First report of Phyllosticta citricarpa and description of two new species, P. paracapitalensis and P. paracitricarpa, from citrus in Europe.

The genus Phyllosticta occurs worldwide, and contains numerous plant pathogenic, endophytic and saprobic species. Phyllosticta citricarpa is the causal agent of Citrus Black Spot disease (CBS), affecting fruits and leaves of several citrus hosts (Rutaceae), and can also be isolated from asymptomatic citrus tissues. Citrus Black Spot occurs in citrus-growing regions with warm summer rainfall climates, but is absent in countries of the European Union (EU). Phyllosticta capitalensis is morphologically similar to P. citricarpa, but is a non-pathogenic endophyte, commonly isolated from citrus leaves and fruits and a wide range of other hosts, and is known to occur in Europe. To determine which Phyllosticta spp. occur within citrus growing regions of EU countries, several surveys were conducted (2015-2017) in the major citrus production areas of Greece, Italy, Malta, Portugal and Spain to collect both living plant material and leaf litter in commercial nurseries, orchards, gardens, backyards and plant collections. A total of 64 Phyllosticta isolates were obtained from citrus in Europe, of which 52 were included in a multi-locus (ITS, actA, tef1, gapdh, LSU and rpb2 genes) DNA dataset. Two isolates from Florida (USA), three isolates from China, and several reference strains from Australia, South Africa and South America were included in the overall 99 isolate dataset. Based on the data obtained, two known species were identified, namely P. capitalensis (from asymptomatic living leaves of Citrus spp.) in Greece, Italy, Malta, Portugal and Spain, and P. citricarpa (from leaf litter of C. sinensis and C. limon) in Italy, Malta and Portugal. Moreover, two new species were described, namely P. paracapitalensis (from asymptomatic living leaves of Citrus spp.) in Italy and Spain, and P. paracitricarpa (from leaf litter of C. limon) in Greece. On a genotypic level, isolates of P. citricarpa populations from Italy and Malta (MAT1-2-1) represented a single clone, and those from Portugal (MAT1-1-1) another. Isolates of P. citricarpa and P. paracitricarpa were able to induce atypical lesions (necrosis) in artificially inoculated mature sweet orange fruit, while P. capitalensis and P. paracapitalensis induced no lesions. The Phyllosticta species recovered were not found to be widespread, and were not associated with disease symptoms, indicating that the fungi persisted over time, but did not cause disease.

Introduction

The genus Phyllosticta was introduced by Persoon (1818), with P. convallariae (nom. cons.) (= P. cruenta) designated as the type species (Donk 1968). Species of Phyllosticta are known as plant pathogens of several hosts and responsible for various disease symptoms including leaf and fruit spots. Species included in the P. ampelicida species complex, which cause black rot disease on grapevines (Wicht et al., 2012, Zhou et al., 2015), and in the P. musarum species complex, which cause banana freckle disease, are economically important plant pathogens (Pu et al., 2008, Wong et al., 2012). Some species of Phyllosticta have also been isolated as endophytes from a wide range of hosts (e.g., P. capitalensis) and as saprobes (Glienke-Blanco et al., 2002, Huang et al., 2008, Thongkantha et al., 2008, Wikee et al., 2011, Wikee et al., 2013b).

Sexual morphs have in the past been named in Guignardia (van der Aa 1973). The name Guignardia was introduced as a replacement for Laestadia by Viala & Ravaz (1892), who applied the name only to Sphaeria bidwellii (= G. bidwellii = P. ampelicida), a species that is different from Laestadia (Bissett 1986). Petrak (1957) included G. bidwellii and related species in Botryosphaeria, an opinion that was initially shared by Barr, 1970, Barr, 1972. Phyllosticta was first monographed by van der Aa (1973) and more recently all species names described in Phyllosticta were re-described by van der Aa & Vanev (2002). Schoch placed Phyllosticta in Botryosphaeriales. Several authors showed that Botryosphaeriaceae contained both Botryosphaeria and Phyllosticta spp., although this relationship remained poorly resolved (Crous et al., 2006, Schoch et al., 2006, Liu et al., 2012).

With the increasing use of molecular data to link asexual and sexual morphs, and the end of dual nomenclature for fungi (Hawksworth , Wingfield ), the oldest and more commonly used name, Phyllosticta, was chosen over that of Guignardia (Glienke et al., 2011, Sultan et al., 2011, Wikee et al., 2011, Wikee et al., 2013b, Wong et al., 2012). Moreover, several studies incorporated DNA sequence data to improve the identification and help resolve the taxonomy of Phyllosticta spp. (Baayen et al., 2002, Wulandari et al., 2009, Glienke et al., 2011, Wikee et al., 2011). Presently, new species of Phyllosticta are described based on a polyphasic approach, incorporating phylogenetic data, morphology and culture characteristics (Crous et al., 2012, Su and Cai, 2012, Wang et al., 2012, Wong et al., 2012, Zhang ). Wikee redefined Phyllosticta, showing that it clusters sister to the Botryosphaeriaceae for which the authors resurrected the family name Phyllostictaceae.

The main morphological characters used to recognise a species of Phyllosticta is the production of pycnidia containing aseptate, hyaline conidia that are covered by a mucoid layer and bearing a single apical appendage (van der Aa 1973). However, the mucoid layer and appendage are not always present. The sexual morph has erumpent, globose to pyriform ascomata, often irregularly shaped, unilocular, and with a central ostiole. Asci are 8-spored, bitunicate, clavate to broadly ellipsoid, with a wide, obtusely rounded or slightly square apex. Ascospores are ellipsoid to limoniform, sometimes slightly elongated, aseptate, hyaline, often with a large central guttule and a mucoid cap at each end. Spermatia produced in culture are hyaline, aseptate, cylindrical to dumbbell-shaped with guttules at both ends (van der Aa 1973).

