Yalpani, Ronald Flannagan, Rafael Herrmann, James Presnail, Tamas Torok, and Nasser

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Lawrence Berkeley National LaboratoryLawrence Berkeley National LaboratoryTitleMicrobial Diversity-Based Novel Crop Protection ProductsPermalinkhttps://escholarship.org/uc/item/58p4w4p8AuthorYalpani, Ronald Flannagan, Rafael Herrmann, James Presnail, Tamas Torok, and NasserPublication Date2010-05-21eScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaIn troduction  Extremophillic  microorganisms have become a valuable  bioprospecting target, as they often possess unusual  physiological properties and the abi lity to produce unique secondary  metaboli tes with potential  commercial  value. These natural products may be advantageous to the microorganisms in their respective environment and defend against predators or competitors. Detailed characterization of microorganisms contributes to a greater understanding of their divers ity, unique  metabolism, and ecological function. Over a 20- year  period, large numbers  of microorganisms were isola ted at contamina ted sites and closed mili tary bases, in deserts and forests in the USA, in Lake Baikal  sediments in Siberia,  and at geothermal and hydrothermal  sites on the Kamchatka peninsula in Russia, as well as in and around the fail ed nuclear  power  plant, and the surrounding  30- km “Exclusion Zone” in Chernobyl, Ukraine.  These environmen tal  field sites were considered extreme in terms of their water activity, xenobiotic contamination, temperature, or biogeochemistry. Published  or novel isolation techniques and growth media were adapted or modified and applied to simulate the eco- physiologi cal  conditions at the respective environmental n iche. Pure cultures of the microorganisms were preserved and stored at ultra- low temperature for long- term maintenance. In a collaborative effort between  Pioneer Hi- Bred International, DuPont Experimen tal  Station, and Lawrence Berkeley National  Laboratory, we screened some 26,000 samples  for novel crop protection products of microbi al origin, and detected many biological activities of mutual interest. Here we focus on novel  antifungal peptides as an examp le of the rich potential the gene pool of extremophil ic microorganisms represents for science and industry. Mat er i a l s and  Method s Microorga nisms.   Field samples were collected at the closed military bases in Alameda and Fort Ord, Californi a, in the Sierra  foothills, in and around Lake Baikal and the Kronotzky National  Park  on the Kamchatka peninsula in Russia, and in and around the fail ed nuclear  power  plant and the surrounding 30- km “exclusion zone” in Chernobyl (ChEZ), Ukraine. Isola tion techniques and conditions targeted for their novel biotechnology potential endospore - forming bacteria, actinobacteria, and filamentous fungi. In short, sub- samples of the environmental materials were air - dried for 10 days at room temperature in a biosafety cabinet under  aseptic conditions. The dry samples  were homogenized with a steri le pestle and stamped with sterile  foam plugs onto a set of ten media routinely used in our labor atory for microbial isolation. The isolation plates were incubated at room temperature for up to 2 months. The plates were examined  for colony formation at weekly interval s. Pure cultures were  preserved and transferred to a - 84°C ultralow- temperature freezer for long- term maintenance. Activity detecti on and identificati on.  Isolated microorganisms were grown under proprie tary conditions. The cell - free supernatants underwent primary screening.  Samples with confirmed insecticidal  or antifungal activities were subjected to HPLC purification. Molecul ar mass of the peptides of interest was determin ed by LC- ESI- TOF- mass spectrometry (LCMS). Ultimately, peptides were sequenced (Figure  1). BLAST analysis revealed novel peptides. Phylogeny of mature amino  acid sequences was calculated using the neighbor joining  method (Figure 2). As an example, Table  1 lists the fungal species that expressed biological  activities of interest. I n  vitro  testing.  Purified and quantified peptides were in vitro tested against target fungal pathogens and insect pests of major  crop plants. If the cell- free supernatants yielded insufficient amount of peptides, the appropriate E. coli  codon- optimized genes were expressed in a proprie tary strain of E. coli . Proprietary in vitro assays scored the inhibition of fungal spore/conidium germin ation and hyphal  growth (Figure 3) or insect lar val growth (Figure  4).  