Volatile organic compounds and microorganisms.

VOLATILE ORGANIC COMPOUNDS AND MICROORGANISMS

Critical Reviews in Microbiology Downloaded from informahealthcare.com by University of Toronto on 07/02/13 For personal use only.

Authors:

Guenther Stotzky Depart men t o f Biology New York University New York, New York Susan Schenck Department of Microbiology and Biochemistry Rutgers University New Brunswick, New Jersey

Referee:

tieorge C. I’apavizas

Agricultural Research Service of Agriculture Be1 tsville, Maryland

U.S.Department

INTRODUCTION Organic volatiles abound in our biosphere and affect all forms of life - from the pheromones of insects, to the attractants of flowers, to the odors of fruits, vegetables, and other food, to the smells of putrefaction, to the perfumes and deodorants used by human beings, to natural and anthropogenic air pollutants. These volatile organics, whether chemical messengers between members of a species (pheromones) or chemicals that affect members of another species (allelochemics),’ are being recognized more and more as being as important as, if not more so than, nonvolatile organics in intraand interspecific reactions. Volatiles have probably played an important role during the evolution of species, as some type of receptor, sensitive to oiily a few molecules of a particular volatile, is present in almost all contemporary species. Because of their volatility, these chemicals are less susceptible to various forms of inactivation (e.g., solvation, sorption, alteration by wet chemical reactions) than nonvolatile chemicals in solution. Con-

sequently, their sphere of influence (“diffusion radius”) is considerably greater. It has also been that lipophilic compounds in the volatile phase may accumulate faster in the plasma membrane of a recipient cell than when in the liquid phase. However, as all metabolizing cells are surrounded by water films, the probability of a cell “seeing” organic volatiles, any more than other gaseous or volatile compounds, requires clarification. As has been suggested,’ 76 the term, “air pollution,” (by extension, including organic volatiles) is a misnomer from a physiological as well as an ecological viewpoint, as cells probably respond to volatiles in solution. The gaseous or volatile phase of a compound should probably be considered only as the “transfer phase.” Once the compound enters the water fiims around cells and then the cytoplasm, there is probably no physiological distinction between volatile and nonvolatile compounds. RegardIess of these subtle, and perhaps semantic, distinctions, it is highly probable that volatiles are important in the activity, ecology, and May 1976

333


population dynamics of microbes in natural habitats, especially in soil. The extent of this importance, however, is poorly defined, even though there is a growing literature on the influence of organic volatiles on microorganisms (e.g., References 2 to 6 ) . Much of this literature, however, has not only been derived from simple in vitro experiments b u t is also scattered and disorganized. Among the purposes of this review is to collate and focus the available information, especially as it may relate to microbial events in soil; t o emphasize the potential importance of organic volatiles in manipulating microbial events in soil; and to stimulate research in this, presumably, important area of microbial ecology. In recent years, much has been learned (and publicized) about the involvement of pheromones and allelochemics in insects, primarily for the purpose of applying this knowledge to their control. Comparable knowledge of the importance of such compounds, whether volatile or not, in microbial events in natural habitats is rudimentary and certainly not as well organized, focused, and publicized. Inasmuch as soil is one of the major repositories (sinks) for all components in our biosphere, including volatile organics, more knowIedge of the effects of volatiIe components on microbes in soil is necessary. As a stimulus to obtaining such knowledge, this review will briefly discuss the types, sources, and possible effects on microbes of organic volatiles that may be present in the soil environment. As will be apparent, the number of types of organic volatiles derived from various sources is vastly greater than the number of organic volatiles known t o affect microbes. However, this may be only a result of what has been studied, and it should perhaps be assumed that all organic volatiles ultimately affect and are transformed by microbes, especially in soil. Furthermore, most of the knowledge of the effects of organic volatiles on microbes has been derived from simple in vitro studies and little is known about their effects in vivo, especially in the complex soil environment. This, however, should not be overly bothersome, as most of what is presumed t o occur to microbes in soil is based on such “inductive leaps.” It s h o u l d , nevertheless, be emphasized that metabolizing microbes in soil are restricted t o microhabitats’ and that extrapolations from pure culture studies to these highly structured and complex environments should be made cautiously. 334

CRC Critical Reviews in Microbiology

Although only organic volatiles (and those on the borderline between organic and inorganic) are discussed, the soil also produces and removes inorganic volatiles that influence microbes in Finally, this review is not all inclusive, and references and acknowledgments are made to other reviews (e.g., References 2 to 7).

SOURCES OF ORGANIC VOLATILES IN SOIL General Comments Organic volatiles present in soil may be derived from a variety o f sources, both internal and external t o the soil. Inasmuch as soil eventually becomes a repository for many materials originating in other environments, the potential contribution of such exogenous organic volatiles, whether of natural or anthropogneic origin, t o the total volatile load in soil must be considered. Although various types of organic volatiles have been detected in soil, their specific sites of synthesis have seldom been unequivocably established. In addition, there is essentially no information on the rate and duration o f their production, on the quantity of a specific volatile present in a soil at any given time, or on the retention time of specific volatiles and the influence of various soil factors on this. Furthermore, it is usually not clear whether the volatiles detected are “primary” compounds (i.e., derived directly from plants, microbes, or other biotic or abiotic sources) or whether they are “secondary” materials resulting from microbial transformations o f primary compounds. Consequently, the attribution of organic volatiles detected in soil becomes blurred between rpimary and secondary sources; this blurring is reflected in the ensuing attempt t o delineate between various sources. Living Plants Various parts of living platns, including fruits, leaves, roots, flowers, and germinating seeds, release organic volatiles. A summary of the types of volatiles produced by different plant parts is presented in Table 1. Additional information, especially on the older literature, can be found in various reviews (e.g., References 11 to 14 and 67).

5

w u, w

4 Q\

\o

c

Y

Soil bacteria

NG

Methane

Alcohols, aldehydes, furfural, a-pincnc Terpenes, aldehydes, (especially nonanal and furfural)

Tomato fruits

Ustilago sp.

NC

Unidentified

Aldehydes

Various plants

Alterriaria brassicola, rust fungi

Puccirria grrrminis var. triciti

NG

Various bacteria

Bacillus tlicrringietisis. Bacillus cereus

NCa

Rhizosphere and phyllospherc organisms

Corn kernels and leaves

glutinosa

Nico tiana

Sclareol

Terpenes

Foliage of Abies balsamra

Wheat

Unidentified

Foliage of various trees

Aromatic shrubs, especially Salvia and A rtemisia Hardwood trees

Various crop plants

Various terpenes (hemi-, mono-, and sesquiterpenes Various terpenes (especially u- and ppinene, myrcene, isoprene) Terpenes

Coniferous and deciduous trees

Aerial Plant Parts

Volatile

Source

Responding microorganism

NG

Gemination of sporidia

Growth and gerniination of uredospores inhibited

S tiniulatcd germination of uredospores

NC

Inhibited, stimulated, or had no effect o n growth or spore germination Differential inhibition

NG

Inhibited growth

NC

Utilized by microbes as carbon source

Effect

Comment

-

Also derived from fungus

I~acultativepathogenic fungi not affected

Also derived from fungus

Effcct dependent on both terpene and bacterial species

Only on poorly drained soils; probably synthesized by Methanobacterium Effect dependent o n plant species

Cineola and camphoi most tonic

Terpenes generally inhibited pathogenic fungi (15) -

Some Examples of Organic Volatiles Released from Living Plants and Their Effect on Microorganisms

TABLE 1


4,41

19

78

75-77

17,45

85

84

50

18-22

17

15, 1 6 , 7 3 , 7 -

Reference

6

e

g-.

s

x3‘

%.

Ei

22.

6 cl

W

Ally1 isothiocyanate

Unidentified

Unidentified

“Hydrocarbons of the ethylene series” Ethylene

Ethylene

Ethylene

Unidentified mercury compounds

Crucifers

Leaves and fruit of apple; leaves of various plants

Onion leaves and bulb scales

Couch grass

Various fruits

Various plants

Injured or infected plant tissue

Various vascular plants

NG

NG

NG

NG

NG

Borryris cinerea (also some other species)

Plasm odiophora brassicae Borryris cinerea (also some other species)

Various fungi Monilinia fmcticola Scleroriuni cepivorum

Penicillium digitarum

C, to C, aldehydes, alcohols, esters, terpenes Aliphatic aldehydes a-Hexenal Alkyl sulfides, allyl alcohol

Citrus fruits

Various fruits Ginkgo biloba leaves AIlium sp.

Pucuccinia cartlrami

Aerial Planr Parrs (continued)

Responding microorganisin

Unidentified

Volatile

Safflower

Source

NG

NG

NG

NG

NG

Inhibited spore germination

Stimulated spore germination Stimulated spore germination

Stimulated growth lnhibited growth Stimulated germination of sclerotia

Stimu la tcd germ ina tion of teliospores Inhibited germination of spores

Effect

Corninen!

Mechanical damage or infection by microbes o r viruses enhances ethylene release Not elemental or dimethyl mercury

Especially during ripening Especially seedlings

Ethyl acetate, malate, and citrate gave similar results as plant volatiles Could be mimicked by high concentrations of ethyl acetate -

n-Propyl and allyl sulfides active components -

Only aldehydes inhibited


TAHLI: 1 (continued)


63a

13,28-32, 34 35-31

13, 33

29

59

59

83

53-58

4,81,82 49

46

80

Referencc

i5

w 4 w

o\

4

W

Y c

NG

Methanol, ethanol, and other alcohols Formic and other shortchain acids Terpenes

Corn and sunflower roots

Ethylene

Acetaldehyde, ethanol, acetone Alkaloids Unidentified

Nodules

Various weed seeds

Tall fescue (Ky. 31) Germinating seeds of bean

Aromatics (e.g., naphthalenes) Unidentified

Potato tubers

Pinus echinata roots

Pinus sylvestris and

Aspergillus jlavipes, Fusariiim oxysporum 1: conglu tinans, Cunningham ella elegans, Penicillium viridicatum, Trichoderma viride

Gelasinospora cerealis Penicillium vermiculatum. Zygorhynchus vuilleminii

NG

No effect

NG Decreased formation of perithecia, conidia, or zygospores

NG

Inhibited spore germination NG

Botrytis cinerea (also some other species) NG

NG

NG

First stimulate and attract fungi to root, than retard spread of mycorrhizal symbiont5

NG

NG

NG

NG

Effect

NC

Boletus variegatus. Rhizopogm roseolus, Fomes annosus. Phy toph th ora cinnam o m i

NG

NG

Alcohols

NG

Corn roots

Corn and buckwheat roots


Subterranean Plant Parts (continued)

Acetaldehyde

Volatile

Wheat roots

Source Comment

-

Effective with 5 bean seeds (2. vuilleminii also with 15 cucumber seeds) -

Produced by nitrogenase in reducing acetylene Inhibited germination of seeds

-

Complex interaction and sequence of volatile production by roots and rootmycorrhizae complex

Excised roots in high nitrate or nitrite Excised root tips grown on glucose Increased release under anaerobic conditions Young seedlings

Some Examples of Organic Volatiles Released from Living PIants and Their Effect on Microorganisms

TABLE 1 (continued)


87

72 87

65

43,44,86

59

71

23.24

70

68.69

51

52

Reference

W W

6z1

8 -.

$

3.

'

m

Ethanol

Pea seed extract

aNG = effect on microbes not given by authors.

Ethanol, methanol, formaldehyde, acetaldehyde, propionaldehyde, acetone, formic acid, ethylene, propylene

Unidentified

Volatile

Germinating seeds of various angiosperms and gymnosperms

Germinating seeds of bean, pea, wheat, corn, cucumber. tomato, lentil, carrot, red pepper, lettuce

Source

I n general, inhibited spore germination, although in some cases germination was increased Served as sole carbon source for growth (did not provide source of nitrogen or growth factors); spores of T. viride germinated better

NC

NC

Effect

Botrytis cinereo. Mucor rocemosus, Trichodermo viride, Verticillium dah lioe, Fusorium oxysporum Pseudomonos fluorescens, Bocillus cereus, Erwinio carotovoro, Agrobocterium tumefociens, Agrobocteriurn radiobocter. Rhizobium japonicum, Mucor mucedo, Fusorium oxysporum. Trichodermo viride, Penicillium verm icu lo iir m

Subterranean Plonr Ports (continued)

Responding microorganism Comment

Active volatile apparently acetaldehyde

Effect differed with species of plant and fungus


TABLE 1 (continued)


66

34,64,89

88

Reference

Among the most frequently reported volatiles from plants are the terpenes. These are cyclic, unsaturated compounds constructed from basic isoprene (C,) units (hemiterpene) to form monoterpenes (C, ’) or sesquiterpenes (C, 5 ) . They are components of the essential oils of plants and have been considered as secondary plant metabolites.’ The major terpenoids emitted by plants, especially by the foliage, are monoterpenes, such as a-pinene, P-pinene, limonene, myrcene, and camphene, although isoprene is apparently also released.’ A primary source of these terpenes is forest trees, with the rates of emission for isoprene from various deciduous species ranging from 0.04 to 2.4 ppb/cm’ /min/l and for monoterpenes from conifers ranging from 0.4 to 3.5 ppb/cm’/min/l. The rates are usually dependent on light, temperature, rate of transpiration, and structural integrity of oil cells and resin glands. The annual production of “reactive” hydrocarbons*, primarily terpenes, from tree foliage on a global basis has been estimated to be 175 X lo6 tons as compared to 27 X l o 6 tons from anthropogenic sources.’ Although forest trees appear to be the major source of volatile terpenes, various crop plants have also been shown to release a-and 0-pinene, myrcene, and isoprene.’ Aromatic shrubs, primarily species of Salvia and Arfemisiu, release volatiles, including the terpenes, camphor and 1,8-cineola.’ In addition to foliage, both intact and crushed, being a source of terpenes, fruits of tomato evolved a-pinene as well as nonterpenoid compounds,’ and roots of pine evolved monoand s e s q u i t e r p e n e ~p.2 4~ ~ inasmuch as essential oils, of which terpenes are a major component, are produced by foliage, cortex, fruit, xylem, secondary phloem, and seed coats of many plant species (especially in the Pinaceae), sources of terpenes are numerous.’ Although not much is known about the entrance of terpenes into soil, the many and varied sources, their wide distribution, and the numerous removal processes active in the atmosphere (e.g., precipitation, adsorption, diffusion) suggest that terpenes are eventually introduced into soil (cf.,


’

‘

’-’’

Reference 6). Furthermore, partial oxidation of terpenes by ozone in the atmosphere results in macromolecules that can be introduced into soil by rain.’ The various biological responses in soil attributed to terpenes indicate that these compounds are present in soil. Another volatile frequently reported to be evolved from plants is ethylene. The interest in this compound and, hence, the frequency of its detection in the biosphere are undoubtedly the result of the demonstration that ethylene is both a natural plant growth hormone (e.g., Reference 13) and a common air pollutant (e.g., References 6, 26, and 27). Ethylene has long been known to be associated with fruits, which release large quantities of this relatively water insoluble volatile during ripening (e.g., Reference 13). Seedlings of many plants, such as wheat, oat, lettuce, and pea, also evolve ethylene,’ ‘4 as do flowers4 and germinating seeds of varieties of bean, corn, cotton, and pea, but not always seeds of cabbage, cucumber, pines, radish, red alder, squash, and tomato. The production of ethylene from germinating seeds was greatest during the first 1 to 3 days after i m b i b i t i ~ n . ~ ~ Although ethylene is produced by many plants during normal growth and development, it is especially associated with injured or infected plants. Tomato, tobacco, and bean plants evolved more ethylene after ozone injury than did uninjured plant^.^ Virus-infected plants produced greater amounts of a volatile, thought to be ethylene, than did uninfected plants, and an increase in leaf necrosis resulted in increased production of the ~ o l a t i l e . ~ Cut carnations evolved ethylene when damaged or infected with fungi; chrysanthemum flowers infected with Ascochyta chrysanthemi, cherry leaves infected with Coccomyces hiemalis, and rose leaves infected with Diplocarpon rosae synthesized greater quantities of ethylene than noninfected p~ants.~ The production of ethylene by plants is related to methionine m e t a b ~ l i s m . ~3 3 Ethylene was produced from methional (methylmercaptopropionaldehyde) in cell-free extracts prepared from cauliflower florets4’ or from pea seedling^,^ and the addition of methionine to pulverized seeds of

