Harnessing endophytes for industrial microbiology

Gary Strobel Department of Plant Sciences Montana State University Bozeman, Montana, 59717

Phone: 406 994 5148 Email: [email protected]

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Summary

Endophytic microorganisms exist within the living tissues of most plant species. They are most abundant in rainforest plants. Novel endophytes usually have associated with them novel secondary natural products and or processes. is a novel endophytic fungal genus that produces bioactive volatile organic compounds (VOC’s). This as well as its VOC’s has enormous potential for uses in agriculture, industry, and medicine. produces a mixture of VOC’s that act synergistically in being lethal to a wide variety of plant and human pathogenic fungi and . This mixture of gases consists primarily of various alcohols, acids, esters, ketones, and lipids. Artificial mixtures of the VOC’s mimic the biological effects of the fungal VOC’s when tested against a wide range of fungal and bacterial pathogens. Many practical applications for “mycofumigation” by M. albus have been investigated and the fungus is now in the market place.

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Introduction

Microorganisms have long served mankind by virtue of the myriad of the enzymes and

secondary compounds that they make [1]. Furthermore, only a relatively small number of

microbes are used directly in various industrial applications i.e. cheese/ wine/ beer making as well as in environmental clean –up operations and for the biological control of

pests and pathogens. It seems that we have by no means exhausted the world for its

hidden microbes. A much more comprehensive search of the niches on our earth may yet

reveal novel microbes having direct usefulness to human societies. These uses can either

be of the microbe itself or one or more of its natural products.

The diversity of microbial life is enormous and the niches in which microbes live is truly

amazing ranging from deep ocean sediments to the earth’s thermal pools [2]. However,

an additional relatively untapped source of microbial diversity is the world’s rainforests.

These forests occupy only 7% of the earth’s land surface and yet represent 50 -70% of all of the species that live here. This is nicely exemplified by the fact that over 30% of all of

the bird species on earth are associated with Amazonia alone. The question is why is so

much diversity focused in such areas? Paul Richards writing in “The Tropical Rainforest”

indicates that “The immense floristic riches of the tropical rainforest are no doubt largely

due to its great antiquity, it has been the focus of plant evolution for an extremely long

time.” In my opinion, the diversity of macro life forms is a template for what probably

has also happened with microorganisms. That is, areas of the earth with enormous

3 diversity of higher life forms are probably also accompanied by a high diversity of microorganisms. It seems that no one has looked!

One specialized and unique biological niche that supports the growth of microbes are the spaces in between the living cells of higher plants. It turns out that each plant supports a suite of microorganisms known as endophytes [3]. These organisms cause no overt symptoms on the plants in which they live [4]. Furthermore, since so little work on these endophytes has been done, it is suspected that untold numbers of novel fungal and bacterial genera exist as plant associated microbes and their diversity may parallel that of the higher plants [3]. Such a specific search is governed by the fact that some endophytes may have coevolved with their respective higher plant thus having an already existing compatibility with a higher life form. Thus, we have begun a concerted search for novel endophytic microbes and the prospects that they may produce novel bioactive products as well as processes that may prove useful at any level. It is obvious that any discovery of novel microbes will have implications in virtually all of the standard processes of industrial microbiology since scale up fermentation of the microbe will be necessary.

Several reviews have discussed the products of endophytic microbes and their promise for use in medicine, agriculture and industry [3,5,6 ] However in order to give the reader a sense of the excitement of discovery that comes with research on rainforest microbes I have concentrated this discussion mostly on the discovery of only one novel endophytic fungal genus – Muscodor. This review discusses the systematic study of its VOC production and its future for human use, biological discovery, and industrial microbiology.

4 Anatomy of the discovery of Muscodor albus

In the late 90’s I was on a collecting trip in the jungles near to the Caribbean coast of

Honduras. I had selected this area to visit since all of Central America is one of the world’s “hot spots of biodiversity”[7]. One modestly sized tree, not native to the new world, was introduced to me as Cinnamomum zeylanicum and I obtained small limb samples. Unfortunately, most plant samples from the tropics are infested with microscopic phytophagous mites. These nasty creatures infest the bench tops and invade parafilm sealed Petri plates. Thus, in order to eliminate this nagging mite problem we decided to place the Petri plates, with plant tissues, in a large plastic box having a firmly fitting lid [3]. This maneuver would make it difficult for the tiny animals to find their way from the bench surfaces to the inside of the box. After a few days most plant specimens had sported endophytic fungal growth. Eventually the plates were removed and the individual hyphal tips were transferred to fresh plates of potato dextrose agar.

After two days of incubation we noted that no transferred endophyte grew except one.

