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).

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. 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. 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. 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. 5/27/2010
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