5/27/2010




                                                                 ‐Project document approved by GEF/UNEP:
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5/27/2010




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5/27/2010




   Entomopathogenic fungi and nematodes                                                     Entomopathogenic...
5/27/2010




         Nematode infiltration and mounting permanent slides of nematodes


                                ...
5/27/2010




                                                                Procaryote diversity: terminal restriction f...
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PK02:Standard methods for sampling and characterization of soil microbial diversity

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A presentation by Prof. Fatima M.S. on the Standard methods for sampling and characterization of soil microbial diversity (LNB, AMF, soil fungi, associative nitrogen fixers, yeasts, nematodes, enchytraeids).

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PK02:Standard methods for sampling and characterization of soil microbial diversity

  1. 1. 5/27/2010 ‐Project document approved by GEF/UNEP: methods were neither  described or mentioned. S LNB AMF ‐Anderson and Ingram, 1993‐ rhizobia (only counting);  vesicular‐ Arbuscular mycorrhiza (methods only presented without details, need updating references), ectomycorriza (idem), culturable bacteria ‐Previous experience in Alternatives for Slash and Burn Project: Previous experience in Alternatives for Slash and Burn Project: microbial biomass, nematodes, arbuscular mycorrhizal fungi, rhizobia Fatima M S Moreira, David E Bignell, E Jeroen Huising (eds) Standard methods for assessment of soil biodiversity and land use practice.Mike Swift and David Bignell (eds) Lecture Note 6B  Nematodes (1 page), rhizobia (6 pages) and mycorrhiza (Half page)  without details  and uncompleted, mainly nematodes and mycorrhiza. Handbook Earthscan It includes methods that were not included in the preceding publications , such as those for entomopathogenic, saprophytic and pathogenic fungi. It provides additional detail for specific functional groups, or the updating of methods to evaluate diversity of nematodes, mycorrhizal fungi and Leguminosae‐nodulating bacteria (LNB) and their host plants. The proposed standard methods accommodate recent technological advances from molecular genetics, such as techniques for the genetic fingerprinting of LNB, where these are appropriate and within Portuguese version of the manual:  + yeasts and  the reach of national laboratories. Methods were discussed and progressively refined in annual meetings between 2002 and 2005, and their Leguminosae nodulating bacteria  evolution is reflected in the reports of the annual (Rhizobia) (a) project meetings of 2002 (Wageningen, the Fatima Moreira Netherlands), 2003 (Sumberjaya, Indonesia), and especially 2004 (Embu, Kenya), and in a number of taxon‐specific or thematic workshops (Molecular techniques with emphasis on T‐RFLP, Cali, Colombia, October 2003; AMF and ectomycorrhiza, Bangalore, India, March 2005; and in several in‐country (b) workshops held since the inception of the project. The definition of standard methods draws upon (c) experience obtained with implementation of the methods in the seven countries participating in the (d) project. 1
  2. 2. 5/27/2010 Saprophytic and plant pathogenic soil‐fungi Ludwig Pfenning and Lucas A Magalhães Soil washing technique for isolation of soil micro‐fungi:  A. Pre‐washing;  B. Sieves with different mesh;  C. Pre‐washed soil;  C Pre washed soil; D. Continued washing procedure;  E. Washed soil particles;  F. Plating on culture medium;  G. Plate with soil particles for incubation;  H. Fungal colonies on culture medium  with growth retardants. 