Introduction
Dothideomycetes represents the largest and most important class of ascomycete fungi, including 23 orders, 110 families, 1,261 genera, and 19,000 species (Wijayawardene et al., 2017). The members of Dothideomycetes comprise both phytopathogenic (Ohm et al., 2012) and non-phytopathogenic fungi with diverse lifestyles (Ruibal et al., 2009) as well as many mycorrhizal fungi (Peter et al., 2016). Among these 110 families, Botryosphaeriaceae is an important and distinctive family. This is because it includes saprobes, endophytes, and phytopathogens, and it is one of the most widely geographically distributed groups of opportunistic plant pathogens. The host range of this family is very wide, and many economically important plants worldwide can be infected by them (Slippers and Wingfield, 2007). These pathogenic fungi can infect plants through wounds or natural openings, such as lenticels and stomata. Once they enter host tissues, they may survive as endophytes to stay at a biotrophic stage for a long time and turn into the destructive necrotrophic stage when the host is stressed (Yan et al., 2013; Morales-Cruz et al., 2015). The members of Botryosphaeriaceae can infect many woody plants and cause serious disease symptoms, such as dieback, branch canker, leaf spots, and fruit and seed rot (Marsberg et al., 2017). But the interaction of some of Botryosphaeriaceae fungi like Botryosphaeria dothidea (B. dothidea) with host plants includes a latent or endophytic phase, which makes the fungal infection easily be neglected (Bostock et al., 2014; Marsberg et al., 2017; Wang et al., 2018). Therefore, it is of substantial biological significance to explore the evolutionary characteristics of Botryosphaeriaceae at the level of Dothideomycetes.
The exceptional feature of Botryosphaeriaceae fungi is that it is difficult to observe their sexual structure under both natural and experimental conditions, but this does not mean that sexual reproduction does not occur for these fungi (Phillips et al., 2013). With more in-depth research being conducted, the mating strategies (homothallism or heterothallism) of increasing members of the Botryosphaeriaceae family have been reported (Bihon et al., 2012a,b, 2014; Billiard et al., 2012; Lopes et al., 2017, 2018), revealing some unresolved questions regarding their sexual reproduction. For example, how conservative are the nucleic acid and protein sequences of the mating type determination genes; how conservative are the genes and their arrangement at the mating type determination loci; how has the mating type evolved, and what is the origin type. In addition, the host range of different Botryosphaeriaceae fungi varies greatly. B. dothidea is a common pathogen with a wide range of hosts. Generally, the infection becomes symptomatic when the host is subjected to drought, physical damage, waterlogging, or freezing stress (Bostock et al., 2014; Marsberg et al., 2017; Wang et al., 2018). The symptoms primarily include canker on young seedlings, branches, and stems; the necrosis of branches; and fruit decay, which may lead to the death of the host in extreme cases (Kim et al., 2005; Tang et al., 2012). Botryosphaeria kuwatsukai (B. kuwatsukai) can also cause symptoms, such as fruit softening and decay, and severe canker of branches and stems (Wang et al., 2021). However, B. kuwatsukai has a relatively narrow host range and primarily infects apple and pear trees (Xu et al., 2015). Therefore, a systematic study of the mating strategies and differences of pathogenicity-related genes in Botryosphaeriaceae fungi will lead to a better understanding of their molecular evolutionary history and pathogenic characteristics.
Currently, genomics technology has been widely used to study many pathogenic fungi of plants, and greatly promoted the understanding of their evolution and pathogenic mechanisms (Islam et al., 2012; Blanco-Ulate et al., 2013; van der Nest et al., 2014; Nagel et al., 2018; Félix et al., 2019; Landi et al., 2020; Meile et al., 2020). For example, gene family expansion associated with virulence factors in wood-colonizing pathogenic fungi in the Botryosphaeriaceae was revealed via phylogenomic comparisons (Garcia et al., 2021); the comparative genome analyses of latent plant pathogens in the Botryosphaeriaceae were conducted to define their genomes (Nagel et al., 2021). In addition, a large-scale comparative genomic analysis can better reveal the physiological characteristics and evolutionary history of fungi (Guttman et al., 2014; Haridas et al., 2020; Miyauchi et al., 2020). Therefore, in this study, 167 Dothideomycetes fungi, including 19 Botryosphaeriaceae species, were fully sequenced, and a comparative genomics approach was used to comprehensively analyze the molecular evolution characteristics, mating strategies and pathogenicity within Botryosphaeriaceae fungi. The sequence differences of related genes benefit our understanding of the evolutionary history of Botryosphaeriaceae fungi and provide useful information for the prevention and control of the diseases caused by these fungi.
Discussion
In this study, we conducted a large-scale whole-genome sequencing of 167 Dothideomycetes fungi, including 19 species, and comprehensively analyzed evolutionary traits, mating strategies and changes in pathogenic genes in Dothideomycetes fungi. Our results can advance our understanding of the evolutionary history of Botryosphaeriaceae fungi.
