Field evaluation
The shot hole disease survey showed highest disease incidence in P. domestica (62.5%) followed by P. persica (54.1%). We found shot hole disease incidence of 30.2, 35.2, and 12.2% in P. armeniaca, P. avium and P. dulcis, respectively. Being deciduous the leaves fall in winter and on the onset of spring the new flush of leaves appear on the tree. The disease incidence in April was low which gradually increased upto June. In the mid of June the lesions started falling down and left holes over the leaves and give ragged appearance to trees.
Diversity in Morphological and pathological characteristics
The fifty isolates cultured on two different media (Asthana hawker’s medium and PDA) showed difference in their colony characteristics. Our goal was to observe difference between the isolates of five different hosts however; the isolates of different hosts overlapped in morphological characteristics. We categorized 50 isolates into five groups (each for Asthana hawker’s medium and PDA) with Group I having velvety growth with uniform margins on both the media. However, the colony colour on Asthana hawker’s medium was brown covered with blackish mass of spores and on PDA it was brownish white. The Group II showed flat to cottony growth with dark golden brown coloration on Asthana hawker’s medium. On PDA they showed fluffy colonies with uniform margins having grayish white centre surrounded by olivaceous regions. The Group III displayed dull white cottony growth on Asthana hawker’s medium and white fluffy cottony colonies with irregular margins on PDA. The Group IV showed brown colonies on Asthana hawker’s medium and cottony dull white to light brown centres surrounded by black mass of spores on PDA. The Group V showed white fluffy growth on Asthana hawker’s medium and grayish fluffy colonies with uniform margins on PDA (Supplementary Table 1.). The pathogen produces light brown to dark brown oval septate (3–5 septa) conidia and conidial size ranges from 15.98–40.20 x 7.20-15.28 µm (Fig. 1.).
Based on the pathogenicity test, incubation time varied from four to seven days for the development of disease symptoms. The lesions developed were brown with circular to slightly irregular shape. The P. persica isolates showed incubation period of 4, 4, 5, 5 and 4 days on Peach, Plum, Apricot, Sweet cherry and Almond hosts respectively. The isolates obtained from P. domesticashowed incubation period of 5, 4, 4, 7 and 6 days on Peach, Plum, Apricot, Sweet cherry and Almond hosts respectively. Isolates collected from P. armeniacaisolates showed incubation period of 5, 5, 4, 7 and 7 days on Peach, Plum, Apricot, Sweet cherry and Almond hosts respectively. Incubation period of 5, 5, 6, 6 and 7 days on Peach, Plum, Apricot, Sweet cherry and Almond hosts, respectively was shown by the isolates collected from P. avium whereas P.dulcisisolates showed incubation period of 5, 4, 6, 6 and 5 days on Peach, Plum, Apricot, Sweet cherry and Almond hosts respectively (Fig. 1). We could not find any significant pathological variability between the isolates of five different hosts that is evident from probability value (p = 0.29).
Genome sequencing
The whole genome sequence of W. carpophilusgenerated total of 9.0 Gb data comprising of 7.9Gbfrom IlluminaHiSeq and 1.1 Gb from PacBio sequencing platforms. The total genome size of W. carpophiluswas found to be 29.9Mb (Accession number: PRJNA791904). Under Biosample number (SAMN24657336) we classified fungus as Ascomycota, Saccharomyceta, Pezizomycotina, Leotiomyceta, Dothideomyceta, Dothideomycetes, Wilsonomyces. We found 130 scaffolds with maximum scaffold length of 2.1 Mb. The N50 (scaffold) of 662.3Kb was calculated in the genome. The total GC content of the genome was calculated to be 49.77%. The minimum read length was observed to be more than 200bp and less than 600bp and the maximum read length was greater than 5000bp (Supplementary Fig. 3.).
Gene prediction and annotation
We predicted a total of 10901 genes with a total gene length of 13.7 Mb. The average gene length was found to be 1263bp with the maximum gene length of 31.8 Kb. The maximum number of genes having gene length between 1000bp to 5000bp and minimum number of genes with gene length between 200 to 300bpwere observed (Supplementary Fig. 4). Gene Ontology (GO) annotation performed for all three GO domains revealed 4584 genes involved in biological processes, 4858 genes were associated with molecular functions and 3551 genes associated with the cellular components (Fig. 2). Under biological process, different categories such as, cellular metabolic process, response to oxidative stress, signaling and others were highly represented. However, other processes that include growth, carbon utilization, and biological adhesion were the least significant. On molecular grounds, the most significant activities were catalytic, binding, transporter, transcriptional regulator, and anti-oxidant activity, whereas, nutrient reservoir and toxin activities were the least represented. In the cellular component class, cell membrane, organelles, proteins containing complex etc. were highly abundant, and extracellular region, supramolecular complex and nucleoid were the least represented.