Several Phyllosticta species have been associated with Citrus spp. worldwide (Baayen et al., 2002, Glienke-Blanco et al., 2002, Everett and Rees-George, 2006, Baldassari et al., 2008, Wulandari et al., 2009, Glienke et al., 2011, Brentu et al., 2012, Wikee et al., 2013a, Er et al., 2014). Citrus black spot (CBS) is a foliar and fruit disease of Citrus spp. caused by P. citricarpa (sexual morph Guignardia citricarpa) (Kotzé, 1981, Baldassari et al., 2008). The pathogen affects fruits and leaves of several citrus hosts causing various symptoms (Kiely, 1948a, Kiely, 1949, Kotzé, 1981, Kotzé, 2000, Snowdon, 1990) with lemons and ‘Valencia’ oranges being more susceptible (Kotzé 2000). Hard spot is the most common symptom characterised by sunken, pale brown necrotic lesions with a dark reddish brown raised border; lesions often containing the pycnidia (asexual sporocarps). Several other kinds of lesions are known: virulent spot, a sunken necrotic lesion without defined borders mostly on mature fruit; false melanose consisting of small black pustules usually in a tear stain pattern; and freckle, cracked or speckled spot. Leaf symptoms are seldom seen except on lemons. They appear as round, small, sunken necrotic spots with a yellow halo (Schubert ). The infected leaves, when fallen on the orchard floor, represent a substrate for the development and maturation of pseudothecia from which the primary inoculum, ascospores, are released for new infections (McOnie 1967). Phyllosticta citricarpa has never been found on plant species outside of the Rutaceae, and can be isolated from asymptomatic citrus tissues (Baldassari ).

Phyllosticta citricarpa is often associated with P. capitalensis, a morphologically similar but non-pathogenic species, previously incorrectly considered as the asexual morph of Guignardia mangiferae (Baayen et al., 2002, Everett and Rees-George, 2006, Glienke et al., 2011). Based on a multi-locus phylogenetic analysis, however, Glienke revealed that P. capitalensis sensu lato was genetically distinct from the reference isolate of G. mangiferae. Phyllosticta capitalensis was initially described on Stanhopea (Orchidaceae) from Brazil (Hennings 1908). Okane attributed the name P. capitalensis to an endophytic species reported on ericaceous plants from Japan, and described the sexual morph as a new species, G. endophyllicola. Subsequently Baayen , based on DNA sequence data of the ITS nrDNA, considered a common endophytic species associated with several plants as morphologically similar to G. endophyllicola, but attributed this species to G. mangiferae, while the asexual morph was referred to as P. capitalensis. Phyllosticta capitalensis is a cosmopolitan fungus that has been reported from plants in 21 different families (Johnston, 1998, Rodrigues and Samuels, 1999, Okane et al., 2001, Baayen et al., 2002, Glienke-Blanco et al., 2002, Rodrigues et al., 2004, Everett and Rees-George, 2006, Meyer et al., 2006, Rakotoniriana et al., 2008, Yuan et al., 2009, Bezerra et al., 2012) and has been found on citrus associated with both CBS affected and asymptomatic plants (Baayen et al., 2002, Everett and Rees-George, 2006, Glienke et al., 2011). Guignardia mangiferae sensu stricto (not P. capitalensis) causes angular leaf spots on mango (Baldassari et al., 2008, Glienke et al., 2011).

The biology and ecology of P. capitalensis differs from that of P. citricarpa. Phyllosticta capitalensis is homothallic, whereas P. citricarpa is heterothallic (Zhang et al., 2015, Wang et al., 2016, Amorim et al., 2017). Phyllosticta capitalensis produces fertile pseudothecia on agar media and P. citricarpa produces them on leaf litter (Kiely 1948a). Moreover, P. capitalensis is an ubiquitous, cosmopolitan endophyte of woody plants (Baayen ) and P. citricarpa is associated only with citrus plants (Glienke ).

Significant progress in species differentiation was achieved with multi-locus phylogenetic analyses performed on a large number of Phyllosticta species, (Wulandari et al., 2009, Glienke et al., 2011, Wang et al., 2012). Using three partial DNA regions, Wulandari revealed three Phyllosticta clades associated with citrus in Thailand, namely P. capitalensis, P. citricarpa and P. citriasiana. Wang described one new species associated with citrus in China, namely P. citrichinaensis, and also distinguished two subclades within P. citricarpa. Sequencing four partial regions of DNA, Glienke distinguished a new species, Phyllosticta citribraziliensis, associated with Citrus sp. in Brazil. Phyllosticta citriasiana causes Citrus Tan Spot disease on Citrus maxima in Asia (Wulandari ). Phyllosticta citrichinaensis is a weak pathogen on various citrus species in Asia, and P. citribraziliensis is non-pathogenic endophyte on citrus in Brazil (Glienke et al., 2011, Wang et al., 2012). A recent study added a sixth Phyllosticta species associated with citrus, namely P. citrimaxima, which was isolated from Citrus Tan Spot on fruit of C. maxima in Thailand (Wikee ).

