Whole genome sequencing of Wilsonomyces carpophilus, an incitant of shot hole disease in stone fruits: insights into secreted proteins of a necrotrophic fungal repository

Shot hole is one of the important fungal diseases in stone fruits viz., peach, plum, apricot and cherry caused by Wilsonomyces carpophilus and almond among nut crops. Fungicides significantly decrease the disease. Pathogenicity studies proved a wide host range of the pathogen infecting all stone fruits and almond among the nut crops, however, the mechanism underlying host-pathogen interaction is still unknown. Molecular detection of the pathogen using polymerase chain reaction (PCR) based simple sequence repeat (SSR) markers is also unknown due to the unavailability of the pathogen genome. We examined the morphology, pathology and genomics of the Wilsonomyces carpophilus. Whole genome sequencing of the W. carpophilus was carried out by Illumina HiSeq and PacBio high throughput sequencing plate-forms through hybrid assembly. Constant selection pressure alters the molecular mechanism of the pathogen causing disease. The studies revealed that the necrotrophs are more lethal with a complex pathogenicity mechanism and little-understood effector repositories. The different isolates of necrotrophic fungus W. carpophilus causing shot hole in stone fruits namely peach, plum, apricot and cherry, and almonds among the nut crops showed a significant variation in their morphology, however, the probability value (p = 0.29) suggests in-significant difference in the pathogenicity. Here, we reported draft genome of W. carpophilus of size 29.9 Mb (Accession number: PRJNA791904). A total of 10,901 protein-coding genes were predicted, including heterokaryon incompatibility genes, cytochrome-p450 genes, kinases, sugar transporters among others. We found 2851 simple sequence repeats (SSRs), tRNAs, rRNAs and pseudogenes in the genome. The most prominent proteins showing necrotrophic lifestyle of the pathogen were hydrolases, polysaccharide-degrading enzymes, esterolytic, lipolytic, and proteolytic enzymes accounted for 225 released proteins. Among the 223 fungal species, top-hit species distribution revealed the majority of hits against the Pyrenochaeta species followed by Ascochyta rabiei and Alternaria alternata. Draft genome of W. carpophilus is 29.9 Mb based on Illumina HiSeq and PacBio hybrid assembly. The necrotrophs are more lethal with a complex pathogenicity mechanism. A significant variation in morphology was observed in different pathogen isolates. A total of 10,901 protein-coding genes were predicted in the pathogen genome including heterokaryon incompatibility, cytochrome-p450 genes, kinases and sugar transporters. We found 2851 SSRs, tRNAs, rRNAs and pseudogenes, and prominent proteins showing necrotrophic lifestyle such as hydrolases, polysaccharide-degrading enzymes, esterolytic, lipolytic and proteolytic enzymes. The top-hit species distribution were against the Pyrenochaeta spp. followed by Ascochyta rabiei.