Had the placement of the endophytes in the large plastic box killed the endophytes by limiting oxygen availability? Quite the contrary, it became obvious that the one endophytic fungus (designated isolate 620) remaining alive was producing volatile antibiotics or volatile organic compounds (VOC’s). The hypothesis that an endophyte can make volatile antibiotic substances with a wide range of biological activity was born. It was quickly learned that although many wood inhabiting fungi make volatile substances, none of these possessed the biological activity of isolate 620 [8]. However some early data supported the observations that Trichoderma sp. produced some VOC’s, however, with only modest biological activity and no attempt was made at that time to identify the

5 VOC’s of this organism [9]. Later, another report on Trichoderma showed the identity of

the VOC’s and pointed out that inhibitory activity was associated with these compounds

[10].

Muscodor albus and its VOC’s

Isolate 620 is a whitish, sterile endophytic fungus possessing hyphal coiling, ropyness,

and right angle branching (Fig.1). Therefore, in order to taxonomically characterize this

organism, the partial regions of the ITS- 5.8 rDNA were isolated, sequenced, and deposited in GenBank as AF 324336 and AF 324337 [11]. Isolate 620 shows some relatedness to several Xylaria spp. at the 82-92% level, but is otherwise unique [11]. The

GC/MS analysis of the fungal VOC’s showed the presence of at least 28 VOC’s [12].

These compounds represented at least five general classes of organic substances

including lipids, esters, alcohols, ketones, and acids with some representative structures

shown in Fig. 2. Final identification of the volatile compounds was done by GC/MS of

authentic compounds obtained from commercial sources or synthesized by us or others

and compared directly to the VOC’s of the fungus [12]. With all of the data in hand we

felt secure in proposing a binomial for this fungus derived from the Latin –Muscodor

(stinky) albus (white) [11] (Fig. 1).

Ultimately, artificial mixtures of the compounds were used in a biological assay system

to demonstrate the relative activity of individual compounds. Although over 80% of the

volatiles could be identified, this seemed to be adequate to achieve an excellent

reproduction of the lethal- antibiotic effects of the VOC’s that were being produced by

the fungus [12]. We then examined each of the five general classes of VOC’s in the

6 bioassay test and each possessed some inhibitory activity with the esters being the most active [12]. Of these the most active individual compound was 1-butanol, 3 methyl-, acetate. However, it is to be strongly stressed that no individual compound or class of compounds was lethal to any of the test microbes which consisted of representative plant pathogenic fungi, Gram positive and Gram negative bacteria, and others [12]. Obviously, the antibiotic effect of the VOC’s of M.albus is strictly related to the synergistic activity of the compounds in the gas phase. We know very little about the mode of action of these compounds on the test microbes thus, this represents an interesting academic avenue to pursue in the future.

Using the bioassay test system the IC50 for each test microbe was determined [12]. Also, the killing ability of the artificial mixture with that of the M. albus VOC’s were compared. One of the most sensitive fungi was Rhizoctonia solani and one of the least sensitive was Fusarium solani. In the later case, F. solani was only inhibited by both the artificial VOC mixture as well as the VOC’s of the fungus. Finally, the viability of all test organisms was also determined after exposure to either the fungus or to its artificial mixture of VOC’s [12]. The artificial mixture generally mimicked the effects of the fungus itself [12]. We have also learned that a minimum of three of the VOC’s

(naphthalene, propanoic acid and butanol, 3-methyl) can quite effectively mimic the killing activity of the fungal VOC’s, thus certainly not all compounds in the fungal

VOC’s are necessary for biological activity [13].

7 Using the original isolate of M. albus as a selection tool, other very closely related isolates of this fungus have been obtained from tropical plants in various parts of the world including Thailand, Australia, Peru, Venezuela, and Indonesia [13,14,15]. They possess high sequence similarity to isolate 620 and produce many, but not all of the same

VOC’s as isolate 620.

Two other species, including M. roseus and M. vitigenus, have also been discovered in

Australia and Peru, respectively [16,17]. The later organism makes only naphthalene as a

VOC and its repellency towards a plant associated insect was demonstrated [18]. Many

temperate areas of the world have been explored and examined for the presence of

Muscodor spp., but with no success.

Physiological aspects of VOC production

The composition of the medium greatly influences the quality and effectiveness of the

VOC’s emitted by M. albus with enriched media being more effective in inhibiting a

suite of plant pathogens used as test microbes [19]. Proton transfer reaction-mass

spectrometry (PTR-MS) was used to acquire on- line quantitative data on VOC

production [20]. The production of VOC’s is temperature dependent with decreased gas

production occurring at higher temperatures [20]. The PTR-MS technique was also

applied to soils containing M. albus along with the plant pathogen Pythium ultimum and

it was possible to show the production of VOC’s from M. albus in situ. An estimation of

the effective range of concentrations of total VOC’s being produced by M. albus is in the

8 order of 100-300 ppb based on the determination of the concentration of propanoic acid,

2-methyl, methyl ester.

