Restoration outplantings of nursery-origin

Californian flora are heavily infested with

Phytophthora

Tyler B Bourret, HK Mehl, DM Rizzo, TJ Swiecki,

EA Bernhardt & JM Hillman 6th SOD Symposium, June 23, 2016

Survey of potentially infested restoration sites

Santa Clara Valley Water District (SCVWD) has routinely performed restoration with nursery-reared native Californian flora

Phytophthora species had been previously isolated from SCVWD outplantings & associated nurseries by Phytosphere (TJ Swiecki & EA Bernhardt), including P. tentaculata #5 on APHIS list of species to keep out of US

Broad survey of 31 sites by Rizzo lab (H. Mehl & T. Bourret) & Phytosphere Sep 2015 - May 2016

31 restoration sites sampled

San Jose

Sample site

36 plant species 193 samples collected Preference for symptomatic plants, especially known P. tentaculata hosts ~1 L soil + roots

Methods - soil sample processing & ID

Baited with green pears & Rhododendron leaves

Strains determined to species using morphotyping & ITS sequences

Pythium s.l. was included but not reported (Mostly Phytopythium & Pythium s.s.)

► Phytophthora recovered

Plant species Common name # samples # positive samples % samples

# Positive sites # Psp.

► Aesculus californica Buckeye 2 1 50 1 1

► Acer negundo Boxelder maple 5 3 60 2 2

► Alnus rhombifolia White Alder 2 1 50 1 1

► Artemisia californica California sagebrush 8 2 25 1 3

► Artemisia douglasiana Mugwort 23 6 26 3 16 Aster chilensis Chilean aster 3 0 0 0 0

► Baccharis glutinosa Salt marsh baccharis 1 1 100 1 2 Baccharis pilularis Coyote brush 3 0 0 0 0

► Baccharis salicifolia Mulefat 3 1 33 1 4

► Ceanothus thyrsiflorus Blue blossom 1 1 100 1 3 Clematis ligusticifolia Virgin's bower 2 0 0 0 0

► Dead plant (unknown sp.) Empty planting site 8 2 25 2 2

► Diplacus aurantiacus Sticky monkey flower 7 5 71 2 6

► Euthamia occidentalis Goldenrod 3 2 67 2 1

► Frangula californica Coffeeberry 9 2 22 2 2 Helenium bignonia Sneezeweed 1 0 0 0 0

► Heteromeles arbutifolia Toyon 18 12 67 9 5

► Juncus sp. Rush (unknown sp.) 6 5 83 4 7 Lepechinia calycina Pitcher sage 1 0 0 0 0

► Platanus racemosa Sycamore 7 1 14 1 2

► Populus fremontii Fremont cottonwood 4 2 50 1 3

► Prunus ilicifolia Holly-leaf cherry 2 1 50 1 1

► Quercus agrifolia Coast live oak 19 5 26 3 5

► Quercus douglasiana Blue oak 3 1 33 1 2

► Quercus lobata Valley oak 12 4 33 1 3 Quercus sp. Oak (unknown sp.) 1 0 0 0 0

► Quercus wislizenii Canyon live oak 1 1 100 1 1 Rhamnus crocea Redberry buckthorn 2 0 0 0 0

► Rosa californica CA wild rose 13 7 54 4 6

► Rubus ursinus CA blackberry 3 1 33 1 1

► Salicornia pacifica Pickleweed 3 2 67 1 2

► Salix sp. Willow (unknown sp.) 1 1 100 1 6

► Sambucus mexicana Blue elderberry 12 4 33 4 4 Sambucus racemosa Mexican elderberry 2 0 0 0 0

► Sambucus sp. Elderberry (unknown sp.) 1 1 100 1 2

► Symphoricarpos laevigatus Snowberry 4 1 25 1 1

► Umbellularia californica Bay laurel 1 1 100 1 2

193 total samples 74 Phytophthora- positive 27 yielded more than one species 36 plant species (28 associated with positive samples)

Plant species Common name # samples # positive samples # Psp. Phytophthora species list

Artemisia douglasiana Mugwort 23 6 16 CAS, CHL, CRA, CRY, HYD, INU, LAC, MUL, PAR, PIN, POLxMAR, ROS, MUG, OAK, THE, THExAMN

