Proton transfer reaction-mass spectrometry as a technique to measure volatile emissions of Muscodor albus
Introduction
The endophytic fungus, Muscodor albus, produces a mixture of volatile organic compounds (VOCs) that are lethal to a wide variety of fungi and bacteria [1], [2]. This mixture of gases has been analyzed using GC/MS and consists primarily of various alcohols, acids, esters, ketones, and lipids [1]. Final verification of the identity of the VOCs were carried out by using artificial mixtures of the putatively identified compounds and showing that the artificial mixture possessed the identical retention times and mass spectral qualities as those of the fungal derived substances. Artificial mixtures of the VOCs nicely mimicked the biological effects of the fungal VOCs when tested against a wide range of fungal and bacterial pathogens [1]. Potential applications for M. albus are currently being investigated and include uses for treating various plant parts, and human wastes. Another promising option includes its use to replace methyl bromide fumigation as a means to control soil-borne plant diseases.
Although many VOCs of M. albus have been identified, the rates and amounts of VOC production have yet to be quantified. This useful information could assist in the development of formulation bases, time and rate of application and the physical conditions most desired for the use of M. albus in “mycofumigation” [3]. It may also provide information regarding what ratios and kinds of VOCs are necessary for a lethal mix. Finally, how various physical and nutritional parameters influence VOC production by M. albus may be determined and more easily monitored [4].
A potentially convenient way to determine the concentrations of the gases released by M. albus is through the use of proton transfer reaction-mass spectrometry (PTR-MS) [5]. The PTR-MS instrument ionizes organic molecules in the gas phase through their reaction with H3O+, forming mostly MH+ molecules (where M is the neutral organic molecule), which can then be detected by a standard quadrupole/multiplier mass analyzer. This process can be run on real air samples without dilution, since the primary constituents of air (nitrogen, oxygen, argon, and carbon dioxide) have a proton affinity less than water and thus are not ionized. Most organic molecules (excepting alkanes) have a proton affinity greater than water and, are therefore, ionized and detected. A further advantage of PTR-MS is by experimentally determining the reaction time, the amount of H3O+ present, and the theoretical reaction rate constant for a proton transfer reaction and ultimately the absolute concentration of constituents in a sample can be quantified [5]. The technique, to date has only seen limited use in biological applications including an examination of volatile compounds produced after wounding of leaves [6]. It seems to never have been used to study VOCs of microbes. Finally, an enormous advantage of PTR-MS is that it can be run on-line while yielding data on the concentrations of specific ions of interest continuously. This report shows how PTR-MS can be applied to the monitoring of M. albus cultures for their VOCs as well as in situ measurements of fungal VOCs in soils treated with M. albus.
Section snippets
Initial identification of the VOCs of M. albus by PTR-MS
The initial identification of the VOCs emitted by M. albus was done previously and the data reported again herein for comparison purposes [1]. For PTR-MS, two Petri plates containing 50 ml of PDA (potato dextrose agar) were prepared. The PDA contained 4 g of potato infusion, 20 glucose and 15 g agar per liter. One was inoculated with M. albus and the other served as a control. The plates were sealed and incubated at 23 °C. After 6 days of fungal growth the headspace of the two Petri plates were
Steps necessary to apply PTR-MS to the analysis of microbial VOCs
In order to effectively utilize the PTR-MS (Fig. 1) in a microbiological application it is necessary to know the identity of the volatiles being produced by the microorganism. In a previous study the principle compounds possessing antimicrobial activities were positively identified (Table 1) [1]. Once the authenticated compounds (Table 1) were obtained from commercial sources or synthesized, authentic mass spectra for most of the compounds were obtained using the PTR-MS. The major product ions
Discussion
Until now virtually all of the VOC work done on fungi has been done using GC/MS from which only qualitative and relative quantitative data have been presented [7], [8], [9], [10], [11], [12]. Since M. albus is currently in use and also is being tested as a biological control agent, it was deemed vital to begin to develop methods that would allow for the estimation of VOC concentrations under conditions in which it may be used. The application of PTR-MS to the VOCs of M. albus has potential
Acknowledgements
This research was supported by Vaadia-BARD Postdoctoral Award No. FI-321-2001 from BARD, The United States–Israel Binational Agricultural Research and Development Fund. Funding was also supplied by the National Science Foundation, The NSF–REU program at MSU, the R&C Board of the State of Montana, the USDA and the Montana Agricultural Experiment Station. Dr. Joe Sears of the MSU Department of Chemistry acquired mass spectral data using GC/MS methods.
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