Pestalotiopsis—morphology, phylogeny, biochemistry and diversity

Sajeewa S. N. Maharachchikumbura; Liang-Dong Guo; Ekachai Chukeatirote; Ali H. Bahkali; Kevin D. Hyde

Phylogenetic evaluation of species nomenclature of Pestalotiopsis in relation to host association. Fungal Diversity 17: 39-55. DNA data from a number of Pestalotiopsis isolates were analysed to investigate whether isolates from the same host are phylogenetically related. The validity of naming species of Pestalotiopsis based on host association was investigated. Regions of the ITS and 5.8S of the rDNA gene were amplified from genomic DNA using PCR. DNA characters were analysed using maximum parsimony (weighted and unweighted) and maximum likelihood criteria. Isolates from the same host were not phylogenetically closely related. A close phylogenetic relationship between isolates possessing similar morphological characters was apparent. Results indicate that the naming of species based on host association is unwise and dispute the assumption that species are host-specific. When new Pestalotiopsis species are described, morphological characters should be taken into account rather than host association. The implications of the results on fungal biodiversity studies are discussed.

Phylogenetic diversity of endophytic Pestalotiopsis species in Pinus armandii and Ribes spp.: evidence from rDNA and β-tubulin gene phylogenies. Fungal Diversity 24: 1-22. The diversity of endophytes associated with bark and needles of Pinus armandii and leaves of Ribes species was investigated and resulted in 132 isolates being identified as Pestalotiopsis species. Isolates were grouped into morphospecies based on morphology and cultural characteristics and named based on morphological characters. To supplement identifications, ribosomal DNA (ITS and 5.8S) and β-tubulin gene sequences from 37 isolates representing diverse morphological and cultural characteristics were analyzed phylogenetically (maximum parsimony and Bayesian criteria). Phylogenies corroborated with morphologies and are largely congruent with previously established taxonomic schemes. Phylogenetic comparison of the two genes reveals that ITS based phylogenies alone were not suitable to infer phylogenetic relationships among Pestalotiopsis species as most of the clades did not receive adequate statistical support. β-tubulin phylogenies were better resolved but a combined dataset was found to be more appropriate. Although results are generally concordant with published studies, there seems to be several inconsistencies in grouping taxa at the species level. The phylogenetic relationships of species in relation to culture morphology, as well as to host and tissue association are discussed.

Fungal Diversity (2011) 50:167–187 DOI 10.1007/s13225-011-0125-x Pestalotiopsis—morphology, phylogeny, biochemistry and diversity Sajeewa S. N. Maharachchikumbura & Liang-Dong Guo & Ekachai Chukeatirote & Ali H. Bahkali & Kevin D. Hyde Received: 8 June 2011 / Accepted: 22 July 2011 / Published online: 31 August 2011 # Kevin D. Hyde 2011 Abstract The genus Pestalotiopsis has received considerable attention in recent years, not only because of its role as a plant pathogen but also as a commonly isolated endophyte which has been shown to produce a wide range of chemically novel diverse metabolites. Classification in the genus has been previously based on morphology, with conidial characters being considered as important in distinguishing species and closely related genera. In this review, Pestalotia, Pestalotiopsis and some related genera are evaluated; it is concluded that the large number of described species has resulted from introductions based on host association. We suspect that many of these are probably not good biological species. Recent molecular data have shown that conidial characters can be used to distinguish taxa; however, host association and geographical location is less informative. The taxonomy of the genera complex remains confused. There are only a few type cultures and, therefore, it is impossible to use gene sequences in GenBank to clarify species names reliably. It has not even been established whether Pestalotia and Pestalotiopsis are distinct genera, as no isolates of the type species of Pestalotia have been sequenced, and they S. S. N. Maharachchikumbura : L.-D. Guo (*) Key Laboratory of Systematic Mycology & Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China e-mail: guold@sun.im.ac.cn S. S. N. Maharachchikumbura : E. Chukeatirote : K. D. Hyde (*) School of Science, Mae Fah Luang University, Thasud, Chiang Rai 57100, Thailand e-mail: kdhyde3@gmail.com A. H. Bahkali : K. D. Hyde College of Science, Botany and Microbiology Department, King Saud University, P.O. Box: 2455, Riyadh 1145, Saudi Arabia are morphologically somewhat similar. When selected GenBank ITS accessions of Pestalotiopsis clavispora, P. disseminata, P. microspora, P. neglecta, P. photiniae, P. theae, P. virgatula and P. vismiae are aligned, most species cluster throughout any phylogram generated. Since there appears to be no living type strain for any of these species, it is unwise to use GenBank sequences to represent any of these names. Type cultures and sequences are available for the recently described species P. hainanensis, P. jesteri, P. kunmingensis and P. pallidotheae. It is clear that the important species in Pestalotia and Pestalotiopsis need to be epitypified so that we can begin to understand the genus/genera. There are numerous reports in the literature that various species produce taxol, while others produce newly discovered compounds with medicinal potential and still others cause disease. The names assigned to these novel compound-producing taxa lack an accurate taxonomic basis, since the taxonomy of the genus is markedly confused. Until the important species have been epitypified with living strains that have been sequenced and deposited in public databases, researchers should refrain from providing the exact name of species. Keywords Epitypify . Host occurrence . Pestalotia . Pestalosphaeria . Pigmentation . Secondary metabolites . Taxol Introduction Pestalotoiopsis Steyaert is an appendage-bearing conidial anamorphic form (coelomycetes) in the family Amphisphaeriaceae (Barr 1975, 1990; Kang et al. 1998, 1999), and molecular studies have shown that Pestalotiopsis is monophyletic (Jeewon et al. 2002, 2003, 2004). Species of Pestalotiopsis are common in tropical and temperate 168 ecosystems (Bate-Smith and Metcalfe 1957) and may cause plant disease (Das et al. 2010), are often isolated as endophytes (Liu et al. 2006; Wei et al. 2007; Watanabe et al. 2010), or occur as saprobes (Wu et al. 1982; Agarwal and Chauhan 1988; Yanna et al. 2002; Hu et al. 2007; Liu et al. 2008a). The genus has received much attention from the scientific community. However, this not because of its pathogenic nature (Hyde and Fröhlich 1995; Rivera and Wright 2000; Yasuda et al. 2003), but rather because its species have been shown to produce many important secondary metabolites (Strobel et al. 1996a, 2002; Ding et al. 2008a, b; Aly et al. 2010; Xu et al. 2010). The aim of the present paper on Pestalotia, Pestalotiopsis and similar genera is to review (1) historical aspects, (2) morphological and molecular studies, (3) life mode of taxa, (4) species numbers and (5) biochemical production by selected species. The problems of understanding the genus are discussed and the work needed to resolve these problems elaborated. In most cases problems arise due to misidentification of taxa and the review illustrates the importance of the correct identification of strains before they are used in biochemical or other studies. History De Notaris (1839) introduced the genus Pestalotia De Not. based on the generic type Pestalotia pezizoides De Not., which occurred on the leaves of Vitis vinifera in Italy. This species is characterized by 6-celled conidia with four deeply olivaceous central cells, distosepta, hyaline terminal cells and simple or branched appendages arising from the apex (Fig. 1.). Steyaert (1949) revised Pestalotia and divided the genus into three main groups based on the Fig. 1 Pestalotia pezizoides De Not. BPI0406483, a Conidia b conidiogenous cells. Scale bars: a–b=20 μm Fungal Diversity (2011) 50:167–187 conidial forms. Steyaert (1949) also introduced two new genera, Truncatella Steyaert for 4-celled conidial forms and Pestalotiopsis Steyaert for the 5-celled forms, while the 6celled forms remained in Pestalotia. Pestalotia was considered to be a monophyletic genus and Steyaert (1949) suggested that the type species could be distinguished from Pestalotiopsis by it cupulate conidiomata and distoseptate median cells. Steyaert (1949) further divided Pestalotiopsis into additional sections based on the number of apical appendages. These were the Monosetulatae, Bistulatae, Trisetulatae and Multisetulatae, which were further divided into subdivisions. Conidia with a single setulae (apical appendage) were included in the Monosetulatae, which was further divided into forms with simple and branched setulae. Conidia