’

93

’

*“Reactive” refers to the ability of a hydrocarbon to react photochemically with other components of the atmosphere to form products considered as air pollutants. “Nonreactive” hydrocarbons. such as methane, do not form such products, although these compounds may be more “reactive” from a microbiological point of view. May 1976

339


pea or corn increased the evolution of e t h ~ l e n e . ’ ~ Ethylene production by pea seedlings was stimulated by either indoleacetic acid or kinetin, but not by gibberellic acid.30i3 The production of ethylene by healthy apple peel was inhibited by rhizobitoxine, an inhibitor of the metabolism of methionine to ethylene, but the production by peel infected with fungi was not inhibited. Although, in both healthy and infected peel, the plant part rather than the fungus was the source of the ethylene, the metabolic pathways appeared to differ.“‘ In addition to ethylene, the olefin, propylene, was also released by some germinating seeds.34 Although there are no reported plant sources of acetylene, there are numerous known fungal and anthropogenic sources (cf. Reference 6), and the reduction of acetylene to ethylene by the enzyme nitrogenase is now used routinely for detecting and quantifying biological dinitrogen fixation. (e.g., References 43 and 44). Although terpenes and ethylene have been the most frequently reported organic volatiles derived from living plants, many other volatiles, which probably have greater effects on microorganisms, have also been reported. Developing fruits of tomato evolved a variety of alcohols and aldehydes, including furfural, in addition to apinene.4 s Volatile components of citrus fruits included aldehydes (primarily CS to C,), alcohols, esters, and t e r p e n e ~ . Aldehydes ~~ and CO have been detected in a variety of plants, including algae,4 7 * 4 8 and leaves of Ginkgo biloba synthesized a-hexenal! Healthy hardwood trees growing on poorly drained soils contained high pressures of methane, although this gas may have resulted from the activities of anaerobic bacteria, primarily Methanobacterium sp., present in the heartwood of the trees.” Alcohols were released from corn root tips during growth on glucose,5 and excised wheat roots grown in solutions containing nitrate or nitrite, which favors active nitrate assimilation, evolved acetaldehyde.’ Ethanol, methanol, and other alcohols, but not acetaldehyde, were released from roots of corn and sunflower, especially under anaerobic conditions! * & and formic and other volatile acids were detected in root exudates of young seedlings of corn and buckwheat.” Unfortunately, there is little else known about organic volatiles released by plant roots, other than these and some mono- and sesquiterpenes from pines” s24 and some aromatic 340


compounds (e.g., naphthalenes) from potato tubers.” Inasmuch as root-derived volatiles are probably important in a variety of root-microbe interactions in soil, more studies are obviously needed in this area. Sulfur-containing organic volatiles are produced by various plant families. Species of Allium released aliphatic disulfides had allyl monosulfide, in addition to allyl alcohol.s3 Seedlings of onion and leek emitted a variety of alkyl sulfides (e.g., n-propyl sulfide and allyl s ~ l f i d e ) , ~and ~ - the ~~ bud scales and leaves of onion were shown, as early as 1922, to evolve volatiles inhibitory to fungal spores.’ Cabbage and other crucifers evolved a variety of sulfur-containing compounds, including methyl mercaptan, dimethyl sulfide, and dimethyl O-‘’ Volatile mercury has also been detected in some vascular plants (e.g.. garlic vine, avocado), which apparently accumulate mercury from soil and then volatilize it. The volatile released was neither elemental nor dimethyl mercury but was c o m p o s e d o f t w o or more unidentified

compound^.^

a

Seeds are probably a major source of organic volatiles in soil. Germinating seeds of a variety of gymnosperms and angiosperms evolved ethanol and many also released methanol, formaldehyde, acetaldehyde, propionaldehyde, acetone, formic acid, ethylene, and propylene. The type and concentration of compounds liberated varied with the species and variety of plant. Most of the compounds were evolved only during the first 3 or 4 days after imbibition, with peak production during the second day, although large quantities of ethanol and methanol were still evolved 8 days after planting. The quantities of volatiles produced appeared to be related to the amount of storage substances present in the seeds. Active seed metabolism was required for the production of volatiles, as dry, nongerminating, or killed seeds did not evolve volatiles, whereas pulverized and wetted seeds did.’ *6 Volatile alkaloids have been demonstrated in seeds of tall fescue,” and the inhibition of germination of buried weed seeds (e.g., morning glory, velvet leaf, wild mustard) was attributed to the production by the seeds of acetaldehyde, ethanol, and acetone and was related to low oxygen concentrations. The greatest inhibitory effect was shown by acetaldehyde, although ethanol was present in higher concentration^.^ The production of ethanol by extracts from pea

’


seeds cultured with starch, glucose, or fructose was by the glycolytic pathway.66 It is apparent, from even this brief discussion, that living plants evolve a wide spectrum of organic volatiles. These volatiles are either released within soil, from roots or germinating seeds, or from aerial plant parts and then, presumably, are introduced into soil, where they may affect the soil microbiota.

Dead Plants Although emanations from living plants are, undoubtedly, important sources of organic volatiles in soil, dead plants (undergoing decomposition in or on soil) are probably sources of larger quantities and more diverse types o f volatile compounds. It is difficult, however, to determine whether volatiles associated ,with dead plant materials arise directly from the cells of the plants (i.e., primary products) or whether the volatiles are evolved by microbes (i.e., secondary products) utilizing the plant materials as energy and carbon sources. Regardless, there is little doubt that the chemical composition of the plants is reflected in the types of volatiles produced. For example, although alcohols and aldehydes appear to be evolved during decomposition of most plant residues in soil, the evolution of sulfur-containing compounds appears t o be restricted to species of Brassica and A Ilium. A variety of volatiles have been identified from plant residues plowed into soil, and some are shown in Table 2 . Methanol, ethanol, acetone,and acetaldehyde, as well as smaller quantities of other alcohols and carbonyls, were evolved during decomposition of cabbage and corn refuse in r6 r9 Sulfur-containing organic volatiles (methyl mercaptan, dimethyl sulfide, dimethyl disulfide, allyl isothiocyanate, butyl isothiocyanate, methyl isothiocyanate, and carbon disulfide) were also produced during the decomposition of crucifer^.^^-^ Aliphatic disulfides, allyl monosulfide, and allyl alcohol were evolved from fresh, chopped onion tissue,’ and n-propyl sulfide and allyl sulfide were found in extracts of onion and garlic.’ 4-5 The volatiles identified in distillates of alfalfa hay included acetaldehyde, isobutyraldehyde, valeraldehyde, isovaleraldehyde, 2-methylbutanol, methanol, and ethanol, although other aldehydes and akohols, with up to six carbon atoms, were y9’

’

also detected.’’ The major components that stimulated soil respiration were acetaldehyde, methanol, and e t h a n 0 1 . ~ ~Vapors - ~ ~ from water distillates of bluegrass clippings, tea leaves, wheat straw, corn leaves, and tobacco also stimulated soil respiration, presumably as a result of these components.’ Not only do plant residues added to soil yield volatiles, but the treatment of grassland soil with a slurry of cow dung and urine resulted in the evolution of ethylene.” Sludge, which is frequently deposited in and on soils, released volatiles, including methane, which injured plant roots.99 The mixed refuse added in landfill areas produced various volatiles, depending on water content, temperature, and aeration.’” Inasmuch as soil is increasingly used as a repository for anthropogenic wastes, the composition and biological effects of the resultant organic volatiles warrant thorough study. Although crop residues and other wastes are major sources of organic additions to soil and, therefore, of potential organic volatiles, there is also considerable natural deposition of wood and other tree components. These dead woody materials result in a variety of volatiles that reflect their chemical composition more than the microbial metabolites usually associated with herbaceous residues. For example, a variety of terpenes are frequently released from resins,’ O4 although aldehydes with as many as nine carbonsIo4 and alkanes with as many as 1 1 carbons’ are also produced.’ N-Nonanal was emitted from rotting wood,” and heat-treated woods of pines volatilized fatty acids (e.g., linoleic and oleic), which were probably auto-oxidation products of the fatty components of the wood.’O4,105 In addition to these compounds associated primarily with trees, the release of species-specific compounds, especially from roots, has been implicated in plant dominance and succession’ and in “soil sickness” and “replant” problems.’ Al though many of these specific allelopathic compounds (e.g., amygdalin, phlorizin, juglone, caffeic, ferulic, coumaric acids) are not highly volatile, their degradation products (some of which are toxic) may be. Some of the organic volatiles present in soil may be derived from the residual soil organic matter rather than from recently added residues.

’

99

9’

’

’’-’

p1

1’

’

7”

9”

’

May1976

341

Reduced root rot Stimulated spore germination Inhibited

Thielaviopsis sp. Plasrnodiophora brassicae Colletotrichum circinans. Botrytis allii Sclerorium cepivorum

Various residues

Hackberry leaf litter

Extracts and tissues of Allium sp.

Aliphatic disulfides, alkyl sulfides Garlic Allicin Caffeic, ferulic, coumaric-acids Cyanogenic glycosides and other alkaloids Soil microbes

Bacteria unidentified

Inhibited production, development, and germination of zoospores, formation of oospores, and growth

A phanomyces eu riches

Methyl mercaptan, dimethyl sulfide, dimethyl disulfide, various allyl isothiocyanates. aldehydes, ketones, and alcohols Ally1 isothiocyanate Mercaptans, sulfides

Usually inhibitory

Stimulated germination of sclcrotia Bactericidal Inhibitory

Reduction in disease Reduction in root rot Increased growth

Rhizocronia solarii Thielaviopsis basicola Rhizocronia solani

Barley residues Various crop residues Plant residues with low C:N ratio Crucifer residues

Unidentified Unidentified Unidentified

Unidentified

Controlled in soil Reduction in disease

Stimulated germination of sclerotia and subsequent growth Stimulated germination of microsclerotia Stimulated germination of conidia Increased respiration and numbers Enhanced decomposition of DDT Increased respiration and numbers; inhibited at higher concentrations

Effect

Increased respiration

Soil microbes

Soil microbes

Fusarium solani

Verticillium dahliae

Sclerorium rolfsii


Phyfophrhora cinnamomi Fusaria pathogenic to onions Soil microbes

Acetaldehyde. isobutyraldchyde, valeraldchydc, isovaleraldehyde, 2-methylbu tanol, methanol, ethanol, other C, to C, aldehydes and alcohols Probably similar to alfalfa hay (above) Unidentified Unidentified

Volatile

Oat Straw

Corn, tea, and tobacco leaves, wheat straw, blue grass clippings Various crop residues

Alfalfa hay

Source

n-Propyl and allyl sulfides are active coniponents May be converted to volatilcs May be converted to volatiles by soil microbes May be microbially converted to HCN and other volatiles by soil microbes

Survival of Verticillium albo-arrurn decreased Volatiles not tested for Altered pigmentation of fungus; active volatile may be ammonia Methyl mercaptan and methyl sulfides appear to be the active components

Active volatile may be ammonia Volatiles not tested for

Anaerobically Acetaldehyde and ethanol probably active components

Acetaldehyde, methanol, and ethanol probably active components

Comment

Some Examples of Organic Volatiles Released from Plant Residues and Their Effect on Microoiganisms

TABLE 2


12, 106, 107

120 108

54-58

63.90 83 119

60-62

118 90 91

117

115 116

92-94,96,91 114 61.92-94

96

95

110-113

References

W

P

W

Fomes annosus

Fatty acids, C, to C, aldehydes, (I-and P-pinene

Various terpenes

Aldehydes

Heat dried conifer woods

Pine oleoresins

Rotting wood Polyponis applanatus and other wood decomposers

Ceratocystis sp.

Fomes annosus,

Wood-rotting fungi (1 1 species) Coniophora cerebella

Fusarium


Unidentified

Volatile

Conifer litter

Source

Stimulated growth

Altered mycelial morphology Inhibited growth and spore germination

Stimulated gcrrnination of chlamydospores Stimulated growth Stimulated growth

Effect

Effect different with individual terpene component and fungal species; n-heptane most effective n-Nonanal was active component

Linoleic and oleic acids apparently active components

Volatiles not tested for

Comment

Some Examples of Organic Volatiles Released from Plant Residues and Their Effect on Microorganisms

TABLE 2 (continued)


81

105 15, 101-103

104

121a

References


For example, a soil sterilized by yradiation evolved ethylene and ten other hydrocarbons. Ethylene was detected within 2 hr of sterilization and was still produced 10 weeks later.'Og Aqueous extracts of four different soil types contained several aromatic hydrocarbons (i.e., benzene, toluene, ethylbenzene, p/m-xylene, 0xylene, naphthalene), but their source was not identified.'09a The extracts were made from the A 0 horizon, suggesting that the hydrocarbons were of aerobic origin; their concentrations were higher in cultivated than podsol soils, suggesting that soil organic matter was involved. Microorganisms Most of the organic volatiles present in soil are probably of microbial origin. Consequently, their production is governed by those environmental factors that influence the activity, ecology, and population dynamics of microbes in soil.' Not only will the environmental factors that affect microbial metabolism influence the types and amounts of volatiles produced, but, as already indicated, the types of substrate play an important role in determining the composition of the resultant volatiles. However, even a single substrate may give rise to a variety of volatiles, depending on whether its utilization is predominantly aerobic or anaerobic. Inasmuch as soil contains both aerobic and anaerobic microhabitats, even under the most apparent ambient aerobic conditions, it is not always possible to determine whether a volatile in soil is the product of aerobic or anaerobic metabolism. Consequently, reliance is often placed on pure culture studies, usually in liquid, to determine which products from a given substrate are produced in the presence or absence of oxygen. Although such a fine distinction may be academic and irrelevant to microbial events in soil in situ, the separation of volatiles produced aerobically and anaerobically has been attempted.

Aerobic A diversity of organic volatiles is produced under aerobic conditions, as indicated in Table 3. Fungi appear to produce a greater variety of volatiles than do bacteria, although this may only be a reflection of the greater number of studies that have been conducted with fungi. Numerous

species of fungi are capable of producing ethylene (e.g., References 121 to 127), but Pseudomonas solanaceamm has been the only bacterium reported to produce ethylene aerobically,' s although some unidentified streptomyces may also d o so.123 Inasmuch as many of the organisms reported to produce ethylene are plant pathogens and because ethylene is a phytohormone, it has been suggested that this volatile may be involved in pathogenesis and disease symptomatology (e.g., References 124 and 128). In addition to ethylene, Penicillium digitatum also evolved the ole fins, acetylene and propylene, as well as the saturated hydrocarbons, ethane and propane.' Although many fungi evolved relatively simple, short-chained (C, to C,) alcohols and aldehydes, several species also produced longer chained and more complex compounds. Several species produced esters (e.g., Dipodasnts aggregatus and Ceratocystis fagaceanim), volatile amines (e.g., TilIetia sp.),* terpenoids (e.g., Cronartium fusiforme and Boletus variegatus), methylated halogens (e.g., Fomes sp.), aromatic aldehydes (e.g., Daedalea juniperina), and aldehydes and alcohols containing more than six carbon atoms (e.g., Puccinia graminis and Aspergillus flavus). It is difficult in some cases, however, to establish whether the fungus actually synthesizes the volatile or only accumulates i t from the substrate or host on which it is growing. For example, the terpenoids present in aeciospores of C. jusijonne may only be accumulated from the host, Pinus elliottii, which produces these compounds. The composition of the volatiles is sometimes dependent on the substrate on which the organisms are grown. Boletus variegatus, an ectomycorrhizal fungus of pine, produced ethanol, isobutanol, isoamyl alcohol, acetoin, and isobutyric acid in pure culture, but, when roots of Pinus sylvestris were infected with the fungus, several terpenes were evolved by the root-fungus combination. Although these terpenes were probably produced by the pine roots, the metabolites produced by the fungus in pure culture were not detected in the combination." 3'4 Ethylene was produced by M u m hiemalis and two unidentified soil yeasts only when cultured on media containing methionine and glucose.' Sulfur-containing volatiles were produced only in

'

'

''

'The production of trimethylamhe by Tilletiu caries is of interest and potentid importance, as this tertiary amine is apparently converted in soil to dimsthylnitrosamine, a compound that is carcinogenic. mutagenic. and teratopenic.' '* 344


s

01 P

w

o\ 4

W c

Y

Schizophyllum commune Ceratocystis sp., Thielaviopsis basicola

Various fungi

Aspergillus f l a w s

Trichodermo sp.

