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Keywords:

  • synergism;
  • volatile antibiotics;
  • Oidium;
  • antibiosis;
  • endophytes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Conclusions
  7. Acknowledgements
  8. References

Oidium sp. has been recovered as an endophyte in Terminalia catappa (tropical chestnut) in Costa Rica. The volatile organic compounds (VOCs) of this organism uniquely and primarily consist of esters of propanoic acid, 2-methyl-, butanoic acid, 2-methyl-, and butanoic acid, 3-methyl-. The VOCs of Oidium sp. are slightly inhibitory to many plant pathogenic fungi. Previous work on the VOCs of Muscodor albus demonstrated that besides esters of small organic acids, a small organic acid and a naphthalene derivative were needed to obtain maximum antibiotic activity. Thus, the addition of exogenous volatile compounds such as isobutyric acid and naphthalene, 1,1′-oxybis caused a dramatic synergistic increase in the antibiotic activity of the VOCs of Oidium sp. against Pythium ultimum. In fact, at elevated concentrations, there was not only 100% inhibition of P. ultimum but killing as well. In addition, a coculture of Muscodor vitigenus (making only naphthalene) and Oidium sp. plus isobutyric acid produced an additive antibiosis effect against P. ultimum. The biological implications of multiple volatile compounds acting to bring about antibiosis in nature are discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Conclusions
  7. Acknowledgements
  8. References

An endophytic fungus producing volatile organic compounds (VOCs) with antibiotic activity is Muscodor albus (Strobel et al., 2001; Worapong et al., 2001). The VOCs represent a mixture of esters, acids, alcohols, lipids, and assorted other volatiles that effectively inhibit, and are usually lethal to, a wide range of target microorganisms including many plant pathogenic fungi and various bacteria including Escherichia coli (Strobel et al., 2001). Using the original isolate of M. albus as a selection tool, at least 12 other isolates of this organism have been found producing biologically active volatiles (Daisy et al., 2002; Sopalun et al., 2003; Ezra et al., 2004; Atmosukarto et al., 2005; Strobel, 2006; Strobel et al., 2007). Because the biological activity of this organism is so effective and broad, it is being used to treat human wastes, and will soon be on the market to treat agricultural soils and fresh produce (Stinson et al., 2003; Mercier & Jimenez, 2004; Strobel, 2006).

Recently, an isolate of Oidium sp. was obtained as an endophyte from tropical chestnut growing in an east coast rainforest of Costa Rica. The isolate produced fruity smells, and upon analysis it was learned that the aromaticity was due to the production of a series of esters of small organic acids by these fungi. Oidium sp., unlike M. albus, only produced a slight inhibition of various microorganisms in a panel of test organisms, which never resulted in the death of these organisms (Strobel et al., 2001). An examination of the VOCs of Oidium sp. revealed that, while inhibitory esters were being produced, two other compounds critical to the bioactivity of M. albus were not found in the volatiles of Oidium sp. (Ezra et al., 2004). These compounds include a small organic acid and naphthalene or a derivative thereof (Ezra et al., 2004). This could effectively explain the relatively low biological activity of the VOCs of this organism. In addition, these two compounds by themselves have only slight inhibitory activity of the primary test organism –Pythium ultimum. However, this report shows there is antagonistic/synergistic enhancement of the antimicrobial activity of the VOCs of Oidium sp., which results from the artificial placement of these compounds, normally found in Muscodor spp., into the atmosphere of Oidium sp. Experiments are presented that also show the additive effects of the biological activity of the VOCs of Oidium sp. and Muscodor vitigenus (only making naphthalene as its sole VOC) against P. ultimum.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Conclusions
  7. Acknowledgements
  8. References

Isolating, culturing, and storing Oidium sp.

The fungal culture used in this study was obtained as an endophyte from small limbs in the crown of Terminalia catappa found in an east coast rainforest of Costa Rica, at 10°14′55″N; 83°51′58″W. Plant samples (stems) were each dipped into a 70% ethanol solution, flamed, and then tissues beneath the epidermal layers removed and plated on water agar. Individual fungi were hyphal tipped from the microorganisms from the tissues. One fungus (designated CR-1), among others, whose structural features were identical to Oidium sp. was recovered from T. catappa. The fungus, by virtue of a simple smell test, yielded a fruity odor, possessed some antifungal activity, and was subjected to further study. The fungus was stored using standard laboratory techniques (Strobel et al., 2007). The fungus was deposited as no. 2331 in the living mycological culture collection at Montana State University.