Juncus sp. Rush (unknown sp.) 6 5 7 CAC, CRY, EUR, INU, JUN, MUL, PIN

Diplacus aurantiacus Sticky monkey flower 7 5 6 CAC, CRA, EUR, INU, MEG, PIN

Rosa californica CA wild rose 12 6 6 CAC, CRA, MEG, QUM, RAM. RIP

Salix sp. Willow (unknown sp.) 1 1 6 CRA, CRY, LAC, PSE, ROS, OAK

Quercus agrifolia Coast live oak 19 5 5 CAC, CRA, OAK, SYR, WAL

Heteromeles arbutifolia Toyon 18 12 5 CAC*, CRA, CRY, HED, OAK

Baccharis salicifolia Mulefat 3 1 4 ASP, KELxCRY, ROS, OAK

Sambucus mexicana Mexican elderberry 12 4 4 CRA, PAL, QUE, RAS

Populus fremontii Fremont cottonwood 4 2 3 ACE, PSE, ROS

Ceanothus thyrsiflorus Blue blossom 1 1 3 CAC, MUL, NIC

Quercus lobata Valley oak 12 4 3 CAC, QUE, PSY

Artemisia californica California sagebrush 8 2 3 CRA, INU, PAL

Acer negundo Boxelder maple 5 3 2 CAC, MUL

Frangula californica Coffeeberry 9 2 2 CAC, MUL

Quercus douglasiana Blue oak 3 1 2 CAC, OCC

Dead plant (unknown sp.) Empty planting site 8 1 2 CAC, ROS

Salicornia pacifica Pickleweed 3 2 2 HUM, HUMxINU

Baccharis glutinosa Salt marsh baccharis 1 1 2 MUL, OAK

Umbellularia californica Bay laurel 1 1 2 MUL, OCC

Sambucus sp. Elderberry 1 1 2 MUL, ROS

Platanus racemosa Sycamore 7 1 2 ROS, OAK

Aesculus californica Buckeye 2 1 1 CAC

Alnus rhombifolia White Alder 2 1 1 CAC

Euthamia occidentalis Goldenrod 3 2 1 CAC

Prunus ilicifolia Holly-leaf cherry 2 1 1 CAC

Rubus ursinus CA blackberry 3 1 1 CAC

Symphoricarpos laevigatus Snowberry 4 1 1 CAC

Quercus wislizenii Canyon live oak 1 1 1 PIN

Aster chilensis Chilean aster 3 0 0

Baccharis pilularis Coyote brush 3 0 0

Clematis ligusticifolia Virgin's bower 2 0 0

Helenium bignonia Sneezeweed 1 0 0

Lepechinia calycina Pitcher sage 1 0 0

Quercus sp. Oak (unknown sp.) 1 0 0

Rhamnus crocea Redberry buckthorn 2 0 0

Sambucus racemosa Red elderberry 2 0 0

Many species from preferentially sampled hosts: - Mugwort - Toyon - Monkey flower - Oak spp.

Also many species from: - Willow - Juncus sp. - Mulefat

P. acerina, ACE; P. asparagi, ASP; P. cactorum, CAC, P. chlamydospora, CHL; P. crassamura, CRA; P. europaea, EUR; P. hedraiandra, HED; P. hydropathica, HYD; P. humicola, HUM; P. humicola x inundata, HUMxINU; P. inundata, INU; P. sp. kelmania, KEL; P. lacustris, LAC; P. megasperma species complex, MEG; P. multivora, MUL; P. nicotianae, NIC; P. occultans, OCC; P. palmivora, PAL; P. parsiana, PAR; P. pini, PIN; P. polonica X aff. "Maryland 8," POLxMAR; P. pseudocryptogea , PCY; P. pseudosyringae, PSE; P. psychrophila, PSY; P. quercetorum, QUM; P. quercina, QUE; P. ramorum, RAM; P. riparia, RIP; P. rosacearum, ROS; P. syringae, SYR; P. taxon casuarina, CAS; P. taxon juncus (sp. nov.), JUN; P. taxon mugwort (sp. nov.), MUG; P. taxon oaksoil, OAK; P. taxon raspberry, RAS; P. taxon walnut, WAL; P. thermophila, THE; P. thermophila

X amnicola, THExAMN *All Phytophthora positive samples included this Psp.

