Sniffing Cryptic Infections: Progress Towards Characterizing the Volatile Signature of Phytophthora-infected Nursery Plants

Transcription

1 Sniffing Cryptic Infections: Progress Towards Characterizing the Volatile Signature of Phytophthora-infected Nursery Plants Richard Bostock Mitch McCartney, Tatiana Roubtsova, Mei Yamaguchi, Takao Kasuga, Susan Ebeler, and Cristina Davis Department of Plant Pathology USDA ARS, Davis Department of Viticulture and Enology Department of Mechanical and Aerospace Engineering UC Davis California Forest Pest Council Annual Meeting University of California, Davis November 15-16, 2017

2 Sudden Oak Death (ramorum blight) Phytophthora ramorum

3 Ramorum blight in a commercial nursery Photos: Jonathan Jones, APHIS-PPQ; Jennifer Parke, Oregon State Univ.

4 SOD/ramorum blight trace-forwards and positive detections July 2004 Map: USDA APHIS-PPQ

5 Detection of Phytophthora species in nursery ornamentals Problem of cryptic, or asymptomatic, infections Time? J. Parke Rhododendron plants cv. Cunningham s White six months after inoculation with Phytophthora ramorum (65% of inoculated plants were positive) Numerous other Phythophthora species of concern! Roubtsova and Bostock, (in preparation)

6 Detection of Phytophthora species in nursery ornamentals AVAILABLE METHODS DIRECT: soil/root/leaf samples or leaf baits on selective medium are time-consuming PHYTOPHTHORA GENUS-SPECIFIC IMMUNOASSAYS (e.g., Agdia, others) rapid, sensitive and reliable, are limited to infected host tissue MOLECULAR DIAGNOSTICS (PCR-BASED) provide high sensitivity and specificity but rely on presence of pathogen DNA in the sample A method that is field portable, non-invasive, and highly sensitive could aid inspection and certification of nurseries and shipments at ports of entry

7 Detection of Phytophthora species in nursery ornamentals OTHER METHODS Phytophthora-infected plants present a distinguishing volatile signature as evidenced by work with canines that can detect diseased material. Can trained canines meet the demand or tolerate all the environmental regimes where detection is needed? Scientific basis? THUNDER

8 Detection of Phytophthora species in nursery ornamentals Developing of VOC Fingerprinting Profile Based on technical advances in sample collection and analytical platforms for detection of volatile organic compounds (VOC) Twister sorbent bar (Gerstel, Inc.) GC/MS

9 Detection of Phytophthora species in nursery ornamentals Objectives Identify chemical biomarkers for detecting asymptomatic Phytophthora infections in nursery ornamentals. Specific objective: identify a Phytophthora signature characteristic of P. ramorum infections in Rhododendrons A method to detect areas of cryptic infections/infestations at critical control points in complex nursery environments to guide direct sampling of material for species-level detection and diagnosis

10 Basic sampling formats

11 Experimental Design: HSSE-GC/MS Headspace Sorptive Extraction (Twisters ) Made of polydimethylsiloxane (PDMS) Unspecific sorption of apolar compounds Image of Twister: gerstel.com All other images: wikipedia.org

