Intraspecific groups of Claviceps purpurea associated with grass species in Willapa Bay, Washington, and the prospects for biological control of invasive Spartina alterniflora

Biological Control 34 (2005) 170–179 www.elsevier.com/locate/ybcon

IntraspeciWc groups of Claviceps purpurea associated with grass species in Willapa Bay, Washington, and the prospects for biological control of invasive Spartina alterniXora Alison J. Fisher a,¤, Joseph M. DiTomaso a, Thomas R. Gordon b a b

Department of Plant Sciences, One Shields Avenue, University of California, Davis, CA 95616-8755, USA Department of Plant Pathology, One Shields Avenue, University of California, Davis, CA 95616-8755, USA Received 23 January 2005; accepted 29 April 2005 Available online 14 June 2005

Abstract Spartina alterniXora is a salt marsh halophyte introduced to the PaciWc Coast of the United States that has become a noxious weed in Willapa Bay, Washington. A sap-feeding insect has been released as part of a biological control program, which has established at multiple sites. A useful complement to this program would be a biological agent that attacks seed, to reduce expansion of the infestation by seedling recruitment. One possibility is the Xoral-infecting, fungal pathogen Claviceps purpurea, which causes the ergot disease. This species is comprised of three intraspeciWc groups, of which one is speciWc to salt marsh habitats, G3. Based on random ampliWed polymorphic DNA markers, all three intraspeciWc groups of C. purpurea were found on grasses in Willapa Bay. The incidence of ergot on Spartina was very low over the three-year sampling period. Genetic diversity was low among G3 isolates, suggesting it may have been recently introduced to the region. Greenhouse tests showed S. alterniXora from Washington to be as susceptible to C. purpurea as S. alterniXora from the Atlantic Coast, where ergot has reached epidemic levels. Neighbor-joining analysis of ampliWed fragment length polymorphism markers suggests that G3 C. purpurea in Washington is most closely related to Southeastern USA G3 C. purpurea. Pairwise comparisons as part of AMOVA showed that Willapa Bay isolates are diVerent from those of all other geographic regions except Argentina. The low intensity of disease in Washington may be due, in part, to a G3 ergot population that is not well adapted to conditions in this area.  2005 Elsevier Inc. All rights reserved. Keywords: Biological control; Spartina alterniXora; Claviceps purpurea; Ergot; Willapa Bay; Host speciWcity; Biological invasion

1. Introduction Spartina alterniXora Loisel. (Poaceae) is a highly invasive salt marsh macrophyte that has successful colonized almost 2000 hectares of mudXats in Willapa Bay, Washington (Garcia-Rossi et al., 2003). In this estuary, previously devoid of vegetation, S. alterniXora eliminates open mudXat habitat to the detriment of shorebirds, Wsh, and invertebrates, including economically important oysters.

*

Corresponding author. Fax: +1 510 559 5737. E-mail address: [email protected] (A.J. Fisher).

1049-9644/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2005.04.014

This noxious weed is currently the focus of an integrated pest management program that incorporates a combination of mechanical (Hedge et al., 2003; Major et al., 2003), chemical (Kilbride et al., 1995; Patten, 2003), and biological controls (Grevstad et al., 2003). The current focus of the biological control program, Prokelisia marginata (Van Duzee), is a planthopper feeding on the vegetative parts of S. alterniXora. To supplement this insect, additional agents, including seed predators, are currently being investigated. One such organism is the fungal pathogen Claviceps purpurea (Fr.) Tul. C. purpurea, commonly know as ergot, is a nonsystemic Xoral pathogen present on S. alterniXora throughout its native range on the Atlantic Coast

