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CURRICULUM VITAE

DR. CASSANDRA LYNN SWETT Department of Plant Pathology Email: [email protected]

University of California at Davis Phone: 530-902-0094

1 Shields Ave, Davis CA

________________________________________________________________________

EDUCATION

2007-2013 PhD, Plant Pathology, University of California, Davis

Advisor: Tom Gordon

2004-2007 MS, Tropical Plant Pathology, University of Hawaii, Manoa

Advisor: Janice Uchida

2000-2003 BS, Plant Sciences with Philosophy minor, University of

California, Santa Cruz

Senior thesis adviser: Greg Gilbert

1997-2000 Associate of Arts, Cabrillo Community College, Aptos, CA

EMPLOYMENT

2013 Post Graduate Researcher (April 2013 to present)

Department of Plant Pathology, University of California, Davis

PI: Doug Gubler, in collaboration with Sunview Vineyards, CA

2013 Assistant Lecturer (Fungal Ecology)

Department of Plant Pathology, University of California, Davis

2006- 2007 Research assistant

Department of Plant and Environmental Protection Sciences,

University of Hawaii , Manoa

Supervisor: Dr. Janice Uchida

PUBLICATIONS

Peer Reviewed

1. Swett, C.L. and Gordon T.R. 2012. First report of grass species (Poaceae) as naturally

occurring hosts of the pine pathogen Gibberella circinata. Plant Disease: 96 (6): 908-

908.

2. Swett, C.L., Porter, B., Fourie, G., Steenkamp, E., Wingfield, M.J., and Gordon, T.R.

2013. Grasses as reservoir hosts for the pitch canker pathogen Fusarium circinatum in

commercial pine stands in South Africa. Submitted to Southern Forests. Accepted, in

revision

3. Swett C.L. and Uchida J.Y. Characterization of emerging Fusarium diseases on

commercially grown orchids in Hawaii. Submitted to Plant Pathology

In preparation

4. Swett, C.L. and Gordon T.R. Characterizing grass symbiosis by Fusarium circinatum,

the cause of pitch canker in pines, based on colonization traits across the range of

infection conditions for two known endophytes in Zea mays. In prep of Fungal Ecology

Cassandra L. Swett Curriculum Vitae

2

5. Swett, C.L. and Gordon T.R. Cryptic infection biology of Fusarium circinatum in

Pinus radiata seedlings. In prep for Phytopathology

6. Swett, CL. and Gordon, T.R. The ecology of cryptic pathogen infection: Physiological

effects of symptomless infection by Fusarium circinatum (the cause of pitch canker) on

fitness and disease resistance of Pinus radiata seedlings. In prep for New Phytologist

Book Chapters

7. Swett, C.L. and Gordon T.R. 2012. Latent infection by Fusarium circinatum

influences susceptibility of Monterey pine seedlings to pitch canker. In: Proceedings of

the Fourth International Workshop on the Genetics of Host-Parasite Interactions in

Forestry: Disease and Insect Resistance in Forest Trees. July 31 to August 5, 2011 –

Eugene, Oregon, USA. Technical Coordinators: R.A. Sniezko, A. D. Yanchuk, J.T.

Kliejunas, K. Palmieri, J. Alexander and S.J. Frankel. U.S. Department of Agriculture,

Forest Service, Pacific Southwest Research Station, Albany, CA General Technical

Report PSW-GTR-240

8. Swett C.L., Huang M., Begovic A., Steenkamp E.T., Wingfield M.J., Gordon T.R. In

press. A new dimension to pitch canker epidemiology: Biology of Fusarium circinatum

as a grass colonist in native and managed pine systems. Proceedings of the 2012 Western

International Forest Disease Work Conference. In press (Invited contribution)

Outreach Publications

Swett, C.L and Gordon, T.R. 2013 Pitch Canker. UC ANR Publication 74107

ORAL PRESENTATIONS

2013 Invited seminar speaker, University of Florida, Gainesville. Complexity in

ecological functions of "hemi-pathogens": Looking at growth, defense

and drought tolerance effects of symptomless infection by the pitch

canker pathogen Fusarium circinatum in Pinus radiata seedlings.

2013 APS-MSA Annual Meeting. Special Session: Plant Pathologists of the

Future: Showcasing the Top Graduate Students from APS Division

Meetings. Dualism in symbiosis: Growth and defense enhancement of

symptomless infection by the pathogen Fusarium circinatum in Pinus

radiata seedlings.

2012 Western International Forest Disease Work Conference (WIFWIC). A new

dimension to pitch canker epidemiology: Biology of Fusarium circinatum

as a grass colonist in native and managed pine systems

2012 American Phytopathological Society (APS) Annual Meeting. Induced

resistance to pitch canker, caused by asymptomatic Fusarium circinatum

infection in seedlings of Pinus radiata

2012 APS Pacific Division. The ecology of tree-seedling immunity:

Symptomless Fusarium circinatum infection as a trigger for enhanced

pathogen resistance in Pinus radiata seedlings


3

2012 Forestry and Agricultural Biotechnology Institute (FABI) research

seminar, University of Pretoria, South Africa (invited talk)

2011 California Forest Pest Council (CFPC). Expanding the host range

of the pitch canker pathogen beyond the Pinaceae: Fusarium circinatum

as a symptomless endophyte of grasses (Poaceae)

2011 APS Annual Meeting. The cryptic dimension of host-pathogen

interactions: Physiological impacts of Fusarium circinatum infection on

symptomless Pinus radiata. (Phytopathology 101:S174)

2011 Disease and Insect Resistance in Forest Trees: Fourth International

Workshop on the Genetics of Host-Parasite Interactions in Forestry.

