Coffee Research at the United States Department of Agriculture. Fernando E. Vega Sustainable Perennial Crops Laboratory Beltsville, Maryland

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1 Coffee Research at the United States Department of Agriculture Fernando E. Vega Sustainable Perennial Crops Laboratory Beltsville, Maryland 1

2 USDA Established by Abraham Lincoln in About 85,000 employees. Agricultural Research Service Principal in-house research agency of the U.S. Department of Agriculture. Workforce: 8,000 total employees 2,100 research scientists About 1,200 research projects. Over 100 locations. Annual budget (FY07): 1.1 billion US$ 2

3 ARS Research Locations (Divided into 8 Regions) International Locations Montpellier, France; Buenos Aires, Argentina; Brisbane, Australia; Beijing, China Over 100 U.S. Locations ARS Research is divided into 4 major National Programs 1. Nutrition, Food Safety/Quality 2. Animal Production and Protection 3. Natural Resources and Sustainable Agricultural Systems 4. Crop Production and Protection 3

4 Instrumentation and Sensing Laboratory 4

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6 COFFEE BERRY BORER Hypothenemus hampei La broca del café 6

7 THE TROPICS TROPIC OF CANCER TROPIC OF CAPRICORN 7

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9 COFFEE BERRY BORER INFESTATION UP TO 60% IN COLOMBIA 58-75% IN JAMAICA 80% IN UGANDA 50-80% IN IVORY COAST ESTIMATED WORLDWIDE LOSSES DUE TO THE COFFEE BERRY BORER $500 MILLION! 9

10 Wolbachia infection in the coffee berry borer (Coleoptera: Scolytidae) - We isolated Wolbachia in coffee berry borers from 11 countries -Wolbachia might be responsible for the 10:1 female to male sex ratio observed in the coffee berry borer Vega et al., Ann. Entomol. Soc. Am. 95: Identification of a coffee berry borer-associated yeast: does it break down caffeine? Pichia burtonii and Candida fermentati Vega et al., Entomo. Exper. Appl. 107:

11 Metaparasitylenchus hypothenemi, a parasite of the coffee berry borer. Poinar et al., J. Parasitol. 90: A new method to evaluate the biocontrol potential of single spore isolates of fungal entomopathogens Posada and Vega, J. Insect Sci.,

12 Mycobiota associated with the coffee berry borer in Mexico. 40 fungal species in 22 genera Fusarium, Penicillium, Candida, Aspergillus Pérez et al., Mycol. Res. 107: Penicillium brocae, a new species associated with the coffee berry borer in Chiapas, Mexico. Peterson et al., Mycologia 95:

13 Penicillium coffeae, a new endophytic species isolated from a coffee plant. Peterson et al., Mycologia 97:

14 Beauveria bassiana: an insect pathogenic fungus Our goal: To introduce the fungus in the plant as an endophyte. ENDOPHYTES Fungi or bacteria inside apparently healthy living plants. Betsy Arnold 14

15 The human intestinal microbiota contains 10,000,000,000,000 to 100,000,000,000,000 microorganisms which contain at least 100 times as many genes as our genome! Donna Mager and Sigmund Socransky...have found 615 different species of bacteria... "In one mouth, the number of bacteria can easily exceed the number of people who live on Earth (more than 6 billion)... 15

16 ENDOPHYTES Mirlohi et al. West Virginia Univ. Betsy Arnold Beauveria bassiana endophytic in: Maize Potato, cotton Tomato Theobroma gileri Opium poppy Coffee berries 16

17 Beauveria bassiana inoculation methods SPRAYING 7 X 10 6 SPORES ML -1 INJECTION 5 X 10 5 SPORES ML -1 IRRIGATION 7 X 10 6 SPORES ML -1 Beauveria bassiana Tissue sterilization for endophyte isolation 0.5% NaOCl 2 min 70% ethanol 2 min Sterile dist. water Arnold et al., 2001, Mycol. Res. 105:

18 Beauveria bassiana recovery: MONTHS POST-INOCULATION % RECOVERY Ribosomal RNA coding gene (rdna( rdna) ITS 1-F SSU (18S) 5.8S LSU (26S) ITS 4 18

19 Fungal endophytes in coffee seedlings 2 and 4 months post-inoculation with Beauveria bassiana Alternaria sp. Arthopyrenaceae Aspergillus tamari Aspergillus westerdijkiae Beauveria bassiana Bionectriaceae Chaetomium sp. Cladosporium cf. sphaerospermum Clavicipitaceae Colletotrichum gloeosporoides complex Cylindrocarpon sp. Exobasidiomycetidae Exophiala sp. Fusarium cf. oxysporum f. sp. vasinfectum Fusarium oxysporum complex (1) Fusarium oxysporum complex (2) Fusarium sp. (1) Fusarium sp. (2) Fusarium sp. (Lateritium clade 1) Hypocreales sp. Macrophomina sp. Paecilomyces sp. Penicillim citrinum Penicillium brevicompactum Penicillium cecidicola Penicillium glabrum Penicillium janthinellum Penicillium sp. near daleae Penicillium steckii Penicillium toxicarium Phyllachoraceae Plectosphaerella sp. Pleosporales sp. Pseudallescheria cf. boydii Rhizopycnis sp. Trichoderma sp. Trichoderma hamatum Trichoderma harzianum Fungal endophytes in green coffee seeds Fungal id Acremonium sp. Aspergillus sumatrense Aspergillus fumigatus Aspergillus fumigatus Aspergillus niger Aspergillus pseudodeflectus Aspergillus pseudodeflectus Aspergillus tubingiensis Aspergillus tubingiensis Clavicipitaceae sp. 1 Clavicipitaceae sp. 2 Aspergillus (Eurotium) ruber Fusarium solani complex Gibberella sp. Penicillium sp., subgenus Biverticillium Penicillium crustosum Penicillium olsonii Country Puerto Rico Puerto Rico India Vietnam Vietnam Papua New Guinea Vietnam Colombia Kenya Puerto Rico Puerto Rico India Vietnam Colombia India Guatemala Colombia 19