Our results also confirmed that the 167 Dothideomycetes fungi can be divided into two subclasses, Pleosporomycetidae and Dothideomycetidae, and Botryosphaeriaceae belongs to Pleosporomycetidae, indicating that it is accurate to classify fungi using phylogenies based on phylogenomics (Haridas et al., 2020). Our results inferred the ancestral lifestyle of Botryosphaeriaceae fungus as saprobe, and these fungi have undergone at least three transitions from saprophytic to phytopathogenic states. This result is consistent with Schoch et al. (2009). They inferred that the Dothideomycetes fungi have experienced multiple transitions from saprophytic pathogens to lichens to phytopathogens, along with multiple transitions from terrestrial to aquatic lifestyles (Schoch et al., 2009). In addition, a larger genome size and a higher number of protein-coding genes are usually associated with saprophytic fungi compared with phytopathogenic fungi. This result is consistent with the findings of Schuelke et al. (2017). They found that in the Geosmithia genus, compared with non-pathogenic fungi, the pathogenic fungi in this family have smaller genomes (Schuelke et al., 2017).
Previous studies have shown that phytopathogenic fungi have a smaller genome and contain fewer protein-coding genes compared with saprophytic fungi, which is related to the expansion and contraction of gene families (Ohm et al., 2012; Haridas et al., 2018). In this study, many gene families of phytopathogenic fungi showed contractions, primarily including genes which contain the Pfam domain tetratricopeptide repeat (TPR). TPR can interact with a variety of proteins, such as the anaphase-promoting complex, NADPH oxidases, and HSP90-binding proteins (Das et al., 1998; Kaneko et al., 2006; Kondo et al., 2010). The TPR protein is also part of the plant hormone signaling pathways (Schapire et al., 2006). The family enrichment results found that multiple members of the TRP family proteins (TPR 1, 3, 8, 10, 11, 12, 16, and 19) contracted in phytopathogenic fungi. This contraction may be due to a reduction of signal related TPR proteins in pathogenic fungi. This is because during the process of pathogenic fungal infection, fungal signal related TPR proteins will be affected by plant hormone signals (Haridas et al., 2020). Some other Pfam domains also contracted significantly in phytopathogenic fungi, including PF17111 (the fungal N-terminal domain of STAND proteins), PF05729 (the NACHT domain), and PF14479 (involved in prion inhibition and propagation). These domains are in heterokaryons and play an important role in incompatibility (Paoletti and Saupe, 2009; Greenwald et al., 2010; Daskalov et al., 2012). The contraction of Pfam domains associated with incompatibility in the heterokaryons suggests that other strategies to reduce the level of incompatibility in heterokaryons also exist to improve the adaptability of pathogenic fungi (Ishikawa et al., 2012). Simultaneously, compared with saprophytic fungi, many GO terms are contracted in phytopathogenic fungi, including protein kinases (GO: 0004672), transcription factors (GO: 0003700), zinc ion binding (GO: 0008270), and regulation of nitrogen utilization (GO: 0006808). Given the complexity of amino acid biosynthetic pathways and energy requirements, fungi rely on the absorption of plant amino acids to conserve their own energy. This could be one of the reasons for the contraction of GO terms in plant pathogens (Staats et al., 2013; Mur et al., 2017).
Botryosphaeriaceae fungi display both types of mating strategies, homothallism and heterothallism, which are determined by the single locus MAT1 (Mur et al., 2017). In this study, the mating type genes of 24 Botryosphaeriaceae fungal isolates (19 species) were analyzed, and we found that these genes were not highly conserved in terms of their nucleotide and amino acid sequences. However, for two subtype genes, two domains, MATalpha_HMGbox (MAT1-1-1) and MATA_HMG-box (MAT1-2-1), are more conservative. This is consistent with the fact that the mating type genes of fungi have almost no differences within a species, but they are highly divergent between species (Turgeon, 1998). In many ascomycete fungi, it is a conservative trend that the MATalpha and MATA_HMG-box regions contain introns (Arie et al., 2000; Paoletti et al., 2005; Duong et al., 2013; de miccolis et al., 2016). In D. sapinea and D. seriata, the introns in the MATA_HMG-box region of the MAT1-2-1 gene were lost, but the amino acid sequences that flank the lost intron site remain intact (Nagel et al., 2018). This phenomenon is consistent with the intron loss model derived from Poly-A primed mRNA (Sharpton et al., 2008). In this study, with the exception of Neofusicoccum, the arrangement of MAT1-1 and MAT1-2 genes is highly conserved in six other genera. They all are located between a collinear region that contains four protein-coding genes and one PIM protein, which is the same arrangement as found in most Phyllostictaceae fungi (Bihon et al., 2014; Wang et al., 2016). The arrangement of mating type genes of Neofusicoccum fungi is exceptional. The locations of the two subtypes of mating type genes are not in conjunction; they are either both located distantly from the collinear region or at different chromosomes (or scaffolds) (Lopes et al., 2017). Similarly, this discontinuous arrangement of mating type genes also exists in Aspergillus nidulans (Galagan et al., 2005), Curvularia cymbopogonis (Galagan et al., 2005), and Neosartorya fischeri (Rydholm et al., 2007).
