Update on wilt, New Species and their Disease Potential

Krishna V. Subbarao Department of Pathology University of California, Davis Verticillium Wilts

Prior to 1995, lettuce was not considered a host of V. dahliae; even when grown in rotation with other diseased crops. of Lettuce

VERTICILLIUM WILT

Foliar Symptoms: • Angular Chlorosis • Necrosis • Wilting • Darkening of Leaf Veins • Acropetal Progression • Collapse of Head Verticillium Wilt of Lettuce

VERTICILLIUM WILT Root Symptoms: • Vascular Discoloration • Occur ~2 wk before foliar symptoms

144 Fields identified ≈ 1085 hectares infested (2,680 acres) Salinas Watsonville

King City Verticillium wilt of lettuce

• First report in 1995 – Extensive damage in Watsonville, CA • Detected in 175 fields from 1995 to 2010 • ~75 fields in 2010 alone • Incidence 70-100% • Random distribution of fields • 2-3 crops following fumigation • Often first observed by field edge • Complete plant death • Pathogen is seedborne Verticillium Wilt of Lettuce

• Monitor Verticillium wilt and soil inoculum density in coastal California. • Assess seed lots of lettuce types for V. dahliae infestation. • Assess the impact of immigrant populations of V. dahliae introduced from spinach seed into soil on crops that follow spinach. • Pre-screen seed of spinach germplasm lines V. dahliae infestation and only screen lines that are pathogen-free for Verticillium wilt resistance in the greenhouse. • Determine the role of pollen in V. dahliae transmission into seed. • Continue the breeding program to identify and develop resistance in crisphead, leaf, and other lettuce types, including germplasm screening against race 2 and screen breeding material for race 1 resistance in infested plots at the USDA Station. • Determine the potential of host-directed evolution of V. dahliae genotypes of differential virulence from a single genotype. • Continue characterization of the spinach and lettuce pathogen populations. FACTS

• is seedborne in lettuce and other crops and weeds. • It is both externally seedborne and internally seedborne. • Infested seed germinates, plants grow and develop wilt. • Seed harvested from infected plants are infested with V. dahliae. Lettuce types (2008-10)

Type Seed lots Infested Range Baby Leaf 34 0 - Crisphead 84 13 0.1-4 Romaine 38 8 0.5-2 Red Romaine 7 1 3 Green Leaf 31 2 0.5-2 Red Leaf 22 2 0.5 Butterhead 5 0 - Other/unknown 44 22 0.5-5 Total 265 48 Where produced (2008-10) Country Seed lots Infested Range USA 195 33 0.1-5 United Kingdom 3 2 0.0-4 China 22 8 0.5-3 Chile 33 5 0.5-2 The Netherlands 5 0 - Australia 7 0 - Total 265 48 Verticillium wilt of lettuce

• Exceedingly high production of microsclerotia – Calculated ~3 million microsclerotia/plant • Two races – Race 1 currently ~75% of population – Resistance for race 1 available Why did lettuce succumb to Verticillium dahliae?

• What is the source of these “apparently” novel isolates? – Endemic; Parasexual cycle (mitotic recombination)? – No teleomorph ever observed. – Exotic; Just another invasive in CA? – How diverse is the V. dahliae population in the Salinas Valley? – Did these isolates wash in from elsewhere during the 1995 floods? • Big implications for disease management & resistance durability. Verticillium Wilt of Lettuce

• Determine whether or not nitrate fertilizers used in strawberries following fumigation render soil Verticillium wilt-conducive relative to ammonium fertilizers. • Assess the impact of immigrant populations of V. dahliae introduced from spinach seed into soil on crops that follow spinach. • Sample spinach seedlings in organic and conventional production systems in Monterey, San Benito, and Santa Barbara counties and assay for Verticillium dahliae. • Continue characterization of the spinach and lettuce pathogen populations. • Determine the potential of host-directed evolution of V. dahliae genotypes of differential virulence from a single genotype. • Create a race 2-infested plot at the USDA Station. Effect of different form of Nitrogen on Verticillium wilt of lettuce

Treatments Ammonium sulfate Ammonium nitrate Calcium nitrate (60 lb N per acre)

Other essential nutrients Hoagland solution without nitrogen was prepared and applied three times per week

