Fig 1.
Scheme of inoculation of grapevine plants and areas analysed.
Image of (A) the inoculation point in the rootstock of plants treated by injection, as well as the four 1.0 cm cylinders (Z1/RI, Z2, Z3 and Z4) analysed once the plants were uprooted, and (B) the zones defined as root insertion point (RI) and root system (RS) also analysed in plants inoculated by immersion in a cell suspension. Note that Z1/RI and RI zones in plants inoculated by injection or immersion are equivalent for analytical purposes.
Fig 2.
RAPD profiles generated by (A) P4 primer and (B) OPA2 primer for rhizosphere (VV/R1 to VV/R5) and endophytic (VV/E1 to VV/E5) Streptomyces sp. strains isolated from the root system of young grapevine plants. MW: GeneRuler 1kb DNA Ladder Plus (Thermo Fisher Scientific). Specific bands amplified from isolates VV/E1 and VV/R4 that were selected for SCAR markers design are indicated by arrows. NC (negative control).
Fig 3.
Representative maps of the characterized RAPD fragments.
(A) Streptomyces sp. VV/E1 and (B) Streptomyces sp. VV/R4. Incomplete ORFs are indicated by grey arrows, whereas black arrows correspond to complete ORFs. The relative positions of the amplified SCAR markers developed are indicated by a black line. GeneBank accession numbers: MH048872 (Streptomyces sp. VV/E1) and MH048873 (Streptomyces sp. VV/R4).
Fig 4.
Representative amplification curves obtained by plotting the mean Ct values with respect to the logarithm of genomic DNA quantity from Streptomyces sp. VV/E1 (black line) and Streptomyces sp. VV/R4 (grey line). Standard curves were generated with 10-fold serial dilutions of genomic DNA concentrations from 5.65 x 106 fg to 56.5 fg (VV/E1 strain) or 5.2 x 106 fg to 52.0 fg (VV/R4 strain) by SYBR green qPCR reactions.
Fig 5.
Quantification in RI and RS DNA samples from the Streptomyces sp. strains inoculated by immersion.
Box plot showing the DNA levels from Streptomyces sp. VV/E1 and Streptomyces sp. VV/R4 strains in plants subjected to inoculation by immersion as compared to control plants in the (A) RI, and (B) RS areas. Black rhombuses show the average values (back transformed) for each treatment. Asterisks indicate existence of significant differences according the LSD-test performed: * p < 0.05; ** p < 0.01; *** p < 0.001. Circles correspond to data outliers (falling outside the Q1-Q3 range).
Fig 6.
Quantification at RI of DNA from Streptomyces sp. strains inoculated by injection.
Box plot showing the levels of DNA from Streptomyces sp. VV/E1 and Streptomyces sp. VV/R4 strains detected at RI in plants inoculated by injection, compared to the amount of DNA detected in control (untreated) plants. Black rhombuses correspond to average values (back transformed). Asterisks indicate significant differences according the LSD test: * p < 0.05; ** p < 0.01; *** p < 0.001. Circles correspond to outliers of the data (data falling outside the Q1-Q3 range).
Fig 7.
Quantification of Streptomyces sp. strain DNA at different zones of the rootstock inoculated by injection.
Box plot corresponding to the DNA amounts of Streptomyces sp. VV/E1 and Streptomyces sp. VV/R4 strains detected in the different zones (Z1/R1 to Z4) analysed in the rootstock of plants inoculated by injection. Note that in this figure, DNA amounts are shown in pg. Black rhombuses correspond to average values (back transformed). Asterisks indicate significant differences according the LSD test: * p < 0.05; ** p < 0.01; *** p < 0.001. Circles correspond to data outliers (outside the Q1-Q3 range).
Fig 8.
Comparison of the effectiveness of inoculation by immersion vs. injection at RI.
Box plot showing DNA amounts at RI of Streptomyces sp. VV/E1 and Streptomyces sp. VV/R4 strains in plants inoculated by immersion or injection as compared to control (untreated) plants. Black rhombuses correspond to average values (back transformed) for each treatment. Asterisks indicate significant differences according to the LSD test: * p < 0.05; ** p < 0.01; *** p < 0.001. Circles correspond to data outliers (outside the Q1-Q3 range).