plant surface microbiology.pdf

24 Microbial Community Analysis in the Rhizosphere 459
compounds (yeast extracts, casamino acids) as described by Hattori and Hattori
(1980). On the other hand, depending on the lower growth rate and a
longer incubation period, exuberant growth of the fast growers was reduced,
giving the slow growing strains a chance to develop (Gorlach et al. 1994; Mitsui
et al. 1997). In addition, minimal media like M9, were supplemented with
compounds described as root exudates, and with soil or root extracts
(Table 2). Plates were incubated at 20 °C for up to 4 weeks. Cell and colony
morphology was recorded and Gram-test, oxidase and catalase tests performed
according to Gerhardt et al. (1994). Genomic DNA of these isolates
was extracted and purified as described previously (Pukall et al. 1998). The
primer pair 27f and 1500r can be used for the amplification of the almost
complete 16S rRNA gene of the bacterial isolates (Lane 1991). PCR-amplification
of a part of the 23S rDNA was performed using the primer pair 2053r and
990 f.
Using this approach, about 70 % of the bacterial isolates from bulk soil and
ectorhizosphere were identified as Gram-positive bacteria using the oligonucleotide
GP (Rheims et al. 1996), whereas their numbers were reduced to 17 %
in the rhizoplane/endorhizosphere compartment of Medicago sativa (Mogge
et al. 2000). A similar result was obtained by Lilley et al. (1996) and Mahaffee
and Kloepper (1997). On the other hand, the numbers of isolates belonging to
the a-, b-, and g-subclasses of proteobacteria were increased in the rhizoplane
Table 2. Composition of media used to retrieve bacteria from bulk soil, ectorhizosphere
and rhizoplane/endorhizosphere samples
Medium Company or reference
King’s B agar; R2A agar; Actinomycete isolation Difco
agar; nutrient agar
CASO agar Merck
Yeast extract mannitol agar Dunger and Fiedler (1997)
Starch agar with and without root extract Dunger and Fiedler (1997)
Cellulose agar supplemented with soil extract Stotzky et al. (1993)
Planctomyces isolation agar(+N-acetylglucosamin) Schlesner (1994)
Hyphomicrobium isolation agar Moore and Marshall (1981)
Caulobacter isolation agar Poindexter (1964)
Glucose-yeast extract malt agar (GYM) Shirling and Gottlieb (1966)
M9 minimal medium a (+ carbon source b / Sambrook et al. (1989, modified)
+ trace elements c )
a Composed of Na2 HPO 4 10.2, KH 2 PO 4 3.0, NaCl 0.6, and NH 4 Cl 1.2 g/l
b 5 g/l carbohydrates (glucose, glucose and vitamin solution No.6 (Staley 1968), fructose,
sucrose, arabinose) or 2 g/l organic acids (fumaric acid, oxal acetic acid)
c 1 ml of sterile filtered trace element stock solution composed of CaCl2 x6 H 2 O 2.7 g,
MgSO 4 x7 H 2 O 15 g, FeCl 3 0.02 g/l