Exploring the Genome of the Endophytic Fungus Botrytis deweyae: Prediction of Novel Secondary Metabolites Gene Clusters: Terpenes and Polyketides

Abstract

Fungi have played a pivotal role in human history, from the dangers of fungal toxins to the revolutionary discovery of penicillin. Fungal secondary metabolites (SMs), such as polyketides (PKs) and terpenes, have attracted considerable interest due to their diverse biological activities. Botrytis deweyae, an endophytic fungus, exhibits behaviors that are notably distinct from those of its necrotrophic relatives within the genus Botrytis. This study explores the importance of terpenes and PK gene clusters and their conservation between species. In addition, new putative biosynthetic gene clusters corresponding to those families were identified. Consequently, the new PKS BdPKS22-26 were also identified in other Botrytis species and other fungi. In addition, those new gene clusters identified in this work show differences in the degree of conservation and are phylogenetically closely related to some of the 21 PKSs previously described in the reference strain Botrytis cinerea B05.10. Moreover, a new gene cluster related to terpenes in B. deweyae B1 and B. cinerea B05.10 was also identified that had never been detected before. This new gene cluster is well conserved among other Botrytis species in many phylogenetically distant fungal lineages. Understanding the genetic basis and conservation of these putative biosynthetic gene clusters sheds light on the metabolic potential and ecological roles of B. deweyae and related fungal species.

1. Introduction

1.1. Fungal Secondary Metabolites

Fungi have a profound connection with humans, highlighted by significant events such as the Turkey X disease outbreak in the 1960s [1] and the discovery of penicillin [2]. The antibiotic era, initiated by penicillin derived from the Penicillium fungus [3,4], transformed healthcare practices worldwide [3].

Fungi exhibit remarkable metabolic adaptability, producing secondary metabolites (SMs) through complex biosynthetic pathways [5,6]. These SMs serve as signaling molecules and defense mechanisms, providing a competitive advantage in natural environments [7]. Their importance extends to applications in medicine, agriculture, and biotechnology [8,9]. Common SM categories include terpenes, polyketides, non-ribosomal peptides, and indoles [10,11].

1.2. The Genus Botrytis

The Botrytis genus comprises ubiquitous fungal pathogens that significantly impact agriculture, horticulture, and ecology [12]. Notably, Botrytis cinerea is infamous for causing gray mold disease, affecting various crops like grapes, strawberries, and vegetables, leading to substantial yield and quality losses [13,14]. This pathogen poses challenges for growers as it can infect plants both pre- and post-harvest [15,16].

Ecologically, Botrytis serves as a necrotrophic pathogen, contributing to nutrient cycling and decomposition [14,17]. Species within this genus exhibit diverse interactions with plant hosts, ranging from benign to virulent pathogenicity, showcasing their ecological versatility [18]. The genetic diversity and metabolic adaptability of Botrytis enable it to evade host defenses and thrive in varying environments [19,20].

Ongoing research into its biology and genetics offers promising strategies for disease management, with advances in genomic tools providing insights into its life cycle and host interactions [14,21].

1.3. Botrytis Deweyae

B. deweyae stands out in the Botrytis genus for its combination of endophytic and facultative necrotrophic behaviors, differing from typical necrotrophic traits [18,22]. Initially isolated from Hemerocallis plants with “spring sickness”, it causes deformities, stunted growth, chlorosis, and necrotic lesions post-winter [22]. While research interest has been limited until recently, reports of its impact date back to the 1970s [22].

Genetic analysis reveals a close relationship between B. deweyae, Botrytis elliptica, and Botrytis squamosa [22]. Although primarily targeting Hemerocallis, it has a polyphagy index of 1, indicating a more restricted host range than its relatives [15]. B. elliptica is the second most polyphagous species after B. cinerea [15]. Recent observations suggest that B. deweyae may be expanding its ecological niche, with reports of gray mold on Polygonatum cyrtonema in China [23].

1.4. Role of Terpenes and Polyketide Synthases in Secondary Metabolism

1.4.1. Terpenes

Terpenoids, derived from isopentenyl diphosphate (IPP), are synthesized via the mevalonate and deoxyxylulose 5-phosphate pathways [24,25]. The mevalonate pathway, predominant in fungi, converts mevalonate to IPP, which is crucial for terpenoid biosynthesis [25]. Terpenoid biosynthesis begins with IPP and dimethylallyl diphosphate (DMAPP), leading to various terpenoid classes through enzymatic transformations [26,27,28,29,30,31]. Terpenoids have diverse biological activities, including hormonal functions and mycotoxin production [32,33,34].

1.4.2. Polyketide Synthases

Polyketides (PKs) are a prominent family of secondary metabolites initiated by the condensation of acetyl-CoA units [35,36]. Polyketide synthases (PKSs) are classified into three major classes based on structure and function: Type I, Type II, and Type III [35,37]. Type I PKSs are multifunctional enzymes resembling fatty acid synthetases, while Type II PKSs are multienzymatic complexes found mainly in prokaryotes [38,39,40,41,42,43]. Type III PKSs are less diverse and primarily involved in plant defense [44,45,46,47]. The structural diversity of PKs is further enhanced by auxiliary enzymes [37].

This study aims to investigate the genomic profiles of B. deweyae to identify gene clusters responsible for secondary metabolite biosynthesis, particularly terpenes and polyketides. By comparing with B. cinerea, we seek to understand genetic variability and phylogenetic relationships of these clusters, alongside their ecological and functional significance, enhancing our knowledge of metabolic diversity within the genus Botrytis and its implications for disease management and biotechnological applications. However, it is important to point out that these results are predictive and, without validation, remain theoretical; therefore, validation would need to be carried out for confirmation.

2. Materials and Methods

2.1. Genome Data Acquisition

The genome data used in this study were sourced from the National Center for Biotechnology Information (NCBI), with accession numbers GCF_000143535.2 for Botrytis cinerea B05.10 [48,49] and GCF_014898535.1 for Botrytis deweyae B1 [50].

2.2. Genome Set Completeness Assessment

We utilized BUSCO v. 5.7.1 in genome modes, employing the fungi_odb10 dataset, which includes 758 BUSCO groups. This analysis assessed the completeness of the predicted protein set and the genome assembly. We compared the BUSCO scores with those obtained for the Botrytis cinerea genome [51]. The alignment of both genomes was visualized by performing a syntenic plot using the TBtools-II tool [52]. The core genes corresponding to the secondary metabolism of B. deweyae that showed collinearity with B. cinerea were represented in the plot (Supplementary Figure S1).

2.3. Secondary Metabolite Gene Cluster Analysis

Secondary metabolite gene cluster analysis was performed using antiSMASH v. 7.0 (fungal version) software [53]. AntiSMASH is an integrated tool designed to identify putative biosynthetic gene clusters (BGCs) within fungal genomes, providing comprehensive annotations that predict the presence and type of secondary metabolites these clusters may produce (Supplementary Tables S1 and S2). We set up default parameters, which facilitates the identification of potential secondary metabolite gene clusters.

2.4. Domain Analysis

Domain analysis was carried out using the Conserved Domain Database (CDD) alongside the Pfam v. 35 database [54]. Pfam is an extensive collection of protein families, represented by multiple sequence alignments and hidden Markov models (HMMs), facilitating the identification and functional characterization of protein domains. When Pfam did not yield results, the InterPro (https://www.ebi.ac.uk/interpro/ (accessed on 30 July 2024)) database was employed. InterPro aggregates predictive models from multiple sources, including Pfam, PRINTS, PROSITE, and SMART, offering a thorough functional analysis of proteins [55,56].

2.5. Phylogenetic Analysis

Phylogenetic relationships were inferred using sequences from Bd22-26 and XP_038807933.1, which served as queries in a BLASTP search against the NCBI non-redundant protein sequence database [57]. Homologous proteins were selected from the resulting list following specific criteria: percent identity (>50%), coverage (>70%), and the bit-score (>50) [58,59]. The maximum likelihood method was employed to infer evolutionary history [60], utilizing 55 homologous proteins, except where fewer than 50 sequences were identified, in which case all sequences were included. The optimal tree was displayed, and evolutionary distances were computed using the Poisson correction method [60]. This analysis involved 57 amino acid sequences, with ambiguous positions removed for each sequence pair (pairwise deletion option). MEGA v. 11 software was used for these evolutionary analyses [61].

2.6. Homologous Protein Identification

Identification of homologous proteins was performed using a BLASTP search with stringent criteria: percent identity (>50%), coverage (>70%), and bit-score (>50) [58,59]. The search targeted the non-redundant protein sequence database, focusing on Botrytis (taxid: 33196), Botryotinia (taxid: 40558), and a broader search across fungi (taxid: 4751). This comprehensive approach ensured the identification of homologous proteins not only within the Botrytis genus but also across a wide range of fungal species, providing a broader context for comparative analysis.

3. Results

3.1. Genome Completeness Assessment

BUSCO (v5.7.1) analysis (Supplementary Table S3) at the genomic level revealed that 99.1% of the fungi dataset was retrieved in full length. Additionally, 0% of the BUSCO genes for fungi, respectively, were identified as duplicated, indicating that the genome was not assembled in a haploid state with no significant evidence of gene duplications (Supplementary Table S3). B. cinerea B05.10 gene content completeness was comparable to that of B. deweyae B1 based on BUSCO metrics even though B. deweyae B1 was a little bit more fragmented.