“Mycofumigation” with Muscodor spp.

The VOC’s of M. albus kill many of the pathogens that affect plants, people and even

buildings [12, 15]. The term “mycofumigation” has been applied to the practical aspects of this fungus [12]. The first practical demonstration of its effects against a pathogen was

the mycofumigation of covered smut infected barley seeds for a few days resulting in

100% disease control [12]. This technology is currently being developed for the treatment

of fruits in storage and transit [21]. Soil treatments have also been effectively used in both field and green house situations [22-24]. In these cases, soils are pretreated with a

M. albus formulation in order to preclude the development of infected seedlings.

AgraQuest, of Davis, Calif., is in the full scale development of M. albus for numerous

agricultural applications with an anticipated release of a product in 2006. The US-EPA

has given provisional acceptance of M. albus for agricultural uses. Also, it appears that

the concept of mycofumigation has the potential to replace the use of otherwise

hazardous substances that are currently applied to crops, soils and buildings and the most

notable of which is methyl bromide for soil sterilization. AgraQuest is expected to have a

product on the market for fruit treatment in 2006. Aspects for the formulation and scale

up of M. albus for these applications are currently being established.

9 Pending questions about the VOC producing fungi

Obviously, because of the impressive biological activity of M. albus and its introduction into practical agriculture/industry, it seems that the fungus should be more fully studied relative to its location and role in nature. Overall, the most pressing question regarding

M. albus is the mode of action of a multitude of volatile compounds and how they act synergistically to cause the death of microbes. Certainly, it seems that knowledge of its host preferences and those factors controlling host preference may eventually allow for the use and development of this organism for hosts that it does not naturally frequent and it could find still more applications and direct uses. This idea may be exemplified by the direct inoculation of plants including those used in agricultural and forestry for their protection against insects and diseases. In addition, it is extremely important to possess information on the distribution, life cycle and other aspects of the chemistry of this organism. Hopefully, the development of information on M. albus will have broad implications for the discovery and development of other rainforest microbes while at the same time adding another impetus for the conservation of the world’s rainforests.

Acknowledgements

The author acknowledges the financial assistance of the NSF, the Montana Board of

Research and Commercialization Technology, the USDA, and the Montana Agricultural

Experiment Station in making this work possible.

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11 12. Strobel GA, Dirksie E, Sears J, Markworth C: Volatile antimicrobials from a

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4031.

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of Muscodor albus, an endophyte from , in Thailand,

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characterization of Muscodor albus I-41.3s, a volatile antibiotic

producing fungus. Plant Sci 2005, 169: 854 861.

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Muscodor roseus anam. sp. nov., an endophyte from Grevillea pteridifolia.

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17. Daisy B, Strobel G, Ezra D, Castillo U, Baird G, Hess WM:

sp.nov. an endophyte from Paullinia paullinoides. Mycotaxon 2002, 84: 39-50.

18. Daisy B, Strobel G, Ezra D, Castillo U, Ezra D, Sears J, Weaver, D, Runyon, J:

Naphthalene, an insect repellent, is produced by Muscodor vitigenus, a novel

endophytic fungus. Microbiol 2002, 148: 3737-3741.

19. Ezra D, Strobel GA: Effect of substrate on the bioactivity of Volatile

antimicrobials emitted by Muscodor albus. Plant Sci 2003, 165: 1229- 1238.

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12 Proton-transfer reaction- mass spectroscopy as a technique to measure

volatile emissions of Muscodor albus. Plant Sci 2004 166: 1471-1477.

21. Mercer J, Jimenez J I: Control of fungal decay of apples and peaches

by the biofumigant fungus Muscodor albus. Post Harvest Biology Tech

2004, 31: 1-8.

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enhancement in green house soilless mix by the volatile-producing fungus

Muscodor albus. Crop Protection 2005, 24: 355-362.

23. Stinson, M., Zidack, N.K., Strobel, G.A., and Jacobsen, B. (2003)

Mycofumigation with Muscodor albus and Muscodor roseus for control of

seedling diseases of sugar beet and verticillum wilt of eggplant. Plant Disease

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24. Jacobsen BJ, Zidack NK, Strobel GA, Ezra D, Grimme E, Stinson AM:

Mycofumigation with Muscodor albus for control of soilborne

microorganisms. IOBC wprs Bulletin 2004, 27: 103-113.

13 Figure Legends:

Fig.1 A culture of Muscodor albus with an SEMs of its hyphae as an insets (below).

Fig.2 Some representative inhibitory volatile compounds produced by Muscodor albus.

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