Based on sequences of the rDNA internal transcribed spacer (ITS) region, the P. citricarpa and P. capitalensis clades are clearly distinct, with each species showing low levels of intraspecific variation (Okane et al., 2003, Rodrigues et al., 2004). Phyllostica citricarpa and P. capitalensis have several morphological and physiological differences: colonies of P. citricarpa produce a yellow halo on oatmeal agar (OA), the growth rate is generally faster in P. capitalensis, conidia are coated with a thicker mucoid layer than observed in P. citricarpa, and there is a higher level of hydrolytic enzyme production in P. citricarpa than in P. capitalensis (Baayen et al., 2002, Glienke et al., 2011, Romão et al., 2011).

Windborne P. citricarpa ascospores produced in pseudothecia (ascocarps) and waterborne conidia produced in pycnidia may cause infection on citrus (Kiely, 1948a, Kotzé, 1963, Kotzé, 1996, Kotzé, 2000). The ascospores are considered the primary source of inoculum in the CBS disease cycle, while conidia may serve for short distance wash-down dispersal by rain (Kiely, 1948a, Whiteside, 1967, Sposito et al., 2011). Alternate wetting and sun drying of leaves and mild to warm temperature fluctuations are favourable conditions for maturation of pseudothecia and ascospore discharge (Kiely, 1948a, Lee and Huang, 1973, Truter, 2010, Fourie et al., 2013, Hu et al., 2014). Subsequently, infection is dependent on the presence of long periods of free surface water and suitable microclimatic conditions (Kiely, 1948a, Kiely, 1948b, Kiely, 1949, Kotzé, 1963, Kotzé, 1981, McOnie, 1967). Leaf litter colonised by P. citricarpa serves as the primary inoculum source. Thus leaf litter plays an important role and its removal or enhanced decomposition results in improved inoculum management (Bellotte et al., 2009, Truter, 2010, Sposito et al., 2011). Pseudothecia develop 40–180 d after leaf fall, releasing mature ascospores during rainfall that are dispersed by wind (Kotzé, 1963, McOnie, 1964, Huang and Chang, 1972, Reis et al., 2006, Fourie et al., 2013, Dummel et al., 2015). Fruits are susceptible for 4–5 mo after petal fall (Kiely, 1948b, Schutte et al., 2003, Schutte et al., 2012, Miles et al., 2004). Therefore, the onset of rain, ascospore release and fruit susceptibility period are strongly correlated in summer rainfall regions resulting in fruit infection (Kotzé, 1963, McOnie, 1964, McOnie, 1967, Whiteside, 1967). Following a long latent period, the onset of symptom expression on fruit coincides with fruit ripening (Kiely, 1948a, Whiteside, 1967, Kotzé, 1981, Spósito et al., 2008).

Phyllosticta citricarpa has been recorded in Australia since the late 19th century, causing CBS disease, specifically in coastal regions of New South Wales and Queensland (Benson, 1895, Kotzé, 1981, Miles et al., 2013), but not from the hot, dry inland citrus orchards, and not in the winter rainfall regions in Australia (Broadbent 1995). Phyllosticta citricarpa has also been recorded in summer rainfall citrus-growing regions in several areas: South America (Argentina, Brazil, Uruguay, Venezuela; Garran, 1996, Kotzé, 2000, European Union, 2000, Paul et al., 2005), Central America (West Indies; Calavan 1960), North America (Dewdney et al., 2012, Schubert et al., 2012, Zavala et al., 2014), Asia (Bhutan, China, India, Indonesia, Philippines, Taiwan; Brodrick, 1969, European Union, 1998, Kotzé, 2000, European Union, 2000) and Africa (Ghana, Kenya, Mozambique, Nigeria, South Africa, Swaziland, Zambia, Zimbabwe; Doidge, 1929, Kotzé, 1981, Kotzé, 2000, European Union, 1998, Baayen et al., 2002, Brentu et al., 2012). Several fruit and leaf diseases caused by different fungi such as Colletotrichum and Alternaria spp. (Vicent et al., 2007, Aiello et al., 2015) are present in the EU citrus-producing countries; however, the CBS disease has not been reported (Baker ). In addition to the general phytosanitary regulations applicable to the import of citrus propagating plant material, the import of citrus fruit into the EU is subject to phytosanitary regulations relating to P. citricarpa (EC2000/29/EC, 2000).

Recent epidemiological studies (Paul et al., 2005, Yonow et al., 2013, Magarey et al., 2015) have shown that the climatic conditions in the citrus growing regions within the EU are unsuitable for establishment of P. citricarpa and development of CBS disease, with only small, restricted Mediterranean coastal areas where the climatic conditions have at most marginal potential suitability. Considering that citrus plants were moved from Asia, where CBS is endemic and also regarded as the centre of origin of citrus, to Northern Africa and other countries around the Mediterranean Sea by traders, as early as the 5th century BC (Ramón-Laca, 2003, Mabberley, 2004, Nicolosi, 2007), it would be expected that P. citricarpa and/or other Phyllosticta spp. may have been introduced to these citrus-growing countries along with the hosts, especially since infected plant material is regarded as the means of long-distance spread of this pathogen (Kiely, 1948b, Kotzé, 1981). Likewise, there is always the possibility of illegal movement of citrus plant propagating material. Therefore, the potential occurrence of Phyllosticta spp. was included in a broad survey of fungal citrus pathogens undertaken in citrus growing regions within EU countries (Guarnaccia et al., 2017, Sandoval-Denis et al., 2018). During 2015–2017, several surveys were conducted in the major citrus production areas of the EU and included the following: (i) surveys of both fresh plant material and leaf litter in commercial nurseries and citrus orchards, gardens, backyards and plant collections, (ii) cultivation of as many Phyllosticta isolates as possible from this material, (iii) subject isolates to DNA sequence analyses combined with morphological characterisation, (iv) compare these results with data from other phylogenetic studies on Phyllosticta, (v) identification of genotypes and mating types of the P. citricarpa isolates found in this study and, (vi) to evaluate potential pathogenicity of the Phyllosticta spp. isolated.

