Agriculture Reference
In-Depth Information
recognized in samples from bulk soil; however, some variations in the TGGE
patterns could be correlated to time of development in the rhizosphere and rhizo-
plane. Nearly, similar findings were reported by Mahaffee and Kloepper (
1997
)
who used fatty acid methyl ester analysis (FAME) to determine the community
shifts in the rhizosphere of cucumber. However, only an altered window of obser-
vations generated by the use of specific primers could possibly reveal a stronger
time-dependent stimulation of certain bacterial groups.
7.5.8 Terminal Restriction Fragment Length Polymorphism
Organisms can also be differentiated according to the patterns derived from cleav-
age of their DNA. Thus, in T-RFLP, the specific fingerprint of a community is
revealed by analyzing the polymorphism of a certain gene. T-RFLP is a high-
throughput, reproducible method that allows the semiquantitative analysis of the
diversity of a particular gene in a community. It requires the extraction of DNA
from a soil sample and its PCR amplification using a fluorescently labeled primer.
T-RFLP yields a mixture of amplicons of the same or similar sizes with a fluores-
cent label at one end. After purification, the amplicon mixture is digested with a
restriction enzyme, which generates fragments of different sizes that are separated
by gel or capillary electrophoresis. The separated, labeled fragments are then
densitometrically detected, and a profile based on fragment lengths is generated.
McSpadden Gardener and Driks (
2004
) studied the population structure of these
two groups by T-RFLP using group-specific primers Ba1F and Ba2R and charac-
terized the plant growth-promoting population of PGPR; only minor differences
were observed in the number and relative abundance of
Bacillus
-like ribotypes
from different sites all the way through Ohio (USA). Despite environmental
constraints and interactions with other microorganisms, some bacteria are able to
colonize the phylloplane with higher frequency than others. Arias et al. (
1999
)
evaluated the diversity and distribution of
Bacillus
spp. from soybean phylloplane
wherein a decline was observed in the population of
Bacillus
spp. from 80 % of total
bacterial isolates in early stages to 0 % at the time of harvesting (Kumar et al.
2011
).
7.5.9 Microbial Resilience
The ability to estimate the relative abundance of each species of microorganisms in
the soil, using the three techniques described above, has led to the suggestion that
the “equitability index” (J) of numbers of individual species is an important
estimation of the resilience of a soil. The use of statistical packages such as Phoretix
enables quantification of both diversity indices and equitability (Girvan et al.
2004
).
The development of approaches that do not require the establishment of microbial
cultures will undoubtedly enhance our knowledge of bio-resources and promote the
