Calonectria in the age of genes and genomes: Towards understanding an important but relatively unknown group of pathogens

Abstract The genus Calonectria includes many aggressive plant pathogens causing diseases on various agricultural crops as well as forestry and ornamental tree species. Some species have been accidentally introduced into new environments via international trade of putatively asymptomatic plant germplasm or contaminated soil, resulting in significant economic losses. This review provides an overview of the taxonomy, population biology, and pathology of Calonectria species, specifically emerging from contemporary studies that have relied on DNA‐based technologies. The growing importance of genomics in future research is highlighted. A life cycle is proposed for Calonectria species, aimed at improving our ability to manage diseases caused by these pathogens.


| INTRODUC TI ON
Calonectria (Ca.) is an ascomycete fungus that resides in one of 47 genera of the Nectriaceae (Lombard et al., 2015b;Rossman et al., 1999). The genus was originally erected in 1867 (De Notaris, 1867) based on the sexual morph of Calonectria daldiniana (a synonym of Ca. pyrochroa; Rossman, 1979) isolated from leaves of Magnolia grandiflora in Italy (Crous, 2002;Vitale et al., 2013). Later, Morgan (1892) described the asexual morph that has branched conidiophores, cylindrical conidia, and stipe extensions with characteristic terminal vesicles (Crous & Wingfield, 1994) in the genus Cylindrocladium (Cy.). For many years, species of Calonectria were best known by their asexual Cylindrocladium morphs. This was largely due to the fact that the asexual state is the one most commonly encountered on diseased plants. The dual nomenclature system applied for pleomorphic fungi meant that both asexual and sexual names were used in the literature, often interchangeably and sometimes inconsistently, resulting in considerable confusion (Hawksworth et al., 2011;Wingfield et al., 2012). For example, Cy. parasiticum is the asexual name for Ca. ilicicola (Crous et al., 1993b;Lechat et al., 2010), while the sexual name for Cy. ilicicola is Ca. lauri (Lechat et al., 2010). This confusing situation continued until the acceptance of the "one fungus, one name" system (Hawksworth et al., 2011;McNeill et al., plant hosts (Crous et al., 2018(Crous et al., , 2019Liu et al., 2020;Wang et al., 2019;Wu & Chen, 2021). Disease symptoms (Figure 1) resulting from these pathogens include seedling and cutting rot, leaf and shoot blight, leaf spot, defoliation, root rot, and stem canker (Crous, 2002;Liu et al., 2021b;Lombard et al., 2010c;Wu & Chen, 2021).
Most studies on Calonectria have focused on the taxonomy, phylogeny, and pathogenicity of species (Lombard et al., 2010c(Lombard et al., , 2016Marin-Felix et al., 2017). However, with the development of molecular biological techniques in recent years, our understanding of Calonectria has changed dramatically. The aim of this review is to illustrate how data relating to genes and genomes have significantly changed and influenced our understanding of the taxonomy and population genetic diversity in Calonectria. We also consider how a F I G U R E 1 Disease symptoms on Eucalyptus spp., including hybrids of E. urophylla with E. grandis (a, b, and d), E. tereticornis (c and f), and E. pellita   100 μm, (f) 20 μm, (g), (h) and (k)-(p) 10 μm, (i) and (j) 50 μm rapidly growing resource of genome data has already impacted, and will increasingly influence, our understanding of the pathogenicity in these important fungi.

