APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1989, p. 100-105 Vol. 55, No. 10099-2240/89/010100-06$02.00/0Copyright © 1989, American Society for Microbiology

Microbial Colonization of Injured Cactus Tissue (Stenocereusgummosus) and Its Relationship to the Ecology of Cactophilic

Drosophila mojavensisJAMES C. FOGLEMAN* AND JOAN L. M. FOSTER

Department of Biological Sciences, University of Denver, Denver, Colorado 80208

Received 28 July 1988/Accepted 15 October 1988

Necrotic tissue of agria cactus (Stenocereus gummosus) serves as a feeding and breeding substrate forDrosophila mojavensis. This fly species is one of the four endemic Drosophila species in the Sonoran Desert.Freeze injuries were created in arms of agria cactus in Mexico to study the events of microbial colonization.Facultative anaerobic bacteria were the first microbes to be detected, and the exclusion of large arthropods bycovering the injuries with netting did not affect bacterial colonization. Yeast growth lagged behind bacterialgrowth by 2 days, and excluding arthropods delayed the detection of yeasts by an additional 2 days. Thus,insects (such as Drosophila species) and other arthropods do play a role in the colonization of agria rots byyeasts. All injuries were attractive to D. mojavensis within 5 days, and these flies were shown to be carryingsignificant densities of both bacteria and yeasts. Analysis of the volatile compounds present in the developingrots over time indicated that the volatile pattern is dynamic. Ethanol and acetic acid were the two volatilesubstances most likely responsible for the initial attraction of the injuries for Drosophila species.

Few ecological systems are as amenable as the cactus-microorganism-Drosophila model system of the SonoranDesert for multidisciplinary approaches to the study ofmicrobial ecology, population biology, ecological genetics,and evolution of groups of organisms. The desert-adaptedDrosophila species endemic to the Sonoran Desert aresaprophytes which breed in necrotic stems of columnarcacti. The resident microorganisms of cactus rots (primarilybacteria and yeasts) serve several functions in the modelsystem. First, their growth and metabolism modify thecactus tissue both physically and chemically, producing ahospitable substrate for a variety of saprophagous insects(9). Second, they serve as a food source for these sa-prophytes. For a Drosophila species to successfully feed andbreed in a cactus rot pocket, it must first be able to detect asuitable host cactus. The flies must also be able to assimilatethe necrotic host tissue and tolerate or otherwise detoxifyantiherbivore compounds present in the plant. Microorgan-isms play a role in both phenomena. Volatile metabolicproducts resulting from the microorganism-cactus interac-tion serve as olfactory cues and may serve to stimulatemating and oviposition (J. C. Fogleman and W. B. Heed, inJ. 0. Schmidt, ed., Special Biotic Relationships in the AridSouthwest, in press). Microorganisms, through their degra-dative action, release the nutrients tied up in complexorganic molecules of the cactus and may break down com-pounds which have antiherbivore function. They may alsoproduce metabolites which are toxic to insects. The rotpockets, a relatively wet microhabitat, are superimposed onthe cacti, which occupy a xeric habitat. Together, thedistribution of the cacti and rot pockets with inherentmicroflora determine the population dynamics of these cac-tophilic Drosophila species.The yeasts which reside in necrotic cactus tissue have

been extensively studied for the past 15 years. This researchhas led to numerous reports on their roles in the modelsystem and specific aspects of their ecology and evolution

Corresponding author.

