Table 1.
Saprolegnia spp. isolates used in the present study.
Fig 1.
Maximum-likelihood phylogenetic tree showing evolutionary relationships of 53 Saprolegnia isolates based on the ITS region.
Isolates examined in this study are shown in boldface. Bootstrap values ≥ 50% (1000 replicates) are given at the branchpoints. The scale bar indicates the number of substitutions per site. P. sojae voucher P6497 and A. euteiches BR694 were used as outgroups.
Fig 2.
Maximum-likelihood phylogenetic tree showing evolutionary relationships of 53 Saprolegnia isolates based on the CoxI gene.
Isolates examined in this study are shown in boldface. Bootstrap values ≥ 50% (1000 replicates) are given at the branchpoints. The scale bar indicates the number of substitutions per site. P. sojae voucher P6497 and A. euteiches BR694 were used as outgroups.
Fig 3.
Concatenation phylogenetic tree based on the sequences of ITS and CoxI.
Isolates examined in this study are shown in boldface. Bootstrap values ≥ 50% (1000 replicates) are given at the branchpoints. The scale bar indicates the number of substitutions per site. P. sojae voucher P6497 and A. euteiches BR694 were used as outgroups.
Fig 4.
Ten-fold dilutions of S. salmonis DNA ranging from 1 fg to 100 ng were amplified in triplicate and the reactions were repeated three times. The iTaq™ Universal SYBR® Green Supermix and qCOXF1+R1 primers were used.
Fig 5.
A. Schematic representation of the ITS region, showing the positioning of the LAMP primers used in the present study. Scale bar represents 100bp. FwdOut = Forward Outer Primer; RevOut = Backward Outer Primer; F2+F1c = Forward Inner Primer; B2c+B1 = Backward Inner Primer; LF = Loop Forward Primer; LB = Loop Backward Primer. B. Positioning and orientation of LAMP primers developed in the present study. Representative ITS sequences from Saprolegnia and non-Saprolegnia oomycetes were aligned and the LAMP primers were positioned on the alignment manually. The LAMP primers amplified the ITS1 regions of the target sequences.
Table 2.
LAMP primers used in the present study.
Fig 6.
Optimization of Mg2+ concentration for LAMP.
The concentrations of Mg2+ ranging from 2 mm-10 mM were tested. Top panel represents positive controls using 2ng/μl S. salmonis gDNA. The bottom panel, No Template Control (NTC) represents negative controls. Color change to green indicates positive reaction, while golden brown indicates negative reaction. The experiments were performed in triplicate and repeated three times.
Fig 7.
Ten-fold dilution S. salmonis DNA ranging from 1 fg to 100 ng were used in the presence of 1000X SYBR Green I. Color changes from golden brown to green indicates a positive reaction. The concentrations of template DNA are indicated, NTC = no template control, represents a negative control. The reactions were performed in triplicate and were repeated three times.
Fig 8.
Direct detection of zoospores.
A. qPCR and B. LAMP. One and ten zoospores were used in each reaction, S. salmonis DNA (10 ng/μl) was used as the positive control while no template control (NTC) was used as the negative control. Each sample was performed in triplicate and was repeated three times.
Table 3.
Specificity of qPCR and LAMP.