| TA XONOMY OF C ALONEC TRIA
For many years subsequent to the first description of the genus, species of Calonectria were identified based on their morphological differences ( Figure 2). Asexual morphs treated in Cylindrocladium were recognized as providing more distinguishing characters than sexual morphs (Crous, 2002;Liu et al., 2020;Lombard et al., 2010bLombard et al., , 2016Rossman, 1979). This especially concerned the shapes and diameters of the vesicles as well as the septation and dimensions of the conidia (Crous, 2002;Lombard et al., 2010bLombard et al., , 2016Schoch et al., 2000a).
However, this approach was complicated by the fact that morphological variation between some species was, at best, subtle, leading to incorrect identification of species and the fact that cryptic species were commonly overlooked (Alfenas et al., 2015;Crous, 2002;Liu et al., 2020;Lombard et al., 2010aLombard et al., , 2010bLombard et al., , 2016Schoch et al., 1999Schoch et al., , 2000b. The biological species concept (BSC) is used to define a taxon as a group of organisms that can successfully interbreed and produce fertile offspring (Ereshefsky, 2007;Sokal & Crovello, 1970); the same biological species can be recognized by sexual compatibility within a species and the reproductive isolation between different species.
This approach was first applied to the taxonomy of Calonectria by Crous et al. (1993a) and it was extensively used for this purpose in later studies (Crous, 2002;Liu et al., 2020;Lombard et al., 2010a;Schoch et al., 1999Schoch et al., , 2000b. Most species of Calonectria are self-sterile and have a biallelic heterothallic mating system (Crous, 2002;Crous et al., 1998;. Consequently, some cryptic species have been recognized based on their mating compatibility (Lombard et al., 2010a;Schoch et al., 1999). However, limitations have arisen in applying the biological species concept to the taxonomy of Calonectria. For example, inducing fruiting bodies in the laboratory for Calonectria spp. is time consuming, requiring up to 2 months (Crous, 2002;Lombard et al., 2010a;Schubert et al., 1989). Sexual recombination is also a complex process related to the genetic properties of strains and is strongly influenced by the environment (Goodenough & Heitman, 2014). Thus, the conditions used to conduct mating studies in the laboratory are not uniformly conducive to achieve reliable results. Moreover, some species lose their ability to recombine and produce fertile progeny under laboratory conditions (Crous, 2002;Lombard et al., 2010a), implying that mating tests fail in culture.

| Gene and gene regions applied in the taxonomy of Calonectria
The emergence of DNA sequencing technology has contributed greatly to our capacity to recognize cryptic species and to identify unknown species. The phylogenetic species concept emphasizes nucleotide divergence between monophyletic lineages, which uses phylogenetic analysis of sequence variation to define species (Cai et al., 2011;Taylor et al., 2000). The Fungal Barcode of Life project has further highlighted the importance of a universal DNA barcode that could be used for the identification of all fungal species (Seifert, 2009).
In 1997, the internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) was applied as the barcoding gene to distinguish between species of Calonectria (Jeng et al., 1997). However, a low number of informative characters was available in the DNA sequence of this genetic region (Crous et al., 2000;Schoch et al., 1999). Later, Crous et al. (2000) found that the β-tubulin (tub2) gene provided better resolution than the ITS for the identification of these fungi. Taylor et al. (2000) established and recommended genealogical concordance phylogenetic species recognition, a high-resolution approach in which multiple gene genealogies could be used to separate isolates into genetically distinct species. Thus, between the years 2000 and 2010, gene genealogy analyses of calmodulin (cmdA), histone H3 (his3), translation elongation factor 1α (tef1), and tub2 sequences were frequently used to identify Calonectria spp. (Crous et al., 2004Lombard et al., 2009Lombard et al., , 2010d. Subsequently,

Lombard et al. (2010b) amplified seven different gene regions for
66 Calonectria species to screen for the best gene regions to delimit species in this genus. These gene regions included actin (act), cmdA, his3, ITS, tef1, tub2, and 28S nuclear ribosomal large subunit (LSU).
The results showed that the cmdA gene region provided the best resolution to distinguish between closely related species of Calonectria.

reconsidered the species boundaries based on 169
Calonectria species using eight gene regions, including act, cmdA, his3, ITS, LSU, DNA-directed RNA polymerase II subunit (rpb2), tef1, and tub2. A combination of six gene regions (tef1, tub2, cmdA, his3, rpb2, and act) was found to provide the best resolution and a stable basis for the identification of Calonectria species. Liu et al. (2020) proposed that these six gene regions be routinely used as effective barcodes for species in the genus.
Approximately 60 novel Calonectria spp. have been identified based on the multigene phylogenetic species concept approach in the last 10 years (Crous et al., 2018(Crous et al., , 2019Liu et al., 2020;Lombard et al., 2010b;Wang et al., 2019). The multigene phylogenetic species concept has thus had profound implications for the taxonomy of species in this genus. However, the phenomenon of conflicting gene trees has emerged as a common problem (Rokas & Carroll, 2006). Debates surrounding the multigene phylogenetic species concept continue and there remain many open questions. These include how many unlinked genes are necessary to reveal cryptic species (Balasundaram et al., 2015;Jeewon & Hyde, 2016;Taylor et al., 2000), how much sequence divergence should there be within a fragment of a gene to define a species (Jeewon & Hyde, 2016;Lukhtanov, 2019), what bootstrap value should be accepted to support new lineages (Hillis & Bull, 1993;Lukhtanov, 2019), and whether all genes selected suitably reflect the evolutionary history of the genus (Hillis & Bull, 1993;Lukhtanov, 2019).