Test microorganisms

All plant-associated microorganisms used in the bioassay test system were obtained from Drs Don Mathre and Nina Zidak of the MSU Department of Plant Sciences. Bacterial cultures were supplied by the MSU Department of Microbiology. All test organisms were grown on potato dextrose agar (PDA) and bacteria on Luria–Bertani agar (LBA) at 23 °C and only freshly transferred cultures (4–7 days old) were used in the bioassay tests.

Scanning electron microscopy (SEM)

Oidium sp. was grown on PDA, and was processed for SEM. Many agar pieces containing the fungus were placed into filter paper packets and suspended in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2–7.4) with Triton X (a wetting agent) as previously described (Ezra et al., 2004). Ultimately, for SEM the fungal material was critical point dried, gold sputter coated, and images were recorded with an XL30 ESEM FEG in high vacuum mode using the Everhart–Thornley detector.

Qualitative analysis of M. albus of the Oidium sp. volatiles

A GC/MS method used to analyze the gases in the airspace above the 10-day-old culture of the CR-1 mycelium growing in Petri plates was comparable to that used on the original isolate of M. albus strain cz-620 (Strobel et al., 2001). The gas chromatograph was interfaced to a Hewlett Packard 5973 mass selective detector (mass spectrometer) operating at unit resolution. Data acquisition and data processing were performed on the Hewlett Packard chemstation software system. Initial identification of the unknowns produced by M. albus was made through library comparison using the National Institute of Standards and Technology (NIST) database.

Comparable analyses were conducted on Petri plates containing only PDA and the compounds obtained therefrom, mostly styrene and its derivatives, were subtracted from the analyses carried out on plates containing the fungus. Tentative identification, based on comparative mass spectral information between observed data and those in the NIST database, was made on the fungal VOCs.

Bioassay tests for volatile antimicrobials and synergy

A bioassay test system was devised that allowed only for VOCs from the fungus being the active agents for any microbial inhibition examined as previously described (Strobel et al., 2001). A 2.5-cm-wide strip of agar was removed from the mid-portion of a standard PDA Petri dish, then CR-1 was inoculated and grown on one side of the plate for varying time periods before testing. The test fungus on a plug of PDA (5 × 5 × 5 mm) was placed on to the agar half-moon strip on the opposite side of the plate. The plate was wrapped with two individual pieces of Parafilm and incubated at 23 °C for 1 day. Eventually, the linear growth of the filamentous fungi (as measured from the edge of the agar inoculum plugs) as well as the viability of each test fungus was evaluated relative to untreated controls. Measurements of mycelial growth made under test conditions were expressed as % growth over that occurring with no treatment. The test with each designated assay organism was repeated at least three times.

Once it was established that CR-1 possessed antagonistic activity of its VOCs, either isobutyric acid or naphthalene, 1,1′-oxybis-, or both, were added to the plates, in small plastic microwell cups. Again, measurements of test organisms were made to assess the biological effects of these added compounds upon the VOC activity. Using a probe (pH), it was determined that the small amounts of isobutyric acid added to the test system did not affect the pH of the agar medium, suggesting that any observed biological effects could not be attributable to a simple pH variation.

Preparation of naphthalene, 1,1′-oxybis-

This compound is the major naphthalene derivative produced by a number of biologically active Muscodor sp. isolates but not available commercially (Ezra et al., 2004). Therefore, it was synthesized by a modification of the procedure reported by Clowes (1968). An intimate mixture of equal weights of 1-naphthol and sodium hydrogen sulfate was fused in an Erlenmeyer flask with magnetic stirring at 200–210 °C for 3 h. At the end of this time an equal volume of Celite was added with continued heating and manual stirring. The resulting reasonably homogeneous solid was immediately removed from the flask, while hot, allowed to cool to room temperature, and then pulverized. The resulting dark solid was extracted by filtration using three portions of hot toluene and the combined organic phases were filtered through a pad of activity 1 alumina. After removal of the solvent in vacuo, the residue was purified by chromatography on activity 1 alumina (benzene for elution). Recrystallization of the residue obtained after removal of the benzene from cyclohexane provided di-1-naphthyl ether (38%). It is to be noted that the compound does not completely vaporize in the solid state when placed in the Petri plate assay system and no attempt was made for it to do so. Thus, all data presented are relative to the amounts of compound that are directly added to the microcup in the Petri plate and not the actual concentration in the atmosphere in the system, which is 50 mL. Isobutyric acid was obtained from Aldrich Chem Co.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Conclusions
  7. Acknowledgements
  8. References

The VOCs and biological activity of Oidium sp.

