Application of the R galegae Specific Probes and Primers for Monitoring

To study the applicability of detection with strain- and species-specific sequences, a greenhouse experiment was conducted.51 Soil devoid of R. galegae was inoculated with different levels of R. galegae HAMBI 1207, which forms an ineffective symbiosis with G. orientalis. Then seeds of G. orientalis, inoculated with the effective strain R. galegae HAMBI 1174, were sown into the soil. The competition between the two Rhizobium strains was assessed by measuring plant dry weights from the plots and inspecting nodules formed on the plant roots. Multiple PCR with the strain- and species-specific primers were used

Fig. 4.2. PCR-RFLP of the R. galegae-specific fragment from genomic DNA of different R. galegae strains with the restriction enzymes HinfI and MspI. The host plant is shown above the samples. Lanes: M, molecular weight marker; 1, reference strain, R. galegae HAMBI 1174; 2-15, other R. galegae strains.

Fig. 4.2. PCR-RFLP of the R. galegae-specific fragment from genomic DNA of different R. galegae strains with the restriction enzymes HinfI and MspI. The host plant is shown above the samples. Lanes: M, molecular weight marker; 1, reference strain, R. galegae HAMBI 1174; 2-15, other R. galegae strains.

Fig. 4.3. Isolation of a strain-specific probe by subtraction hybridization. HAMBI 1174 and HAMBI 1207 are genetically closely related strains of R. galegae.

directly from crushed nodules (Fig. 4.4). The results were in good agreement with those of dilution platings on selective media, in which the two strains could be counted.

The specific primers were also used for the amplification of DNA extracted from soil and peat.61 After developing a suitable method for DNA extraction and purification from the peat samples (for methods see Chapter 3), the PCR assay proved to be very useful for quality control (i.e., confirmation of the inoculum srain) of commercial peat inoculants.

1 2

3 4 5 6 7 8 9 10 111213 14 IS 1617181920 21 2223 24 25 26 - \

bp

2645 -

«*

-

1198-

wt

676-

t" 5?

s

350 -

222 -

Fig. 4.4. PCR amplification of the R. galegae species-specific fragment (upper band) and the R. galegae HAMBI 1174 strain-specific fragment (lower band) from crushed nodules. Lanes: X, molecular weight marker; 1, R. galegae HAMBI 1174 genomic DNA; 2, R. galegae HAMBI 1207 genomic DNA; 3-26, nodules from G. orientalis inoculated with R. galegae; -, negative control without template DNA. Reprinted with permission from Tas E, Leinonen P, Saano A et al. Appl Environ Microbiol 1996; 62:529-535.

Fig. 4.4. PCR amplification of the R. galegae species-specific fragment (upper band) and the R. galegae HAMBI 1174 strain-specific fragment (lower band) from crushed nodules. Lanes: X, molecular weight marker; 1, R. galegae HAMBI 1174 genomic DNA; 2, R. galegae HAMBI 1207 genomic DNA; 3-26, nodules from G. orientalis inoculated with R. galegae; -, negative control without template DNA. Reprinted with permission from Tas E, Leinonen P, Saano A et al. Appl Environ Microbiol 1996; 62:529-535.

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