3.2. Comparative Analysis of Secondary Metabolite Gene Clusters in B. deweyae and B. cinerea

The comparison of secondary metabolite gene clusters between B. deweyae B1 and B. cinerea B05.10 reveals distinct differences in their metabolic capabilities. B. deweyae B1 contains a greater number of polyketide synthase (PKS) clusters, with 11 identified gene clusters compared to 10 in B. cinerea B05.10. Similarly, the number of terpene biosynthesis clusters is higher in B. deweyae B1, with six gene clusters, compared to five in B. cinerea B05.10 (Figure 1).

These differences indicate a higher diversity in the secondary metabolism of B. deweyae. The identified PKS clusters in B. deweyae include several novel clusters that have not been previously described, suggesting potential new pathways for secondary metabolite production. The analysis of these gene clusters provides insights into the unique metabolic capabilities of B. deweyae, which may contribute to its ecological roles and interactions with host plants.

Figure 1 illustrates the distribution of secondary metabolite gene clusters identified in both B. deweyae and B. cinerea. The increased number of clusters in B. deweyae highlights its potential for producing a broader range of secondary metabolites, which could be crucial for its adaptation and survival in diverse environmental conditions.

3.3. Terpene Gene Clusters in B. deweyae

In this section, we focus on the identification and characterization of terpene gene clusters in B. deweyae. The analysis revealed several key findings, highlighting the diversity and potential functional roles of these clusters.

Table 1. Comparison of Terpene Gene Clusters in B. deweyae and B. cinerea.

GC	Gene ID B. deweyae	Protein ID B. deweyae	Protein Length in B. deweyae	Gene ID B. cinerea	Protein ID B. cinerea	Protein Length in B. cinerea	% Similarity	Annotation
1	EAE98_002948	XP_038813097.1	482	BCIN_06g02400	XP_001560441.1	482	93.57%	Bcerg9
2	EAE98_003830	XP_038811923.1	437	BCIN_04g03550	XP_001546971.2	441	79.18%	Bcstc4
3	EAE98_006162	XP_038810179.1	278	BCIN_11g06510	XP_024551950.1	321	80.37%	Bcstc7
4	EAE98_008016	XP_038807933.1	394	BCIN_14g01170	XP_024552819.1	381	96.33%	Unknown
5	EAE98_008221	XP_038807710.1	399	BCIN_12g06390	XP_024552383.1	399	87.72	Bcbot2
6	EAE98_010975	XP_038805111.1	611	BCIN_01g04560	XP_024546243.1	610	90.51%	Bcphs1

GC: Gene Cluster. Protein lengths are measured in amino acids (aa). Gene IDs and Protein IDs are sourced from the NCBI database.

presents a detailed comparison of the terpene gene clusters identified in B. deweyae and B. cinerea. The table includes gene IDs, protein IDs, protein lengths, and percentage similarities, providing a comprehensive overview of the similarities and differences between these species.

This table highlights that B. deweyae contains a higher number of unique terpene gene clusters compared to B. cinerea, suggesting a more diverse metabolic potential. Notably, the presence of novel gene clusters in B. deweyae indicates potential new pathways for secondary metabolite production, which could play crucial roles in the organism’s ecological interactions and adaptability.

3.3.1. Description of Terpene Gene Clusters

The identified terpene gene clusters in B. deweyae B1 include several well-conserved genes, such as Bcerg9, Bcstc4, Bcstc7, Bcbot2, and Bcphs1, along with an unannotated gene. Bcerg9 encodes for a key enzyme in the ergosterol biosynthesis pathway, essential for maintaining cell membrane integrity [62]. Bcstc4 and Bcstc7 are involved in the biosynthesis of specific sesquiterpenoids: (3R,6E)-nerolidol and (+)-α-bisabolol (for Bcstc4 gene) and (+)-4-epi-eremophil-9-en-11-ol (for Bcstc7 gene) [63,64]. Bcbot2 is associated with the synthesis of botrydial, a phytotoxic sesquiterpene produced by B. cinerea [65]. Bcphs1 plays a role in the biosynthesis of retinal, an aldehyde form of vitamin A [66]. These clusters are crucial for the biosynthesis of various terpenoid compounds, which are known for their diverse biological activities.

In B. cinerea B05.10, gene clusters corresponding to the sesquiterpene family such as Bcstc2 (BCIN_08g02350) and Bcstc5 (BCIN_01g03520) have been detected. However, the gene Bcstc2 is not present in the genome of B. deweyae B1, while the protein BcSTC5 has two homologous proteins, EAE98_005930 and EAE98_005931, with 36% and 63% identity, respectively. The union of these two proteins gives rise to BcSTC5 in B. cinerea B05.10, indicating a putative annotation error in the case of B. deweyae B1.

Figure 2 illustrates the phylogenetic relationships of all the genes that present the IPR008949—Isoprenoid synthase domain superfamily, terpene synthases identified in B. deweyae and their homologs in B. cinerea. The phylogenetic tree was inferred using the maximum likelihood method via MEGA 11 software, with bootstrap values from 1000 trials indicated at each branch node.

BdSTC4, BcSTC3, BdBOT2, BcSTC5, BcERG9, and BdSTC7 are closely related, forming part of the same subclade. On the other hand, BcPAX1, BcCOQ1, BcERG20, and BdUnannotated (XP_038807933.1, which forms a cluster in both B. cinerea B05.10 and B. deweyae B1 according to the antiSMASH cluster prediction tool) are found in another subclade belonging to the same clade 1 (Figure 2). Likewise, for the protein named BcUnannotated the most closely related protein is BcStc2 (Figure 2).

The phylogenetic analysis reveals that the terpene synthases in B. deweyae are closely related to those in B. cinerea, suggesting a shared evolutionary history. However, the presence of unique genes in B. deweyae indicates species-specific adaptations and potential new putative biosynthetic capabilities.

3.3.2. Description of EAE98_008016 Gene in B. deweyae

The EAE98_008016 gene in B. deweyae presents a unique aspect of the species’ genomic architecture, differentiating it from B. cinerea. This gene, identified via the AntiSMASH tool, is part of a cluster predicted to encode an unannotated terpene (BdUnannotated protein) (Figure 3). Notably, this gene cluster does not contain additional genes in either B. deweyae or B. cinerea B05.10 (Figure 3).

The EAE98_008016 gene encodes a protein (XP_038807933.1) with two polyprenyl synthetase domains located between amino acids 316-870 and 858-1097. It shows a 96.33% similarity to the BCIN_14g01170 gene, which encodes the protein XP_024552819.1, in B. cinerea (Table 1).

Comparative analysis reveals homologous proteins across various Botrytis species, indicating conservation of this gene. This protein has homologs in all Botrytis species whose genomes have been sequenced and annotated (

Table 2. Distribution of homologous proteins to XP_038807933.1 of B. deweyae within the Botrytis genus. * means that there are no homologous proteins in this species annotated, but there is a region on its draft genome that could encode the XP_038807933.1 protein in this species.

Botrytis Species	Strain	GeneBank Genome Reference	Protein Accession Number	Number of Amino Acids	Percentage of Identity with B. deweyae
B. cinerea	T4	GCA_000227075.1	CCD33657.1	394	96.70%
B. cinerea	BcDW1	GCA_000349525.1	EMR82980.1	394	96.45%
B. cinerea	Bc448	GCA_037039525.1	KAK6598033.1	381	96.59%
B. cinerea	B05.10	GCA_000143535.4	XP_024552819.1	381	96.33%
B. elliptica	Be9612	GCA_014898555.1	KAF7925222.1	394	99.75%
B. sinoallii	Bc 23	GCA_014898435.1	XP_038758944.1	394	99.24%
B. hyacinthi	Bh0001	GCA_004786245.1	TGO37690.1	394	98.22%
B. aclada	633	GCA_014898285.1	KAF7953262.1	381	98.95%
Botryotinia convoluta	MUCL 11595	GCA_004786275.1	TGO54160.1	381	98.95%
B. byssoidea	MUCL 94	GCA_014898295.1	XP_038734128.1	381	98.95%
B. fragariae	BVB16	GCA_013461495.1	XP_037193855.1	381	98.69%
B. tulipae	Bt9001	GCA_004786125.1	TGO06850.1	381	98.69%
B. paeoniae	Bp0003	GCA_004786145.1	TGO30745.1	381	98.43%
Botryotinia globosa	MUCL 444	GCA_014898425.1	KAF7887299.1	381	98.43%
Botryotinia narcissicola	MUCL 2120	GCA_004786225.1	TGO61103.1	381	98.43%
Botryotinia calthae	MUCL 2830	GCA_004379285.1	TEY75198.1	381	97.11%
B. porri	MUCL 3234	GCA_014898465.1	XP_038765908.1	381	95.80%
B. porri	MUCL 3349	GCA_004786265.1	TGO84949.1	381	95.54%
B. squamosa	MUCL 31421	GCA_014898485.2	RCTC02000009.1 *	-	94.40%
B. medusae	B555	GCA_019395255.1	JAHXJL010000116.1 *	-	93.66%
B. pseudocinerea	BP362	GCA_019395245.1	JAHXJK010000109.1 *	-	92.16%
B. fabae isolate	DLY-16-612	GCA_004335055.1	RSAG01000202.1 *	-	91.42%

). B. cinerea, one of the most studied species in the genus, is represented by multiple strains including T4, BcDW1, Bc448, and B05.10. These strains exhibit high percentages of identity with B. deweyae, ranging from 96.33% to 96.70%, suggesting close genetic relationships. B. elliptica strain Be9612 shows an even higher percentage of identity (99.75%), indicating a very close genetic similarity to B. deweyae B1 (Table 2). Other strains, such as B. sinoallii (Bc 23 strain) and Botrytis hyacinthi (Bh0001 strain), also display high levels of identity, with percentages of 99.24% and 98.22%, respectively (Table 2). Several strains from different species exhibit identities above 98%, including Botrytis aclada (633 strain), Botryotinia convoluta (MUCL 11595 strain), Botrytis byssoidea (MUCL 94 strain), and Botrytis fragariae (BVB16 strain), among others. In contrast, species such as Botryotinia calthae (MUCL 2830 strain), Botrytis porri (MUCL 3234 and MUCL 3349 strains), and B. squamosa (MUCL 31421 strain) exhibit lower percentages of identity, ranging from 91.42% to 97.11% (Table 2).