Phytophthora species recovered, by site

P. acerina, ACE; P. asparagi, ASP; P. cactorum, CAC, P. chlamydospora, CHL; P. crassamura, CRA; P. europaea, EUR; P. hedraiandra, HED; P. hydropathica, HYD; P. humicola, HUM; P. humicola x inundata, HUMxINU; P. inundata, INU; P. sp. kelmania, KEL; P. lacustris, LAC; P. megasperma species complex, MEG; P. multivora, MUL; P. nicotianae, NIC; P. occultans, OCC; P. palmivora, PAL; P. parsiana, PAR; P. pini, PIN; P. polonica X aff. "Maryland 8," POLxMAR; P. pseudocryptogea , PCY; P. pseudosyringae, PSE; P. psychrophila, PSY; P. quercetorum, QUM; P. quercina, QUE; P. ramorum, RAM; P. riparia, RIP; P. rosacearum, ROS; P. syringae, SYR; P. taxon casuarina, CAS; P. taxon juncus (sp. nov.), JUN; P. taxon mugwort (sp. nov.), MUG; P. taxon oaksoil, OAK; P. taxon raspberry, RAS; P. taxon walnut, WAL; P. thermophila, THE; P. thermophila X amnicola, THExAMN

Site ID Year(s) planted Total

samples Positive samples

% positive samples

Number Psp. Phytophthora spp.

GUAD6 2011-2012 3 3 100 13 CHL, CRA, HYD, INU, LAC, PAR, PCY, POLxMAR, ROS, CAS, OAK, THE, THExAMN

GU1A 1994-1995 7 7 100 11 ACE, ASP, CRA, KEL, LAC, MUL, PCY, PSE, ROS, OAK, WAL

UPC 2012 8 7 88 6 CAC, CRA, INU, MEG, PAL, PIN

GRCE 2013 5 4 80 5 CAC, HED, MUL, PIN, OAK

STMC 2002 10 8 80 5 CAC, MUL, PIN, PSY, QUE, OAK

ADOB5 2008 12 6 50 4 CAC, MUL, NIC, RAS

CARA 2012 11 4 36 4 CAC, EUR, MUL, PIN

JACB 2009 6 2 33 3 CAC, MUL, OCC

STSU 2012 4 2 50 3 CAC, CRA, HED

COPAR 2006-8 5 3 60 3 EUR, MUL, JUN

PAJ 2007-8 3 3 100 3 INU, RAM, RIP

TOMA 2013 9 3 33 3 MUG, QUM, ROS

CSS 2011 8 3 38 2 CAC, PCY

STRP 2002-2003 5 2 40 2 CAC, HED

ADOB 2005 17 3 18 2 CAC, CRA

PENT 2014 3 2 67 2 CRA, SYR

COYM 1998-9 3 2 67 2 HUM, HUMxINU

JACA 2009 8 1 12 1 CAC

STWA 2013 2 2 100 1 CAC

STWB 2013 3 1 33 1 CAC

STWC 2013 5 2 40 1 CAC

TAR 2008 4 1 25 1 CAC

CCR2 1980’s 3 2 67 1 MUL

CALE 2013 6 0 0 0

CCR3 1990’s 3 0 0 0

GCBH 2013 2 0 0 0

LEND1 2011 3 0 0 0

LEND2 2009 1 0 0 0

LEND3 2009 6 0 0 0

TIC 2010 4 0 0 0

TOCA 2012 8 0 0 0

P. cactorum most

common and widespread species

Phytophthora species recovered, by site

P. acerina, ACE; P. asparagi, ASP; P. cactorum, CAC, P. chlamydospora, CHL; P. crassamura, CRA; P. europaea, EUR; P. hedraiandra, HED; P. hydropathica, HYD; P. humicola, HUM; P. humicola x inundata, HUMxINU; P. inundata, INU; P. sp. kelmania, KEL; P. lacustris, LAC; P. megasperma species complex, MEG; P. multivora, MUL; P. nicotianae, NIC; P. occultans, OCC; P. palmivora, PAL; P. parsiana, PAR; P. pini, PIN; P. polonica X aff. "Maryland 8," POLxMAR; P. pseudocryptogea , PCY; P. pseudosyringae, PSE; P. psychrophila, PSY; P. quercetorum, QUM; P. quercina, QUE; P. ramorum, RAM; P. riparia, RIP; P. rosacearum, ROS; P. syringae, SYR; P. taxon casuarina, CAS; P. taxon juncus (sp. nov.), JUN; P. taxon mugwort (sp. nov.), MUG; P. taxon oaksoil, OAK; P. taxon raspberry, RAS; P. taxon walnut, WAL; P. thermophila, THE; P. thermophila X amnicola, THExAMN

Site ID Year(s) planted Total

samples Positive samples

% positive samples

Number Psp. Phytophthora spp.