Materials and methods

Sampling and isolation

The initial surveys were carried out in 2015 and 2016 covering a total of 95 sites located in some of the main citrus-producing regions of Europe (Table 1). Evaluations were conducted by sampling approx. 25 fruits, 25 twig portions, 50 living leaves and 50 leaves from the litter layer from each Citrus host present in each site investigated. Samples were collected from Andalusia, Mallorca, Valencia (Spain), Apulia, Calabria, Sicily (Italy), Algarve (Portugal), Crete, Mesolongi, Nafplio (Greece), Gozo and La Valletta (Malta) areas. Investigated citrus species included Australasian lime (Citrus australasica), citranges (Citrus sinensis × Poncirus trifoliata), citrons (C. medica, C. medica var. sarcodactylis), kumquat (C. japonica), limequats (Citrus ×floridana), calamondin (×Citrofortunella microcarpa), mandarins (C. reticulata), tangelo (C. ×tangelo), oranges (C. ×aurantium, C. ×bergamia, C. ×sinensis), pummelo (C. maxima), grapefruit (C. paradisi), limes (C. ×aurantifolia, C. ×hystrix, C. ×latifolia) and lemons (C. ×limon). New surveys were performed during December 2016 and January 2017 at the sites where P. citricarpa and P. paracitricarpa were found during the initial surveys (Table 1) to confirm these findings and to assay the presence of symptoms on fruit, leaves and twigs.

Table 1

Location and characteristics of the investigated sites.