On the other hand, some strains, such as Botrytis medusae (B555 strain), Botrytis pseudocinerea (BP362 strain), and Botrytis fabae (DLY-16-612 strain), have not been fully characterized in terms of amino acid sequences (Table 2). However, regions on the draft genomes of B. pseudocinerea, B. fabae, and B. medusae homologous to the XP_038807933.1 protein show percentages of identity with the XP_038807933.1 protein of B. deweyae ranging from 91.42% to 93.66% (Table 2).

3.3.3. Comparative Analysis of EAE98_008016 Protein with Other Fungal Species

In addition to the comparative analysis within the Botrytis genus,

Table 3. Distribution of the XP_038807933 protein (EAE98_008016) gene across the Fungi Kingdom.

A. Phylum Ascomycota.
Clade	Class	Order	Number of Proteins	Number of Organisms
Sordariomyceta	Leotiomycetes	Heliotales	75	61
		Unclassified Leotiomycetes	1	1
		Leotiales	1	1
		Leotiomycetes incertae sedis	27	23
		Erysiphales	15	11
	Sordariomycetes	Togniniales	3	2
		Diaporthales	32	18
		Magnaporthales	26	7
		Sordariales	93	67
		Coniochaetales	6	5
		Chaetosphaeriales	2	1
		Ophiostomatales	8	5
		Cephalothecales	2	1
		Xylariales	227	122
		Unclassified Xylariomycetidae	2	2
		Xylariomycetidae incertae sedis	4	1
		Hypocreales	546	259
		Glomerellales	159	88
		Microascales	7	6
		Sordariomycetes incertae sedis	2	1
		Lulworthiales	1	1
Leotiomyceta	Lecanoromycetes	Acarosporales	4	3
		Rhizocarpales	2	1
		Lecanorales	22	12
		Peltigerales	5	4
		Caliciales	3	2
		Teloschistales	47	20
		Unclassified Lecanoromycetidae	1	1
		Ostropomycetidae incertae sedis	2	1
		Umbilicariales	4	3
		Trapeliales	15	12
		Pertusariales	3	3
		Sarrameanales	2	2
		Ostropales	4	4
		Lecanoromycetes incertae sedis	1	1
	Coniocybomycetes	Coniocybales	3	2
	Xylonomycetes	Xylonales	2	1
	Candelariomycetes	Candelariales	4	4
	Sareomycetes	Sareales	1	1
	Lichinomycetes	Lichinales	4	4
	Xylobotryomycetes	Xylobotryales	2	1
	Geoglossomycetes	Geoglossales	5	4
	Eurotiomycetes	Eurotiales	1116	230
		Onygenales	110	70
		Chaetothyriales	95	41
		Verrucariales	3	2
		Phaeomoniellales	1	1
	Unclassified Eurotiomycetes		1	1
Dothideomyceta	Dothideomycetes	Mycosphaerellales	251	45
		Dothideales	138	20
		Myriangiales	7	6
		Cladosporiales	6	4
		Capnodiales	2	2
		Trypetheliales	8	3
		Botryosphaeriales	47	16
		Acrospermales	2	1
		Lineolatales	1	1
		Dothideomycetes incertae sedis	13	8
		Eremomycetales	2	1
		Patellariales	1	1
		Phaeotrichales	1	1
		Mytilinidiales	4	3
		Pleosporomycetidae incertae sedis	2	2
		Pleosporales	216	109
		Aulographales	2	2
		Venturiales	15	5
	Arthoniomycetes	Arthoniales	2	2
Saccharomyceta	Pezizomycotina incertae sedis	Thelocarpales	2	2
		Vezdaeales	2	2
		Pezizomycotina incertae sedis	2	2
	Pezizomycetes	Pezizales	34	26
	Orbiliomycetes	Orbiliales	35	15
	Saccharomycetes	Saccharomycetales	45	23
Taphrinomycotina incertae sedis			3	1
-	Taphrinomycetes	Taphrinales	3	2
B. Phylum Basidiomycota.
Class		Order	Number of Proteins	Number of Organisms
Microbotryomycetes		Leucosporidiales	1	1
		Sporidiobolales	20	10
		Unclassified Microbotryomycetes	1	1
		Microbotryales	6	4
		Kriegeriales	1	1
		Microbotryomycetes incertae sedis	1	1
Mixiomycetes		Mixiales	3	1
Cystobasidiomycetes		Cyphobasidiales	1	1
Pucciniomycetes		Pucciniales	40	17
Atractiellomycetes		Atractiellales	1	1
Agaricomycetes		Agaricomycetes incertae sedis	196	135
		Agaricales	233	140
		Jaapiales	1	1
		Boletales	62	34
		Atheliales	2	2
		Amylocorticiales	2	1
		Geastrales	2	1
		Hysterangiales	3	1
		Gomphales	2	2
Tremellomycetes		Filobasidiales	6	4
		Trichosporonales	18	8
		Tremellales	99	73
		Cystofilobasidiales	3	1
Dacrymycetes		Dacrymycetales	2	2
Exobasidiomycetes		Exobasidiales	3	2
		Tilletiales	13	5
Ustilaginomycetes		Ustilaginales	23	12
		Violaceomycetales	1	1
Malasseziomycetes		Malasseziales	14	9
Wallemiomycetes		Wallemiales	6	1
C. Phylum Mucoromycota.
Class		Order	Number of Proteins	Number of Organisms
Mucoromycetes		Mucorales	194	57
Umbelopsidomycetes		Umbelopsidales	13	5
Endogonomycetes		Endogonales	3	2
Mortierellomycetes		Mortierellales	88	51
Glomeromycetes		Glomerales	21	9
		Diversisporales	18	10
		Paraglomerales	3	2
		Archaeosporales	3	3
		Entrophosporales	12	2
D. Phylum Zoopagomycota.
Class		Order	Number of Proteins	Number of Organisms
Basidiobolomycetes		Basidiobolales	1	1
Entomophthoromycetes		Entomophthorales	7	2
Kickxellomycetes		Kickxellales	160	137
Dimargaritomycetes		Dimargaritales	11	6
Harpellomycetes		Harpellales	3	3
Kickxellomycotina incertae sedis		Ramicandelaberales	1	1
Zoopagomycetes		Zoopagales	4	4
E. Phylum Chytridiomycota.
Class		Order	Number of Proteins	Number of Organisms
Chytridiomycetes		Synchytriales	4	2
		Spizellomycetales	14	8
		Rhizophlyctidales	3	2
		Chytridiomycetes incertae sedis	3	3
		Cladochytriales	3	2
		Chytridiales	7	7
		Lobulomycetales	2	2
		Rhizophydiales	9	5
		Polychytriales	2	1
Monoblepharidomycetes		Monoblepharidales	3	3
Neocallimastigomycetes		Neocallimastigales	5	5
F. Phylum Chytridiomycota.
Class		Order	Number of Proteins	Number of Organisms
Blastocladiomycota		Blastocladiomycetes	10	5
		Physodermatomycetes	1	1

provides a comprehensive overview of the taxonomic distribution of fungi, focusing on the phyla Ascomycota, Basidiomycota, Chytridiomycota, Mucoromycota, Zoopagomycota, and Blastocladiomycota, in relation to the unannotated gene EAE98_008016 from B. deweyae. This breakdown reveals the diversity of fungal species and their distribution across various taxonomic levels, including phyla, classes, and orders. Additionally, the table includes the number of proteins and organisms associated with each taxonomic group, providing valuable insights into the functional and ecological diversity of fungi.

Ascomycota is the largest phylum represented, with 3547 protein sequences across 1419 organisms. The Sordariomyceta clade stands out with 1239 identified proteins and 683 organisms (Table 3A). Within this clade, the Leotiomycetes class includes significant orders such as Heliotales, with 75 proteins in 61 organisms, and Erysiphales (Table 3A). The Sordariomycetes class shows high protein diversity, especially in orders like Xylariales, Hypocreales, and Glomerellales, which have 227, 546, and 159 proteins, respectively, across 122, 259, and 88 organisms (Table 3A).

The Leotiomyceta clade follows closely, with 1462 proteins in 431 organisms. The class Eurotiomycetes is prominent, particularly the order Eurotiales, which has 1116 proteins in 230 organisms (Table 3A). Other notable orders within Eurotiomycetes include Onygenales and Chaetothyriales, which contribute significantly to the protein diversity with 110 and 95 proteins, respectively (Table 3A). Additionally, less common orders like Verrucariales and Phaeomoniellales are represented by fewer proteins and organisms (Table 3A).