including their distribution relative to the cactus species,physiological characteristics, community structure, and nu-tritional value to drosophilids (5, 15, 16, 18, 20-22). Thepaucity of knowledge pertaining to the bacterial residents ofnecrotic cactus tissue is conspicuous, and yet much of theinformation which has been gathered about the model sys-tem points to the bacterial component as having a primaryrole. Since the bacteria interact at all levels of organizationin this model system, they are, in many respects, the mostimportant component. For example, they are thought to bethe first microorganisms in a necrosis (15), and their degra-dative action makes yeast and Drosophila growth possible.The volatile metabolic products produced by bacteria (low-molecular-weight alcohols, esters, and volatile fatty acids)are, most likely, the chemical basis of host plant selection bythe Drosophila species (Fogleman and Heed, in press).These same fermentation products may represent an energysource for the yeasts and Drosophila species since allcactophilic yeasts can use ethanol as a carbon source andadult D. mojavensis (one of the four endemic Drosophilaspecies) experience increased longevity (ca. twofold) undercertain concentrations of atmospheric ethanol (19). Differ-ences between cacti, either in bacterial species or in thespectrum of their fermentation products, may be involved inthe maintenance of certain genetic polymorphisms, e.g., thealcohol dehydrogenase polymorphism in D. mojavensis (2).Bacteria are ingested in large numbers by both adults andlarvae of Drosophila flies and may, therefore, affect thefitness of these insects through their nutritional quality.Finally, bacteria-Drosophila coevolution is possible in thissystem because flies vector bacteria (11, 13) which, in turn,makes the cacti usable and attractive to the Drosophilaspecies.Knowledge of the interactions of bacteria with the other

components is essential to a thorough understanding of thismodel system as a whole as well as contributing to microbialecology as a discipline. In this communication, we report theresults of experiments which were designed to elucidate the

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MICROBIAL COLONIZATION OF CACTUS TISSUE 101

events of microbial colonization of human-induced injuriesin agria cactus (Stenocereus gummosus).The specific questions that are being addressed in this

report are as follows. Which microorganismic component,bacteria or yeasts, is the first to colonize injured agria tissue?With respect to temporal succession, what are the charac-teristics of the growth curves for bacteria and yeasts duringthe initial stages of colonization in a natural environment?What effect do large insects (Drosophila species) have on thepattern of microbial colonization? How quickly are Droso-phila species attracted to a developing agria necrosis giventhat flies are present in the area? Which volatile compounds(fermentation products) are present in the rot when it be-comes attractive to Drosophila species and what are theirconcentrations? What is the pattern of volatile productionduring microbial colonization? Aspects of microbial coloni-zation and succession that relate to the ecology of D.mojavensis, the resident drosophilid for this cactus species(4), were also examined.

MATERIALS AND METHODS

Sampling procedures. Field experiments were conductedduring December 1986 in the state of Sonora, Mexico, at asite approximately 30 km north of Kino Bay (Bahia de Kino)on the west coast of mainland Mexico. Four agria plantswere chosen, with one arm of each plant being selected onthe basis of similarity of age, size, general condition, andorientation. All four arms were situated within 3 m ofexisting rots being utilized by Drosophila species. Spineswere removed from a 30- by 10-cm area of each arm, and thearea was surface sterilized with ethanol. A pair of holes (2.5cm in diameter, 5.0 cm deep, and 20 cm apart) wereasepticallay drilled in each arm. A tissue sample was takenfrom each hole prior to the induction of freeze injury byliquid nitrogen. One of each pair of holes was covered withsterile nylon netting to exclude Drosophila species and otherlarge arthropods. The netting was secured to the arm withtacks. Samples were taken for analysis of microbial densityand volatile concentration on days 2, 4, 6, 8, and 11postinjury. The arms were then severed from the plant,brought back to the laboratory, and placed in an environ-mental chamber which had been set to mimic field condi-tions. Two additional samples (days 16 and 27) were takenbefore the tissue became too dry to sample.