The culture of Oidium sp. (given a designation of CR-1 from Terminalia catapa – tropical chestnut) used in this study was positively identified on the basis of its colony morphology (flat oppressed whitish colonies with a sheen-like appearance) as well as its spore and hyphal characteristics (Fig. 1). The arthrospores (c. 3.0 × 5.0 μm) are produced by budding of the hyphae and take on the appearance of small barrels (Fig. 1). The VOCs of this fungus were identified by GC/MS and they mainly consisted of an assorted variety of esters of propanoic acid, 2-methyl-, butanoic acid, 2-methyl-, and butanoic acid, 3-methyl- (Table 1). A comparative examination of the VOCs of M. albus revealed that three compounds held in common with CR-1 were 2-phenylethyl alcohol, propanoic acid, 2-methyl-, methyl ester, and propanoic acid, 2-methyl-, ethyl ester (Strobel et al., 2001). For optimum inhibitory activity of CR-1, a free acid and a naphthalene derivative was missing (Ezra et al., 2004). Therefore, because CR-1 produces a plethora of esters (Table 1), it seemed reasonable to assume that providing an artificial supplement of an organic acid and or a naphthalene derivative, as per the M. albus VOCs, to an assay test organism would enhance the biological activity of the VOCs of CR-1. Experiments were devised to determine whether the addition of an exogenous free acid (isobutyric) and a naphthalene derivative (naphthalene, 1,1′-oxybis-) could influence the biological activity of the VOCs of CR-1.

image

Figure 1.  Scanning electron micrographs of the hyphae and arthrospores of Oidium sp. isolated as an endophyte from Terminalia catappa in a Costa Rican rainforest. The left side (a) shows the arthrospores of Oidium sp. and the right side (b) illustrates the sporulating hyphae of this fungus.

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Table 1.   A GC/MS analysis of the VOCs of Oidium sp., an endophytic fungus of Terminalia catappa in Costa Rica
Retention time (min)Total area (%)Possible compoundM- Da
  1. The gases were trapped in the airspace above a 10-day-old culture with a ‘Solid phase micro extraction’ syringe (Supelco). The data are reported as the actual retention time, total area of the eluting peak and the actual mass-daltons (M-Da) for each compound. The compounds best fitting the mass data in the NIST data bank are reported as –‘ possible compound.’ Compounds appearing in the gases of the control PDA plate were subtracted from the plate supporting fungal growth. In addition, compounds that occupied only a small total area were not included in this table, thus the total is 88.1%.

5:260.61Propanoic acid, 2-methyl-, methyl ester102.07
6:281.58Propanoic acid, 2-methyl-, ethyl ester116.08
7:463.41Butanoic acid, 2-methyl-, methyl ester, (+/−)116.08
8:1410.20Butanoic acid, 3-methyl-, methyl ester116.08
9:063.26Butanoic acid, 2-methyl-, ethyl ester130.1
9:1114.66Butanoic acid, 3-methyl-, ethyl ester130.1
10:031.86Propanoic acid, 2-methyl-, butyl ester144.12
12:125.86Butanoic acid, 2-methyl-, 2-methylpropyl ester158.13
12:185.862-Butenoic acid, 3-methyl-, methyl ester114.07
13:0110.38Butanoic acid, 3-methyl-, 2-methylpropyl ester158.13
13:212.02Methyl butenoate (E isomer) (methyl tiglate)114.07
14:231.072-Butenoic acid, 3-methyl-, ethyl ester128.08
14:350.60Hexanoic acid, ethyl ester144.12
14:141.712-Butenoic acid, 2-methyl-, ethyl ester128.08
15:134.34Butanoic acid, 2-methyl-, 3-methylbutyl ester172.15
16:0610.25Butanoic acid, 3-methyl-, 3-methylbutyl ester172.15
17:571.242-Butenoic acid, 2-methyl-, 2-methylpropyl ester, (E)-156.12
31:038.722-Phenylethyl alcohol122.07
32:560.52Butanoic acid, 3-methyl-, 2-phenylethyl ester206.13