City (country)	GPS coordinates	Site	Plant age (years)	Condition3
Acitrezza (Italy)	37.561077, 15.161086	Backyard	20–30	Cultivated
Agia (Greece)	35.465979, 23.921240	Orchard	5–10	Cultivated
Algemesi (Spain)	39.207638, −0.449773	Orchard	5–10	Cultivated
Algemesi (Spain)	39.196895, −0.470823	Orchard	5–10	Cultivated
Alginet (Spain)	39.260069, −0.458032	Orchard	10–15	Cultivated
Alginet (Spain)	39.251407, −0.416424	Orchard	5–10	Cultivated
Alhaurin El Grande (Spain)	36.645374, −4.677086	Orchard	15–25	Unkept
Alhaurin El Grande (Spain)	36.664689, −4.698184	Orchard	15–25	Cultivated
Alikianos (Greece)	35.456657, 23.908632	Orchard	15–25	Cultivated
Alikianos (Greece)	35.462384, 23.904367	Orchard	10–15	Unkept
Alikianos (Greece)	35.446440, 23.919798	Orchard	10–15	Unkept
Alikianos (Greece)	35.466216, 23.945558	Orchard	10–15	Cultivated
Almeria (Spain)	36.834637, −2.402932	Experimental orchard	15–25	Cultivated
Almeria (Spain)	36.828832, −2.402892	Experimental orchard	15–25	Cultivated
Alzira (Spain)	39.156963, −0.490723	Orchard	10–15	Cultivated
Amfilochia (Greece)	38.961381, 21.171635	Orchard	10–15	Cultivated
Argo (Greece)	37.628645, 22.742179	Orchard	10–15	Cultivated
Argo (Greece)	37.655558, 22.739309	Orchard	10–15	Cultivated
Argos (Greece)	37.686587, 22.661719	Orchard	10–15	Cultivated
Arta (Greece)1	39.161719, 20.929585	Backyard	30–40	Unkept
Arta (Greece)	39.155661, 20.903791	Orchard	15–25	Cultivated
Arta (Greece)	39.160465, 20.918257	Orchard	5–10	Cultivated
Barcellona P.G. (Italy)	38.110560, 15.136794	Nursery	1–3	Cultivated
Brucoli (Italy)	37.294823, 15.110518	Orchard	15–25	Cultivated
Canicattì (Italy)	37.358434, 13.840898	Backyard	20–30	Cultivated
Carruba (Italy)	37.684625, 15.190943	Orchard	15–25	Unkept
Castellò (Spain)	39.903922, −0.086197	Orchard	10–15	Cultivated
Castellò (Spain)	39.883861, −0.088225	Orchard	10–15	Cultivated
Castellò (Spain)	39.884013, −0.090945	Orchard	10–15	Cultivated
Cefalù (Italy)	38.029481, 14.012267	Backyard	20–30	Unkept
Chania (Greece)	35.493153, 24.051141	Orchard	10–15	Cultivated
Chania (Greece)	35.477894, 23.948060	Orchard	10–15	Cultivated
Comiso (Italy)	36.943980, 14.637159	Orchard	15–25	Unkept
Conceicao (Portugal)	37.048481, −7.916927	Orchard	15–25	Cultivated
Curiglia (Italy)	38.767729, 16.203763	Orchard	20–30	Unkept
El Ejido (Spain)	36.795207, −2.719992	Orchard	20–30	Cultivated
Estellencs (Spain)	39.653504, 2.481876	Backyard	30–40	Unkept
Faro (Portugal)	37.108457, −7.916805	Orchard	20–30	Unkept
Faro (Portugal)	37.062641, −7.917432	Orchard	10–15	Unkept
Giarratana (Italy)	36.883438, 14.974420	Orchard	10–15	Cultivated
Gouria (Greece)	38.454977, 21.257646	Orchard	15–25	Cultivated
Gozo (Malta)	36.049069, 14.259299	Backyard	20–30	Unkept
Gozo (Malta)	36.037531, 14.260120	Orchard	10–15	Unkept
Gozo (Malta)	36.049646, 14.279360	Orchard	15–25	Cultivated
Gozo (Malta)2	36.055138, 14.259907	Backyard	60–70	Unkept
Gozo (Malta)	36.058166, 14.284453	Backyard	60–70	Unkept
Grotte (Italy)	37.679925, 15.177006	Orchard	15–25	Cultivated
Guardia (Italy)	37.662709, 15.175918	Orchard	15–25	Cultivated
Kirtomados (Greece)	35.478749, 23.916661	Orchard	15–25	Cultivated
Leni (Italy)	38.044422, 14.597517	Backyard	30–40	Cultivated
Leni (Italy)	38.552889, 14.827128	Backyard	30–40	Cultivated
Lentini (Italy)	37.320577, 15.020901	Orchard	15–25	Cultivated
Malaga (Spain)	36.761761, −4.427060	Botanical garden	40–50	Unkept
Mascali (Italy)	37.767684, 15.192503	Nursery	1–3	Cultivated
Mascali (Italy)	37.768258, 15.194639	Nursery	1–3	Cultivated
Massafra (Italy)	40.544756, 17.144112	Orchard	10–15	Cultivated
Mastro (Greece)	38.430287, 21.280539	Orchard	15–25	Cultivated
Mesquita (Portugal)	37.213673, −8.289493	Orchard	10–15	Cultivated
Mesquita (Portugal)	37.204525, −8.297812	Orchard	20–30	Unkept
Mineo (Italy)	37.350719, 14.690858	Orchard	15–25	Cultivated
Moncada (Spain)	39.588547, −0.394583	Experimental orchard	10–15	Cultivated
Monchique (Portugal)	37.332409, −8.514506	Backyard	20–30	Unkept
Monchique (Portugal)	37.336226, −8.503686	Backyard	20–30	Unkept
Monchique (Portugal)	37.332239, −8.492232	Backyard	20–30	Unkept
Monchique (Portugal)2	37.326195, −8.526232	Backyard	30–40	Unkept
Motta S. Anastasia (Italy)	37.482099, 14.886016	Orchard	15–25	Cultivated
Motta S. Anastasia (Italy)	37.469713, 14.954161	Orchard	15–25	Cultivated
Nafplio (Greece)	37.589312, 22.785267	Orchard	10–15	Unkept
Nafplio (Greece)	37.575095, 22.695589	Orchard	15–25	Cultivated
Nafplio (Greece)	37.582292, 22.696803	Orchard	10–15	Cultivated
Nafplio (Greece)	37.588798, 22.874844	Backyard	10–15	Cultivated
Nicolosi (Italy)	37.611273, 15.029477	Backyard	5–10	Cultivated
Niscemi (Italy)	37.139783, 14.393402	Backyard	15–25	Cultivated
Noto (Italy)	36.846497, 15.095445	Orchard	15–25	Unkept
Pachino (Italy)	36.720032, 15.086993	Backyard	15–25	Unkept
Pachino (Italy)	36.722328, 15.089408	Orchard	15–25	Unkept
Pedara (Italy)	37.608708, 15.066544	Backyard	30–40	Cultivated
Pizzo Calabro (Italy)	38.760390, 16.226005	Orchard	15–25	Cultivated
Ribera (Italy)	37.497113, 13.241850	Orchard	5–10	Cultivated
Ribera (Italy)	37.504423, 13.252070	Orchard	5–10	Cultivated
Riposto (Italy)	37.696470, 15.199345	Nursery	1–3	Cultivated
Rocca Imperiale (Italy)	40.108385, 16.617951	Orchard	10–15	Cultivated
San Gregorio (Italy)	37.562297, 15.100965	Backyard	60–70	Cultivated
Scordia (Italy)	37.281526, 14.869149	Orchard	15–25	Cultivated
Seville (Spain)	37.508538, −5.962815	Orchard	15–25	Cultivated
Seville (Spain)	37.482978, −5.954910	Orchard	15–25	Unkept
Sikoula (Greece)	39.085933, 21.083398	Orchard	10–15	Cultivated
Silves (Portugal)	37.164148, −8.390841	Orchard	15–25	Unkept
Soller (Spain)	39.764529, 2.709609	Botanical garden	30–40	Cultivated
Soller (Spain)	39.770115, 2.726600	Orchard	20–30	Cultivated
Terme Vigliatore (Italy)	38.145801, 15.163235	Nursery	1–3	Cultivated
Torremolinos (Spain)	36.672722, −4.504134	Orchard	30–40	Cultivated
Trebisacce (Italy)2	39.910122, 16.564824	Backyard	20–30	Cultivated
Trebisacce (Italy)	39.906711, 16.560634	Orchard	3–6	Cultivated
Zurrieq (Malta)2	35.823845, 14.505099	Backyard	15–25	Unkept

Site where P. paracitricarpa isolates were found associated with leaf litter sampled.

Sites where P. citricarpa isolates were found associated with leaf litter sampled.

Cultivated: Plants kept under constant agronomical management. Unkept: Plants abandoned.

