L of 72 plants (eight reisolation times after inoculation multiplied by nineL of 72 plants

L of 72 plants (eight reisolation times after inoculation multiplied by nine
L of 72 plants (eight reisolation times after inoculation multiplied by nine replicates) was used to investigate vascular colonization, and 20 plants (four plants per each time point) were used for RNA extraction and transcriptomic analysis.Vascular colonizationAfter inoculation, seedlings were monitored for fungal colonization along the stem by reisolation. The experiment was concluded at 21 dpi, when all plants undergoing the compatible interaction (Charentais Fom-2 Pinometostat molecular weight inoculated with race 1,2) displayed obvious and severe wilting symptoms. Nine plants for each strain were cut at the stem base 1, 2, 4, 8, 14, 16, 18 and 21 dpi, and were defoliated. After discarding the basal 15 mm, the stems were cut into 12 sections, each 5 mm in length, to a maximum height of 75 mm. Sections were placed on PDA, incubated PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 at room temperature for 8 days and examined every day for the appearance of fungal outgrowths. A completely randomized distribution was adopted for the melon plants kept in the greenhouse as well as for the Petri-dishes with stem sections incubated in the laboratory.Data analysistotal of the four classes. Follow-up tests of the differences between times were performed by considering all possible pairwise contrasts. In this case, to avoid inflation of the type I error rate, a Bonferroni-corrected significance level of P 0.0018 (P < 0.05 divided by 28, which is the total number of possible contrasts between the eight reisolation times) was calculated and used as minimum nominal P-value to obtain an actual P 0.05 value. The statistical results refer to the analysis performed on the total of the four height classes for each strain. To characterize the continuity of the distribution of the fungus along the stem, a continuity index was calculated based on the reisolation data. The index was determined for each plant by considering the presence or absence of the fungus in the pairs of subsequent stem sections and assigning a value of 1 when the fungus was reisolated or not reisolated in both sections and a value of 0 when it was reisolated only in one of the two sections. The index was then calculated by averaging the obtained values.RNA extraction procedureVascular colonization was scored according to the frequency of successful reisolation in stem sections arranged in four height classes measured from the stem base: 15-30 mm, 30-45 mm, 45-60 mm and 60-75 mm. Percentage values grouped in the four height classes were subjected to a two-way ANOVA for each height class and for the total of the four classes. The two factors considered were strain and time (dpi). The data did not match the parametric ANOVA requirements (normal data distribution and homogeneous variance) with any transformation, so the non-parametric Monte Carlo permutation test was used instead. The probabilities of the main effects of each factor were generated by restricting permutations within the levels of the other factor [80], whereas the interaction between strain and time was tested by unrestricted permutations after the calculation of residuals [81]. The statistical test used for the main factors was the sum of squares between groups (Q b ), whereas the test used for interaction was the pseudo F-ratio (F = Q b /Q w , where Q w is the sum of squares within groups). Because of interactions between factors present in all five two-way ANOVA tests, the effect of time was tested separately for each strain in a one-way ANOVA either for each height class or for theFor each pla.