GUAD6 2011-2012 3 3 100 13 CHL, CRA, HYD, INU, LAC, PAR, PCY, POLxMAR, ROS, CAS, OAK, THE, THExAMN

GU1A 1994-1995 7 7 100 11 ACE, ASP, CRA, KEL, LAC, MUL, PCY, PSE, ROS, OAK, WAL

UPC 2012 8 7 88 6 CAC, CRA, INU, MEG, PAL, PIN

GRCE 2013 5 4 80 5 CAC, HED, MUL, PIN, OAK

STMC 2002 10 8 80 5 CAC, MUL, PIN, PSY, QUE, OAK

ADOB5 2008 12 6 50 4 CAC, MUL, NIC, RAS

CARA 2012 11 4 36 4 CAC, EUR, MUL, PIN

JACB 2009 6 2 33 3 CAC, MUL, OCC

STSU 2012 4 2 50 3 CAC, CRA, HED

COPAR 2006-8 5 3 60 3 EUR, MUL, JUN

PAJ 2007-8 3 3 100 3 INU, RAM, RIP

TOMA 2013 9 3 33 3 MUG, QUM, ROS

CSS 2011 8 3 38 2 CAC, PCY

STRP 2002-2003 5 2 40 2 CAC, HED

ADOB 2005 17 3 18 2 CAC, CRA

PENT 2014 3 2 67 2 CRA, SYR

COYM 1998-9 3 2 67 2 HUM, HUMxINU

JACA 2009 8 1 12 1 CAC

STWA 2013 2 2 100 1 CAC

STWB 2013 3 1 33 1 CAC

STWC 2013 5 2 40 1 CAC

TAR 2008 4 1 25 1 CAC

CCR2 1980’s 3 2 67 1 MUL

CALE 2013 6 0 0 0

CCR3 1990’s 3 0 0 0

GCBH 2013 2 0 0 0

LEND1 2011 3 0 0 0

LEND2 2009 1 0 0 0

LEND3 2009 6 0 0 0

TIC 2010 4 0 0 0

TOCA 2012 8 0 0 0

19 species between

two sites on the Guadalupe river

Juncus sp: P. multivora, P. pini Willow sp: P. crassamura, P. pseudocryptogea, P. lacustris, P. pseudosyringae, P. rosacearum, P. taxon oaksoil

Juncus sp: P. pseudocryptogea

Toyon: P. cactorum, P. crassamura, P. hedraiandra

Mugwort: P. chlamydospora, P. crassamura, P.

hydropathica, P. inundata, P. lacustris, P. polonica X aff. "Maryland 8", P. taxon casuarina, P. thermophila X amnicola

38 Phytophthora species

All Phytophthora species # sites # Plant species Clade P. acerina 1 1 2 P. asparagi 1 1 6 P. cactorum 15 16 1 P. chlamydospora 1 1 6 P. crassamura 5 7 6 P. europaea 2 2 7 P. hedraiandra 3 2 1 P. humicola 1 1 6 P. humicola X inundata 1 1 6 P. hydropathica 1 1 9 P. inundata 3 4 6 P. lacustris 2 2 6 P. megasperma species complex 1 2 6 P. multivora 8 8 2 P. nicotianae 1 1 1 P. occultans 1 2 2 P. palmivora 1 2 4 P. parsiana 1 1 9 P. pini 4 4 2 P. polonica X aff. "Maryland 8" 1 1 9 P. pseudocryptogea 3 4 8 P. pseudosyringae 1 2 3 P. psychrophila 1 1 3 P. quercetorum 1 1 4 P. quercina 1 2 1 P. ramorum 1 1 8 P. riparia 1 1 6 P. rosacearum 3 7 6 P. syringae 1 1 8 P. sp. kelmania 1 1 8 P. taxon casuarina 1 1 6 P. taxon juncus (sp. nov.) 1 1 ? P. taxon mugwort (sp. nov.) 1 1 ? P. taxon oaksoil 4 7 6 P. taxon raspberry 1 1 6 P. taxon walnut 1 1 6 P. thermophila 1 1 6 P. thermophila X amnicola 1 1 6

28 “good” species - 6 described since 2014 - 2 described since the project started in August 5 described but provisional 3 putative hybrids 2 novel species P. cactorum, P. multivora and P. crassamura most widespread

About 1/4 of the ~150 species of Phytophthora

All subgeneric clades represented except 5 and 10

Big, lingering question: How many of our 38 species truly came from the nurseries?