The mating type gene MAT1 has two subtypes—MAT1-1 and MAT1-2. When two subtypes are both harbored in the genome, the mating strategy is hom, and if only one is present, it is het (Idnurm, 2011). The repetitive sequence-mediated deletion of one or more mating type genes can cause non-directional changes in mating types, such as the transition from self-fertility to self-sterility (Wilken et al., 2014; Xu et al., 2016; Yun et al., 2017). Such repetitive sequences were not observed in this study. Therefore, Botryosphaeriaceae fungi are unlikely to undergo a non-directional change regarding mating types (Nagel et al., 2018). In addition, to further understand the evolutionary characteristics of the fungal mating type in Botryosphaeriaceae, we reconstructed the ancestral state of the Botryosphaeriaceae fungal mating type and found that the het mating strategy is the ancestral type. Moreover, the fungi in this family experienced a number of transitions to the homothallism strategy, a shift that is common in ascomycete fungi (Inderbitzin et al., 2005; Yokoyama et al., 2006; Nygren et al., 2011; Gioti et al., 2012).
In this study, many gene families of the Botryosphaeriaceae fungi have shown significant expansions and contractions, and this change is conducive to the adaptation of fungi to the living environment (Alkan et al., 2013). These contracted gene families include genes that encode secondary metabolite synthases, secreted proteins, and carbohydrate active enzymes. In the family of genes that encode secondary metabolite synthases, NPRS and PKS gene clusters are responsible for the synthesis of toxic peptides and the production of naphthalenone pentaketides, respectively, in Botryosphaeriaceae fungi (D. seriata, L. theobromae and N. parvum) and other pathogenic fungi (A. fumigatus, Diaporthe ampelina, Phaeomoniella chlamydospora, and Togninia minima) (Andolfi et al., 2011; Paolinelli-Alfonso et al., 2016). Although the gene clusters of secondary metabolites are not regulated during infection, a large number of products of these gene clusters, which are significantly expanded in the genome, may be involved in the induction of disease symptoms and host adaptation (Yan et al., 2018). Cytochrome P450 is a superfamily of monooxygenases. In addition to participating in the post-synthesis modification of a variety of metabolites, it can also promote the adaptation of fungi to specific ecological niches by altering potentially harmful chemicals in the environment (Siewers et al., 2005). Here, we found the expansion of this gene family, which can explain the wide host range of Botryosphaeriaceae fungi. This is because these genes are also associated with some physiological characters; thus, their expansions are likely to promote pathogenic evolution (Nierman et al., 2005). In this study, expansions are obvious for the genes that encode ABC transporters in Botryosphaeriaceae fungi, indicating that these fungi have evolved stronger virulence and capacity against plant defense compounds (Han et al., 2001; Luini et al., 2010). Sugar synergistic transporters belong to the MFS family, and they play important roles in fungal spore formation, intercellular communication, and pathogenicity (Gaur et al., 2008). In the Botryosphaeriaceae fungi, we found that the sugar synergistic transporter gene family showed different degrees of expansion, which probably makes it more adaptive to different host environments (e.g., different pH values), and more resistant when interacting with various plant pathogens (Zhang et al., 2012; Yin et al., 2015).
Previous studies have showed that secreted proteins play important roles in the infection process of pathogenic fungi (Cobos et al., 2010; O’Connell et al., 2012; Fernandes et al., 2014; Félix et al., 2016, 2019). In this study, the secretory protein gene families of Botryosphaeriaceae fungi have expanded to different degrees, but there are large differences between different genera, which may be related to the different infection ranges of Botryosphaeriaceae fungi (Wang et al., 2018). In addition, the gene families of carbohydrate active enzymes in Botryosphaeriaceae fungi also showed different degrees of expansion. Among these families, the glycoside hydrolase family GH33 is composed of sialidase, which can hydrolyze the glycosidic bonds of terminal sialic acid residues in oligosaccharides. Sialidase can function as a pathogenic factor, facilitating the adaption to the host by evading host recognition or inhibiting host defense responses (Alviano et al., 2004). The cell wall of most dicotyledonous plants is composed of approximately 35% pectin. Pectin-degrading enzymes contribute to the degradation of the cell wall. This local degradation of the cell wall is necessary for fungi to enter the plant cytoplasm and replicate in the host tissue (ten Have et al., 1998). Similar to the highly pathogenic Colletotrichum higginsianum, Neofusicoccum, and Botryosphaeria both possess a larger number of genes encoding pectin-degrading enzymes, which may explain the difference in pathogenicity between Botryosphaeriaceae fungi (Wang et al., 2018). Many genes encoding cellulase (AA9 and GH12) and hemicellulase (GH31 and GH43) have been significantly expanded in Neofusicoccum and Botryosphaeria fungi. These expansions may explain the rapid infection and colonization of Botryosphaeriaceae fungi in woody plants (Yan et al., 2018).