A.J. Fisher et al. / Biological Control 34 (2005) 170–179

of North America (Eleuterius and Meyers, 1974; Mobberley, 1956). Historically studied as an agricultural pathogen, C. purpurea also includes strains speciWc to grasses in salt marsh habitats. This group is referred to as ‘G3’ or salt marsh C. purpurea (Paqoutová et al., 2000). This habitat-specialized group within C. purpurea may have potential as a biological control agent for the control of S. alterniXora in Willapa Bay. Spartina alterniXora was introduced to Willapa Bay, Washington in the late 19th century from the Atlantic Coast of the United States (USA), presumably from seed that originated in the northeastern USA (Davis et al., 2004; Stiller and Denton, 1995). Population growth remained relatively stable until the mid 1900s when increased seed recruitment led to rapid expansion (Feist and Simenstad, 2000; Sayce, 1988). MudXat colonized by S. alterniXora increased by 60% between 1994 and 1997 according to aerial photographs (Reeves, 1999). Expansion into uninvaded areas of the bay occurs by seedling recruitment (Feist and Simenstad, 2000) resulting from either outcrossing or self-fertilization (Davis et al., 2004). Once seedlings become established, plants grow vegetatively until separate individuals coalesce to form Spartina meadows; as density of plants increases, so does seed production, the result of a weak Allee eVect (Davis et al., 2004). The importance of seed recruitment in establishing new populations, relative to rhizome fragmentation, has also been described in another estuary invaded by S. alterniXora, the San Francisco Bay, California. High rates of seedling establishment in the San Francisco estuary are due to high seed production, high seed germination and little competition at lower intertidal elevations (Callaway and Josselyn, 1992; Daehler and Strong, 1994). In the Wrst use of classical biological control to manage an invasive exotic grass, P. marginata was introduced to Willapa Bay in 2000. Since its introduction, P. marginata has become established at multiple sites and reduced plant biomass by 50% in Weld cages (Grevstad et al., 2003). In greenhouse studies, S. alterniXora from Willapa Bay shows less resistance to herbivore attack than S. alterniXora from the Atlantic Coast and San Francisco Bay, increasing the potential for agent eVectiveness (Daehler and Strong, 1997). In this study, Willapa Bay S. alterniXora exposed to P. marginata averaged 12 and 30% of the aboveground biomass of herbivore-free control plants. By comparison, S. alterniXora from San Francisco Bay exposed to P. marginata averaged 77 and 83% of the biomass of herbivore-free controls and plants from Maryland had the same aboveground biomass as controls. Reduced resistance to herbivores is unique to Willapa Bay S. alterniXora, which has grown herbivore-free for over a century. However, variability in resistance could lead to failure of control over time (Garcia-Rossi et al., 2003). Regardless, a seed predator is needed as a supplement to the insect herbivore to decrease episodic seed recruitment.

171

Claviceps purpurea (ascomycete) is a parasite of grass gynoecia. When a Xoret becomes infected, the fungus replaces the ovary with sphacelial tissue that later develops into a sclerotium, or ergot. Infected Xowers are unable to produce seed. Three groups within C. purpurea are recognized: G1, on terrestrial grasses; G2, in ‘wet and shady’ habitats, and G3, in salt marsh habitats on cordgrass species of the genus Spartina (Paqoutová et al., 2000). Distinct from G1, the pathogen of cereal grains, G3 ergot showed distinct asexual spore size, and alkaloid proWle and could be diVerentiated using random ampliWed polymorphic DNA (RAPD) markers. In addition, G2 can be diVerentiated from G1 and G3 because it lacks an EcoRI site in the 5.8S rDNA. While RAPDs are used to diVerentiate G1, G2, and G3, ampliWed fragment length polymorphism (AFLP) markers diVerentiate G3 populations from diVerent geographic regions (Fisher et al., 2005). Along with C. purpurea found in intermediate habitats (G2), G3 sclerotia Xoat in deionized water, an adaptation presumably to aid in survival in aquatic and riparian habitats. Although G3 C. purpurea is not host speciWc under controlled conditions (Paqoutová et al., 2002a), this group has a very limited host range in the Weld, where it has been identiWed only on Spartina species and the closely related saltgrass Distichlis spicata (L.) Greene (Fisher et al., 2005). The current known ecological host range of G3 is narrow, however more research is necessary to conWrm there is little potential for spread to commercially important turfgrasses, which are grown throughout the PaciWc Northwest. High rates of disease caused by C. purpurea have been observed in both native and invasive Spartina populations. An epidemic has been described on S. alterniXora from the Atlantic Coast of the USA (Eleuterius and Meyers, 1974) and on S. anglica Hubbard in Poole Harbour in the south of England (Gray et al., 1990; Raybould et al., 1998). Spartina foliosa Trin. on the outer coast of the San Francisco Bay supports a heavy infestation as well (A. Fisher, personal observation). In addition to documenting high rates of infection, Eleuterius (1970) estimated that C. purpurea infection reduced seed production by 68% in impacted marshes. Gray et al. (1990) observed that C. purpurea on S. anglica went from rare or unnoticed in the 1960s to epidemic levels in the late 1980s, with all populations averaging 85% infected by 1988. During these years seed production was often very low. A comparison of infected and uninfected inXorescences in 1985 showed that infected inXorescences produced 21% less seed than uninfected inXorescences. Combining the data from Gray et al. (1990) with new data from the 1990s, Raybould et al. (1998) determined that >10% of S. anglica Xorets within an inXorescence must be infected before seed production is reduced. In S. foliosa, even low rates of infection by G3 C. purpurea reduce both seed quantity and seed quality (A. Fisher, unpublished data). In a review of the