Fusllrium oxysporum. Rhizopus stolonver

Ceratocystis fimbriata Penicillium digitorurn, Mumr hiemalis Aspergillus clavorus. 2 unidentified soil yeasts Bhstomyces dennotitdis 58 fungal species Penicillium digitoturn

Volatile producer

NG lnhibited spore germination

NG

F. oxysporum, R . srolonifer,

Acetylene, propylene, ethane, propane Aldehydes, alcohols, organic acids, esters

121 123 129

125-121

124 122, 1 4 7 , 1 4 8

Reference

S-containing volatiles essentially only produced in presence of methionine and glucose

Methylation of inorganic sulfate -

NG

NG NG NG NG

-

144 158, 159

133, 136-1 38, 140

157

Acetaldehyde appears t o be ac149-152 tive component; produced by mycelium; (mostly C, to C, compounds) Acetaldehyde and ethanol may be 153-155 active components; greatest effect due to CO, 156

Ethylene produced on living plant tissue or on media containing methionine; seldom on other standard media

Comments

NC

NG

Caused oospore formation by selfing NG

Phyfophrliom sp.

Unidentified

3-Methyl butanol, 3-octanone. 3-octano1, 1-octen-3-01, I-octanol, cis-2-octen-1-01 Methyl mercaptan, dimethyl sulfide, dimethyl disulfide, various nonsulfur containing compounds Methyl mercaptan Ethanol, formaldehyde, acetaldehyde, furfural, 2-hexenal, 2-methyl propanal, 2-methyl butanal, acetone, 2-propanone, 2-heptanone, ethyl acetate

Inhibited growth of fungi; no effect o n bacteria

Various fungi and bacteria

Acetaldehyde, ethanol, acetone, ethylene

Cunningharnella elegans

NG

Effect

NGa

Fur igi


Ethylene

Volatile

Some Examples of Organic Volatiles Released by Microbes tinder Aerobic Conditions and Their Effect on Microorganisms

TABLE 3


P

W

3

QI

Aspergillus niger. Chaetomium globosurn Various fungi and bacteria

Arm illaria niellea

Hexa-l.3,S-triyene

Hydrogen cyanide

Ethanol

Fomes annoms

Fomes scutellatus, Marasmius oreacles, Pholiota aurea, Clitocybe geotropa, unidentified basidiomycetes Aerobasidium pullulans

Fomes sp.

Methyl chloride, methyl bromide, ethanol, acetaldehyde, isobutanol

Aspergillus niger

Acetaldehyde ,ethy1 acetate, ethanol, npropanol, isobutanol, isopentanols Ethanol, isobutanol, isoamyl alcohol, acetoin, isobutyric acid

Saccharonivces cerevisiae

Boletus variegatus

Pestalo trb rhododendri

Aldehydes, alcohols, ethyl acetate, ethyl propionate

Dipodascus aggregatus

NG

NG

NG NG

Junipal, anisaldehyde Various terpenes

Daedalea juniperina Cronartium fusiforme

NG

Responding microorganism Fungi (continued)

Methyl and ethyl benzoate, methyl salicylate, benzyl alcohol, phenyl ethyl alcohol, monoterpene alcohol

Volatile

Phellinus sp.

Volatile producer

NG NG

NG

Effects varied with producer and responding species and with investigator

Same effect with exogenous ethanol Methyl chloride and methyl bromide produced primarily by F. pomaceus

Rhizomorph production stimulated NG

-

Formed in pure culture, but not in roots of Pinus sylvestris; mycorrhizal combination evolved terpenes

2, 168

301 - 303

2, 169-174

161. 168

23,24

166

162-165

161 131

160

Reference

Present in aeciospores; may be accumulated from pine host Growth inhibited at high concentrations of some compounds; n-propanol may be active component Acetaldehyde and ethanol were active components in inhibiting growth but not sporulation

-

-

Comments

Inhibited growth and sporulation in some fungi: bacteria generally not affected

Inhibits

NG

Inhibited growth and sporu. lation

Enhanced growth and sporulation at low concentrations

Effect

Some Examples of Organic Volatiles Released by Microbes under Aerobic Conditions and Their Effect on Microorganisms

TABLE 3 (continued)


z

P 4

w

QI

. I

u l

w

Y

NG NG NG

NG NG NG

2,3-Dime th yl-1-pen tene

Agar icus campestris

Ethylene Ethylene Methyl mercaptan, dimethyl sulfide, dimethyl disulfide

Stimulation of spore germina t ion Stimulation of spore germination

A. b isporus

lsovaleric acid

Pseudomonas solanacearum Unidentified streptomyces Various bacteria

Inhibition of teliospore germination

T. caries and T. controversa

Trimethylamine, other volatile amines

Tilletia caries, T. foetida, and T. confroversa Agaricu s biSpOruS

Bacteria

Morphological change in germination process

Ustilago sp.

Unidentified

A . canipestris

Transmissible lysis

G. graminis

Unidentified

Stimulated growth lnduced zygospore formation in unlike mating types Stimulated zygospore production lnhibited or stimulated germination of uredospores

Inhibited, stimulated, or had no effect o n mycelial growth and spore germination

Effect

Gaeumannomyces graminis var. tritici Ustilago sp.

P. gram itiis

R. sexualis

Rli izop us sexualis. Chaeromium glo bosurn, St ere um h irsutum, Aspergillus niger, Botrytis cinerea Pliytophrhora citroph fhora M.mucedo

Fungi (conrinued)


Trimethyle thylene, n-nonanal, acetaldehyde, furfural, methyl ferulate, terpenes

Unidentified Precursors of trisporic acids Unidentified

Unidentified

Volatile

Puccinia graminis var. tritici

Rhizopus sexualis

Mucor spinosus Mucor mucedo

30 Fungi Imperfecti)

62 fungi (13 Phycomycetes, 1 3 Ascomycetes, 6 Basidiomycetes,

Volatile producer

123 133, 139, 141 Essentially only produced in presence of methionine and glucose

128

-

190

188, 189

186, 187

-

Volatile produced by mycelium

Active volatile is probably not trimethylamine; produced by teliospores Volatile produced by mycelium

79

185

179-184

75-77,

178

177

Temperature-dependent synthesis of stimulator Volatiles produced by uredospores; active inhibitor is probably methyl ferulate; active stimulators are probably nonanal and furfural Also causes some growth stimulation prior t o lysis May also be derived from host tissues

176 -

175

Reference

-

Effects varied with producer and responding species

Comments

Some Examples of Organic Volatiles Released by Microbes under Aerobic Conditions and Their Effect on Microorganisms

TABLE 3 (continued)


Fusarium oxysporum f. conglu tinans, Gelasinospora cerealis, Pen icill ium viridicatum, Trichoderma viride. Zygorhynchus vuilleminii

Unidentified

Unidentified

Unidentified

Pseudomonas sp.

Microbes in mushroom substratum Agrobacterium radiobacter, Agrobacterium rhizogenes. Bacillus cereus, Enterobacter aerogenes, Escherichia coli, Micro coccus lut eus, Nocardia corallina, Proteus vulgaris, &rcina lutea, Serratia marcescens

"NG = effect on microbes not given by authors.

Verticillium malthousei

Unidentified

Streptomyces sp. Agaricus bisporus

Gleoesporiumaridum Sclerorium cepivorum, Rhizoctonia solani Various fungi

NG NG

ESSect

Inhibition of growth and spore production; alterations in colony and hyphal morpholOgY

Inhibited spore germination

Inhibited growth and spore germination Induction of fruiting bodies

Reduced sporulation lnduced formation o f sclerotia

Bacteria (continued)


Unidentified

Hydrogen cyanide

Volatile

Pseudomonas sp., Chromobacterium sp. Streptomyces griseus

Volatile producer

Effects varied with producer and responding species; both inhibitors and stimulators present; effects are concentration dependent

Effect is temperature dependent

-

May be a soil fungistatic factor

-

-

Comments

Some Examples of Organic Volatiles Released by Microbes under Aerobic Conditions and ?heir Effect on Microorganisms

TABLE 3 (continued)


87, 197, 198

196

194, 195

4, 192. 193

191

146

Reference


the presence of sulfur-containing substrates, and the types of volatiles depended on the form of sulfur contained in the substrate. For example, Saccharomyces cerevisiae evolved methyl mercaptan when using methyl cysteine as a substrate, whereas ethyl mercaptan was emitted when grown on they1 cysteine.' 3 z A variety of volatile sulfur compounds are produced by microorganisms, but many of these are either inorganic or formed under anaerobic conditions.' 3 3 Although there is controversy about whether dimethyl sulfide' or hydrogen sulfide (e.g., Reference 135) is the primary form in which biogenic sulfur is transported through the atmosphere, the global emission of dimethyl sulfide from soil has been computed to be about 10' g/yr.' Dimethyl sulfide has been reported as a volatile product of the metabolism of methionine by Scupulariopsis brevicatrlis, along with methyl mercaptan.' The degradation of cruciferous plants in soil also resulted in the evolution of dimethyl sulfide, as well as of dimethyl disulfide and methyl mercaptan.6 Numerous bacteria and fungi also evolved methyl mercaptan and dimethyl disulfide when grown aerobically on methionine.' '-I4 In general, the first product was methyl mercaptan, a part of which was then further oxidized to dimethyl disulfide. With the exception of certain soil bacteria (e.g., species of Pseudomonas, Achromobacter, and Flavobacterium) which could use methionine as a sole source of carbon, nitrogen, and sulfur, another source of organic carbon was necessary for growth and the transformation of the amino acid. Most of the sulfur contained in various organic compounds (e.g., cysteine, cystine, and taurine), as well as in methionine, was c o n v e r t e d t o i n o r g a n i c f o r m s of s u l f u r . ' 3 8 ~ ' 4 z ~ ' 4 3The wood-rotting fungus, Schizophyllum commune, is apparently unique, as it can methylate inorganic. sulfate to produce methyl mercaptan.144 The development of highly sensitive gas chromatographic techniques for the identification of organic sulfur volatiles in soil' 4 s may result in the detection of additional organic sulfur volatiles. Because of the broad toxicity of hydrogen cyanide and its production by various microbes, it has been included in Table 3. Volatile hydrogen cyanide has been shown to be produced by several fungi (cf., Reference 3) but by only a few species of bacteria, primarily in the genera Pseudomonas and Chromobacterium The production of hydro-

''

'

2'

cyanide by bacteria is inhibited under anaerobic conditions, and this compound appears to be a secondary metab01ite.I~~ gen

Anaerobic Anaerobic metabolism usually results in more metabolic products than does aerobic metabolism. This increased diversity is reflected by the data in Table 3. Inasmuch as filamentous fungi are essentially obligate aerobes, anaerobic and facultative bacteria are the primary producers of volatile organics in anaerobic environments. Although several volatiles have been reported to be formed under both aerobic and anaerobic conditions (e.g., ethylene and short-chain aliphatic aldehydes and alcohols), most volatiles produced under anaerobic conditions are known products of h e terofermentative processes. The types of volatiles are dependent on the environmental conditions, the types of substrate, and the types af microbes involved. For example, although methane has often been detected in soils, especially when anaerobic, its synthesis is restricted to methanogenic bacteria.' ' The types of volatiles produced in the same soils under anaerobic conditions differed markedly with the type of organic amendment: e.g., alcohols and esters were detected in soils amended with only glucose but not in soils amended with both glucose and peptone; organic sulfur volatiles were detected in soils amended with glucose and peptone or with glucose and methionine, but not in soils amended with glucose and cystine, cysteine, or glutamate. The types of volatiles produced in soils differing in texture, pH, and other characteristics, but amended with the same substrates, also differed markedly: e.g., an alkaline loam soil evolved only ethanol when amended with glucose, whereas a more acidic clay soil amended with glucose yielded a variety of alcohols, esters, carbonyl compounds, and dimethyl sulfide.' *' In addition to saturated hydrocarbons with one to four carbons, olefmic hydrocarbons have also been detected in soil.' 29*201-208 The formation of these volatiles is apparently the result of microbial activity, as they are produced in much greater quantities in nonsterilized than in sterilized soil. The small amounts of these compounds evolved from soils sterilized by 7-radiation have been attributed to possible residual enzymic activity,'03 to the formation of free radicals, and to other abiological factors.'0g Among the olefinic hydrocarbons, ethylene has May1976

349


received considerable attention, primarily because of its effects on plants.203’204I t has also been suggested that ethylene is not only a cause of fungistasis in soil but that i t regulates most microbiological processes in soil.’ 0 6 - ’ 0 Although numerous soil-borne microbes have been shown to produce ethylene in vitro, both aerobically and anaerobically, it has been suggested that anaerobic spore formers, specifically species of Clostridium, are the main producers of ethylene in soil.2o8 Inasmuch as ethylene is produced both aerobically and anaerobically, its formation in aerobic soils by obligate anaerobes is presumably within anaerobic microhabitats. An oxygen-ethylene cycle that mediates the respective development of aerobic and anaerobic microbes has been postulated to explain the presumed regulatory effect of ethylene on microbial activities in This postulate, however, has not been unequivocably demonstrated. The presence of acetylene in soil could also result in the formation of ethylene by nitrogenase, a reaction that is extensively used in assaying dinitrogen f i x a t i ~ n . ~ Furthermore, during the reduction of acetylene by detached soybean nodules, acetaldehyde, acetone, and ethanol were also detected.86 Consequently, the presence of one organic volatile in soil may result in the formation of various other volatile organics. Although there is little doubt that organic volatiles are produced by microbes in soil, under both aerobic and anaerobic conditions, there have been surprisingly few studies to identify and quantitate these volatiles, despite the availability of such sensitive techniques as gas chromatography a n d mass spectrometry. These techniques, however, have been used to detect microbes in various environments (including subclinical infections) and to identify and distinguish species and strains of closely related It should be realized, however, that not all the compounds detected by these methods, which have been called “fingerprinting,” are volatile under natural conditions, inasmuch as derivatives are usually prepared from solvent extracts of cells, media, sera, etc. prior to pyrolysis or high temperature chromatography. However, as some of the microorganisms investigated by these methods are also present in soil, some of their potentially volatile compounds are included in Tables 3 and 4. These data again demonstrate the potential of microbes to produce l4

350

CRC Critical Reviews in Microbioiogy

a variety of volatilizable compounds; that different species, even of the same genus, and different strains of the same species produce different volatiles; and that the types of volatiles produced are substrate dependent. Although the spectrum of volatiles produced by microbes is broad (as indicated in Tables 3 and 4), the data are essentially qualitative, as quantitative data are seldom presented. Even when the relative amounts of each volatile in a sample are indicated, the amounts are dependent on both substrate and environment and, therefore, would be expected to fluctuate greatly in soil. Furthermore, the types and quantities of volatiles detected are dependent on the type and sensitivity of the analytical equipment (e.g., types of columns and detectors and injection and operating temperatures). This variability in techniques probably also explains, in part, the difference in volatiles detected by different investigators from the same organisms. Miscellaneous In addition to organic volatiles in soil derived from plant emanations and from microbial transformations of plant and other refuses (e.g., animal wastes), there are also organic volatiles that arise from abiotic sources and from microbial action on these and on inorganic compounds. Although a discussion of the mechanisms of formation of organometal compounds is beyond the scope of this review, their production and presence in soil and their apparent importance in pollution of the biosphere warrant their mention. The reader is referred elsewhere for more detail (e.g., Reference 223). The inorganics that have been studied most extensively are mercury, selenium, tellurium, and arsenic, as they are c a p a b l e o f being methylated by microorganisms’ 2 4 (Table 5). Methylation not only causes these inorganics to become volatile, although metallic mercury is also but it also changes their potential volatile,* 2 5 ecological significance. The altered properties of organometal compounds that have been suggested to affect their ecological impact include their tendency for complex formation, their relative and absolute water and lipid solubility, their valence state, and their distribution due to their volatility.” T h e primary microorganisms capable of methylating arsenic, tellurium, and selenium are fungi,’ 3 6 , 2 2 4 , 2 2 7-2 2 9 although a species o f

’

TABLE 4 Some Examples of Organic Volatiles Released by Microbes under Anaerobic Conditions

Volatile producer


Clostridium sp.