Synergy experiments with CR-1 and exogenous organics

Initially, a number of test assay organisms were studied to determine whether the VOCs of CR-1 possessed any inhibitory activity against test organisms (seven test fungi and two bacteria) and the results generally varied between 0% and 20% growth inhibition as compared with untreated controls. This is in sharp contrast to the response of these same test organisms to the VOCs of M. albus, wherein 100% growth inhibition generally occurs followed by death of the organism (Strobel et al., 2001). Then, to determine whether it was possible to enhance the VOC activity of CR-1 cultures, two exogenous VOCs including propanoic acid, 2-methyl- (isobutyric acid), and naphthalene, 1,1′-oxybis- alone and in combinations with various concentrations, were added. Whereas Phytophthora cinnamoni showed a promising increased inhibitory response, other test fungi including Rhizoctonia solani, Sclerotinia sclerotiorum, Aspergillus fumigatus, Geotrichum candidum, Botrytis cinerea, Trichoderma viridi, E. coli, and Bacillus subtilis either did not respond to the added organics or only gave hints of responding in either an additive or a synergistic manner (data not shown).

Ultimately, the best test organism showing an enhanced response to the VOCs of CR-1 and the two exogenously added compounds was P. ultimum. For instance, when CR-1 alone was tested, it produced about a 26±3.0% inhibition of P. ultimum (Fig. 2). Similarly, the presence of naphthalene, 1,1′-oxybis- or isobutyric acid alone also resulted in about a 20–25% inhibition of P. ultimum at the level of 0.25 mg per assay plate (Fig. 2). However, CR-1 combined with naphthalene, 1,1′-oxybis- resulted in no increase in activity at the lowest concentration (0.25 mg) but the activity was increased at higher concentrations, and CR-1 with isobutyric acid at all levels increased the percentage of inhibition of P. ultimum (Fig. 2). Most interestingly, the combination of CR-1, naphthalene, 1,1′-oxybis- and isobutyric acid at the low concentration of 0.25 mg per plate (of each compound) resulted in 98–99% inhibition of P. ultimum. Thus, this near total inhibition is more than the additive effect of each of the inhibitors and CR-1, and a chemical synergism seems to be occurring. However, there was no synergistic activity observed between the combination of isobutyric acid and naphthalene, 1,1′-oxybis- at any concentration (Fig. 2). Furthermore, at the higher concentrations of naphthalene, 1,1′-oxybis-, isobutyric acid, and CR-1, there was 100% inhibition of P. ultimum, which resulted in the death of the test organism (Fig. 2).

image

Figure 2.  The antibiotic effects of CR-1 [Oidium sp. VOCs alone – represented by the clear spotted bar (left)] and either isobutyric acid and/or naphthalene, 1,1′-oxybis- in a bioassay test against Pythium ultimum. The concentrations of either isobutyric acid or naphthalene, 1,1′-oxybis- alone or together are represented by the bar codes on the right side of this figure as follows: clear spotted bar=CR-1 alone; slashed bar=25 μL of isobutyric acid or 0.25 mg naphthalene, 1,1′-oxybis- per plate or both; the gray bar=50 μL isobutyric acid or 0.50 mg naphthalene, 1,1′-oxybis- per plate or both, and white dots on a gray background are at a level of 0.75 μL of isobutyric acid or 75 mg naphthalene, 1,1′-oxybis- per plate or both.

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Antibiosis effects of Oidium sp. with M. vitigenus

Experiments were performed by devising a coculture technique of Oidium sp. along with M. vitigenus on the same plate of PDA. The latter organism, M. vitigenus, makes only naphthalene as its sole VOC (Daisy et al., 2002). These two organisms together produced a 45±18% inhibition of P. ultimum and, in contrast, Oidium sp. alone only produced a 26±3% inhibition of P. ultimum. However, when 0.25 μL of isobutyric acid was added to the coculture, a 93±7% inhibition of P. ultimum resulted. Isobutyric acid alone at this concentration produced only a 20±5% inhibition of P. ultimum. It appears that there is a synergistic action of the two fungi plus the low concentration of isobutyric acid against P. ultimum.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Conclusions
  7. Acknowledgements
  8. References