Density of microorganisms. The densities of bacteria andyeasts per gram of tissue were determined by viable cellcounts performed at the field site in a mobile laboratory.Serial dilutions were generated, with two dilutions of eachsample being plated on separate media for bacteria andyeasts. The lowest dilution that could be plated represented100 CFU/g of tissue. Bacterial densities were determinedfrom counts on plates of Trypticase soy agar (BBL Micro-biology Systems, Cockeysville, Md.) supplemented with 100mg of cycloheximide (Sigma Chemical Co., St. Louis, Mo.)and 10 mg of amphotericin B (Sigma) per liter of medium toinhibit the growth of fungi. Yeast densities were determinedfrom counts on plates of yeast extract-malt extract (DifcoLaboratories, Detroit, Mich.) agar acidified to pH 3.7 with 1N phosphoric acid. This pH inhibits the growth of mostbacteria. All plates were incubated at ambient temperatureand under aerobic conditions since preliminary experimentsfailed to demonstrate significant densities of obligate anae-robes (J. L. M. Foster, unpublished data).The densities of bacteria and yeasts being carried by D.

mojavensis which were attracted to the developing necroses

were also estimated by viable cell counts. Five days postin-jury, flies were collected in or around the developing necro-ses. Ten flies per plant were collected and homogenized, anda dilution series of the homogenate was plated on both thebacteria and yeast media. The lowest dilution used yieldedan estimate of the number of CFU per fly.

Analysis of dissolved volatile compounds. The concentra-tions of volatile substances in tissue samples from thedeveloping necroses were determined by quantitative gaschromatography with a Varian 3700 gas chromatograph(with dual flame ionization detectors) linked to an Apple IIecomputer through an analog/digital interface (AI13; Interac-tive Structures). Duplicate glass columns (2 m by 2-mm innerdiameter by 6.35-mm outer diameter) packed with 6.6%Carbowax 20M on 80/120 Carbopack B AW (catalog no.

2-3845; Supelco, Inc., Bellefonte, Pa.) were used. Nitrogendelivered at a flow rate of 20 ml/min was used as the carriergas. Individual runs were temperature programmed frorm 70to 140°C at 3°C/min and then held at 140°C until termination(30 min). Injector ports and detectors were set at 210°C.Samples for volatile analysis were collected as part of the

samples for microbial density determinations (see above).Approximately 1 g of each sample was placed in a Nunccryotube (catalog no. 5151-m39; Thomas Scientific, Swedes-boro, N.J.), frozen in liquid nitrogen, and returned to thelaboratory for analysis. Gas chromatography involved thaw-ing the samples, separating the tissue and rot liquid bycentrifugation in a tabletop clinical centrifuge, and injecting3 to 5 pl of the supernatant directly into the gas chromato-graph column. Peak identification was based on retentiontime as compared with retention times of known com-

pounds, with the R, of acetic acid serving to standardizedifferent runs. Quantification was based on empirically de-rived regression equations relating peak area and injection ofstandards of specific concentration.

Statistical analyses. Data on both microbial densities andvolatile concentrations were analyzed primarily by two-wayanalysis of variance tests (14) in which covered versus

uncovered injuries represented one major factor and time (indays postinjury) was the other major factor. For microbialdensities, the data were log transformed prior to statisticalanalysis.

RESULTS

The log1o of the densities of bacteria and yeasts (per gramof sample) in each injury (covered and uncovered) over timeare presented in Table 1. Bacteria were not detected in thecovered injuries until day 2 postinjury. The same is true ofthe uncovered injuries except for Agria-2. Given that the day0 sample of Agria-2 (uncovered) was taken prior to liquidnitrogen treatment and given the closeness of the densitymeasurement to the limit of detection, it is likely that thepresence of bacteria on this plate was the result of airbornecontamination. Data on bacterial density in two samples(Agria-3, uncovered, day 4, and Agria-3, covered, day 6) are

missing owing to a dilution problem which resulted inuncountable plates. Agria-3 (uncovered) was not sampled on

day 27 because of substrate desiccation. Two-way analysisof variance on bacterial colonization of injured agria tissuedid not indicate any significant difference between coveredand uncovered injuries (Fs = 1.859; df = 1, 39; P > 0.1). Thisanalysis included data from days 2 to 27.