Mixed bacterial cultures; various pure cultures of bacteria

Unidentified bacteria

Anaerobic soil

Volatile

Comment

Dimethyl sulfide, acetic and propionic acids Formic, acetic, caproic, isocaproic, valeric, isovaleric, acrylic, bu tyric, isobutyric, propionic, crotonic acids; 2,3-butanediol, methanol, ethanol, isobutanol, isopentanol; acetoin Acetic, formic, propionic, butyric, valeric, caproic, isovaleric, lactic, succinic, isobutyric acids; ethyl acetate, acdtaldehyde, acetoin, diacetyl, ethanol, 2,3-bu tanediol, unidentified Methane, ethane, ethylene, acetylene, propylene, methyl mercaptan Methane, ethane, ethylene propylene, n-butane, isobutane, propane, butene Acetaldehyde; acetone; methyl ethyl ketone; rnethylpropyl ketone; diacetyl; cthanol, n - and isopropanol; n-, 2-, and isobutmol; acetic acid; butyric acid; ethyl acetate; ethyl butyratc; butyl acetate; butyl butyrate; methyl mercaptan; dimethyl sulfide; d i m e thy I d isul lid c

Methanobacterium has been shown to synthesize d i m e t h ~ l a r s i n e . ~ ~Both fungi and bacteria, however, are able to methylate m e r c ~ r y , ~ ’and, although methylation occurs both aerobically and a n a e r o b i c a l l y , m o r e is p r o d u c e d aerobi~ally.~~ Although the toxicity of these volatile methylated inorganics to plants and animals, including human beings, is relatively well defined, there have been few studies on the effects of these materials on microorganisms. Inasmuch as these volatiles are present in soil, their potential influence on the activity and ecology of microbes in soil requires investigation. Another group of volatile compounds that is present in soil (but a thorough discussion of which is also beyond the scope of this review) is pesticides. Inasmuch as many pesticides are volatile and others are introduced into the atmosphere during application, they often enter soils considerably distant from the target soils or plants. This translocation, not only over short distances in

’

Reference

Isolated from anaerobic mud; grown on dimethyl$-propiothetin Differed with species and substrates; determined by “fingerprinting”

239

209,215, 217,220

Various laboratory and natural substrates (e.g., sewage, milk); qualitative differences between species and strains

209.21 1, 213, 214, 216,240

Various natural habitats; methane produced only by methanogcnic bacteria Ethylene (from Clostridium sp.) may be microbiostatic factor (208) Type of volatiles produccd dependent on organic amcndments and on soil physicochemical characteristics; organic S-containing volatiles only produced with methione or pcptone amcndments

129,201

204,206-208

’

143,200

agricultural areas but also transcontinentally, has been repeatedly demonstrated and is considered to be a serious pollution problem (cf. References 6 and 26). As most of these air-borne pesticides are eventually introduced into soil, they must be considered among the volatiles that have a potential impact on microbes in soil. Another source of organic volatiles in soil from agricultural operations is the hydrocarbons resulting from the burning of crop residues. In addition to such controlled burning, wildfires in forests add significant quantities t o the ambient concentrations of hydrocarbons. The contribution of hydrocarbons to the atmosphere from such burning approaches that produced by automobile emissions.26 p 2 3 Even though a sizeable portion o f t h e s e hydrocarbons is photochemically converted in the atmosphere to other compounds, which are of concern as air pollutants, some of these hydrocarbons enter soil and affect the soil microbiota.’ In addition to the incomplete combustion of May 1976

351

N

01

w

Monomethyl and dimethyl mercury

Dimethyltellurite

Dimethylselenide

Dimethylarsine, trimethylarsine

Methyl chloride

Methyl bromide

Tellurium

Selenium

Arsenic

Chloride

Bromide

Organic product

Mercury

Inorganic substrate

Pure culture

Fomes pomaceus

Pure culture, wall paper

Pure culture, sewage soil

Pure culture

Pure culture, sewage,

sewage, pure culture

Soil, sediment,

Environment

Penicillium sp., Scopulariopsis brevicaulis. Schizophyllum commune Penicillium sp., Aspergillus sp.. Scopulariopsis brevicaulis. Candida humicola. Schizophyllum commune, unidentified soil microbes Methanobacterium sp., Candida humicola Scopulariopsis brevicaulis Fomes sp.

Various bacteria and fungi

Converting microbe

-

able t o methylate chloride

Only 6 species out of 37 tested were

Dimethylarsine formed anaerobically

Produced both aerobically and anaerobically; more efficient production aerobically

Comments

Some Examples of Inorganic Compounds Converted t o Volatile Organic Compounds by Microbes

TABLE 5


2

224,227,228 2,3

230

136,224,241

23 1-234

References


fuels as an anthropogenic source of hydrocarbons, oil spills and leaks from pipelines and sewers constitute other abiotic sources of volatile hydrocarbons in There has been little attempt at direct detection and quantitation of hydrocarbons from these sources in soil, but the apparent enrichment of microbes capable of utilizing these compounds in soil surrounding leaks (e.g., Reference 237) and the ability of soil to remove some of these v o l a t i l e ~ ~ indicate ”~~ that soil microbes are affected by such hydrocarbons. Even this cursory review indicates that there are numerous sources of organic volatiles in the biosphere and that many, if not all, are present, at some time, in soil. The sources of these volatiles may be biotic, including microbes themselves, or abiotic; they may be of natural or anthropogenic origins. Regardless of their source, the presence of organic volatiles in soil suggests that they must have some effect, either beneficial or detrimental, on soil microorganisms. Unfortunately, these effects are not sufficiently defined.

SOIL AS A SINK FOR 0 RGANIC VO LATI LES In addition to being a major source of volatile organics, the soil is also an important sink for these materials. The scavenging processes of soil involve adsorption and absorption (by both biotic and abiotic components), chemical conversion, a n d m i c r o b iol og ica 1 transformations (cf., References 5 and 6 ) . A detailed discussion of the former processes are beyond the scope of this review; emphasis, therefore, will be restricted to those processes involving microorganisms. The reader is referred elsewhere for additional information on the nonbiological removal of both organic and inorganic volatiles by soil.’ One method used to distinguish between biological and abiological scavenging of volatiles by soil has been to compare rates of removal by sterilized and nonsterilized soil. By this simple comparison, it has been shown that, for example, sulfur dioxide, nitrogen dioxide, hydrogen sulfide, and methyl mercaptan were adsorbed by abiological processes,* ,2 y 2 whereas the disappearance of carbon monoxide, ethylene, and acetylene was due to biological factors.’ ,24 a Similarly, the ability of soil to scavenge volatiles appears to differ under aerobic and anaerobic conditions; e.g., the uptake of ethylene’ or carbon

’ ‘

monoxide243 - 2 4 4 did not occur under anaerobic conditions, whereas the stimulation of the decomposition of DDT by volatiles (e.g., aldehydes and ethanol) from alfalfa hay occurred only under anaerobic conditions.’ l 4 Although there is surprisingly littIe information on the adsorption of inorganic volatiles by soil (cf., References 6 and 9 ) or by specific particulates present in soil (e.g., ammonia on silicate^,'^ hydrogen sulfide on oxides of calcium and rnagne~iurn,’~‘nitric oxide and nitrogen dioxide by calcareous soils247 T 2 4 and clay minerals,249 and ozone on soil particulates’ ,” ), there is even less known about the adsorption of organic volatiles. Short-chain alcohols, acids, and aldehydes appear to be adsorbed on magnesium oxide surfaces,2s and sterile soil differentially removed some unidentified volatiles produced by different bacteria that were either inhibitory or stimulatory to growth and sporulation of various f ~ n g i . ~ ’Conversely, neither sterile soil nor the clay minerals, kaolinite and montmorillonite, adsorbed the short-chain volatiles evolved from germinating seeds that supported microbial growth.” Inasmuch as the adsorption and binding of some nonvolatile organics by clay minerals appear to protect them from microbial degradation (cf., Reference 7), the adsorption, by both organic and inorganic soil particulates, and the subsequent degradation of various types of organic volatiles in soil merit extensive investigation. In addition to the direct effect on soil microbes of the scavenging of volatiles by soil, the indirect effect of the uptake of volatiles by plants and their subsequent introduction via plant residues into soil might be important. Although vegetation appears to be an important sink for various inorganic volatiles (e.g., hydrogen fluoride, sulfur dioxide, chlorine, nitrogen dioxide, nitric oxide, ozone, 3-2 and carbon monoxide, and ammonia)’ models for simulating sorption of gaseous pollutants by plants have been proposed,’ 5 4 there are essentially no data on the uptake of organic volatiles by plants. Seedlings of various species (e.g., cucumber, squash, cabbage, radish, pea, bean, and pine) can apparently reduce the concentration of aldehydes (primarily formaldehyde), ethylene, acetylene, and some other unidentified organic air pollutants in ambient atmospheres and in atmospheres amended with these and other. organic ~ o l a t i l e s .The ~ ~ fate of these volatiles, however, is not known. Similarly, although damage to plants by various organic air

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May1976

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pollutants has been often demonstrated (cf., References 5, 6 , 2 6 , 27, and 258), it is also not known whether these volatiles enter the plant, are metabolized, or are eventually introduced into soil. Inasmuch as many of these organic volatiles can exert a marked effect on microbes, the potential, albeit indirect, influence of organic air pollutants on microbial events in soil should be clarified. Another abiotic mechanism that may remove organic volatiles from the soil atmosphere, and one that requires investigation, is the dissolving of volatiles in the soil water and their subsequent removal by leaching. The amounts of volatiles that will dissolve in soil water depend on the characteristics of the volatiles and on numerous environmental factors (e.g., temperature and the ionic composition and strength of the soil solution). Many water-soluble volatiles will, of course, be rapidly decomposed by the soil microbiota (e.g., ethanol, acetaldehyde, and acetate).96p25 9-26 The primary factor that accounts for the efficacy of soil as a sink for volatile organics is the presence o f microorganisms, some of which are capable of degrading one or another of the various organic volatiles that enter soil. Microbes are capable of utilizing a variety of hydrocarbons, whether of biotic or abiotic origin.8p26l a N u m e r o u s bacteria (e.g., Methanomonas methanica, Mycobacterium methanicum, Pseudomonas methanica, Methylococcus capsulatus, and Methylosinus t r i c h o s p o r ~ m ) 7, ,~2~6 2 7 2 6 fungi (e.g., some species of A ~ r e m o n i u m . ~ Cephalosporium,26 Gmphiwn, Phialophora,266a and Penicillium),26 and a green alga (Chlorella sp.)266 have been shown to be capable of oxidizing and utilizing methane, although the predominant methane utilizers appear to be Cram-negative, aerobic bacteria.26 A soil isolate of Brevibacterium sp. metabolized propane;26 species of Mycobacterium, Flavobacterium, and Alcaligenes utilized ethane;' species of A r t h r e bacrer and Brevibacterium, as well as some fungi (Penicillium nigricans, Allescheria boydii, Graphium cumeifemm, and possibly a Gliocladium sp.) utilized n-butane;' and species of Mycobacterium oxidized propane, n-butane, n-pentane, and n-hexane. The transformation of the latter hydrocarbons by mycobacteria resulted in the production of other hydrocarbons (e.g., methyl ketones), which, in turn, could be used as sole carbon sources by the bacteria.270

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Components of the aerobic microbiota in soil are capable of degrading natural gas, which contains primarily methane, ethane, and propane, with traces of n-butane, isobutane, n-pentane, and isopentane. The mixed microbiota in soil is apparently necessary for these transformations, as isolates of hyphomycetes capable of growing on natural gas utilized ethane, propane, or n-butane but would not grow on methane alone.27' Similarly, 15 strains of Arthrobacter and Brevibacterium and four fungal species isolated from soil in areas of natural and domestic gas seepage grew on n-butane, isobutane, propane, or ethane, but none grew on methane.26 In addition to the metabolism of volatile saturated hydrocarbons in soil, aerobic microbial activity removes approximately 7 X 1O6 tonsfyear of ethylene from the atmosphere in the U.S. alone.8 Acetylene is probably also removed in appreciable quantities by soil, primarily by aerobic and anaerobic organisms able to fix dinitrogen and which convert acetylene to e t h ~ l e n e . *~4 4 The soil microbiota is also capable of utilizing a variety of saturated and unsaturated hydrocarbons from dilute automobile as well as benz(a)pyrene particulates emitted into the a t m o s p h e r e by aircraft engines and oil refineries.2 * Not only d o soil microbes metabolize hydrocarbons of relatively simple structure, but microbes in soil, in litter, and on plant surfaces also utilize terpenes and other volatiles emanating from plants. Organic volatiles from tropical foliage were utilized as the sole carbon source by f ~ n g i . ~It ~ has ~ * been ~ ~ postulated ~ that microbes, especially in the rhizosphere and phyllosphere, constitute a major global sink for the removal of naturally occurring organic ~ o l a t i l e s . ' ~ The spermosphere should, perhaps, be included in this 'postulate, as volatile compounds from germinating seeds supported growth of a variety of aerobic bacteria and fungi in the absence of any other carbon s o u ~ c e ' ~as well as influenced the formation' and germination" of fungal spores. Little is known about either the origin or fate of volatile nitrogen-containing organics in soil. Although a variety of volatile alcohols, aldehydes, acids, and esters were produced in soils amended with amino acids, no volatile organics containing nitrogen were detected.' 4 3 Polyamines associated with decaying animal cells (e.g., cadaverine and putrescine) and compounds associated with fecal


material (e.g., skatole and indole) are not usually included in lists of nitrogenous volatiles detected in soil (e.g., References 275 and 276), suggesting that they are either adsorbed and/or rapidly degraded or they have not been sufficiently searched for in soil. Small concentrations of such volatile organic nitrogen compounds have been detected around animal wastes (e.g., References 277 to 279), but their ultimate fate, especially in soil, has apparently not been determined. Passage of sewer gases through soil removed nitrogencontaining organic volatiles from the a t m o sphere,238 but their final fate also has not been established. More is known about the origin o f volatile organic sulfur compounds in soil than about organic nitrogen compounds, but there is little information on the degradation of volatile sulfur compounds in soil. These volatiles, however, are p r o b a b l y d e c o m p o s e d readily in 3 3 ,I 4 2.23 8,280

Although organic volatiles can serve as a sole energy and carbon source for some microbes, the presence~ofother carbonaceous materials is sometimes necessary for utilization of these volatiles (e.g. volatiles from alfalfa enhanced the anaerobic degradation of DDT;’ decomposition of hydrocarbons was improved by the presence of simple substrate^;^^ Craphiurn sp. would not grow on only methane but cooxidized methane in the presence of ethane).’66 Furthermore, the heterogeneous microbial population in soil is more effective in the degradation of some organic volatiles than are pure cultures (e.g., mixed soil inocula degraded hydrocarbons more extensively and completely than did inocula of individual species).’ In addition to microbial methylation of inorganics in soil, degradation and, thereby, detoxification of such compounds also appear to occur in Although there have been few studies on the decomposition of organometallics in soil in siru, studies with aquatic sediments and with pure cultures isolated from soil show that organic mercurials are readily degraded. Incubation of lake sediments with inorganic mercury resulted first in an increase and then in a decrease of methyl mercury and in the evolution of elemental mercury. The organisms responsible for this appeared to be species of Pseudomonas, which in pure culture rapidly converted methyl mercury to elemental mercury and methane.’ * A pseudo-

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monad isolated from soil contaminated with phenylmercuric acetate, a slimicide, decomposed phenylmercuric acetate, ethylmercuric phosphate, and methylmercuric chloride to yield volatile elemental mercury and, respectively, benzene, ethane, and methane.’82 Hence, the decomposition of organic mercurials also results in the volatilization of hydrocarbons. The bacteria capable of degrading organic mercurials appear to b e more resistant to mercury than nondegraders,2 J J 8 4 and this resistance appears to be plasmid related in Pseudomonas aemginosa, as well as in Escherichia coli and Staphylococcus aureus.” The ability to volatilize elemental mercury from cationic or organomercurials, the presumed mechanism for mercury resistance, is apparently determined by inducible R-factor systems in enteric bacteria.’ Volatile elemental mercury has also been produced abiotically in aqueous solutions containing humic acid by the chemical reduction of mercuric ions.286 Soil, therefore, is clearly a sink for volatile organics as it is for most other organics in the biosphere (cf., Reference 7). Some of the organisms and metabolic pathways responsible for the transformation of these volatiles are known, but they are not as well defined as they are for most nonvolatile organics. Furthermore, the rates and capacities for the utilization of various volatile organics in soil are not known. Because of the important role of soil as a sink for air pollutants, of both natural and anthropogenic origins, such data should be obtained.