Latent infection by Fusarium circinatum influences susceptibility of

Monterey pine seedlings to pitch canker

POSTER PRESENTATIONS

2012 APS Annual Meeting. Grasses as a new cryptic host of the pitch canker

pathogen Fusarium circinatum.

2010 CFPC. Symptomless infection of Fusarium circinatum in Monterey pine:

Evidence for a hemibiotrophic association, and economic importance to

worldwide pine production

2009 APS Annual Meeting. Colonization of corn (Zea mays) by the pitch

canker pathogen, Fusarium circinatum: Insights into the evolutionary

history of a pine pathogen (Phytopathology 99: S126-S127)

2007 XXIV Fungal Genetics Conference. Evaluating genetic diversity of

Fusarium proliferatum from orchids in Hawaii

2006 International Mycology Conference. Four Fusarium species causing

diseases on orchids in Hawaii, including a potentially undescribed species

in the Gibberella fujikuroi species complex

2006 APS/MSA Annual Meeting. Four Fusarium species causing disease on

orchids in Hawaii

GRANTS, SCHOLARSHIPS and FELLOWSHIPS

2012 UCD and Humanities Graduate Research Award, UC Davis

2012 Raymond J. Tarleton Student Fellowship. (APS)

2011 California Native Plant Society research grant

2011 California Forest Pest Council student scholarship

2008-2010 Jastro-Shields Research Grant, UC Davis

2009 Hewitt Fellowship, UC Davis

2009 Forest Fungal Ecology Research Award (MSA)

HONORS and AWARDS

2012 Western International Forest Disease Work Conference Travel Award

2012 First Place, APS Pacific Division meeting student paper competition

2012 Gloria and Jesse Dubin Travel Award, UC Davis


4

2011 Second Place, APS Pacific Division student paper competition. APS/IPPC

meeting

2011 APS Foundation Student Travel Award

2011 APS Pacific Division Student Travel Award

2009 Leach Travel Award, UC Davis

2006 First Place, Graduate Student Poster Presentation in Plant and Fungal

Pathogens session. 8th International Mycology Congress, Cairns Australia

2006 Chancellors Excellence in Student Research Award (Masters level, 1

awarded/year campus-wide), University of Hawaii, Manoa

2006 Graduate Student Organization Student Travel Award. UH, Manoa

2006 First Place, Master’s Student Oral Presentation, 18th Annual CTAHR

Student Research Symposium, UH, Manoa

2006 The Janell Stevens Johnk and Dennis H. Hall Student Travel Award (APS)

RESEARCH COLLABORATIONS

2013 Post-doctoral collaborator with Mathew Smith, University of Florida,

Gainsville. Co-author on grant proposal to study management of truffle

production in pecan orchards.

2013 Post-doctoral collaborator with Tom Gordon, UC Davis. Biology of the

pitch canker pathogen Fusarium circinatum.

2012 Visiting Graduate Student Researcher. Forestry and Agricultural

Biotechnology Institute University of Pretoria, South Africa. Drs. Mike

Wingfield and Emma Steenkamp. Analysis of grasses as cryptic hosts of

the pitch canker pathogen in nurseries and plantations

2010-2012 Pitch canker monitor in bishop pine populations at the Point

Reyes National Seashore. Supported by the US Forest Service

2010 Visiting Graduate Student Researcher. Lawrence Berkeley National Lab,

Berkeley, CA, Gary Andersen. Microarray analysis of bacterial

endophyte community composition in Pinus radiata needles.

2006 Visiting Graduate Student Researcher. Department of Plant Pathology,

Kansas State University, Dr. John Leslie. AFLP analysis of population

diversity of Fusarium proliferatum strains from commercially grown

orchids in Hawaii

SPECIALIZED TRAINING

2005 National Fusarium Laboratory Workshop, Kansas State University

OUTREACH AND EXTENSION

2012 Workshop: methods for isolation and identification of Fusarium

species. FABI, University of Pretoria, South Africa

2012 Workgroup on the Pitch Canker Fungus in South African Pine

Production, FABI, University of Pretoria, South Africa

2011-2012 Outreach Services. Presidio National Park, San Francisco, CA Supporting

cypress canker management trials.


5

2008-2012 Disease diagnosis, UC Davis plant disease clinic

2011-2012 UC Davis, Plant Disease Clinic Co-Director, in collaboration with the UC

Davis Arboretum

2008-2011 UC Davis, Plant Disease Clinic Director, in collaboration with the UC

Davis Arboretum

2006 Orchid disease workshop, Oahu. Hawaii Orchid Growers Association

TEACHING EXPERIENCE

2012 Assistant Lecturer. Fungal Ecology (UC Davis) Average evaluations (scored out of 5.0): Your overall evaluation of the instructor: 5.0 Knowledge and command of subject matter : 4.9 Organization and clarity of presentation: 4.8 Enthusiasm for teaching and ability to make the subject interesting: 5.0

2012 Teaching assistant (TA). Mushroom Cultivation (UCD)

2011 Co-instructor. Perennial Crop Pathology (Graduate course).

Principle instructor: Dr. Bruce Kirkpatrick. (UCD)

2010-2012 TA. Mushrooms, Molds and Society (UCD)

2009 TA. Introductory Plant Pathology (UCD)

2008 TA. Introductory Mycology (UCD)

2008-2012 Guest Lecturer. (8 total). Plant pathology, mycology and disease-oriented

courses, at lower and upper division, and graduate levels.