Yeasts were not detected in appreciable numbers until day4 and then only in the uncovered injuries (Table 1). In thecovered injuries, yeasts were not detected in all four plants

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APPL. ENVIRON. MICROBIOL.102 FOGLEMAN AND FOSTER

TABLE 1. Loglo of the CFU per gram of sample in both coveredand uncovered injuries in the four agria plants sampled over time

Log1o CFU/gDays postinjury Covered injuries Uncovered injuries

and sample __________

Bacteria Yeasts Bacteria Yeasts

0NDaNDNDND

4.354.605.294.60

6.424.956.074.48

9.377.62

8.53

10.379.549.4611.02

ND NDND 2.90ND NDND ND

ND 5.16ND 2.48ND 3.79ND 6.21

ND4.68NDND

4.306.314.825.26

6.228.144.307.15

4.708.56

b

7.74

7.229.203.007.62

9.369.266.798.47

log1o 7 CFU/g. No significant changes were noted in celldensity for any treatment between day 11 and the last twosample periods (days 16 and 27). For this reason, the datafrom the last two samples were omitted from the figure. Thisfigure clearly demonstrates that there is a 2-day lag betweenbacterial colonization and the earliest yeast colonization(uncovered injuries), at least during the initial stages of rotdevelopment. Within the yeast data, there is also a 2-day lag,with the uncovered injuries being colonized sooner thanthose which excluded large insects.The first flies to visit an injury were observed in the

uncovered hole of Agria-2 on day 1. By day 5, D. mojavensisflies were observed in or around the uncovered injuries of allfour plants. Table 2 contains the densities of both bacteriaand yeasts that were being carried by D. mojavensis flieswhich visited the developing rots on day 5. The averagedensity of bacteria (per fly) exceeded that of yeasts in threeof the four data sets. It is evident from Table 2 that bothbacteria and yeasts are being vectored by the flies.The average concentrations of the volatile compounds (±

standard deviations) that were detected in the samples arepresented in Table 3. Twelve volatile compounds weredetected at an average concentration of 0.1 mM or greater onat least one day during the experiment. Two-way analysis ofvariance was performed on all but three volatile compounds:2-propyl acetate, 1-propyl acetate, and n-butyric acid. Thesecompounds were excluded because they were not present ina sufficient number of the samples to warrant statisticalanalysis. Analysis of variance for the remaining nine com-pounds did not indicate significant differences between cov-ered and uncovered injuries. Four of the nine compounds(methanol, ethanol, acetic acid, and propionic acid) showedsignificant differences in concentration over time (P < 0.05).

11Agria-1Agria-2Agria-3Agria-4

16Agria-1Agria-2Agria-3Agria-4

27Agria-1Agria-2Agria-3Agria-4

a ND, None detected.b -, Missing data.

10.039.54

10.309.48

9.799.939.489.09

10.149.469.368.99

6.918.065.787.75

8.117.965.947.33

8.247.757.766.57

9.369.438.48

10.38

10.109.389.369.62

10.598.54

7.53

6.958.117.728.90

until day 6. Statistical analysis of the data from days 4 to 27demonstrated that there is a significant difference betweencovered and uncovered injuries with respect to yeast density(Fs = 5.681; df = 1, 35; P < 0.05). Yeasts were slower tocolonize damaged tissue when large insects (e.g., Drosophilaspecies) were excluded.The growth curves of both bacteria and yeasts in the

covered and uncovered injuries are shown in Fig. 1. Thecurves for bacteria are very similar, and carrying capacityfor both curves was essentially reached on day 8 at aboutlog1o 9 CFU/g of sample. The growth curves for yeasts showthat carrying capacity was reached for both treatments on

day 11 and that the density at carrying capacity was around

DISCUSSION

The stems of columnar cacti may become necrotic for a

variety of reasons. Although not particularly common,freeze injury is a natural phenomenon in the northern part ofthe Sonoran Desert. Other major causes of necrosis includelightning, wind (mechanical damage), herbivores, and senes-

cence.Prior to this study, the only published evidence pertaining

to the question of whether bacteria or yeasts were the firstmicrobes to colonize injured cactus tissue was that ofStarmer (15). In his studies on yeast community structure,he reported that although most rots had both bacteria andyeasts, some (young) agria rots had bacteria but no yeasts.Rots with yeasts and no bacteria were never observed. Ourresults confirm, in a much more direct way, that bacteria are

the initial colonizing microbes. The fact that the growthcurves for bacteria and yeasts in the developing necroses