However, there have been no phylogenomic analyses for species of
Calonectria, although it is inevitable that this situation will change in the near future.

| P OPUL ATI ON G ENE TI C S AND THE D IS E A S E C YCLE
Even though Calonectria has been known since the mid-19th century, very little work has been done to determine the population structure and diversity of populations for even the most important species.
The earliest such study was conducted by Schoch et al. (2001), and study used inter-simple sequence repeat markers to determine the genetic diversity of Ca. pteridis (Freitas et al., 2019). This provided the first evidence that genome data is emerging as an important tool to promote an enhanced understanding of the population biology of Calonectria species that cause important plant diseases.
All previous studies concerning the population biology of patho-  (Burgess & Wingfield, 2017;Wingfield et al., 2001). This also reflects trends in agriculture, forestry, and the ornamental plant trade that contribute deeply to the global distribution of pathogens (Roy, 2016;Santini et al., 2018;Wingfield et al., 2001), including Calonectria species, via international trade and travel.
Once a pathogenic Calonectria species has been accidentally introduced into a new environment (Figure 3), it is able to rapidly colonize and adapt by producing a large number of asexual propagules (conidia, chlamydospores, and microsclerotia) in a short period of time (Ashu & Xu, 2015;Crous et al., 1991;Vitale et al., 2013). This is consistent with the fact that conidia of the asexual stage of Calonectria species are often observed on diseased plant tissues.
Conidia are the propagules that enable direct penetration of healthy plant tissue (Crous, 2002;West, 2014). Chlamydospores usually occur in clusters and form microsclerotia, which are specialized longterm survival structures that can be found in soil and host tissue, enabling the fungus to resist harsh environments (Pérez-Sierra et al., 2007;Phipps et al., 1976). When conditions are suitable for growth to occur, microsclerotia germinate to produce hyphae and conidia that then infect plants (Avenot et al., 2017;Dart et al., 2015).
The asexual propagules of Calonectria species are able to disperse over short distances aided by rain splash or in irrigation water, in wind currents, via insect vectors, and farming tools, and thus to cause local disease epidemics (Crous, 2002;Crous et al., 1991;Vitale et al., 2013). In addition, once individuals of opposite mating type in a heterothallic Calonectria species are introduced into the same area and come into contact, the pathogen is able to produce new and potentially more aggressive genotypes via sexual recombination, which can be dispersed over longer distances (Crow, 1994;Heitman et al., 2013;Lumley et al., 2015). This then increases their adaptability to the environment and their ability to break down resistance genes in plant hosts, leading to disease outbreaks (Ashu & Xu, 2015;McDonald & Linde, 2002).
Calonectria spp. can have one of two modes of sexual reproduction (Alfieri et al., 1982;Schubert et al., 1989). Thus, some species are heterothallic whereas others are homothallic (Crous, 2002;Lombard et al., 2010c). In heterothallic species, individual isolates, derived from a single spore, will have either one of the two mating type

F I G U R E 3 Putative life cycle of Calonectria species. (a) Calonectria pathogens spread between regions via infected plant germplasm or contaminated soil. (b) Once
Calonectria is introduced, the propagules germinate to form mycelium on the surface of infected plants under suitable environmental conditions. Mycelium can rapidly initiate the asexual cycle by forming a large number of conidiophores in a short period of time. (c) Under unfavourable conditions, the pathogen can enter the sexual cycle by the union of individuals of opposite mating type (heterothallism) or self-fertilization (homothallism). After mate recognition, cell-cell fusion, and diploid zygote formation, gametes are generated via meiosis and ploidy changes via mitosis (Ni et al., 2011;Wilson et al., 2019). The haploid ascospores are formed and dispersed by wind or rain splash to penetrate healthy plant tissue. (d) Infections usually begin from the base of a tree or seedling and lead to various disease symptoms (Chen et al., 2011;Rodas et al., 2005). The long-term survival structures are microsclerotia that can be found in plant debris and soil (Crous, 2002;Phipps et al., 1976). When conditions are suitable for growth to occur, microsclerotia germinate to infect roots and the disease cycle is repeated    The genome assembly quality was evaluated using the abyss-fac function of ABySS (Jackman et al., 2017) in this review.
[Correction added on 23 April 2022, after first online publication: the 'Country' column in Table 1 has been deleted in this version.]