with two setulae or on average two setulae were included in the Bistulatae. Conidia with three setulae or on average three setulae were included in the Trisetulatae, which was further divided by concolorous or versicolorous conidia, fusiform or claviform conidia and spatulate or nonspatulate setulae. Conidia with more than three setulae were included in the Multisetulatae. Steyaert (1949) reduced Monochaetia (Sacc.) Allesch. from its generic state and placed species with single setula in section Monosetulatae of Pestalotiopsis and Truncatella. Steyaert (1949) provided descriptions of 46 species and Pestalotiopsis guepinii (Desm.) Steyaert was considered to be the type species of the newly introduced genus. Pestalotiopsis guepinii is characterized by 4-euseptate and fusiform conidia with a hyaline basal cell. Steyaert’s introduction of the genus Pestalotiopsis was not supported by Moreau (1949), Servazzi (1953) and Guba (1956, 1961). Steyaert (1953a, b, 1961, 1963), however, published further evidence in support of his new genus with answers to the criticisms made by others. Fungal Diversity (2011) 50:167–187 The primary work on Pestalotia was carried out by Guba (1961) in his “Monograph of Monochaetia and Pestalotia”. Guba (1961) divided the genus into the sections quadriloculate, quinqueloculatae and sexloculatae for 4-celled conidia, 5-celled conidia and 6-celled conidia respectively. For his sections, Guba (1961) used a simple but very effective system as proposed by Klebahn (1914), which was based on the number conidial cells. Guba (1961) further subdivided the sections into different categories, mainly on the basis of conidial form, colour, and the position, and character of the setulae. Monochaetia was retained as a distinct genus based on its single apical appendage, while Pestalotiopsis and Truncatella, the new genera proposed by Steyaert (1949), were synonymised with Pestalotia. Guba (1961) described 258 species of Pestalotia in his monograph. Steyaert (1956) argued that the retention of Monochaetia as a distinct genus based on a single character, a single apical appendage was incorrect, while other genera (Pestalotiopsis, Truncatella and Pestalotia) were differentiated from each other based on a set of characters. Sutton (1961, 1980) gave more weight to conidiogenesis when considering Pestalotia and Pestalotiopsis, and he identified three major problems relating to their taxonomy. According to the Steyaert system, Sutton (1980) concluded that a large number of species that should be included in Pestalotiopsis are still placed in Pestalotia by some authors. In their studies, Guba (1961), Steyaert (1949, 1953a, b, 1955, 1956, 1961) and most other workers used primarily dried herbarium material. Sutton (1980) pointed out that when species were grown in artificial culture, they show more variability and species limits overlap. Therefore, identification of species from culture and the application of names based on herbarium taxonomy present a confusing situation. Sutton (1980) used the investigation of Griffiths and Swart (1974a, b), which showed the differentiation of conidial wall development in two species of Pestalotiopsis, P. funerea (Desm.) Steyaert and P. triseta (Moreau & V. Moreau) Steyaert and in Pestalotia pezizoides to support Steyaert’s opinions. Griffiths and Swart (1974a, b) electron microscopic study was carried out to establish the relationship among Pestalotia and Pestalotiopsis and other allied generic members of Monochaetia and Seimatosporium Corda. The minute zonation in conidial wall structure of P. pezizoides was thought to separate it from Pestalotiopsis (Griffiths and Swart 1974a, b). Until 1990, phylogenetic understanding of the taxonomy associated with Pestalotiopsis and allied genera was based mainly on conidial characters (Steyaert 1949; Guba 1961; Nag Rag 1993), conidiogenesis (Sutton 1980) and teleomorph association (Barr 1975, 1990; Metz et al. 2000; Zhu et al. 1991). Morphological characters used to differentiate species of Pestalotiopsis and similar genera are limited (Hu et al. 2007); the morphological characters are plastid and morphological 169 markers vary between host and environment (Egger 1995). Hu et al. (2007) showed that colony morphology (colour, growth rate and texture) is highly variable within single isolates of Pestalotiopsis; this phenomenon can be easily observed through repeated subculturing. Also within a single species, conidial morphology (shape and colour of the median cells), growth rate and fruiting structure, may vary (Jeewon et al. 2003). Satya and Saksena (1984) observed Pestalotiopsis glandicola (Castagne) Steyaert and P. versicolor var. polygoni and found that the intensity of the median cells varied with culture and host and concluded that colour of median cells cannot be used to judge their taxonomic position. Dube and Bilgrami (1965) observed Pestalotiopsis darjeelingensis Dube, Bilgrami & H.P. Srivast. and showed morphological variation of conidia in culture (dimension, length of the setulae, shape, number of cells and the colour of the cells). Similar observations were made by Purohit and Bilgrami (1969) when studying more than 100 pathogenic strains. Conidiogenesis is also confusing when used for species separation; Watanabe et al. (1998), showed that Pestalotiopsis neglecta (Thüm.) Steyaert and P. guepinii having similar acervuli development. Jeewon et al. (2003) and Tejesvi et al. (2009) compared morphology with sequence data and showed that species of Pestalotiopsis display considerable diversity in morphology and that isolates grouped together based on similarities in conidial morphology. Hu et al. (2007) found that conidial characters such as conidial length, median cell length, conidial width and colour of median cells were stable characters within Pestalotiopsis; however, the length of the apical and basal appendages varied. Jeewon et al. (2003) evaluated the morphological characters that could be used to differentiate species of Pestalotiopsis. He suggested that melanin granule deposition within the cell matrix providing pigmentation to the median cells has taxonomic value; this agreed with the findings of Griffiths and Swart (1974a, b). He suggested that the colour of median cells was useful for distinguishing species of Pestalotiopsis. Tejesvi et al. (2009) also agreed that species of Pestalotiopsis can be distinguished on the basis of morphological characters rather than host-specificity or geographical location. Liu et al. (2010a) proposed that instead of using “concolorous” and “versicolor” as proposed by Steyaert (1949) and Guba (1961), “brown to olivaceous” and “umber to fuliginous” median cells can be a key character in distinguishing species in Pestalotiopsis. However the pigmentation can be effected by environmental conditions, different stages of spore maturity and the observer’s expertise (Liu et al. 2010a), hosts, medium, and even different generations through subculturing (Purohit and Bilgrami 1969; Satya and Saksena 1984; Hu et al. 2007). The pigmentation of the median cell however, can be stable even within a successive subculture; when using standard conditions and 170 culture on autoclaved carnation leaf segments (Liu et al. 2010a). ‘The teleomorph of a whole fungus has been traditionally classified and named separately from their anamorphs. Each of the morphs of anamorphosis was also given different binomials as if they were different species. As a result, a whole fungus finds itself in two classification and nomenclature systems against the principle of natural classification’ (Shenoy et al. 2007). The gene responsible for the expression of teleomorph and anamorph evolve at different rates; anamorph characters tend to be morphologically divergent even with the monophyletic groups while teleomorph characters are highly conserved (Chaverri et al. 2003; Dodd et al 2003). The teleomorph characters can thus be used as a precise taxonomic marker for Pestalotiopsis. However the anamorph of Pestalotiopsis is Pestalosphaeria M.E. Barr and only twelve species are known as compared to the asexual state (235 species names). Pestalotiopsis has been linked to Neobroomella Petr. one species and was described by Petrak (1947) and Pestalosphaeria (12 species), the genus being described by Barr (1975). As such, the earliest name is Neobroomella, but this state has rarely been recorded. Pestalotia De Not. has been linked to Broomella Sacc. (1883) which has 20 species. Since Pestalotiopsis is the most commonly used name, we therefore suggest that this name be adopted for the anamorph and teleomorph forms. However, if Pestalotia is found to incorporate species of Pestalotiopsis in future studies, then this name would be used to represent Broomella, Neobroomella and Pestalosphaeria. Morphological characters use in the differentiation of species Conidial morphology (Fig. 2.) is the most widely used taxonomic character for the genus Pestalotiopsis. Most species are divided into different groups based on the size of the conidia. The length and width are good taxonomic markers for the genus and stable within