2005 Plant Bacteriology (UH Manoa)

2004 Introductory Plant Pathology (UHM)

LEADERSHIP POSITIONS

2008-2012 Director/Co-Director, Graduate Student Plant Disease

Clinic, UC Davis (http://pdc.ucdavis.edu/)

2009- 2010 Vice President, UC Davis Plant Pathology Graduate Students

PROFESSIONAL SERVICES

2013 Professional Society (APS) and Meeting (APS-MSA). Special session

coordinator and moderator: Interactions and Mechanisms of Symptomless

Plant Symbioses

2012-2013 Professional Society (APS). Member. Mycology Committee

2012-2013 Professional Society (APS). Member. Phyllosphere Microbiology

Committee

2012-2013 Manuscript review: Plant Disease, Plant Pathology

2012 Meeting (APS). Session moderator

2009-present University. Internship mentor. Present total: 10 interns.

Typical project length: 2-6 quarters (maximum: 9 quarters)

2011 Meeting (Disease and Insect Resistance in Forest Trees). Session

Moderator


6

2011 Professional Society (APS). Member. Committee for

Entrepreneurship and Innovation

2011 Department. Member. Graduate program application committee

2009 Meeting (MSA). Volunteer

2011 University. Member. Student Travel Award Committee

2009-2012 Department. Coordinator. Summer Social

2009- 2010 Department. Member. Curriculum Committee

2009, 2010 Department. Member. Retreat Committee

2009, 2010 Department. Co-coordinator. Prospective Student Open House

2007 Meeting (ESA). Volunteer

2007 Professional Society (APS). Student Travel Award Committee

2004- 2006 University. Member. Graduate Grant Review Committee

PROFESSIONAL AFFILIATIONS

Mycology Society of America

American Phytopathological Society (APS) and APS Pacific Division

REFERENCES

Primary references:

Thomas R. Gordon

Department of Plant Pathology

UC Davis, Davis, CA 95616

(530) 754-9893

[email protected]

David M. Rizzo



(530) 754-9255

[email protected]

Richard M. Bostock



(530) 752-0308

[email protected]

W. Doug Gubler



(530) 752-0304

[email protected]

Plant Disease June 2012, Volume 96, Number 6 Page 908 http://dx.doi.org/10.1094/PDIS-02-12-0136-PDN Disease Notes

First Report of Grass Species (Poaceae) as Naturally Occurring Hosts of the Pine Pathogen Gibberella circinata

C. L. Swett and T. R. Gordon, Department of Plant Pathology, University of California, Davis 95616

Gibberella circinata (anamorph Fusarium circinatum) causes pitch canker in pines and is not known to have any hosts outside the Pinaceae. However, G. circinata is closely related to and interfertile with G. subglutinans, which is associated with grasses both as a pathogen and a commensal endophyte. Furthermore, studies under controlled conditions have shown that G. circinata can colonize corn (Zea mays) without inducing symptoms (4). To determine if G. circinata can also infect grasses under natural conditions, plants were collected in proximity to trees with symptoms of pitch canker in native stands of Pinus radiata (Monterey pine) on the Monterey Peninsula and P. muricata (bishop pine) at Pt. Reyes National Seashore on the California coast during July and August of 2011. Leaves and stems were rinsed in 0.1% Tween 20, immersed in 70% ethanol for 30 s followed by 1 min in 1% NaOCl, and placed on a Fusarium selective medium (FSM) (1). Single-spore subcultures of colonies growing from cultured plant material were transferred to 0.6% KCl agar and identified as G. circinata based on morphological criteria as described by Gordon et al. (2). G. circinata isolates were recovered from Holcus lanatus and Festuca arundinacea on the Monterey Peninsula and H. lanatus at Pt. Reyes National Seashore. Three isolates from each of these sources (nine total) and one known G. circinata isolate from pines (GL 17) were tested for virulence by inoculating 1-year-old, greenhouse-grown Monterey pine trees; three trees were inoculated, once for each isolate. Trees were inoculated by depositing 250 spores in a wound on the main stem by the method described by Gordon et al. (3). Two weeks later, all grass isolates had induced resinous branch cankers with lesions comparable in length (17 to 24 mm) and appearance to those caused by GL 17. Similar results were obtained when inoculations were repeated. One isolate from F. arundinacea and one from H. lanatus (collected at Pt. Reyes National Seashore) were tested and shown to be somatically compatible with tester strains for vegetative compatibility groups C6 and C1, respectively, both of which are associated with isolates previously recovered from diseased pines (2). GL 17 and one isolate each from F. arundinaceae and H. lanatus were tested for their ability to infect F. arundinaceae cv. Fawn. For each isolate, 20 14-day-old seedlings (10 pots with two plants per pot) were sprayed to runoff with an aqueous suspension of 106 spores per ml. All inoculations were repeated once. Two weeks after inoculation, leaves and stems were rinsed briefly in 0.1% Tween 20, immersed for 10 s in 70% ethanol, followed by 30 s in 1% NaOCl, and cultured on FSM. All tested isolates were recovered from at least some

of the inoculated plants (range 20 to 100%), from living stems and leaves, as well as from senescing tissue. These results show that grass species can be symptomless hosts for G. circinata, constituting the first documentation of any host for this pathogen outside the Pinaceae. Studies are underway to further characterize the host range of G. circinata and assess the epidemiological implications of grasses as alternate hosts for the pitch canker pathogen.

References: (1) B. J. Aegerter and T. R. Gordon. For. Ecol. Manag. 235:14, 2006. (2) T. R. Gordon et al. Mycol. Res. 100:850, 1996. (3) T. R. Gordon et al. Hortscience 33:868, 1998. (4) C. L. Swett and T. R. Gordon. Phytopathology (Abstr.) 89:S126, 2009.