Pathogen Verticillium dahliae

Time of sampling for N analysis 0 days after fertigation 3 days after fertigation 7 days after fertigation Data Disease incidence Disease severity Levels of Ammonium and Nitrate in Sand

NH4 NO3

450 450 400 0 DAF 400 350 350

300 3 DAF 300 250 250

200 7 DAF 200 0 DAF 150 150 3 DAF 100 100 7 DAF

50 50

Level of NH4 (mg/kg of (mg/kg soil) NH4 Level of Level soil) NO3 Level of(mg/kg of 0 0 Ammonium Ammonium Calcium Ammonium Ammonium Calcium sulfate nitrate nitrate sulfate nitrate nitrate Effects of Nitrogen form on Verticillium wilt

100

90

80

70

60

50

40

30

20 Percent disease incidencediseasePercent

10

0 Ammonium Ammonium Calcium Control sulfate nitrate nitrate

5

4

3

2

Diseaseseverity 1

0 Ammonium Ammonium Calcium Control sulfate nitrate nitrate Verticillium Wilt of Lettuce

• Determine whether or not nitrate fertilizers used in strawberries following fumigation are a factor in rendering soil Verticillium wilt-conducive relative to ammoniacal fertilizers. • Sample spinach seedlings in Monterey, San Benito, and Santa Barbara counties and assay for Verticillium dahliae. • Assess the impact of immigrant populations of V. dahliae introduced from spinach seed into soil on crops that follow spinach. • Continue characterization of the spinach and lettuce pathogen populations. • Determine the potential of host-directed evolution of V. dahliae genotypes of differential virulence from a single genotype. • Create a race 2-infested plot at the USDA Station. Number of fields Surveyed: Monterey= 23; Santa Barbara = 25; Santa Cruz = 6; and

San Benito = 15

120 140 129 104 99 V. tricorpus (petiole) 100 120 108 V. tricorpus (root) 100 V. dahliae (petiole) 80

V. dahliae (root) spp. recovered

80 60 60 40 40 34 35 Vertiillium 13 20 20 3

No. of of No. 0 0 V. V. V. dahliae V. dahliae V. V. V. dahliae V. dahliae tricorpus tricorpus (petiole) (root) tricorpus tricorpus (petiole) (root) (petiole) (root) (petiole) (root)

Monterey Santa Barbara 16 15 14 12

10 spp. recovered spp.

8 7 6 4 2

Vertiillium 2 0 of 0 V. V. V. dahliae V. dahliae

No. No. tricorpus tricorpus (petiole) (root) (petiole) (root) San Benito

Distribution of Verticillium spp. on spinach in four California counties

spp. spp. recovered

Verticillium Number Number of

Monterey Santa Barbara Santa Cruz San Benito Verticillium spp. recovered per field

8

V. t (petiole) V. t (root) 6.93 7 6.6 V. d (petiole) V. d (root)

6

5.16 spp. spp. recovered

5 4.32 4 3.17 3

2.5 Verticillium

2 1.36 1.4 1 1 0.87 0.47 Number Number of 0.2 0.33 0 0 0.13 0

Monterey Santa Barbara Santa Cruz San Benito Acres of Spinach Grown in Monterey and Santa Barbara Counties from 1993

18000

16000

14000

12000

10000 Acres Monterey 8000 Santa Barbara

6000

4000

2000

0

2002 1993 1994 1995 1996 1997 1998 1999 2000 2001 2003 2004 2005 2006 2007 2008 2009 2010 Year PD595: anemone, Japan PD596: eggplant, Japan PD709: opium poppy, Ukraine Gibellulopsis PD711: opium poppy, Ukraine nigrescens PD710: opium poppy, Ukraine (outgroup) 100/94 PD743: potato, Canada PD712: opium poppy, Ukraine