the different media and the generations in most cases (Hu et al. 2007). Colour of the median cells is still a widely used character, and all species separate into three groups based on this- concolorous, versicolorous umber olivaceous and versicolorous fuliginous olivaceous. Molecular evidence indicates that it is more precise to group species according to concolorous and versicolorous rather than the above three groups (Jeewon et al. 2003). The length of the apical appendages and the number of the apical appendages are also widely used characters for species identification. Some species can also be identified by the presence of knobbed apical Fungal Diversity (2011) 50:167–187 Fig. 2 Some commonly use conidial characters for Pestalotiopsis b species identification (1) colour of the median cells a light concolorous b dark concolorous c versicolorous (2) size of the conidia d small conidia e large conidia f relatively long conidia g relatively broad conidia (3) number of apical appendages h two apical appendages i three apical appendages j five apical appendages (4) presence or absence of knobbed apical appendages k apical appendages without knobbed apical appendages l apical appendages with knobbed apical appendages (5) length of the apical appendages m relatively short apical appendages n relatively large apical appendages (6) branched or unbranched apical appendages o branched apical appendages (7) position of the apical appendages attached to the apical cell p attached to the top of the apical appendages q attached to the middle of the apical appendages r some attached to the bottom of the apical cell (8) presence or absence of basal appendages s presence of apical appendages t absence of apical appendages. Scale bars: a–b=20 μm appendages. The apical appendages can arise from the top, middle, bottom or different positions in the apical hyaline cells and such characters are widely used in species identification. Furthermore the apical appendages can be divided into branches; in some species presence or absence of the basal appendages is another character for species diagnosis. Recent molecular data Hu et al. (2007) showed that the ITS gene is less informative than the β-tubulin gene in differentiating endophytic species of Pestalotiopsis in Pinus armandii and Ribes spp. When gaps in the ITS region are treated as a missing data, the total number of informative characters is 5% and this results in difficulty in separating taxa and low statistical support. When β-tubulin gene data are used and gaps are treated as missing data, the number of informative characters is about 11%, and when gaps are treated as newstate, it is more than 15%. Thus, Hu et al. (2007) pointed out that the β-tubulin genes resolves Pestalotiopsis phylogeny better than the ITS gene. A combination of both the β-tubulin and ITS genes gave better phylogenetic resolution, and they suggested that at least two genes should be used to resolve the phylogeny of species of Pestalotiopsis. However, Liu et al. (2010a) disagreed with Hu et al. (2007) concerning the ITS region as being less informative when compared to the β-tubulin region. They indicated that proper analysis and alignment of the ITS region can be a useful character in grouping Pestalotiopsis to different types of pigmentation, which can be used as a key character for the phylogeny of the species. Random amplification of polymorphic DNA (RAPD) can also be used to detect genetic diversity in species of Pestalotiopsis (Tejesvi et al. 2007a). Tejesvi et al. (2009) showed that the Fungal Diversity (2011) 50:167–187 171 172 ITS region is more informative than internal transcribed spacer—restriction fragment length polymorphism (ITSRFLP). They used five restriction enzymes (Alu I, Hae III, Ava II, Hpa II and Taq I) in their ITS-RFLP analysis and showed that ITS-RFLP profiles were distinctly different in P. virgatula (Kleb.) Steyaert and P. theae (Sawada) Steyaert and intraspecific polymorphism highly variable in P. microspora (Speg.) G.C. Zhao & N. Li. Based on the ITS sequence, pathogenic and endophytic strains clustered into distinct groups and these clusters were irrespective of the host, parts of the host or location. Life cycle in Pestalotiopsis A disease cycle of a pathogen may be closely related to its life cycle, and the former refers to the emergence, development and maintenance of the disease (Agrios 2005) but is not discussed further here. Species of Pestalotiopsis are not highly host-specific and taxa may have the ability to infect a range of hosts (Hopkins and McQuilken 2000; Keith et al 2006). Species of Pestalotiopsis cause a variety of disease in plants, including canker lesions, shoot dieback, leaf spots, needle blight, tip blight, grey blight, scabby canker, severe chlorosis, fruit rots and leaf spots (Pirone 1978; Kwee and Chong 1990; Xu et al. 1999; Tagne and Mathur 2001; Sousa et al. 2004; Espinoza et al. 2008). Pirone (1978) considered that species of Pestalotiopsis are weak or opportunistic pathogens and may cause little damage to ornamental plants; however, Hopkins and McQuilken (2000) pointed out that some species of Pestalotiopsis may cause serious damage to pot grown plants and the number of known infected plant species is generally increasing. Pathogenic species of Pestalotiopsis initially make contact with the host where the infection occurs (inoculum), probably by means of the conidia or fragmented spores (Espinoza et al. 2008). These inocula may survive during harsh weather conditions and may cause primary infections. Secondary inoculum produced on diseased tissue may cause secondary infections and increase the severity of the disease. The source of the inoculum can be wild plantations (Keith et al. 2006), flowers (Pandey 1990), crop debris, disease stock plants, used growing media, soil and contaminated nursery tools (McQuilken and Hopkins 2004), splashed water droplets (Hopkins and McQuilken 1997; Elliott et al. 2004) and also spores in the air (Xu et al. 1999). Species of Pestalotiopsis have constantly been isolated as endophytes from plant tissues (Wei and Xu 2004; Liu et al. 2006; Wei et al. 2005, 2007; Tejesvi et al. 2009; Watanabe et al. 2010). We suspect that many endophytic species remain as dormant symptomless inhabitants of plants until the plant is stressed, and then Fungal Diversity (2011) 50:167–187 the endophytes become pathogens. This is thought to occur in other pathogenic genera (Gehlot et al. 2008). The pathogenic phase may be triggered by a combination of environmental factors, plant susceptibility and the virulence of the pathogen. However, further research is needed to prove the endophytic pathogenic relationship in the genus. Pestalotiopsis is also considered to be a weak pathogen (Madar et al. 1991), and most weak pathogens penetrate the host through natural openings such as stoma, lenticels and hydathodes (Agrios 2005). Wright et al. (1998) stated that species of Pestalotiopsis only infect wounded or stressed plants, so pruning wounds or other physical means play important roles in disease development (Elliott et al. 2004; McQuilken and Hopkins 2004; Keith et al. 2006). Plants may also be stressed due to insect, pesticide or sun damage (Hopkins and McQuilken 2000). High temperature, high rainfall and human activities may also trigger infections, and this may lead to disease development (Tuset et al. 1999; Hopkins and McQuilken 2000; Elliott et al. 2004). The anamorph-teleomorph relationships and life cycles are not well known for most species, as the sexual stage does not often develop (Armstrong-Cho and Banniza 2006). Therefore, conidia therefore appear to play a key role in providing the inocula. A general disease cycle for Pestalotiopsis is illustrated in Fig. 3. The spore of Pestalotiopsis is considered to be a dry spore. Watanabe et al. (2000) studied conidial adhesion and germination of spores of P. neglecta and showed that infection occurs in four stages. At the beginning, the lower median cell germinates and becomes firmly attached to the substrate. Future successive infections can be achieved by two upper median cells. In the first stage, weak adhesion is achieved by the mucilaginous matrix coating the conidia. A second weak adhesion occurs at the bases of the pedicel. The next two stages provide a strong attachment by release of fibrillar adhesive substances. In the third stage, fibrillar adhesive substances are produced along the length of the pedicel to the apex of the basal cell and at times a smaller amount of fibrillar material is released from the apical appendages. The fourth stage involves the release of fibrillar material at the point of germ tube emergence. Nag Rag (1993) described conidiomata of the genus as variable, ranging from acervuli to pycnidia. Conidiomata can be immersed to erumpent, unilocular to irregularly plurilocular with the locules occasionally incompletely divided and dehiscence by irregular splitting of the apical wall or overlying host tissue (Nag Rag 1993). Conidiophores partly or entirely develop inside the conidiomata, and they can be reduced to conidiogenesis cells