Phytophthora

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)].

In order to find substantial expansions and contractions of gene families observed in other eukaryotes, we used the PANTHER Classification System [49 (link),137 (link),138 ]. We first scored all predicted proteins from the P. ultimum genome against the PANTHER HMMs, and created a tab-delimited file with two columns: the P. ultimum protein identifier and the PANTHER HMM identifier from the top-scoring HMM (if E-value < 0.001). We created similar files for three Phytophthora genomes (Ph. infestans, Ph. ramorum, and Ph. sojae), and a diatom genome (P. tricornutum) for comparison. We removed protein families of probable viral origin or transposons (PTHR19446, PTHR10178, PTHR11439, PTHR23022, PTHR19303). This left 7,762 P. ultimum proteins in PANTHER families, 8,169 from Ph. infestans, 7,667 from Ph. ramorum and 7,701 from Ph. sojae. We then uploaded the tab-delimited files to the PANTHER Gene List Comparison Tool [137 (link),139 ] and analyzed the list for under- and over-representation of genes with respect to molecular functions, biological processes, and pathways. For each class that was significantly different (Bonferroni-corrected P < 0.05) between P. ultimum and all of the Phytophthora genomes, we determined the protein family expansions or contractions that made the biggest contributions to these differences (Table 1). Finally, we determined likely gene duplication and loss events that generated the observed protein family expansions and contractions by building phylogenetic trees of each of these families using the 48 genomes included in the trees on the PANTHER website [140 ], in addition to the five stramenopile genomes above (P. ultimum, Ph. infestans, Ph. ramorum, Ph. sojae, P. tricornutum). Phylogenetic trees were constructed using the GIGA algorithm [141 (link)], which infers the timing of likely gene duplication events relative to speciation events, allowing the reconstruction of ancestral genome content and lineage-specific duplications and losses. Using v3 of the annotation (MAKER output without manual curation), P. ultimum genes orthologous to genes in Ph. infestans, Ph. sojae and Ph. ramorum were identified using PHRINGE ('Phylogenetic Resources for the Interpretation of Genomes') [103 ] in which the evolutionary relationships among all oomycete protein families are reconstructed.

Preliminary screening assay was performed to estimate inhibitory effect of AgNPs against 12 phytopathohenic fungi, namely Alternaria alternata IOR 1783 (isolated from kohlrabi), Botrytis cinerea IOR 1873 (isolated from tomato), Colletotrichum acutatum IOR 2153 (isolated from blueberry), Fusarium oxysporum IOR 342 (isolated from pine), Fusarium solani IOR 825 (isolated from parsley), Phoma lingam IOR 2284 (isolated from rape), Sclerotinia sclerotiorum IOR 2242 (isolated from broccoli), and oomycetes, such as Phytophthora cactorum IOR 1925 (isolated from strawberry), Phytophthora cryptogea IOR 2080 (isolated from Lawson cypress), Phytophthora megasperma IOR 404 (isolated from raspberry), Phytophthora plurivora IOR 2303 (isolated from Quercus petraea) using agar well-diffusion method (Magaldi et al., 2004 (link)), with some modifications. Briefly, fungal colonies grown on potato dextrose agar (PDA, Becton Dickinson) in Petri plates for 14 days at 26°C were washed with 10 ml of sterile distilled water to release fungal spores/sclerotia. Their suspensions were collected and filtered through a sterile cotton wool syringe filter to remove mycelia. The concentration of fungal spores/sclerotia were estimated using cell counting chamber (Brand, Germany) and diluted to adjust concentration of 10⁶ spores mL⁻¹. One milliliter of such suspension was added into 6 ml of sterile melted PDA and spread on the surface of sterile medium in Petri plates, as a second layer. Subsequently, the wells (Ø =5 mm) were cut in the inoculated plates using sterile cork borer and filled with 50 μl of AgNPs solution at concentration of 3 mg mL⁻¹. Then, inoculated plates were incubated for 7 days at 26°C and zones of inhibition of fungal growth around wells were measured in mm.

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.

Monitoring activities were conducted during spring 2022 on five natural Alnus glutinosa stands located in the central part of Portugal, the districts of Aveiro and Guarda (Table 1). The altitude of survey sites ranged from 9 to 750 m. a.s.l.
At each site, mature alder trees were visually checked for the presence of typical Phytophthora disease symptoms, including wilting of foliage, shoot and twigs dieback, sudden death, bleeding cankers, and root and collar rot. In Sites 2 and 3, four linear transects of 50 m were randomly established to evaluate disease incidence and mortality rate, expressed as the number of symptomatic trees out of the total number of trees (DI = n/N × 100) and the number of dead trees out of the total number of trees (M = d/N × 100), respectively [19 ].
At each site, representative trees were randomly chosen for sampling (Table 1). Rhizosphere soil samples (about 1 L of soil and fine roots) were collected around the collar of 38 declining alder trees. Among these, eight trees were chosen for the collection of bark tissue samples, taking small fragments from the border of bleeding cankers on the stem. In Sites 2 and 3, the occurrence of Phytophthora species was also monitored in the water streams using nylon mesh bags containing 10 young cork oak (Quercus suber L.) leaves as bait [10 (link),20 ]. The nylon mesh bags were positioned near the root systems of the selected alder trees.