Recently, an endophytic isolate of Oidium sp. (CR-1) was obtained from T. catappa growing in the eastern rainforest of Costa Rica (Fig. 1). The fruity smells, produced by this fungus, are primarily attributed to a complex mixture of esters of propanoic acid, 2-methyl- and butanoic acid, 2-methyl- and butanoic acid, 3-methyl- (Table 1). Unlike the VOCs of M. albus isolates, no free organic acids, azulene, or naphthalene derivatives were detectable in the VOCs of CR-1 (Table 1) (Strobel et al., 2001). The biological activity of M. albus greatly surpasses that of CR-1 (Strobel et al., 2001). One of the most biologically active compounds among the VOCs of M. albus is 1-butanol, 3-methyl-, acetate. It alone produced complete inhibition of some of test assay fungi (Strobel et al., 2001). Interestingly, only a few compounds, as taken from the list of VOCs made by M. albus, are needed to produce inhibition and death of many of the test assay fungi (Strobel et al., 2001). These include 1-butanol, 3-methyl-, acetate, a simple organic acid such as propanoic or isobutyric, and either naphthalene or one of its derivatives in an appropriate ratio (Ezra et al., 2004). Therefore, because CR-1 produces a plethora of esters, it seemed reasonable to assume that providing an artificial supplement of an organic acid and or a naphthalene derivative to an assay test organism would enhance the biological activity of the fungal VOCs. In fact, there was increased biological activity and also the activity was greater than the collective activity of each of the test ingredients and the phenomenon of synergism seemed to be in effect (Fig. 2). Furthermore, the addition of M. vitigenus, as the producer of naphthalene, along with isobutyric acid and CR-1 also caused an increased inhibitory effect against P. ultimum, the test organism. This work collectively suggests that the VOCs of different endophytic fungi may act both additively and synergistically to bring about inhibition of other symbiotic and or pathogenic microorganisms inhabiting the same plant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Conclusions
  7. Acknowledgements
  8. References

The authors appreciate the financial assistance of the REU–NSF program in MSU Department of Chemistry and an NSF grant to G.A.S. Assistance was also provided by the Montana Department of Commerce-Board of Research and Commercialization Technology and the Dole Fruit Company and the Montana Agricultural Experiment Station.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Conclusions
  7. Acknowledgements
  8. References
  • Atmosukarto I, Castillo U, Hess WM, Sears J & Strobel G (2005) Isolation and characterization of Muscodor albus I-41.3 s, a volatile antibiotic producing fungus. Plant Sci 169: 854861.
  • Clowes GA (1968) Studies of the Scholl reaction: oxidative dehydrogenation involving 1-ethoxynaphthalene and related compounds. J Chem Soc (C): 25192526.
  • Daisy BH, Strobel GA, Castillo U, Sears J, Weaver DK & Runyon JB (2002) Naphthalene production by Muscodor vitigenus, a novel endophytic fungus. Microbiology 148: 37373741.
  • Ezra D, Hess WM & Strobel GA (2004) New endophytic isolates of Muscodor albus, a volatile antibiotic-producing fungus. Microbiology 150: 40234031.
  • Mercier J & Jimenez JI (2004) Control of decay of apples and peaches by the biofumigant fungus Muscodor albus. Postharvest Biol Technol 31: 18.
  • Sopalun K, Strobel GA, Hess WM & Worapong J (2003) A record of Muscodor albus, an endophyte from Myristica fragrans, in Thailand. Mycotaxon 88: 239247.
  • Stinson AM, Zidack NK, Strobel GA & Jacobsen BJ (2003) Effect of mycofumigation with Muscodor albus and Muscodor roseus on seedling diseases of sugarbeet and Verticillium wilt of eggplant. Plant Disease 87: 13491354.
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  • Strobel GA, Kluck K, Hess WM, Sears J, Ezra D & Vargas PN (2007) Muscodor albus E-6, an endophyte of Gauzuma ulmifolia, making volatile antibiotics: isolation, characterization and experimental establishment in the host plant. Microbiology 153: 26132620.
  • Worapong J, Strobel GA, Ford EJ, Li JY, Baird G & Hess WM (2001) Muscodor albus anam. nov. an endophyte from Cinnamomum zeylanicum. Mycotaxon 79: 6779.