Computational protocol: Cladosporium cladosporioides and Cladosporium pseudocladosporioides as potential new fungal antagonists of Puccinia horiana Henn., the causal agent of chrysanthemum white rust

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Protocol publication

[…] The three gene sequences from each antagonist, Cladosporium cladosporioides and C. pseudocladosporioides, were aligned with the sequences available in GenBank (NCBI, USA). Our sequences were manually edited by CLCbio (Qiagen, USA). Sequence data obtained from Bensh et al. [,] were used as reference data for the alignments (). Multiple alignments were performed by ClustalW software and best nucleotide model determinate by jModelTest v. 2.1.7 [] using BIC criteria for each locus and then incorporating it in the analysis. A Bayesian phylogenetic inference tree was generated based on data from each partition sequence of the three genes on BEAST v.1.8.1 [] and Markov Chain Monte Carlo analysis, from four chains started from random tree topology and taken over 80 000 000 generations. Three independent runs were combined by LogCombiner v.1.8.1. Trees were saved each 1 000 generations, resulting in 80 001 saved trees. Using Tracer v.1.6, burn-in was set at 15 000 000 generations, after which the likelihood values were stationary. The coalescent algorithm with GTR+G+I substitution model and a lognormal uncorrelated relaxed clock was selected for the data. Maximum clade credibility tree was visualized by Fig Tree v. 1.4.2. For the stability and robustness of each species, Neighboring-Joining analysis was performed for each data partition, using MEGA 6.0 [] and 1000 replications using bootstrap. The ITS region has limited resolution for species in Cladosporium, therefore results for the ACT and EF-1α regions were used for comparison of clade stability (). [...] For the antagonism test, leaves infected (severity up 30%) and non-infected by chrysanthemum white rust, were collected from 20 different 30-day-old Chrysanthemum × morifolium cv. Polaris plants in a commercial greenhouse at Texcoco, México. The leaves were disinfested by immersion in sodium hypochlorite 3% for 3 min and then triple washed with sterilized water. There were five treatments: 1) antagonist 1 vs P. horiana (conidia were applied on pustules); 2) antagonist 2 vs P. horiana; 3) antagonist 1, conidia applied on healthy chrysanthemum leaves; 4) antagonist 2, conidia applied on healthy chrysanthemum leaves; and 5) control, P. horiana infected leaves treated with sterile water. Ten leaves per treatment were put into a humid chamber, each leaf representinged a repetition. A spore suspension (2×105 conidia mL-1) of each antagonist was sprayed onto pustules on diseased leaves and onto healthy chrysanthemum leaves. All treatments were incubated at 24°C and 12 h light/dark. When signs of antagonists appeared, a sample of the fungus was cultured on synthetic PDA (Bioxon, Mexico) to confirm that it was the fungus originally inoculated.After 96 h of incubation, antagonism percentages, measured as the proportion of pustules of P. horiana colonized by C. cladosporioides and C. pseudocladosporioides, were recorded. Differences in percentage were statistically tested by one-way ANOVA. To meet ANOVA assumptions, normal distribution was assessed by a Shapiro-Wilk test [P>0.05] and homogeneity of variance was evaluated by Levene’s test [P>0.05]. The differences among treatments were tested by post hoc Ryan-Einot-Gabriel-Welch based on an F test (REGW-F; P = 0.05). All statistical analyses were carried out using SPSS Statistics 21.0. […]

Pipeline specifications

Software tools CLC Assembly Cell, Clustal W, jModelTest, BEAST, MEGA, SPSS
Applications Miscellaneous, Phylogenetics
Chemicals Carbon