1

Association of the pitch canker pathogen Fusarium circinatum with grass hosts in 1

commercial pine production areas of South Africa 2

3

CL Swetta*

, B Porterb, G Fourie

b, ET Steenkamp

b, TR Gordon

a, and MJ Wingfield

b 4

5

a Department of Plant Pathology, University of California Davis, 1 Shields Ave, Davis, 6

California, USA 7

b Forestry and Agricultural Biotechnology Institute, Department of Microbiology and 8

Plant Pathology, University of Pretoria, Pretoria 002, South Africa 9

* Corresponding author, email: [email protected] 10

11

The pitch canker pathogen, Fusarium circinatum, has major impacts on production in 12

pine nurseries and plantations in South Africa. Thus far, efforts to reduce local spread 13

have focused on rouging of infected pines and sanitation to eliminate local sources of 14

inoculum. Although the host range of F. circinatum was thought to be limited to pines 15

and Douglas-fir, recent studies in California indicate that this fungus is capable of 16

infecting grasses as a symptomless endophyte. Consequently, it is possible that grasses 17

represent a reservoir of inoculum that influences the occurrence of disease in South 18

African pine nurseries and plantations. The objectives of this study were to survey a wide 19

range of grass species in both nurseries and plantations in South Africa for the presence 20

of F. circinatum. In all, 22 species of grass were sampled at a nursery in Mpumulanga 21

and in a plantation on the Western Cape. Isolates obtained from grasses were identified 22

based on morphological criteria and DNA sequence data. Fusarium circinatum was 23

2

recovered from vegetative tissues of four grass species including Briza maxima, Ehrharta 1

erecta var. erecta, Pentameris pallida, and one species that could not be identified. All 2

isolates were pathogenic to pines, and comparable in virulence to a known F. circinatum 3

isolate that was included as a positive control. These studies indicate that grasses may 4

constitute inoculum reservoirs that could facilitate infections of pines in nurseries and 5

plantations. They may also provide a means for the pathogen to move between widely 6

separated pine stands, where grass hosts occur in intervening areas. 7

8

Keywords: pitch canker, Fusarium circinatum, Pinus, Poaceae, grasses, alternate hosts 9

10

Introduction 11

12

Fusarium circinatum is one of the most destructive pathogens of pines worldwide, 13

especially on certain highly susceptible species desirable in forestry, such as Pinus 14

radiata and P. patula (Wingfield et al. 2008, Gordon 2012). This fungus has major 15

impacts on pine production in many countries, including the US, Chile, Spain and South 16

Africa (Wingfield et al. 2008, Gordon 2012). In South Africa, F. circinatum is both a 17

nursery and plantation production issue (Wingfield et al. 2008, Mitchell et al. 2011, 18

Mitchell et al. 2012). In nurseries, it is responsible for seedling mortality, primarily 19

associated with girdling lesions at the root collar (Mitchell et al. 2012, Morris 2010). In 20

plantations, it can severely reduce post-planting survival, often in association with cryptic 21

infections in planting stock (Morris 2010, Mitchell et al. 2012). In addition, trees can 22

3

suffer reduced growth and mortality from branch and trunk cankers, and in seed orchards, 1

infections can result in contaminated seed (Wingfield et al. 2008, Morris 2010). 2

3

Since its initial description in 1946, F. circinatum has been considered a specialized 4

pathogen, with all known hosts being in the Pinaceae (Pinus species and Douglas-Fir) 5

(Hepting and Roth 1946, Dwinell et al. 1985, Gordon et al. 2006). However, recent 6

studies in native P. radiata and P. muricata forests in California have revealed that F. 7

circinatum can also infect grasses (family Poaceae) within pitch canker infested stands 8

(Swett and Gordon 2012). Grass-associated isolates were shown to be pathogenic on 9

pines and somatically compatible with isolates obtained from pines (Swett and Gordon 10

2012, Swett et al. in press). Studies using Zea mays (corn) as a model system have shown 11

that F. circinatum can establish infections through both horizontal and vertical modes of 12

transmission, and is capable of infecting root, shoot and developing ear tissue (Swett and 13

Gordon 2009). Corn plants show no symptoms or measurable reduction in biomass as a 14

consequence of infection by F. circinatum. 15

16

In South Africa, known sources of inoculum in nurseries may include contaminated 17

planting trays or irrigation water, airborne inoculum from other infected pines, and/or 18

infested seed (Morris 2010). In plantations, other infected pines are considered the 19

primary inoculum source. In both systems, grass-host reservoirs of F. circinatum could 20

be significant contributors to disease development in pines. 21

22

4

The objectives of this study were to survey a wide range of grass species in both nurseries 1

and plantations for the presence of F. circinatum. If these were found, a further aim was 2

to evaluate the virulence of grass associated isolates on pines. These studies could 3

provide foundational knowledge from which to evaluate the potential impact of grasses as 4

reservoirs for the pitch canker pathogen in the country. 5

6

Materials and methods 7

8

Sampling 9

All collections were made between 11 March and 5 April 2012. Grasses were sampled 10

from two sites: (1) a nursery in Ngodwana in the Mpumalanga province, which grows 11

Pinus species (primarily P. patula) and Eucalyptus species and (2) a Pinus radiata 12

plantation near Cape Town in the Western Cape province. In the Ngodwana nursery, 13

grasses were sampled both beneath planting benches and at the periphery of the shade 14

cloth, within 3 meters of areas with recent pitch canker contamination. Within the 15

plantation, grasses were collected along roadsides and, when possible, beneath the 16

canopy, within three meters of symptomatic trees. 17

18

Grass specimens were collected only if floral structures were present (either actively 19

flowering or recently senesced). All above ground parts were collected, including 20

flowers, stalks/stems and leaves. Where possible, species of grasses were identified 21

following collection by staff of the Mercer Arboretum and Botanical Gardens, Pretoria. 22