20 Fungal endophytes in coffee plants Sampling of Coffea arabica L. Colombia Hawaii Mexico Puerto Rico 20

21 Tissue sterilization for endophyte isolation 0.5% NaOCl 2 min 70% ethanol 2 min Sterile dist. water Arnold et al., 2001, Mycol. Res. 105:

22 Ribosomal RNA coding gene (rdna( rdna) ITS 1-F SSU (18S) 5.8S LSU (26S) ITS 4 Colombia Endophytes recovered 267 ITS genotypes 113 Hawaii Mexico Puerto Rico Total

23 Colombia n = 267 Leaves Roots Stems Crown Skin Seed Peduncle Flower 113 genotypes Dominant genus: Colletotrichum (106 isolates, 25 genotypes) Dominant genotype: Colletotrichum sp. 2 (25 isolates) Beauveria bassiana, Cladosporium sp., Clonostachys sp Hawaii n = 393 Leaves Roots Stems Crown Skin Seed Peduncle Flower 126 genotypes Dominant genus: Colletotrichum (121 isolates, 20 genotypes) Dominant genotype: Penicillium olsonii (32 isolates) Acremonium sp., Beauveria sp., Cladosporium sp., Paecilomyces sp. 23

24 Mexico 32 genotypes Dominant genus: Fusarium (68 isolates, 8 genotypes) Dominant genotype: Fusarium sp. 16 (48 isolates) Paecilomyces sp. n = 109 Leaves Roots Crown Skin Seed Peduncle 35 Puerto Rico n = 74 Leaves Crown Skin Seed Peduncle genotypes Dominant genus: Fusarium (24 isolates, 8 genotypes) Dominant genotype: Fusarium sp. 2 & sp. 18 (8 Cladosporium sp., Paecilomyces sp. 24

25 Luis Mejia Fungal endophyte Acremonium alternatum Acremonium sp. Beauveria bassiana Beauveria bassiana Beauveria bassiana Beauveria bassiana Cladosporium cf. cladosporioides Cladosporium cf. cladosporioides Cladosporium cf. sphaerospermum Cladosporium sp. 1 Cladosporium sp. 2 Cladosporium sp. 3 Cladosporium sp. 4 Cladosporium sp. 4 Clonostachys rosea Paecilomyces cf. fumosoroseus Paecilomyces cf. javanicus Paecilomyces sp. 1 (near C. inflatus) Paecilomyces sp. 2 (near C. lilacinus) Coffee tissue Skin (berry) Root Seed Skin (berry) Peduncle Crown Leaves Leaves Leaves Peduncle Seeds Leaves Skin (berry) Crown Leaves Crown Peduncle Skin (berry) Seedling - root Collection site Colombia Hawaii Colombia Colombia Colombia Colombia Puerto Rico Hawaii Maryland Hawaii Hawaii Colombia Colombia Colombia Colombia Puerto Rico Colombia Mexico Hawaii 25

26 Survival ( % ) B. bassiana C. rosea Control Control Days after inoculation COFFEE BACTERIAL ENDOPHYTES Vega et al., 2005, J. Basic Microbiol. 45:

27 Coffee Bacterial Endophytes Bacillus cereus Bacillus megaterium Bacilus subtilis Bacillus megaterium Burkholderia cepacia Burkholderia gladioli Burholderia glathei Burkholderia pyrrocina Cedecea davisae Chromobacterium sp. Clavibacter michiganense insidiosum Curtobacterium flaccumfaciens Enterobacter asburiae Enterobacter cancerogenus Enterobacter gergoviae Escherichia vulneris Gordona sp. Klebsiella planticola Klebsiella pneumoniae Klebsiella trevisanii Kocuria kristinae Methylobacterium radiotolerans Micrococcus sp. Pantoea agglomerans Pseudomonas chloroaphis Pseudomonas putida Rhodococcus equi Salmonella typhimurium Serratia liquefaciens Stenotrophomonas maltophila Variovorax paradoxus Xanthomonas sp. Vega et al., 2005, J. Basic Microbiol. 45:

28 Jack Armstrong, USDA, ARS Hilo, Hawaii Ozone fumigation for coffee imports. The FAS coffee production numbers in the Tropical Products circular. Utilizes the in-depth analysis from 14 overseas offices. Tony Halstead has replaced Debra Pumphrey. 28

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32 SUMMARY Increase awareness of the enormous breadth of scientific expertise at ARS that might be used to solve other problems of interest to the coffee industry. ARS is an important player in agricultural research. ARS has a strong coffee berry borer research program. International collaborative research has always been an important component in our research programs. There are opportunities for short term visits to ARS laboratories: Cochran Fellowship and Borlaug Fellows Program. 32

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