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eVects of pathogens in wild plant populations, Jarosz and Davelos (1995) concluded that ergot is unlikely to impact Spartina population growth in established cordgrass estuaries with little area for new colonization because of the limited role of seed recruitment. On the other hand, in Willapa Bay, where thousands of hectares of mudXats are available for Spartina colonization and seed recruitment is a major contributor to population growth, the role of Xoral pathogens such as ergot could be decisive. The objectives of this study were to: (i) determine which of the three subgroups of C. purpurea are present in Willapa Bay, Washington, (ii) characterize the population of C. purpurea on S. alterniXora in terms of genetic diversity and its relationships to geographically distinct populations of this species, (iii) determine if S. alterniXora from Washington is as susceptible to G3 C. purpurea as S. alterniXora from the Atlantic Coast, and (iv) quantify the rates of infection in Willapa Bay.

bered isolates were obtained as pure cultures from S. Paqoutová (Institute of Microbiology, Czech Republic) including: G1 isolates 165, 204, 374, 428, and 478; G2 isolates 236 and 434; and G3 isolates 500 and 538 (Paqoutová et al., 2000, 2002b). All other isolates listed in Table 2 were cultured and analyzed by Fisher et al. (2005).

2. Materials and methods

2.2.2. EcoRI analysis The polymerase chain reaction (PCR) was used to amplify the region containing 5.8S rDNA, using ITS1 and ITS4 primers (White et al., 1990) as described in Fisher et al. (2005). PCR was carried out using a PCT100 thermocycler (MJ Research, Boston, MA, USA) with the following protocol: an initial denaturing step at 94 °C (2 min) followed by 34 cycles of 94 °C (30 s), 55 °C (30 s), and 72 °C (1 min), and a Wnal elongation step at 72 °C (7 min). Following digestion, products were separated by electrophoresis on 1.5% agarose gels, which were stained with ethidium bromide and photographed under UV illumination.

2.1. Isolate collection and culturing Willapa Bay isolates of C. purpurea and their origin are listed in Table 1. Sclerotia were collected during the fall of 2001 and 2002 and brought to University of California, Davis (UC Davis) for culturing. Sclerotia were surface sterilized, cultured, dried, and refrigerated as described by Fisher et al. (2005). Isolates included in previously published analyses are listed in Table 2. Num-

Table 1 Host and origin of Weld-collected isolates from Willapa Bay, Washington used in this study Code G1 WFA 1 WLS 1 WLL 1 G2 WAB 1 WAB 2 WCN 2 WDG 1 WDS 1 WHS 1 WPA 1 G3 WKL 1 WNC 1 WTA 1 WWR 1

Origin

Year collected

Host

Nahcotta Nahcotta Leadbetter State Park

2002 2002 2001

Festuca arundinacea Lolium spp. Leymus mollis

Long Beach Long Beach Willapa Bay Leadbetter State Park Willapa River Long Beach Leadbetter State Park

2002 2002 2002 2002

Ammophila breviligulata A. breviligulata Calamagrostis nutkaensis Dactylis glomerata

2002 2002 2002

Deschampsia caespitosa Holcus lanatus C. nutkaensis

KaVe Lewis North Cove Tarlatt Slough Willapa River

2002 2002 2002 2002

Spartina alterniXora S. alterniXora S. alterniXora S. alterniXora

2.2. ClassiWcation using EcoRI and RAPD analyses 2.2.1. DNA extraction Tissue for DNA extraction was obtained by growing isolates on cellophane overlaid on PDA as described by Fisher et al. (2005). Total DNA was extracted using the methods described by Daehler et al. (1999). After DNA was recovered in solution for the Wnal time, 75
l of supernatant were withdrawn and mixed with 200
l Tris–EDTA buVer (10 mM Tris, 1 mM EDTA (pH 7.8)), and DNA concentration was quantiWed by spectrophotometry.

2.2.3. RAPD marker development RAPD primers 1CR, 206, 257 (Paqoutová et al., 2000), OPA02, OPA03, OPA04, OPA08, OPE01, OPE04, and OPE14 (Operon, Alameda, CA, USA) were used to diVerentiate between intraspeciWc groups of C. purpurea as described by Fisher et al. (2005). Polymerase chain reaction was carried out on an Eppendorf Mastercycler gradient 5331 (Eppendorf, Cologne, Germany) using a protocol with an initial denaturing step at 94 °C (4 min) followed by 35 cycles of 93 °C (20 s), 46.6 °C (1 min), and 73 °C (1 min), and a Wnal elongation step at 73 °C (6 min). PCR products were separated by electrophoresis on 1.5% agarose gels, which were stained with ethidium bromide and photographed under UV illumination. All monomorphic and polymorphic RAPD markers were scored using a binary system (zero, absent and one, present). The resulting matrix from RAPD data was used to create phenetic cladograms using unweighted pair-group method with arithmetic averaging (UPGMA) algorithms in PAUP 4.0b10. Percent similarity between isolates was calculated by dividing the