In total, 22 species of grass were collected: 12 at the Ngodwana nursery, and 12 at the 23

5

plantation on the Cape (two species occurred at both sites), with five to ten plants of each 1

species collected at each site, for a total of approximately 200 samples. 2

3

Isolation procedures 4

Samples were stored at 4 °C and processed within two to twelve days after collection. 5

Seven to ten stems, between 10 and 30 cm in length, were processed for each species. 6

Leaves, flowers and nodes (or, if no nodes, three to five internodal segments) were 7

detached from each stem, cut into 5 cm segments and placed together in a polyvinal mesh 8

bag. Tissue in bags was rinsed in 0.1% Tween 20, surface disinfested by immersion for 9

10 seconds in 70% EtOH followed by 30 seconds in 0.1% NaOCl, and then aseptically 10

transferred to paper towels to remove residual bleach. Tissue was divided into different 11

plant parts (leaves, flowers and stems segments or nodes), aseptically placed on a 12

Fusarium selective medium (FSM) (Aegerter and Gordon 2006), and incubated at 25°C. 13

14

Morphological identification 15

Between four and ten days after preparation, all sporulating colonies were examined 16

under a light microscope at 100 x magnification for the presence of polyphialides and 17

spores in false heads, but not in chains, as described by Leslie et al. (2006). For all 18

cultures meeting these criteria, a single hyphal tip from a recently germinated spore was 19

transferred to 0.5% Potassium chloride agar, on which spores in chains could readily be 20

distinguished from false-heads. Records were also taken for all other Fusarium species 21

recovered from grasses, which could be putatively identified based on morphology. All 22

6

isolates are maintained in the culture collection (CMWF) of the Forestry Agricultural 1

Biotechnology Institute (FABI), University of Pretoria, South Africa. 2

3

DNA sequence analyses 4

The identities of all putative Fusarium circinatum isolates were confirmed by BLAST 5

search analysis against the Fusarium-ID database 6

(http://isolate.fusariumdb.org/index.php) and phylogenetic comparison with a subset of 7

members from the Gibberella fujikuroi species complex. Fungal DNA was extracted 8

from pure cultures, using the PrepMan Ultra DNA extraction kit (Applied Biosystems). 9

10

The TEF1-! region was amplified by PCR using primers EF1 and EF2 (O'Donnell et al., 11

1998), on an Applied Biosystems 2720 Thermal Cycler (reaction mixtures: ~5 ng/l DNA, 12

0.3 M of each primer, 250 M dNTPs (Fermantas, Nunningen, Switzerland), 0.04 U/l Taq 13

DNA polymerase (Roche Molecular Biochemicals, Manheim, Germany) and PCR buffer 14

with MgCl2 (Roche). Thermal cycler conditions were as follows: 5 min 95°C, followed 15

by 35 cycles at 92°C for 1 min, 53°C 1 min, and 72°C 1 min, with a final extension at 16

72°C 10 min. The amplicons were sequenced on an ABI PRISM® 377 DNA sequencer 17

(Applied Biosystems, Foster City, CA) in both directions with the original primers. 18

Taxon identity was assigned based on a BLAST match of 98% or greater homology with 19

one or more accessions in the database. Maximum likelihood analyses were performed in 20

PhyML version 2.4.3 (Guidon and Gascuel 2003) using the best fit substitution model 21

TIM2 with gama correction (Tavare 1986)as determined by j Modeltest (Posada 2008). 22

Bootstrap confidence values were based on 1000 replications. 23

7

1

Pathogenicity tests 2

Pathogenicity tests were conducted on six-month old Pinus patula seedlings, grown from 3

seed obtained from a multi-clonal, open pollinated orchard. To prepare inoculum, fungal 4

isolates identified as F. circinatum, as described above, were grown for a minimum of 14 5

days on potato dextrose agar (39 g DIFCO Bacto PDA, 1L Di H2O), after which spores 6

were suspended in 15% glycerol, and concentration adjusted to 5 x 104 spores/ml. 7

8

Seedlings were inoculated by removing shoot tips with sterile pruning shears, and placing 9

a 1 ml droplet of spore suspension on the cut surfaces (Porter et al. 2013). Following 10

inoculation, plants were maintained under greenhouse conditions and watered daily. 11

Inoculation trials were arranged in a completely randomized design, with 30 replicates 12

per isolate. Each trial included trees inoculated with the known virulent isolate, FCC 13

3579, as a positive control. As a negative control, trees were wounded as described above 14

but inoculated with 15% glycerol instead of a spore suspension. 15

16

Lesion length measurements were taken at 50 days post inoculation. Re-isolation of the 17

pathogen was accomplished by detaching the leading margin on the stem, surface 18

disinfesting the tissue in 10% NaOCl and placing it on FSM. Resulting cultures were 19

identified using F. circinatum specific primers (Schweigkofler et al. 2004). 20

21

Results 22

23

8

In total, six isolates recovered from grasses were identified as F. circinatum based on 1

morphological criteria and a TEF-1! sequence that was a 98-98.6% match (560-630 base 2

pairs) with one F. circinatum isolate in the Fusarium ID database (NRRL 26432). 3

Phylogenetic placement of these isolates within the Gibberella fujikuroi species complex 4

