DOI: 10.25403/upresearchdata.26262473
title: In vitro and in planta data on the phosphonate sensitivity and fitness of Phytophthora nicotianae isolates from citrus orchards and nurseries across South Africa

description: 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 <em>In vitro</em> isolates" contains only the <em>Phytophthora nicotianae</em> isolates that were screened for their <em>in vitro</em> 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.
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The sheet named "2 <em>In vitro</em> trials" contains the data collected from the <em>in vitro</em> phosphonate sensitivity trials conducted by an agar dilution method, using 50 identified <em>P. nicotianae</em> 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 EC<sub>50</sub> from the regression equation. 
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The data in the sheet named "2 <em>In vitro</em> trials EC<sub>50</sub>" represents the post-hoc analysis, specifically the Fisher’s protected t-LSD (Least Significant Difference) that was calculated to compare the EC<sub>50</sub> 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 <em>In planta</em> trials soil CFU" contains data from the <em>in planta</em> phosphonate sensitivity trials on soil colony forming units of <em>P. nicotianae</em> 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 <em>In planta</em> trials root collars" contains data from the <em>in planta</em> 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 <em>In planta</em> trials soil CFU data. 
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The sheet named "2 <em>In planta</em> trials qPCR" contains the data on <em>P. nicotianae</em> DNA concentrations in harvested plant roots for the same treatments listed under the <em>in planta</em> trials above. DNA concentrations refer to concentrations of the ras-related protein (<em>Ypt</em>1) 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<em> in planta</em> trials, were calculated by multiplying the calculated concentrations by the dilution factor. Statistical analysis was performed on the actual concentrations. The sheet named "2 <em>In planta</em> trials temperatures" contains the temperature data recorded with a HOBO data logger throughout the <em>in planta</em> trials. The sheet named "3 <em>In vitro </em>mycelial growth", contains data from the <em>in vitro</em> 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'. 
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The sheet named "3 <em>In vitro</em> sporangia", contains data from the <em>in vitro </em>assay of sporangium production capacity of the same six isolates from the <em>in vitro</em> 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 <em>In vitro</em> stability", contains data from the <em>in vitro </em>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 EC<sub>50</sub> values to ammonium phosphite determined by an agar dilution method, as previously described. The sheet named "3 <em>In planta</em> soil CFU" contains soil CFU data from the <em>in planta </em>trials that compared the fitness of the same six isolates (137, 121, 146, 89, 302, 193). However, due to <em>Fusarium oxysporum </em>overgrowth (column G) on the media, no reliable data was generated for this variable. The sheet named "3 <em>In planta</em> root reisolati", contains data on the reisolation of <em>P. nicotianae </em>from root pieces from the <em>in planta </em>trials that compared the fitness of the same six isolates. However, due to <em>Fusarium oxysporum </em>overgrowth (column H) on the media, no reliable data was generated for this variable. 
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The sheet named "3 <em>In planta</em> <em>Fusarium</em> id" contains data on the identity of the <em>Fusarium </em>contaminant mentioned above. The translation elongation factor 1-α (<em>tef1</em>) gene was sequenced and compared to sequences in the Genbank database using the BLASTn tool. The sheet named "3 <em>In planta</em> root mass coll" contains data on root wet mass and root collar diameter measurements taken during the <em>in planta </em>trials that compared the fitness of the aforementioned six isolates. There were two plants (subsamples) per treatment (isolate) per block. The sheets named "3 <em>In planta</em> qPCR Trial 1" and "3 <em>In planta</em> qPCR Trial 2" contain the data on <em>P. nicotianae</em> DNA concentrations in harvested plant roots for the same <em>in planta </em>trials that compared the fitness of the aforementioned six isolates. DNA concentrations were determined by qPCR, as previously described. The sheet named "3 <em>In planta</em> temperatures" contains the temperature data recorded with a HOBO data logger throughout the <em>in planta</em> trials that compared the fitness of the aforementioned six isolates.