Introduction
With the expansion of our knowledge about threats posed by the plant pathogens, it is necessary to elucidate the molecular basis of plant disease mechanism. The use of fungicide is a common practice and management of plant diseases alone costs around $220 billion dollars annually [1]. Besides cost, fungicide resistance is a major concern to the plant pathologists [2]. Therefore, to devise an ecofriendly management capsule, it is mandatory to understand the underlying molecular mechanism and signaling events during the disease development. There are many studies related to secretome and virulence strategies of biotrophs [3], however, there is dearth of information about the necrotrophic genome repository. Although, the necrotrophs are far more damaging than biotrophs and cause tremendous production loss in different crops [4]. Among others, the Wilsonomyces carpophilus is a necrotrophic plant pathogenic fungus that cause notable damage to majority of Prunus species such as P. persica (Peach), P. domestica (Plum), P. armeniaca (Apricot), P. avium (Sweet cherry) and P. dulcis (Almond). The trees infected with the fungus show 30 to 90% production loss [5,6]. On the fruits, the disease symptoms appear as pinkish to reddish necrotic lesions. On leaves, reddish to purplish lesions are formed, which fall down and generate shot holes, giving the tree a ragged appearance due to the abscission layer production [7]. The disease has been documented from Africa, Asia, North, South, and Central America, Australia, and Oceania [8], and recently reported from Western Tianshan Mountains of China [9]. In India, the disease was reported for the first time from Kumaon (Uttarakhand) on different stone fruits such as peaches, apricots and cherries [10] and on almonds [11]. The shot hole disease from Kashmir was reported by Munjal [12]. Shot hole is a devastating disease on apricots and peaches during the rainy period (March and April) in Kashmir [13]. The disease is a major threat to stone fruit industry in Kashmir valley and becomes destructive under favourable weather conditions. The disease incidence of about 60-80 per cent was reported on leaves and developing fruits of cherry in Kashmir [14]. The disease showed darker coloration on infected buds than normal ones, and on twigs, initial spots develop as raised, small and purplish in colour which later expand into elongated necrotic areas [15]. Taxonomically, W. carpophilus belongs to the Dothideomycetes, Pleosporomycetidae, Pleosporales, Dothidotthiaceae group of fungi [16]. The taxonomy of the genus has been controversial over the long time and the pathogen is considered to have number of synonyms such as Stigmina carpophila, Coryneum beijerinkii, Clasterosporium carpophilum, Thyrostroma carpophilum, Sciniatosporium carpophilum and Sporocadus carpophilus [17,18], however, recent multigene phylogeny considered Wilsonomyces as a separate genus [16].
Despite of huge damage by the pathogen and taxonomic crises, there are only few studies in the literature. There is no information on fungal biology, secretome, SSRs (simple sequence repeats) and other metabolites related to pathogenicity. Furthermore, the pathogen necrotrophic life style and lack of host specificity necessitate the identification of pathogenicity arsenals, toxins, enzymes, and other secreted compounds for the disease development. It has been discovered that the metabolites or toxins produced by the necrotrophs excite rather than prevent programmed cell death (PCD), which can provide the pathogen with metabolizable substrate [19]. From this perspective, defining the role of pathogen-encoded secreted proteins in stimulating or dampening the plant defense system becoming essential.
To gain the insights about the poorly studied W. carpophilus fungus, we used the high throughput sequencing toolbox for better understanding of life-style and developmental dynamics of the fungus. In present work, we studied plant pathogenic fungus associated with various Prunus species at morphological, pathological and molecular level to discern the metabolites, toxins and enzymes released by the pathogen and to ascertain its necrotrophic behavior. Although, we provide an arrayed list of genes that might play a crucial role during the successful host colonization but many questions related to patho-system oblige additional research. However, recent optimization of random insertional mutagenesis protocol of the pathogen [20] can be used for functional validation of these pathogenicity arsenals that we reported in the present study. The present draft genome assembly is the first genome draft of W. carpophilus and will be helpful in understanding the host-pathogen interface in future.

Disease status and site of investigation
The survey was conducted to record the shot hole disease status on P. persica, P. domestica, P. armeniaca, P. avium and P. dulcis in the University orchard of Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Shalimar, Srinagar, Jammu and Kashmir (34.0837° N, 74.7973° E). We randomly selected the stone fruit trees and each tree was divided into north, south, east and west directions. About, thirty leaves were collected from all the directions of each host species and disease incidence was calculated by following formula: Number of inf ected leaves T otal number of leaves examined × 100

Isolation of the fungus from diseased samples
Fifty samples were brought to the laboratory to raise the fungal cultures. The infected leaves were thoroughly washed under running tap water to remove any dirt. After drying, the pathogen was isolated on Asthana and Hawker's medium following the procedure given by Nabi et al. [5]. Each Petri plate was incubated at 24 ± 1ºC under a 12-hour photoperiod in a light incubator for seven days. A 5 mm disc from each fungal culture was transferred to Asthana and Hawker's medium as well as on potato dextrose agar (PDA) medium [21]. To purify the cultures, we used single spore isolation technique described previously by Tuite [22].

Assessment of morphological characteristics
Fifty isolates from five different hosts were used for morphological characteristics such as growth pattern, colony colour and texture. The cultures incubated at 24 ± 1ºC under the 12-hour photoperiod were evaluated every seven days after inoculation. Conidial characteristics such as shape, size, colour and septation of different isolates were assessed using microscope previously calibrated with ocular and stage micrometer.

Pathogenicity test
Pathogenic variability of five isolates from five different hosts was studied under in vitro. A total of 25 treatment and treatment combinations for pathogenicity tests were conducted by inoculating each isolate on their respective hosts and on other host species following detached leaf technique [23]. The freshly collected healthy leaves were thoroughly washed with sterilized water and placed in a 200 × 200 mm moist chamber. Inoculations were carried out with spore (conidial) suspensions containing 10 5 spores/ ml by drop placement method [24] with the help of 20 µl micropipette. The inoculated leaved in moist chambers were then incubated at 24 ± 1ºC with 12-hour photoperiod. The control groups were mock inoculated with distilled water. The experiment was laid in a Complete Randomized Design (CRD) and the one-way ANOVA (Analysis of variance) was used to determine the pathological variability between the isolates of five hosts.