which are discrete or integrated, cylindrical, smooth, colourless and invested in mucus (Nag Rag 1993). Pycnidia can mostly be seen with the unaided eye as a black or brown spore masses with copious conidia. Fungal Diversity (2011) 50:167–187 173 Fig. 3 Disease cycle of the genus Pestalotiopsis (References: revised and redrawn; Von Arx 1974; Nag Rag 1993; Kobayashi et al. 2001) Control strategies are needed for serious Pestalotiopsis disease, and therefore, knowledge of the causal agent and the disease cycle is important. Precise knowledge of the plant/ pathogen interaction and its functional variation according to the environmental factors are important for integrated disease management using cultural, biological and chemical methods. Elliott et al. (2004) stated that Pestalotiopsis may produce large numbers of spores which 174 are easily dispersed in air or by water splash, thus sanitation and disease management are critical. They suggested that water management strategies, such as elimination of overhead irrigation, decreasing wetness of leaves, increasing the spacing of plants and increasing the air circulation, can reduce disease in palm plantations. Different harvesting factors also directly affected disease development in tea plantations. Sanjay et al. (2008) showed that highest disease incidence occurred in continuously shear-harvested fields and least in hand-plucked plantations, and they evaluated systemic fungicide and biocontrol agents such as a Trichoderma, Gliocladium and Pseudomonas for use in controlling grey blight disease in tea. Fungal Diversity (2011) 50:167–187 not always clear that the two stages found are definitely the same biological species and therefore molecular evidence is needed to link them. In the laboratory species of Pestalotiopsis rarely develop sexual forms (Metz et al. 2000). Zhu et al. (1991) induced Pestalosphaeria accidenta P.L. Zhu, Q.X. Ge & T. Xu and P. jinggangensis P.L. Zhu, Q.X. Ge & T. Xu to form on potato dextrose agar (PDA). However, this took 5 to 6 months of incubation. Metz et al. (2000) obtained the sexual state of P. microspora, an endophytic isolate that produced taxol. The asexual stage formed after 3–6 weeks on water agar with dried yew needles when incubated at 16–20 C with 12 h of light per day and was identified as Pestalosphaeria hansenii Shoemaker & J.A. Simpson. The twelve sexual states known for species of Pestalotiopsis are listed in Table 1. Mode of life Species of Pestalotiopsis commonly cause disease in a variety of plants (Hyde and Fröhlich 1995; Hopkins and McQuilken 2000; Tagne and Mathur 2001), are commonly isolated as endophytes (Kumar and Hyde 2004; Wei and Xu 2004; Wei et al. 2005, 2007; Liu et al. 2006; Tejesvi et al. 2009; Watanabe et al. 2010) and some species likely have endophytic and pathogenic stages in their life cycle (Wei et al. 2007: Tejesvi et al. 2009). Species have also been recorded as saprobes (Guba 1961; Wu et al. 1982; Agarwal and Chauhan 1988; Yanna et al. 2002; Liu et al. 2008a) where they are recyclers of dead plant material (Okane et al. 1998; Osono and Takeda 1999; Tokumasu and Aoiki 2002) and even rarely cause disease in humans (Sutton 1999) Sexual and asexual forms One fifth of all known anamorphic fungi lack known sexual states (Shearer et al. 2007), and out of 2,873 anamorphic genera names, 699 genera and 94 anamorph-like genera are linked to a sexual state (Hyde et al. 2011). The links between sexual and asexual stage are mostly from indirect evidence, with some links known through experimental or molecular data (Kendrick 1979; Reynolds 1993; Shenoy et al. 2007; Hyde et al. 2011). Pestalotiopsis is a species-rich anamorphic genus with species mostly lacking sexual morphogenesis, unlike the coelomycetous genera Colletotrichum and Phyllosticta (Armstrong-Cho and Banniza 2006; Wulandari et al. 2009) and Penicillium (Cannon and Kirk 2000). The sexual states or teleomorphs of Pestalotiopsis species have been identified as Pestalosphaeria (Barr 1975) and Neobroomella (Kirk et al. 2008). The asexual Pestalotiopsis state and ascomycetous sexual state have rarely been recorded in the same host plant (Barr 1975; Nag Raj 1985; Hyde 1996). However, it is Pestalotiopsis Steyaert as a plant pathogen Pestalotiopsis is a relatively important plant pathogenic genus known mostly from the tropics, where it causing leaf blights (Guba 1961) in many plant species (Hyde and Fröhlich 1995; Xu et al. 1999; Das et al. 2010). Species may also cause rots of fruit and other post harvest disease (Ullasa and Rawal 1989; Korsten et al. 1995; Xu et al. 1999). It has been estimated that in southern India grey blight disease of tea (Camellia sinensis) caused by Pestalotiopsis has resulted in 17% production loss (Joshi et al. 2009) and 10–20% yield loss in Japan (Horikawa 1986). Five species of Pestalotiopsis - have been recorded from tea (Agnihothrudu 1964), although P. longiseta (Speg.) H.T. Sun & R.B. Cao and P. theae are considered to be the major species causing grey blight (Joshi et al. 2009). Pestalotiopsis sydowiana (Bres.) B. Sutton causes foliage, root and stem-base browning disease in container-grown ericaceous plants, resulting in plant losses and reduced plant quality (McQuilken and Hopkins 2004). Antheraea assamensis, a silkworm endemic to the north eastern part of India that depends on Perseabombycina as the primary food plant, is endangered due to grey blight disease cause by Pestalotiopsis disseminata (Thüm.) Steyaert (Das et al. 2010). Pestalotiopsis funerea was found to cause leaf spots of Hakea sericea, a plant that is considered as an invader of natural habitats in northern Portugal, and this may allow its use in biological control (Sousa et al. 2004). P. menezesiana (Bres. & Torrend) Bissett and P. uvicola (Speg.) Bissett causes postharvest disease of grape (Xu et al. 1999) and P. clavispora (G.F. Atk.) Steyaert, P. disseminata and P. microspora cause scab in Guava in Hawaii (Keith et al. 2006). The economically important blueberry fruit from Chile is infected by pathogenic P. clavispora and P. neglecta, which cause canker and twig dieback (Espinoza et al. 2008). Fungal Diversity (2011) 50:167–187 175 Table 1 List of anamorphs with known teleomorphs Asexual form Sexual form Pestalotiopsis Pestalotiopsis Pestalotiopsis Pestalotiopsis Pestalotiopsis Pestalotiopsis Pestalotiopsis Pestalotiopsis baarnensis Steyaert sp. sp. guepinii var macrotricha (Kleb.) B. Sutton sp. eugeniae (Thüm.) S. Kaneko neglecta microspora Pestalosphaeria Pestalosphaeria Pestalosphaeria Pestalosphaeria Pestalosphaeria Pestalosphaeria Pestalosphaeria Pestalosphaeria accidenta alpiniae P.K. Chi & S.Q. Chen austroamericana Nag Raj & DiCosmo concentrica M.E. Barr elaeidis (C. Booth & J.S. Robertson) Aa eugeniae P.K. Chi & S.M. Lin gubae Tak. Kobay., Ishihara & Yas. Ono hansenii Pestalotiopsis Pestalotiopsis Pestalotiopsis Pestalotiopsis podocarpi (Dennis) X.A. Sun & Q.X. Ge sp. maculiformans (Guba & Zeller) Steyaert besseyi (Guba) Nag Raj Pestalosphaeria Pestalosphaeria Pestalosphaeria Pestalosphaeria jinggangensis leucospermi Samuels, E. Müll. & Petrini maculiformans Marinc., M.J. Wingf. & Crous varia Nag Raj In Sicily, the economically important plant Laurus nobilis is infected by P. uvicola, which causes causing leaf spots and stem blights (Vitale and Polizzi 2005). Chlorosis and reduction of growth were recorded in maize fields in the Cameroons when the plants were infected by P. neglecta (Tagne and Mathur 2001). The medicinally important ornamental shrub Lindera obtusiloba, which grows wild in the mountain areas of the Korean Peninsula, is infected by P. microspora, and the affected leaves initially have grey or dark brown lesions, surrounded by yellowish halos; these enlarge, coalesce and become entire at a later stage, finally causing full leaf blight (Jeon et al. 2007). Affected leaves of Hymenaea courbaril show symptoms of leaf spots and the pathogen was identified as a P. subcuticularis (Guba) J.G Wei & T. Xu (Fail and Langenheim 1990). Pathogenic P. funerea infects conifer species and causes necrosis on infected tissues and sometimes death of the plants involved (Bajo et al. 2008). The medicinal and ornamental Carapa guianensis is infected by P. macrochaeta (Speg.) J. Xiang Zhang & T. Xu, and foliar blight has been observed in the lower canopy of the plants (Halfeld-Vieira and Nechet 2006). Species of Pestalotiopsis also have the potential to cause leaf and/or fruit spots on ginger, rambutan, lychee and orchid (Keith and Zee 2010) Pestalotiopsis glandicola is a postharvest pathogen on mango in Bangalore; the disease can be observed on the leaves throughout the year and it provides the inoculum for mature fruits, which develop postharvest decay during storage (Ullasa and Rawal 1989). Fruit rot of grapevine is caused by P. menezesiana and P. uvicola, and the pathogens were not only isolated from diseased and healthy fruits but also from the airspora in grape orchards; thus, the authors pointed out that latent infection or conidial attachment to the barriers in the field will lead to postharvest disease in grapes (Xu et al. 