Fungal isolates were obtained using two procedures. In the first, leaf and fruit sections (5 × 5 mm) were aseptically cut and surface-sterilised in a sodium hypochlorite solution (10 %) for 20 s, followed by 70 % ethanol for 30 s, and rinsed three times in autoclaved water. The sections were dried on autoclaved tissue paper, placed on malt extract agar (MEA; Crous ) amended with 100 μg/mL penicillin and 100 μg/mL streptomycin (MEA-PS) and incubated at 25 °C until characteristic Phyllosticta colonies were observed. In the second procedure, leaf litter, living leaves, fruits and twig portions were incubated in moist chambers at room temperature (25 °C ± 3 °C) for up to 14 d and inspected daily for fungal sporulation. Sporulating pycnidia obtained through both procedures were collected and crushed in a drop of sterile water and then spread over the surface of MEA-PS plates. After 24–36 h germinating spores were individually transferred onto MEA plates. The isolates used in this study are maintained in the Westerdijk Fungal Biodiversity Institute (CBS culture collection), Utrecht, The Netherlands, and in the working collection of Pedro Crous (CPC), housed at the Westerdijk Institute. In addition, two Phyllosticta isolates collected in Florida, USA (CPC 25312, CPC 25327) and three from China (ZJUCC200933, ZJUCC200937, ZJUCC200952) were included in the phylogenetic analyses. Sequences from additional species were retrieved from NCBI's GenBank nucleotide database. A total of 111 Phyllosticta (incl. 64 European) isolates were included in the study (Table 2), of which 100 (incl. the outgroup, Neofusicoccum mediterraneum CBS 121718) were used in the phylogenetic analysis.

Table 2

Collection details and GenBank accession numbers of isolates included in this study.