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EFFECTS OF ORGANIC VOLATILES ON MICROBES IN SOIL General Comments Soil is both a source and a sink for volatile organics, with the soil microbiota being actively involved in both the production and transformation of these materials. Many of these volatile organics, regardless of their origin, appear to influence the activity, ecology, and population dynamics of microbes in soil. These influences will be discussed in this section, which has been divided into subsections on the basis of the source of the volatiles involved. Volatiles from Living Plants (Table 1) There is limited information on the use of volatiles from plants as nutrient sources by May 1976

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microbes in soil. However, as many of the components of plant emanations are readily utilized in solution by microbes, both in pure culture and in soil, there is little doubt that the volatiles are so used in soil. For example, short-chain (C, to C4) alcohols, aldehydes, and acids have been shown to be decomposed rapidly in soil.4 ,93 ,94 ,96 ,9 7 , 1 5 9-1 6 1 Volatile organic compounds evolved from germinating seeds of slash pine, bean, cabbage, corn, cucumber, and pea served as sole carbon sources for the growth of bacteria (i.e., Pseudomonas fluorescens, Bacillus cereus, Erwinia carotovora, Agrobacterium tumefaciens, A . mdiobacter, and Rhizobium japonicum) and fungi (i.e., Mucor mucedo, Fusarium oxyspomm f. conglu tinans, Trichodenna viride, and Penicillium venniculatum) in mineral salts media. As few as five pea seeds provided sufficient volatiles to support the growth of P. fluorescens. The volatiles did not support growth of Serratia marcescens or Sarcina lutea, which are more fastidious heterotrophs and require various growth factors, nor did the volatiles provide a source of nitrogen.'' Although the seeds evolved a variety of volatiles (e.g., short-chain aldehydes, ketones, alcohols, acids, ole fin^),^ /j4 the microbes appeared to use primarily aldehydes (especially acetaldehyde) as the substrate, even though alcohols (especially ethanol) were evolved in substantially higher quantities. The volatiles supporting microbial growth were not absorbed by water or adsorbed by clay minerals or organic matter, as passage through water, sterile soil, or clay minerals (i.e., kaolinite and montmorillonite) did not reduce the ability of the volatiles to support growth. Passage of the seed volatiles through chemical solutions, that absorb either oxidizable compounds or specifically aldehydes, or over charcoal or nonsterile soil significantly reduced their ability to support growth of the test organisms. These observations suggest that volatile organics evolved from germinating seeds move outwards from the seed through the soil and, as they are not appreciably sorbed by soil particulates or water, their influence on the soil microbiota probably encompasses an area larger than of nonvolatile organic excretions.' In addition to short-chain volatiles of relatively simple structure, hydrocarbons liberated by the canopy of tropical forests were utilized by microbes in the rhizosphere and phyllosphere of surrounding plants and in litter.73 356


When used as nutrients, plant volatiles appear to be used primarily as carbon and energy sources, as there have been no reports that either nitrogenor phosphorous-containing organic volatiles are released by plants or that the sulfur of sulfurcontaining volatiles is used as a nutrient source. More studies are obviously needed to define the role of volatiles from living plants as nutrient sources for microbes in soil. Many more studies have been conducted on the effects of organic volatiles from plants on the stimulation or inhibition of microbial growth and development than on the use of volatiles as nutrients. Small amounts of ethanol increased the growth of some fungi on carbon sources that the fungi did not use efficiently, suggesting that the volatile acted as a metabolic regulator or a growth factor (cf. Reference 4). Similarly, volatiles from germinating seeds enhanced growth of A g o bacterium tumefaciens, a nonfastidious hetkrotroph, in the presence of glucose and mineral salts.s9 Ethylene, which is evolved by various plant parts, has also been implicated as a regulator of microbial development in soil.' ,*O 8 The germination of teliospores of Puccinia carthami was stimulated by unidentified volatiles from its host plant, safflower." The germination of uredospores of another rust fungus, P. graminis, was stimulated by a variety of terpenes and aldehydes produced by the f ~ n g u s . ~ ' However, such volatiles are also produced by plants, and these may exert a similar stimulatory effect on the germination of uredospores. The germination of spores of Botrytis cinerea was stimulated by volatiles from apple leaves and fruit, from crushed leaves of a variety of woody and herbaceous plants, and from rose petals.59 Volatiles from germinating seeds of pea provided sufficient substrates, probably aldehydes, to induce germination of spores of Trichoderma viride. 8 9 Onion and leek seedlings evolved organic sulfides (e.g., n-propyl and allyl sulfides) that stimulated the germination of sclerotia of Sclerotium cepivomm in and cnicifers evolved allyl isothiocyanate which stimulated the g e r m i n a t i o n of spores of Plasmodiophora brassicae.8 3 These stimulatory effects of sulfurcontaining organics were in contrast to the reported effects of such volatiles on Aphanomyces euteiches. o-6 Although the relatively few studies on the stimulatory effects of plant volatiles have been conducted primarily with plant pathogens, there

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have been, surprisingly, even fewer studies on the effects of these volatiles on the attraction of microbial pathogens or symbionts to plants or on the actual infection process. Zoospores of Phytophthora cinnamomi were attracted to ethanol in glass capillaries with dimensions presumably similar to those of soil pores.2 The infectivity of bean leaves inoculated with spores of Botrytis cinerea was enhanced when leaves of Amomum cardomomum or Choisya ternata, both of which release volatiles that stimulate germination of these spores, were placed nearby.” Conversely, exposure of sweet potato root tissue to 8 ppm ethylene for 2 days induced resistance to infection by Ceratocystis fimbriata. 2 8 ’ a There was no relation observed between the amounts or times of maximum production of oxidizable (by KMn04) volatiles by seeds of different varieties of pea and the resistance or susceptibility of these varieties to Fusarium oxyspomm f. pisi. The involvement of volatiles in mutualistic relations between plants and microbes has also received little attention. The symbiotic relation between Pinus sylvestris and the mycorrhizal fungi, Boletus vanegatits and Rhizopogan roseolus, appears to involve a complex sequence of volatile production. The root system of the conifer releases a variety of volatile and nonvolatile materials that are stirnulatory and attractive to fungi, both mycorrhizal and nonmycorrhizal. The mycorrhizal fungi produce antibiotic substances that retard the growth of nonmycorrhizal competitors, thereby, facilitating infection of the roots by the mycorrhizal symbionts. The tree responds to infection by the production of volatile (e.g., terpenes) and nonvolatile (e.g., phenolic) substances, which retard the spread of the fungal symbionts within the host and enable a balanced mutualistic relation. The volatiles produced by the host are only fungistatic to the mycorrhizal fungi, as normal growth of the fungi resumes, in vitro, when the concentrations of individual terpenes (mono- or sesquiterpenes) are reduced below i n h i b i t o r y levels.23 9 2 4 A similar selective mutualistic relation occurs in orchids, which produce orchinol that prevents penetration of orchid tubers by nonmycorrhizal fungi but permits penetration by mycorrhizal fungi.” The importance of volatile organics from plants in overcoming fungistasis, and perhaps bacteriostasis, in soil also requires more investigation. As will be discussed later, there is considerable

evidence to indicate that volatile factors are responsible for the imposition of fungistasis in soil, but there are relatively few data that show the effects of organic volatiles from living plants on the release from fungistasis. Inasmuch as fungistasis can be reversed by the addition of soluble carbonaceous nutrients to soil (e.g., Reference 289, 290, and 291), it seems reasonable to suspect that volatile organics would have the same effect, especially in microhabitats near their source where concentrations would be sufficiently high. It appears that more study is needed to define the stirnulatory effects of organic volatiles from living plants on microorganisms, especially in soil. There is currently more known about the inhibitory effects of such volatiles on microbes, although many of these studies have, again, been conducted with plant pathogens. Volatiles from as few as five germinating bean seeds per Petri dish inhibited the formation of conidia in Penicillium vermiculatum, zygospores in Zygorhynchus vuilleminii, and perithecia in Gelasinospora cerealis, but they did not affect sporulation in Aspergillus jlavipes, Fusarium oxyspomm f. conglutinans, Cunninghamella elegans, I? viridicatum, or Trichoderma viride. Volatiles from 15 germinating seeds of cucumber a l s o inhibited zygospore formation in Z. vuilleminii 8 7 Volatiles from germinating seeds of various vegetable and cereal plants inhibited the germination of spores of Bottytis cinerea, Mucor ra c ern ostis. Trichoderma viride, Verticillium dahliae, and, with only some seeds, Fusarium o~yspontm.The effects of the volatiles were more a function of the species of fungus than of the plant, as volatiles from one species of plant either inhibited, stimulated, or had no effect on the germination of spores from different fungi. Whei the fungi were exposed to the volatiles in the presence of KOH, spore germination was reduced even further, presumably because of the absorption of CO?. When KMn04 was present, the i n h i b i t o r y e f f e c t s of the volatiles were decreased,8 suggesting that the inhibitory factors were oxidizable volatiles.8y The germination of spores of Penicillium digitatum was inhibited by various aldehydes (C, to C,) that are volatile components of citrus fruits, whereas other components (e.g., alcohols, esters, and terpenes) did not inhibit germinat i ~ n The . ~ ~isolation of cu-hexenal from leaves of Ginkgo biloba by homogenization and distillation May 1976

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suggested that the resistance of this plant to fungi is related to the presence of this fungitoxic aldehyde in sufficiently high concentration (75 ~ p m ) . ~ Volatile sulfur compounds from cruciferous plants inhibited the germination of the zoospores of Aphanomyces euteiches o-6 In addition to the inhibitory effects of plant volatiles on fungi, volatiles from foliage of both coniferous and deciduous trees affected growth and spore germination of Bacillus thuringiensis and B. cereus. The volatiles either inhibited, stimulated, or had no effect, depending on the plant speciesa4 The active components were assumed to be terpenes, and studies with individual terpenes (a- and P-pinene, limonene, phellandren, fenchone, and thujone) from Abies balsama showed that these compounds were differentially active against various Bacillus sp. as well as other Gram-positive and negative bacteria.8 Leaves of Salvia leucophylla evolved terpenes which inhibited the growth of soil bacteria.” Ethylene, which is produded by living plants, may be responsible for both fungistasis and bacteriostasis in soil.zo6-20 Volatile terpenes from a variety of plants have been shown to be toxic to various fungi.” Terpenes from crude resin of Pinus ponderosa were inhibitory t o the growth of Fumes annusus and four species of Ceratocystis. The degree of inhibition differed with both the type of terpene and the species of fungus.’03 The volatiles from the heartwood of most Cupressaceae were more toxic to fungi that were volatile oils from Pinaceae, presumably due to the presence of phenolic compounds in the former.’ It has been suggested that a possible function of terpenes in wood is to retard the growth of fungi until the plant can synthesize and accumulate sufficient quantities of phenols, which are more effective than terpenes against fungi.’ 7 1 The infection of roots of Pinus sylvestris and P. echinata by mycorrhizal fungi often results in an increase in terpenes, which restrict not only the further proliferation of these fungi but also of soil-borne, root-infecting pathogens, such as Fomes annosus a n d Phytuphthora The role of these terpenes in cinnamomi ” conifer roots on the maintenance of a balanced mutualistic relation and on the resistance to invasion by pathogens has already been discussed. The specificity of terpenes against fungi is further demonstrated by the diterpene, sclareol,

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which exists as an epimeric mixture and constitutes 10% of the surface exudate of Nicofiana glutinosu. This terpene affects neither the spore germination nor mycelial production of several species of facultative pathogenic fungi in vitro, whereas the growth of Alternaria brassicola and the germination of uredospores of various rust fungi (e.g., species of Puccinia and Uromyces) were inhibited.’* It is apparent, from even these relatively few studies, that volatiles from living plants play a major role in plant pathogenesis by either inhibiting or stimulating the propagules of the pathogen. Unfortunately, essentially nothing is known about the effect of these volatiles on altering the susceptibility or resistance of the plant host to infection and disease development (e.g., to what extent do volatiles function as phytoalexins). The importance of such allelochemical reactions in the interrelations between species of different kingdoms of organisms’ applies, however, not only t o plants and their microbial pathogens or symbionts, but also to interactions between microbes. The selective inhibition or enrichment by plant-derived volatiles of specific microorganisms, whether they are pathogens or not, will ultimately result in changes in the ecology of microbes in soil. The mechanisms whereby such changes can be effected (e.g., by selective stimulation or inhibition; by alterations in morphology and reproductive potential, which render the organism more or less suited to the prevailing environment; or by attraction to a favorable host) require extensive study. Similarly, the influence of ambient environmental conditions (e.g., the pH, E,, temperature, aeration status, etc. of the soil; the health of the host) on the influence of plant-derived organic volatiles on microbes in soil requires clarification. Furthermore, the detection of an organic volatile, derived from living plants, in soil does not confirm that the volatile is affecting microbes in soil, even though the same volatile can be demonstrated to have marked effects on soilderived microbes in vitro (e.g., Reference 46,49, and 59). Although a volatile in vivo must probably also be dissolved in the liquid phases around and inside microbial cells to exert its effect, the amount of volatile dissolved depends, in part, on its concentration in the vapor phase and on its solubility coefficient in the specific liquid phase. Consequently, the concentrations of volatiles in soil, in


situ, probably vary and have a concentration gradient away from the site of production. Inasmuch as the effects of volatiles on microbial events appear to be concentration dependent (e.g., the germination of spores of Botrytis cinerea was inhibited by saturated solutions but stimulated by 10% saturated solutions of ethyl acetate, malate, and citrate, the presumed volatiles emitted by leaves and fruits of apple and from numerous other plant species and parts),' general statements about the effects of volatiles from living plants on microbes must be accepted cautiously, especially in the absence of any data on the concentrations present in the target area. Despite the absence of such detailed information, it appears clear that volatile organics emanating from living plants influence microbes in soil. This influence may be restricted close to the origin of the volatile or, depending on its concent r a t ion and the prevailing physicochemical conditions of the soil, may be exerted at some distance. Volatiles from Plant Residues (Table 2) Plant residues probably constitute the major source of nutrition for microbes in soil. However, as with volatiles emanating from living plants, there are sparse data on the nutritional value and utilization of volatiles from these residues. Volatiles from plant residues with a low carbon:nitrogen ratio increased growth of Rhizoctonia ~ o l a n i , ~ 'and volatiles from distillates of corn leaves, wheat straw, alfalfa hay, bluegrass clippings, and tea and tobacco leaves increased both the respiratory rate of the soil microbiota' and the numbers of fungi and bacteria isolated.9z>97 ' " The components used by the microbes were primarily acetaldehyde (which stimulated fungi more than bacteria) and ethanol, even though methanol, isobu tyraldehyde, 2-met hylbutanol, and/or valeraldehyde were also components of the ~ o l a t i l e s . $9 ~ When C-ethanol was added to soil, respiration increased, and 80 to 90% of the evolved COz was labeled.96 As the concentration of the volatiles increased, they became inhibitory to the microbe^.^' The use of ethanol as a primary substrate in these studies is of interest, as soil isolates grown in vitro on volatiles from germinating seeds, which also released large quantities of e than01 ,3 appeared to use primarily acetaldehyde as the carbon source.89 As with volatiles from living plants, organic T~~

