YEAR % LDaV TESTED TOMBUS

PRE 2011* 17 100 29 2011 9 100 78 2012 12 100 67 • Identified 2013 4 0 75 2014 24 17 58 association 2015 11 33 91 between LDaV, 2016 6 0 67 tombusviruses, 2017 17 0 65 2018 4 0 75 and lettuce TOTAL 108 dieback disease

*Freezer RNA extracts were confirmed viable by RT-PCR re- *ALLamplification RNA EXTRACTS of the WEREtombusvirus CONFIRMED, a highly TO stable BE plant virus VIABLE BY RT-PCR RE-AMPLIFCATION OF THE TOMBUSVIRUS, A HIGHLY STABLE PLANT VIRUS LDaV Genome Organization

Lettuce Dieback associated Virus (LDaV) Genome organization showing each of the three virus RNAs (black lines) including their length in nucleosides (nts), proteins produced from the RNAs (colored boxes) including their length in number of amino acids (aa) and mass (kDa). Relationship of LDaV to other known plant viruses Alignment of conserved motifs within the virus replication protein suggest relationships with other viruses

Virus (genus) Premotif A Motif A Motif B LDaV DQHGGNREIYVL DASKWSQCH GMMQGILH End Sequences of LDaV “L RNA” indicate relationships within the Phenuiviridae

LDaV 5’ -ACACAAAGACCACCAAC...... UGGUGGGUCUUUGUGU

What does the genome tell us about LDaV? • The virus is related to members of the family Phenuiviridae, order Bunyavirales, and a broad group of viruses with icosahedral, enveloped virus particles. • Current known plant viruses in this family infect monocots (maize, rice, etc.). It is likely this virus will fall into its own genus. • Most known members of the Phenuiviridae are insect- transmitted; this does not seem highly likely for LDaV but cannot be ruled at out this time, again suggesting divergence from known members of the family. – WCLaV—a relative of LDaV, shares limited relationships to ophioviruses, including those that cause big vein and lettuce ring necrosis. Could this suggest a soil-borne vector? • Seed transmission seems unlikely based on related viruses. LDaV differs in symptoms on test plants to tombusviruses.

Progression of symptoms on N. benthamiana following passage from symptomatic lettuce Comparison of the RdRp protein sequences of LDaV with those of its closest known

19052304 Monterey relatives 13-06 Fresno County 16-18 South Monterey County

69 18-15 Monterey CTY-Santa Maria 12-26 Salinas 19053003-3 Monterey

72 Isolates Coastal 67 19092401-1 Monterey

99 19041801 Monterey Coastal and desert isolates RNA1 NGS 68 share only about 95% 19081401 Monterey 98 identity but are closely 19070307 San Luis Obispo 19052903 Greenfield related within-region 20022109 Yuma

100 18-01 Arizona 19-43 Arizona

56 20020402 Yuma 58 20-5-3 Imperial Valley 15-50 Arizona

51 Romaine12-2 Yuma

100 Isolates Desert 20-01-A Yuma 20-5-1 Imperial Valley 71 20-5-2 Imperial Valley

100 MK728654.1 Grapevine Muscat rose virus isolate K335 segment RNA1 MK728657.1 Grapevine Garan dmak virus isolate 332 segment RNA1 100 KY781184.1 Watermelon crinkle leaf-associated virus 1 isolate KF-1 segment RNA1 RdRp gene 100 KY781187.1 Watermelon crinkle leaf-associated virus 2 isolate KF-15 segment RNA1 RdRp gene Genetic comparisons 957 nt RdRp gene region of LDaV among isolates collected from coastal regions with original Salinas Valley isolate Sample Number Date Location LDaV Tombus % identity 19041801 4/18/19 Monterey + - 98.93 19052304 5/23/19 Monterey + - 99.41 19052903 5/29/19 Greenfield + - 96.92 19053003-3 5/30/19 Monterey + - 99.17 San Luis 19070307 7/3/19 Obispo + - 97.39 19081401 8/14/19 Monterey + - 99.05 19092401-1 9/24/19 Monterey + - 99.05 12_26 Salinas + 99.05 13_06 Fresno County + 99.41 16-18 S. Mntry Cty + 99.29 Monterey CTY-Santa 18-15 Maria + 99.41 MEAN 98.92 Genetic comparisons 957 nt RdRp gene region of LDaV among isolates collected from desert regions with original Salinas Valley isolate Sample Number Date Location LDaV Tombus % identity Romaine 12-2 12/2/19 Yuma + - 94.91 20020402 2/4/20 Yuma + - 94.79 20022109 2/22/20 Yuma + - 94.67 Imperial 20-5 1 02/21/2020 Valley + 94.79 Imperial 20-5 2 02/21/2020 Valley + 94.79 Imperial 20-5 3 02/21/2020 Valley + 94.91 20-01-A 01/16/2020 Yuma + 94.91 15-50 Arizona + 94.67 18_01 Arizona + 95.38 19-43 Arizona + 94.79 MEAN 94.86 Determining if LDaV directly causes lettuce dieback disease

Symptoms induced by LDaV Symptoms of lettuce rub-inoculation to romaine in dieback on LDaV-positive the greenhouse* romaine in the field

*Only transmitted once. Need to repeat multiple times. Next Steps: • Continue evaluation of additional fields for LDaV, TBSV and MPV and determine further sequence variability among isolates (yes, we still want samples). • Isolate the new virus from N. benthamiana and re- introduce to lettuce to prove causative relationship between LDaV infection and disease. Need to increase transmission efficiency as it is low with current methods. • Determine performance of Tvr1 gene against LDaV in direct greenhouse tests. • What impact do field environmental conditions have on disease development and accumulation of LDaV in lettuce? Downstream research: • Develop antiserum for routine detection of LDaV. – Not in proposal, but plan to begin this work to facilitate identification • Assuming LDaV is a causative agent for lettuce dieback or “the real” causative agent, what is the role of MPV and TBSV, which were widely associated with this disease through 2014. • Does LDaV have a vector? What would it be? How does the virus get into lettuce plants in the field? Partners and Appreciation

Bill Wintermantel Lab Project Partners and USDA-ARS, Salinas Cooperators • Art Cortez • Steve Koike, UCCE and TriCal • Laura Jenkins Hladky • Richard Smith, UCCE • Vivian Camelo • Sarah Mosakowski • Several company representatives, • Aaron Rocha PCAs, and growers • Misael Saenz Rodriguez • Dimitre Mollov, USDA-ARS, • Jeff Wasson Beltsville

• Zhangjun Fei Lab, Boyce Thompson Institute, Ithaca, NY Thanks to the California Leafy Greens Research Program for the Support!