Abstract Extremophilic  microorganisms are  adapted to survive  in ecological  niches with high temperatures, extremes of pH, high salt concentrations, high pressure, radiation, etc. Extremophiles produce unique  biocatalysts and natural products that function under extreme conditions comparab le to those prevailing in various  industrial processes. Therefore, there is burgeoning interest in bioprospecting for extremophiles  with potential immed iate use in agr iculture, the food, chemical, and pharm aceutical industries, and environmental biotechnology. Over the years,  several thousand extremophilic  bacteria, archaea, and filamentous fungi were collected at extreme environmental sites in the USA, the Chernobyl Exclusion Zone surrounding the failed nuclear  power  plant in Ukraine, in and around  Lake Baikal  in Siberia, and at geothermal  sites on the Kamchatka peninsula  in Russia.  These organisms were  cultured under  proprietary  conditions, and the cell - free supernatants were screened for biolog ical activities against plant pathogenic fungi and major  crop damaging insects. Promising peptide lead molecules were isolated, characterized, and sequenced. Relatively high hit rates characterized the tested fermentation broths. Of the 26,000 samples screened, over  thousand contained biological activity of interest. A fair number  of microorganisms expressed broad- spectrum antifungal or insecticidal  activity. Two- dozen broadly  antifungal peptides (AFPs) are alr eady patent protected, and many more  tens are under  further investigation. Tapping the gene pool of extremophil ic microorganisms  to provide novel ways of crop protection proved  a successful strategy. Acknow l edgements  We thank V. Zakharchenko, N. Zhdanova, and I.  A. Ellanskaya (Zabolotny Institute of Microbiol ogy and Virology, Kiev, Ukraine) for fungal collection and identification, and Dr. J. Hunter- Cevera  (UMBI) for her input on proprietary isolation and cultivation methods. Work on expression of AFPs in E. coli was performed by J. English and A. Wong (Pioneer). AFPs were purified by D. Altier and J. Gilliam  (Pioneer). C. Simmons (Pioneer) provided bioinformatics support. Program  support was provided by R. Herrmann, B. McCutchen and E. Krebbers at DuPont and G. Dahlbacka at LBNL. The US Department of Energy GIPP  program  supported the project under  LBNL- T2-0193. Absidia spinosa Doratomyces microsporus Mycelia sterilia (white) Peyronella sp.Acremonium rutilum Embellisia helianthi Myrothecium cinctum Phoma fimetiAcremorium flavum Emericella nidulans Myrothecium roridum Phoma glomerataAcremorium strictum Emericella variecolor Myrothecium  sp. Phoma  sp.Alternaria tenuissima Emericella violacea Myrothecium verrucaria Sclerotinia riciniApiospora montagnei Epicoccum purpurascens Nectria inventa Scolecobasidium tshawytschaeArthrinium phaeospermum Eupenicillium alutaceum Nigrospora sphaerica Scopulariopsis brevicaulisarthrinium state of Apiospora montagnei Eupenicillium erubescens Paecilomyces lilacinus Scopulariopsis candidaAspergillus alliaceus Eupenicillium javanicum Paecilomyces marquandii Scopulariopsis koningiiAspergillus deflectus Eupenicillium lapidosum Penicillium  citrinum Stachybotrys bisbyiAspergillus flavipes Eurotium halophilicum Penicillium   sp. Thermoascus aurantiacusAspergillus flavus Eurotium halophilicum Penicillium canescens Tolypocladium inflatumAspergillus fresenii Exophiala  sp. Penicillium chrysogenum Trichoderma albumAspergillus ochraceus Fusarium acuminatum Penicillium citreonigrum Trichoderma hamatumAspergillus rhizopodus Fusarium avenaceum Penicillium claviforme Trichoderma harzianumAspergillus sclerotiorum Fusarium bullatum Penicillium clavigerum Trichoderma koningiiAspergillus  sp. Fusarium culmorum Penicillium dierckxii Trichoderma polysporumAspergillus sydowii Fusarium culmorum Penicillium expansum Trichoderma pseudokoningiAspergillus terreus Fusarium equiseti Penicillium frequentans Trichoderma sp.Aspergillus ustus Fusarium equiseti Penicillium funiculosum Trichoderma virensAspergillus versicolor Fusarium heterosporum Penicillium glandicola Trichotecium roseumAureobasidium pullulans Fusarium