A.J. Fisher et al. / Biological Control 34 (2005) 170–179

173

Table 2 Host and origin of additional C. purpurea isolates used in this study Codea

Origin

Year collected

Host

2001 2000 2001

Secale cereale L. Secale cereale Leymus mollis

165 204 374b 428b 478

MacDoel, CA, USA Aberdeen, ID, USA L’Anse aux meadows, Newfoundland, Canada Zubli, Czech Republic Lauderdale, AL, USA Altamont, AL, USA Hohenheim, Germany Bøezno, Czech Republic

1994 1996

Poa pratensis Festuca arundinacea F. arundinacea Secale cereale Dactylis glomerata

G2 236b 434b

Vlèí Pole u Bousova, Czech Republic Phillipsreuth, Germany

Molinia coerulea L. 1998

Dactylis spp.

G3 ADI 1 ARG 1 ARG 2 ARG 3 CDE 1 CDE 2 CDE 3 CDE 4 CDE 5 CDE 6 CDE 7 CDE 8 CDE 9 CDE 10 CDE a1 CDE a3 CDE a4 CNB 21 CPE 10 CSL 1 CSL 2 CSL 3 CSL 4 CSL 5 CSL 6 CSL 7 CSL 8 CSL 9 CSL 10 CSM 10 FSA 1 GML 1 IRE 3 IRE 12 NYF 1 NYH 1 RIH 1 RIH 2 WDI 1 WLT 1 WPR 1 500b 538b

Dolphin Island, AL, USA Argentina Celpa Marsh, Argentina Argentina Celpa Marsh, Argentina Argentina Celpa Marsh, Argentina Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Point Reyes NS, CA, USA Bolinas Lagoon, CA, USA Palo Alto, CA, USA Mountain View, CA, USA Mountain View, CA, USA Mountain View, CA, USA Mountain View, CA, USA Mountain View, CA, USA Mountain View, CA, USA Mountain View, CA, USA Mountain View, CA, USA Mountain View, CA, USA Mountain View, CA, USA San Mateo, CA, USA St. Augustine, FL, USA Marsh Landing, GA, USA Dublin, Ireland Dublin, Ireland Flax River, NY, USA Hempstead Bay, NY, USA Rhode Island, USA Rhode Island, USA Willapa River, WA, USA Leadbetter State Park, WA, USA Palix River, WA USA Southriver, NJ, USA Marchwood, UK

2000 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2001 2001 2002 2002 2002 2002 2002 2002 2002 2002 2002 2001 2001 2000 2000 2001 2001 2001 2001 2001 2001 2002 2001 2002 1998 1999

S. alterniXora S. densiXora S. densiXora S. densiXora Spartina foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. alterniXora S. alterniXora S. alterniXora S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. foliosa S. alterniXora S. alterniXora S. anglica S. anglica S. alterniXora S. alterniXora S. alterniXora S. alterniXora Distichlis spicata S. alterniXora S. alterniXora S. alterniXora S. alterniXora

G1 CMD 1 IA 1 NGE 1

a

Numbered isolates were generously supplied by S. Pazoutová Institute of Microbiology, Czech Academy of Sci., Prague, Czech Republic (CR). Isolates deposited in CCF (Culture Collection of Fungi, Department of Botany, Faculty of Sciences, Charles University, Prague, CR) (Paqoutová et al., 2000, 2002b). b

174

A.J. Fisher et al. / Biological Control 34 (2005) 170–179

number of shared presence/absence characters by the total number of presence/absence characters and multiplying by 100. Analysis of molecular variance (AMOVA, ExcoYer et al., 1992) was used to partition the total genetic variation among and within populations of G3 C. purpurea. AMOVA was performed using Arlequin 2.0 (Schneider et al., 2000). 2.3. Identifying relationships among populations using AFLP markers AmpliWed fragment length polymorphism markers were developed according to the method described by Vos et al. (1995). Thermocycler conditions began with an initial denaturing step at 72 °C (1 min) followed by 94 °C (3 min). Next were 9 cycles of 65 °C (30 s) decreasing by 1 °C each cycle followed by 35 cycles at 56 °C (30 s); and a Wnal elongation step at 72 °C (1 min). A Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) was used for all AFLP ampliWcations. Following ampliWcation, reaction mixtures were loaded onto a 4% acrylamide gel buVered in TBE (1.35 M Tris, 0.45 M boric acid, 25 mM EDTA) for approximately 2 h at 1800–1900 V using a Bio-Rad Sequi-General GT Sequencing Cell (Bio-Rad Laboratories, Hercules, CA, USA), as described by Douhan et al. (2002). Gels were silver-stained using the method described by Bassam et al. (1991) and allowed to dry before visual scoring of bands. All monomorphic and polymorphic AFLP markers were scored using a binary system (zero, absent; one, present). The matrix from AFLP data was used to create a dendrogram using neighbor-joining algorithms in PAUP 4.0b10. 2.4. Testing for variation in susceptibility 2.4.1. Preparation of plants Seeds of S. alterniXora were collected in the fall of 2000 from its native range on the Atlantic Coast of the USA at two sites on Sapelo Island, Georgia (University of Georgia Marine Institute) and two sites in Florida (Levy County and Saint Augustine). Seeds and plugs of introduced S. alterniXora were collected from 11 sites throughout Willapa Bay, Washington. Seedlings and plugs were grown in a greenhouse at UC Davis. From May to November, 2002, plants were inoculated with G3 C. purpurea spores using the technique described below. After inoculations, half the plants were maintained in the greenhouse at UC Davis in central California and half were taken to a greenhouse at the Bodega Marine Laboratory, on the California coast. To increase the number of genotypes, additional plants were collected in 2002. Cuttings of S. alterniXora