(Fig. 1) are essentially parallel argues against the possibilitythat the observed results were due to simultaneous inocula-tion of the injuries with bacteria and yeasts coupled with aslower growth rate for the yeasts.Although it was not the intent of this study to determine

the species composition of the colonizing bacterial and yeastcommunities, some general statements can be made. Thetypical yeast species of necrotic agria tissue are well known(15, 21). These include Pichia cactophila, P. amethioninavar. amethionina, P. mexicana, Candida sonorensis, C.eremophila, C. mucilagina, C. deserticola, C. ingens, andCryptococcus cereanus. Seven of the nine species listedwere detected among the yeasts isolated from our injuries bya standard selective plating technique (15). The other two

2

4

6

8

Agria-1Agria-2Agria-3Agria-4

Agria-1Agria-2Agria-3Agria-4

Agria-1Agria-2Agria-3Agria-4

Agria-1Agria-2Agria-3Agria-4

Agria-1Agria-2Agria-3Agria-4

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MICROBIAL COLONIZATION OF CACTUS TISSUE 103

12

10

01i

Li.C)

0

C,0-J

8

6

4

21-

0 2 4 6 8 10 12

TIME (daysiFIG. 1. Growth curves for both bacteria and yeasts in injured agria tissue. Each line represents the average of four replicate experiments.

Symbols: 0, bacteria in covered injuries; 0, bacteria in uncovered injuries; *, yeasts in covered injuries; O, yeasts in uncovered injuries.

species (C. eremophila and C. deserticola) are not as easilydetected by selective plating, and their presence among theisolates is uncertain. The species identity of bacterial iso-lates from cactus rots has not been studied as extensively asthat of the yeasts. A list of 24 bacterial species identified infour unpublished surveys is presented by Fogleman andHeed (in press). In these surveys and in our own work, thevast majority of bacteria isolated from necrotic cactus tissuebelong to the family Enterobacteriaceae and are thereforefacultative anaerobes.The initial source of the bacterial inocula is probably dust

or dirt particles blown into the injuries by wind. Even underthe harsh conditions of the desert, the bacterial density in thetop soil layers has been estimated in the range of 106 viablecells per g (J. L. M. Foster, unpublished data). Yeasts, onthe other hand, are not typically transported by passivemeans such as by wind. They are typically vectored byinsects and other arthropods which pick them up at existingrots and carry them into developing necroses. Several Dro-sophila species have been shown to be capable of transport-ing and inoculating both bacteria and yeasts into new sub-strates (6, 8). Our results (Table 2) have added D. mojavensisto this list.The fact that the growth curves for bacteria and yeasts are

similar with respect to rate of increase (Fig. 1) was unex-pected since, in general, bacteria replicate much faster thanyeasts. Furthermore, the rates of increase for both microbecategories within the developing agria necroses were much

TABLE 2. Log10 of the CFU per fly

Log10 CFU/flyaPlant

Bacteria Yeasts

Agria-1 6.05 4.71Agria-2 6.62 5.28Agria-3 6.05 4.66Agria-4 3.78 5.52

a Each number is the average of 10 D. mojavensis caught on each of the fouragria plants.

lower than they are under more idealized conditions (e.g.,complete media). The carrying capacities, however, do notappear to be significantly reduced compared with idealizedconditions. These facts suggest that some component of thecactus tissue is inhibitory to microbial growth. The mostlikely candidates for this inhibitory effect are the pentacyclictriterpene glycosides which are present in high concentra-tions in fresh agria tissue (10). These compounds have beenshown to inhibit the growth of some cactophilic yeasts (12,20) and bacteria (J. C. Fogleman and L. Armstrong, J.Chem. Ecol., in press).

