Source: MONTANA STATE UNIVERSITY submitted to NRP
THE BIOLOGY AND UTILITY OF MUSCODOR ALBUS IN AGRICULTURE (MICROBE /PLANT INTERACTIONS)
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0070380
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2002
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Plant Sciences and Plant Pathology
Non Technical Summary
This project deals with various aspects of the associations of Muscodor albus, a novel endophytic microbe, with higher plants. We will study how this organism can be utilized in numerous biological control mechanisms to the benefit of agriculture. The development of Muscodor albus as a biological control agent has the potential to replace harful and toxic biocides that are now used in agriculture. This endophytic fungus is novel, has been patented, and licensed to numerous companies for agricultural use. This project will examine other uses for this interesting fungal organism.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2120640104010%
2120640116010%
2122410104010%
2122410116010%
2124020104010%
2124020116020%
2150640104010%
2150640116010%
2152499116010%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
4020 - Fungi; 2410 - Cross-commodity research--multiple crops; 0640 - Tropical forests; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1160 - Pathology;
Goals / Objectives
In the past few years we have made some chemical and biological observations on several rainforest microbes that are truly unique and have a marked potential in expanding our understanding of how certain microbes in the rainforest may compete and interact within the confines of their environment by utilizing volatile and other chemical defense mechanisms. The organisms making these volatiles are endophytic fungi, belonging to the genus Muscodor, and as such are causing no overt damage to their host plant as described above, but live in concert with the host plant. Obviously, these organisms gain access to energy and nutrient sources via the plant host. On the other hand, their contribution to the host remains a mystery. It is assumed that the role of these biologically active compounds exist to protect the host plant by warding off pathogens or other harmful agents such as insects or herbivores. At the present time, however, no evidence exists for the actual in vivo production of any of these compounds in the microbe-host interaction, thus the interaction remains entirely undefined. However, can these observations be used for the benefit of agriculture? Project Goals Goal A. To more fully establish the general distribution of Muscodor species in selected forest ecosystems throughout the world, to acquire new isolates of Muscodor, and to specifically learn if Muscodor occurs naturally in the USA. Goal B. To better comprehend the nature of the M. albus plant host relationship, to learn more of the life cycle of Muscodor spp. and to better comprehend its host range Goal C. To isolate and characterize the non volatile substance (s) from Muscodor that possess antimicrobial activities and to ascertain the combination of the minimum number of volatile organic compounds required for maximum antibiotic activity.
Project Methods
Approach We shall use the specific techniques to do this work that are outlined in a comprehensive proposal on this topic that was submitted to the Agricultural Experiment Station. For instance new isolates of the fungus will be obtained by sampling plant tissues in various rainforest locations and then subjecting them to Petri plates containing mature cultures of Muscodor as a specific selection tool. Only isolates of Muscodor will grow on these plates and this technique we have used in the past to acquire isolates of this organism. We now have succeeded in getting a successful infection of Muscodor albus into its natural host and this is a first for this organism. The host is Guazuma and the new isolate of Muscodor does invade tissues of this plant. This will enable us to proceed to learn more of its relationship to its host as described in the proposal. Other bioactive non volatile products will be isolated by standard procedures involving chromatographic methods and bioassay guided fractionation. The end result of the work is visualized as the use of Muscodor in agriculture. Already several patents have issued and the United States government has approved Muscodor for release into the environment. Its planned uses are for soil treatment as well as for treating seeds, fruits, vegetables and other plant parts. Agricutlural implements, buildings and industrial places that may get fungal infestations are also targets for Muscodor treatment applications. This organism is likely to replace harmful products that are currently in use and are not friendly to either people or the environment including methyl bromide.

Progress 10/01/02 to 09/30/12

Outputs
OUTPUTS: Muscodor spp. are newly described endophytic fungi that have been isolated from a variety of tropical trees on five continents. These organisms are characterized as having ropy, and coiled hyphae, are sterile, and can be isolated from the stems and fruits of the plants that in which they live. All members of the genus Muscodor make and exude volatile bioactive substances. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Thus far, three species of this organism have been described and they all have 18S rDNA sequence similarities in the 96-99% range. Isolate 620 of M. albus seems destined for commercialization and it makes over 25 volatile compounds. Most of these have been identified by GC/MS and their identity confirmed by the acquisition of comparable MS and GC retention data of authentic standards prepared by organic synthesis or from chemical supply houses. Recently at least 8 other new isolates of M. albus have appeared from the jungles of Indonesia and the monsoonal rainforest of the Northern Territory of Australia and the jungles of Bolivia. Muscodor albus has a wide range of microbes that it both inhibits and kills. Microbes that are extremely sensitive to the volatiles of M. albus including Phytium spp., Phytophthora spp., Stachybotrys spp., Rhizoctonia solani, Aspergillus fumigatus, Escherichia coli, Erwinia spp. and Vibrio cholera. No adverse effects of it have been noted in plants or animals. Artificial mixtures of the volatiles nicely mimic the biological effects of the fungus. Uses and applications in agriculture, and industry are being developed for both Muscodor spp and its volatile compounds. Other work on endophytes during the course of the project has also been accomplished. The publication list is shown below and starts at year 2001 to the present.

Publications

  • Mallette, N., Knighton, W.B. Strobel, G.A., Carlson, R.P., and Peyton, B.M. (2012) Resolution of volatile compound profiles from Ascocoryne sarcoides: a comparison by proton transfer reaction and gas chromatography-mass spectrometry. AMB express (in press).
  • Mends, M.T.,Yu, E.,Strobel, G.A., Hassan, S.R. U.,Booth, E., Geary, B., Sears, J., Taatjes,C.A., Hadi, M. (2012) ). An endophytic Nodulisporium sp. producing volatile organic compounds having bioactivity and fuel potential. J. Petrolem and Envir. Biotech. (in press).
  • Strobel, G.A. 2012. Muscodor albus- the anatomy of an important biological discovery. Microbiology Today (in press).
  • Strobel, G.A. 2011. Bioprospecting Endophytic Microbes. Ed B. Ponman, "In Realizing Nature's Potenial: Proceeding of the Wm L. Brown symposium Honoring Dr G Cragg." Mo. Bot. Garden Proc. 159 -179. 192 p.
  • Benham-Pyle, B., Strobel,G., Geary, B., Vargas, P.N., and Strobel, S.A., Vietor, 2011. A novel Hypocrealous genus producing manumycin A. MycoScience (in review).
  • Zhao, K., Penttinen, P., Guan,T., Xiao, J., Chen, Q., Xu, J., Lindstrom, K., Zhang, l., Zhang, X., and Strobel, G., 2011. The diversity and antimicrobial activity of endophytic actinomycetes isolated from medicinal plants in Panxi Plateau, China. Curr Microbiol. 62: 182-190.
  • Verma, V.C., Gond, S.K., Kumar, A., Kharwar, R.N., Boulanger, L.A., and Strobel, G.A. 2011. Endophytic Fungal Flora from Roots and Fruits of an Indian Neem Plant Azadirachta indica A. Juss., and Impact of Culture Media on their Isolation. Indian J. Microbiology. 51: 469-476.
  • Singh, S.,K., Strobel, G.A., Knighton, B., Geary, B., Sears, J., Ezra, D. 2010. An endophytic Phomopsis sp. possessing bioactivity and fuel potential with its volatile organic compounds. Microbial Ecology. 61: 729-739.
  • Strobel, G.A., Singh, S.K., Riyaz-Ul - Hassan, R., Mitchell, A., Geary, B., and Sears. 2011. An Endophytic/Pathogenic Phoma sp. from Creosote Bush Producing Biologically Active Volatile Compounds Having Fuel Potential. FEMS letters 320: 87-94.
  • Kudalkar, P., Strobel, G.A., Riyaz-Ul Hassan, S., Geary, B. and Sears, J. 2011. Muscodor sutura, a novel endophytic fungus with volatile antibiotic activities. Mycoscience (in press).
  • Booth, E., Strobel, G., Knighton, B., Sears, J., Geary, B., and Avci, R. 2011. A rapid column technique for trapping and collecting volatile fungal hydrocarbons. Biotechnology Letters 33:1963-1972. Strobel, G.A. 2011. Muscodor albus and its biological promise. Phytochemistry Reviews 10:165-172.
  • Kharwar, R.N. and Strobel, G.A. 2011. Fungal endophytes an alternative source of bioactive compounds. Natural Products in Pest Management ed. NK Dubey CAB International, Wallingford, England.
  • Kokaew, J., Manoch, L., Worapong, J., Chamswarng, C., Singburaudom, N., Visarathanonth, N., Piasai, O., and Strobel, G. 2012. Coniochaeta ligniaria an endophytic fungus from Baeckea frutescens and its antagonistic effects against plant pathogenic fungi. Thai journal of Agricultural Science. (in press).
  • Hassan, R., Strobel, G.A., Booth. E., Knighton, B., Floerchinger, C. and Sears, J. 2012. Modulation of Volatile Organic Compound Formation in the Mycodiesel producing endophyte- Hypoxylon sp C1-4. Microbiology 158: 465-473. Harper, J., Strobel, G., Arif, A.M., 2012. 1H (3-carbamoyl-quinoxalinium) chloride. Acta Crystallographica Section E 68: 79-80.
  • Strobel, G.A (2012) Genetic diversity of microbial endophytes and their biotechnical applications. In Genomics Applications for the Developing World [Nelson ed]. Springer Co.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: A novel fungal genus is described that produces extremely bioactive volatile organic compounds (VOC's). The initial fungal isolate was discovered as an endophyte in Cinnamomum zeylanicum in a botanical garden in Honduras. This endophytic fungus was named Muscodor albus because of its odor and its white color. This fungus produces a mixture of VOC's that are lethal to a wide variety of plant and human pathogenic fungi and bacteria. It also is effective against nematodes and certain insects. The mixture of VOC's has been analyzed using GC/MS and consists primarily of various alcohols, acids, esters, ketones, and lipids. Final verification of the identity of the VOC's was carried out by using artificial mixtures of the putatively identified compounds and showing that the artificial mixture possessed the identical retention times and mass spectral qualities as those of the fungal derived substances. Artificial mixtures of the available VOC's mimicked some but not all of the biological effects of the fungal VOC's when tested against a wide range of fungal and bacterial pathogens. Other species and isolates of this genus have been found in various tropical forests in Australia, Bolivia, Ecuador, and Thailand. The most recent discovery is Muscodor crispans whose VOCs are active against many plant and human pathogens. Potential applications for "mycofumigation" by members of the Muscodor genus are currently being investigated and include uses for treating plant diseases, buildings, soils, agricultural produce and many more. This report will describe how the fungus was discovered, identified, and found potentially useful to agriculture, medicine and industry. PARTICIPANTS: Participants working on the project included A. Mitchell, undergraduate student; Drs. Hess and Robison, BYU professors; Joe Sears, professional mass spectroscopist, Pasco, WA; and P.N. Vargas, Peruvian botanist. TARGET AUDIENCES: There have been a number of target audiences including those reading the reviews that have been written. Several companies also have been interested in the results including Phillips Environ Products of Belgrade Mt, and Jeneil Biotechin Saukville , Wisc. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The outcomes from this project are numerous and include many publications and patents on the topic. They are listed under publications. In summary, several novel muscodors have been found including M. crispans, M. vitigenus and the newest one is M sutura. Each one is biologically unique and yet related by virtue close gene sequence identity (ITS region of rDNA). Work is underway to commercialize these organisms and their biologically active products. The products are active against a wide array of plant and human pathogens.