16 SrDNA sequencin Arbuscular mycorrhizal fungi Spore extraction Joseph D. Bagyaraj & Sidney L. Stürmer Saprophytic and plant pathogenic soil‐fungi Ludwig Pfenning and Lucas A Magalhães cont. 750 µm 45 µm Isolation of zoosporic fungi from environ‐ mental  Samples   (Peronosporomycetes, Cytridiomycetes):  Wet sieving A. Soil sample with baits;  B. Water sample with baits;  B Water sample with baits; C. Pure culture on bait;  D. Sporangia of Phytophthora;  E. Oogonium and antheridum of Pythium;  F. Pythium liberating zoospores Slide to identification 20%/60% sucrose gradient centrifugation Collect spores Arbuscular mycorrhizal fungi Entomopathogenic fungi and nematodes Trap plants Joseph D. Bagyaraj & Sidney L. Stürmer Alcides Moino‐Jr and Ricardo S Cavalcanti 25.0 Soil sample from field, homoge- neized with roots A B C D Mix field soil with sterile sand Collect sample to extract spores and Procedures to isolate entomopathogenic fungi on culture media: identify species Place mixture in a pot and A) serial dilution of the fungal aqueous suspension;  heavilly seed with host B) inoculation of the suspension (0.1 ml aliquot) into Petri dish with culture medium;  C) incubation under controlled conditions in BOD chamber;  D) Permanent freezer storage of conidia in Eppendorf tubes. Grow for 3-4 months 2
  3. 3. 5/27/2010 Entomopathogenic fungi and nematodes Entomopathogenic fungi and nematodes Alcides Moino‐Jr and Ricardo S Cavalcanti cont. Alcides Moino‐Jr and Ricardo S Cavalcanti cont. A B D C Procedure for the use of the White trap use for the isolation of entomopathogenic nematodes: Beauveria bassiana: purified cultures growing in PDA medium.  A) petri dish with filter paper (dry chamber); B) Galleria mellonella larvae dead after  Separation from contaminants was achieved by collecting a small portion  contact  with the soil sample;  of any fungal colonies of interest with a needle and transferring this portion C) larvae showing typical pathology of infection by nematodes;  to three points of inoculation in a fresh Petri dish with PDA culture medium. D) emergence of infective juveniles in the White trap. Extraction of nematodes Soil nematodes •Nematodes extracted from 300 cc soil, by the combined methods of sieving and sugar Juvenil E. Cares & Shiou Pin Huang floatation techniques Sampling for nematodes •Pass nematode  suspension trough  •Grid system‐ 1 sample at crossing lines screen with oppening  •One compound sample made of 12 soil cores (0 to 20cm) of 0.25 mm + screen of  37 μm  37 μm  0.25 mm  Killing, fixing and counting nematodes a. Nematodes were gently killed in water (60° C during 1 min.) b. Nematodes fixed with formalin 4% c. Total nematodes counted in each sample •Centrifugation of nematode suspension at 3000 rpm for 5 min. •Centrifugation of nematode sediments at 1000 rpm for 1 min in sugar solution (sucrose 456 g/L). 3
  4. 4. 5/27/2010 Nematode infiltration and mounting permanent slides of nematodes Functional groups added to the Portuguese version of the Handbook a. Nematodes  gradually  infiltrated with glycerin b. Nematodes manually  picked and mounted  in  microscope slides for   identification c. 100 nematodes  identified through light  microscope to the genus  and trophic levels Enchytraeid (Enchytraeidae, Oligochaeta, Annelida) Isolation and identification of yeasts Cintia Carla Niva,  Jörg Römbke, Rüdiger Maria Schmelz e George Gardner Brown Disney R. Dias and Rosane F. Schawn Sampling Extraction in water Associative N2‐fixers Functional groups studied‐methods not added to Handbook  Mainly grasses and palm species Soil or root samples inoculated in Isolated strain N-free media Positive growth pelicle formation 4
  5. 5. 5/27/2010 Procaryote diversity: terminal restriction fragment length  polymorphism (T‐RFLP) DNA extraction from  PCR with fluorescent  marked soil communities il iti primers i Digestion of PCR products 1 2 Azospirillum lipoferum – from rhizosphere ofPaspalum plicatum 1- Cells contrast phase microscope 2 –Colonies in potato agar medium Detection of fluorescent  Separation of  DNA fragments  fragments with different lengths by sequencing 5

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