12 Experimental Design: SBSE-GC/MS Stir-bar Sorptive Extraction (Twisters )

13 Results: Rhododendron VOC Profile - HSSE 78 total compounds putatively ID ed Farnesene Caryophyllene Ledol Pinene Plant 17 Plant 22 Plant 30 Plant 519 Compound AVG RSD AVG RSD AVG RSD AVG RSD ß-Elemene enantiomer % % % % γ-maaliene % % % % Naphthalene-D % % % % (E,E)-α-Farnesene % % % % (E,Z)-α-Farnesene % % % % Humulene % % % % γ-muurolene % % % % (+)-epi-bicyclosesquiphellandrene % % % % α-pinene % % % % α-selinene % % % % Caryophyllene % % % % Sandaracopimaradiene % % % % cis-ß-ocimene % % % % ß-Thujene % % % % Ylangene % % % % (+)-ß-Selinene % % % % Furan, 3-(4-methyl-3-pentenyl) % % % % (-)-ß-Elemene % % % % Naphthalene, decahydro-1,1,4α-trimethyl-6-methylene-5-(3-methyl-2,4-pentadienyl)-, [4aS- (4a.alpha.,5.alpha.,8a.beta.)] % % % % ß-Myrcene % % % % 3-Hexen-1-ol, acetate, (Z) % % % % (+)-Sabinene % % % % cis-linaloloxide % % % % Kaur-16-ene, (8.beta.,13.beta.) % % % % Eucalyptol % % % % α-dendrolasin % % % % Methyl salicylate % % % % Linalool % % % % Kaur-16-ene % % % % α-bulnesene % % % % cis-3-hexenyl-.alpha.-methylbutyrate % % % % Eremophilene % % % % Germacrene B % % % % α-cubebene % % % % (+)-Cyclosativene % % % % trans-ß-ocimene % % % % γ-terpinene % % % % Furfural % % % % δ-elemene % % % % n-valeric acid cis-3-hexenyl ester % % % % (-)-α-cadinene % % % % Pyridine, 3,4-dimethyl % % % % δ-limonene % % % % α-terpineol % % % % Alloaromadendrene % % % % α-guaiene % % % % Hibaene % % % % Camphene % % % % o-cymene % % % % trans-linalool 3,7-oxide % % % % Selinα-3,7(11)-diene % % % % Naphthalene, decahydro-1,6-bis(methylene)-4-(1-methylethyl)-, (4.alpha.,4a.alpha.,8a.alpha.) % % % % Neointermedeol % % % % Isogermacrene D % % % % α-terpinolene % % % % 1-Octen-3-ol % % % % (1S,4aR,7R)-1,4α-Dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthalene % % % % Cyclohexanemethanol, 4-ethenyl-.alpha.,.alpha.,4-trimethyl-3-(1-methylethenyl)-, acetate, [1R- (1.alpha.,3.alpha.,4.beta.)] % % % % Styrene % % % % 1-Octanol % % % % γ-ionone % % % % (-)-Cyperene % % % % Elemene % % % % α-calacorene % % % % Camphor % % % % α-thujene % % % % ß-Elemene % % % % Globulol % % % % Ledol % % % % trans-linalool oxide (furanoid) % % % % (-)-Phyllocladene % % % % (-)-Atisirene % % % % 3-Hexen-1-ol, (E) % % % % RSD: (16.53 ± 3.36)% Elemene Eucalyptol Kaur-16-ene n-valeric acid cis-3-hexenyl ester

14 % Composition of VOC Profile Results: Rhododendron VOC Profile 75.0% 62.5% 50.0% 70.4% At 4 months after inoculation, only 3 compounds significantly different, and lower than the controls 37.5% 25.0% 12.5% 0.0% 11.1% 10.4% 4.3% 2.6% 0.7% (C 15 H 24 ) (C 10 H 16 ) (C 20 H 32 )

15 Results: Rhododendron VOC Profiles by HSSE and SBSE SBSE HSSE 24 SBSE 115 VOCs HSSE 78 VOCs

16 SBSE HSSE Results: Rhododendron VOC Profiles by HSSE and SBSE Noninoc Mock Inoc Inoculated (E)-3-Hexen-1-ol cis-3-hexenyl pentanoate Linalool Heat map of 33 differentially expressed (p 0.05) Rhododendron volatiles between healthy (non- and mock-inoculated) and asymptomatic P. ramoruminoculated plants Camphene Octen-3-ol Pinocarvone Cintronellol cis-3-hexenyl pentanoate Caryophyllene ,4-Dimethyl-3-(3-methylbut-3-enylidene)-2-methylenebicyclo [4.1.0]heptane ,9-Dehydroneoisolongifolene β-gurjurene β-vatirenene Isogermacrene D Alloaromadendrene β-chamigrene γ-muurolene (+)-ß-Selinene α-selinene Ledene ,5,9,10-Dehydroisolongifolene Caryophyllene oxide I (+)-Spathulenol II Ledol Globulol Ledene oxide (+)-Selin-7(11)-en-4α-ol Agarospirol Sesquiterpene oxide I Aristolone Diterpene I Diterpene II Labd-14-ene Diterpene III

17 Experimental Design: SPME-GC/MS Water Runoff Samples 69 d.p.i. Solid Phase Microextraction 84 d.p.i. Results: Four unique VOCs in water runoff of P. ramorum-inoculated Rhododendrons CAS# KI (Calc) KI (Lit) Compound (Z)-11-Hexadecenoic acid (Z)-9-Hexadecenoic acid Cyclic octaatomic sulfur (Z)-9-Octadecenoic acid

18 Detection of Phytophthora species in nursery ornamentals Preliminary data are encouraging. Water runoff samples suggest unique VOCs are produced. Seeking support to continue investigations, determine specificity, and to test this in the field within the containment nursery at NORS-DUC Publication: McCartney, M. et al. Effects of Phytophthora ramorum on volatile organic compound emissions of Rhododendron using gas chromatography-mass spectrometry. Analytical and Bioanalytical Chemistry (accepted)

19 Acknowledgements Thanks to Dr. Russ Bullock and APHIS-PPQ for funding our pilot study, and to USDA-NIFA/NPDN (R. Bostock) and NSF (C. Davis) for partial support