A New Phytophthora Species in ITS Clade 2 Killing Ceanothus Grown for Rehabilitation of Disturbed Forest Sites Paul W

A new Phytophthora species in ITS Clade 2 killing Ceanothus grown for rehabilitation of disturbed forest sites Paul W. Reeser1, Wendy Sutton1, Everett M. Hansen1, and Ellen M. Goheen2 1-Department of Botany and Plant Pathology, Oregon State University, Corvallis OR 97331 USA, [email protected] 2-USDA Forest Service, Forest Health Protection, Central Point, OR USA.

DISCOVERY PATHOGENICITY TESTING Symptomatic ceanothus plants being grown for native landscape restoration were discovered in a Healthy seedlings of C. sanguineus and C. velutinus were used for inoculations. Isolates (three of nursery in southwestern Oregon. Container-grown Ceanothus sanguineus, C. velutinus, and C. P. taxon Ceanothus, one of P. cactorum, one of P. pini) were grown on 2% V8 agar for 8 days. Bark integerrimus showed leaf wilt associated with stem lesions developing above diseased roots. was wounded by inserting a push-pin around 6–8 cm above the soil line. A 5 mm colonized agar Stems were disinfested by wiping with 95% ethanol. Bark pieces were taken from the stem lesion plug was placed over the pin hole, sealed with Parafilm®, and wrapped in aluminum foil. Sterile 2% margin and from the root collar and plated in CARP (corn meal agar amended with 10 ppm V8 agar was used as a control. Plants were incubated in the greenhouse at ca. 20–22 C until natamycin, 200 ppm Na-Ampicillin, and 10 ppm Rifamycin SV). Colonies resembling Phytophthora symptoms started to develop. Date of wilting and lesion length were recorded, and lesions were were purified by subculturing hyphal tip selections onto fresh CARP. Purified isolates were grown sampled apically and basally to re-isolate the Phytophthora. Purified re-isolations were grown out on CMAβ (corn meal agar amended with 20 ppm beta-sitosterol) for storage (plugs in water with and on CMAβ and culture morphology was compared with isolates used for inoculation to verify identity. without hemp) and DNA extraction.

Ceanothus velutinus turnerphotographics.com

Symptoms on C. sanguineus 14 d after inoculation. Left: Symptoms on C. velutinus 14 d after inoculation. P. taxon Ceanothus. Right: Control. Left,Center: P. taxon Ceanothus. Right: Control. Ceanothus is a genus comprising around 60 species of small trees and shrubs which are important Three homothallic Phytphthora species were isolated and shown to be pathogenic to Ceanothus. components of many wildland Details of the pathogenicity test results are shown in the following table. Ceanothus sanguineus ecosystems in North America. Ceanothus integerrimus Michiganflora.net Florafinder.com Days to wilt Average lesion Host Phytophthora n 1417212431no wiltlength (mm) Isolates were identified by morphology and DNA sequence analysis using the mitochondrial cox C. sanguineus CONTROL 5 5 0 spacer region and the rDNA ITS region. A previously undescribed species, P. taxon Ceanothus, cactorum 5 3 2 139 was most frequently isolated from the infected nursery plants, as shown in the following table. taxon Ceanothus 15 4 4 5 2 167 pini 53 11 185 Ceanothus Phytophthora sanguineus integerrimus velutinus C. velutinus CONTROL 3 3 0 taxon Ceanothus 822 cactorum 3 2 1 105 pini 100 taxon Ceanothus 9 9 145 cactorum 011 pini 3 3 157

P. taxon Ceanothus MORPHOLOGY On V8 agar, the ceanothus Phytophthora produced a flame-like radiate colony reminiscent of P. citrophthora. Radial growth rate was relatively fast on V8 agar, at ca. 7 mm/d at 22 C. Sporangia produced from agar plugs placed in filtered stream water were papillate and caducous, with shapes ranging from ovoid to bizarre, including many bifurcate, trifurcate, and compound shapes similar to those depicted for P. himalsilva (Vettraino et al. 2011). Sporangiophores were simple or sympodial. Oogonia and antheridia were produced in single culture on V8 agar. Oogonia were smooth and globose, averaging around 27 µm diameter. Oospores were globose and slightly aplerotic, averaging around 24 µm diameter. Antheridia were predominately paragynous with about 20 percent amphigynous.

P. taxon Ceanothus HISTORY A search of Phytophthora DNA sequence databases in Oregon revealed that ITS sequence matching the ceanothus Phytophthora occurred in 11 isolates recovered over about 10 years from ornamental nursery plants and planting media (Grunwald, personal communication). Four of these isolates were associated with boxwood (Buxus sp.) soil and roots. In addition, isolates from Rhododendron in 2002 and boxwood in 2005 obtained from the OSU Plant Clinic were found to match the ceanothus Phytophthora by cox spacer and ITS sequence. It is interesting to note that the one GenBank deposit that exactly matches the ITS sequence of our ceanothus Phytophthora is a homothallic Phytophthora isolate from boxwood (B. sempervirens) from Poland. Perhaps there is a special association between boxwood and the ceanothus Phytophthora which should be explored.

DNA ANALYSIS ITS sequences for all P. taxon Ceanothus cultures are identical. The sequence similarity to P. citrophthora and P. himalsilva make it difficult to distinguish between them. The COX1 gene however is more useful and gives a clear separation between species.

HM752784-P.himalsilva AY129173-P.colocasiae P.taxon Ceanothus ITS HM752800-P.himalsilva COX1

AF266785-P.citrophthora P.taxon Ceanothus

AF266786-P.colocasiae HM752781-P.citrophthora Cautionary Tale

FJ237526-P.citricola”s.s.” AY129166-P.capsici This work dramatizes two challenges facing the Phytophthora community: 1. Nursery-grown plants present a high risk for carrying exotic Phytophthora species (and other AF266787-P.capsici FJ237512-P.citricola”s.s.” plant pathogens) into new landscapes (in this case P. taxon Ceanothus, P. cactorum, and P. pini). 0.01 0.01 Implementation of Best Management Practices that improve control of pathogens in nursery production should reduce the chances that new or non-native pathogens will be outplanted into 15 24 26 70 77 46 67 94 163 220 238 244 250 277 337 373 463 natural environments along with their hosts. Resource specialists charged with forest and wildland AF266785-P.citrophthora - G G T C HM752781-P.citrophthora A C T T A A T C T T A G P.taxon Ceanothus A G G - T P.taxon Ceanothus A C T T A A T A A T A A restoration must demand healthy stock from nurseries in the same manner as regeneration foresters HM752784-P.himalsilva A - - - C HM752800-P.himalsilva T T A A G T A A T A T A now demand quality from forest tree nurseries. 511 535 631 715 718 784 814 862 988 1012 1030 2. Phytophthora identification remains a challenge. P. taxon Ceanothus (homothallic) has been HM752781-P.citrophthora C T C C G A A T T T C P.taxon Ceanothus T C C C G A A A C T C mis-identified as P. citrophthora (heterothallic) by those relying only on ITS sequence. It might also HM752800-P.himalsilva T T T A T T C C C C T be mis-identified as P. himalsilva by relying only on morphology without sequencing

Acknowledgements: We thank the Oregon Department of Forestry and the USDA Forest Service, Pacific Southwest Experiment Station and Southwest Oregon Forest Insect and Disease Center for participation in, and support of, this work.