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volatiles from plant residues appear to be sources of only carbon and energy, as there are no data which suggest that these volatiles are used as sources of other nutrients. Furthermore, as with volatiles from living plants, most studies on volatiles from dead plants have been concerned primarily with stimulatory or inhibitory effects, especially on plant pathogens. The sclerotia of Sclerotiwn rolfsii were stimulated to germinate when exposed to volatiles from alfalfa hay,' O-' and subsequent mycelial growth was enhanced.' When the sclerotia were exposed to these volatiles in nonsterile soil, they again germinated, but the mycelium was rapidly Iysed as a result of the increased activity and numbers of other soil microbes.' 91 Volatiles from alfalfa hay also stimulated the germination of microsclerotia of Verticillium dahliae, but, as the volatiles also resulted in an increase in bacteria and actinomycetes in the soil, the population of V. dohiiae rapidly decreased to below its original level .9 The amendment of soil with residues of oat or alfalfa increased soil respiration but decreased the survival of Verticillium albo-atmm; presumably because the other soil microbes inhibited germination of the pathogen."7 Fusaria pathogenic to onion were also reduced in soils amended with crop residues,' as was Rhizoctonia solani (one of the fungi involved in the pinto bean root rot c o m p l e x ) i n soils amended with barley residues.' * Although volatiles were not shown to be directly involved in these events, the results of other studies would suggest that organic volatiles from the residues played some role. The reduction of bean root rot, caused by Thielaviopsis basicoh, after the addition of crop residues was suggested to be caused, in part, by volatiles stimulating the germination of the propagules of the pathogen, which were then lysed by the soil microbiota before a susceptible host that the fungus could invade was present?' Volatiles from alfalfa hay stimulated the germination of conidia of Fusarium ~ o l a n i and ,~~ conifer litter stimulated the germination of chlamydospores of Fusariurn.'Z a Volatiles from various decomposing plant tissues affected pigmentation, growth, and survival of R. solani." ' In these examples, the plant residues and the volatiles evolved appeared to be nonspecific in that residues from various plant species stimulated a variety of plant pathogenic fungi as well as the

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heterotrophic soil microbiotia. The primary stimulants appeared to be acetaldehyde and ethanol.'' ,96 The stimulation of germination of the sclerotia of Sclerorium cepivorum by volatiles from species of Allium appeared, however, to be a specific s t i m u l a t i ~ n . ' ~ -Plants ~~ of this genus contain a variety of sulfoxides which, although themselves active against microbes,' give rise to organic sulfides5 that stimulate the germination of S. cepivorum in soil. Mustard oil, allyl isothiocyanate, present in crucifers also stimulated the g e r m i n a t i o n of spores of Plusmodiophom brussicue. Sulfur-containing organic volatiles from plant residues, however, have also been shown to inhibit plant pathogens in soil. Decomposing crucifers, especially cabbage, released a variety of sulfurcontaining volatiles (e.g., methyl isothiocyanate, allyl isothiocyanate, butyl isothiocyanate, methyl mercaptan, dimethyl sulfide, and dimethyl disulfide) which inhibited the production, development, and germination of zoospores and the growth of Aphunomyces euteiches.60-6 Similar effects have been observed with Thieluviopsis sp.63*90 The active components appeared to be methyl mercaptan and the methyl sulfides. Allicin, formed enzymically when garlic plants are crushed, is one of several sulfoxides that is bactericidal.'" The crucifers and a few other plant families contain mustard oil glycosides that are enzymically hydrolyzed upon cell damage to release volatile mustard oils such as allyl isothiocyanate (cf., Reference 1). These and other cyanogenic glycosides are further decomposed in soil to yield hydrogen cyanide (cf. References 12, 106, and 107). Although plant residues incorporated in soil appear to reduce the incidence and severity of diseases of crops subsequently planted in the amended soil, the mechanisms involved and the importance of organic volatiles in this reduction are not always clear. For example, the control of root rot of avocado, caused by Phytophthom cinnumomi, by the incorporation of alfalfa meal in soil was apparently caused by the high levels of ammonia produced.' Similarly, it is not clear in all cases whether the organic volatiles produced from residues arise directly from the residues or whether they are products of microbial transformations of the residues. With one type of plant residue, namely woody


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tissue, it appears that the active volatiles arise from the plant tissues. When volatiles from heatdried wood of red pine were tested for their effects on Fomes annosus, the growth of the wood-inhabiting basidiomycete was stimulated by fatty acids, especially by linoleic and oleic acids, whereas a series of aldehydes (C, to C l o ) and terpenoids (e.g., a- and 0-pinene) had no e f f e ~ t . ' ' ~Eleven other wood-rotting fungal species showed increased growth when exposed to volatiles from heat-treated wood of Pinus sylvestris, and Coniophoru cerebella exhibited altered mycelial morphology." Volatile terpenes, from a variety of woods, generally inhibited the growth and spore germination of the test fungi.'y2'4" O 3 It has been suggested that one of the beneficial effects of the controlled burning of forests, or of permitting natural fires to continue, is the fumigation o f soils with volatile phenols from the burning wood, which provides some control of outbreaks ot pathogenic fungi.' The lack of effect of aldehydes and pinenes from red pine on F. annosus' O 4 is interesting, as many fungi (including wood decomposers such as Pu~vporusupplunutus) have been shown to be stimulated by nonanaIa 78 and other aldehydes (e.g., References 89, 92, and 96) and inhibited by pinenes and other terpenes (e.g.. Reference 15). These differences in effects with the same types of volatiles may have been the result of differences in the concentration of the volatiles used. Unfortunately, the vapor phase concentrations of the volatiles used are seldom indicated, and, inasmuch as the volatiles are usually extracted from the plant and applied as solutions to fdter paper discs, it is not possible to compare the concentrations of the individual compounds that actually interact with the assay organisms. The importance of concentration is further emphasized by studies which showed that high concentrations of volatiles from a variety ot crop residues inhibited, rather than stimulated, the respiration and numbers of soil microbes.92 There is little doubt that volatiles from dead plants influence microbes in soil; but, as with volatiles from living plants, there are numerous aspects that require further investigation. Although some of these volatiles appear to be able to release resting fungal propagules from fungistasis the specificity, both in terms of the types ot volatiles responsible and the spectrum of fungi affected, requires clarification. The need for such

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clarification is especially important if crop residues, and the volatiles from these, are t o be used more extensively as a method of biological c o n t r o l o f s o i l - b o r n e p l a n t pathogens. Furthermore, better definition o f the mechanisms whereby these volatiles exert such biological control (e.g., b y directly affecting the pathogen through influencing its growth, sporulation, or spore germination or by affecting the pathogen indirectly by stimulating the amensalistic, lytic, or competitive activities o f the nonpathogenic soil microbiota) should sharpen the focus on the most effective types and amounts o f residues to use. The effects of soil type and environmental conditions (e.g., aeration status, pH, and temperature) on the types and amounts of volatiles produced and on their microbiological effects also require clarification. Similarly, being able to distinguish between the potential functions of an "active" volatile (e.g., as a nutrient, as a germination inhibitor or stimulator, as a metabolic regulator) should help in choosing the most effective plant residues and in deciding whether any other materials (e.g., inorganic nutrients) should be concurrently added. Furthermore, studies of this type might indicate i f and which other organic residues (e.g., animal and anthropogenic wastes) might function as well or better than plant residues in affecting microbial events in soil. Volatiles from Microorganisms (Table 3) Although organic volatiles from living and dead plants, from animals, and from abiotic sources undoubtedly affect microbes in soil, the most important sources, with the greatest potential influences, are probably organic volatiles evolved by microorganisms. This, however, is a presumption, inasmuch as data on actual volatile production by microbes in soil are sparse and data on their effects even more sparse. Consequently, extrapolations are usually made from in vitro studies to what presumably occurs in soil. Until more precise techniques are developed to enable the study of microbial events in soil in situ, evaluations o f the effects of microbially produced volatiles on the same or other microbes in soil will be restricted t o such extrapolations and surmises. However, in vitro, many microbes known to be soil inhabitants are able t o produce organic volatiles that affect other soil-derived microbes. Consequently, it is probable that such or similar volatiles are also produced in soil and that they

affect microbes in the same and adjacent microhabitats, similar t o the presumed production and effects of nonvolatile metabolites (cf. Reference

7). Most o f the studies on the effects of volatiles from microbes on other microbes have been concerned primarily with broad inhibitory or stimulatory effects, and there have been few studies on the importance of these volatiles as a source of nutrient^.^^^'^-^^^ However, as some of the components present in microbially produced volatiles have been shown in vitro t o serve as nutrients for microbes (e.g., acetaldehyde and ethanol), it is probable that they serve a similar function in soil. It has also been suggested that some vitamins or precursors of vitamins are constituents of the volatiles found in soil atmospheres and that they are of microbial origin (cf. References 293 and 295). The effectiveness of volatiles as nutrients' or growth factors is, undoubtedly, dependent on their concentration at the sites of microbial development, which, in turn, is dependent on the physicochernical characteristics of the soil (cf. Reference 7). The volatiles produced by microbes can be either stimulatory or inhibitory t o microbes of other species or t o propagules of the same species. Although the mechanisms o f these responses have not been clarified in most interactions (cf. Reference 4), the latter situation represents an interesting mechanism of self-regulation. The germination of microconidia of Fusurium oxysporum a n d sporangiospores of Rhizopus stulonifer was inhibited by volatiles (especially acetaldehyde) produced by their respective mycelium.' Y-' " The teliospores of Tifletia caries, T. foetida, and T. contrvversa released volatiles (e.g., ammonia, trimethylamine, and two unidentified amines) which inhibited other spores of the same species. The active volatile has not been identified, and trimethylamine was inhibitory only at concentrations that probably would not accumulate in vivo. " The uredospores of Pziccinia graminis var. tritici produced volatiles that inhibited their 809'82y183 The active volatile was thought to be trimethylethylene' 79 or acetaldehyde,' although more recent studies indicate that it is a mixture of the cis- and trans-isomers of methyl ferulate (methyl'

' *' *'

4-hydroxy-3-rnethoxycinnamate).'* Volatiles from an organism may also stimulate the production or germination of propagules of May 1976

361


that organism. Zygospore production in young cultures of Rhizopus sexualis was stimulated by volatiles from mature cultures of the same species,’ 7 8 and volatiles produced by mycelium of Mucor mucedo induced zygospore formation in unlike mating types of the fungus.’ ” The uredospores of P. graminis var. tritici produced volatiles that not only overcame self-inhibition or inhibition caused by other agents, but they also retarded growth of the germ tube while stimulating development of appressoria and infection hyphae.’ Although the uredospores released a variety of volatiles (e.g., aldehydes of various chain lengths, terpen~ids),”~’8 2 the active agent appeared to be n-nonana17 8 4 and, possibly, f ~ r f u r a l .Volatiles ~~ from their own sporidia or mycelium, as well as from host plants, caused sporidia of some Ustilago sp. to germinate by producing a germ tube rather than additional ~ p o r i d i a The . ~ ~ germination of spores of Agaricus bisporus’ a a and A. campestris.I appears to be stimulated by volatiles (e.g., 2,3-dimethyl-lpentene for A. campestris and isovaleric acid for A. bisporus) evolved by their own mycelium. Volatiles also appear to be responsible for the attraction and aggregation of the ameboid cells of slime molds (e.g., References 296 and 297). Although self-regulation by the production of organic volatiles is interesting, the importance of this phenomenon in soil is unknown. Many of the known examples of this phenomenon involve fungi normally found on the aerial parts of plants, where the environment and the general microbial composition differ from those in soil. Consequently, the role of volatiles in the self-regulation of microbes in soil, as both inhibitors and stimul a t o r s , requires further study before any meaningful extrapolations to the soil environment can be made. An example of the involvement of volatiles in self-regulation of an organism in soil is the “decline” phenomenon of the “take-all” disease of grains caused by Gaeumannomyces graminis var. tritici. The fungus apparently releases a volatile(s) which triggers a mechanism that causes lysis of the organism, with the concomitant release of additional volatile which remains in the soil and induces further lysis. This volatile, as yet unidentified, causes transmissible lysis only in some strains of G. graminis and none in other species of fungi. The volatile also has a growth-stimulating effect, even on strains of G.graminis that are not induced y’

1‘

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CRC Critical Reviews in Microbiolog-v

to lyse, that precedes the appearance of lysis. The lytic factor appears to be extremely stable, as it is not inactivated by heat, nucleases, filtration, 01 UV-irradiation.’ ” A phenomenon more common than selfregulation by volatiles appears to be the stimu. latory or inhibitory effects of volatiles from one species ofl another. However, it should again be emphasized that most demonstrations of suck effects have also been in vitro. Interspecific stimulatory effects have been observed primarily with fungi. Volatiles frorr Mucor spinoszis stimulated mycelial growth of Phytophthora citrophthora. ’I Zygospore production by Rhizopus sexualis was stimulated by volatiles not only from the same organism’ 7 R bui also from Fomes annosus, Ceratocystis coem lescens, and Miicor plumbeus. ’I A1though thc growth of Pestalotia rhododendri appeared not tc be influenced by organic volatiles,‘ the’ pro. duction of conidia was stimulated by a variety 01 volatile organic acids, alcohols, esters, anc carbonylcontaining compounds,’ all of whick were released by Dipodascus aggregarus. Tht stimulation by one component, n-propanol, wa: temperature dependent, suggesting that thi: component was involved in a sequence of enzymic reactions that resulted in conidiation of P. rhodo dendri. Volatile compounds evolved by microbe: present in the substratum used for mushroon production induced the formation of sporocarp: of Agaricus bisporus. 9 4 Pseudomonas pu ridL and related species, w h c h apparently producec these volatiles, were, in turn, stimulated b) volatiles released by A. bisporus. I 9 4 In addition tc this reciprocal interspecific stimulation, volatile: from the substratum inhibited, at certair temperatures, the germination of spores of Verti cillium malthousei, a pathogen of A . bisporus. 9 t The spores of A . bisporus were also stimulated bl volatiles evolved from its own mycelium.’ * Volatiles, derived either intra-or interspeci fically, also appear to be important in the repro duction of some fungi. Both positive and negativc zygotropism between zygophores of plus an( minus mating types4 J and zygospore formatior in Mucor rnucedo could be induced betweer unlike mating types, even in the absence of direc contact or contiguous water films between them Consequently, trisporic acids (which are pre sumably produced by the mycelium and necessaq

‘

‘

‘

‘

‘’

‘

‘


'or zygophore formation) were not involved, as hey are not volatile. However, some precursors of risporic acids are volatile, and these appeared t o >e responsible for both zygophore initiation and {ygotropic responses in this fungus.' ' Similarly, cygospore production in young cultures of Rhizopus sexualis appeared to be influenced by volatiles. The inhibition of zygospore formation by low temperatures (10°C) could be relieved by passing air from a mature culture growing a t room temperature onto a young one, indicating that a volatile (possibly methylamine), whose synthesis was inhibited by low temperature, was an inducer of zygospore formation.' Voiatiles from various species o f Trichoderma stimulated oospore formation by selfing in single A2 isolates of various species of Phytophthora. Although this fungus is normally heterothallic, the volatiles from Trichoderma may be chemically related t o something produced by the A l mating type in A l X A2 pairings.' sf, This response also apparently occurs in soil,2 suggesting a method for both the survival and genetic variation of heterothallic Phytuphrhora species in V ~ V O O. 0~ Interestingly, the ability of various Trichoderma isolates to stimulate formation of sex organs was correlated with their ability to produce volatile "antibiotics" against Phytophfhora. ' sf, Although some microbially derived volatiles stimulate microbes of the same or other species, most of these organic volatiles appear to exert inhibitory effects, at least as indicated by the greater number of reports of the latter than of the former phenomena. The germination of spores of Cunninghamella elegans was inhibited by volatiles from Fusarium oxysporum. The compounds present in the volatiles were acetaldehyde, propionaldehyde, n-bu tyraldehyde, n-propanol, isobutanol, ethyl acetate, isobutyl acetate, and acetone, but acetaldehyde was considered to be the active inhibitor.I4 52 Some isolates of Trichoderma produced volatiles that inhibited the mycelial growth of various other fungi, with acetaldehyde again being considered the active c o m p o n e n t . ' 5 3 Studies with Trichoderma harzianum indicated that the volat iles responsible for the inhibition of growth and sporulation of Aspergillus niger and Pestalotia rhododendri were primarily C 0 2 and ethanol, with COz having the greatest effect.' s 4 Similar results were obtained when volatiles from various species of Trichuderma were evaluated against a spectrum of other fungi.