Species	Culture no.1	Host	Country	Mating type idiomorph	GenBank no.2
					ITS	actA	tef1	gapdh	LSU	rpb2
Neofusicoccum mediterraneum	CBS 121718	Eucalyptus sp.	Greece	–	GU251176	KY855639	GU251308	KY855694	KY855754	KY855815
Phyllosticta aloeicola	CPC 21020 = CBS 136058	Aloe ferox	South Africa	–	KF154280	KF289311	KF289193	KF289124	KF206214	KY855816
	CPC 21021	Aloe ferox	South Africa	–	KF154281	KF289312	KF289194	KF289125	KF206213	KY855817
P. bifrenariae	CBS 128855 = VIC30556	Bifrenaria harrisoniae, leaf	Brazil	–	JF343565	JF343649	JF343586	JF343744	KF206209	KY855818
	CPC 17467	Bifrenaria harrisoniae, leaf	Brazil	–	KF170299	KF289283	KF289207	KF289138	KF206260	KY855819
P. capitalensis	CBS 226.77	Paphiopedilum callosum, leaf spot	Germany	–	FJ538336	FJ538452	FJ538394	JF343718	KF206289	KY855820
	CBS 100175	Citrus sp.	Brazil	–	FJ538320	FJ538436	FJ538378	JF343699	KF206327	KY855821
	CBS 101228	Nephelium lappaceum	Hawaii	–	FJ538319	FJ538435	FJ538377	KF289086	KF206325	KY855822
	CBS 114751	Vaccinium sp., leaf	New Zealand	–	FJ538349	FJ538465	FJ538407	KF289088	EU167584	KY855823
	CBS 123373	Musa paradisiaca	Thailand	–	FJ538341	FJ538457	FJ538399	JF343703	JQ743604	KY855824
	CBS 123374	Citrus aurantium	Thailand	–	FJ538332	FJ538448	FJ538390	JF343702	KY855755	KY855825
	CBS 128856 = CPC 18848	Stanhopea sp.	Brazil	–	JF261465	JF343647	JF261507	JF343776	KF206304	KY855826
	CPC 14609	Zyzygium sp.	Madagascar	–	KF206184	KF289264	KF289175	KF289081	KF206280	KY855827
	CPC 20259	Orchidaceae	Thailand	–	KC291340	KC342537	KC342560	KF289104	KF206244	KY855828
	CPC 20263	Magnoliaceae	Thailand	–	KC291341	KC342538	KC342561	KF289085	KF206241	KY855829
	CPC 20268	Hibiscus syriacus	Thailand	–	KC291343	KC342540	KC342563	KF289117	KF206236	KY855830
	CPC 20275	Polyalthia longifolia	Thailand	–	KC291347	KC342544	KC342567	KF289107	KF206230	KY855831
	CPC 20278	Euphorbia milii	Thailand	–	KC291347	KC342544	KC342567	KF289107	KF206230	KY855832
	CPC 20508	Ixora chinensis	Thailand	–	KF206198	KF289302	KF289185	KF289111	KF206225	KY855833
	CPC 25327	Citrus sinensis	Florida	–	KY855585	KY855640	KY855914	KY855695	KY855756	KY855834
	CPC 27059	Citrus limon, leaf	Italy	–	KY855586	KY855641	KY855915	KY855696	KY855757	KY855835
	CPC 27060	Citrus limon, leaf	Italy	–	KY855587	KY855642	KY855916	KY855697	KY855758	KY855836
	CPC 27061	Citrus limon, leaf	Italy	–	KY855588	KY855643	KY855917	KY855698	KY855759	KY855837
	CPC 27062	Citrus limon, leaf	Italy	–	KY855589	KY855644	KY855918	KY855699	KY855760	KY855838
	CPC 27084 = CBS 141345	Citrus aurantifolia, leaf	Italy	–	KY855590	KY855645	KY855919	KY855700	KY855761	KY855839
	CPC 27085	Citrus aurantifolia, leaf	Italy	–	KY855591	KY855646	KY855920	KY855701	KY855762	KY855840
	CPC 27086	Citrus aurantifolia, leaf	Italy	–	KY855592	KY855647	KY855921	KY855702	KY855763	KY855841
	CPC 27087	Citrus aurantifolia, leaf	Italy	–	KY855593	KY855648	KY855922	KY855703	KY855764	KY855842
	CPC 27786	Citrus limon, leaf	Greece	–	KY855594	KY855649	KY855923	KY855704	KY855765	KY855843
	CPC 27787	Citrus limon, leaf	Greece	–	KY855595	KY855650	KY855924	KY855705	KY855766	KY855844
	CPC 27788	Citrus limon, leaf	Greece	–	KY855596	KY855651	KY855925	KY855706	KY855767	KY855845
	CPC 27789	Citrus limon, leaf	Greece	–	KY855597	KY855652	KY855926	KY855707	KY855768	KY855846
	CPC 27825 = CBS 141346	C. medica var. sarcodactylis, leaf spot	Italy	–	KY855598	KY855653	KY855927	KY855708	KY855769	KY855847
	CPC 27826	C. medica var. sarcodactylis, leaf spot	Italy	–	KY855599	KY855654	KY855928	KY855709	KY855770	KY855848
	CPC 27827	C. medica var. sarcodactylis, leaf spot	Italy	–	KY855600	KY855655	KY855929	KY855710	KY855771	KY855849
	CPC 27828	C. medica var. sarcodactylis, leaf spot	Italy	–	KY855601	KY855656	KY855930	KY855711	KY855772	KY855850
	CPC 27917 = CBS 141347	Citrus limon, leaf	Malta	–	KY855602	KY855657	KY855931	KY855712	KY855773	KY855851
	CPC 27918	Citrus limon, leaf	Malta	–	KY855603	KY855658	KY855932	KY855713	KY855774	KY855852
	CPC 27919 = CBS 141348	Citrus limon, leaf	Portugal	–	KY855604	KY855659	KY855933	KY855714	KY855775	KY855853
	CPC 27920	Citrus limon, leaf	Portugal	–	KY855605	KY855660	KY855934	KY855715	KY855776	KY855854
	CPC 28124	Citrus limon, leaf	Spain	–	KY855606	KY855661	KY855935	KY855716	KY855777	KY855855
	CPC 28125	Citrus limon, leaf	Spain	–	KY855607	KY855662	KY855936	KY855717	KY855778	KY855856
	CPC 28126	Citrus limon, leaf	Spain	–	KY855608	KY855663	KY855937	KY855718	KY855779	KY855857
P. citriasiana	CBS 120486	Citrus maxima, fruit	Thailand	–	FJ538360	FJ538476	FJ538418	JF343686	KF206314	KY855858
	CBS 120487	Citrus maxima, fruit	China	–	FJ538361	FJ538477	FJ538419	JF343687	KF206313	KY855859
	CBS 123370	Citrus maxima, fruit	Vietnam	–	FJ538355	FJ538471	FJ538413	JF343689	KF206310	KY855860
P. citribraziliensis	CBS 100098	Citrus sp., leaf	Brazil	–	FJ538352	FJ538468	FJ538410	JF343691	KF206221	KY855861
	CPC 17464	Citrus sp., leaf	Brazil	–	KF170300	KF289280	KF289224	KF289159	KF206263	KY855862
	CPC 17465	Citrus sp., leaf	Brazil	–	KF170301	KF289281	KF289225	KF289160	KF206262	KY855863