(GFSC) confirmed they are most closely related to F. circinatum (Figure 1). In addition, 5

of the twenty isolates putatively identified as F. circinatum, based on morphology, 6

fourteen had a TEF-1! sequence that was most similar to other Fusarium species, 7

including F. anthophilum (Fusarium-ID accession number: FD 01297) and an 8

undescribed species in the GFSC (NRRL 25807). 9

10

All six isolates of F. circinatum originated from one of four grass species: Briza maxima, 11

Ehrharta erecta var. erecta, Pentameris pallida and one unidentified species, all of which 12

were collected at the Tokai plantation (Table 1). The fungus was found in association 13

with all vegetative plants parts (leaves and stems), but was never recovered from floral 14

tissue (Table 1). No isolates were recovered from samples collected at the nursery in the 15

Mpumulanga province (Table 1). 16

17

Inoculations confirmed that all six isolates of F. circinatum were pathogenic to P. patula 18

seedlings, with symptoms and lesion lengths similar to those caused by the positive 19

control isolate (Figure 2 and 3). Fusarium circinatum was successfully recovered from 20

lesions induced by each of the six isolates (Figure 4). 21

22

Discussion 23

9

1

The results of this study have shown for the first time that grass species in South Africa 2

can be infected by F. circinatum. These findings support the earlier discovery that grasses 3

collected below infected pines in California can become infected with the pathogen 4

(Swett and Gordon 2012). The fact that F. circinatum can colonize grasses is perhaps not 5

surprising, given that it is a close relative of other Fusarium spp. that are well known 6

commensal and pathogenic associates of corn, wheat and other species in the grass family 7

(Kuldau and Yates 2000, Desjardins 2003). An important result of the present study is 8

that some of the Fusarium spp. isolated from grasses in pine plantations cannot be 9

distinguished from F. circinatum based only on morphology. It is thus imperative that 10

identifications of these fungi are based on careful DNA sequence comparisons. Given the 11

importance of making rapid and accurate identifications, a simple and reliable PCR-test 12

should be developed and verified for this purpose. 13

14

It was interesting that only grasses collected from a plantation in the Western Cape and 15

not those from the nursery in Mpumulanga were infected with F. circinatum. This is 16

possibly due to the fact that inoculum of this fungus would be more abundant in the 17

plantation than in the nursery environment. For example, a recent study (Fourie et al. 18

2013) has shown that air-borne inoculum in the nursery where the present study was 19

conducted is small and strongly localized. 20

21

More extensive surveys are needed to establish the geographic range over which 22

colonization of grasses can occur in South Africa. Furthermore, it would be useful to 23

10

know the time during the year when infections become established and to link these to an 1

understanding of the epidemiology of pitch canker. The low recovery of F. circinatum 2

from the plantation site (recovered from up to 2/10 samples per species) and absence of 3

the pathogen from samples collected at the nursery in Ngodwona indicate that more 4

intensive sampling may be needed to establish infection frequencies where population 5

density may be low. In addition, greenhouse trials with common native and introduced 6

grass species are required to better characterize the host range of F. circinatum within the 7

grass family. Such information will help to guide efforts to manage potential inoculum 8

sources in nurseries and plantations. 9

10

The risk posed by infected grasses to pine plantation forestry in South Africa and 11

elsewhere will depend on the extent to which F. circinatum can produce inoculum on 12

grass hosts. Preliminary studies have shown that F. circinatum will sporulate on 13

senescing grass tissue under controlled conditions (Swett et al. in press). If this occurs 14

under natural conditions, grasses could facilitate infection of pines in nurseries and 15

plantations. In addition, grass savannas, which are a dominant ecosystem in many areas 16

where pines are grown, could provide a bridge for spread of F. circinatum from infested 17

to un-infested stands. It is important to also recognize that the potential for F. circinatum 18

to infect species in the grass family may offer opportunities for global movement of this 19

pathogen in association with agronomic crops as well as ornamental taxa. This will be of 20

particular concern if vertical transmission occurs, allowing grain crops to serve as carriers 21

of F. circinatum. 22

23

11

Acknowledgements 1

2

We acknowledge Caroline Mashau and Lynn Fish from the Mercer Arboretum and 3

Botanical Gardens, Pretoria for identifying grass species. We are also grateful to the 4

members of the Tree Protection Co-operative Programme (TPCP) and the Department of 5

Science and Technology/National Research Foundation Centre of Excellence in Tree 6

Health Biotechnology (CTHB) for providing funding for this study. 7

8

9

10

12

1

Figure 1. Maximum likelihood tree derived from analysis of a partial Gibberella fujikuroi 2

TEF 1! dataset. Bootstrap values above 75% are indicated at nodes. 3

4

13

1

2

3

4

Table 1. Summary of grass species collected and F. circinatum recovery data 5

Grass species sampled

Locations

collecteda

F. circinatum

recovered Plant Partb

1 Avena sp. CT (-) na

2 Briza maxima CT (+) L

3 Chloris pycnothrix Ng (-) na

4 Cynosurus echinatus CT (-) na

5 Digitaria sanguinalis CT (-) na

6 Digitaria ternata Ng (-) na

7 Digitaria unkn. sp. Ng (-) na

8 Ehrharta erecta var. erecta CT (+) L, SN

9 Ehrharta rehmannii subsp.

Subspicata

CT (-) na

10 Eleusine coracana subsp.

Africana

Ng (-) na

11 Eragrostis biflora Ng (-) na

12 Eragrostis curvula CT (-) na

13 Eragrostis mexicana subsp.

Virescens

Ng (-) na

14

14 Eragrostis pilosa Ng (-) na

15 Eragrostis trichophora Ng (-) na

16 Melinis repens subsp.

Repens

Ng (-) na

17 Panicum maximum Ng (-) na

18 Paspalum dilatatum Ng, CT (-) na

19 Pennisetum clandestinum CT (-) na

20 Pentameris pallida CT (+) L

21 Sporobolus africanus Ng, CT (-) na

22 Unknown CT (+) L, SN

a Locations: Ng=Nursery in Ngodwona; CT= Plantation in Cape Town. 1

b Plant part from which F. circinatum was recovered; L = leaves and SN = stem 2

nodes. 3

4

5

6

7

8

9

10

11

12

13

15

1

2

Figure 2. Pinus patula seedlings used to test pathogenicity of Fusarium circinatum 3

isolates obtained from grasses, showing the condition of plants at the end of the trial (a), 4