1999). Pestalotiopsis fruit rot is one of the serious postharvest diseases of rambutan fruit in Thailand (Sangchote et al 1998). Pestalotiopsis psidii (Pat.) Mordue is considered to be the causal agent of scabby fruit canker of guava in India and infection results in rapid yield loss and affects the postharvest quality of the fruits (Kaushik et al. 1972). Pestalotiopsis as an endophyte Most resent Pestalotiopsis research is based on endophytic isolates (Liu et al. 2006; Wei et al. 2007; Watanabe et al. 2010; Aly et al. 2010) and has resulted in a four new species being described. These are P. hainanensis A.R. Liu, T. Xu & L.D. Guo, P. jesteri Strobel, J. Yi Li, E.J. Ford & W.M. Hess, P. kunmingensis J.G. Wei & T. Xu and P. pallidotheae Kyoko Watanabe & Yas. Ono. Most endophytic studies have used morphological characters and either gene sequence data (Hu et al. 2007; Liu et al. 2007; Wei et al 2007) or RFLP technique (Tejesvi et al. 2007a) or a combination of gene sequence and RFLP techniques (Tejesvi et al. 2009) to distinguish species. The distribution of the endophytic species of Pestalotiopsis is ubiquitous and is not largely influenced by geographical factors (Wei et al. 2007; Tejesvi et al. 2009). Tejesvi et al. (2005) stated that the endophytic species of Pestalotiopsis dominant in the winter season and their colonization are comparatively low in the monsoon season. The colonization frequency of species of Pestalotiopsis increased with the increasing the age of the host plant and colonization frequency was variable (Wei et al. 2007). Some endophyte studies in which species of Pestalotiopsis have been recovered are listed in Table 2. Pestalotiopsis as a saprobe Species of Pestalotiopsis have been repeatedly isolated as saprobes from dead leaves, bark and twigs (Guba 1961). 176 Fungal Diversity (2011) 50:167–187 Table 2 List of endophytes and associated host Species Host References P. clavispora Camellia oleifera, C. sinensis, Terminalia arjuna, Podocarpus macrophyllus Lithocarpus glabra, C. nitidissima Catharanthus roseus Podocarpus macrophyllus Camellia japonica, C. oleifera, Castanopsis sclerophylla, Cephalotaxus fortunei, Podocarpus macrophyllus, Lithocarpus glabra, Fragraea bodenii Camellia japonica, C. sasanqua Podocarpus macrophyllus Camellia japonica, C. reticulate, C. sasanqua, Podocarpus nagi Azadirachta indica, Camellia sinensis, Maytenus ilicifolia, Podocarpus macrophyllus Terminalia arjuna, T. chebula, Taxus wallichiana, Taxodium distichum, Camellia sinensis, C. nitidissima, Podocarpus macrophyllus, P. nagi, Taxus chinensis, T. yunnanensis Camellia sasanqua, Podocarpus macrophyllus, Camellia nitidissima, Podocarpus macrophyllus Camellia sasanqua, Cephalotaxus fortune, Ginkgo biloba, Podocarpus macrophyllus, Taxus yunnanensis Pieris japonica Camellia japonica, C. sasanqua, Podocarpus macrophyllus P. nagi, Taxus chinensis, Acer palmatum Camellia sasanqua, Taxus yunnanensis, T. chinensis, Equisetum sp., Lyonia ovalifolia Camellia nitidissima, C. sinensis, Holarrhena antidysenterica, Podocarpus macrophyllus, Terminalia arjuna Tamarindus indica Liu et al. 2007; Tejesvi et al. 2007a, 2009; Wei et al. 2007 Wei et al. 2005, 2007 Srinivasan and Muthumary 2009 Liu et al. 2007 Wei et al. 2005, 2007; Liu et al. 2007 P. conigena (Lév.) G.C. Zhao & N. Li P. funerea P. hainanensis P. heterocornis (Guba) Y.X.Chen P. jesteri P. karstenii (Sacc. & P. Syd.) Steyaert P. kunmingensis P. mangifolia (Guba) J. Xiang Zhang & T. Xu P. microspora P. neglecta P. olivacea (Guba) G.C. Zhao & J. He P. oxyanthi (Thüm.) Steyaert P. paeoniae (Servazzi) Steyaert P. palliditheae P. photiniae (Thüm.) Y.X. Chen P. subcuticularis P. submersa Sati & N. Tiwari P. theae P. versicolor (Speg.) Steyaert Many species have been isolated from soil, polluted stream water or are associated with the deterioration of wood, paper, fabrics and decay of wool (Guba 1961). For an example, P. bicolor (Ellis & Everh.) A.R. Liu, T. Xu & L.D. Guo, P. funerea, P. monochaetioides (Doyer) Steyaert, P. montellica (Sacc. & Voglino) Tak. Kobay., P. disseminata, P. foedans (Sacc. & Ellis) Steyaert, P. versicolor and P. virgatula are common species recorded either from decaying leaves or bark. Several saprobic species of Pestalotiopsis are listed in Table 3. Strobel et al. 2000 Liu et al. 2007; Wei et al. 2007 Wei et al. 2007 Liu et al. 2007; Wei et al. 2007 Li et al. 1996; Strobel et al. 1996a, b; Wei et al. 2005, 2007; Gomes-Figueiredo et al. 2007; Liu et al. 2007; Tejesvi et al. 2007a, 2009 Liu et al. 2007; Wei et al. 2007 Liu et al. 2007; Wei et al. 2007 Liu et al. 2007; Wei et al. 2007 Wei et al. 2005, 2007 Liu et al. 2007 Watanabe et al. 2010 Wei et al. 2005, 2007; Liu et al. 2007 Liu et al. 2007; Wei et al. 2007 Sati and Belwal 2005 Liu et al. 2007; Tejesvi et al. 2007a, 2009; Wei et al. 2007 Liu et al. 2007, 2010a only one fungal species, while some have additional species. In most cases these additional fungal species are parasitic while few are parasymbiont. A parasymbiont is a secondary fungus present in the lichen thallus, growing in intimate association with the primary symbionts without causing them any apparent harm (Sun et al. 2002). Pestalotiopsis maculans (Corda) Nag Raj is considered to be the dominant parasymbiont in the North American lichen species Cladonia rangiferina, C. subtenuis, C. mitis, C. leporina, Parmotrema perforatum and Usnea strigosa (Sun et al. 2002). Pestalotiopsis as a parasymbiont Pestalotiopsis as potential human and animal pathogens Lichen symbiosis is an association between a fungus (the mycobiont) and an alga or a cyanobacterium (the photobiont) (Schwendener 1868). Most lichens associate with Species of Pestalotiopsis are also known to cause human and animal disease. Pestalotiopsis has been isolated from Fungal Diversity (2011) 50:167–187 177 Table 3 List of recently recorded saprobes with their host/substrata Species Host/ substrate References Pestalotiopsis sydowiana Dead leaves of Calluna vulgaris, Erica sp., Rhododendron ponticum, R. hybridum, Prunus laurocerasus Dead leaves of Rhododendron sp, Chamaecyparis sp., Cupressus sp., Pinus sp., Juniperus sp. Seeds of Diospyros crassiflora Decaying leaves of Dracaena loureiri Dead culms of Schoenoplectus triqueter Dennis 1995; Ellis and Ellis 1997 P. funerea P. theae P. guepinii P. palmarum the human sinuses, fingernails, a bronchial biopsy, eyes, scalp and feet with corneal abrasions (Sutton 1999). One isolated from cotton was tested in a toxicity bioassay, which indicated that it caused reduction in weight, pathological abnormalities and even mortality in rats (Diener et al. 1976) Pestalotiopsis in extreme environments Some species of Pestalotiopsis have also been isolated from extreme environments and these isolates have been shown to produce bioactive metabolites (Tejesvi et al. 2007b). Pestalotiopsis microspora isolated from Taxus sp. from the foothills of Himalayas produced taxol (Strobel et al. 1996a), P. microspora isolated from Sepik River drainage system in Papua New Guinea produced isopestacin (Strobel et al. 2002) and Pestalotiopsis sp. obtained from the gut of a grass hopper (Chondracris rosee) produced two new phytotoxic g-lactones, pestalotines A and B (Zhang et al. 2008). Endophyte-pathogen relationships Lee et al. (1995) was able to show that P. microspora has an endophyte-pathogen relationship with the North American endangered tree Torreya taxifolia. They demonstrated that P. microspora inhabits the inner bark of the tree without causing symptoms. However, physiological or environmental factors trigger the fungus to become pathogenic. Typical symptoms include needle spots, needle death and stem cankers. The pathogenic ability of the fungus depends upon it producing phytotoxins, pestalopyrones, hydroxypestalopyrones and pestalosides. At the same time antifungal activity by the fungus produces exudates of pestaloside; this competes with other fungi. Pestalotiopsis subcuticularis naturally inhabits Hymenaea courbaril (Leguminosae) and remains dormant until leaves become mature. Fail and Langenheim (1990) stated that when leaves become mature the fungal hyphae spread and enter in to the intracellular spaces of the leaves. When the plant tissues are damaged due to Dennis 1995; Ellis and Ellis 1997 Douanla-Meli and Langer 2009 Thongkantha et al. 2008 Wu et al. 1982 mechanical injury such as insect feeding, active infection by the fungus occurs. The typical symptoms of infected leaves included serious leaf blight. Phylogenetic analysis of existing data in GenBank ITS sequences of 48 species of Pestalotiopsis were downloaded from GenBank and aligned using Clustal X. The alignment was optimized manually to allow maximum alignment and maximum sequence similarity. Gaps were treated as missing data. Phylogenetic analysis was carried out based on the aligned dataset using PAUP* 4.0b10 (Swofford 2002). Ambiguously aligned regions were excluded from all analyses. Trees were inferred using the heuristic search option with TBR branch swapping and 1,000 random sequence additions. Maxtrees were unlimited, branches of zero length were collapsed