were taken from plants originally collected on the Atlantic Coast of the USA at two sites on Long Island, New York and one site in North Carolina (Carteret County). Plants were inoculated from April to September 2003 and returned only to the greenhouse at UC Davis. The proportion of inoculations resulting in infection were compared between S. alterniXora from the Atlantic Coast and Willapa Bay. The null hypothesis of no association, or independence, was tested by computing the
2 statistic (Sokal and Rohlf, 1995)

where m D a + b, n D c + d, r D a + c, and s D b + d. The theoretical
2 value for 1 and 2 degrees of freedom at the 0.05 probability level are 3.84 and 5.99, respectively. Willapa Bay plants were tested for genotype diVerences in susceptibility in the summer of 2003. In October 2002, 0.5 m2 plugs of S. alterniXora were collected at Wve sites spanning the plant’s current distribution in the Bay: Jensen Spit, Leadbetter State Park, Naselle River, North Cove, and Willapa River. Each plug was separated into 10 clonal divisions at UC Davis. Plants were further divided into either two or three in March of 2003 for a total of approximately 25 clones of each genotype. One month after inoculation (see below), inXorescences were harvested. The
2 statistic was used to compare infection rates among Willapa genotypes. The theoretical
2 value for 4 degrees of freedom at the 0.05 probability level is 9.49. 2.4.2. Inoculation protocol Claviceps purpurea isolate CDE 1 from S. foliosa collected in Point Reyes, California was used for all inoculations. Stored, dried Wlter paper was used to establish PDA cultures (Fisher et al., 2005). Cultures were maintained at 24 °C for 14 days before conidia were collected in 0.5% KCl. For comparing susceptibility between S. alterniXora from the Atlantic Coast and the PaciWc Coast, plants were inoculated with a suspension of 106 spores/ml. When comparing the eVect of genotype on susceptibility (Willapa Bay S. alterniXora only) the suspension concentration was at least 2 £ 106 spores/ml. Approximately 50 spores/100
l were plated onto PDA before each inoculation to conWrm spore viability. Inoculations were accomplished by dipping S. alterniXora inXorescences into the spore suspension just prior to anthesis. Control plants were dipped into a solution that contained only 0.5% KCl. Following inoculation, inXorescences were wrapped in polyvinyl chloride (plastic wrap) and placed in a growth chamber for 24 h with day temperature of 17 °C, night 14 °C, photoperiod day length 15 h, light level Max PAR 1100 microeinsteins/ m2/s, and relative humidity 80%. After 24 h, the plastic wrap was removed and plants were returned to the

A.J. Fisher et al. / Biological Control 34 (2005) 170–179

greenhouse (see above). Average daily greenhouse temperature and relative humidity for Davis were 24 °C, and 75%, respectively; average daily greenhouse temperature and humidity for Bodega Marine Laboratory were 16 °C, and 75%, respectively. InXorescences were checked weekly for signs of infection. InXorescences with Xorets producing honeydew, the Wrst visible sign of infection, were scored as positive for the disease (Paqoutová et al., 2002a). Claviceps purpurea honeydew contains abundant conidia, asexual spores between 5 and 12
m in length, in a sugary liquid produced from plant sap. These conidia are produced asexually from sphacelia in infected Xorets. 2.5. Rates of infection on S. alterniXora in Willapa Bay Spartina alterniXora marshes around the perimeter of Willapa Bay were sampled for C. purpurea infection during fall of 2001, 2002, and 2003. Sampling dates ranged from September 23 to October 26 due to yearly variation in inXorescence development. At each site, 0.25 m2 quadrats were placed every 10 m along two randomly selected 100-m transects for a total of 20 quadrats per site. For each quadrat, the number of inXorescences with and without ergots was counted. Infection rates are deWned here as the percentage of inXorescences per quadrat with at least one ergot. At each site, a mean infection rate was calculated from infection rates for individual quadrats. The relationship between infection and inXorescence density was analyzed using ANOVA. Statistics were carried out using SAS Institute software 8.2.