Publications

  • Strobel, G.A. 2010. Muscodor spp., endophytes with biological promise. Phytochemistry Reviews. (in press).
  • Mitchell, A.M. Strobel, G.A., Moore, E., Robison, R., and Sears, J. 2010 Volatile antimicrobials from Muscodor crispans. Microbiology 156: 270-277.
  • Mitchell, A.M., Strobel, G.A., Hess, W.M., Vargas, P.N., and Ezra, D. 2008. Muscodor crispans, a novel endophyte from Ananas ananassoides in the Bolivian Amazon. Fungal Diversity. 31: 37-43.
  • Strobel, G.A., Kluck, K., Hess, W.M., Sears, J., Ezra, D. and Vargas, P.N. 2007. Muscodor albus E-6, an endophyte of Guazuma ulmifolia, making volatile antibiotics: isolation, characterization and experimental establishment in the host plant. Microbiology. 153: 2613-2620.
  • Strobel, G.A. 2006. Harnessing endophytes for industrial microbiology. Current Opinions in Microbiology. 9: 240-244.
  • Strobel,G.A. 2006. Muscodor albus and its biological promise. Journal of Industrial Microbiology and Biotechnology. 33: 514:522.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Muscodor crispans (isolate B-23) was described as a new species of Muscodor. It is a deuteromycetous (Mycelia Sterilia) endophytic fungus residing within the stem tissues of Ananas ananassoides, a wild pineapple in the Bolivian Amazon Basin. This strain was characterized by the production of a pinkish felt-like mycelium on potato dextrose agar (PDA) and other media under lighted conditions, but developing a whitish mycelium in the dark. The fungus produces no fruiting structures or spores of any kind when incubated on multiple synthetic or natural media. On PDA and other common laboratory media, its hyphae develop into regular undulating patterns and associated with them are cauliflower-like structures (3.5-14 microns). Analysis of the volatile organic compounds was done by GC/MS and showed that M. crispans primarily produces a number of esters, alcohols, and small molecular weight acids but no naphthalene or azulene derivatives as per other members of this genus. The volatiles possess antibiotic properties making this organism potentially useful in a number of venues. A molecular genetic analysis of the ITS1, 5.8S rDNA, and ITS2 regions showed 100% similarity to Muscodor albus. Muscodor albus, M. roseus and M. vitigenus are each genetically related to the Xylariaceous fungi by virtue of ca. 95% sequence similarity to this group. Justification for the designation of a new species is primarily based on the novel phenotypic characters of isolate B-23 including its peculiar hyphal growth patterns (undulating hyphae), its reddish pigment production in the light, the odd cauliflower-like structures associated with its hyphae, and its unusual gaseous products. In spite of its 100% genetic similarity to the rDNA regions of M. albus, this organism is considered distinct because of the number and kind of its unusual phenotypic characteristics including its ability to inhibit and kill many pathogenic microorganisms. In addition, an isolate of Gliocladium roseum was discovered that makes some of the ingredient components of diesel fuel. It was discovered as an endophyte in the tree ulmo growing in South America and was recovered from a selection plate containing Muscodor albus. Other products form endophytes include a new immunosuppressive agent called Colutellin A and phomodione -an antifungal agent. Endophytes have also been isolated from Azadirachta indica and Catharanthus roseus (L.). Javacinin has been isolated and characterized from Chloridium sp. PARTICIPANTS: Several undergraduate studnets worked on this project from Montana State University and Yale University. Their names appear on the publications that are listed. Partner organizations were Yale University, university of Utah, several institutions in India, and Portland State University TARGET AUDIENCES: There has been an enormous public interest in mycodiesel project with over 20 countries and hundreds of people, magazines, articles, television programs,newspaper articles on this discovery. Please see the new gary strobel web site and Montana State university for all details on this development. PROJECT MODIFICATIONS: We have begun to examine other gas producing fungi for their abilities to make products that are needed and wanted by the general public including high energy compounds, and volatile antibiotics. These technologies all involve the use of Muscodor albus as a selection tool. The techniques and goals of the original project do continue to remain the same but with an expanded base.

Impacts
The results of the work described above have resulted in 6 provisional patents.These patents all relate to the production of myco-diesel by Gliocladium roseum. In the meantime three other patents on products from endophytic microbes have issued this past year.

Publications

  • Hoffman A.M., Strobel, G.A., Hess, W.M., Sovocool, G.W., Grange, A.H., Harper, J.K., Arif, A.M., and Grant, D.M. 2008. Purification, identification and activity of phomodione, a furandione from an endophytic Phomopsis species. Phytochem. 69: 1049-1056.
  • Verma, V.C., Gond, S.K., Kumar, A., Kharwar, R.N. and Strobel, G.A. 2008. Endophytic fungi from root and fruits of Azadirachta indica (Meliaceae). Current Science. 95: 28-33.
  • Ren,Y., Strobel, G.A., Graff, J.C., Jutila, M., Park, S.G., Gosh,S., Teplow,D., Condron, M.,Eric Pang, E., and Hess, W.M., Moore, E. 2008. Colutellin an immunosuppressive peptide from Colletotrichum dematium. Microbiology 154: 1973-1979
  • Kharwar, R.N., Strobel, G. A., and Ezra, D. (2008). The Endophytic Fungal Complex of Catharanthus roseus Current Science 95: 228-233.
  • Strobel, G.A. Spang, S., Kluck, K., Hess, W.M., Sears, J., and Livinghouse, T. (2008). Synergism Among Volatile Organic Compounds Resulting in Increased Antibiosis in Oidium sp. FEMS Lett 283: 140 -145.
  • Strobel, G., Knighton, B., Kluck, K., Ren, Y., Livinghouse, T., Griffen, M., Spakowicz, D., and Sears, J. (2008) The production of mycodiesel hydrocarbons and their derivatives by the endophytic fungus Gliocladium roseum. Microbiology 154: 3319-3328.
  • Mitchell, A.M., Strobel, G.A., Hess, W.M., Vargas, P.N., and Ezra,D. (2008) Muscodor crispans, a novel endophyte from Ananas ananassoides in the Bolivian Amazon. Fungal Diversity. 31: 37-43.
  • Smith, S.A., Tank, D.C.,Boulanger, L.A., and Strobel, G.A. Bioactive endophytes support intensified exploration and conservation PloS 1 biology Published on line August 25, 2008.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: A new isolate of Muscodor albus was obtained from Guazuma ulmifolia growing in the dry forest on the pacific coast of Ecuador. The fungus was characterized by morphological and molecular biological methods. seeds of the host plant were germinated and developed iot seedling plants. These plants could be successfully inoculated with the M. albus E-6 isolate and the organism established itself in it s host. this is the first instance of the successful re introduction of any Muscodor spp. into its host or any plant for that matter. This discovery will enable further investigations into the relationship of M albus with its host at the molecular level. Presently, Muscodor spp. are being developed for a wide variety of agricultural applications by AgraQuest of Davis, California. The bioactive organic compounds found in the volatiles of Muscodor sp. have been used in test plot systems and the results have been positive. In addition, several bioactive endophytic streptomycetes have been isolated and characterized from Nothofagus spp in Patagonia. These Streptomyces spp each have antibiotic properties and several are specific to certain plant pathogenic fungi. Extracts of some of these organisms are now being tested in field conditions for the control of certain plant diseases. In addition, a whole series of bioactive antibiotic producing streptomycetes were isolated and characterized from a series of medicinal plants growing on the Malay peninsula. Finally, the endophytic microbes associated with the neem tree have been isolated and characterized. Several textbooks on topics dealing with biotechnology and endophytes now have chapters contributed by the PI. PARTICIPANTS: Several groups of students at Yale university in the Scott Strobel Hughes Professor program are now involved inthe search for more isolates of Muscodor. in 2007 the group, having 15 students, found at least four new isoaltes of thsi organism and they have characterized them. TARGET AUDIENCES: A DVD video has been produced entitled Jewels of the Jungle and it has been shown nationally on PBS. The film was made with funds provided by the natioanl science foundation. the production,editing and all other aspects of it have been done by the montana state university msater's program in film making in science. It can be seen on the jewels of the jungle web site.

Impacts
At least three patents have issued on varoius aspects relating to the uses of muscodor for agricultural and industrial purposes. Agraquest of Davis California is planning on releasing a product containing muscodor as a replacement for chemical sterilants during this calendar year. Other products involving muscodor will be used for the treatment of fruits in shipment and greenhouse soil treatment. Phillips environmental Productsof Belgrade Montana is already using muscodor a an item used in their WAG bags. Muscodor has also been used to find a Gliocladium isolate that makes diesel fuel and this work is pushing forward with natioanl science foundation funding.