(b) and symptoms on trees inoculated with a grass isolate. 5

6

7

8

9

10

11

12

13

14

15

16

17

18

(b) (a)

16

1

Figure 3. Mean lesion length 50 days after inoculation with the positive control (FCC 2

3579) and six F. circinatum isolates from grasses (C4: CMWF1232; C16: CMWF1235; 3

C34: CMWF1243; C43: CMWF1294; C64: CMWF1256; and C70: CMWF1295). 4

5

6

7

8

9

10

11

12

13

14

15

17

1

Figure 4. Amplicons obtained from DNA extracts of fungi recovered from symptomatic 2

pine tissue, using F. circinatum specific primers. M: 100 bp ladder; C4-C 70: Fusarium 3

circinatum isolates from grasses (C4: CMWF1232; C16: CMWF1235; C34: 4

CMWF1243; C43: CMWF1294; C64: CMWF1256; and C70: CMWF1295); +1 and +2: 5

known F. circinatum; -ve: negative control (ddH2O instead of DNA template) 6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

18

References 1

2

Aegerter BJ, Gordon TR 2006. Rates of pitch canker induced seedling mortality among 3

Pinus radiata families varying in levels of genetic resistance to Gibberella circinata 4

(anamorph Fusarium circinatum). Forest Ecology and Management 235:14-17. 5

Desjardins AE. 2003. Gibberella from A (venaceae ) to Z(eae). Annual Review of 6

Phytopathology 41:177–98. 7

Dwinell LD, Barrows-Broaddus JB, Kuhlman EG. 1985. Pitch Canker - a Disease 8

Complex of Southern Pines. Plant Disease 69: 270-276. 9

Fourie G, Wingfield MJ, Wingfield BD, Jones NB, Morris AR, and Steenkamp ET. 2013. 10

Culture-independent detection and quantification of Fusarium circinatum in a pine 11

seedling nursery. Southern Forests this volume 12

Geiser DM, Jimenez-Gasco MM, Kang S, Makalowski I, Veeraraghavan N, Ward TJ, 13

Zhang N, Kuldau GA, O’ Donnell K. 2004. FUSARIUM-ID v.1.0: A DNA sequence 14

database for identifying Fusarium. European Journal of Plant Pathology 110:473-15

479. 16

Gordon TR. 2006. Pitch canker disease of pines. Phytopathology, 96: 657-659 17

Gordon TR. 2012. Biology and management of Gibberella circinata, the cause of pitch 18

canker in pines. In: Alves-Santos FM and Diez J (eds), Control of Fusarium Diseases. 19

Research Signpost. pp 195-208. 20

Guidon S, and Gascuel O. 2003. A simple, fast and accurate algorithm to estimate large 21

phylogenies by maximum likelihood. Systematic Biology 52: 696-704. 22

19

Hepting GH Roth ER. 1946. Pitch canker, a new disease of some southern pines. Journal 1

of Forestry 55:742–744. 2

Kuldau GA, Yates IE, 2000. Evidence for Fusarium endophytes in cultivated and wild 3

plants, in: Bacon CW and White JF. (eds), Microbial Endophytes. Marcel Dekker Inc. 4

pp 85-120. 5

Leslie JF, Summerell BA, Bullock S. 2006. The Fusarium Laboratory Manual. Oxford: 6

Blackwell Publishing. 7

Mitchell RG, Coutinho T., Steenkamp E, Herbert M, Wingfield MJ. 2012. Future 8

outlook for Pinus patula in South Africa in the presence of the pitch canker fungus 9

(Fusarium circinatum). Southern Forests 74: 203-210. 10

Mitchell RG, Steenkamp ET, Coutinho TA, Wingfield MJ. 2011. The pitch canker 11

fungus, Fusarium circinatum: implications for South African forestry. Southern 12

Forests 73: 1-13. 13

Morris A. 2010. A review of pitch canker fungus (Fusarium circinatum) as it relates to 14

plantation forestry in South Africa. Forest research, Shaw Research Centre, Sappi. 15

O’Donnell K., Kistler HC, Cigelnik E and Ploetz RC. 1998. Multiple evolutionary origins 16

of the fungus causing Panama disease of banana: Concordant evidence from the 17

nuclear and mitochondrial gene genealogies. Proceedings of the National Academy of 18

Science of the United States of America 95: 2044-2049. 19

Porter B, Wingfield MJ, and Coutinho TA. 2013. Evaluation of techniques to screen pine 20

seedlings and cuttings for tolerance to infection by Fusarium circinatum. Southern 21

Forests this volume 22

20

Schweigkofler W, O’Donnell K, Garbelotto M. 2004 Detection and quantification of 1

Fusarium circinatum, the casual agent of pine pitch canker, from two California sites 2

by using a real-time PCR approach combined with a simple spore trapping method. 3

