Pythium

The P. ultimum genome annotations were created using the MAKER program [110 (link)]. The program was configured to use both spliced EST alignments as well as single exon ESTs greater than 250 bp in length as evidence for producing hint-based gene predictions. MAKER was also set to filter out gene models for short and partial gene predictions that produce proteins with fewer than 28 amino acids. The MAKER pipeline was set to produce ab initio gene predictions from both the repeat-masked and unmasked genomic sequence using SNAP [111 (link)], FGENESH [112 (link)], and GeneMark [113 (link)]. Hint-based gene predictions were derived from SNAP and FGENESH.
The EST sequences used in the annotation process were derived from Sanger and 454 sequenced P. ultimum DAOM BR144 ESTs [31 (link)] considered together with ESTs from dbEST [114 (link)] for Aphanomyces cochlioides, Phytophthora brassicae, Phytophthora capsici, Phytophthora parasitica, Ph. sojae, Ph. infestans, and Pythium oligandrum. Protein evidence was derived from the UniProt/Swiss-Prot protein database [115 (link),116 (link)] and from predicted proteins for Ph. infestans [28 (link)], Ph. ramorum [27 (link)], and Ph. sojae [27 (link)]. Repetitive elements were identified within the MAKER pipeline using the Repbase repeat library [117 (link)] and RepeatMasker [45 (link)] in conjunction with a MAKER internal transposable element database [118 (link)] and a P. ultimum specific repeat library prepared for this work (created using PILER [119 (link)] with settings suggested in the PILER documentation). Ab initio gene predictions and hint-based gene predictions [110 (link)] were produced within the MAKER pipeline using FGENESH trained for Ph. infestans, GeneMark trained for P. ultimum via internal self-training, and SNAP trained for P. ultimum from a conserved gene set identified by CEGMA [110 (link)].
Following the initial MAKER run, a total of 14,967 genes encoding 14,999 transcripts were identified, each of which were supported by homology to a known protein or had at least one splice site confirmed by EST evidence. Additional ab initio gene predictions not overlapping a MAKER annotation were scanned for protein domains using InterProScan [120 (link)-122 (link)]. This process identified an additional 323 gene predictions; these were added to the annotation set, producing a total of 15,290 genes encoding 15,322 transcripts (referred to as v3). Selected genes within the MAKER produced gene annotation set were manually annotated using the annotation-editing tool Apollo [123 (link)]. The final annotation set (v4) contained 15,297 genes encoding 15,329 transcripts, including six rRNA transcripts.
Putative functions were assigned to each predicted P. ultimum protein using BLASTP [124 (link)] to identify the best homologs from the UniProt/Swiss-Prot protein database and/or through manual curation. Additional functional annotations include molecular weight and isoelectric point (pI) calculated using the pepstats program from the EMBOSS package [125 (link)], subcellular localization predicted with TargetP using the non-plant network [126 (link)], prediction of transmembrance helices via TMHMM [127 (link)], and PFAM (v23.0) families using HMMER [128 ] in which only hits above the trusted cutoff were retained. Expert annotation of carbohydrate-related enzymes was performed using the Carbohydrate-Active Enzyme database (CAZy) annotation pipeline [68 (link)].

