Synthesis and Chemistry of Agrochemicals IV - American Chemical

Chapter 39

Antimicrobial Properties of Natural Volatile Compounds 1

1,2

2,3

T. R. Hamilton-Kemp , C. T. McCracken, Jr. , R. A. Andersen , and D. F. Hildebrand 2

Downloaded by MICHIGAN STATE UNIV on February 21, 2013 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0584.ch039

1

2

Department of Horticulture and Department of Agronomy, University of Kentucky, Lexington, KY 40546 Agricultural Research Service, U.S. Department of Agriculture, University of Kentucky, Lexington, KY 40546 3

Macerated foliage from several plant species including tomato emits volatile compounds which inhibit germination of pollen and growth of hyphae of the economically important plant pathogenic fungi Alternaria alternata and Botrytis cinerea. The compounds emitted from the wounded tomato tissue consist of lipoxygenase -lyase derived aldehydes and alcohols and also terpene hydrocarbons as shown by Tenax headspace vapor trapping and GC-MS analysis. Unsaturated aldehydes such as (E)-2-hexenal, at sub-μmol / L air concentrations, inhibited fungal growth in bioassays whereas the terpenes tested did not. The aldehydes appear to account for the antifungal activity exhibited by vapors from the wounded leaf tissue. Lipoxygenase-lyase derived volatile compounds also inhibited growth of plant pathogenic bacteria (Pseudomonas species) and E. coli. (E)-2-Hexenal was active against Pseudomonas syringae pv angulata at a sub-μmol / L air concentration. (E)-2-Hexenal was relatively toxic to Salmonella typhimurium tester strains in the Ames test but did not appear to be mutagenic when tested in the vapor phase. Possible applications of volatile compounds, which are human dietary constituents, to reduce microbial populations on foods and plants are discussed.

It is well-known that volatile organic compounds are ubiquitous in plant species and are emitted by leaves, flowers, fruits, stems, and roots. Hundreds of volatile compounds have been identified as plant components including substances such as alcohols, aldehydes, ketones, esters, lactones, ethers, amines, and carboxylic acids. These compounds are important constituents of food flavor (2,2) and are thought to function in biological processes such as pollination (3) and host-pest 0097-6156/95/0584-0449$12.00/0 © 1995 American Chemical Society

In Synthesis and Chemistry of Agrochemicals IV; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


450

SYNTHESIS AND CHEMISTRY OF AGROCHEMICALS IV

interactions including the growth of pathogenic microorganisms on plants (4,5). The role of volatiles in flavor chemistry and physiology has been investigated more thoroughly than their roles in pollination or plant-pest interactions. In this chapter an overview is presented of several studies which we have conducted and published (6-8) on the inhibitory effects of natural volatile compounds, emittedfrom"wounded" leaves, on the germination of pollen and the growth of certain fungi and bacteria. The studies summarized herein were initiated as a follow-up to a report by French and coworkers (9) that certain synthetic volatile compounds stimulated germination of pollen from several species of pine. We investigated the effects of volatile compounds emitted from both intact and crushed flowers and leaves on germination of apple pollen. None of the plant material tested had an effect on pollen, except for crushed or wounded leaves, which partially or completely inhibited germination. Since economically important fungi and bacteria which cause serious diseases may come in contact with vapors from damaged or wounded plant material, studies were conducted to determine the effects of commonly occurring natural volatile compounds on representative pathogenic microorganisms which attack plants and their products. Experimental Bioassays - Pollen, Fungi, Bacteria. The bioassay system has been described previously (7, 8). Briefly, it consisted of a 5 cm Petri dish containing a block of water agar placed within a 9 cm Petri dish. Apple pollen grains or fungal (Alternaria altemata, Botrytis cinerea) spores were placed on the surface of the agar block. Leaves or flower petals, crushed or intact, were placed in the 9 cm dish around the edges of the 5 cm dish. Solutions of synthetic compounds were tested by placing them in a sample dish contained within the 9 cm dish. The lid of the 9 cm dish was then placed over the assembly (120 ml volume). Pollen germination or fungal hyphal length after an exposure period (1.5 hrs for pollen; 5-10 hrs for fungi) was measured using a microscope with a net micrometer ocular. Bacterial bioassays using Pseudomonas syringae pv angulata, P.s. pv tabaci and E. coli TB 1 were carried out as above except that overnight broth cultures were placed on nutrient agar. Bacteria exposed to compounds for 22.5 hrs were serially diluted, plated, and the number of colony forming units was counted. Isolation of Volatile Compounds. The method for isolation of headspace compounds has been described earlier (6). Briefly, crushed tomato leaves were placed in a flask and high purity air was used to entrain the emitted volatile compounds which were subsequently trapped on Tenax. The Tenax was washed with hexane and the tomato components were then separated on a polar capillary column using GC. Compounds were identified by GC-MS analysis and cochromatography. Direct headspace quantitation of components in the atmosphere within the Petri dish bioassay system (fitted with a septum in the lid) was achieved by