Lecanoromycetes, another class within Ascomycota, includes orders such as Teloschistales, Lecanorales, and Trapeliales, each showing varied protein counts and organism numbers (Table 3A). Other classes such as Coniocybomycetes, Xylonomycetes, and Candelariomycetes exhibit lower but still notable protein counts and organism diversity (Table 3A).

The Dothideomyceta clade, with 720 proteins and 232 organisms, represents a diverse group of fungi that includes many plant pathogens, saprobes, and lichenized fungi. The class Dothideomycetes covers a wide range of orders with varying protein counts and organism numbers (Table 3A). For instance, Mycosphaerellales has 251 proteins in 45 organisms, while Dothideales has 138 proteins across 20 organisms. Other orders like Myriangiales, Cladosporiales, and Trypetheliales have fewer proteins and organisms but still contribute to the overall diversity (Table 3A).

Saccharomyceta, the smallest clade in terms of both protein count and organism number, comprises 126 proteins distributed among 73 organisms (Table 3A). This clade, encompassing various orders within the Pezizomycotina subphylum, demonstrates diverse protein counts and organism numbers. Notably, Pezizales has 34 proteins in 26 organisms, and Orbiliales has 35 proteins across 15 organisms (Table 3A). Saccharomycetales exhibits 45 proteins in 23 organisms, showcasing the range of diversity within Saccharomyceta (Table 3A).

Taphrinomycotina incertae sedis, a class within the Taphrinomycotina subphylum, shows limited protein diversity and organism representation, with only three proteins identified in a single organism (Table 3A). In contrast, Taphrinomycetes in the order Taphrinales demonstrates slightly higher protein diversity with three proteins in two organisms (Table 3A). Despite its modest representation, this class highlights the presence of lesser-known fungal taxa, emphasizing the need for further exploration and study.

Basidiomycota, the second-largest phylum, includes classes such as Microbotryomycetes, Agaricomycetes, and Tremellomycetes (Table 3B). Agaricomycetes is particularly diverse, featuring orders like Agaricales, Boletales, and Tremellales (Table 3B). Agaricales is especially prominent, with a high number of proteins and organisms, indicating its ecological and functional importance within the phylum (Table 3B).

Within Microbotryomycetes, various orders demonstrate diverse protein counts and organism numbers (Table 3B). Sporidiobolales stands out with 20 identified proteins across 10 organisms, showing significant protein diversity (Table 3B). Other orders like Leucosporidiales, Microbotryales, and Kriegeriales exhibit lower protein counts and organism numbers (Table 3B).

The class Tremellomycetes includes orders such as Filobasidiales, Trichosporonales, and Tremellales. Tremellales shows higher protein diversity with 99 proteins across 73 organisms. Other orders within Tremellomycetes exhibit varying protein counts and organism numbers, contributing to the overall diversity within the class (Table 3B).

Other phyla, although less represented, still exhibit significant diversity. Chytridiomycota and Mucoromycota include notable orders like Mucorales with 194 proteins in 57 organisms and Mortierellales with 88 proteins in 51 organisms (Table 3C,E). Chytridiomycetes includes orders such as Spizellomycetales and Rhizophlyctidales, each contributing to the overall diversity.

Zoopagomycota and Blastocladiomycota, though less studied, also show interesting diversity (Table 3D,F). Kickxellales within Zoopagomycota stands out with 160 proteins in 137 organisms. The order Blastocladiales in Blastocladiomycota contains 10 proteins in five organisms, adding to the functional variety within these phyla.

In summary, the taxonomic analysis of the EAE98_008016 protein across different fungal phyla reveals extensive diversity. This diversity reflects the wide range of ecological roles and metabolic capabilities present within the fungal kingdom, providing valuable insights into their functional and ecological significance.

On the other hand, regarding the phylogenetic relationship, the XP_038807933 protein (encoded by the EAE98_008016 gene; Supplementary Table S4) from B. deweyae B1 is closely related to proteins of species from the genus Sclerotinia and Stromatinia, with both proteins located in the same subclade (Figure 4). Additionally, XP_038807933 is phylogenetically related to proteins from fungi of the genera Ciborinia, Monilinia, Rustroemia, Chlorociboria, Bisporella, Claussenomyces, Coleophoma, Amorphotheca, Xylogone, Hyphodiscus, Hyaloscypha, Diplocarpon, Marssonina, Drepanopeziza, Rhynchosporium, Cadophora, Rhexocercosporidium, Leptodontidium, Lachnellula, Glarea, Amylocarpus, Venustampulla, Halenospora, and Hymenoscyphus (Figure 4). Moreover, the top 55 sequences selected for the phylogenetic analysis belong to species from the Helotiales order. Among these top hits, one sequence corresponds to an unclassified Leotiomycetes species, Xylogone sp. PMI_703. Another sequence is from Leotiomycetes sp. MPI-SDFR-AT-0126 is closely related to species of the genus Cadophora. Lastly, Claussenomyces sp. TS43310 is classified within the Leotiales order.

3.4. Polyketide Gene Clusters in B. deweyae

In this section, we focus on the identification and characterization of polyketide gene clusters in B. deweyae. The analysis revealed several key findings, highlighting the diversity and potential functional roles of these clusters.

Table 4. Comparison of Polyketide Gene Clusters in B. deweyae and B. cinerea.

GC	Gene ID in B. deweyae	Protein ID in B. deweyae	Protein Length in B. deweyae	Gene ID B. cinerea	Protein ID B. cinerea	Protein Length in B. cinerea	% Similarity	Annotation
1	EAE98_002293	XP_038813652.1	2542	-	-	-	-	Unknown
2	EAE98_005009	XP_038811280.1	2579	BCIN_05g08400	XP_024549041.1	2571	83.33%	Bcpks21
3	EAE98_006691	XP_038809559.1	2575	BCIN_16g05040	XP_024554044.1	2575	88%	Bcpks16
4	EAE98_007035	XP_038808800.1	499	BCIN_13g02130	XP_001555327.1	499	93.99%	Bcchs1
5	EAE98_009027	XP_038807018.1	2537	-	-	-	-	Unknown
6	EAE98_009190	XP_038806877.1	2346	-	-	-	-	Unknown
7	EAE98_009473	XP_038806567.1	2007	BCIN_05g06220	XP_024548906.1	2126	84.49%	Bcpks15
8	EAE98_010322	XP_038805735.1	2415	BCIN_13g01510	XP_001557060.1	2417	94.08%	Bcpks10
9	EAE98_010696	XP_038805323.1	2143	BCIN_03g08050	XP_001547095.2	2138	96.08%	Bcpks13
10	EAE98_010906	XP_038805042.1	2208	-	-	-	-	Unknown
11	EAE98_010943	XP_038805079.1	2353	-	-	-	-	Unknown

GC: Gene Cluster. Protein lengths are measured in amino acids (aa). Gene IDs and Protein IDs are sourced from the NCBI database.

presents a detailed comparison of the polyketide gene clusters identified in B. deweyae and B. cinerea. The table includes gene IDs, protein IDs, protein lengths, and percentage similarities, providing a comprehensive overview of the similarities and differences between these species.

This table reveals that B. deweyae harbors six putative polyketide gene clusters not found in B. cinerea, indicating a greater metabolic diversity. These unique clusters (gene IDs: EAE98_002293, EAE98_009027, EAE98_009190, EAE98_010906, and EAE98_010943) suggest the presence of new pathways for secondary metabolite production.

3.4.1. Description of Polyketide Gene Clusters

Previous studies by [20] identified 21 polyketide synthases in B. cinerea, of which only BcBOA6/BcBOA9, BcPKS13, BcPKS1, BcPKS21, BcPKS16, BcCHS1, BcPKS10, BcPKS8, BcPKS2, and BcPKS15 form gene clusters [20]. In B. deweyae, some of these clusters, specifically BcBOA6/BcBOA9, BcPKS1, BcCHS11, BcPKS8, and BcPKS2, were not identified, indicating differences in gene cluster formation between these species.

For the non-cluster-forming polyketide synthases in B. cinerea, no homologous proteins were identified in B. deweyae for BcPKS3, BcPKS7, BcPKS9, BcPKS11, BcPKS17, BcPKS19, and BcPKS20. However, although BcPKS5 was not found as a homologous protein, a genomic region in B. deweyae suggests the presence of this polyketide synthase, indicating its potential yet unannotated presence. This identifies the possible presence of this gene in the genome of this fungus, but it is not properly annotated for this protein.

Additionally, previous studies by [11] proposed several new potential polyketide synthases (PKSs) in B. cinerea B05.10. These genes, including BCIN_09g06350, BCIN_01g00450, BCIN_04g00210, BCIN_03g06470, BCIN_09g06360, BCIN_08g02570, BCIN_08g02560, and BCIN_12g03250, were identified as candidates based on the presence of various indicative domains [11]. However, many of these genes either lack complete PKS domains or are present as fragments. For instance, BCIN_09g06350 contains only the IPR042104 domain with 104 amino acids and lacks other typical PKS domains (Supplementary Table S5). Similarly, BCIN_03g06470, despite having multiple domains such as the zinc-binding dehydrogenase domain, a KR domain, and a phosphopantetheine attachment site domain, does not exhibit the full domain architecture expected of classical PKS genes (Supplementary Table S5). Further analysis reveals that homologous proteins in other fungal species with over 50% identity similarity to these genes often have significantly more amino acids than those in B. cinerea, suggesting that these PKS candidates might be incomplete or fragmented (Supplementary Table S6). In contrast, BcFAS2 (BCIN_01g00440) in B. cinerea is a well-characterized gene, encoding a protein with 1860 amino acids and containing several complete PKS Type I domains. These include Fas_alpha_ACP, FAS_I_H, ACPS, cond_enzymes superfamily, ketoacyl-synt_C superfamily, and NADB_Rossmann superfamily domains, ensuring its functionality as a full polyketide synthase (Supplementary Table S5). The comprehensive domain structure of BcFAS2, along with its relevance and completeness, justifies its selection for inclusion in the phylogenetic analysis. Homologous proteins from other Botrytis species and fungi exhibit similar lengths and domain structures, further supporting the use of BcFAS2 in comparative studies (Supplementary Table S6).