3. Results 3.1. Characterization of Willapa Bay C. purpurea Random ampliWed polymorphic DNA markers showed that all three C. purpurea intraspeciWc groups, those adapted to terrestrial (G1), intermediate (G2), and salt marsh (G3) habitats are present on grasses in Willapa Bay, Washington. Willapa Bay isolates grouped with known G1, G2, and G3 samples using UPGMA supported by bootstrap values of 100, 98, and 100%, respectively (tree not shown). Comparison of Willapa Bay isolates to preidentiWed isolates showed that terrestrial (G1) C. purpurea was found on Festuca arundinacea Schreb., Lolium spp., and Leymus mollis (Trin.) Pilg.; intermediate (G2) C. purpurea was found on Ammophila breviligulata Fern., Calamagrostis nutkaensis (Presl) Steud., Deschampsia cespitosa (L.) Beauv., Dactylis glomerata L., and Holcus lanatus L.; and G3 C. purpurea was found on S. alterniXora and D. spicata. G1 and G3 isolates tested positive for the presence of an EcoRI restriction site, while G2 isolates lacked this site, consistent with distinctions among these groups described by

175

Paqoutová et al. (2000). Willapa Bay G3 isolates collected from S. alterniXora and D. spicata shared 89% of RAPD and AFLP makers, an indication of low genetic diversity in this population. 3.2. Identifying relationships among isolates using AFLP markers Neighbor-joining analysis resulted in a dendrogram in which G3 C. purpurea from Washington, California, and the Southeastern USA (Alabama, Florida, and Georgia) were in the same cluster, with 95% bootstrap support (Fig. 1). AmpliWed fragment length polymorphism data are consistent with RAPD results that grouped isolates from Willapa Bay with known G1, G2, and G3 isolates, earning bootstrap values of 100, 99, and 100%, respectively (Fig. 1). An AMOVA of G3 populations showed signiWcant genetic variation among geographic regions (Fst D 0.846, P D 0.000) (Table 3). A large proportion of the genetic variation was partitioned among (84.6%), rather than within groups (15.4%). Pairwise comparisons showed that Willapa Bay isolates were signiWcantly diVerent from populations in all other geographic regions except Argentina (Table 4). 3.3. Comparing susceptibility to C. purpurea infection Spartina alterniXora from Washington and the Atlantic Coast were equally susceptible to C. purpurea infection in our greenhouse studies. There was no diVerence in infection rates between Atlantic Coast plants (
2 D 1.87) or Washington plants (
2 D 0.49) brought to UC Davis or Bodega Marine Laboratory in 2002, so data from the two locations were combined. There was no diVerence in susceptibility between S. alterniXora plants from the Atlantic Coast and Willapa Bay in either 2002 (
2 D 0.005) or 2003 (
2 D 0.03) (Table 5). In addition, there was no diVerence in susceptibility among Wve S. alterniXora genotypes collected in Willapa Bay (
2 D 7.57, P < 0.05) (Table 6). 3.4. Infection rates in the Weld Field incidence of C. purpurea infections ranged from 0 to 7.3% throughout Willapa Bay over the three years sampled (Table 7). The average rate of infection was 2.8% (SD D 2.1) in sites where infected inXorescences were observed. Palix Meadow had the highest rates with 7.3% of inXorescences infected in 2002. In 2001, one heavily infected inXorescence (>10 ergots per inXorescence) was observed at Palix Meadow. This was the only highly infected inXorescence sampled in any site. C. purpurea was absent during most sampling periods. Eight sites sampled had no infection. Three of the Wve sites sampled for three consecutive years (North Cove, Lead-

176

A.J. Fisher et al. / Biological Control 34 (2005) 170–179

Fig. 1. Neighbor-joining analysis on AFLP generated markers from three intraspeciWc groups of Claviceps purpurea (G1, G2, and G3). Bootstrap values (>90%) show clusters formed by isolates from California, Washington, Alabama, Florida, and Georgia.

Table 3 Analysis of molecular variance (AMOVA) for 67 isolates of G3 Claviceps purpurea from six geographic regions Sources of variation

df Sum of Variance % Total Fst squares

Among populations 5 320.586 10.218 Within populations 41 76.073 1.855

84.63 15.37

P value

cence density and infection over all 620 quadrats. For the 40 quadrats with at least one infected inXorescence, there was no relationship between inXorescence density and the proportion of inXorescences infected per quadrat (P < 0.3738).