and all multiple parsimonious trees were saved. Trees are figured in Treeview (Page 1996). An example of the confusion which results from molecular data is shown in Fig. 4. In this phylogram we downloaded 44 selected strains of eight species which have high number of ITS sequences in GenBank plus 4 sequences from ex-type cultures available in GenBank (Table 4). According to Jeewon et al. (2003) and Liu et al. (2010a), pigmentation is a highly weighted character in the lineage of species of Pestalotiopsis and which can be differentiated into two main groups based on the colour of the median cells. This recent finding was previously supported in the separation of species by Guba (1961) and Steyaert (1949), based on versicolorous median cells as well as those species characterized by concolorous median cells. Jeewon et al. (2003) showed that species such as P. theae with dark colored concolorous median cells with knobbed apical appendages should be included in the versicolorous group. Jeewon et al. (2003) argued that the arrangement of Guba (1961) that groups the versicolorous assemblages of species into umber olivaceous and fuliginous olivaceous depends on the color intensity of the median cells. This statements was followed by Liu et al. (2010a) and they proposed the use of “brown to 178 Fungal Diversity (2011) 50:167–187 Fig. 4 Maximum parsimony phylogram generated from ITS sequence analysis of selected sequences from selected species of Pestalotiopsis including P. clavispora, P. disseminata, P. microspora, P. neglecta, P. photiniae, P. theae, P.virgatula and P. vismiae downloaded from GenBank with other related taxa. Data were analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Type sequences of Pestalotiopsis pallidotheae, P. hainanensis, P. jesteri and P. kunmingensis are in black and bold olivaceous” and “umber to fuliginous” colour median cells as valid for the taxonomy of the genus instead of the use of the “concolorous” and “versicolor” median cells grouping system proposed by Steyaert (1949) and Guba (1961). Pestalotiopsis clavispora, P. disseminata, P. microspora, P. neglecta, P. photiniae, P. theae, P. virgatula and P. vismiae can be divided into two groups depending mainly on the colour of the median cells. One group is the versicolorous group, consisting of P. clavispora, P. photiniae and P. virgatula, and dark concolorous median cells with knobbed apical appendages containing the P. theae group. The other group consists of species with concolorous median cells (i.e., P. disseminata, P. microspora, P. neglecta and P. vismiae. Almost all strains that separate into two main clades depend on the concolorous and versicolor system, and only P. microspora strains AY924295 and FJ478120 cluster in the wrong clade. However, within the two main groups, the respective species distributions are scattered and most species overlap with each other. Because of the limitation of characters used to differentiate species (Hu et al. 2007) and many overlapping characters (Sutton 1980), identification to species in Pestalotiopsis is presently difficult. For an example according to Guba (1961), P. disseminata, P. microspora, P. neglecta and P. vismiae within the concolo- Fungal Diversity (2011) 50:167–187 Table 4 Isolates and GenBank accession numbers of taxa used to generate the phylogram. Type species are marked in bold 179 Species GenBank accession numbers Species GenBank accession numbers P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. clavispora clavispora clavispora clavispora disseminata disseminata disseminata disseminata disseminata disseminata disseminata disseminata hainanensis jesteri kunmingensis AY682928 AY924263 DQ812921 GU362540 AY687870 DQ001000 DQ195782 EF055196 HM535728 HM535738 HM535752 HM535759 GQ869902 AF377282 AY373376 P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. EU342212 FJ037759 GU595050 AB482220 AY682937 AY682943 AY682946 DQ812939 EU030345 AY924281 DQ812936 DQ813436 HM535725 EF055220 EF055221 P. P. P. P. P. P. P. P. P. microspora microspora microspora microspora microspora microspora neglecta neglecta neglecta AY924278 AY924285 DQ000996 FJ459945 FJ478120 FJ487936 AY682930 DQ812935 EF055209 P. vismiae P. vismiae P. vismiae P. vismiae P. vismiae P. theae P. theae P. theae Truncatella angustata rous group have the same conidia size (18–26×5–8 μm). Pestalotiopsis vismiae can be differentiated as it has two apical appendages, while Pestalotiopsis microspora is differentiated from P. neglecta and P. dissementa by the length of the apical appendages. Pestalotiopsis neglecta and P. dissementa can be distinguished from each other only by the shape of the conidia. Most of above characters vary when in culture and following successive subculturing (Hu et al. 2007). Within the versicolorous group, P. clavispora and P. photiniae are morphologically very similar (conidia size 19–26×6–8.5 μm), while P. virgatula can be differentiated from P. clavispora and P. photiniae by its relatively small conidia (17–23×6–8 μm). However, these characters overlap and thus identification to these species is rather difficult. For this reason, naming of species is difficult and highly subjective and many sequences for Pestalotiopsis deposited in GenBank are likely to be wrongly named. Species numbers According to Index Fungorum (http://www.indexfungorum. org/names/names.asp; accession date, 2010.10.21) there are 235 Pestalotiopsis names, while in MycoBank (www. mycobank.org/mycotaxo.aspx; accession date, 2010.10.21) neglecta neglecta neglecta pallidotheae photiniae photiniae photiniae photiniae photiniae virgatula virgatula virgatula virgatula vismiae vismiae EF055222 EU273510 EU326213 HM535710 HM535751 AY924265 DQ812917 EF423551 DQ093715 there are 232 names. The reason for the large number of names is historical and may not reflect the actual number of species (Jeewon et al. 2004). As with other pathogenic genera such as Colletotrichum (Cai et al. 2009), species of Pestalotiopsis were historically named according to the host from which they were first observed. If a new host occurrence was found a new species was described. For example, Venkatasubbaiah et al. (1991) isolated a species of Pestalotiopsis from leaves of Oenothera laciniata and described the new species P. oenotherae Venkatas., Grand & Van Dyke. The new species was justified because no species of Pestalotiopsis had been described previously from Oenothera and its morphological characters clearly distinguished it from other species found on any member of the family Onagraceae (Venkatasubbaiah et al. 1991). Kohlmeyer and Kohlmeyer (2001) described Pestalotiopsis juncestris Kohlm & Volkm.-Kohlm which was isolated from the host Juncus roemerianus; the taxon is morphologically similar to P. versicolor and several other species of Pestalotiopsis, but the taxon was described as a new species based on the host occurrence. Similarly, Pal and Purkayastha (1992) and Singh (1981) described the new species P. agallochae A.K. Pal bis and Purkay and P. arborei N.I. Singh, respectively based on host occurrence. As recently as 2002, Chen et al. (2002) described P. afinis Y.X. Chen & G. 180 Wei, P. alpiniae Y.X. Chen & G. Wei, P. antiaris Y.X. Chen and G. Wei, P. dilleniae Y.X. Chen & G. Wei, P. kuwangsiensis Y.X. Chen and G. Wei, P. nelumbinis Y.X. Chen & G. Wei, P. schimae Y.X. Chen & G. Wei and P. synsepali Y.X. Chen & G. Wei based on the host association. More recently, some new species have been introduced based on host occurrence, plus morphological and molecular data. Wei and Xu (2004) isolated an endophytic species of Pestalotiopsis (P. kunmingensis J.G. Wei & T. Xu) from Podocarpus macrophyllus (Thunb.) Sweet and described it as a new species, supported by both morphological and molecular evidence. An endophytic species isolated from the Japanese plant Pieris japonica Thunb. L. was named as Pestalotiopsis pallidotheae Kyoko Watanabe and Yas. Ono; its conidial morphology is quite similar to P. theae but molecular data showed it to be distinct (Watanabe et al. 2010). Similarly, Strobel et al. (2000) and Liu et al. (2007) described P. jesteri Strobel, J. Yi Li, E.J. Ford & W.M. Hess and P. hainanensis A.R. Liu, T. Xu & L.D. Guo, respectively, using the same considerations. Species status and host-specificity within the genus Pestalotiopsis has been questioned previously or investigated (Zhu 1989; Jeewon et al. 2004; Wei et al. 2005, 2007; Hu et al. 2007). These authors showed that different species isolated from the same host may not be phylogenetically closely related (Jeewon et al. 2004; Wei et al. 2007). Wei et al. 2007 investigated endophytic species of Pestalotiopsis associated with plant species in the families Podocarpaceae, Theaceae and Taxaceae. The endophytic species of Pestalotiopsis associated with these host families were not generally host-specific, occurring on a range of hosts. For example, P. neglecta (Thüm.) Steyaert and P. photiniae were isolated from all the host plants in three plant families. Tejesvi et al. (2007a) isolated endophytic species of Pestalotiopsis associated with the medicinal plants Azadirachta