DNA isolation for genome sequencing
We selected P. persica (peach) isolate for whole genome sequencing based on its virulence and minimum incubation period for disease development. The DNA of the pathogen isolate was extracted using XcelGen DNA isolation Kit (Xceleris, Ahmedabad, India) according to the manufacturer's instructions. The quality and quantity of extracted DNA was ascertained using a Qubit 2.0 Fluorimeter (Life Technologies Ltd., Paisley, UK). The integrity of DNA (DIN) was checked using Bioanalyser 2100 (Agilent Technologies, Santa Clara, CA).

Library preparation and genome sequencing
The DNA Library was prepared using NEBNext Ultra DNA Library Prep Kit (Biolabs, England). The library preparation process was initiated with 200ng DNA. The adapters were ligated to both ends of the DNA fragments. These adapters contain sequences essential for binding dual-barcoded libraries to a flow cell for sequencing and PCR amplification. To ensure maximum yield from a limited amount of starting material, a high-fidelity amplification step was performed using PCR Master Mix. The whole genome of plant pathogenic fungus W. carpophilus was decoded using Illumina HiSeq and PacBio high throughput sequencing technologies. De Novo assembly of high quality paired end reads was accomplished using Velvet v1.2.10 [25] and the assembly was optimized at Kmer-79 ( Supplementary Fig. 1). Further, scaffolding was performed on pre-assembled contigs taking long reads of PacBio using SSPACE-LongRead v1.1 [26]. We aligned Illumina short reads on PacBio long reads (a hybrid approach) using PBJelly software [27] and GapCloser v1.12 to increase the precision of base calling.

Gene prediction and annotation
The assembled genome was subjected to gene prediction using Augustus v2.5.5 for the identification of coding sequences. The predicted protein coding genes were subjected to similarity search against NCBI's non-redundant (nr) database using Uniprot, KOG and Pfam database of BLASTp algorithm with an e-value threshold of 1e-5. Simultaneously, all the proteins were searched for similarity against BLASTp with an e-value threshold of 1e-5. Comparative analysis of gene annotation in different database was carried out using http://www.interactivenn.net/. Gene Ontology (GO) annotation was obtained using nr database through Blast2GO command line v-1.4.1. GO sequence distributions helps in specifying all the annotated nodes comprising of GO functional groups. Genes associated with the similar functions were assigned to same GO functional group. The GO sequence distribution was analyzed for all the three GO domains i.e. biological processes, molecular function and cellular components. lesions started falling down leaving a shot hole appearance on the leaves and give ragged appearance to the trees.

Diversity in morphological and pathological characteristics
The fifty isolates cultured on two different media (Asthana and Hawker's and PDA) showed difference in their colony characteristics. Our goal was to observe difference between the isolates from five different hosts, however, the isolates from different hosts overlapped in morphological characteristics. We categorized 50 isolates into five groups (each for Asthana and Hawker's and PDA medium) with Group I having velvety growth and uniform margins on both the media. However, the colony colour on Asthana and Hawker's medium was brown covered with blackish mass of spores and on PDA, it was brownish white in colour. The Group II showed flat to cottony growth with dark golden brown coloration on Asthana and Hawker's medium. On PDA, the isolates showed fluffy colonies with uniform margins having grayish white centre surrounded by olivaceous regions. The Group III displayed dull white cottony growth on Asthana and Hawker's medium and white fluffy cottony colonies with irregular margins on PDA medium. The Group IV showed brown colonies on Asthana and Hawker's medium and cottony dull white to light brown centres surrounded by black mass of spores on PDA medium. The Group V showed white fluffy growth on Asthana and 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 to 40.20 × 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 were brown with circular to slightly irregular in 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. domestica showed incubation period of 5, 4, 4, 7 and 6 days on peach, plum, apricot, sweet cherry and almond hosts, respectively. Pathogen isolates collected from P. armeniaca 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. dulcis isolates 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 was evident from probability value (p = 0.29).

Simple sequence repeats
A high-throughput SSR search using MIcroSAtellite (MISA) was performed to identify mono to hexa nucleotide SSR motifs (http://pgrc.ipk-gatersleben.de/misa/download/ misa.pl). The default parameters were used so that di-nucleotide pattern appearing at least six times, whereas tri, tetra, penta and hexa nucleotide motifs appearing five times.

Pathway analysis and identification of tRNAs and rRNAs
Pathway analysis, ortholog assignment and mapping of genes to the biological pathways were performed using KEGG automatic annotation server (KAAS). All the gene sequences were compared against the KEGG database using BLASTp with threshold bit-score value of 60 (default).
To identify probable tRNA genes, we used tRNAscan-SE that allows detection of unusual tRNA species with accurate prediction of secondary structures. It includes both prokaryotic and eukaryotic selenocysteine tRNA genes, tRNA-derived repetitive elements and pseudogenes. The RNAmmer 1.2 was used for rRNA gene identification.