0.846 0.00

4. Discussion better State Park, and Palix River) varied from low to no infection. 3.5. Pattern of infection Too few infected inXorescence were identiWed to determine if there was a relationship between inXores-

All three intraspeciWc groups of C. purpurea are present in Willapa Bay, Washington. Isolates collected on S. alterniXora were consistently identiWed as G3 C. purpurea according to RAPD and AFLP analyses. G3 C. purpurea on S. alterniXora from Willapa Bay was very similar in these molecular systematic indices to isolates

A.J. Fisher et al. / Biological Control 34 (2005) 170–179

177

Table 4 Pairwise genetic distances (below diagonal) and signiWcance (above diagonal) between G3 Claviceps purpurea populations Populationa

California

California Argentina Southeast USA Western Europe Northeast USA Washington

— 0.930¤ 0.876¤ 0.927¤ 0.897¤ 0.610¤

Argentina

Southeast USA

Western Europe

Northeast USA

Willapa, WA

0.000 — 0.738 0.773 0.713¤ 0.898

0.000 0.769 — 0.723 0.687¤ 0.803¤

0.000 0.769 0.231 — 0.385 0.897¤

0.000 0.000 0.000 0.077 — 0.840¤

0.000 0.769 0.000 0.000 0.000 —

a

Southeast USA includes isolates from Alabama, Florida, and Georgia; Western Europe includes isolates from UK and Ireland; and Northeast USA isolates include New Jersey, New York, and Rhode Island. ¤ SigniWcant diVerences at P D 0.01.

Table 5 Number (and percentage) of inoculated plants infected by G3 C. purpurea in 2002 and 2003a

Table 7 Percentage of inXorescences infected per quadrat in sites surrounding Willapa Bay, Washington over a three-year period

Year

Site

2001

2002

2003

Bay Center Cedar River Diamond Slough Jensen Spit KaVee Lewis Leadbetter State Park Nahcotta Naselle River Nemah River Niawiakum North Cove Palix Meadow Palix River Seal Slough Tarlatt Slough Tower Slough Willapa River

— — — — 0.8 0 0 — 0 — 4.4 5.4 3.8 — — — —

— — 0 0 0 0 0 2.4 0 — 1.0 7.3 0 0 0 0.9 2.3

0a 0 — — — 1.9 0 0 0 0.5 0 — 2.7 — 0 — —

G3 isolate CDE 1 (Infected/total)

Control (Infected/total)

S. alterniXora (Atlantic Coast) 2002 3/5 (60%) 2003 9/16 (56.3%)

0/6 (0%) 1/3 (33.3%)

S. alterniXora (Washington) 2002 14/24 (58.3%) 2003 15/28 (53.6%)

2/21 (9.5%) 1/18 (5.6%)

a Spartina alterniXora seeds and plants were collected from populations on the Atlantic Coast and on the PaciWc Coast in Washington state.

Table 6 EVect of genotype on susceptibility of S. alterniXora from Willapa Bay, Washington in 2003 S. alterniXora origin

G3 isolate CDE 1 (Infected/total)

Control (Infected/total)

Jensen Spit Leadbetter State Park Naselle River North Cove Willapa River

7/13 (53.8%) 15/22 (68.1%) 5/10 (50%) 10/18 (55.6%) 17/19 (89.5%)

0/5 (0%) 0/6 (0%) 0/3 (0%) 0/8 (0%) 0/4 (0%)

from California, which suggests PaciWc Coast isolates have a common origin. Neighbor-joining analysis indicated that PaciWc Coast G3 isolates were most closely related to isolates from the Southeastern USA. Alternatively, an AMOVA using the same data showed that G3 C. purpurea from Willapa Bay was diVerent from all other geographic regions except Argentina. These relationships suggest, but do not prove, that there was movement of infected plant material between one of these geographic regions and Willapa Bay. The presence of G3 C. purpurea in Willapa Bay leaves two options for using this pathogen as a biological control agent: augmentative or inundative control using existing genotypes or classical biological control with an introduced isolate better adapted to Northwest coastal conditions, after suYcient host range testing and analysis of risks to nontarget species such as D. spicata.

a

Sites not sampled in a particular year are marked by a dash (—).