javanicum Penicillium griseolum Trimmatostroma sp.Bartalinia  sp. Fusarium oxysporum Penicillium griseum Ulocladium botrytisBipolaris australiensis Fusarium poae Penicillium herquei Ulocladium consortialeBipolaris sorokiniana Fusarium sambucinum Penicillium hirsutum Undetermined agonomyceteChaetomium aureum Fusarium sambucinum var.  ossicolum Penicillium islandicum Undetermined agonomyceteChaetomium cochliodes Fusarium semitectum Penicillium janczewskii Undetermined ascomyceteChaetomium dolichotrichum Fusarium solani Penicillium janthinellum Undetermined ascomyceteChaetomium globosum Fusarium tricinctum Penicillium lanosocoeruleum Undetermined ascomyceteChaetomium homopilatum Fusarium tumidum Penicillium lanosum Undetermined ascomyceteChaetomium nigricolor Gliocladium solani Penicillium luteum Undetermined ascomyceteChaetomium subspirale Gliocladium varians Penicillium miczinskii Undetermined ascomyceteChrysosporium pannorum Gliocladium viride Penicillium oxalicum Undetermined ascomyceteChrysosporium sp. Helicorhoidion pulchrum Penicillium purpurogenum Undetermined ascomyceteChrysosporium  sp. Humicola grisea Penicillium restrictum Undetermined ascomyceteCladosporium cladosporoides Monascus ruber Penicillium rugulosum Undetermined ascomyceteConiothyrium fuckelii Mortierella vinacea Penicillium simplicissimum Undetermined ascomyceteCorynascus sepedonium Mucor hiemalis Penicillium sp. Undetermined ascomyceteCunninghamella echinulata Mucor plumbeus Penicillium variabile Undetermined basidiomyceteCunninghamella elegans Mucor racemosus Penicillium varians Undetermined coelomyceteCurvularia inaequalis Mycelia sterila Penicillium vermiculatum Wardomyces anomalusDichobotrys sp. Mycelia sterilia (orange) Penicillium verrucosum var. cyclopiumWardomyces columbinusPenicillium waksmani Zygorhynchus moelleriFungal speciesRe sul ts and  Di scussion Over 26,000 primary samples were screened over a three- year  period and some 1,000 showed insecticidal  or antimicrobial  activities. These samples were produced under  proprie tary cultivation conditions by over 74 bacterial and 86 fungal species (Table  1). Many microorganisms  expressed broad-spectrum antifungal or insecticidal activities (Table  2). As an example, we show that activity- guided purification led to the identification of novel antifungal peptides (AFP) from several  filamentous fungi (Figure  1). These peptides were characterized, sequenced, phylogene tically anal yzed (Figure  2), and their antifungal activity validated. They were shown to be inhibitory to fungal pathogens of maize that cause stalk rot and ear  mold. These cysteine- rich and basic peptides have 55 to 61 residues respectively,  and can be aligned as one protein family  together with some previously characterized AFPs. DNA sequence of several  of these indicates that they are synthesized as preproproteins  from which the mature AFP is cleaved. Promising AFPs were filed for patent protection in the USA and abroad.  Bioprospecting has elevated the search for microorganisms with novel capabil ities to a safe, ethical, and meaning ful conduct that equally  benefits science and industry. Extremophilic  microorganisms  repre sent an immensely r ich and mostly untapped genetic pool of novel  bioactive capabil ities. Microb ial  divers ity- based new crop protection products and technologies are replacing traditionally  used synthetic chemicals and agr icultural practices. Transgenic plants expressing heterologous genes of extremophilic  microorganisms may provide a secure solution to  agr icultural production of major crop plants and industrial  feedstocks. Figure 1.  Example antifungal peptide (AFP) isolation and identification Figure 2.  Phylogenetic analysis of AFPs (neighbor joining  method of Saitou and Nei with a utility in Vector NTi –AlignX) Figure 3.  In vitro antifungal activity assay (Ps-AFP1; 48 h) Figure 4.  In vitro insecticidal activity assay Table 1.  List of fungal species that expressed biological activities of interest Table  2.  Spectrum characteristics of microbial  natural products in primary  screens  Activity Strains with 4  targets Strains with 3  targets Strains with 2  targets Strains with 1  target Antifungal 48 73 151 524 Insecticidal*  21 30 200 961 *includes non- protein hits 

Microbial Diversity-Based Novel Crop Protection Products

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