Figure 1 also demonstrates the effect of the exclusion oflarge arthropods on microbial colonization. There is noapparent effect on bacterial colonization since bacteria donot rely exclusively on arthropods for transport. There is,however, a large effect on yeast colonization. As shown inthe figure, covering the injuries with netting produced a2-day lag in the appearance of yeasts in the developing rots.These results certainly lend credence to our previous state-ments on the vectoring of yeasts by Drosophila species andthe arrival of bacteria in an injury by more passive means.Drosophilids, however, are not the only yeast vectors in thissystem since yeasts were eventually detected in the coveredinjuries as well. In these cases, the sources of yeast inoculaare probably phoretic mites belonging to the genus Gama-sellus. These mites, which are typical residents of cactusrots, are small enough to pass through the screen and wereactually observed in the covered (as well as uncovered)injuries. Since they are saprophytic, one would reasonablyexpect them to be vectoring yeasts as well as bacteria. Theremaining question, then, is how did the mites get to theinjury sites? Phoretic mites hitch rides on the larger insectsincluding drosophilids. Desert Drosophila species carryingone or more mites are common, if not frequent, and miteloads can be as high as seven to eight mites per individual.When a mite-carrying drosophilid enters a new substrate,some mites may release their grasp and inoculate the sub-strate with microbes by walking on the substrate surface.The timing of the appearance of drosophilids at a devel-

oping rot depends on several factors including the size of the

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104 FOGLEMAN AND FOSTER

TABLE 3. Concentration of volatiles compounds produced indeveloping agria rots over time (average of the four plants)"

Volatile mM (mean + SD)compound on

day: Covered Uncovered

Methanol2 0.0 0.0 0.1 0.24 0.3 0.4 0.1 0.26 0.6 0.4 0.3 0.2

11 1.3 0.1 1.1 0.616 0.6 0.7 0.4 0.527 1.2 0.5 1.8 1.6

Ethanol2 1.9 0.4 1.8 0.24 2.7 1.5 3.0 1.66 3.8 0.7 3.1 2.4

11 9.1 10.1 8.4 8.816 5.1 6.3 2.8 1.927 2.1 1.1 1.4 1.4

2-Propanol2 0.1 0.2 0.1 0.14 0.1 0.1 0.1 0.36 0.3 ± 0.2 0.3 0.3

11 0.2 ± 0.2 0.1 0.116 0.3 ± 0.3 0.0 + 0.027 0.7 0.6 0.2 0.4

1-Propanol2 0.0 + 0.0 0.0 + 0.04 0.0 + 0.0 0.0 + 0.06 0.0 + 0.0 0.0 + 0.011 0.3 0.3 0.1 0.216 0.3 0.3 0.3 0.327 0.4 ± 0.5 0.0 + 0.0

Acetoin2 0.0 ± 0.0 0.0 0.04 0.0 ± 0.0 0.0 0.06 0.0 ± 0.0 0.0 ± 0.0

11 1.4 ± 2.6 0.3 ± 0.216 1. ± 2.3 0.2 ± 0.327 0.8±0.9 0.1±0.1

Acetic Acid2 0.4 + 0.5 1.0 ± 1.54 1.3 + 1.5 1.0 + 1.16 2.3 1.8 1.2 + 2.1

11 7.9 3.5 7.2 2.216 12.7 4.2 9.1 + 4.427 33.8 + 15.3 24.5 + 26.3

Propionic acid2 0.0 ± 0.0 0.0 ± 0.04 0.0 ± 0.0 0.0 ± 0.06 0.0 ± 0.0 0.0 ± 0.0

11 0.7 ± 1.4 0.4± 0.916 2.1 ± 1.8 1.4 1.327 4.1 ± 2.4 2.3 2.0

2,3-Butanediol2 0.0 ± 0.0 0.0 0.04 0.0 0.0 0.0 0.06 0.0 + 0.0 0.0 + 0.0

11 1.0 1.4 1.1 0.516 1.7 1.2 1.8 0.627 4.9 4.8 1.5 1.5

Other volatiles: 2-propyl acetate and 1-propyl acetate were detected onday 27 in trace amounts (0.1 to 0.2 mM), acetone was detected on days 16 and27 in trace amounts, n-butyric acid was detected on day 27 in trace amountsin the covered rots and at 1.6 ± 3.2 mM in the uncovered rots.