Field evaluation
The shot hole disease survey showed the highest disease incidence on P. domestica (62.5%) followed by P. persica (54.1%). We found shot hole disease incidence of 30.2, 35.2, and 12.2% on P. armeniaca, P. avium and P. dulcis, respectively. Being deciduous in nature, the leaves fall in winter and on the onset of spring, the new flush of leaves appears on the tree. The disease incidence was low in the month of April which gradually increased upto June. After the second week of June, the maximum number of genes having gene length between 1000 and 5000 bp and minimum number of genes with gene length between 200 and 300 bp were observed (Supplementary Fig. 4). Gene Ontology (GO) annotation performed for all the 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 the 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, membrane enclosed lumen were highly abundant, and extracellular region, supra-molecular complex and nucleoid were the least represented (Fig. 2).

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

Genome sequencing
The whole genome sequence of W. carpophilus generated a total of 9.0 Gb data comprising of 7.9 Gb from Illumina HiSeq and 1.1 Gb from PacBio sequencing platforms. The total genome size of W. carpophilus was found to be 29.9 Mb (Accession number: PRJNA791904). Under Bio-sample 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 scaffold length distribution was ranging between < 200 bp and > 5000 bp. The maximum scaffolds (90-100) were in read length greater than 5000 bp followed by 20-25 scaffolds in length ranging between > 1000 and < 5000 bp, and 4-5 scaffolds were in read length of > 200 bp but < 600 bp, whereas the least scaffolds were observed between read length > 600 bp and < 1000 bp (Supplementary Fig. 3).

Gene prediction and annotation
We predicted a total of 10,901 genes with a total gene length of 13.7 Mb. The average gene length was found to be 1263 bp with the maximum gene length of 31.8 Kb. The

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 bp. 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 di-nucleotides, 1362 tri-nucleotides, 265 tetra-nucleotides and few penta-and hexa-nucleotides. Tri-nucleotide 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 150 bp flanking region. analysis of W. carpophilus secretome. Out of 10,901 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, carboxyl-esterases, laccases, endo-1, 4-beta-galactosidases, rhamnogalacturonan lyases, rhamnogalacturonases, pectin-esterases, lipolytic protein, endoglucanases, phospholipases and phytases. Besides CWDEs, we found chaperone proteins, actin-like proteins, mycelial catalase, ricin beta lectin, cathepsin, histidine kinases, galactose oxidases, carbonic anhydrases, aliphatic nitrilases and FAD binding domain-containing protein in the genome (Supplementary Table 2). These findings indicate that W. carpophilus is a necrotrophic plant pathogenic fungus and possess a set of enzymes that is suitable for degradation of cell wall components in the host plants.

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, protein families, secondary metabolites, and xenobiotics. We found 328 genes involved in carbohydrate metabolism followed by amino acid and lipid metabolism in which 287 and 173 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

Comparison with other fungal genomes
A total of 10901predicted proteins of different genes were identified, out of which, 9427 proteins showed hits in nr database. 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, antimicrobial peptides (AMP) binding genes, adh-shorts, heterokaryon incompatibility genes and kinases. Comparative analysis showed that the genes annotated in UniProt, Pfam and KOG databases were in concordance with nr database.  Table 4). We predicted thirty-three 8 S rRNA, fifteen18S rRNA, three 28 S rRNA and one 5 S 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 all the scaffolds, genes, tRNAs and rRNAs of W. carpophilus genome in a circos plot (Fig. 6).