indica, Holarrhena antidysenterica, Terminalia arjuna and T. chebula. They showed that isolates obtained from a single plant were genetically diverse, while the same species occurred in most plants. According to Guba (1961), most species of the Pestalotia were listed from a range of hosts. For example, Pestalotia microspora was listed from several different host plants (i.e., Ananas comosus, Araucaria sp., Carya sp., Hedera helix, Juniperus bermudiana and Platanus occidentalis). Hu et al. (2007) tested the relationships of endophytic Pestalotiopsis strains from two tissues of Pinus armandii and found that even strains isolated from the same tissue type were not phylogenetically related. Zhu (1989) used artificial cross inoculation studies to show that pathogenic species of Pestalotiopsis may not be specific to the single host. Jeewon et al. (2004) pointed out that host-specificity of Pestalotiopsis is not supported by the large number of species recorded on one host. They also argued that many taxa used in literature can be misinterpretations or synonyms Fungal Diversity (2011) 50:167–187 of species with wide host ranges. Jeewon et al. (2004) used analysis of ITS and 5.8S rDNA to show that isolates taken from the same host were not phylogenetically related and that taxa with similar morphological characters were phylogenetically related. Up to this time, most phylogenetic research on Pestalotiopsis has shown that Pestalotiopsis is not highly hostspecific and that species are found on a range of hosts (Jeewon et al. 2004; Wei et al. 2005, 2007; Hu et al. 2007). The diseases caused by species of Pestalotiopsis have been recorded in different ecosystems and infect a diverse range of unrelated plant taxa. Isolation of endophytic Pestalotiopsis strains for bioprospecting for new biochemical compounds have shown that the same species can be found in a range of hosts. Therefore, most of the species recorded in checklists and the literature may not reflect what actually occurs. As in other related plant pathogenic genera such as Colletotrichum, the Pestalotiopsis species concept depends mostly on the conidial characteristics. It has been shown that most of the key conidial characters used in species level separation are not stable and vary with host range, generation, culture and other environmental conditions (Hu et al. 2007). The arrangement of species by Steyaert (1949) and Guba (1961) in various coloured groupings is problematic because this character has been shown to be variable within a species (Liu et al. 2010a). Thus, most species in the above arrangements may be confused and many species are probably synonyms. Due to the fact that (1) species of Pestalotiopsis are generally not host-specific, (2) conidial characters vary and species limits overlap, and (3) species arrangements in Steyaert (1949) and Guba (1961) are problematic, then the actual number of species in Pestalotiopsis is likely to be much lower than presently recorded in databases (e.g., Index Fungorum, MycoBank) and the literature (Kirk et al. 2008). For example, according to Guba (1961), Pestalotiopsis breviseta (Sacc.) Steyaert, P. eugeniae, P. ilicicola T., P. microspora, P. podocarpi and P. sinensis (C.I. Chen) P.L. Zhu, Q.X. Ge & T. Xu have very similar, overlapping morphological characters and these species were justified mainly according to the host association. Also the above six species vary from P. carissae Guba, P. disseminata, P. neglecta, and P. olivacea by the length of the apical appendages. We question whether these names are synonyms of a single biological species. Furthermore, the versicolorous umber olivaceous group which comprises 40 species and versicolorous fuliginous olivaceous group comprising 56 species. These groups are differentiated depending on the intensities of the median cells, while most species have similar conidial measurements and thus are likely to be synonyms. We suspect that the actual number of biological species may be fewer than 50. The scientific community, however, uses many more names when diagnosing disease and in phylogenetic studies and biochemical Fungal Diversity (2011) 50:167–187 181 studies. Therefore, modern research approaches are needed for species of Pestalotiopsis in order to establish the acceptable names. Species numbers and accepted species When species are morphologically distinct and molecular evidence shows they are monophyletic, then such species can be considered as a distinct and valid species in a particular genus. Based on their distinct morphological characters, we suggest that the 20 species listed in Table 5 can be considered as good species in the genus at this time. Furthermore some other species (Table 6) which have considerable value because of their economic roles (in bioactive metabolites production, frequent pathogens, or frequently isolated endophytes) are possibly good species. We suggest that type material of these species should be reexamine and epitypified with fresh collections. With the help of ex-type living cultures and sequence data, a robust species concept can be developed for the genus Pestalotiopsis. Novel Pestalotiopsis biochemistry Species of Pestalotiopsis have been well-studied because of the diverse array of novel compounds that they have been shown to produce. As such, they are thought to be a rich source for bioprospecting when compared to those of other fungal genera (Aly et al. 2010; Xu et al. 2010). Strobel and Long (1998) described Pestalotiopsis as the ‘E. coli of the temperate and tropical rainforest systems’. Species of Pestalotiopsis may have an important role in forest ecosystems; they have a cosmopolitan geographical distribution and are found almost everywhere (Tejesvi et al. 2007a). Moreover, species of Pestalotiopsis have been found to produce an enormous number of secondary metabolites that may have medicinal, agricultural and industrial applications. The majority of compounds have been discovered from endophytic strains of Pestalotiopsis (Lee et al. 1996; Strobel et al. 1996a, b; Li and Strobel 2001) plus some pathogenic strains (Kwon et al. 1996). Species of Pestalotiopsis have been shown to produce bioactive alkaloids, terpenoids, isocoumarin derivatives, coumarins, chromones, quinones, semiquinones, peptides, xanthones, xanthone derivatives, phenols, phenolic acids, and lactones with a range of antifungal, antimicrobial, and antitumor activities (Xu et al. 2010). Xu et al. (2010) reviewed 130 different compounds isolated from species of Pestalotiopsis. In the present review, we discuss some selected species and their bioactive potential. Pestalotiopsis microspora is a common species present in tropical and subtropical plants and is a widespread saprobe of bark and decaying plant material (Metz et al. Table 5 Morphologically distinct Pestalotiopsis species with their host and location Species with distinct morphological characters Host and location P. gaurae Guba P. multiseta (Speg.) Guba P. trevoae Speg. Pestalotiopsis bicolor P. distincta (Guba) K. Yokoy. On stem of Gaura parviflora in Hays, Kansas, United States On fallen leaves of Iris germanica in Conegliano, Italy On dead decaying branches of Trevoa trinervia in Santiago, Chile Isolated from the dead leaves of Salix sp. in Tuskegee, Alabama, United States On leaves of Castanopsis cuspidate in Japan P. P. P. P. P. P. P. funerea guepinii hughesii Steyaert karstenii leucopogonis Nag Raj macrospora (Ces.) Steyaert maculans P. P. P. P. P. P. P. P. monochaetioides montellica palustris Nag Raj perseae Nag Raj pseudomontellica Nag Raj smilacis (Schwein.) B. Sutton tecomicola Nag Raj trichocladi (Laughton) Steyaert On dead leaves of Thuja sp. in Paris, France On stem and leaves of Camellia japonica in France. On stems of Cyperus articulate in Gold Coasts in West Africa On leaves of Camellia japonica in United States On leaves of Leucopogan lanceolatus in Australia On fronds of Pteridium aquilinum in Italy On leaves of Camellia japonica and Camellia sp. in Czechoslovakia, France, Germany and United States On dead twig of Chamaecyparis lawsoniana in Naarden, Holland On dead leaves of Quercus rubra in Canada On Euphorbia palustris in Italy On leaves of Persea borbonea in United States On leaves of Lithocarpus densiflora in United States On stem of Smilax rotundifolia in United Sates On Tecoma radicans in United States On leaves of Trichocladus crinitus in South Africa 182 Fungal Diversity (2011) 50:167–187 Table 6 Economically important Pestalotiopsis species with their host and location Economically important species Host and location Pestalotiopsis adusta On leaves of Prunus cerasus in (Ellis & Everh.) Steyaert Newfield, New Jersey, United States P. clavispora On leaves of Quercus sp. in Auburn, Alabama, United States P. disseminata On dead leaves of Eucalyptus globules in Coimbra, Portugal P. fici Steyaert On Ficus sp. in Kiagwe, Uganda P. foedan (Sacc. & Ellis) On decaying bark of Thuja occidentalis Steyaert in Newfield, New Jersey, United States P. heterocornis On leaves of Anarcardium occidentale in Cantanduva, São Paulo, Brazil P. longiseta On leaves