Applied and Environmental Microbiology 70: 3512--3520. 4

Swett C. and Gordon T. 2009. Colonization of corn (Zea mays) by the pitch canker 5

pathogen, Fusarium circinatum: Insights into the evolutionary history of a pine 6

pathogen. Phytopathology 99: S126-S127. 7

Swett CL Gordon TR. 2012. First Report of Grass Species (Poaceae) as Naturally 8

Occurring Hosts of the Pine Pathogen Gibberella circinata. Plant Disease 96: 908-9

908. 10

Swett CL, Huang M, Begovic A, Steenkamp ET, Wingfield MJ, Gordon TR. In press A 11

new dimension to pitch canker epidemiology: Biology of Fusarium circinatum as a 12

grass colonist in native and managed pine systems. Proceedings of the 2012 Western 13

International Forest Disease Work Conference. 14

Tavare S. 1986. Some probabilistic and statistical problems in the analysis of DNA 15

sequences. Lectures on Mathematics in the Life Sciences 17:57-86. 16

Wingfield MJ, Hammerbacher A, Ganley, RJ, Steenkamp ET, Gordon TR, Wingfield 17

BD, Coutinho TA. 2008. Pitch canker caused by Fusarium circinatum—a growing 18

threat to pine plantations and forests worldwide. Australasian Plant Pathology 37: 19

319-334. 20

21

22

Proceedings of the 4th International Workshop on Genetics of HostParasite Interactions in Forestry

159

Latent Infection by Fusarium circinatum Influences Susceptibility of Monterey Pine Seedlings to

Pitch Canker

Cassandra L. Swett1 and Thomas R. Gordon1 Pitch canker, caused by Fusarium circinatum, is a serious disease affecting Pinus radiata D. Don (Monterey pine) in nurseries, landscapes, and native forests. A typical symptom of pitch canker is

canopy dieback resulting from girdling lesions on terminal branches (Gordon et al. 2001). More

extensive dieback can result from coalescing lesions on large branches or on the main stem of the tree.

The severity of disease depends, in part, on susceptibility of the individual tree. Some will suffer no

more than a few infected branch tips, whereas others sustain extensive damage and may ultimately

die from the disease, often in conjunction with other forms of stress. However, some trees that

become severely diseased eventually recover, with the absence of new infections attributed to

systemic induced resistance (Gordon et al. 2011). To date, induced resistance in Monterey pine has

been examined only in mature trees, but the disease can also affect seedlings, with potentially

significant impacts on regeneration. Although the pitch canker pathogen can be a cause of mortality

in seedlings, those that are not killed may remain infected without showing symptoms (Gordon et al.

2001, Storer et al. 2001). The present study was undertaken to determine if seedlings with

symptomless infections manifest systemicinduced resistance to pitch canker.

To establish symptomless infected seedlings, seed was sown in sand infested with either 100 or

1,000 propagules per gram, referred to as the low and high inoculum treatments, respectively. Control

seedlings were grown in noninfested sand. Six months after sowing, symptomless seedlings

representative of each treatment were challenge inoculated by depositing a suspension of 1.25 x 104

spores per ml into a 1.0 mm diameter wound on the main stem. Susceptibility to pitch canker was

quantified as the length of the lesion developing at the site of inoculation.

The results showed that resistance was significantly increased in seedlings previously exposed to

the pathogen (P < 0.001). Stem lesions were 32 to 54 percent shorter than controls in the low inoculum induction treatment and 63 percent shorter in the high inoculum treatment (fig. 1). In

addition, a greater proportion of plants appeared healthy in the high inoculum treatment, compared to

untreated plants (P = 0.033) (fig. 2).

Figure 1—Lesion sizes on inoculated trees (n=60) 19 days after inoculations. 1 Department of Plant Pathology, University of California, Davis, CA 95616. Corresponding author: [email protected].

GENERAL TECHNICAL REPORT PSWGTR240

160

Figure 2—Percent of plants (n = 60) appearing healthy 19 days after inoculation. Similar results were obtained in experiments using 18monthold seedlings, suggesting that

systemicinduced resistance can persist as seedlings mature. Together, these results indicate that

symptomless root infections can induce systemic resistance in seedlings, potentially enhancing

survival rates.

The growthdefense balance hypothesis predicts that increased expression of secondary metabolic

pathways associated with disease resistance will decrease allocation of resources to growth. Contrary

to this prediction, plant growth was not reduced in induced plants (fig. 3).

Figure 3—Effect of induced resistance on plant growth. This is the first study to describe systemicinduced resistance in tree seedlings, and offers insight

into the ecological role of Fusarium circinatum as an endophyte. If subsequent studies confirm these findings, we aim to determine if similar effects can be documented to occur under natural conditions.

If so, it will be of interest to know what factors determine whether infections at the seedling stage

result in death or a longer lasting association that may enhance resistance to subsequent challenge by

the pitch canker pathogen.

Proceedings of the 4th International Workshop on Genetics of HostParasite Interactions in Forestry

161

Literature Cited Gordon, T.R.; Kirkpatrick, S.C.; Aegerter, B.J.; Fisher, A.J.; Storer, A.J.; Wood, D.L. 2011. Evidence for the natural occurrence of induced resistance to pitch canker, caused by Gibberella circinata, in populations of Pinus radiata. Forest Pathology. 41: 227–232.

Gordon, T.R.; Storer, A.J.; Wood, D.L. 2001. The pitch canker epidemic in California. Plant Disease. 85: 1128–1139.

Storer, A.J.; Wood, D.L.; Gordon, T.R.; Libby, W.J. 2001. Restoring native Monterey pine forests in the presence of an exotic pathogen. Journal of Forestry. 99: 14–18.

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