The differences in the amount of inhibition of the assay fungi were related to the amounts of COz produced by the Trichoderma sp., although acetaldehyde and ethanol, but not ethylene or acetone, were implicated in the inhibition of some species.' " A discussion of the inhibitory effects of C02,which have been relatively well defined (cf., References 3 and 7), is beyond the scope of this review. A variety of volatiles (e.g., alcohols, aldehydes, olefins, methyl chloride, and hydrogen cyanide) is produced by different species of Fornes, and these have variable effects on other fungi, ranging from 8 i ' 74 Among none to marked the components present in the volatiles, hexa1,3,5-triyne' a and hydrogen cyanide' 7 4 were considered to be the most active. An unidentified basidiomycete also produced hydrogen cyanide, which again had a vzriable effect on the growth and sporulation of a spectrum of other fungi. O Cultures of Saccharomyces cerevisiae evolved acetaldehyde and ethanol, which were inhibitory to the growth and sporulation of Aspergillus niger. 6 6 There are numerous other examples of the inhibition of one fungus by volatiles from another, (e.g., References 3 and 4), as well as numerous examples of volatiles from a specific fungus either stimulating, inhibiting, or having no effect on wrious other fungi (e.g., References 175) (Table 3). The apparent involvement of acetaldehyde and ethanol in the inhibition of some fungi by volatiles from the same or other species is interesting, inasmuch as the same compounds released by p l a n t residues92996 or by germinating seeds34*6478g appeared to stimulate fungal growth. Whether this apparent contradiction is the result of differences in concentration (e.g., more aldehydes and alcohols may accumulate in the head space of closed culture vessels than in soil or in continuously aerated systems) requires clarification, especially as higher concentrations of volatiles from alfalfa hay inhibited microbial 9'

'

'

3 ' '

Although most studies on volatiles from microbes have been concerned with interactions between fungi, volatiles from bacteria also affect fungal growth and development. Unidentified volatiles from Streptomyces griseus reduced sporulation in Cleoesporium aridurn and induced formation of sclerotia in Sclerutium cepivonm and May 1976

363

Rhizoctonia solatzj. I Other claims have also been made for the evolution of volatile antifungal antibiotics by various Srrepromyces s p . 4 9 1 9"2 " Species of Pseudomonas evolved unidentified volatiles that induced the formation of fruiting bodies in Agaricus bisporris. Volatiles from ten species of bacteria (both Gram-positive and -negative) inhibited mycelial growth and sporulation of Fusarium oxysporum f. conglutinans, Penicillium viridicarttm, Trichoderma viride, Zygorhy nchu s vu illeminii, and Gelasinospora cerealis The degree of inhibition varied among the different bacterium-fungus combinations, suggesting that several active volatiles were involved.' Fu rthermore, the degree of inhibition was concentration dependent, and both stimulatory and inhibitory volatiles were present. In general, heavy bacterial development always inhibited growth and/or sporulation of all test fungi, whereas lighter bacterial growth either inhibited, did not alter, or stimulated growth and/or sporulation, depending on the bacteriumfungus c ~ m b i n a t i o n . ' ~Differential sorption of the volatiles by KOH, KMn04, charcoal, or soil further indicated that both inhibitory and stimulatory volatiles were produced. Although the chemical composition o f these volatiles has not been determined, the inhibitory components appeared to be both oxidizable and nonoxidizable compounds, whereas the stimulatory components, which were apparently present in lower concentrations, appeared to be primarily nonoxidizable compounds. In contrast to the marked effects of these bacterial volatiles on fungi, there appeared to be no effect of the fungi on the bacteria.' A similar lack of effect on bacteria was observed with volatiles from Fomes sailellatus' 7 4 and Trichoderma harzianum,' 5 4 although volatiles from both these fungi had marked inhibitory effects on other fungi. However, because relatively few studies have apparently been conducted to test the effects of microbially derived volatiles on bacteria, it is not possible to conclude that, in bacterium-fungus combinations, it is always only the fungus that is affected. Volatiles from bacteria also induced morpho-


' ''

'

'

logical abnormalities in fungi, such as shortening of condiophores, thin-walled vesiclelike swellings, increased septation, hyphal branching and distortion, and altered colony pigmentation and shape. In general, the extent of morphological change was correlated with the amount of inhibition of growth and/or sporulation induced by the b a c t e r i a l ~ o l a t i l e s . ~ 7' i~1 9' '~ Rhizom orph production in Armillaria mellea was stimulated by low concentrations of ethan01,~O 1 9 3 either added exogenously or produced in dual cultures Other morphowith Aureobasidium pullulans. logical alterations in fungi, primarily in sex organs, caused by volatiles from the same or different species have already been discussed. The advantages or disadvantages of such morphological responses to volatiles in the survival of the responding fungus in natural habitats is not known. With bacteria, changes in cell morphology are often beneficial responses to unfavorable environmental conditions (cf. Reference 7). Although must studies on the effects of microbially derived volatiles on microbes have been conducted in vitro, a phenomenon much observed in soil, and which may involve volatiles, is fungistasis. Soil fungistasis, or mycostasis, is a condition found in natural soils containing living microorganisms, in which fungal propagules that would normally germinate and grow vegetatively are prevented from doing so unless sufficient nutrients are present or unless the soil microbiota is inhibited. Although it has been suggested, and partially demonstrated, that fungistasis is primarily the result of the depletion of nutrients from the surrounding soil and from the propagules them~ e l v e s8,9~ evidence is accumulating that indicates the existence of fungistatic factors in soil and that these factors may be volatiles produced by other microorganisms.' The germination of conidia of Aspergillus flavus and Fusarium solani f. citctlrbirae was retarded when suspended over soil,3o4 as was the germination of various spore types of other fungi,'93~305*306 The addition of chtin to the soil increased the amount of fungistasis, presumably by increasing microbial numbers and activity, which caused more volatiles to be evolved.*' 9 3 O 4 When activated charcoal was

'

J

*Volatiles causing fungistasis of sclerotia o f Macrophomina phoseo/ina were not detected in a variety o f soils, except in the Colorado soil used by Baker et al. and sent to Beltsville, Maryland for examination. This soil apparently contains unusually high concentrations of ammonia. Although the addition of chitin increased fungistasis attributable to volatiles, the effect lasted only a few weeks.' '' 364

CRC Critical R e v i e w in Microbiology


added t o soil, fungistasis was eliminated,” and autoclaving of inhibitory water extracts from soil also eliminated the inhibition.” N e i t h e r sterilized soils nor these soils recolonized by various bacteria and fungi (except by TricSzoderma sp.) exhibited fungistasis. However, sterile soils recolonized by actinomycetes, especially Srrepromyces sp., or Trichoderma sp. were fungistatic.‘ y 3 The atmospheres above cultures of Streptomyces sp. were also fungistatic, further indicating that actinomycetes were, in part, responsible for the volatile fungistatic factors in soil.’ ” The fungistatic effect was greatest in alkaline soils, which was originally thought t o be a result of the better growth of actinomycetes at the higher p H values.’ However, a volatile fungistatic substance extracted with water from alkaline soil volatilized at pH 7.6 but not at lower pH values. This substance has been identified as ammonium chloride. The biological activity of extracts from alkaline soils and of solutions of ammonium chloride was identi~al.~ Numerous other studies, however, indicate that various organic volatiles are involved in soil fungistasis. Many of these volatiles have been shown to be effective in vitro and were discussed previously. The addition to soil of solutions of silver nitrate or mercuric perchlorate, compounds which form c o m p l e x e s with unsaturated hydrocarbons, reduced fungistasis.’ I I These observations have been supported by studies which indicate that ethylene is the fungistatic factor in soil. Although ethylene in soil can result from various plant and microbial sources (as discussed previously), it has been suggested that the ethylene responsible for fungistasis is produced primarily by anaerobic bacteria metabolizing in anaerobic microhabitats in soil and that an oxygen ethylene cycle is operative in soil to control microbial development.*08 It is interesting, however, that ethylene is seldom detected in control soils during acetylene reduction assays for dinitrogen fixation. This either suggests the lack o f a role of ethylene in controlling microbial activity in soil or emphasizes the importance of localized effects within the microhabitats. The occurrence of sufficient quantities of ethylene to affect microbes in aerobic soils (i.e., soils which were grossly - as contrasted t o individual microhabitats - aerobic) has also been questioned, inasmuch as little ethylene (less than ‘I’

’

0.1 vpm) was detected until the oxygen concentration was reduced t o less than 0.1% of the soil a t m ~ s p h e r e . ~ It ” ~has also been suggested that anaerobiosis, rather than the ethylene produced, is the cause of fungistasis. Although ethylene produced in anaerobic microhabitats could “diffuse” to and inhibit microbes in adjacent aerobic sites, the concentration of ethylene may be reduced below effective levels by diffusion and metabolism,’ I I a which could explain the low or absent background levels of ethylene in control soils during acetylene reduction assays. Additional studies, especially in soil, are obviously needed to define the importance of ethylene in soil fungistasis, especially the concentrations required to inhibit different species, the rates of diffusion from sites of formation and the resultant concentration gradients, and the rates of metabolism by nonsensitive species before the ethylene reaches sensitive species. Although much progress has been made in identifying the factors responsible for fungistasis in soil, the mechanisms involved are still not resolved. Because so many diverse factors ranging from nutritional considerations to nonvolatile and volatile compounds, both organic and inorganic - have been implicated, it is probable that no single factor is responsible for this phenomenon in all soils. I t is apparent, however, that microbial activity is invoIved in most instances, although fungitoxins of nonmicrobial origin have been ~ u g g e s t e d . ~ ,3 I 2 Similarly, the causes for the apparent occurrence o f bacteriostasis in soil require further study. This phenomenon also appears t o be the result of microbial action, even though organic volatiles have not yet been i m p l i ~ a t e d ‘3-31 .~ Furthermore, in both types of stasis the role of organic volatiles in reversing the inhibition requires clarification, especially as nonvolatile carbonaceous nutrients appear t o release microbes from such inhibition .Z I9-2 9 1 There can be little doubt that organic volatiles produced by microbes affect other microbes. What are not always known are the mechanisms of action (e.g., nutrition, metabolic and gene regulation, and alterations in permeability) (cf. References 3 and 4), the substrate and concentration dependence, the species specificity, the reciprocity o f the effect, and, perhaps most important, the influence of the soil environment on both the production and action of the volatiles. Although May1976

365


there is considerable information on the effects of pH, temperature, aeration status, amount and types of substrates, mineralogical composition, and other soil characteristics on the general microbial activity in soil (e.g., Reference 7), little is known about the influence of these factors, singly and in combination, on the production and activity of organic volatiles in soil. Inasmuch as soil appears differentially to remove components of volatiles produced by bacteria that are either inhibitory or stimulatory to fungi87 and some volatile microbial metabolites apparently become more toxic in soil under conditions of poor the physicochemical properties of aeration: soil undoubtedly are important. However, until more information on the effects of these properties is available, care must be taken in extrapolating from observations derived in simple systems in vitro to the complex in vivo soil system.

'

Volatiles from Animals Many animals, both invertebrates and vertebrates, release various organic volatiles as either attractants, repellents, signals, products of normal food digestion, etc. ( e g , Reference 1). The effects of such volatiles, especially from soil-inhabiting animals, on soil microbes have not been extensively studied, even though some o f the volatiles produced by such animals, especially by insects, are similar to some plant- and microbederived volatiles known to affect microbes. Among these compounds are lower aliphatic acids, aldehydes, ketones, esters, lactones, phenols, quinones, and hydrogen cyanide.' J A commonly reported volatile from insects is 2-hexenal, which is also released by some plants and fungi and is inhibitory to various fungi. This aldehyde appears to be the active component of the volatiles released by Scaptocoris talpa, an insect associated with the roots of some bananas and which, in culture, inhibited Fusarium oxysporum f. cubense, a soil-borne root-infecting pathogen of banana, as well as a variety of other soil-inhabiting fungi. It was originally suspected that the presence of this insect and, therefore, of the volatile inhibitory to the pathogen in some soils but not in others was responsible for the differential spread of Fusarium wilt of banana in various soils.3' Further studies, however, did not sustain this early correlation, and the differential spread of the disease appeared to be correlated with the presence (slow spread) or absence (rapid spread) in soils of a specific clay mineral having

'

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CRC Critical Reviews in Microbiologv

X-ray diffraction and other characteristics similar to montmoril1onite.' The scent glands of various pentatomid insects also contain 2-hexenal. When the storage sacs of these scent glands were explanted to agar plates seeded with species of Fusarium, Pythium, Phytophthora, and Rhizoctonia, growth of these fungi was r e d ~ c e d . ~ ' Trap formation by certain species of nematode-trapping fungi can be induced by soluble substances derived from these worms and from some other sources. These substances have been called, collectively, n e ~ n i n . ~A ~recent study to test the possibility that trap formation could be induced by volatiles released by the nematodes indicated that no such volatiles were produced in the system used (Monacrosporiumrutgeriensis and Panagrellus redi~ivus).~'a However, as another nematode (Ascaris lumbricoides) has been shown to release volatile fatty acids in axenic culture?22 more studies are needed before volatiles derived from nematodes can be excluded as inducers of morphogenetic changes in these fungi. Because microorganisms are in intimate contact with animals (especially arthropods) in soil, the potential effects of organic volatiles released by the soil microfauna on the activity and ecology of microbes in soil require further study. T~

'

'

'

Volatiles from Abiotic Sources (Table 6 ) Some organic volatiles from abiotic sources are similar to some from biological sources which affect microbes. Consequently, an increased concentration of such compounds in the biosphere could alter normal microbiological responses to these volatiles. Some relatively recent anthropogenic organic volatiles have no natural counterparts. Therefore, as microbes have not had much opportunity to evolve or adapt in response to these, their effects should be known, especially as some of these volatiles are present chronically and in relatively high concentrations in the atmosphere (e.g., References 5 and 6 ) . The effects of various inorganic air pollutants on microbes have been discussed elsewhere,' and only a brief discussion of the influence of some organic volatiles from abiotic sources will be presented here. A major abiotic source of organic volatiles is the incomplete combustion of fossil fuels, which releases various hydrocarbons into the atmosphere. Some of these hydrocarbons are further altered in the atmosphere by photochemical reactions with inorganic air pollutants to yield other compounds. p6

m

\o -4

c

$!.

