In vitro and in planta data on the phosphonate sensitivity and fitness of Phytophthora nicotianae isolates from citrus orchards and nurseries across South Africa

All sheet names preceded by a number '2' contain data from Chapter 2, while those preceded by a number '3' contains data from Chapter 3. The data from the sheet named "2 All isolates" includes the identity of all isolates from the sample and supplementary information on the province from where they were sourced (MP = Mpumalanga; WC = Western Cape; EC = Eastern Cape; NW = North West; LP = Limpopo; NC = Northern Cape; Unknown = Samples from South Africa with unknown province of origin). The identity was determined by PCR Restriction fragment length polymorphism and sequencing of the internal transcribed spacer (ITS) region. The sheet named "2 In vitro isolates" contains only the Phytophthora nicotianae isolates that were screened for their in vitro sensitivity to phosphonates using an agar dilution method. Additional data provided for these isolates include the BLASTn identity of their respective ITS sequences, their province of origin, and previous phosphonate exposure history. The history of phosphonate exposure was ascertained through interviews with growers at sampled orchards and nurseries.

The sheet named "2 In vitro trials" contains the data collected from the in vitro phosphonate sensitivity trials conducted by an agar dilution method, using 50 identified P. nicotianae isolates, two different phosphonates namely ammonium phosphite (NH4-P) and potassium phosphite (K-P), five different concentrations of these phosphonates (0, 25, 75, 150, 600, 1250 ug/ml), three replicates and two trials. The average of two perpendicular measurements of radial colony diameter (minus the 8mm diameter agar plug) was used to calculate percentage inhibition, which was then logit transformed and regressed against the concentrations to calculate EC₅₀ from the regression equation. 

The data in the sheet named "2 In vitro trials EC₅₀" represents the post-hoc analysis, specifically the Fisher’s protected t-LSD (Least Significant Difference) that was calculated to compare the EC₅₀ means of significant effect. Means that do not share t-groupings, or t-grouping ranges, are significantly different at the 5% significance level. The sheet named "2 In planta trials soil CFU" contains data from the in planta phosphonate sensitivity trials on soil colony forming units of P. nicotianae in different pots (subsamples) per block of different treatments (NH4-LS = Ammonium phosphite sprayed plants inoculated with the pooled least sensitive isolates; NH4-MS = Ammonium phosphite sprayed plants inoculated with the pooled most sensitive isolates; NH4-uninoculated = Ammonium phosphite sprayed plants that are not inoculated; K-LS = Potassium phosphite sprayed plants inoculated with the pooled least sensitive isolates; K-MS = Potassium phosphite sprayed plants inoculated with the pooled most sensitive isolates; K-uninoculated = Potassium phosphite sprayed plants that are not inoculated; Unsprayed-LS = Unsprayed plants that have been inoculated with the pooled least sensitive isolates; Unsprayed-MS = Unsprayed plants that have been inoculated with the pooled most sensitive isolates; Unsprayed-Uninoculated = Unsprayed plants that have not been inoculated). The sheet named "2 In planta trials root collars" contains data from the in planta phosphonate sensitivity trials on root dry mass, root wet mass, and root collar diameter measurements of two plants (subsamples) per block for the same treatments listed under the In planta trials soil CFU data. 

The sheet named "2 In planta trials qPCR" contains the data on P. nicotianae DNA concentrations in harvested plant roots for the same treatments listed under the in planta trials above. DNA concentrations refer to concentrations of the ras-related protein (Ypt1) gene, as determined by qPCR. The given concentrations (column F) were those of standards used in standard curve construction. The quantification cycle (Cq) (column D), calculated concentration (column G), and % variation (column H) data, as well as the slope and y-intercept, were generated by Q-qPCR version 1.0.2. The sample DNA was diluted by a factor of five (column I) to reduce the effect of qPCR inhibitors. The actual concentrations (column J) of three single-sample biological replicates of the same treatments listed under the in planta trials, were calculated by multiplying the calculated concentrations by the dilution factor. Statistical analysis was performed on the actual concentrations. The sheet named "2 In planta trials temperatures" contains the temperature data recorded with a HOBO data logger throughout the in planta trials. The sheet named "3 In vitro mycelial growth", contains data from the in vitro assay of mycelial growth rate. The average of two perpendicular measurements of the radial colony growth (minus the 8mm agar plug diameter) was calculated for each of three petri dishes (subsamples), together representing a replicate. Isolates 137, 121, and 146 were categorised as 'Sensitive' and isolates 89, 302, and 193 as 'Reduced Sensitivity'. 

The sheet named "3 In vitro sporangia", contains data from the in vitro assay of sporangium production capacity of the same six isolates from the in vitro assay of mycelial growth rate. The number of sporangia at the edge of the upper surface of an agar plug (subsample), of which there were 4 on a microscope slide (representing a replicate), were counted. The sheet named "3 In vitro stability", contains data from the in vitro assay of the stability of reduced phosphonate sensitivity. The same six isolates from the previously described sheet were subcultured onto new non-selective media for 10 successive generations and subsequently, these 10th generation isolates, along with their original 'parent' strains (indicated by a letter 'P' following the isolate number), had their EC₅₀ values to ammonium phosphite determined by an agar dilution method, as previously described. The sheet named "3 In planta soil CFU" contains soil CFU data from the in planta trials that compared the fitness of the same six isolates (137, 121, 146, 89, 302, 193). However, due to Fusarium oxysporum overgrowth (column G) on the media, no reliable data was generated for this variable. The sheet named "3 In planta root reisolati", contains data on the reisolation of P. nicotianae from root pieces from the in planta trials that compared the fitness of the same six isolates. However, due to Fusarium oxysporum overgrowth (column H) on the media, no reliable data was generated for this variable. 

The sheet named "3 In planta Fusarium id" contains data on the identity of the Fusarium contaminant mentioned above. The translation elongation factor 1-α (tef1) gene was sequenced and compared to sequences in the Genbank database using the BLASTn tool. The sheet named "3 In planta root mass coll" contains data on root wet mass and root collar diameter measurements taken during the in planta trials that compared the fitness of the aforementioned six isolates. There were two plants (subsamples) per treatment (isolate) per block. The sheets named "3 In planta qPCR Trial 1" and "3 In planta qPCR Trial 2" contain the data on P. nicotianae DNA concentrations in harvested plant roots for the same in planta trials that compared the fitness of the aforementioned six isolates. DNA concentrations were determined by qPCR, as previously described. The sheet named "3 In planta temperatures" contains the temperature data recorded with a HOBO data logger throughout the in planta trials that compared the fitness of the aforementioned six isolates.