99/100 PD341, PD610, PD611, PD612, PD613, PD660 , PD661: lettuce, CA; PD367: hairy nightshade, CA PD618: tomato, UK; PD619: soil, Canada Verticillium 96/100 PD343: artichoke, CA; PD437: lettuce, CA isaacii Figure. Phylogenetic relationships PD752, PD753: spinach seed, WA of the ten Verticillium species based PD347: artichoke, CA; PD401 , PD458: lettuce, CA Verticillium on the combined ACT, EF, GPD PD407, PD657, PD658, PD659: lettuce, CA klebahnii and TS dataset of 2658 characters PD593: potato, Japan; PD594: tomato, Japan Verticillium and 77 taxa, with Gibellulopsis 73 PD685: larkspur, Japan;PD690 : tomato, UK /- PD703: carnation, Netherlands tricorpus nigrescens as outgroup. One of the 86/100 PD586, PD740: stock, Japan; PD733, most parsimonious trees is shown. PD736 , PD737, PD738: lettuce, Japan PD731, PD734: lettuce, Japan Verticillium Species in bold are newly described 98/100 PD735: lettuce, Japan zaregamsianum in this study, strain identifiers in bold PD739: stock, Japan represent ex-type strains. Numbers Flavexudans Clade PD670: potato, WI; PD746, PD747 , Verticillium by the branches are parsimony and PD748: soil, Canada PD693: potato, UK albo-atrum Bayesian and likelihood support PD621: UK; PD702: potato, UK; PD741, values above 70. PD742 : soil, UK Verticillium nubilum

PD338, PD353: alfalfa, PA; PD489:?; PD620: Verticillium alfalfa, Canada; PD681: alfalfa, USA; PD682, 84/- PD683: alfalfa, Japan alfalfae

PD592 : potato, Japan; PD616, PD626: hop, UK; PD744: soil, Cuba; PD745: potato, Verticillium Canada; PD808, PD809, PD811: hop, nonalfalfae Slovenia; PD810: petunia, Slovenia PD348: , CA; PD356: Inderbitzin, Bostock, Davis, Platt, Usami, & horseradish, IL;PD687 : horseradish, V. longisporum allele A1 Subbarao. 2011. PLoS One: e28341 Germany 100/- PD348: cauliflower, CA Verticillium longisporum allele D1

PD356: horseradish, IL PD327: bell pepper, CA Verticillium dahliae / 95/100 PD502: maple, WI V. longisporum alleles

PD687 : horseradish, Germany FlavnonexudansClade 50 changes D2, D3 PD322 : lettuce, CA Verticillium tricorpus: Is now three species, V. tricorpus, V. isaacii and V. klebahnii, that are morphologically indistinguishable. And they may differ in pathogenicity and virulence. Which of these are present in CA?

Also of interest, V. zaregamsianum, a lettuce pathogen in Japan. It looks similar to V. dahliae, forms mostly microsclerotia, only a few brown-pigmented hyphae, and has yellow-pigmented hyphae. However, the pigmented hyphae may not be formed in culture, and thus V. zaregamsianum may be confused with V. dahliae. Or if judged solely by the yellow pigmentation, it could be confused with species in the V. tricorpus group. Is V. zaregamsianum present in CA?

All these species can be differentiated by DNA sequencing, or more easily, by PCR.

Verticillium tricorpus

Verticillium zaregamsianum Verticillium klebahnii Verticillium isaacii V. isaacii V. tricorpus V. dahliae V. klebahnii V. zaregamsianum

500 bp 500 bp

200 bp 200 bp

Actin EF-1a EF-1a Actin GPD ITS By 2 weeks PI 6 to 8 weeks PI

Determinative Phase or

Leading edge of Advancing mycelia systemic Colonization of lateral advancing into Colonization of root Systemic colonization colonization root tip the taproot via a elongation zone of lateral root along lateral root Close-up of colonized lateral taproot exhibiting root vascular Initial 48 hours PI discoloration

? 8 to 10 weeks PI

?

Microsclerotia Germinating conidium with germtube and appresorium Conidiophores ? Eruption of Advance of Discoloration of mycelia from mycelia into vascular tissues 10+ weeks PI xylem vessels taproot via in the taproot in into surrounding xylem vessels advance of cortical tissues of mycelia taproot

14+ weeks PI

Mature Colonization of Taproot colonization microsclerotia taproot and coincides with foliar Anthropogenic dispersal of embedded in microsclerotia symptom microsclerotia throughout taproot tissues development development soil

Lettuce - Verticillium dahliae Systemic spread Colonization with Internal colonization of via hyphal sporulation on achene limited to Disease Cycle budding in stem achenes in a maternal tissues; tissues dehiscent capitulum seedborne phase