Ca. pseudoreteaudii is the causal agent of Calonectria leaf blight on
Eucalyptus in plantations of China and Southeast Asia (Crous et al., 2012;Li et al., 2017;Liu et al., 2021b;Lombard et al., 2010dLombard et al., , 2015aWang & Chen, 2020;Ye et al., 2017Ye et al., , 2018. Thus, understanding the biology and particularly the mechanisms underlying pathogenicity in Calonectria species is emerging as an important topic for future research, particularly as this relates to disease prevention and control. There have been few studies on Calonectria species regarding the mechanisms underpinning pathogenicity that have used genome sequencing technologies. The only such investigations have been those of Ye et al. (2017Ye et al. ( , 2018 and Santos et al. (2020), who analysed the mechanisms of pathogenicity in Ca. pseudoreteaudii on Eucalyptus. They suggested that the establishment of Calonectria leaf blight is associated with toxin and cell-wall-degrading enzymes (Santos et al., 2020;Ye et al., 2017Ye et al., , 2018. Clearly, there are many opportunities to better understand the biology of Calonectria species and their modes of pathogenicity. In this regard, future studies to consider these factors will depend on the availability of wholegenome sequences for Calonectria species. These are rapidly emerging and it is realistic to expect that genomes of most species of Calonectria spp. will become available for study in the near future. An important first step towards understanding the molecular basis of fungal pathogenicity in plants is the availability of a reliable and meaningful inoculation protocol. For Calonectria species, conidial suspensions or mycelial plugs placed on whole plants or detached leaves are commonly used to test for pathogenicity (Alfieri et al., 1972;El-Gholl et al., 1993;Graça et al., 2009;Guo et al., 2016;Richardson et al., 2020;Wang & Chen, 2020). These techniques are beset by a number of challenges. For example, various species of Calonectria fail to sporulate in culture, making inoculation using spores impossible. In these cases, it is common to use agar plugs overgrown with mycelium or mycelial fragments in inoculation tests.
It remains unclear whether the latter approach mirrors the natural situation and consequently studies considering genetic responses to infection could be compromised. Thus, future studies on the molecular mechanisms underpinning the pathogenicity of Calonectria species will require refined techniques to inoculate plants.  (Santos et al., 2020;Ye et al., 2017Ye et al., , 2018. We anticipate that the genomes of all Calonectria species available in culture will become available for study in the relatively near future. This will substantially promote our understanding of these fungi.

| CON CLUS IONS
While species of Calonectria have been collected and studied where they are associated with diseases of crop plants, there has been a strong bias towards particular environments such as their presence in Eucalyptus plantations Lombard et al., 2010b). This is linked to the fact that they have emerged as important constraints to Eucalyptus plantation forestry, particularly where these trees are propagated as non-natives (Alfenas et al., 2015;Freitas et al., 2019;Li et al., 2017Li et al., , 2021Liu et al., 2021b;Schoch et al., 2001;Wang & Chen, 2020;Wu & Chen, 2021;Ye et al., 2017Ye et al., , 2018. Where they have emerged on Eucalyptus, most species have probably originated as part of the natural soil environment. This suggests a significant gap in our knowledge and a need to sample soils in natural forests and other ecosystems. Sampling crop environments other than those linked to forestry and also the many parts of the world where these fungi have not been considered should be a priority in the future.
While genome sequencing has already impacted substantially on Calonectria research, next-generation sequencing and metagenomic studies have not been undertaken with a focus on these pathogens.
As with other fungi (Stewart et al., 2018;Tremblay et al., 2018;Vaz et al., 2017), such studies will make it possible to more deeply interrogate questions relating to the presence of Calonectria species in the soil and other environments where they might not easily be detected using culture-dependent approaches. They will also improve quarantine protocols and reduce accidental introductions of pathogenic Calonectria species into new environments.

ACK N OWLED G EM ENTS
This work was initiated through the bilateral agreement between

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed.