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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
Cassandra L. Swett Curriculum Vitae
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
Cassandra L. Swett Curriculum Vitae
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.
Cassandra L. Swett Curriculum Vitae
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
Cassandra L. Swett Curriculum Vitae
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
David M. Rizzo
Department of Plant Pathology
UC Davis, Davis, CA 95616
(530) 754-9255
Richard M. Bostock
Department of Plant Pathology
UC Davis, Davis, CA 95616
(530) 752-0308
W. Doug Gubler
Department of Plant Pathology
UC Davis, Davis, CA 95616
(530) 752-0304
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
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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
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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
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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
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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.