Prediction and secretome analysis
For successful infection, the plant pathogenic fungi release arsenal of secreted proteins, particularly effectors. A comprehensive pipeline was designed to carry out the prediction analysis of W. carpophilussecretome. Out of 10901 protein encoding genes, we found 225 potentially secreted proteins in W. carpophilus genome. We found abundant cell wall degrading enzymes (CWDE) such as cellulases, pectate lyases, cutinases, glycosidases, glucanases, carboxylesterases, laccases, endo-1, 4-beta-galactosidase, rhamnogalacturonanlyase, pectin-esterase, lipolytic protein etc. Besides CWDEs, we found chaperone proteins, mycelial catalase, ricin beta lectin, cathepsin, histidine kinases etc. in the genome (Supplementary Table 2)., These findings indicate that W. carpophilusis a necrotrophic plant pathogenic fungus and possess a set of enzymes that is suitable for degradation of cell wall components in the host plants.
Simple sequence repeats
Microsatellites (SSRs) have an active role in upholding genetic variations in genome evolution. These are the tandem repeats of nucleotide motifs of 2–6 base pair (bp) size. They are highly polymorphic and ubiquitously present in all the known genomes. SSRs were identified in different scaffold sequences with the help of MISA perl script. We identified 2851 SSRs with 937 dinucleotides, 1362 trinucleoids, 265 tetranucleotides and few penta and hexanucleotides. Trinucleotide motifs were the most abundant in number comprising of 47.7% of total SSRs in the genome. SSRs with 150 bp flanking region (upstream as well as downstream) were fetched with in-house python script which can be further used for primer designing. We found 2592 SSRs out of 2851 having 150bp flanking region.
Comparison with other fungal genomes
A total of 10901predicted proteins of different genes were identified, out of which, 9427 proteins showed hits in nrdatabase. Among 223 selected fungal species, the top-hit species distribution revealed that the majority of hits were found to be against the Pyrenochaeta species followed by Ascochyta rabiei (Fig. 3.). The Pyrenochaeta species and A. rabiei showed 1812 and 1391 hits, respectively against W. carpophilus genome. In Uniprot database we found 6331 protein hits, in KOG database 4378 protein hits were obtained and only 4272 proteins hits were observed in case of Pfam database (Fig. 4). The most abundant proteins identified in Pfam database were cytochrome p450, AMP (Anti-microbial peptides) binding genes, adh-shorts, heterokaryon incompatibility genes and kinases etc. Comparative analysis showed that genes annotated in UniProt, Pfam and KOG databases are in concordance with nr database. Among 10901 genes coding proteins, only 3142 genes were common to all databases (Fig. 5).
Scrutiny of metabolism
The proteins mapped to biological pathways represented several metabolic pathways of major biomolecules such as carbohydrates, lipids, nucleotides, amino acids, glycans, cofactors, vitamins, terpenoids, polyketides etc. We found 328 genes involved in carbohydrate metabolism followed by amino acid and lipid metabolism in which 287 and173 genes were involved respectively. However, very less number of genes were involved in terpenoid and polyketide metabolism. In case of genetic information of the pathogen genome, the high number genes were involved in translation, degradation, replication and repair. We also found a significant number of genes that play an important role in signal transduction, transport, catabolism and many other cellular processes of the pathogen (Supplementary Table 3).
Uncovering non-coding RNA species within the genome
We predicted a total of 146 tRNAs out of which only fifteen tRNAs were found on the scaffold one and rest of the tRNAs on the scaffold two (Supplementary Table. 4). We predicted thirty-three 8S rRNA, fifteen18S rRNA, three 28S rRNA and one 5S rRNA. We found these rRNA genes on 1, 10, 12, and 124 scaffolds respectively. In addition, we also predicted pseudogenes in the genome. We represented the all scaffolds, genes, tRNAs and rRNAs of W. carpophilusgenome in a circos plot (Fig. 6).