Baiting a comparable amount of bulk soil from Big Sur forests resulted in 6 species (TBB, unpublished)

A combined effort over more than a decade of nursery, landscape and riparian isolations across Europe produced a list of ~60 species (Jung et al. 2014)

So many species

P. asparagi & P. crassamura from declining “maquis”

Declining native vegetation on Maddalena Archipelago (Scanu et al. 2015)

Dieback of Quercus ilex, Asparagus albus, Cistus sp., Juniperus phoenicea, J. oxycedrus, Pistacia lentiscus and Rhamnus alaternus

Several species of Phytophthora associated

P. asparagi most commonly isolated & most virulent on Juniperus seedlings

P. crassamura and P. ornamentata described as novel species

crassamura means “thick walled” (Scanu et al. 2015)

Fig 1. Symptoms of decline on Mediterranean maquis vegetation caused by Phytophthora spp.: a. Dead and dying trees of Juniperus phoeniceae with abnormal production of epicormic shoots; b. Mature tree of Juniperus oxycedrus showing severe wilting and red discoloration; c. Young tree of J. oxycedrus with red/bronze colour of foliage over the entire crown; d. Basal phloem lesion on a juniper tree extending up from below ground level; e. Collar and root rot on a young juniper tree; f. Extensive dieback and mortality of Pistacia lentiscus across site; g. Dieback and wilting of Asparagus albus.

(Scanu et al. 2015)

Phytophthora quercina Species first isolated from declining oaks in Central & Southern Europe in the 1990s

Most common Phytophthora in declining Central European oak forests, especially where trees die rapidly and in groups

Fine-root pathogen, specific to the genus Quercus, but found in nursery soil with other plant species

Aggressive on all Quercus species tested

Balci et al. found a close relative, P. sp. ohioensis from eastern US oak stands, but P. quercina has not been isolated in North America

Declining Q. robur showing different levels of crown dieback and chlorosis (specific symptom on calcaric soils due to advanced fine root destructions caused by P. citricola and P. quercina).

Aggressiveness of P. quercina to Q. robur roots in a soil infestation test after 6 months; left: control, right: infested soil.

APHIS ranked listed P. quercina as the #1 Phytophthora species of concern for introduction into the US

P. quercina isolation in Santa Clara Co.

Isolated from four Valley Oaks (Q. lobata); site planted in 2002

Initial isolation is the only documented baiting of P. quercina by pear; subsequent isolations were only successful with Q. robur and Q. ilex leaf bait

This represents the first detection of P. quercina in the US, confirmed by USDA

Ongoing work

Follow-up, including treatment of most critical areas (SCVWD & Phytosphere taking the lead)

Collaboration with “CalPhytos” working group and other researchers

Culture-independent techniques with frozen soil samples DNA has been extracted from most soil samples

Oomycete DNA can be detected w/ specific primers in all samples tested

Systematic studies of interesting strains

Two novel species of Phytophthora were isolated

P. taxon juncus

P. taxon mugwort

Neither could be unambiguously placed into a subgeneric clade, even with multiple (up to 6) loci

The nature of the project raises important questions about their status as native or exotic pathogens

Cla

de

7

Cla

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1

Sporangia of P. taxon mugwort

The big picture

Increased regulations following SOD have been largely successful for foliar Phytophthora species, less so for soil-borne species Move differently, symptoms less diagnostic Regulated differently

Well-meaning environmental regulations may have led to poor choices for restoration in regards to root-associated Phytophthora Choice of nursery-origin transplants rather

than seeds or natural regeneration Year-round irrigation of plants adapted to

Mediterranean climates Direct movement of soil for the purposes of

moving unculturable plant-mutualistic microbes

Oomycete diversity remains undersampled at many scales

Acknowledgments

Santa Clara Valley Water District & employees

Noah Luecke & Miles lab (CSUMB)

Rino Oguchi & Valerie Wuerz, UC Davis