39. HAMILTON-KEMP ETAL.

Natural Volatile Compounds

451


withdrawing an air sample using a syringe. The sample was analyzed directly using a GC with a capillary column. A similar sampling method was used to measure the concentration of (E)-2-hexenal in the Ames bioassay desiccator system described below. Ames Bioassay. Five strains of histidine deficient Salmonella typhimurium, TA 97a, TA 98, TA 100, TA 102, and TA 104, were used in the bioassays which were carried out following the procedures of Maron and Ames (70) as modified for volatile compounds using a 9 L desiccator according to Simmon (77). 2Aminofluorine, activated with S-9 was used as a positive control for the tester strains. Results and Discussion Plant Vapors and Pollen Germination. The flowers and the intact leaves bioassayed did not affect apple pollen germination. However, vapors emitted by crushed tomato, apple, and strawberry leaves did inhibit germination whereas chrysanthemum leaf components did not (6,12). Five grams of 'Mountain Pride* tomato leaves completely inhibited pollen germination during the 90 minute bioassay whereas the control pollen germinated (data not shown). Identification of Volatile Compounds from Crushed Tomato Leaves. To determine the types of volatile compounds which were emitted from damaged leaves that inhibited pollen germination, a dynamic headspace apparatus was set up to isolate the leaf vapors. Compounds were entrained in air and trapped on Tenax to obtain large enough quantities for analysis by gas chromatography-mass spectrometry. A chromatogram of the compounds emitted from tomato leaves is shown in Figure 1. Sixteen compounds were identified (Table I) which fall into two main categories. These comprise six-carbon aldehydes and alcohols derived from the lipoxygenase-hydroperoxide lyase pathway and terpene hydrocarbons (mono- and sesquiterpenes) formed from the mevalonic acid pathway. The concentrations of several of these were subsequentiy determined by direct sampling of the atmosphere within the Petri dish bioassay system using a gas-tight syringe followed by GC analysis (Table I). Pollen Bioassays using Individual Components of Crushed Tomato Leaf Vapor. Plant components identified, except a-phellandrene and a-terpinene, were tested individually against pollen at three vapor phase concentrations. The test concentrations were selected to include a vapor phase level corresponding to that measured in vapor emitted from crushed tomato leaves in the bioassay system. Plots of the vapor phase concentration versus the percent germination of pollen were made from the bioassays and an ED value was estimated for each tomato component evaluated (Table II). These data indicated that the most inhibitory compounds from crushed tomato leaves were the lipoxygenase-lyase products (£)2-hexenal and (Z)-3-hexenal. The estimated ED for (£)-2-hexenal, for example, was 0.25 pmol / L air which was close to the vapor phase concentration of this compound, 0.19 pmol / L air, measured from crushed tomato leaves in the 50

50



10

2

0) C

Time, min.

30

40

T3 u

a

Figure 1. Chromatogram of crushed tomato leaf headspace compounds obtained using a 60 m X 0.32 mm Supelcowax GC column.

20

CNJ

-C

0

X

O c

0 c = a o >>


50

£

o c

4t

39. HAMILTON-KEMP ET AL.


Table I.


453

Identification and Quantitation of Headspace Compounds from Crashed Tomato Foliage

recovery from Tenax, % of total

direct sampling of concn above leaves in bioassay, pmol / L air 0

compound

evidence*

hexanal

MS,RT

5.3

0.34 ±0.19

(£)-2-hexenal

MS,RT

9.0

0.1910.08

(Z)-3-hexenal

MS,RT

14.9

0.29 ± 0.15

1-hexanol

MS,RT

1.4

(£)-2-hexen-l-ol

MS,RT

0.7

(Z)-3-hexen-l-ol

MS,RT

8.9

(Z)-3-hexenyl acetate

MS,RT

0.2

2-carene

MS,RT

6.1

0.21 ± 0.02

limonene

MS,RT

6.3

0.10 ± 0.01

a-phellandrene

MS,RT

1.5

b

0.07 ± 0.01

p-phellandrene

MS

d

27.2

0.42 ± 0.08

a-pinene

MS,RT

0.5

0.05 ± 0.01

a-terpinene

MS,RT

1.4

caryophyllene

MS,RT

3.0

a-humulene

MS,RT

0.6

benzyl alcohol

MS,RT

0.1

0.03 ± 0.01

identification based on comparison of mass spectral and GC retention time data of plant components with those of authentic compounds, ^enax trapping period was 2 hrs; total yield of volatile compounds trapped was 24 pg/g of leaves. Results from direct headspace sampling, using syringe, of atmosphere in bioassay system containing 5 g of crushed tomato leaves 1 hr after set up of assembly. Spectrum consistent with that published by Buttery et al. (24). (Reproduced in part from Ref. 6\ Copyright 1991, American Chemical Society.) c

d



454


Table II. Estimated ED s of Compounds Assayed for Activity against Apple Pollen (Malus x domestica cv Red Delicious)* 50