Publications

  • Strobel, G., and Castillo, U. 2007. Sourcing Natural Products from Endophytic Microbes In Bioactive Natural Products: Detection, Isolation and Structural Determination, editors S.M Colegate and R.J Molyneux.Second Edition. CRC Press. Boca Raton.
  • Verma, V.C., Gond, S.K., Kumar, A., Kharwar, R.N., and Strobel, G.A. 2007. The endophytic mycoflora of bark, leaf, and stem tissues of Azadirachta indica A. Juss from Varanasi. Microbial Ecology 54: 119-125.
  • Grimme, E., Zidack, N.K., Strobel, G.A., and Jacobsen, B.J. 2007. Comparison of Muscodor albus volatiles with a biorational mixture for control of seedling diseases of sugar beet and root knot nematode on tomato. Plant Disease 91: 220-225.
  • Strobel, Scott and Strobel, Gary A. 2007. Plant endophytes as a platform for discovery-based undergraduate science education. Nature Chemical Biology 3: 356-359.
  • Strobel, G.A., Kluck, K., Hess, W.M., Sears, J., Ezra, D. and Vargas, P.N. 2007. Muscodor albus E6, an endophyte of Guazuma ulmifolia, making volatile antibiotics: isolation,characterization and experimental establishment in the host plant. Microbiology.153: 2613- 2620.
  • Strobel, G.A. 2007. Biotechnology for Beginners ed. R. Renneberg and A.Demain. Chapter on M. albus. Elsevier, N.Y. ed
  • Strobel, Gary. 2007. Plant Associated Microorganisms (Endophytes) as a New Source of Bioactive Natural Products in Medical Plant Biotechnology ed. O Kayser & W.J. Quax Wiley VCH Verlag, Weinheim, Germany.
  • Zin, N.M., Sarmin, N.I.M., Ghadin,N., Basri, D.F., Sidik, N. M., Hess, W.M. and Strobel, G.A. 2007.Bioactive Endophytic Streptomycetes from the Malay Peninsula. FEMS Microbiol. Lett. 274: 83-88.


Progress 01/01/06 to 12/31/06

Outputs
Munumbicins E -4 and E-5 were isolated from an endophytic streptomycete associated with snakevine, a plant native to Australia. These new compounds possessed a wide spectrum of biological activity including pathogenic fungi and bacteria. The compounds were shown to be novel in their chemical structure and have been demonstrated to be useful as antimicrobial agents. Muscodor albus, an endophytic fungus, makes volatile antibiotics. A synthetic mixture of many of these compounds, in the proportion that they occur in the fungus, very nicely mimics the biological activity of the fungal volatiles. Placement of the mixture in soil not only exerted a positive effect in controlling fungal diseases, but nematode caused diseases as well. A search for novel bioactive endophytes was focused in Patagonia. From mainly representative members of the plant family - Fagaceae, we were able to recover a number of unique endophytic streptomycetes from Southern Patagonia. Interestingly, No endophytic streptomycetes were obtained from any plant species (non fagaceae) studied in Northern Patagonia. However, from several plant species, each isolate, as studied by scanning electron microscopy (SEM), has small hyphae, some produce typical barrel shaped spores in culture and each has some characteristic hyphal surface structures. Interestingly, although none has any detectable antibacterial killing properties, each had demonstrable killing activity against one or more pathogenic fungi including organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis and Rhizoctonia solani. Molecular biological studies on the 16 S r DNA of each isolate revealed that they are distinct from all other genetic accessions of streptomyectes in GenBank. However, isolate C-2 from Chiliotrichum diffusum (Compositae) is identical, in all respects, to isolate C-4 obtained from Misodendrum punctulatum (Loranthaceae). Endophytic streptomycetes represent a novel source of biologically active microorganisms. A discussion is presented indicating that Gondwanaland plants may serve as a reservoir for bioactive endophytic streptomycetes.

Impacts
Already several rainforest fungi or other microbes and or their products have been patented and licensed to several US companies. This includes the pseudomycins from Pseudomonas syringae for the control of Sigatoka diseae in bananas, Muscodor albus for treating human and animal wastes, and others that are in progress including the munumbicins that are described in the references in this report. The discovery of novel bioactive streptomycetes is also proceeding along the avenues of both patenting and publication.

Publications

  • Castillo, U., Strobel, G.A. 2006. Munumbicins: novel broad spectrum antibiotics from Streptomyces 3052. Fed European Microbiol.Soc.Lett. 255: 296-300.
  • Strobel,G.A. 2006. Muscodor albus and its biological promise. Journal of Industrial Microbiology and Biotechnology. 33: 514:522.
  • Strobel, G.A. 2006. Harnessing endophytes for industrial microbiology. Current Opinions in Microbiology. 9: 240-244.
  • Strobel, G.A. 2006. Socotra, the Galapagos of the Arabic Sea. The Explorers Journal. 84 : 10-15.
  • Strobel, G., and Castillo, U. 2006. Sourcing Natural Products from Endophytic Microbes In Bioactive Natural Products: Detection, Isolation and Structural Determination: Second Edition
  • Castillo,U.F., Browne,L., Strobel, G.A. 2007. Biologically active endophytic streptomycetes from Nothofagus spp. and other plants in Patagonia. Microbial Ecology 53: 12-19.


Progress 01/01/05 to 12/31/05

Outputs
Soils of all types and locations have generally served as the major sources of streptomyceteous bacteria. These organisms supply about eighty percent of the antibiotics in the world. Now, it is realized that Streptomyces spp. can exist as endophytes within the interstices of some higher plants. While it is sometimes possible to isolate one or two different streptomycetes from certain plants, most plants are free of these organisms. However, the snakevine (Kennedia nigriscans) of the Northern Territory of Australia has yielded at least 39 different endophytic streptomycetes. Most of these isolates possessed no detectable antibiotic properties while at least 7 had impressive antibacterial and antifungal activities. Examination by regular scanning electron microscopy (SEM) as well as environmental scanning electron microcopy (ESEM) revealed unusual patterns, structures and features of the spores and hyphae of the endophytic streptomycetes. For instance, for the first time, it has become obvious, as revealed by ESEM, that extremely fine hairs exist on the spores and hyphae of some endophytic streptomycetes. These were not evident in regular SEM. It is obvious that Both SEM and ESEM can be effectively used as tools in identification and the elucidation of the biology of these organisms. In addition, it was obvious that unusual colony morphology, observed with the naked eye can very easily be used to distinguish some of these isolates since characteristic donut and pseudo-horn shaped colonies did appear in culture. Muscodor albus is an endophytic fungus of tropical tree species that produces volatile organic compounds that have antibiotic activity. A new isolate of this organism has been obtained from a small, unidentified vine, generally used by the indigenous people of the Tesso Nilo region in Central Sumatra, Indonesia, to treat snakebites. This unique organism produces a number of volatile antibiotics not previously observed in other M. albus isolates including tetrohydofuran, 2-methyl furan; 2-butanone; aciphyllene, and large amounts of an unusual azulene derivative. Noticeably absent from the volatile antibiotic mixture was 1-butanol, 3-methyl-. Scanning electron micrographs of the organism showed a unique fishnet-like deposit of what appears to be a biopolymer covering the hyphae. The DNA partial sequence data showed ninety nine identity to the original M. albus strain. In addition, an artificial mixture of some of the volatile antibiotics produced by this new isolate generally mimicked the inhibitory as well as lethal effects of the fungal volatile antibiotics on the test microorganisms. One of the most sensitive test fungi was Stachybotrys chartarum, an organism associated with the toxic mold syndrome of buildings. Fungi belonging to the Muscodor genus regularly appear in tropical rainforests throughout the world and these isolates appear to have chemical, biological, and structural characteristics that make them potentially useful in medicine, agricultural and industrial applications. In 2005, a US patent was issued on M. albus which is 6,911,338.Also issued was 6, 926,892 on oocydin A and serrotamolide, antifungal agents.

Impacts
Muscodor albus has been patented and licensed to AgraQuest of Davis Calif. The EPA has approved this organism as a soil treatment and as a way to treat fruits and vegetables in shipment and storage. It will be on the market in 2006/2007. In additions the organism already is a product with Phillips Environmental Products of Belgrade Mt. It is being used to treat human wastes. A new patent on waste treatment using this organism for human and animal wastes will issue in 2006. The basis for the activity of this organism is related to its ability to produce volatile antibiotics. New antibiotics have been isolated form endophytic streptomycetes and they are both patented, published, and soon to be licensed. A film on how we isolate endophytes was released in 2005 and it will show on public TV around the country in 2006. The support for the film production was NSF. The film is called Jewels of the Jungle. Information on it can be accessed at: www.jewelsofthejungleweb.com

Publications

  • Strobel, G., Daisy, B., and Castillo, U. 2005. The Biological Promise of Microbial Endophytes and Their Natural Products. Plant Pathology Journal. 4: 161-176.
  • Atmosukarto, I., Castillo, U., Hess, W.M., Sears, J., and Strobel, G. 2005. Isolation and characterization of Muscodor albus I-41.3s, a volatile antibiotic producing fungus. Plant Science. 169: 854-861.
  • Strobel, G.A. Daisy, B.H., Castillo, U., and Harper, J. 2005. Natural Products ed. L Zhang and A. Demain. Natural Products from Rainforest Endophytes. Humana Press Book Chapter. p.329-352.


Progress 01/01/04 to 12/31/04

Outputs
We have established the techniques and methods for the application of proton transfer mass spectroscopy for the on line and immediate detection of volatile emissions from Muscodor albus in culture and in soil conditions. This technology adds immediate insight into the detection and quantitation of antibiotic volatiles being emitted by this biocontrol fungus. In addition, we have found and describe 7 novel isolates of M. albus from various parts of the Northern Territory, Australia. Each of these isolates is biologically active and capable of killing other important microbes including bacteria and plant pathogenic fungi. Other searches have revealed similar microbes in the jungles of Venezuela. Muscodor albus appears to be a widely distributed endophytic microbe and each isolate, while genetically related to the original 620 isolate does make volatiles that are distinctly different form the original 620 wild type organism. The organisms are being developed for agricultural commercialization by Agraquest of Davis, California. In addition, we have isolated and characterized a novel endophytic streptomycete that produces an anti- pythium peptide antibiotic-coronamycin. Coronamycin also has phenomenal biological activity against the malarial parasite. Hundreds of other endophytic streptomycetes have been isolated and are being characterized. A brief survey was donme of the endopyhtes in the endemic plants of the Yemeni island of Socotra in the Arabic Sea and many microbves were found having biological actrivites.