Furthermore, new gene clusters were identified in B. deweyae that do not have homologous gene clusters in the genome of B. cinerea. These new gene clusters, named BdPKS22-26 (following the numbering after the 21 polyketides previously described in B. cinerea), are reported here for the first time, not having been identified in any Botrytis genome before (Figure 5).

The phylogenetic distribution of these new polyketide synthases, along with the 21 previously described in B. cinerea, reveals their arrangement into two distinct clades (Figure 6).

In the first clade, BdPKS22 can be identified in subclade 1, closely related to BcPKS1 and BcPKS21. BdPKS21 shares a subclade with BcPKS2, BcPKS8, as well as BcPKS10 and BcPKS11, which are phylogenetically related to each other. More distantly related within the same subclade is BcBOA9, which is closely related to BdPKS23, BdPKS24, and BdPKS26. Additionally, subclade 2 of clade 1 shows that BcBOA6 is closely related to BcPKS3 and BcPKS7, while BcPKS4 and BcPKS5 are more distantly related to BcBOA6 within the same subclade. It is noteworthy that the gene clusters of the polyketide synthases BcBOA6 and BcBOA9, responsible for the biosynthesis of botcinins, belong to different subclades. BcBOA6 is closely related to other polyketide synthases described in the genome of B. cinerea, while BcBOA9 is closely related to novel gene clusters encoding polyketide synthases in B. deweyae.

In the second clade, two subclades are identified. Subclade 1 contains closely related polyketide synthases BcPKS12–BcPKS15. In subclade 2, BdPKS25 is closely related to BcPKS16, which are on the same branch as BcPKS18 and BcPKS19. Additionally, BcPKS17 and BcPKS20 are more phylogenetically distant within the same clade.

A point of particular interest is the genetic diversity exhibited by the newly described polyketide synthases in B. deweyae. Most of these new PKSs are identified within the same clade and subclade (except for BdPKS22, which belongs to another subclade), with BdPKS25 being the most phylogenetically distant among the newly identified PKSs.

3.4.2. Description of EAE98_002293 Gene in B. deweyae—Bdpks22

The gene cluster containing the EAE98_002293 gene, referred to as Bdpks22 in this study, consists of a single putative biosynthetic gene encoding the protein XP_038813652.1 (Figure 5). The XP_038813652.1 protein, encoded by EAE98_002293, is composed of 2542 amino acids and contains multiple functional domains: beta-ketoacyl synthase N-terminal (aa 21-257, pfam00109), beta-ketoacyl synthase C-terminal (aa 267-382, pfam02801), ketoacyl-synthetase C-terminal extension (aa 386-430, pfam16197), acyl transferase (aa 555-902, cl08282), Hotdog superfamily (aa 956-1242, cl00509), methyltransferase (aa 1453-1558, pfam08242), medium chain reductase/dehydrogenase-like family (aa 1990-2081 and 2044-2165, cl16912), KR domain (aa 2189-2344, pfam08659), and phosphopantetheine attachment site (pfam00550).

BdPKS22 is also found as a homologous protein in B. elliptica, B. convoluta, B. galanthina, B. porri, and B. tulipae. Strains Be9612 of B. elliptica and MUCL 31421 of B. squamosa exhibit high conservation percentages of 97.01% and 94.42%, respectively. However, strains MUCL 3234 and MUCL 3349 of B. porri show lower conservation percentages of 88.08%. For B. squamosa, a specific annotated protein was not identified, but a genomic region (RCTC02000012.1) was found that corresponds to the BdPks22 amino acid sequence (

Table 5. Identification of homologous proteins from BdPKS22-26 in other Botrytis species. * The protein accession number refers to the region of the genome that matches the amino acid sequence of the protein obtained from the T-BLAST result.

PKS	Botrytis Species	Strain	Protein Accession Number	Number of Amino Acids	Identity with B. deweyae
EAE98_002293 (BdPKS22)	Botrytis elliptica	Be9601	TGO80363.1	2513	96.73%
	Botrytis elliptica	Be9612	KAF7928242.1	2443	97.01%
	Botrytis convoluta	MUCL 11595	TGO65325.1	2633	90.31%
	Botrytis galanthina	MUCL 435	THV55759.1	2616	89.79%
	Botrytis porri	MUCL 3234	XP_038774872.1	2601	88.08%
	Botrytis porri	MUCL 3349	TGO92077.1	2601	88.08%
	Botrytis tulipae	Bt9001	TGO09113.1	2330	89.98%
	Botrytis paeoniae	Bp0003	TGO26315.1	2527	88.98%
	Botrytis squamosa	MUCL 31421	RCTC02000012.1 *	-	94.42%
EAE98_009027 (BdPKS23)	Botrytis elliptica	Be9601	TGO77401.1	2547	96.43%
	Botrytis elliptica	Be9612	KAF7911819.1	2562	95.87%
	Botrytis sinoallii	Bc23	XP_038753118.1	2543	95.77%
	Botrytis fragariae	BVB16	XP_037188393.1	2552	94.07%
	Botryotinia convoluta	MUCL 11595	TGO52589.1	2547	93.87%
	Botrytis hyacinthi	Bh0001	TGO38645.1	2562	92.70%
	Botrytis aclada	633	KAF7946733.1	2544	91.97%
	Botrytis paeoniae	Bp0003	TGO27521.1	2258	84.85%
	Botrytis galanthina	MUCL 435	THV49893.1	1884	86.19%
	Botrytis squamosa	MUCL 31421	RCTC02000008.1 *	-	98.43%
EAE98_009190 (BdPKS24)	Botrytis elliptica	Be9601	TGO80020.1	2335	97.29%
	Botrytis elliptica	Be9612	KAF7941341.1	2359	94.43%
	Botrytis squamosa	MUCL 31421	RCTC02000005.1 *	-	83.28%
	Botrytis aclada	633	RCSV01000004.1 *	-	78.53%
	Botrytis porri	MUCL 3234	RCTA01000006.1 *	-	72.12%
	Botryotinia globosa	MUCL 444	RCSZ01000002.1 *	-	75.22%
EAE98_010906 (BdPKS25)	Botrytis paeoniae	Bp0003	TGO20404.1	2621	91.95%
	Botryotinia globosa	MUCL 444	KAF7896114.1	2528	84.54%
	Botrytis elliptica	Be9601	TGO71777.1	2133	95.90%
	Botrytis elliptica	Be9612	KAF7923671.1	1808	97.11%
	Botrytis squamosa	MUCL 31421	RCTC02000002.1 *	-	84.75%
	Botrytis sinoallii	Bc 23	RCTB01000005.1 *	-	50.05%
EAE98_010943 (BdPKS26)	Botrytis elliptica	Be9601	TGO73554.1	2327	98.77%
	Botrytis elliptica	Be9612	KAF7923645.1	2327	98.64%
	Botrytis sinoallii	Bc 23	XP_038763137.1	2073	87.33%
	Botrytis galanthina	MUCL 435	THV54946.1	1623	86.56%
	Botrytis squamosa	MUCL 31421	RCTC02000002.1 *	-	95.29%
			RCTC02000005.1 *		54.23%
	Botrytis medusae	B555	JAHXJL010000007.1	-	89.03%
	Botryotinia globosa	MUCL 444	RCSZ01000001.1	-	86.14%

). This was the only case among the 10 Botrytis genomes where a homologous protein to BdPks22 was not found (Table 5).

The distribution of BdPKS22 in other fungi spans the Sordariomyceta, Leotiomyceta, and Dothideomyceta clades. In Sordariomyceta, PKS proteins are found in the Leotiomycete and Sordariomycete orders. Within Leotiomycete, Heliotales hosts one PKS protein in one organism, and Xylariales contains four PKS proteins among three organisms. Leotiomycetes incertae sedis also harbors one PKS protein. In Leotiomyceta, orders such as Teloschistales and Pertusariales exhibit PKS proteins. Teloschistales displays three PKS proteins across three organisms, while Pertusariales contains one PKS protein. In Eurotiomycetes, Eurotiales includes ten PKS proteins across seven organisms. In Dothideomyceta, Pleosporales stands out with thirteen PKS proteins identified within two organisms (

Table 6. Distribution of homologous proteins from BdPKS22-26 with an identity percentage > 50% and a coverage > 70% in fungi from genera other than Botrytis.