Discussion
In present study, we investigated the Wilsonomyces carpophilus on morphological, pathological, and genomic level to understand the pathogenicity mechanism of the pathogen and discover its necrotrophic fungal features. The pathogen displayed morphological variations such as colony colour, texture and sporulation on Asthana and Hawker's and potato dextrose agar media, which is usually attributed to differences in geographic locations and environmental effects; however, we collected the isolates from only one location and assume that it could be due to the differences in plant age, plant resistance, and the presence of different pathotypes [28]. The another possible reason could be repeat induced point (RIP) mutation in the genes that leads to variation in isolates and is evident from the presence of pseudogenes in the W. carpophilus genome [29,30]. The pathogen isolates collected from five different hosts were subjected to cross pathogenicity tests which showed difference in incubation period. The incubation period varied from 4 to 7 days 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   [9]. Besides, we explored the pathogen on genomic grounds using HTS technology (Illumina HiSeq and PacBio) to gain insights on the complex molecular mechanism of the pathogen. We found 10,901 protein coding genes using gene prediction algorithm in the genome. When these genes were blasted against the nr database, most of the tophits were against the Pyrenochaeta spp. followed by Ascochyta rabiei. The protein-coding genes in W. carpophilus are almost similar in number (10,901) compared when inoculated on their respective hosts and on other host species. The peach isolate showed the least incubation period when compared to other isolates collected from other host species using two-way ANOVA. Our results are in conformity with the reports given by different researchers who showed either minimum or varied incubation period incubation period in the pathogen on peach host [20,31]. On the basis of incubation period, the peach isolate was selected for whole genome sequencing. The pathological variability in the pathogen indicates that it can Fig. 6 Schematic representation assembled genome. Circle A and Circle B represents gene predicted in the positive frame. Circle C represents gene predicted in the negative frame, Circle D represents tRNA (red tiles) and rRNA (blue tiles). The inner E circle represents distribution of GC content. The red shade in the E circle represents regions of low GC contents (less than 35%) precursor that plays an important role in lignin de-polymerization of the infected host [44] and number of other enzymes such as endo-1, 4-beta-galactosidase, cutinase, rhamnogalacturon amlyase, pectin-esterase, pectate lyase, lipolytic protein, galactose oxidase, carbonic anhydrase and aliphatic nitrilase play a decisive role in the pathogenicity mechanism. However, these pathogenicity determinants need further characterization and validation. The present study revealed the number of pathogenicity genes that can be easily targeted to render pathogen ineffective. Our research opened new opportunities for the comprehensive genomic study of a variety of biological, metabolic and pathological aspects that make the W. carpophilus a successful necrotrophic pathogen. Therefore, it is an opportune time to go beyond the conventional neutral genetics by identifying, analyzing, site specific targeting of pathogenicity determinants and re-modelling the core effector repositories.
to the genome of the necrotrophic fungal pathogen Ascochyta rabiei (10,596) [3], however, Pyrenochaeta spp. have relatively higher protein coding genes and it could be due to wide range of species in the genus infecting both plants and humans [32,33].
In nature, the sexual stage of W. carpophilus rarely exists [34,35]. It has been observed that the pathogen with broader host range loose sexuality over a time and exhibit a greater degree of asexuality than those with narrow host ranges [36]. Thus the variability in the fungus could be potentially due to the vegetative hyphal fusion or anastomosis, and is evident from the presence of abundant heterokaryon incompatibility (HET) genes in the W. carpophilus genome. The viable heterokaryon is formed when individuals have same set of HET genotype, whereas individuals with different HET genotypes form incompatible vegetative heterokaryon [37]. The other key genes deployed in the pathogen genome were antimicrobial peptide (AMP) binding genes that are used by the pathogen to overcome host defense. These AMPs are the part of plant's innate immunity system against the pathogen attack. The pathogen has an ability to bind these genes in order to surpass plant defense system. The abundant AMP binding genes in the pathogen suggest that it encounters many host AMPs that ultimately facilitates pathogen to infect wide range of species [38]. The other abundant genes in the pathogen genome were cytochrome P450, pKinases, sugar transporters and adh-shorts. Among them, cytochrome P450 genes are involved in the degradation of plant derived toxins and therefore plays an important role in fungal colonization eventually in the pathogenesis [39]. Higher number of CYPs also indicates the wide host range of the pathogen requiring more toxins to overcome the phytoalexins [3]. The presence of abundant sugar transporters in the pathogen genome suggests accumulation of sugars [40] which in turn increases abscisic acid levels in an infected cell [41] that can be the possible reason for abscised shot holes symptoms on the leaves of host though it needs an additional research for validation. The protein kinases in the pathogen genome plays key role in various processes of the fungal life cycle such as growth direction, nutrient uptake, stress responses and reproduction [42] thus can be an important factor for survival of the pathogen.
Nowadays, necrotrophs are gaining more attention due to new theories put forth on the mechanism of the host pathogen dialogue. The W. carpophilus can be an excellent model to study pathogenicity mechanism of the wide host range necrotrophs. The presences of cell wall degrading enzymes such as glycosidases, glucanases, pectatelyases, cellulases, endo-glucanases, phospholipases, rhamno-galacturonases and phytases in the W. carpophilus genome are more often predicted in necrotrophs and shows its stronger resemblance to the necrotrophic plant pathogens. Other secretory proteins such as chaperone proteins called as heat shock proteins play an important role in maintaining the integrity of the pathogen during the adverse conditions, thus making it more viable for the host infection [43]. Laccasse