We identified gene families in S. japonica by performing an all-against-all BLASTp search against the protein sequences of 25 algae and plants with available whole-genome information. Among these algae, 13 belonged to Chromalveolata (S. japonica, E. siliculosus, Nannochloropsis gaditana, Nannochloropsis oceanica, Aureococcus anophagefferens, Phaeodactylum tricornutum, Thalassiosira pseudonana, Albugo laibachii, Phytophthora infestans, Pythium ultimum, Saprolegnia parasitica, Emiliania huxleyi and Guillardia theta CCMP2712); 1 was Rhizaria (Bigelowiella natans); 1 was Glaucophyta (Cyanophora paradoxa); 5 were Rhodophyta (Chondrus crispus, Cyanidioschyzon merolae, Galdieria sulphuraria, Porphyridium purpureum and Pyropia yezoensis); 3 were Chlorophyta (Chlamydomonas reinhardtii, Coccomyxa subellipsoidea and Volvox carteri); and 2 were plants (Arabidopsis thaliana and Physcomitrella patens).
The global protein identities of each BLAST match were calculated using InParanoid56 (link) to filter out matches exhibiting poor similarities (<20%) or poor gene coverage (<50%). Gene families were identified by MCL57 (link) with the ‘inflation' option as 1.2. Gene numbers were counted for each family and for each species, and were compared by using the K-means algorithm in R. To obtain a robust species tree, redundant sequences (90% identity or more) from the same organism were removed using CD-HIT58 (link), then, homologue clusters were predicted by comparing each pair of the 25 algal and plant genomes and further summarized by InParanoid and QuickParanoid. Subsequently, the clusters containing single-copy genes from each organism and clusters with species-specific duplications were selected for further consideration. For each cluster, multiple alignments were then performed using MUSCLE v3.8.31 (ref. 59 (link)) with the default parameters and were further trimmed using trimAl v1.4 (ref. 60 (link)) with the options ‘-gt 0.1 -resoverlap 0.75 -seqoverlap 80'. RAxML61 (link) was employed to reconstruct a maximum-likelihood phylogenetic tree for each cluster with an evolutionary model specified as ‘PROTCATJTT' and to perform a bootstrap significance test with 100 replicates. TreSpEx62 (link) was then applied to the tree of each cluster to evaluate taxa with long branches and to the trees of the 41 clusters with both long branch score heterogeneity and upper quartiles smaller than 30. The according clusters were selected for concatenation of their sequences within the same species into super genes.
A Dollo analysis was conducted on the homologue clusters of the 25 algal and plant proteomes using the Dollop tool in the PHYLIP package63 and custom Java scripts (available on request). To examine the evolutionary relationships between the duplicated homologues in the S. japonica genome before or after its divergence from E. siliculosus, synonymous (Ks) and non-synonymous (Ka) substitution rates were calculated using KaKs_Calculator64 (link). A gene function-enrichment analysis for the clustered genes was performed using Fisher's exact test to compare all of the genes in the S. japonica genome.

Phylogenetic trees of the isolated P. cactorum strain Pca-NJ-1 with other oomycetes including several Phytophthora pathogens and Pythium were constructed based on the ITS sequence using the maximum-likelihood (ML) method in MEGA 7.0 software.

The bacterial strains of E. coli (MTCC-118) and P. aeruginosa (MTCC-1035) were inoculated in Luria broth and were kept at 37 °C overnight to obtain an exponential growth phase to use for further study.
The THY-1 sequence was submitted to GenBank by Paul, B and Voglmayr, H (2013) under the GeneBank accession of KF806442. This strain was isolated from Nagpur, India, by Prof. Paul, where the aforementioned professor has graciously consigned it to the author. Although the oomycete strain THY-1 was named Pythium siamicum, it was never described as a new species. A BLAST search shows that it closely resembles Pythium nunn (97.74% identity, Globisporangium nunn). THY-1 was used for the microbial studies, and the strains were cultured in PDA slants, while fresh potato dextrose broth (PDB) was used for the inoculation process. All experiments were performed at 25 °C. Fungal species confirmation was performed using an 18s rDNA sequence. A fragment of amplified 18s rDNA was compared to the NCBI GenBank database.

A total of 166 core genes in the genomes of 10 Pythium insidiosum strains and 5 other Pythium species (Table S1) were used for phylogenetic analysis. These genes were selected by scanning through our homologous gene cluster data for genes present in all 15 genomes that did not possess length variations of more than 45% relative to the longest representative gene in each cluster (or each row in the Pythium Gene Table). Nucleotide sequences of each gene from all genomes were aligned using ClustalW version 2 with default parameters [36 (link)]. All ClustalW alignment results were then subjected to gap removal and concatenated to produce a single multiple-sequence alignment file. FastTree2 was then used to create a maximum-likelihood tree [37 (link)]. A bootstrap analysis was carried out to test the reliability of the tree. Finally, the phylogenetic tree was visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/; accessed on 4 July 2022).