PKS	Phylum	Clade	Class	Order	Number of Proteins	Number of Organisms
BdPKS22	Ascomycota	Sordariomyceta	Leotiomycete	Heliotales	1	1
				Leotiomycetes incertae sedis	1	1
			Sordariomycete	Xylariales	4	3
		Leotiomyceta	Lecanoromycete	Teloschistales	3	3
				Pertusariales	1	1
			Eurotiomycete	Eurotiales	10	7
		Dothideomyceta	Dothideomycetes	Pleosporales	13	2
	TOTAL				33	18
BdPKS23	Ascomycota	Sordariomycelta	Sordariomycete	Heliotales	9	7
				Xylariales	32	25
				Xylariomycetidae incertae sedis	4	1
				Diaporthales	4	3
		Leotiomyceta	Lecanoromycetes	Lecanorales	2	2
				Teloschistales	5	4
				Peltigerales	1	1
				Pertusariales	1	1
			Eurotiomycetes	Eurotiales	3	2
		Dothideomyceta	Dothideomycetes	Dothideomycetes incertae sedis	1	1
	TOTAL				62	47
BdPKS24	Ascomycota	Leotiomyceta	Lecanoromycete	Sarrameanales	1	1
				Trapeliales	1	1
				Pertusariales	1	1
				Teloschistales	2	2
			Eurotiomycete	Onygenales	1	1
				Eurotiales	2	1
		Sordariomyceta	Sordariomycete	Xylariales	3	2
	TOTAL				11	9
BdPKS25	Ascomycota	Sordariomyceta	Leotiomycete	Heliotales	8	7
				Leotiomycetes incertae sedis	1	1
			Sordariomycete	Hypocreales	6	5
				Microascales	3	2
				Xylariomycetidae incertae sedis	4	1
				Sordariales	6	6
		Dothideomyceta	Dothideomycetes	Dothideomycetes incertae sedis	5	3
				Pleosporales	4	3
			Arthoniomycetes	Arthoniales	1	1
		leotiomyceta	Eurotiomycetes	Eurotiales	5	4
			Lecanoromycetes	Lecanorales	1	1
	TOTAL				44	34
BdPKS26	Ascomycota	Sordariomyceta	Leotiomycete	Helotiales	8	6
			Sordariomycetes	Hypocreales	12	9
				Glomerellales	10	5
				Xylariales	53	39
				Sordariales	1	1
				Diaporthales	2	1
				Magnaporthales	1	1
		Leotiomyceta	Lecanoromycetes	Trapeliales	2	2
				Teloschistales	1	1
			Eurotiomycetes	Eurotiales	27	13
				Onygenales	36	2
		Dothideomyceta	Dothideomycetes	Mytilinidiales	2	1
				Pleosporomycetidae incertae sedis	1	1
				Pleosporales	1	1
				Botryosphaeriales	2	1
	TOTAL				159	84

).

Of the 18 homologous hits to BdPKS22 (Figure 7 and Supplementary Table S7), the phylogenetic distribution shows that BdPKS22 is closely related to Sclerotinia nivalis within the same subclade (subclade 2 of clade 1). All species in clade 1 belong to Sordariomyceta, except Biscogniauxia marginata and Annulohypoxylon truncatum from Leotiomyceta, closely related to Oidiodendron maius, Penicillium occitanis, and Talaromyces rugulosus. Additionally, BdPKS22 shares a clade with species of the genera Xanthoria, Teloschistes, and Aspergillus (Figure 7).

3.4.3. Description of EAE98_009027 Gene in B. deweyae—Bdpks23

The gene cluster containing the EAE98_009027 gene, referred to as Bdpks23 in this study, consists of six putative biosynthetic genes (Figure 5). The gene EAE98_009027 encodes the protein XP_038807018.1, which is composed of 2537 amino acids and contains multiple functional domains: beta-ketoacyl synthase N-terminal (aa 16-266, pfam00109), beta-ketoacyl synthase C-terminal (aa 275-393, pfam02801), ketoacyl-synthetase C-terminal extension (aa 398-528, pfam16197), acyl transferase (aa 556-887, cl08282), polyketide synthase dehydratase (aa 944-1251, pfam14765), methyltransferase (aa 1444-1548, pfam08242), alcohol dehydrogenase GroES-like (aa 1866-1916, cl17172), zinc-binding dehydrogenase (aa 1981-2072, pfam00107), KR domain (aa 2190-2348, pfam08659), and phosphopantetheine attachment site (aa 2467-2525, pfam00550).

Additionally, this cluster includes other genes: EAE98_009028 (543 amino acids, FAD binding domain, aa 101-239, pfam01565), EAE98_009029 (303 amino acids, crotonase/enoyl-CoA hydratase superfamily domain, aa 37-263, cl23717), EAE98_009030 (506 amino acids, FAD binding domain, aa 66-206, cl19922), EAE98_009031 (552 amino acids, cytochrome P450 domain, aa 103-514, cl12078), and EAE98_009032 (438 amino acids, major facilitator superfamily domain, aa 53-391, pfam07690).

The comparative analysis of protein sequence identities among various Botrytis species strains relative to the reference protein XP_038807018.1 (BdPKS23) reveals intriguing insights into the genetic diversity within this fungal genus. B. squamosa strain MUCL 31421 exhibits the highest identity at 98.43%, indicating a close genetic relationship to the reference protein. Despite the lack of an annotated protein in B. squamosa, a genomic region likely involved in the biosynthesis of this protein was identified. B. elliptica strains Be9601 and Be9612 follow closely with identities of 96.43% and 95.87%, respectively, suggesting substantial genetic similarity to BdPKS23. B. sinoallii strain Bc23 and B. fragariae strain BVB16 exhibit identities of 95.77% and 94.07%, respectively. However, B. paeoniae strain Bp0003 shows the lowest identity at 84.85%, indicating greater genetic divergence (Table 5).

The distribution of BdPKS23 in other fungi spans the Ascomycota phylum, specifically within the Sordariomyceta, Leotiomyceta, and Dothideomyceta classes. In Sordariomyceta, PKS proteins are found in the Heliotales (9 PKS proteins in 7 organisms) and Xylariales (32 PKS proteins in 25 organisms) orders. Xylariomycetidae incertae sedis and Diaporthales each contain four PKS proteins found in one and three organisms, respectively. In Leotiomyceta, Lecanorales contains two PKS proteins found in two organisms, Teloschistales exhibits five PKS proteins in four organisms, and Peltigerales and Pertusariales each contain one PKS protein in one organism. In Eurotiomycetes, Eurotiales includes three PKS proteins in two organisms. In Dothideomyceta, Dothideomycetes incertae sedis contains one PKS protein in one organism (Table 6).

The phylogenetic distribution of BdPKS23 is noteworthy as it shows that among the 51 top protein sequences (Figure 8 and Supplementary Table S8), all belong to the Sordariomyceta clade except for three Leotiomyceta species (genera Talaromyces and Letrouitia) and one species, Zopfia rhizophila, belonging to Dothideomyceta. These species are found in a different clade from the one in which BdPKS23 is located, sharing a clade with species of the genera Lachnellula, Hypoxylon, Cudoniella, and Xylaria, among others. In clade 2, BdPKS23 is closely related to species of the genera Mollisia, Xylariaceae, Icmadophila, Teloschistes, Mycoblastus, Alectoria, and Rutstroemia (Figure 8).

3.4.4. Description of EAE98_009190 Gene in B. deweyae—Bdpks24

The gene cluster containing the EAE98_009190 gene, referred to as Bdpks24 in this study, consists of three putative biosynthetic genes (Figure 5). The gene EAE98_009188 encodes a protein with 311 amino acids, containing the domains metallo-beta-lactamase superfamily (aa 70-232, pfam00753) and hydroxyacylglutathione hydrolase C-terminus (aa 233-310, pfam16123). The gene EAE98_009190 encodes the protein XP_038806877.1, which is composed of 2346 amino acids and contains multiple functional domains: beta-ketoacyl synthase N-terminal (aa 11-249, pfam00109), beta-ketoacyl synthase C-terminal (aa 258-376, pfam02801), ketoacyl-synthetase C-terminal extension (aa 380-514, pfam16197), acyl transferase (aa 543-856, cl08282), polyketide synthase dehydratase (aa 931-1228, pfam14765), alcohol dehydrogenase GroES-like (aa 1644-1696, cl17172), zinc-binding dehydrogenase (aa 1759-1849, pfam00107), KR domain (aa 1991-2163, pfam08659), and phosphopantetheine attachment site (aa 2277-2334, pfam00550). Additionally, the gene EAE98_009191 encodes a protein with 551 amino acids, featuring an amidase domain (aa 81-531, cl18951).

The comparative analysis of protein sequence identities among various Botrytis strains highlights the genetic diversity within this fungal genus. B. elliptica strains Be9601 and Be9612 show high sequence identities of 97.29% and 94.43%, respectively, compared to the reference protein XP_038806877.1 (BdPKS24). However, B. squamosa strain MUCL 31421 exhibits a lower identity of 83.28%, indicating greater genetic divergence. Similarly, B. aclada strain 633, B. porri strain MUCL 3234, and B. globosa strain MUCL 444 display identities of 78.53%, 72.12%, and 75.22%, respectively. These similarities were identified based on draft genome sequences, as no homologous proteins for BdPKS24 were annotated in the genomes of B. squamosa, B. aclada, B. porri, and B. globosa (Table 5).

The distribution of BdPKS24 protein across the Ascomycota phylum, specifically within the Leotiomyceta and Sordariomyceta classes, reveals various taxonomic orders harboring these proteins. In the Leotiomyceta clade, the orders Sarrameanales, Trapeliales, Pertusariales, and Teloschistales each contain one PKS protein in one organism, suggesting a diverse distribution of PKS enzymes within this class, reflecting potential ecological adaptations and metabolic diversity. In the Eurotiomyceta clade, the Onygenales order contains one PKS protein in one organism, while the Eurotiales order harbors two PKS proteins in one organism, indicating a comparatively lower abundance of PKS proteins within Eurotiomycetes. In the Sordariomyceta clade, the Sordariomycete order Xylariales exhibits three PKS proteins across two organisms, suggesting a moderate abundance of PKS enzymes within this order (Table 6).