ED , pmol / L air compound 50

compound

ED^, pmol / L air

(Z)-3-hexenal

0.19

(£)-2-hexen-l-ol

5.0

(£)-2-hexenal

0.25

hexanal

7.8

(Z)-3-hexenyl acetate

3.6

1-hexanol

8.3

(Z)-3-hexen-l-ol

4.8

a

ED s estimated from plots of vapor-phase concentrations of compounds measured by GC versus percent pollen germination. Tests with the terpene hydrocarbons, 2-carene, limonene, and a-pinene, at vapor-phase concentrations similar to those of the lipoxygenase-lyase products, did not inhibit pollen germination. (Reproduced in part from Ref. 6, Copyright 1991, American Chemical Society.) 50



39. HAMILTON-KEMP ET AL.


455

bioassay system (Table I). Thus the lipoxygenase-lyase derived aldehydes appear to contribute to the inhibition of pollen germination observed. With regard to natural occurrence, (E)-2-hexenal appears to be a ubiquitous compound found in all vegetative tissues examined, in varying amounts, and is referred to as leaf aldehyde. (Z)-3-Hexenal is a more labile natural product and deteriorates relatively rapidly. In contrast to the results with the aldehydes, the terpene hydrocarbons had little or no effect on pollen germination even at the highest concentrations evaluated. The highest concentrations of the monoterpenes, (D)-limonene, 2carene, and (D)-a-pinene tested, which were 4.8, 7.6, and 9.9 pmol / L air, respectively, did not significantly inhibit pollen germination. Likewise, the sesquiterpenes caryophyllene and a-humulene at 0.6 and 0.7 pmol / L air, respectively, had no effect on germination. These concentrations exceeded those detectedfromcrushed tomato leaves. Based on these results it was concluded that of the two major groups of compounds emitted from wounded tomato leaves, the lipoxygenase-lyase unsaturated aldehydes effectively inhibited pollen germination whereas the terpene hydrocarbons had no significant effect. Effects of Volatile Compounds on Fungal Pathogens. Following the investigations of the marked inhibitory effects caused by leaf wound vapors on pollen germination, studies were undertaken to determine if these natural compounds affect another important type of micropropagule, that is, spores of pathogenic fungi which parasitize plants. Two fungal pathogens were chosen for this study, Botrytis cinerea which causes gray mold disease on fruit and flowers and Alternaria alternata which causes a foliar disease. Spores of these fungi were placed on an agar block as in the pollen experiments and exposed to volatile compounds from leaves. Vapors emitted from wounded tomato leaves inhibited hyphal growth of Alternaria alternata as shown in Table III. Tests with two representative terpenes, 2-carene and (D)-limonene, revealed that neither of these compounds inhibited growth of A. alternata. The concentrations of the terpenes tested exceeded the concentrations measured above crashed tomato leaves (Table I). In contrast, the aldehydes tested inhibited hyphal growth which corresponded to the results obtained with pollen. (£)-2-Hexenal inhibited hyphal growth at a concentration (0.23 pmol / L air) which was in the same range as that detected in crashed tomato leaves (0.19 pmol / L air). Hexanal also inhibited the growth of A. alternata. The nine-carbon analogs, (E)-2-nonenal and nonanal which are also naturally occurring plant constituents, caused inhibition similar to that observed with the six-carbon aldehydes (Table III). Bioassays using the same aldehydes and terpenes described above and Botrytis cinerea as the test fungus gave similar results (Table III) as were obtained with A. alternata. The a, P-unsaturated aldehydes were more inhibitory than were the saturated aldehydes. At the lowest concentrations tested, the saturated aldehydes, hexanal and nonanal, appeared to stimulate hyphal growth in B. cinerea. This is consistent with the results obtained with saturated aldehydes and other volatile synthetic flavor compounds using certain other fungi, for example, Puccinia species which cause rust disease on grains such as wheat [see review by French (13)1


456


Table HI. Effects of Natural Volatile Compounds on Hyphal Lengths of the Fungal Pathogens Alternaria alternata and Botrytis cinerea source

concn, umol / L air

100 A*

100 A

18 B

20 B

0

100 A

100 A

4.8

100 A

110 A

0

100 A

100 A

7.6

92 A

108 A

0

100

100

0.02

94

23

0.23

25

no leaf


crushed tomato leaf (5g) (D)-limonene

2-carene (E)-2-hexenal

4.6 hexanal

(£)-2-nonenal

nonanal

relative hvphal length A. alternata B. cinerea

6L

0 b

0L

0

100

100

0.04

100

132

0.32

67

15

12.8

Synthesis and Chemistry of Agrochemicals IV - American Chemical

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