Augmentative or inundative control would entail growing large quantities of a single genotype in culture and spraying S. alterniXora while in Xower. Large-scale Weld inoculation techniques have been successful infecting Weld grown rye (Lewis, 1945). Higher infection might be achieved with G3 genotypes better adapted to local conditions. In costal Mississippi, 96% of inXorescences were infected in 1968 with an estimated 68% reduction in seed set (Eleuterius, 1970) (Fig. 2). Sampling in all Gulf and East Coast states except New York, Connecticut, and Maine found ergot in most marshes sampled with rates of infection ranging from 10 to 100% (Eleuterius and Meyers, 1974). In Poole Harbour, an 85% infection rate was sustained for over 10 years (Raybould et al., 1998). For classical biocontrol, a Northeastern USA isolate might be better adapted to the cool Northwestern climate than the form of G3 C. purpurea currently present. Intermediate (G2) C. purpurea was found on grasses bordering the estuary and terrestrial (G1) C. purpurea was found on grasses typical of upland habitats. At Leadbetter State Park, on the north end of the peninsula, all three ergot groups were found within an area of

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A.J. Fisher et al. / Biological Control 34 (2005) 170–179

Fig. 2. Geographic range of native (circles) and introduced (squares) S. alterniXora. Spartina anglica (S. alterniXora £ S. maritima) in England is marked with a triangle. Ergot populations with published incidence levels are lettered.

about 20 m2, in close proximity: G3 C. purpurea was found on S. alterniXora, intermediate, G2, on D. glomerata and C. nutkaensis, and terrestrial, G1, on L. mollis. Spartina alterniXora grows intermixed with L. mollis at the high edge of the marsh and the species sometimes intermingle at the border. A host-range study of G3 C. purpurea showed the pathogen to be capable of infecting Arrhenatherum elatius (L.) Presl., D. glomerata, Helictotrichon pubescens (Huds.) Pilger., H. lanatus, Phalaris arundinacea L., Poa annua L., Poa nemoralis L., and Poa pratensis L. under controlled conditions (Paqoutová et al., 2002a). Our study showed that in nature, D. glomerata is infected by G2, even though G3 C. purpurea was found on adjacent S. alterniXora plants. Thus, our results suggests that the ecological host range of G3 is more limited than what might be inferred from studies in controlled environments, an inference also made by Paqoutová et al. (2000, 2002a). Similarly, studies comparing the ecological and physiological host range of insects have shown that hostrange experiments accurately predict an insect’s physiological host range but not necessarily the ecological range (Louda et al., 2003a,b; Pemberton, 2000). In the Weld, G3 has only been found on Spartina spp. and D. spicata although host-range experiments show additional grasses are susceptible. Spartina and Distichlis are closely related members of the same subfamily (Chloridoideae) and tribe (Cynodonteae). Perhaps not surprisingly, species closely related to the target are at the greatest risk of attack (Pemberton, 2000) by introduced biological control agents. Research is needed to assess the impacts of the pathogen on D. spicata. Claviceps purpurea on S. alterniXora in Willapa Bay is currently rare. Yet, our results show that S. alterniXora from Washington is highly susceptible to the pathogen under greenhouse conditions. Several factors may contribute to the observed low incidence of ergot in Willapa

Bay including suboptimal climate conditions and limitations on dispersal due to a low incidence of suitable vectors. Currently, introduced S. alterniXora in Willapa Bay hosts few resident insects (Garcia-Rossi et al., 2003; Grevstad et al., 2003). If the introduced, vascular feeding planthopper insect, P. marginata, becomes more abundant, it may become a signiWcant factor in the spread of C. purpurea within the estuary. Insect-associated movement of the pathogen would result from incidental contact with conidia (asexual spores), which are produced in great numbers in honeydew on infected plants. In the absence of insects, conidia are limited to dispersal over a shorter range via rain splash or direct contact between plants. Prokelisia marginata aggregates on inXorescences, disperses by Xight for long distance, and must be considered a prime candidate vector of G3 C. purpurea spores. Flies, wasps, and other insects attracted to cordgrass inXorescences and the honeydew produced by C. purpurea are also candidate vectors. The cool wet climate in Willapa Bay is well suited for C. purpurea infection. However, disease development requires an overlap of spore production, Xowering, environmental conditions suitable for sclerotia development, and perhaps animal vectors. This study considered factors that may be limiting disease incidence in Willapa Bay, all of which have relevance to the potential for C. purpurea to serve as an agent of biological control. If this approach is to be pursued, further study is needed to fully characterize the pathogen’s host range and its impact on seed production under Weld conditions.

Acknowledgments We thank S. Paqoutová for providing C. purpurea isolates; K. Sayce for Weld samples and plant identiWcation;

A.J. Fisher et al. / Biological Control 34 (2005) 170–179

F. Grevstad and D. Strong for comments on this manuscript; D. Ayres, G. Douhan, and D. Rizzo for training and use of laboratory equipment; H. Davis, J. Lambrinos, and J. Bando for Weld help; B. Aegerter, R. Bhat, J. Petersen, S. Kirkpatrick, and G. Kyser for research advice and analysis. This study was Wnancially supported by Jastro Shields and Humanities awards from the University of California, Davis and NSF Biocomplexity DEB 0083583 to A. Hastings, University of California, Davis.

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