The phylogenetic distribution of BdPKS24 is noteworthy as, among the four top protein sequences (Figure 9 and Supplementary Table S9), all belong to the Leotiomyceta clade except for Monosporascus sp. mg162, which belongs to the Sordariomyceta clade along with Botrytis deweyae. BdPKS24 is also related to species of the genera Loxospora, Trapelia, and Penicillium (Figure 9).

3.4.5. Description of EAE98_010906 Gene in B. deweyae—Bdpks25

The gene cluster containing the EAE98_010906 gene, referred to as Bdpks25 in this study, consists of a single putative biosynthetic gene, EAE98_010906, which encodes the protein XP_038805042.1 with 2208 amino acids (Figure 5). This protein contains multiple functional domains: beta-ketoacyl synthase N-terminal (aa 122-288, pfam00109), beta-ketoacyl synthase C-terminal (aa 296-411, pfam02801), acyl transferase (aa 565-826, cl08282), Hotdog superfamily (aa 979-1214, cl00509), phosphopantetheine attachment site (aa 1280-1338 and 1384-1439, pfam00550), helix–turn–helix (aa 1497-1580, pfam18558), methyltransferase (aa 1682-1782, pfam08242), and BD-FAE (aa 1894-2155, pfam20434).

The comparison of protein sequence identities among various Botrytis strains provides insights into the genetic diversity within this fungal genus. B. paeoniae strain Bp0003 exhibits a sequence identity of 91.95% to the reference sequence XP_038805042.1 (BdPKS25), indicating a relatively close genetic relationship. Similarly, B. globosa strain MUCL 444 shows a sequence identity of 84.54% to its reference sequence KAF7896114.1. In contrast, B. elliptica strains Be9601 and Be9612 demonstrate high sequence identities of 95.90% and 97.11% to their respective reference sequences (TGO71777.1 and KAF7923671.1). B. squamosa strain MUCL 31421 displays a sequence identity of 84.75% to its reference sequence RCTC02000002.1. However, in B. squamosa and B. sinoallii, the similarity was based on draft genome sequences that correspond to the BdPKS25 protein, as no homologous protein for BdPKS25 is annotated in their genomes (Table 5).

The distribution of BdPKS25 protein within the Ascomycota phylum, focusing on the Sordariomyceta and Dothideomyceta clades, demonstrates a diverse array of taxonomic orders harboring these proteins. In the Sordariomyceta clade, the Leotiomycete order Heliotales contains eight PKS proteins across seven organisms, and Leotiomycetes incertae sedis hosts one PKS protein in one organism. Within the Sordariomycete order Hypocreales, six PKS proteins are identified across five organisms, while Microascales exhibits three PKS proteins in two organisms. The Xylariomycetidae incertae sedis order contains four PKS proteins found in one organism. Sordariales contains six PKS proteins across six organisms. In the Dothideomyceta clade, Dothideomycetes incertae sedis contains five PKS proteins in three organisms, and Pleosporales exhibits four PKS proteins in three organisms. Additionally, the Arthoniomycetes order Arthoniales contains one PKS protein in one organism. In the Leotiomyceta clade, Eurotiomycetes order Eurotiales contains five PKS proteins across four organisms, suggesting significant metabolic diversity. Lecanoromycetes of order Lecanorales also contains one PKS protein in one organism (Table 6).

Regarding the phylogenetic relationship of BdPKS25, among the top 25 protein sequences (Figure 10 and Supplementary Table S10), all belong to the Sordariomyceta clade except for Bathelium mastoideum, Viridothelium virens, and Bipolaris maydis, which belong to the Dothideomyceta clade, and Aspergillus sp., which belongs to the Leotiomyceta clade. These latter three are listed as independent branches within the phylogenetic tree. BdPKS25 is closely related to Ciborinia camelliae and shares a subclade with species of the genera Lachnellula and Rutstroemia sp. NJR-2017a BVV2 (Figure 10).

3.4.6. Description of EAE98_010943 Gene in B. deweyae—Bdpks26

The gene cluster containing the EAE98_010943 gene, referred to as Bdpks26 in this study, consists of six putative biosynthetic genes (Figure 5). The gene EAE98_010940 encodes a protein with 294 amino acids, containing a short-chain dehydrogenase domain (aa 5-191, pfam00106). The gene EAE98_010941 encodes a protein with 513 amino acids, showing an acyltransferase family domain (aa 68-448). The gene EAE98_010943, named in this work as Bdpks26, encodes the protein XP_038805079.1, which has 2353 amino acids and contains multiple functional domains: beta-ketoacyl synthase N-terminal (aa 14-265, pfam00109), beta-ketoacyl synthase C-terminal (aa 274-392, pfam02801), ketoacyl-synthetase C-terminal extension (aa 396-517), acyl transferase (aa 546-878, cl08282), polyketide synthase dehydratase (aa 933-1236, pfam14765), KR domain (aa 1967-2145, pfam08659), and phosphopantetheine attachment site (aa 2272-2328, pfam00550). Additionally, the gene EAE98_010945 encodes a protein with 252 amino acids, containing a Rossmann-fold NAD(P)+-binding protein domain (aa 8-200 and 32-246, cl21454), and the gene EAE98_010949 encodes a protein with 576 amino acids, featuring a flavin-binding monooxygenase-like domain (aa 12-554, cl30939).

The comparison of protein sequence identities among various Botrytis strains provides valuable insights into the genetic diversity within this fungal genus. B. elliptica strains Be9601 and Be9612 exhibit remarkably high sequence identities of 98.77% and 98.64% to their respective reference sequences (TGO73554.1 and KAF7923645.1). B. sinoallii strain Bc23 shows a sequence identity of 87.33% to its reference sequence XP_038763137.1. B. galanthina strain MUCL 435 displays a sequence identity of 86.56% to its reference sequence THV54946.1. B. squamosa strain MUCL 31421 demonstrates a high sequence identity of 95.29% to its reference sequence RCTC02000002.1 and a lower identity of 54.23% to the second reference sequence RCTC02000005.1. However, in B. squamosa, the similarity was based on draft genome sequences that correspond to the BdPKS26 protein, as no homologous protein for BdPKS26 is annotated in its genome (Table 5).

The distribution of BdPKS26 protein within the Ascomycota phylum, focusing on the Sordariomyceta, Leotiomyceta, and Dothideomyceta clades, reveals a diverse array of taxonomic orders harboring these proteins. In the Sordariomyceta clade, within the Leotiomycete order Helotiales, eight PKS proteins are distributed across six organisms. The Sordariomycetes order Hypocreales exhibits the highest abundance of PKS proteins, with 12 identified across nine organisms. Additionally, Glomerellales contains 10 PKS proteins in 5 organisms, while Xylariales exhibits 53 PKS proteins across 39 organisms, indicating its significant role in secondary metabolite production. Other orders such as Sordariales, Diaporthales, and Magnaporthales also contain a smaller number of PKS proteins. In the Leotiomyceta clade, Lecanoromycetes order Trapeliales and Teloschistales each contain two and one PKS proteins, respectively, distributed in the same number of organisms. In the Eurotiomycetes class, Eurotiales contains 27 PKS proteins across 13 organisms, while Onygenales exhibits 36 PKS proteins in only 2 organisms. Within the Dothideomyceta clade, Dothideomycetes contains two PKS proteins in Mytilinidiales and one PKS protein each in Pleosporomycetidae incertae sedis, Pleosporales, and Botryosphaeriales (Table 6).

The phylogenetic distribution of BdPKS26 with the top 55 homologous proteins (Figure 11 and Supplementary Table S11) shows a predominance of species belonging to the Sordariomyceta clade, except for Pleomassaria siparia (subclade 1 from clade 1), Mytilinidion resinicola (subclade 1 from clade 1), and Glonium stellatum (clade 2) from the Dothideomyceta clade. Additionally, species of the genus Aspergillus (subclades 1 and 2 from clade 1) and Ophidiomyces ophidiicola (clade 2) belong to the Leotiomyceta clade. BdPKS26 is closely related to Rutstroemia sp. NJR-2017a BVV2 and Bisporella sp. PMI 857 in the same subclade (Figure 11).

4. Discussion

The comparative analysis of secondary metabolites between B. deweyae B1 and B. cinerea B05.10 reveals significant insights into their metabolic diversity, especially in polyketide and terpene biosynthesis. B. deweyae B1 demonstrates a higher number of polyketide clusters (11 gene clusters) compared to B. cinerea B05.10 (10 gene clusters), suggesting potential differences in their secondary metabolite profiles. Similarly, B. deweyae B1 exhibits more clusters related to terpene biosynthesis (six gene clusters) compared to B. cinerea B05.10 (five gene clusters), indicating a nuanced metabolic variation between these closely related species.

Within the terpene biosynthesis clusters of B. deweyae B1, several key genes have been identified, such as Bcerg9, Bcstc4, Bcbot2, and an unannotated terpene gene. While most of these genes have counterparts in B. cinerea B05.10, notable differences exist in their regulatory roles. For instance, Bcstc7 and Bcbot2 in B. cinerea B05.10 regulate the synthesis of eremophilane and botryane metabolites, respectively [63,65]. In contrast, B. deweyae B1 lacks Bcstc2 and Bcstc5 but possesses an unannotated terpene gene also identified in B. cinerea B05.10.

