Optimization of the Headspace Solid-Phase Microextraction Gas Chromatography for Volatile Compounds Determination in Phytophthora Cinnamomi Rands

Phytophthora cinnamomi (P. c) is a plant pathogenic oomycete that is capable of damaging plants in commercial production systems and natural ecosystems worldwide. The most common methods for the detection and diagnosis of P. c infection are expensive, elaborate and time consuming. This study was carried out to examine whether species specific and life cycle specific volatile organic compounds (VOCs) can be absorbed by solid-phase microextraction fibers and detected by gas chromatography that are produced by P. c and another oomycete Pythium dissotocum. A headspace solid-phase microextraction (HS-SPME) together with gas chromatography (GC) method was developed and optimized for the identification of the VOCs released by P. c. The optimized parameters included type of fiber, exposure time, desorption temperature and desorption time. Optimization was achieved with the analytes of P. c+V8A and V8A alone. To perform the HS-SPME, six types of fiber were assayed and compared: 7μm Polydimethylsiloxane (PDMS), 100μm Polydimethylsiloxane (PDMS), 50/30μm Divinylbenzene/CarboxenTM/Polydimethylsiloxane DVB/CAR/PDMS), 65μm Polydimethylsiloxane/Divinylbenzene (PDMS/DVB), 85μm Polyacrylate (PA) fibre and 85μm CarboxenTM/ Polydimethylsiloxane (Carboxen™/PDMS). In a comparison of the efficacy of the fibers, the bipolar fiber DVB/CAR/PDMS had a higher extraction efficiency than the other fibers. An exposure time of 16h with DVB/CAR/PDMS fiber in the sample headspace was enough to reach the maximum extraction efficiency. A desorption time of 3min in the GC injector with the desorption temperature of 250°C was enough for the fiber to desorb the compounds of interest. The chromatograms and morphology study confirmed that the VOCs from P. c+V8A had distinct differences from V8A alone, as did different life cycle stages of P. c and different taxa such as Pythium dissotocum. The study proved that P. c has species and life cycle specific VOCs, which in turn demonstrated the feasibility of this method as means of

Authors:

• Rui Qiu
• Giles Hardy
• Dong Qu
• Robert Trengove
• Manjree Agarwal
• YongLin Ren

Keywords:

• Gas chromatography
• headspace solid-phase microextraction
• optimization
• volatile compounds.

References:

[1] A. R. Hardham, "Pathogen profile Phytophthora cinnamomi," Mol. Plant.
Path., vol. 6, no. 6, pp. 589-604, Nov. 2005.
[2] R. D. Rand, "Streepkanker van Kaneel, veroorzaakt door Phythophthora
cinnamomi n. sp. (Stripe canker of cinnamon caused by Phytophthora
cinnamomi n. sp.)," Meded. Inst. Plantenziekten, vol. 54, pp. 1-53, 1922.
[3] D. M. Cahill, J. E. Rookes, B. A. Wilson, L. Gibson and K. L. McDougall,
"Turner Review No. 17. Phytophthora cinnamomi and Australia-s
biodiversity: impacts, predictions and progress towards control," Aust J
Bot., vol. 56, no. 4, pp. 279-310, Jun. 2008.
[4] S. R. Shea, B. L. Shearer, J. T. Tippett and P. M. Deegan, "Distribution,
reproduction, and movement of Phytophthora cinnamon on sites highly
conducive to jarrah dieback in south Western Australia," PLANT DIS.,
vol. 67, pp. 970-973, 1983.
[5] G. M. Waterhouse, F. J. Newhook and D. J. Stamps, "Present criteria for
classification of Phytophthora," in Phytophthora: its biology, taxonomy,
ecology, and pathology, D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao,
Eds. St. Paul, MN, APS, 1983, pp. 139-147.
[6] L. Ferraris, F. Cardinale, D. Valentino, P. Roggero and G. Tamietti,
"Immunological discrimination of Phytophthora cinnamomi from other
Phytophthorae pathogenic on chestnut," J. Phytopathol., vol. 152, no. 4,
pp. 193-199, Apr. 2004.
[7] J. Schn├╝rer, J. Olsson and T. Börjesson, "Fungal volatiles as indicators of
food and feeds spoilage," Fungal Genet Biol. J., vol. 27, no. 2-3, pp.
209-217, Jul-Aug. 1999.
[8] F. Van Lancker, et al., "Use of headspace SPME-GC-MS for the analysis
of the volatiles produced by indoor molds grown on different substrates,"
J. Environ Monit., vol. 10, no. 10, pp. 1127-1133, Oct. 2008.
[9] P. M. Miller, "V8juice agar as a general purpose medium for fungi and
bacteria," Phytopathology, vol. 45, pp. 461-462, 1955.
[10] S. Risticevic, H. Lord, T. G├│recki, C. L. Arthur and J. Pawliszyn,
"Protocol for solid-phase microextraction method development," NAT
PROTOC, vol. 5, no. 1, pp. 122-139, Jan. 2010.
[11] B. R. Grant, W. Greenaway and F. R. Whatley, "Metabolic changes
during development of Phytophthora palmivora examined by Gas
Chromatography/Mass Spectrometry," J. Gen Microbio., vol. 134, no. 7,
pp. 1901-1911, Mar. 1988.