injury, the season (and other season-dependent climaticfactors), whether the cactus stem has broken open, whichallows volatile compounds to escape versus having the rotcontained within the cactus skin, and proximity to other rotsbeing utilized by Drosophila species. In nature, it is notunusual to observe rots in close proximity to each other ondifferent stems of the same plant. In our experiments, theflies responded to the injuries very rapidly and, as previouslymentioned, were first observed in one of the induced injuries24 h after the start of the experiment. D. mojavensis flieswere observed in all uncovered injuries by day 5 and wereusing these injuries as oviposition substrates as indicated bythe presence of larvae in subsequent samples. Therefore, theconditions under which these experiments were performedcan be considered close to ideal for drosophilid-mediatedmicrobial colonization and were ecologically realistic aswell.The fact that some Drosophila species are attracted to the

volatile products of fermentation has been well established(3, 7, 23) and dates back to 1907 (1). In these studies, ethanoland acetic acid are typically among the most attractivecompounds. In specific studies on the cactus-microorgan-ism-Drosophila model system, the volatile profile of matureagria rots and the fact that D. mojavensis flies are attractedto the volatile substances produced during microbial fermen-tation of cactus tissue and can differentiate cactus speciesbased on the volatile pattern have also been established (17;Fogleman and Heed, in press). However, the volatile pat-terns that are involved in the initial attraction of D. moja-vensis to developing necroses had never been characterized.On day 4, only four volatile compounds were present inconcentrations of 0.1 mM or greater. These compounds weremethanol, ethanol, 2-propanol, and acetic acid. Of thesefour, ethanol and acetic acid were present in the greatestconcentrations (Table 3) and were the most consistentlypresent over replicate injuries. These facts, coupled withlaboratory experiments demonstrating the attractiveness ofethanol and acetic acid to D. mojavensis (J. C. Foglemanand D. A. Davis, unpublished data), suggest that these twocompounds play an active role in the initial attraction ofdeveloping necroses to D. mojavensis. Since the uncoveredinjuries were attractive prior to significant yeast growth, weconclude that the initial volatile pattern is produced mainlyby bacteria.

In comparing the previously published data on the averageand maximum concentrations of volatile substances in moremature agria rots with the data in Table 3, several differencescan be noted. First, as a rot develops, the number of volatilecompounds that are present increases. In the volatile profilebased on seven naturally occurring agria rots (Fogleman andHeed, in press), 15 different compounds had an averageconcentration of 0.1 mM or greater (11 of which had maximagreater than 5.0 mM). This phenomenon can also be seen inTable 3, in that 8 of the 12 compounds listed appear on day11 or later. The second difference concerns the concentra-tions of ethanol and acetic acid. The average concentrationsof these two volatile compounds in natural (mature) agriarots have been reported as 5.0 and 35.4 mM, respectively,while the maxima were 25.1 and 82.1 mM, respectively. Notonly is the volatile profile a dynamic characteristic thatchanges drastically over time, but also, the initial attractionof D. mojavensis to agria rots is based on fewer volatilesubstances and much lower concentrations than had beenpreviously suspected.

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MICROBIAL COLONIZATION OF CACTUS TISSUE 105

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant GM

34820 from the National Institute of General Medical Sciences toJ.C.F.We gratefully acknowledge Richard Thomas for the mite identifi-

cation.

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22. Vacek, D. C. 1982. Interactions between microorganisms andcactophilic Drosop/liia in Australia, p. 175-190. In J. S. F.Barker and W. T. Starmer (ed.), Ecological genetics and evo-lution: the cactus-yeast-Drosophilal model system. AcademicPress, Sydney.

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