Impacts
We anticip[ate that M albus will be on the market as an agricultural product within by the end of 2005. It will be used to decontaminate soils, cut flowers, fruit and grain in shipment and storage. Currently it is on a limited market for use in the decontamiantion of human wastes by Phillips Environmetal Products of Belgrade Mt. We also anticipate that the new antibiotcs munumbicins, kakadumycins will eventually find use in plant oriented agriculture for the treatment of plant diseases. The new antioxidants isopestacin and pestacin will also have a place in the market since they are more active than trolox as antioxidants and they possess no cytotoxicity.These compounds have recenly been flund to have anticancer activity. teh new compound-coronamycin will also be licensed this year to a biotech company.

Publications

  • Ezra, D., Jasper, J., Rogers, T., Knighton, B., Grimsrud, E., and Strobel, G.A. 2004. Proton-transfer reaction- mass spectroscopy as a technique to measure volatile emissions of Muscodor albus. Plant Science. 166: 1471-1477.
  • Strobel,G., Ezra, D., Castilo, U., and Hess, W.M. 2004. A question concerning the identity of Streptomyces MSU 2110. Microbiology. 150: 3094-3096.
  • Ezra, D., Hess, W.M. and Strobel,G.A. 2004. Unique wild type endophytic isolates of Muscodor albus, a volatile antibiotic producing fungus. Microbiology 150:4023-4031.
  • Browne, L., Castillo, U., Ford, E., Ezra, D., Suliman, A. S., Hess, W.M., and Strobel, G.A. 2004. Some endophytic microorgamisms from Socotra Island( Republic of Yemen).Yemeni J. of Science. 6: 47-57.
  • Strobel,G. A. 2004. Microbial Diversity and Bioprospecting, ed. Alan T. Bull, ASM Press, Washington, D.C., 2004. 496p. Quarterly Review of Biology. 79: 309-310.
  • Jacobsen, B.J. N.K. Zidack, G.A. Strobel, D. Ezra, E. Grimme and A.M. Stinson. 2004. Mycofumigation with Muscodor albus for control of soilborne microorganisms. IOBC wprs Bulletin Vol 27: 103-113.
  • Ezra, D., Castillo, U., Strobel, G.,. Hess, W.M., Porter, H., Jensen, J., Condron, M., Teplow,D.B., Sears, J, Maranta, M., Hunter, M., Weber, B., and Yaver, D. 2004. Coronamycins, peptide antibiotics produced by a verticillated Streptomyces sp. (MSU-2110) endophytic on Monstera sp. Microbiology. 150: 785-793.
  • Strobel, G.A. Daisy, B.H., Castillo, U., and Harper, J. 2004. Natural Products from Endophytic Microorganisms. J. Nat. Prod. 67: 257-268.


Progress 01/01/03 to 12/31/03

Outputs
A novel antioxidant compound which is a a 1,3 -dihydrobenzofuran was isolated and completely characterized from the endophytic fungus Pestalotiopsis microspora obtained as an endophyte from Papua New Guinea. The mode of action was also ascertained and it is related to a reactive carbon at position 1 in the molecule. In addition, the composition of substate was shown to control the nature of the volatile products being emitted from Muscodor albus. M. albus was also discovered and reported from Thailand. M. albus has been shown to be effective as a mycofumigant in extensive greenhouse trials. Kakadumycin A and B were found and reported as novel antibiotics from an endophytic streptomycete obtained from fern-leafed Grevillea in Australia. These compounds were especially active against the malaria parasite, and many Gram positive pathogenic bacteria. Acremostatins AB and C were isoalted from Acremonium sp. Gliocladium sp was found as an endophyte in Chile and it also is a producer of volatile antibiotics including [8] annulene. Finally, an entirely new method has been devised to aid in the structural determination of organic molecules and this is solid state NMR. The technique was devised using ambuic acid as a model compound. Ambuic acid is a novel antifungal product that we isolated from Pestalotiopsis microspora obtained as an endophyte from many rainforest locations.

Impacts
We anticip[ate that M albus will be on the market as an agricultural product within two years. It will be used to decontaminate soils, cut flowers, fruit and grain in shipment and storage. Currently it is on a limited market for use in the decontamiantion of human wastes by Phillips Environmetal Products of Belgrade Mt. We also anticipate that the new antibiotcs munumbicins, kakadumycins will eventually find use in plant oriented agriculture for the treatment of plant diseases. The new antioxidants isopestacin and pestacin will also have a place in the market since they are more active than trolox as antioxidants and they possess no cytotoxicity.

Publications

  • Harper, J.K., Barich, D.H., Hu, J.Z., Strobel, G.A., and Grant, D.M. 2003. Stereochemical Analysis by Solid-State NMR. J. Org. Chem. 68: 4609-4614.
  • Harper, J.K., Ford, E.J., Strobel, G.A., Arif, A., Grant, D.M.,et al. 2003. Pestacin from Pestalotiopsis microspora possessing antioxidant and antimycotic activities. Tetrahedron 59: 2471-2476.
  • Saxena, S., and Strobel,G.. 2003. Green chemicals; prospects and future in designing new drugs In Green Chemistry. Ed. R. Sanghi and M.M. Srivastava. Narosa Publ. House. New Dehli, India. p. 281-293.
  • Ezra, D., and Strobel, G.A. 2003. Substrate affects the bioactivity of volatile antimicrobials emitted by Muscodor albus. Plant Science 165: 1229-1238.
  • Stinson, A.M., Zidack, N.K., Strobel, G.A., and Jacobsen, B.J. 2003. Effect of mycofumigation with Muscodor albus and Muscodor roseus on seedling diseases of sugarbeet and Verticillium wilt of eggplant. Plant Disease. 87: 1349-1354.
  • Strobel, G. 2003. Endophytes as sources of bioactive products. Microbes and Infection. 5: 535-544. .
  • Castillo, U., Harper, J.K., Strobel, G.A., Sears, J.et al. 2003 Kakadumycins, novel antibiotics from Streptomyces sp. NRRL 30566, an endophyte of Grevillea pteridifolia FEMS Letters. 224: 183-190. Strobel, G.A. and Daisy, B., 2003. Bioprospecting for microbial endophytes and their naturalproducts. Microbiology and Molecular Biology Reviews 67: 491-502..
  • Sopalun, K., Strobel, G.A., Hess, W.M., and Worapong, J. 2003. A recordof Muscodor albus, an endophyte from Myristica fragrans, in Thailand, Mycotaxon.88: 239-247
  • Stinson, M., Ezra, D., Hess W.M., Sears J., and Strobel, G.A. 2003. An endophytic Gliocladium sp. of Eucryphia cordifolia producing selective volatile antimicrobial compounds. Plant Science 165: 913-922.
  • Castillo, U., Ezra, D., Strobel, G., and Hess, W.M. 2003. Use of ESEM/FEG to study newly discovered endophytic fungi. Scanning ( J of Scanning Microscopies) 25: 84.
  • Worapong, J., Inthararaungsorn, S., Strobel, G.A., and Hess, W.M. 2003. A new record of Pestalotiopsis theae, existing as an endophyte on Cinnamomum iners in Thailand. Mycotaxon 88: 365-375.


Progress 01/01/02 to 12/31/02

Outputs
Two novel antioxidant substances have been isolated from the endophytic fungus Pestalotiopsis microspora. These compounds are psetacin and isopestacin and they have in vivo and invitro activity. Additionally, a totally novel fungus - Muscodor vitigenus that makes biologically active amounts of naphthalene has been isolated and totally characterized. A whole new family of peptide antibiotics has been isolated and characterized.These compounds are active against plant and human pathogenic fungi and bacteria. Muscodor albus has been studied formthe standpoint of its usefullness in agriculture, industry and forestry.

Impacts
many of the inventions and discoveries have been patented and licensed to interested companies, including the M albus M roseus fungi for use in treating human wastes and in treating infested soils. The antioxidants have been provisionally patented and we are in the process of licensing HMV on their discovery. The new antibiotcs have been provisionally patented and we expect to license a major biotech firm so that these may be developed for human and agricultural use.

Publications

  • Worapong, J., Strobel, G.A., Daisy, B., Castillo,U., Baird, G., and Hess, W.M. 2002. Muscodorroseus anna. nov. an endophyte from Grevillea pteridifolia. Mycotaxon 81: 463-475.
  • Strobel, G.A. 2002. Microbial gifts from the rainforest. Can. J. Phytopath. 24:14-20.
  • Worapong, J Ford, E., Strobel, G., and Hess, W. M., 2002. UV- light induced conversion of Pestalotiopsis microspora to mutant biotypes with multiple conidial forms. Fungal Diversity 9:179-194.
  • Strobel, G.A. 2002. Rainforest endophytes and bioactive products. Critical Reviews in Biotechnology. 22: 315-333.
  • Castillo, U, Strobel, G.A., Ford, E.J.,. Hess, W.M., Porter, H., Jensen, J.B., Albert, H.,Robison, R., Condron, M.A., Teplow, D.B., Stevens, D., and Yaver, D. 2002.Munumbicins, wide spectrum antibiotics produced by Streptomyces munumbi, endophytic on Kennedia nigriscans. Microbiology 148: 2675-2685.
  • Daisy, B., Strobel, G., Ezra, D., Castillo, U., Baird, G., and Hess, W.M. 2002.Muscodor vitigenus sp.nov. an endophyte from Paullinia paullinoides. Mycotaxon 84: 39-50.
  • Daisy, B.H., Gary A. Strobel, G.A., Castillo,U., Sears, J., Weaver, D.K., and Runyon, J.B. 2002. Naphthalene production by Muscodor vitigenus, a novel endophytic fungus. Microbiology 148: 3737-3741.
  • Strobel, G.A. 2002. Collecting Biological Materials. Science. 297: 519-520.
  • Degenkolb, T., Heinze, S., Schlegel, B., Strobel, G., and Grafe, U. 2002. Formation of new lipoaminopeptides, acremostatins A,B, and C, by co-cultivation of Acremonium sp. Tbp5 and Mycogone rosea DSM 12973. Biosci. Biotechnol. Biochem. 66: 883-886.
  • Strobel, G.A. 2002. Useful Products from Rainforest Microorganisms I. endophytes and taxol. Agro-food industry Hi -Tech 13(2): 30-32.
  • Strobel, G.A. 2002. Useful Products from Rainforest Microorganisms II. unique bioactive products from endophytes. Agro-food Industry Hi - Tech 13: 12-17.
  • Strobel, G.A., Ford, E., Worapong, J., Harper, J.K., Arif, A.M., Grant, D.M., Fung, P., and Chau, R. M. W. 2002. Isopestacin, a unique isobenzofuranone from Pestalotiopsis microspora possessing antifungal and antioxidant properties. Phytochem. 60:179-183.