Further analysis of the relationships between these genes reveals interesting clustering patterns. Genes like Bdstc4, Bcstc3, Bdbot2, and Bcstc5 are closely related, forming part of the same subclade, indicating functional similarities in terpene biosynthesis. Conversely, genes like Bcpax1, Bccoq1, Bcerg20, and the unannotated gene form another subclade, suggesting potential functional diversification within the terpene biosynthesis pathway.

The presence of putative unannotated genes in B. deweyae B1 suggests the existence of novel metabolic pathways yet to be characterized. The analysis of these unannotated clusters reveals polyprenyl synthetase domains, indicating potential roles in specialized metabolite synthesis. However, validation would need to be carried out for confirmation. These genes have homologs in other Botrytis species, suggesting conservation and importance across the genus.

Comparative genomic analysis with other Botrytis species highlights the genetic relationships and evolutionary divergence within the genus. Strains like B. cinerea T4, BcDW1, and B05.10 exhibit high genetic identity with B. deweyae B1, indicating close evolutionary ties. However, strains from different species show varying levels of identity, reflecting the genetic diversity within the Botrytis genus.

The taxonomic distribution of fungi, particularly within the Ascomycota and Basidiomycota phyla, provides a comprehensive overview of fungal diversity and the distribution of the unannotated gene EAE98_008016 from B. deweyae. The abundance of proteins and organisms within different classes, orders, and unclassified groups underscores the functional and ecological diversity of fungi. This diversity highlights evolutionary complexity and the potential for discovering novel metabolites with unique biological activities. In addition, this putative unannotated gene is closely related to established terpene biosynthetic pathways, as evidenced by a similarity score of 0.18 to BGC0000673, which is involved in the synthesis of pimara-8(14),15-diene in Aspergillus nidulans. It also shows a similarity score of 0.17 to BGC0002320, responsible for producing conidiogenone in Penicillium rubens. Furthermore, a score of 0.12 connects it to BGC0002604, associated with sartorypyrone A in Aspergillus felis. These connections suggest that the gene may play a significant role in terpene biosynthesis, highlighting its potential importance in the ecological and metabolic dynamics of B. deweyae.

The distribution of polyketide synthase (PKS) gene clusters in B. deweyae reveals significant genetic diversity and evolutionary history compared to B. cinerea. Previous studies identified 21 PKS genes in B. cinerea, with only some forming gene clusters [11,20]. These include Bcboa6/Bcboa9, Bcpks13, Bcpks1, Bcpks21, Bcpks16, Bcchs1, Bcpks10, Bcpks8, Bcpks2, and Bcpks15. However, in B. deweyae, clusters corresponding to Bcboa6/Bcboa9, Bcpks1, Bcchs11, Bcpks8, and Bcpks2 were not identified. Additionally, several non-cluster-forming PKS genes in B. cinerea had no homologs in B. deweyae, except for Bcpks5, which showed potential presence but lacked proper annotation.

New potential PKS genes were identified in B. cinerea B05.10, including BCIN_09g06350, BCIN_01g00450, BCIN_04g00210, BCIN_03g06470, BCIN_09g06360, BCIN_08g02570, BCIN_08g02560, and BCIN_12g03250 [11]. These genes showed various domain compositions, with some lacking typical PKS domains. Homologous proteins for these genes were identified, showing varying degrees of similarity across different fungal species.

In B. deweyae, new putative gene clusters named Bdpks22-26 were discovered, not previously found in any Botrytis genome. These clusters, along with the 21 described in B. cinerea, form two distinct phylogenetic groups. The phylogenetic analysis reveals the presence of novel PKS genes in B. deweyae, indicating evolutionary divergence and potential new metabolic pathways. Within the first clade, Bdpks22 is closely related to Bcpks1, Bcpks21, and other PKS genes in B. cinerea. This clade also includes Bcboa9, Bdpks23, Bdpks24, and Bdpks26, suggesting functional similarities and evolutionary relationships. In the second clade, Bdpks25 is closely related to Bcpks16, Bcpks18, and Bcpks19, indicating potential functional similarities. Other PKS genes in this clade, such as Bcpks17 and Bcpks20, show more distant relationships, suggesting diverse roles in secondary metabolism.

The genetic diversity of PKS genes in B. deweyae highlights its varied metabolic functions and adaptive capabilities. High sequence identities were observed in some strains, indicating close genetic relationships, while others exhibited lower identities, suggesting greater genetic divergence. This diversity underscores the potential for varied functions and roles in secondary metabolism, critical for ecological success and adaptability.

The Bdpks22 in question is likely involved in polyketide biosynthesis, as suggested by its similarity to characterized gene clusters. With a score of 0.32 to BGC0002191 (producing prolipyrone B and gibepyrone D in Fusarium graminearum), and 0.30 to BGC0002155 (nectriapyrone compounds in Pyricularia oryzae), it may contribute to metabolites with diverse biological activities. Additionally, the connection to BGC0001252 indicates potential for producing bioactive polyketides that impact ecological interactions. The gene Bdpks23 is likely involved in polyketide biosynthesis, as indicated by its similarity scores. With a score of 0.33 to BGC0002525, associated with compounds like fusarubin and lucilactaene in Fusarium sp., it suggests a potential role in producing bioactive metabolites. Additionally, the scores of 0.33 and 0.32 with BGC0002194 (epipyrone A in Epicoccum nigrum) and BGC0002191 (prolipyrone B and gibepyrone D in Fusarium graminearum) further support its implication in polyketide biosynthesis. The gene Bdpks24 is likely associated with the biosynthesis of polyketides and non-ribosomal peptides, as indicated by its similarity scores. With a score of 0.29 to BGC0000046, which produces depudecin in Alternaria brassicicola, it suggests potential involvement in generating bioactive metabolites. Additionally, there are scores of 0.28 and 0.27 with BGC0002228 and BGC0002227, linked to non-ribosomal peptides in Colletotrichum incanum and Aspergillus luchuensis, respectively. The gene Bdpks25 is likely involved in polyketide biosynthesis, as suggested by its similarity scores. With a score of 0.20 to BGC0001284, which is associated with alternariol in Parastagonospora nodorum, it indicates a potential role in producing bioactive compounds. Additionally, the score of 0.20 with BGC0002175 (YWA1 in Aspergillus oryzae) and BGC0000107 (naphthopyrone in Aspergillus nidulans) further supports its involvement in diverse metabolic pathways. Finally, the Bdpks26 gene is likely associated with the biosynthesis of polyketides and terpenes, as indicated by its similarity scores. With a score of 0.26 to BGC0001264, linked to betaenones A, B, and C in Phoma betae, it suggests a role in producing bioactive polyketide compounds. Additionally, the score of 0.24 with BGC0002266, associated with calidoustene A, B, and C in Aspergillus calidoustus, highlights its potential involvement in terpene biosynthesis. However, this analysis is a predictive study where further analyses are necessary to identify the metabolites in which these new putative biosynthetic clusters present in B. deweyae are involved.

On the other hand, comparative analysis of PKS sequences among various Botrytis strains highlighted genetic diversity within the genus. Strains of B. deweyae showed varying degrees of similarity to reference sequences, with some strains exhibiting high sequence identity similarities, indicating close relationships, and others showing lower identity similarities, suggesting greater divergence. The phylogenetic distribution of PKS proteins across different fungal species provided insights into their evolutionary relationships. Botrytis PKS proteins were found within the Sordariomyceta, Leotiomyceta, and Dothideomyceta clades, indicating wide distribution across different taxonomic orders. This broad distribution reflects evolutionary complexity and diverse ecological roles of these enzymes.

5. Conclusions

The distribution of PKS and terpene gene clusters in B. deweyae provides valuable insights into the genetic diversity and evolutionary history of this fungus. The absence of certain clusters found in B. cinerea and the discovery of new putative clusters in B. deweyae suggest divergent evolutionary paths in secondary metabolism between these two species. The genetic diversity observed within B. deweyae PKS genes highlights the potential for varied metabolic functions and adaptive capabilities within this species.

Comparative analysis and phylogenetic distribution underscore the evolutionary relationships and wide distribution of PKS genes across different fungal taxa. Understanding the genetic basis of secondary metabolism in Botrytis species has implications for disease management strategies in agriculture and provides opportunities for the discovery of novel bioactive compounds.

The identification of novel putative PKS and terpene biosynthesis gene clusters in B. deweyae highlights its potential for producing unique secondary metabolites. These metabolites could have various applications in agriculture, medicine, and biotechnology. Understanding the biosynthesis pathways of these compounds could lead to the development of new fungicides or pharmaceuticals.

The genetic diversity observed in B. deweyae suggests that this species may have a greater capacity for adaptation and survival in diverse environmental conditions. Future research should focus on characterizing the functions of the unannotated genes and novel clusters identified in B. deweyae. Functional genomics approaches, such as gene knockouts and overexpression studies, could elucidate the roles of these genes in secondary metabolism. Additionally, comparative genomics with other Botrytis species and related fungi could uncover the evolutionary origins and diversification of these metabolic pathways.

Comparative analysis of secondary metabolite gene clusters in B. deweyae and B. cinerea has revealed significant genetic and metabolic diversity. The discovery of novel PKS and terpene biosynthesis gene clusters in B. deweyae underscores its potential for producing unique secondary metabolites. These findings enhance our understanding of the genetic basis of secondary metabolism in Botrytis species and provide a foundation for future research into the ecological and biotechnological applications of these compounds.