of Rubus caesius in Susegana, Conegliano, Italy P. microspora On leaves of Hedera helix in Botanical garden, College of Argentina, Buenos Aires, Argentina P. neglecta P. pauciseta (Sacc.) Y.X. Chen P. photiniae P. theae P. uvicola On leaves of Euonymus japonicas in Coimbra, Portugal On leaves of Litsea glutinosa in Mount Makiling, near Los Banos, Laguna province, Philippine On leaves of Photinia serrulata in Istria, Australia On leaves of Camellia sinensis in Japan On Gaura parviflora and Vitis vinifera in Italy 2000). The species has most commonly been isolated as an endophyte associated with rainforest plants (Strobel et al. 2002) or as a pathogen (Keith et al. 2006). Pathogen associations include scab disease on Psidium guajava (Keith et al. 2006), leaf blight of Lindera obtusiloba (Jeon et al. 2007) and as an endophyte on Terminalia morobensis (Womersley 1995). Pestalotiopsis microspora has the potential to be a model organism for biological and biochemical studies in the laboratory (Metz et al. 2000). Isolates of this species (or possibly species complex) show diverse genetic variation and thus each individual isolate is generally unique in the substances that it produces (Harper et al. 2003). Long et al. (1998) have shown that under laboratory conditions it can take up heterologous DNA, add telomeric DNA, express heterologous DNA and can replicate independently of chromosomal DNA. Such genetic diversity would be useful to the species in nature, helping it adapt to a new plant by incorporating plant DNA into its own genome (Strobel et al. 1996a; Li et al. 1996). Bioactive compounds such as the anti-cancer Economically importance Bioactive metabolites Li et al. 2008b Plant pathogen, Common endophyte Plant pathogen, Bioactive metabolites Bio active metabolites Bio active metabolites Keith et al. 2006; Espinoza et al. 2008; Wei et al. 2007; Liu et al. 2007 Das et al. 2010; Keith et al. 2006; Deyrup et al. 2006 Liu et al. 2008a, b, 2009b Ding et al. 2008a Common endophyte Wei et al. 2007; Liu et al. 2007 Plant pathogen, Bioactive metabolites Plant pathogen, Common endophyte, Bioactive metabolites Joshi et al. 2009; Nagata and Ando 1989; Nagata et al. 1992; Xu et al. 2010 Strobel et al. 1996a, b, 2000; Metz et al. 2000; Keith et al. 2006; Jeon et al. 2007; Womersley (1995); Harper et al. 2003; Lee et al. 1995; Kai et al. 2003 Plant pathogen, Endophyte Tagne and Mathur 2001; Espinoza et al. 2008; Wei et al. 2007; Liu et al. 2007 Bioactive metabolites Gangadevi et al. 2008 Bioactive metabolites Ding et al. 2009 Plant pathogen, Endopyte, Bioactive metabolites Li et al 2008a; Nagata et al. 1992; Shimada et al. 2001; Tuset et al. 1999; Worapong et al. 2003; Joshi et al. 2009; Muraleedharan and Chen 1997; Ding et al. 2008b; Shimada et al. 2001 Vitale and Polizzi 2005; Xu et al. 1999 Plant pathogen drug taxol, jesterone, ambuic acid, torreyanic acid, pestaloside, pestalotiopsins and 2-a hydroxydimeniol (Strobel et al. 2002), hetero-polysaccharides (Kai et al. 2003) have been obtained from P. microspora. The multimillion dollar anti-cancer drug, taxol was obtained from an endophytic strain of P. microspora isolated from Taxus wallachiana (Strobel et al. 1996a) and Taxodium distichurn (Strobel et al. 1996b). Kai et al. (2003) found that P. microspora can metabolize various monosaccharides and the composition of hetero-polysaccharides depends on the type of monosaccharide in the media. Harper et al. (2003) investigated the production of pestacin, a 1,3-dihydro isobenzofuran with moderate anti-fungal properties and high anti-oxidant activity when compared with the vitamin E derivative trolox from endophytic strains of P. microspora. The anti-oxidant activity works mainly by cleavage of an unusually reactive C–H bond. Lee et al. (1995) obtained several anti-fungal compounds such as pestaloside, an aromatic glucoside, and two pyrones (pestalopyrone and hydroxypestalopyrone) Fungal Diversity (2011) 50:167–187 from a strain of P. microspora isolated from the endangered North American tree Torreya taxifolia. When Pestalotiopsis microspora is cultured on media containing various monosaccharides as a carbon source, different polysaccharides are produced and this mainly depends on the monosaccharide used as the carbon source (Kai et al. 2003). Whether all these strains were in fact P. microspora is yet to be determined, since the identifications were based on morphology or comparison with GenBank sequence data, which itself may be erroneously named. This species is in need of epitypification. Pestalotiopsis theae is an economically important species that has been reported from all major tea growing countries of the world (Muraleedharan and Chen 1997) and also as an endophyte (Worapong et al. 2003). Pestalotheols A–D, four new metabolites isolated from endophytic Pestalotiopsis theae, and pestalotheol C showed an inhibitory effect against HIV-1LAI replication in C8166 cells (Li et al. 2008a). Three new compounds, pestalamides A–C and two known metabolites, aspernigrin A and carbonarone A, were obtained from the same fungus isolated from the branches of tea (Ding et al. 2008b). The newly isolated pestalamide B inhibited HIV-1 replication in C8166 cells with EC50 of 64.2 μM and antifungal activity against Aspergillus fumigatus. Chloroisosulochrin and chloroisosulochrin dehydrate were obtained from the culture filtrate of Pestalotiopsis theae, and these compounds can be used as plant growth regulators (Shimada et al. 2001). This species is obviously important as a producer of novel medicinal metabolites. The generic type of Pestalotiopsis is P. guepinii, a plant pathogen that causes disease in important crop plants (Karaca and Erper 2001). Strains of Pestalotiopsis guepinii isolated as an endophyte from the plant families Anacardiaceae, Apocynaceae, Leguminosae and Palmae were tested for their in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity, using Ellman’s colorimetric method adapted for thin layer chromatography (Rodrigues et al. 2005). Pestalotiopsis guepinii from Anacardium giganteum inhibited both enzymes in the TLC polar region and a strain isolated from Myracroduon urundeuva and Spondias mombin showed selective inhibition of AChE. Parshikov et al. (2001) suggested that P. guepinii may be a useful model for the mammalian transformation of fluoroquinolones. They obtained the metabolites N-acetylciprofloxacin (52%), desethylene- N-acetylciprofloxacin (9.2%), Nformylciprofloxacin (4.2%), and 7-amino-1-cyclopropyl6-fluoro- 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.3%) by specific culture of P. guepinii dosed with ciprofloxacin (300 μM). In addition, by dosing with norfloxacin (313 μM) and the metabolites N-acetylnorfloxacin (55.4%), desethylene-N-acetylnorfloxacin (8.8%), 183 N-formylnorfloxacin (3.6%), and 7-amino-1-ethyl-6fluoro- 4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.1%) were obtained. Liu et al. (2008b) isolated five new cyclohexanone derivatives, pestalofones A–E, with the known compounds isosulochrin, isosulochrin dehydrate, and iso-A82775C, from cultures of the plant endophytic fungus Pestalotiopsis fici. Pestalofones A and B were inhibitory against HIV-1 replication in C8166 cells, pestalofones C showed antifungal activity against Aspergillus fumigatus while pestalofones E showed both the above effects. Chloropestolide A extracted from the scale-up fermentation extract of Pestalotiopsis fici showed significant inhibitory effects on growth of two human cancer cell lines, HeLa and HT29 (Liu et al. 2009). Liu et al. (2010b) obtained chloropupukeanolides A and B (unprecedented spiroketal peroxide) and chloropupukeanone A (three highly functionalized metabolites featuring a chlorinated pupukeanane core) from an endophytic strain of Pestalotiopsis fici. The compound chloropupukeanolide A showed significant anti-HIV-1 and cytotoxic effects. These findings will most likely trigger further studies on total synthesis. Whether Pestalotiopsis is unique amongst endophytes or coelomycetes in producing large numbers of secondary metabolites with medicinal and pathogenic control significance has yet to be established. Taxonomic confusion and way forward Pestalotiopsis is taxonomically poorly understood both at the inter- as well as the intraspecific level. It is not clear whether Pestalotia is really distinct from Pestalotiopsis, since stains of the type of the former have not been sequenced. Nomenclature of the genus is confusing and most host based names in databases may be synonyms. Molecular data have still not been successfully applied for species-level differentiation and names applied to data in GenBank are doubtful, as they are not linked to any type materials. Epitypification with molecular work is therefore needed to understand the species and what distinguishes them. Re-examination of type materials and establishment of epitypes with living cultures is essential for real progress (Hyde and Zhang 2008), and sequence data are needed to develop a strong species-based taxonomic system for the genus Pestalotiopsis. 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