Progress 01/01/01 to 12/31/01

Outputs
The concept of mycofumigation has arisen through the discovery of two newly described endophytic fungi- Muscodor albus and M.roseus. These organisms are the first to be shown to make volatile antibiotics. There are over 25 compounds in the volatile phase made by these organisms. The volatiles have been acquired, synthesized and made into artificial mixtures that are effectivley mimicking the gas effects of these fungi. In collaboration with Drs Jacobsen and Zidak both the fungi and the artifical mixtures have been applied to solid and vermiculite in greenhouse conditions and excellent control of Pythium and Rhizcotonia caused diseases of plants have been achieved. Eventually these endophytic fungi are being considered as replacements for methyl bromide in agricultural settings. Other applications to human waste control are being made of the gas producing fungi. In addition, several new antifungal compounds including ambuic acid, jesterone and hydroxy jesterone have been isolated and characterized from endophytic fungi. A biochemical mechanism for the detoxification of thaxtomin has been described and a totally new method has been developed for the stereochemical determination of chemcial structure. The telomerases of C. albicans also have been described. Other discoveries include new fungal genus from the tepuis of Venezuela and new compounds form endophytic fungi and higher plants. Finally, there has been success in acquiring endophytic streptomycetes from woody plants and these have yielded a whole series of novel bioactive compounds. These compounds are active against a representative group of plant pathogenic oomycetes , ascomycetes and basidiomyetes.

Impacts
This MAES project already has and will continue to have multiple impacts on our society by virtue of the development of new knowledge, national and international patents that have and will be licensed to pharmaceutical, agricultural and industrial based companies interested in novel applications to their businesses. This knowledge will be generated from a myriad of projects dealing with the biology of the associations of microbes with plants.

Publications

  • Li, J.Y., Harper, J.K., Grant, D.M., Tombe, B., Bashyal, B., Hess.W.M., and Strobel,G.A. 2001.Ambuic acid, a highly functionalized cyclohexenone with bioactivity from Pestalotiopsis spp. and Monochaetia sp. Phytochem. 56: 463-468.
  • Strobel, G.A., Hess,W.M., Baird, G., Ford, E., Li, J.Y., and Sidhu, R.S. 2001. Stegolerium kukenani gen. nov. an endopyhtic, taxol producing fungus from the Roraima and Kukenan Tepuis of Venezuela. Mycotaxon 78: 353-361.
  • Acuna, I.A., Strobel, G.A., Jacobsen, B.J., and Corsini, D.L. 2001. Glucose conjugation as a mechanism of resistance to thaxtomin A in potatoes. Plant Science 161:77-88.
  • Shrestha, K., Strobel, G.A., Prakash, S., and Gewali, M. 2001. Evidence for paclitaxel from three new endophytic fungi of Himalayan yew of Nepal. Planta Medica 67: 374 -376.
  • Li, J.Y., and Strobel, G.A. 2001. Jesterone and hydroxy-jesterone antioomycete cyclohexenenone epoxides from the endophytic fungus- Pestalotiopsis jesteri. Phytochemistry 57: 261-265.


Progress 01/01/00 to 12/31/00

Outputs
Biological strategies for the control of spotted knapweed , using pathogenic fungi, have been undertaken. Two fungi, Rhizoctonia solani in combination with Fusarium avenaceum can be added to soils as inoculum on sterilized barley seed. These organisms bring about a 95-100% reduction in the subsequent stand of knapweed. Tests done in both the field and greenhouse confirm this finding. Cryptocin, is a new natural product that has outstanding activity against Pyricularia oryzae. Its structure, and bioactivity has been determined. Three new cytochlasins have been isolated and their structures determined by NMR and mass spectral methods. These compounds are from rainforest endophytic fungi. In addition, other new bioactive compounds have been isolated and their structures determined. They are active against the oomyceteous fungi and they include ambuic acid, jesterone, and hydroxy-jesterone. The x-ray structure of the bioactive product terrein has also been determined. The fate of the toxin, thaxtomin, in potatoes has been determined and its metabolism is related to the ability of the plant to convert it to a glycoside. The maculosin relatives have been synthesized and tested for activity against spotted knapweed. New endophytes have been found and the influence of host compounds on their sexual stage have been studied.

Impacts
The discovery of both new natural products that have biological activity as well as new organisms that produce them is of some economic as well as environmental importance. Al of the new compounds that we have discovered are patented and licensed to US companies. The organsims that we have found for biological control purposes are being considered by Montana companies for sale.

Publications

  • Strobel, G.A. 2000. "Taxol" in Encyclopedia of Life Sciences. Macmillan Press. London.
  • Bobylev, M.M., Bobyleva, L.I., and Strobel, G.A. 2000. Effects of synthetic congers of the natural product phytotoxins- maculosin- 1 and -2 on growth of wheat coleptiles ( T. aestivum L.) International Conference on Biological Control, Bozeman, Mt 1999.
  • Metz, A., Haddad, A., Worapong, J., Long, D., Ford, E., Hess,W.M., and Strobel, G.A. 2000. Induction of the sexual stage of Pestalotiopsis microspora, a taxol producing fungus. Microbiol. 146: 2079-2089..
  • Wagenaar, M., Corwin, J., Strobel, G.A., and Clardy, J. 2000. Three new chytochalasins produced by an endophytic fungus in the genus cladiella. J.Nat. Prod.63: 1692-1695.
  • Strobel, G.A., Ford, E., Worapong, J., and Hess, W.M. 2000. Pestalotiopsis jesterii sp. nov. an endophyte from Fragraea bodeni from the southern highlands of Papua New Guinea. Mycotaxon. 76: 257-266.
  • Li, J., Strobel, G.A. Harper, J., Lobkovsky, E., and Clardy, J. 2000. Cryptocin, a potent tetramic acid antimycotic from the endophytic fungus- Cryptosporiopsis quercina. Org Lett. 2: 767-770.
  • Acuna, I.A., Strobel, G.A., Jacobsen, B.J., and Corsini, D.L. 2000. Glucose conjugation as a mechanism of resistance to thaxtomin A in potatoes. Plant Science (in press).
  • Shrestha, K., Strobel, G.A., Prakash, S., and Gewali, M. 2000. Evidence for paclitaxel from three new endophytic fungi of Himalayan yew of Nepal. Planta Medica 66: 1-2.
  • Bobylev, M. M., Bobyleva L. I., Cutler H. G., Cutler S. J., and Strobel G. A. 1999. Growth regulating activity of maculosin analogs in the etiolated wheat coleoptile bioassay (Triticum aestivum L. cv. Wakeland). PGRSA Quarterly. 27(4), 105-118.
  • Li, J.Y., and Strobel, G.A. 2000. Jesterone and hydroxy-jesterone antioomycete cyclohexenenone epoxides from the endophytic fungus- Pestalotiopsis jesteri. Phytochemistry (in press).
  • Harper, J., Arif, A.M., Li, J.Y., Strobel, G.A., Grant, D.M. 2000. Terrein. Acta. Crystallographica. C56, e570-e571.
  • Li, J.Y., Harper, J.K., Grant, D.M., Tombe, B., Bashyal, B., Hess.W.M., and Strobel, G.A. 2000. Ambuic acid, a highly functionalized cyclohexenone with bioactivity from Pestalotiopsis spp. and Monochaetia sp. Phytochem. (in press).


Progress 01/01/99 to 12/31/99

Outputs
Various biological control measures were investigated for such agricultural pests as spotted knapweed and several plant diseases caused by pathogenic oomycetes. Both Fusarium avenaceum and Rhizoctonia solani caused seed deacy and seedling blight of emerging knapweed seedlings. Field tests gave a positive indication that, if properly formulated, these fungi have the potential to be used in a biological control scheme for spotted knapweed. More field and greenhouse testing is underway. Another potentially useful microbe for biological control of oomycetous fungal diseases is a specific strain of S. marscens which produces a novel chlorinated macrocyclic lactone that has potent antioomyceteous activity. Both the compound as well as the bacterium, are in extensive field, greenhouse and lab testing schemes. In addition, a novel antifungal agent called cryptocandin has been discovered. It has potent activity against several economically important fungi. Several new fungal genera and species have been described from various rain forest ecosystems. These fungi are producing taxol, another antioomyceteous compound that has implications for cancer treatment. Sphaeric acid, anovel antifungal agent has been described from Sphaeropsis sp. and a new anti-leafy spurge compound produced by Septoria sp. is described. In the meantime, many chemical derivatives of maculosin have been prepared and tested against spotted knapweed with some success.

Impacts
Several biological systems in this station project are being developed for use in agriculture. These systems have the potential to be both effective and environmentally safe. One is for the biological control of spotted knapweed and the other is the biolgical control of pythium, phytophthora and aphanomyces. In the process of developing these agents for agriculture, we also test our newly insolated microbes for their medicinal qualitities.

Publications

  • Wilkinson,R.A., Strobel, G.A., and Stierle, A., 1999. Sphaeric acid, a new succinic acid derivative from Sphaeropsis sp. J. Nat. Prod. 62: 358-360.
  • Strobel, G., J.Y., Sugawana, F., Koshino, H., Harper, J., and Hess, W. M. 1999. Oocydin A, a chlorinated macrocyclic lactone with potent anti-oomycete activity from Serratia marcesens. Microbiol 145: 3557-3564.
  • Bobylev, M.M., Bobyleva, L. I., and Strobel,G.A. 1999. Natural products containing phenylalanine as potent herbicides in Biological Active Natural Products: ed. H.G. and S.J. Cutler CRC Press Boca Raton Florida 299p.
  • Bashyal, B., Li, J.Y., Strobel, G.A., Hess,W.M. 1999. Seimatoantlerium nepalense, an endophytic taxol producing coelomycete from Himalayan yew (Taxus wallachiana). Mycotaxon72: 33-42.
  • Sugawara, F., Uzawa, J., Esumi, Y., Suzuki, M. Yoshida, S., Strobel, G. Steiner, J.L. and Clardy, J. 1998. Phytotoxins from the Septoria spp. Plant Pathogenic Fungus on Leafy Spurge. Biosci. Biotech. Biochem. 62: 638-642.
  • Kim, S-U, Strobel, G., Ford, E. 1999. Screening of total producing endophytic fungi: from Ginko biloba and Taxus cuspidata in Korea. Agric. Chem. Biotechnol. 42: 97-99.
  • Strobel, G.A., Ford, E., Li, J.Y., Sears, J., Sidhu, R.S. and Hess, W.M. 1999. Seimatoantlerium tepuiense gen. nov. A unique epiphytic fungus producing taxol from the Venezuelan Guayana. System.Appl. Microbiol 22: 426-433.
  • Strobel,G.A., Miller,R.V., Miller,C., Condron,M., Teplow, D.B., and Hess, W.M., 1999. Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina Microbiol. 145: 1919-1926.


Progress 01/01/98 to 12/31/98

Outputs
Biological control measures for Montana's major weed- spotted knapweed are being tested, developed and established. A rapid method for the testing of any potential fungal pathogen of knapweed involves an agar plate test utilizing water agar, the potential pathogen and knapweed seeds. Results, that are statistically well correlated with greenhouse and field trials, appear after two weeks. Formulation, of the biocontrol fungi with extremely low levels of pesticides and growth hormones have been successfully tried. New endophytic /plant relationships have been discovered. Periconia sp, an endophyte of Torreya sp. has been demonstrated to produce taxol in an inducible manner. Extracts of the host plant facilitate enhanced production of taxol. Other naturally occurring compounds, such as p-hydroxybenzoic acid act at micromolar levels to induce taxol formation in this fungus. This appears to be the first report of this phenomenon in fungi. Taxol has also been found in Cornylus, the first report for an angiosperm. Using fluorescence imaging spectroscopy it has been possible to demonstrate toxin induced damage on leaves well before any visible symptoms appear suggesting that this method will be useful as an important tool in predicting and measuring plant loss very early during the course of an epidemic. Genetic transformation of P. microspora has been possible. Surprisingly, the fungus is easily transformed with a hygromycin resistant plasmid. Apparently, the fungus opens the plasmid and then proceeds to add telomeric sequences to either end of the DNA, allowing for the heteologous DNA to independently replicate (free of the fungal chromosomes ). The finding has implications for making artificial human chromosomes since the telomeric sequences are identical to those of humans. Several new biologically active compounds have been isolated and characterized including the antifungal agent ecomycin. More scientific details have also been forthcoming on the pseudomycins.

Impacts
(N/A)

Publications

  • Strobel, G.A. and Long, D. 1998. Endophytic Microbes Embody Pharmaceutical Potential. ASM News 64: 263-268.
  • Sugawara. F., J. Uzawa, Y. Esumi, M. Suzuki, S. Yoshida, G.A. Strobel, J.L. Steiner, and J. Clardy. 1998. Phytotoxins from the Septoria spp. Plant Pathogenic Fungus on Leafy Spurge. Biosci. Biotech. Biochem. 62:638-642.
  • Strobel, G.A. 1998. Biology in Pictures. Chemistry & Biol. 8:295.
  • Strobel, G.A. 1998. Fungal Endophytes. Proc. Of the 1997 Biotechnology of Microbial Products - Developments in Ind. Microbiology 35: 69-71.
  • Martinez-Miller, C., R.V. Miller, D. Garton-Kinney, B. Redgrave, J. Sears, M. Contron, D. Teplow, and G.A. Strobel. 1998. Ecomycins, unique antimycotics form Pseudomonas viridiflava. J. Applied Microbiology 84:937-944.
  • Long, D.M., E.D. Smidmansky, A.J. Archer, and G.A. Strobel. 1998. In vivo additon of telomeric repeats to foreign DNA generates chromosomal DNAs in the taxol-producing fungus Pestalotiopsis microspora. Fungal Genetics & Biol. 24: 335-344.
  • Li, J.Y., R.S. Sidhu, E. Ford, W.M. Hess, and G.A. Strobel. 1998. The induction of taxol production in the endophytic fungus - Periconia sp. from Torreya grandifolia. J. Ind. Microbiol. 20:259-264.
  • Li, J.Y., R.S. Sidhu, A. Bollon, and G.A. Strobel. 1998. The stimulation of taxol production in liquid cultures of Pestalotiopsis microspora Ne32. Mycol. Res. 102: 461-464.
  • Hoffman, A., W. Khan, J. Worapong, G.A. Strobel, D. Griffin, B. Arbogast, D. Borofsky, R.B. Boone, L. Ning, P. Zheng, and L. Daley. 1998. Bioprospecting for taxol in Angiosperm plant extracts. Spectroscopy 13:22-32.
  • Bowyer, W.J., L. Ning, L.S. Daley, G.A. Strobel, G.E. Edwards, and J. Callis. 1998. In vivo fluorescent imaging for detection of damage to leaves by fungal phytotoxins. Applied Spectroscopy 13:36-44.
  • Strobel, G.A., and W.M. Hess. 1996. A scanning electron microscopy study of taxus leaves as related to taxonomy. Scanning microscopy 10:1111-1126. (This paper, although dated 1996 just appeared in November 1998).


Progress 01/01/97 to 12/31/97

Outputs
Biocontrol measures for spotted knapweed have been explored. Two fungi, Fusarium avenaceum 1003, and Rhizoctonia solani show excellent pathogenic effects to the seeds and seedlings of C. mculosa. A lab test has been perfected to assist in the preliminary screening for potential bio control agents of this weed. Both greenhouse and field testing have demonstated the increased efficacy of these biocontrol agents when combined (formulated) with gibberellic acid or picloram. Endophytic fungi from higher plants have been tested for antifungal substances. Both leucinostatin A and its novel leucinostation AB-di-o-gluoside have been found in acremonuim sp. Acremonium is an endophyte of Taxus baccata. Leucinostatin A has outstanding antifungal and anticancer properties. Leaves of Taxus spp. (all spines) the souce of taxol, have been examined by SEM and the leaf pattern of stomates, and papiliform cells can be associated with the specific species from which they originated. Novel imaging spectroscopy, using short near infrared light, has been advised for use in the early detection of plant disease development. Endophytes of Cephalotaxies sp have been isoloated and a Phoma sp procedures antifunal substances. Work on the regulation of taxol production by various endophytic fungi is in progress. Exgenous factors that affect taxol products are being studied.

Impacts
(N/A)

Publications

  • STROBEL, G.A., R. TORCZYNSKI, and A. BOLLON. 1997. ACREMONIUM sp. a Leucinostatin A producing endophyte of European yew - Taxus baccata. Plant Science 128:97-108.
  • STROBEL, G.A. and W.M. HESS. 1997. Glucosylation of the peptide leucinostatin A produced by an endophytic fungus of European yew, may protect the host from leucinostatin toxicity. Chemistry & Biology
  • NING, LI, L.S. DAYLEY, W.J. BOWYER, E.H. PIEPMEIER, G.A. STROBEL, and J.B. CALLIS, 1997. Five novel applications of imaging visible and short near-infrared spectro photometry and fluorometry in the plant sciences - Part II: Non invasive in vivo applications. Spectroscopy
  • LI, J.Y., G.A. STROBEL, W.M. HESS, and E. FORD. 1997. An endophytic fungus from cephalotaxus: Phoma sp. which produces antifungal substances. Microscopy and microanalysis. Vol 3 (Supplement 2)
  • CZEMBOR, E., and G.A. STROBEL. 1997. Limitations of exotic and indigenous isolates of Fusarium avenaceum for the biological control of spotted knapweed - Centaurea maculosa. World Journal of


Progress 01/01/96 to 12/30/96

Outputs
During the past year we have made important studies understanding the role of endophytes in plants. A broad collection of fungal endophytes has been isolated that have the capability of producing the important anticancer drug, taxol. P. microspora an endophyte, pathogen of Florida Torreya was shown to be a toxin producer and a microbe having a significant threat to this rare plant. The structure of each of the major pseudomycins has been completed and these compounds shown to be unique lipopeptides having broad antifungal activities. These drugs are now under development by Eli Lilly Co. Special measurements made on infected and toxin treated leaves show remarkable-measurable effects before visible symptoms can be seen. Important strides have been made in the biocontrol of spotted knapweed using various endemic pathogens such as F. avenacem. Extensive greenhouse tests have demonstrated the proof of principle for further testing of this organism in the field.

Impacts
(N/A)

Publications

  • Lee, J., X. Yang, M. Schwartz, G. Strobel, and J. Clardy. 1996. The relationship between the rarest tree in North America and an endophytic fungus. Chemistry and Biology 2:721-727.
  • Ning, L., L.S. Daley, W.J. Boyer, E.H. Piepmeier, G.A. Strobel, and J.B. Callis.1996. Spectroscopic imaging of water in living plant leaves. Spectroscopy 11:34-41.
  • Stierle, A., D. Stierle, and G. A. Strobel. 1996. The search for taxol producing microorganisms among the endophytic fungi of the Pacific yew, Taxus brevifolia. J. Nat. Products 58:1315.
  • Ning, L., L.S. Daley, W.J. Bowyer, E.H. Piepmeier, G.A. Strobel, and J.B. Callis. 1996. Spectroscopic imaging of water in living plant leaves - Part II Challenges, Uses and Advantages of in vivo absorbance methods for the analysis of biolog
  • Lee, J.C., G.A. Strobel, E. Lobokousky, and J.C. Clardy. 1996. Torreyanic acid: a selectively cytotoxic Quione dimer from the endoyphytic fungus Pestalotiopsis microspora. J. Org. Chem. 61:3232-3233.
  • Strobel, G., X. Yang, J. Sears, R. Kramer, R.S. Sidhu, W.M. Hess. 1996. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiol 142:435-440.


Progress 01/01/95 to 12/30/95

Outputs
Our efforts to learn the structure of pseudomycins have been fruitful. Each of the pseudomycins has a cyclic peptide component with a c-14 fatty acid side chain. The lipodepsipeptides are effective against a wide range of fungal pathogens of both plants and animals. Our taxol work has concentrated on both its formation in trees (yew) and the microbes associated with yews (endophytes). Some of the endophytes make antibiotics as well as taxol. Stimulator of taxol biosynthesis include chlorocholine chloride and other related compounds. We have also examined the cause of the decline of the rarest tree in N. America, the Florida Tonya. The fungus associated with this decline is Pestaliotopis micropora. A fluorescence method to detect plant damage was devised and used in the tonya experiments. We also studied the endophyte relationship between Phoma spp. and Taxus wallachiana. The phytotoxin maculosin in spotted knapweed the fate has also been determined.

Impacts
(N/A)

Publications


    Progress 01/01/94 to 12/30/94

    Outputs
    We have discovered that Septoria cirscii is a potent pathogen of Canada thistle.Greenhouse tests confirmed our field observations. Toxicology studies demonstrated that the fungus is capable of causing disease symptoms by virtue of an extremely potent toxin which we crystallized and identified as nitropropionic acid. This fungus is native to Italy and should be considered for eventual study for introduction to the U.S. In this regard, we have done extensive studies on the pathogenic fusaria of spotted knapweed. Of the fungi studied in greenhouse tests, the Montana isolate 1003 was the most pathogenic as compared to an isolate 365 obtained from central Europe. Many greenhouse tests are underway to evaluate these organisms as potential biocontrol microbes. An examination of factors that may stimulate taxol production in vitro and in vivo showed that a number of plant growth regulators are capable of this phenomenon. Chlorocholine chloride and alar were the best stimulators. Taxol biosynthesis was also studied in virtually all known species of Taxus. Taxol was found in Taxus wood and only a small amount of it was made in the ray parenchyma of the sapwood. Taxol synthesis was greatest in the cambial/phloem region of the plant Taxol has been discovered in a number of endophytic fungi of a wide range of yew species. Some make it to an extent that is 1000 times more than Taxomyces andreanae. This microbe produces two antibiotics that were isolated and crystallized and shown to play a role in the assoc.

    Impacts
    (N/A)

    Publications


      Progress 01/01/93 to 12/30/93

      Outputs
      Work on this plant/microbe project has focused on the biological control of weedy plants with pathogens and their phytotoxic compounds, the involvement of phytotoxins in the plant disease process, and the possibe presence of new bioactive compounds from microbes associated with plants. As examples, we isolated and characterized a whole series of zinniol related componds from a Montana isolate of Alternaria cichorii. New analytical methods for working with phytotoxins have been outlined. We have discovered a novel and exceedingly pathgenic strain of F. avenaceum on spotted knapweed. Nearly all of its phytoxins have been identified. In addition, a whole series of novel triticones. Our most interesting discovery has been a novel fungal organism associated with Pacific yew that makes toxol. The organism if Taxomyces andreanae. Taxol was unequivocally isolated and identified by a series of spectral analyses. This project will continue to be productive especially as new approaches are being made in studying host/microbe relationships.

      Impacts
      (N/A)

      Publications


        Progress 01/01/92 to 12/30/92

        Outputs
        After years of effort we have been able to isolate and characterize a novel set of antimycotic substances produced by a Montana strain of Pseudomonas syringae. These compounds are a series of 4 cyclic peptides that possess several odd amino acids such as hydroxy aspartic acid, a diaminbutyric acid. The compounds have a wide range of antifungal activity including many plant, animal and human fungal pathogens. The compounds were facilitated for antimycotic production by selective transpositional mutagenesis. In addition, a unique system for studying "in vitro" taxol biosynthesis has been established and is being used. Other progress was made on identifying a novel pathogen of spotted knapweed and the isolation of visinic acid amide (antifungal) agent from C. hennigsii. 3 U.S. Patents: 1) A Taxol Bioreactor - filing date 02/92, 2) Microbes Producing Taxol - filing date 04/92, 3) Pseudomycins-novel antimycotics - filing date 12/92.

        Impacts
        (N/A)

        Publications


          Progress 01/01/91 to 12/30/91

          Outputs
          This past year was almost unprecedented in terms of publications from our work on host-parasite relationships. We have isolated and characterized all of the phytotoxins of M. fijiensis causing Black sigatoka disease in banana. These are now being used, in various parts of the world to screen for disease resistance. New disease causing organisms of yellow and purple nutsedge were isolated and some toxins described. Many companies, federal agencies, etc. have inquired about these results especially in a Scientific American article on biological control. Cercosporamide was isolated and characterized as a broad spectrum antibiotic and phytotoxin. Petasol and gigantenone were described as to their mode of action, role in disease and potential usefulness in tissue culture systems.

          Impacts
          (N/A)

          Publications


            Progress 01/01/90 to 12/30/90

            Outputs
            Several novel pathogens of purple and yellow nutsedge have been described and these include Phoma and Ascochyta. Both organisms produce phytotoxins that are currently being characterized. Extensive biological control tests on Dutch elm disease have been conducted in Holland in conjunction with lab work at MSU. The results were generally positive. P. syringae produced at least 2 years' protection in field elms from the effects of artificial C. ulmi infections. Several new bioactive compounds have been found in cultures of Pyrenophora teres. One, pyrenolide A, shows some specificity toward the more susceptible lines of barley and no reaction to the more resistant lines. It might be used to screen for resistance at the tissue culture or cell levels. Extensive studies on the structure and mode of action of the eremophilanes have been undertaken. A comprehensive study relating structure to bioactivity reveals that important oxygen substitution arrangements are needed before the eremophilane can be active. Many other studies on such things as M. fijiensis, V. alboatrum, and other fungal bioactive compounds are underway.

            Impacts
            (N/A)

            Publications


              Progress 01/01/89 to 12/30/89

              Outputs
              We have examined a number of important fungal pathogens from weed species for the production of phytotoxins. A. alternata, on spotted knapweed, produces several toxins including maculosin, some perylene quinnones and tenuzonic acid. Curvulin and its methyl ester is produced by D. indica on purslane. Giganteneone, a novel phytohormone mimic, is produced by D. gigantea. It causes a green island effect in monocots and necrosis in dicots. Several novel phytotoxins were described from A. cassiae on sickle pod. Extensive biocontrol studies on Dutch elm disease as it relates to P. syringae have been conducted under field conditions in Holland with backup lab studies at MSU. Further studies on the potential usefulness of A. rhizogenes have been conducted. Seedling olives, treated with A. rhizogenes, produced more vigorous growth when planted in the field. Also, coinoculation of seedling legumes with Rhizobium produced more nodules than with Rhizobium alone.

              Impacts
              (N/A)

              Publications


                Progress 01/01/88 to 12/30/88

                Outputs
                Several novel toxic ophiobolins were isolated and characterized from Drechslera oryzae. Their bioactivity was determined and plant tissue culture experiments using these compounds as screening agents were initiated. A new class of bioactive compounds was isolated from a number of phytopathogenic fungi including D. tritici on wheat. These compounds are spirocyclic lactams and known as the triticones. A flow cytrometric process was established in order to better assay protoplasts for toxin sensitivity. Maculosin, a host specific toxin of spotted knapweed, was isolated from Alternaria alternata. Several phytotoxins were isolated from D. indica. Factors affecting the root forming activity of Agrobacterium rhizogenes were established. The mode of action of the triticones was determined to be a Michael addition reaction into free thio group.

                Impacts
                (N/A)

                Publications


                  Progress 01/01/87 to 12/30/87

                  Outputs
                  (1) The major ophiobolins (phytotoxins) were isolated and characterized from D. maydis, the causal agent of the Southern corn blight epidemic of 1970. One of these ophiobolins, 6-epi-ophiobolin A (6-epi A), was unequivocally characterized by NMR spectroscopy after ophiobolin I, a novel ophiobolin, was crystallized and structurally determined by x-ray analysis. The 6-epi A possessed extreme host specificity to corn lines bearing tms cytoplasm. Other ophiobolins were also toxic, but did not possess selectivity. Some of these ophiobolins were also isolated from D. sorghicola, D. oryzae and D. heveae. (2) Tryptophol was isolated from D. nodulosum and shown to be phytotoxic. A series of genetic experiments were done on P. syringae in a greenhouse test for the biological control of Dutch elm disease. Transposon mutants were generated via Tn903. An off-mutant and an on-mutant were injected into trees and the bacteria as well as the fungus (challenge) were followed over two years. The mutant making the antibiotic-pseudomicin protected the trees from the effects of Dutch elm disease. (3) Toxin production was studied in A. helianthi.

                  Impacts
                  (N/A)

                  Publications


                    Progress 01/01/86 to 12/30/86

                    Outputs
                    The scalding symptoms of barley caused by R. secalis are associated with severalphytotoxic compounds called the rhynchosporosides. We have been able to synthesize rhynchosporosides by means of organic chemistry. In conjunction with our research on phytotoxins from crop plants we have begun to seriously examine toxins produced by pathogens on weeds infecting field crops worldwide. Knowledge of the structures of these molecules has the potential to serve industry in developing models for more biologically dependable and more selective herbicides. We have worked with fungi attacking grasses and have succeeded in isolating and characterizing several very novel compounds and characterizing them by x-ray analysis. The compounds are bipolaroxin from B. cynodontis, dihydropyrenophorin from D. avenae, and exerohilone from E. holmii. Some of these toxins are now actually being modeled by industry and tested for their herbicidal activity. Studies on Phoma macdonaldi have shown that it produces phytotoxins, one of which is zinniol, which for the first time has been shown to be within any infected plant, in this case sunflower. The toxin preparations are now being used by a commercial enterprise to screen sunflower callus tissue for toxin resistance. We now understand that D. montia when properly inoculated into lodgpole pine trees will cause yellowing, necrosis and eventual death of the tree. The Ri plasmid of A. rhizogenes has been successfully transferred to R.