Oxidants

Peroxyacetyl nitrate

Photooxidation of incompletely combusted fuels

Olefin-ozone mixtures

Ambient atmospheres

Irradiated auto exhaust plus ozone Laboratory mixtures

Dilute auto exhaust

NG

Photooxidation products of cis-2-butene and NO, Irradiated mixtures of 1-hexene and NO, Irradiated mixtures of various hydrocarbons and NO, Irradiated fuel exhausts, containing 30-40% olefins NGa

Laboratory mixtures and ambient polluted air Laboratory mixtures

Auto exhausts

Volat ile

Source

Serratia marinorubro

Mutagenic

Inhibited autotrophic and heterotrophic growth

N o effect Toxic

Toxic

Eschericliia coli, Micrococcus albus

Toxic

No effect

Inhibited

Toxic

Escliericlria coli Escherichia coli, Serratia marcescetis. Pasteurella tularetisis, Bnrcella suis, Staphylococcus epidermidis. Envinia amylovora, Streptococcus sp., Coliphage T7, Semliki Forest virus, Vaccinia virus Chlamydomotias reitihardtii

Effect Reduction in luminescence and viability Inhibited; increase in pigmentation Inhibited

Esclierichia coli

Escherichia colr

Esctierictria coli

Esctrericlria coli

Esclierichia coli

Serraria marcescetis

Plrotobacteriutn ptiosptiorrurn


Probably due to HNO,

Photosynthesis more sensitive than respiration

Exposed o n microthreads (spider webs) Active compound probably an olefin-ozone complex Mixtures of ozone and 2-pentene, 2hexene, 2-heptene, or 2-octene Ring olefins (cyclopentene, cyclohexene, cycloheptene) more toxic than open chain olefins Low oxidant concentration Active compound is probably a zwitterion resulting from reaction of 0, with olefins (337)

Active product may be peroxyacetyl nitrate; mixtures of 1butene and NO, most toxic Removal of particulates eliminated inhibition

Active product may be peroxyacetyl nitrate or ozone -

Comment

Some Examples of Organic Volatiles from Abiotic Sources and Their Effects o n Microorganisms

TABLE 6


342

34 1

332 335.337-340

337

3 36

335

335

330,331

327-239

326

323-325

References

Various Va r iou s

Various Various

Various

Escherichia coli

Various

Escherichia coli. Bacillus rnegaterium, Bacillus licheniform is, Staphylococcus aureus Rhizobium rnelilori Euglena gracilis

Formaldehyde

Formaldehyde, propionaldehyde Paraffinic, olefinic, and fluorinated hydrocarbons (C, to C, ) Various (e.g., vinyl chloride) Hydrocarbons

Bacillus rnegaterium

Bacteriophage S5

DNA from Serraria rnarcescens, Bacillus subriiis


Carcinogenic arene-type hydrocarbons

Volatile

aNG = effect on microbes not given by authors.

Oil spills, leaks, sewers, etc. Burning Pesticldes

Industrial processes and fossil fuel combustion Industrial processes

Source Comment

Not defined

Not defined

N o t defined

Inhibited Reduced photosynthesis and respiration Essentially only fluorinated compounds reduced viability and induced some mutation Not defined

-

Some microbes inhibited; some can decompose Some microbes inhibited; some can decompose

Some microbes can decompose

Requires investigation

Vinyl fluoride and carbon tetrafluoride most active

Aerosolized cells

Reduced transforming activity, Probably d u e t o HNO, produced melting temperature, and viscosity; during breakdown of PAN modified bases Reduced infectivity for Serratia rnarcescens Growth suppression; Fiant Noncarcinogenic arenes have little cell formation; increased effect; active both aerobically and metabolic activity anaerobically Inhibited; extended lag Effect was function of concentraphase of growth tion and exposure time

Effect

Some Examples of Organic Volatiles from Abiotic Sources and Their Effects on Microorganisms

TABLE 6 (continued)


8, 1 5 , 2 6 , 2 3 5 cf., 5,6,26

8,236-238

341,348

345 346

330,331,333

343,344

342

342

References


Despite the world-wide occurrence of these materials, with higher concentrations being present in areas of intense urbanization and industrialization, there have been relatively few studies on the effects of these volatiles on microbes. Part of this paucity may reflect the technical difficulties involved in studying the effects of air pollutants on microbes (cf. Reference 6). Although some studies have been conducted with artificial smogs (e.g., mixtures o f gasoline vapors and ozone, ozonated olefins, and mixtures of olefins and nitrogen oxides), few have been conducted with natural ambient atmospheres. When the luminescent bacterium, Photobacterium phosphoreum, was exposed to the products of the photochemical oxidation of cis-2-butene and either nitric oxide or nitrogen dioxide, luminescence and viability decreased. The decreases were dependent on the length of exposure and on the ratio o f the initial concentrations of the reactants. When the identifiable oxidation products (e.g., ozone, nitrogen dioxide, formaldehyde, acetaldehyde, and peroxyacetyl nitrate) were evaluated individually, the effects were most pronounced with peroxyacetyl nitrate and ozone, with the aldehydes having no effect on viability. Similar reductions were also observed with ambient pollut2 3-3 2 5 Exposure of Serrafia marcescens to ed irradiated mixtures of nitrogen dioxide and 1-hexene resulted in some inhibition and in an increase in p i g m e n t a t i ~ n . ’ ~ ~ The growth o f C‘scherichia coli exposed to artificial smogs, produced by the irradiation of gaseous mixtures of nitrogen oxides and hydrocarbons with fluorescent lights, was reduced. The inhibitory product may have been peroxyacetyl nitrate, although methanol, acetaldehyde, acetone, and nitromethane were also identified in the photochemical reaction mixture^.^' 7 - 3 2 9 Irradiated fuel exhausts, containing olefin concentrations between 30 and 40%, were also inhibitory to E. coli. However, if the particulates were removed from the exhaust, no inhibition occurred.’ 3 0 Exposure of E. coli to ambient Los Angeles atmospheres during periods of low oxidant concentrations had no effect on the bacterium,’ ” whereas exposure to synthetic pollutant mixtures containing oxidants was inhibitory.’ 3 3 When E. coli was suspended on microthreads (i.e., the microbes were sprayed on ultrafine, inert, silk spider thread^)"^ and exposed t o dilute automobile exhaust, no deleterious effects were 9’

’

noted. However, when ozone was added and the nitric oxide content of the exhaust was oxidized by irradiation, the resultant product, probably an ozone-ole fin complex, was highly toxic.’ Similar toxicities were observed when E. coli, suspended on microthreads, was exposed to mixtures o f ozone and olefins (2-pentene, 2-hexene, 2-heptene, and 2-octene).’ An ozone-olefin complex, probably a zwitterion,’ has also been postulated as being responsible for the toxic effects of ambient night air on E. coli, Serratia marcescens,Pasteurella tularensis, Brucella suis, Staphylococcus epidermidis, Streptococcus sp. group C , Erwinia amylovora, coliphage T7, Semliki Forest virus, and vaccinia virus.’ 8-340 Olefin-ozone mixtures containing ring olefins (e.g., cyclopentene, cyclohexene, and cycloheptene) were more bactericidal (e.g., to E. coli and Micrococcus albtis) than mixtures containing open chain olefins (e.g., propylene and cis-2-butene).’ Another family of atmospheric photochemical products that have been shown to affect microbes are the peroxyacyl nitrates, especially peroxyacetyl nitrate (PAN). Ehe exposure of the green alga (Chlamydomonas reinizardtii.) to PAN inhibited both autotrophic and heterotrophic growth. Photosynthesis was inhibited more than respiration, and chlorophyll “b” was more stable t o P A N than chlorophyll “a” and the c a r ~ t e n o i d s . ’ ~The infectivity of bacteriophage SS for its host, Serratia marcescens, was reduced by exposure to P A N , w h i c h was also mutagenic for S. marinorubra.34’ The exposure of DNA from S. marcescens and Bacillus subtilis to PAN reduced the transforming activity, melting temperature, and viscosity of the DNA, as well as modified nucleic acid bases. The susceptibility of the bases to attack by PAN was thymine >guanine > uracil > cytosine > adenine. These genetic effects were probably the result of the nitrous acid produced during decomposition of the PAN.’ Arene-type hydrocarbons, some of which have been shown t o be carcinogenic to humans, are also released into the atmosphere by industrial processes and affect microorganisms. The exposure of Bacillus megaterium, both aerobically and anaerobically, to benz(a)pyrene, 20-methylcholanthrene, or 7,12-dimethylbenzanthraceneresulted in growth suppression and in the formation of giant cells containing vacuoles and granulati on^.^^' J~~ Wh en exposed aerobically, the cells

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also exhibited enhanced lipogenesis, glycolysis, and methylene blue reductase activity.34 Under anaerobic conditions, these hydrocarbons caused accelerated oxidase activities, especially of reduced d i p h o s p h o p y ri d i n e n u c l eotide ~ x i d a s e . ’ ~ ~ Noncarcinogenic, polycyclic aromatic hydrocarbons did not elicit these responses, which, even with the carcinogenic hydrocarbons, were temporary and disappeared within 24 hr after exposure.343 Some microbes in soil are capable of degrading benz(a)pyrene, although neither the organisms involved have been identified nor have the potential effects of the volatile on them been studied.’ 7’ A group of volatile compounds in the atmosphere that are produced both biotically and abiotically are aldehydes. The exposure to volatile formaldehyde, but not to aerosols of potassium chloride, was inhibitory t o E. coli, and exposure to combinations of formaldehyde and potassium chloride was even more inhibitory, suggesting a synergistic e f f e ~ t’O. ~> 3 Exposure t o formaldehyde also extended the lag phase of growth of E. coli, Bacillus megaterium, B. lichenifom is, and Staphylococcus aurcus. The length of the lag phase was a function of the concentration of the volatile and the exposure time.333 The viability of aerosolized Rhizobium meliloti was also reduced and the exposure of Euglena by gracilis to formaldehyde or propionaldehyde reduced the rates of both respiration and photo~ y n t h e s i s4. 6~ Various bacteria in aqueous suspension were exposed to homologous paraffinic (alkanes) and olefinic (alkenes) hydrocarbons, ranging from one to four carbons, as well as t o their partially fluorinated (genetrons) and perfluorinated (freons) analogues. Although most of these gases somewhat reduced the survival of various strains of E. coli, only vinyl fluoride had a significant effect (e.g., reduced viability by 94%). Exposure t o freons, especially carbon tetrafluoride (tetrafluoromethane), increased the number of penicillin-resistant mutants capable of fermenting various carbohydrates. Although the data from these studies347 9 3 4 8 are difficult to interpret and are inconclusive, they d o indicate that supposedly inert gases (such as fluorinated hydrocarbons), which are increasingly emitted into the atmosphere, affect microorganisms. Unfortunately, there appear t o be n o data on the effects of other volatiles derived from industrial processes, such as vinyl chloride, on micro-

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organisms, even though some of these volatiles appear t o be harmful to plants and animals. Among other organic volatiles of abiotic origin present in the atmosphere and which eventually are introduced into soil, where they may influence microbes, are pesticides. As mentioned earlier, however, a discussion of the microbiological effects of pesticides, as well as of hydrocarbons derived from burning, oil spills, pipeline leaks, sewers, and other anthropogenic sources, is beyond the scope of this review. (cf. References 5 and 6). Nevertheless, volatile organic compounds from these sources, as well as from the combustion of fossil fuels, are increasing in our atmosphere. Inasmuch as these volatiles appear to affect the growth, reproduction, morphology, and other characteristics of microbes, their potential influence on the activity, ecology, and population dynamics of microbes in soil and other natural habitats should be determined and then continually monitored. As all life in our biosphLre is ultimately dependent on the activities of microorganisms, it is imperative that the potential effects of organic volatiles from abiotic, especially anthropogenic, sources be clarified and, if found to be detrimental, controlled.

CONCLUSIONS A major purpose of this review is to focus attention on the potential effects of organic volatiles on microorganisms, especially in soil. However, volatiles from microbes also affect plants (e.g., References 2 t o 4 , 15, 104, 154, 166, 171 t o 174, 202, 204, 306, 308, and 349 to 351) and animals (e.g., References 3 , 4, 15, and 158); volatiles from plants affect other plants (e.g., References I 1 t o 15, 19, 20, 2 2 , 3 6 , 6 5 , 104, 106 t o 108, and 352) as well as animals (e.g., References 15, 158, and 353 to 356) even when the effect is indirect, e.g., on the rumen microbiota (e.g., References 357 and 358); and volatiles from animals affect other animals (cf. References 1 and 317) and probably plants (cf. Reference 1). Although these interactions are interesting, they are beyond the scope of this review. Nevertheless, as some o f these interactions have been more extensively studied than those involving microorganisms and as some of the volatiles involved also affect microbes (e.g., terpenes, short-chain


aldehydes and alcohols, ethylene, and hydrogen cyanide), they should be noted. There is little doubt that a vast variety of organic volatiles, whether derived from living or dead biotic sources or from abiotic sources, is present in our biosphere. There is also little doubt that most, if not all, of these volatiles are eventually introduced into soil. Components of the soil microbiota are probably capable of transforming most o f these volatiles t o their ultimate end-products (e.g., carbon dioxide, water, and inorganic salts) or to some intermediate products. The latter may be decomposed further by other microbes, leached from the soil, or accumulated. Based on the results of numerous studies, there can also be little doubt that many of these organic volatiles differentially affect a variety of microorganisms. Unfortunately, however, most of these microbiological responses have been determined in simplified in vitro experiments, and relatively little is known about the effects of these volatiles on microbes in soil in sini. The effects of organic volatiles on microorganisms appear to be dependent, in part, on their concentration, as some are stimulatory at one concent ration but inhibitory a t higher concentrations. This concentration dependence may also explain why the same volatiles from different sources have been reported to have either inhibitory, stimulatory, or no effects on the same or similar microorganisms. Although i t is difficult to measure accurately the vapor-phase concentration of an organic volatile in a simple laboratory experiment, especially if the compound is applied as a liquid, it is immensely more difficult to measure its concentration in soil, especially in the microhabitat wherein the volatile may be exerting its effect. Until such measurements are made, however, progress in elucidating the effects of organic volatiles on microbes in soil will be slow. Such measurements will also provide information on the rates of movement of a volatile from its source and on any differential microbiological effects that may occur along its concentration gradient.

The physicochemical characteristics of the soil probably influence not only the production or introduction of a particular volatile, but also its movement, retention time, and accumulation in microbial habitats. Soil factors may also potentiate, amplify, or decrease the activity of a volatile in soil as compared t o its activity in experiments conducted without soil. The microbiological composition of the soil, especially in individual microhabitats, will ultimately determine both the effects and fate of a volatile. Because of the enzymic complexity of the soil microbiota, a volatile that has a marked effect on an organism in simple dual-culture experiments may never reach that “target” organism in soil, as it may be intercepted and degraded by nonsensitive microbes. Similarly, a volatile that inhibits an assay organism in vitro may stimulate or inhibit various other microorganisms in vivo, possibly resulting in completely unexpected ecological. perturbations. Consequently, the influence of the physicochemical and biological characteristics of soils must be evaluated before results from simple experiments on the apparent effects of organic volatiles on microbes are extrapolated t o the soil environment. Inasmuch as organic volatiles, especially from natural sources, may provide an ecologically sound alternative to other methods of manipulating microbes in soil, especially t o the use of nonbiodegradable chemicals, such data should be obtained, certainly before hopes are raised too high for the use of such volatiles for biological control.

ACKNOWLEDGMENTS The authors are grateful t o Harvey J. Babich for providing references and concepts and for critically proofreading this manuscript. Some of the studies reported herein were supported, in part, by Grant R-800671 from the U.S. Environmental Protection Anencv.

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High-resolution gas chromatographic profiles of volatile organic compounds produced by microorganisms at refrigerated temperatures.

Identification of microorganisms based on headspace analysis of volatile organic compounds by gas chromatography-mass spectrometry.

Metal organic frameworks as sorption media for volatile and semi-volatile organic compounds at ambient conditions.

The network of plants volatile organic compounds.

Effect on microorganisms of volatile compounds released from germinating seeds.

Fungal volatile organic compounds and their role in ecosystems.

Indoor volatile organic compounds and chemical sensitivity reactions.

Methods in plant foliar volatile organic compounds research.

Removal of volatile organic compounds using amphiphilic cyclodextrin-coated polypropylene.

Volatile and semi-volatile organic compounds of respiratory health relevance in French dwellings.

First Characterisation of Volatile Organic Compounds Emitted by Banana Plants.

New graphene fiber coating for volatile organic compounds analysis.

Exposure to volatile organic compounds in healthcare settings.

Volatile organic compounds as non-invasive markers for plant phenotyping.

Volatile organic compounds emitted by Trichoderma species mediate plant growth.

Are Some Fungal Volatile Organic Compounds (VOCs) Mycotoxins?

Diagnosing gastrointestinal illnesses using fecal headspace volatile organic compounds.

Urinary Volatile Organic Compounds for the Detection of Prostate Cancer.

Geographical variation in the exhaled volatile organic compounds.

Factors controlling volatile organic compounds in dwellings in Melbourne, Australia.

Can volatile organic compounds be markers of sea salt?

Enhanced Volatile Organic Compounds emissions and organic aerosol mass increase the oligomer content of atmospheric aerosols.

Detection of volatile organic compounds by weight-detectable sensors coated with metal-organic frameworks.

Succession of microorganisms in a plate-type air treatment biofilter during filtration of various volatile compounds.