                    Impacts
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                    Publications


                      Progress 10/01/84 to 09/30/85

                      Outputs
                      We havesucceeded in finding that the plasmid pRK 2013 serves as a vehicle for introducing transposons into certain plant associated pseudomonads. This finding has many practical implications in bacterial genetics, pathology and antibiotic studies. Several follow up studies in each of these areas is planned. A high school student, using this system, received a national award at the National Science Fair in New Orleans, 1985. We have learned that A. rhizogenes has some potential to be useful in agriculture. Olive and almond seedlings inoculated with a mutant of this bacterium put on much better growth than their control counterparts in the Negev Desert in Israel. The Ri plasmid has been successfully introduced into Rhizobium meliloti. The transconjugant now causes more nodulation on its alfalfa host and slightly more N2 fixed. This was true in the lab (growth chamber) but not always in the field. Our efforts continue on phytotoxins as we have learned that dehydrocurvularin is the major phytotoxin component from Alternaria macrospora. Alteichin, a novel perylene quinnone was isolated and crystallized from Alternaria eichorniae and its structure determined by X-ray analysis. The compound is phytotoxic. A novel glycopeptide was isolated form Rhynchosporium secalis. It too is phytotoxic and also acts as to aggllutinate red blood cells of certain types.

                      Impacts
                      (N/A)

                      Publications


                        Progress 01/01/84 to 09/30/84

                        Outputs
                        We have examined, by transposon mutagenesis, the idea that genes for host specificity are carried on the Ri plasmid of Agrobacterium rhizogenes. Using Tn5 we have provided ample evidence, both physical and biological, that genetic information for host specificity is encoded on the Ri (root inducing) plasmid of A. rhizogenes. We have just concluded a study demonstrating the presence of several new phytotoxins associated with the Alternaria blight organism of sunflower. These compounds have been called deoxyradicinin and deoxyradicinol. Deoxyradicinin has been detected in plants infected by A. helianthi suggesting its involvement in the blight symptoms. Both of these toxins arise via an acetate condensation reaction as shown by 13C nmr. In addition, we have demonstrated that zinniol, a common phytotoxin, has properties of a phytohormone in certain cereals. It causes the retention of chlorophyll in barley. This is the first demonstration of a substance that is a phytotoxin as having properties of a cytokinin. It suggests that pathogens of cereals causing green island effects may be producing substances related to zinniol. Studies on weed pathogens have been continued. We are seeking to isolate and extract the broth cultures of these organisms as a means of studying the phytotoxins that they produce. Such phytotoxins may prove useful herbicides, or herbicide models.

                        Impacts
                        (N/A)

                        Publications


                          Progress 07/01/76 to 09/30/83

                          Outputs
                          During the course of this project we examined the possibility that the causal agent of Dutch elm disease, C. ulmi, could be inhibited by another microbe. Members of the group Pseudomonas syringae produce an antibiotic which kills C. ulmi. When P. syringae was placed into elm seedlings it established in the xylem and did not cause a noticeable harmful effect on the elm seedling. Furthermore, the bacteria would grow on elm sap and produce its antibiotic. When bacteria inoculated trees were challenged with C. ulmi, in most cases there was no development of the fungus in the tree - nor did trees show symptoms of the disease. The conditions for optimal growth and production of the antibiotic have been demonstrated, i.e., presence of Fe+++, histidine, and glucose. In addition, we have continued a strong research effort on understanding the genetics of control of antibiotic production by P. syringae. We have found that the plasmid pRK 2013 serves as a transposon in certain strains of P. syringae. Several papers are in the preparation stage on this topic.

                          Impacts
                          (N/A)

                          Publications


                            Progress 01/01/81 to 12/30/81

                            Outputs
                            Phytotoxins of Alternaria sp. are derivatives of radicinin. We established the unequivocal structure of radicinin by X-ray spectroscopy. It was isolated from Alternaria chrysanthemi along with radicinol. Furthermore, both compounds were obtained from plants having lesions caused by A. chrysanthemi. Two new phytotoxic compounds were isolated from Alternaria helianthi on sunflower. They were characterized deoxyradicinic and deoxyradicinol. Phytotoxins were also isolated from Alternaria cineraria, a pathogen of several weed hosts, and also Drechslera turcica. The structure of helminthosporoside, a host specific toxin, has been revised using the new techniques of FAB and 13C spectroscopy. A novel glycolipid was isolated from sugarcane and shown to activate toxin production in Helminthosporium sacchari. Research continued on the nature and effect of Pseudomonas syringae antagonism against the Dutch elm disease fungus. The requirements for Fe+++, histidine and glucose in Pseudomonas syringae cultures for antibiotic production was demonstrated. Our efforts on the involvement of the Hr plasmid in the hairy root disease continue. Studies are underway to determine if "host specificity genes" are carried on the Hr plasmid. Other studies on the genetics of P. syrinage, relative to antibiotic production, are underway.

                            Impacts
                            (N/A)

                            Publications


                              Progress 01/01/80 to 12/30/80

                              Outputs
                              When the toxin binding protein from sugarcane was isolated free of lipids or lipid-like substance it behaved in an anomalous manner, that is its binding activity decreased when protein concentration was increased from 2 mg/ml to 100 mg/ml. The protein forms large multimeric associations which explains this behavior. These multimers are not as active as tetramers that form at lower protein concentrations. This information has been used to explain the relative resistance of young cane plants to eye spot disease. Several phytotoxins have been isolated and further characterized. These include helminthosporoside from H. sacchari and a series of oligo-glucosyl derivatives from Rhynchosporium secalis. Biosynthesis work on the rhynchosporoside toxin has also been completed. A cooperative project with I. Barash in Israel on phytotoxins associated with Mal-seceo disease has resulted in the successful isolation and characterization of some compounds with biological activity. Several projects dealing with such things as Dutch elm disease, hairy root disease, and weed pathogens are in progress and reports on this progress have been written.

                              Impacts
                              (N/A)

                              Publications


                                Progress 01/01/79 to 12/30/79

                                Outputs
                                A plasmid was isolated from Agrobacterium rhizogenes that is responsible for thepathogenicity of this organism to the roots of numerous plants. This plasmid is 1.1 x 10(8) daltons and has many EcoR(1) restriction sites. The plasmid is vital to the infection process as manifested by: 1) loss of infectivity of A. rhizogenes when the plasmid is lost, 2) successful conjugational transfer to plasmidless strain making them pathogenic and, 3) modification of the p plasmid by mutagen treatment results in loss of pathogenicity with concomitant changes in the EcoR(1) restriction sites. Studies have continued on the mechanism faction of the specific toxin--helminthosporoside. Heating of cane leaves 4h at 35 degrees C renders the resistant to the toxin. Inhibitors of membrane ATPase protect the plants from the effects of the toxin and protoplasts of susceptible cane are rendered insensitive to the effects of the toxin in a medium deficient in K + and Mq + +. These data collectively indicate that sensitivity of cane to the toxin is mediated by membrane ATPase. Site studies have been done on rhynchosporoside, a host selective toxin produced by R. secalis (barley). The membrane protein that interacts with rhynchosporoside has been isolated and partially characterized.

                                Impacts
                                (N/A)

                                Publications


                                  Progress 01/01/78 to 12/30/78

                                  Outputs
                                  Several phytotoxic glycopeptides were isolated from ceratocystis ulmi, causal agent of Dutch Elm Disease. These compounds were characterized as to their molecular weight, biological activity, and residue composition. More work on the mode faction and toxin isolation and characterization is under way. Protoplasts of potato were regenerated into whole plants. Plants derived from these protoplasts were shown to vary in their susceptibility to alternaria solani, the early blight pathogen. Toxins isolated from A. solani were shown to be host-specific and were lipoidal in nature, at least 2 compounds (toxins) were required for any biological activity to be expressed by toxin preparations. Rhynchosporoside, a host selective toxin produced by R. secalis, a barley pathogen, was isolated and characteraized. The compound was shown to be a glycoside of 1, 2 propanediol. Its biological activity was limited to certain barley varieties and this activity was not identical to the host range of R. secalis. Serinol, an amino alcohol was described as a pathogenicity activating factor of H. saccahi. The metabolic pathogen leading to the formation of serinol was described. It is formed via the transanimation of dihydroxyacetone phosphate-yielding serinol phoshate which is then dephosphorylated to yield serinol. Work on a number of other interesting host parasite systems is underway.

                                  Impacts
                                  (N/A)

                                  Publications


                                    Progress 01/01/77 to 12/30/77

                                    Outputs
                                    Compounds involved as chemoattractants of Rhizobium to the legumes were isolatedfrom Birds-foot Trefoil root exudates. The principle compound in trefoil exudate is a glycoprotein with a molecular weight of 70,000. Work on its biology, chemistry, and role in nodulation is currently in process. The chemotactic behaviour of Rhizobium was studied in some detail. A binding protein for the toxins of H. maydis was discovered in corn hybrids, both susceptible and resistant to H. maydis. A binding assay was developed and the role of the protein in disease resistance and susceptibility was discussed. A toxic glycopeptide from Ceratocystis ulmi was isolated and characterized. The compound has a molecular weight of 250,000 and is 5% peptide and 95% sugars. The nature of the glycosidic bond and the carbohydrate backbone was determined. Work is underway on the toxin from Rhynchosporium secalis.

                                    Impacts
                                    (N/A)

                                    Publications


                                      Progress 07/01/76 to 12/30/76

                                      Outputs
                                      This project was begun at mid-year. In the past six months we have successfully demonstrated the presence of a toxin in cultures of Rhynchosporium secalis, the casual agent of barley scald. The toxin has been isolated and partially characterized. It appears to be the beta-glucoside of a small molecular weight unsaturated secondary alcohol. Compounds identical or similar to the toxin are currently being synthesized by organic means. A compound with similar chromatographic and spectroscopic properties was isolated from barley plants infected with R. secalis. The toxin causes initial leaf wilt followed by marginal tip death and a leaf flecking. The compound appears to have some host specificity. Work has also begun on the toxin of Phorma trachaephila on a cooperative US-Israeli grant. Work has continued on the chemistry and biology ofthe Ceratocystis ulmi phytotoxic glycopeptide.

                                      Impacts
                                      (N/A)

                                      Publications