P. citricarpa	CBS 122482	Citrus sinensis	Zimbabwe	MAT1-2-1	FJ538317	KF289265	FJ538375	KF289146	KF306230	KY855864
	CBS 127452	Citrus reticulata	Australia	MAT1-2-1	JF343581	JF343665	JF343602	JF343769	KF206307	KY855865
	CBS 127454	Citrus limon	Australia	MAT1-2-1	JF343583	JF343667	JF343604	JF343771	KF206306	KY855866
	CPC 16151	Citrus sp.	South Africa	MAT1-1-1	KF170291	KF289267	KF289221	KF289156	KF206276	KY855867
	CPC 16586	Citrus limon	Argentina	MAT1-1-1	KF170293	KF289269	KF289220	KF289155	KF206274	KY855868
	CPC 16603	Citrus limon	Uruguay	MAT1-1-1	KF170295	KF289274	KF289213	KF289147	KF206269	KY855869
	CPC 16609	Citrus sp.	Argentina	MAT1-1-1	KF170298	KF289277	KF289217	KF289152	KF206266	KY855870
	CPC 25312	Citrus sinensis	Florida	MAT1-2-1	KY855609	KY855664	KY855938	KY855719	KY855780	KY855871
	CPC 279093 = CBS 141349	Citrus limon, leaf litter	Italy	MAT1-2-1	KY855610	KY855665	KY855939	KY855720	KY855781	KY855872
	CPC 279103	Citrus limon, leaf litter	Italy	MAT1-2-1	KY855611	KY855666	KY855940	KY855721	KY855782	KY855873
	CPC 279113	Citrus limon, leaf litter	Italy	MAT1-2-1	KY855612	KY855667	KY855941	KY855722	KY855783	KY855874
	CPC 279123	Citrus limon, leaf litter	Italy	MAT1-2-1	KY855613	KY855668	KY855942	KY855723	KY855784	KY855875
	CPC 279133 = CBS 141350	Citrus sinensis, leaf litter	Malta	MAT1-2-1	KY855614	KY855669	KY855943	KY855724	KY855785	KY855876
	CPC 279143	Citrus sinensis, leaf litter	Malta	MAT1-2-1	KY855615	KY855670	KY855944	KY855725	KY855786	KY855877
	CPC 279153	Citrus sinensis, leaf litter	Malta	MAT1-2-1	KY855616	KY855671	KY855945	KY855726	KY855787	KY855878
	CPC 279163	Citrus sinensis, leaf litter	Malta	MAT1-2-1	KY855617	KY855672	KY855946	KY855727	KY855788	KY855879
	CPC 281043 = CBS 141351	Citrus sinensis, leaf litter	Portugal	MAT1-1-1	KY855618	KY855673	KY855947	KY855728	KY855789	KY855880
	CPC 281053 = CBS 141352	Citrus sinensis, leaf litter	Portugal	MAT1-1-1	KY855619	KY855674	KY855948	KY855729	KY855790	KY855881
	CPC 281063	Citrus sinensis, leaf litter	Portugal	MAT1-1-1	KY855620	KY855675	KY855949	KY855730	KY855791	KY855882
	CPC 281073	Citrus sinensis, leaf litter	Portugal	MAT1-1-1	KY855621	KY855676	KY855950	KY855731	KY855792	KY855883
	CPC 311713	Citrus sinensis, leaf litter	Malta	MAT1-2-1	–	–	–	–	–	–
	CPC 311723	Citrus sinensis, leaf litter	Malta	MAT1-2-1	–	–	–	–	–	–
	CPC 311733	Citrus sinensis, leaf litter	Malta	MAT1-2-1	–	–	–	–	–	–
	CPC 311743	Citrus sinensis, leaf litter	Malta	MAT1-2-1	–	–	–	–	–	–
	CPC 312793	Citrus sinensis, leaf litter	Portugal	MAT1-1-1	–	–	–	–	–	–
	CPC 312803	Citrus sinensis, leaf litter	Portugal	MAT1-1-1	–	–	–	–	–	–
	CPC 312813	Citrus sinensis, leaf litter	Portugal	MAT1-1-1	–	–	–	–	–	–
	CPC 312823	Citrus sinensis, leaf litter	Portugal	MAT1-1-1	–	–	–	–	–	–
	ZJUCC200952	Citrus reticulata, leaf	China	MAT1-2-1	JN791635	JN791556	JN791480	KY855732	KY855793	KY855884
P. citrichinaensis	CBS 129764 = ZJUCC2010100	Citrus reticulata, leaf	China	–	JN791598	JN791527	JN791453	KY855733	KY855794	KY855885
	CBS 130529 = ZJUCC201085 = CGMCC3.14302	Citrus maxima, leaf	China	–	JN791597	JN791526	JN791452	KY855734	KY855795	KY855886
P. citrimaxima	CPC 20276 = CBS 136059 = MFLUCC10-0137	Citrus maxima, fruit	Thailand	–	KF170304	KF289300	KF289222	KF289157	KF206229	–
P. cordylinophila	CPC 20261 = MFLUCC10-0166	Cordyline fruticosa	Thailand	–	KF170287	KF289295	KF289172	KF289076	KF206242	KY855887
	CPC 20277 = MFLUCC12-0014	Cordyline fruticosa	Thailand	–	KF170288	KF289301	KF289171	KF289075	KF206228	KY855888
P. cussonia	CPC 14873	Cussonia sp.	South Africa	–	JF343578	JF343662	JF343599	JF343764	KF206279	KY855889
	CPC 14875	Cussonia sp.	South Africa	–	JF343579	JF343663	JF343600	JF343765	KF206278	KY855890
P. eugeniae	CBS 445.82	Eugenia aromatica	Indonesia	–	AY042926	KF289246	KF289208	KF289139	KF206288	KY855891
P. hypoglossi	CBS 434.92	Ruscus aculeatus	Italy	–	FJ538367	FJ538483	FJ538425	JF343695	KF206299	KY855892
P. paracapitalensis	CBS 173.77	Citrus aurantiifolia	New Zealand	–	KF206179	KF289244	FJ538393	KF289100	KF306231	KY855893
	CPC 26517 = CBS 141353	Citrus floridana, leaf	Italy	–	KY855622	KY855677	KY855951	KY855735	KY855796	KY855894
	CPC 26518	Citrus floridana, leaf	Italy	–	KY855623	KY855678	KY855952	KY855736	KY855797	KY855895
	CPC 26700 = CBS 141354	Citrus floridana, leaf	Italy	–	KY855624	KY855679	KY855953	KY855737	KY855798	KY855896
	CPC 26701	Citrus floridana, leaf	Italy	–	KY855625	KY855680	KY855954	KY855738	KY855799	KY855897
	CPC 26805	Citrus floridana, leaf	Italy	–	KY855626	KY855681	KY855955	KY855739	KY855800	KY855898
	CPC 26806	Citrus floridana, leaf	Italy	–	KY855627	KY855682	KY855956	KY855740	KY855801	KY855899
	CPC 28120 = CBS 141355	Citrus limon, leaf	Spain	–	KY855628	KY855683	KY855957	KY855741	KY855802	KY855900
	CPC 28121	Citrus limon, leaf	Spain	–	KY855629	KY855684	KY855958	KY855742	KY855803	KY855901
	CPC 28122	Citrus limon, leaf	Spain	–	KY855630	KY855685	KY855959	KY855743	KY855804	KY855902
	CPC 28123	Citrus limon, leaf	Spain	–	KY855631	KY855686	KY855960	KY855744	KY855805	KY855903
	CPC 28127 = CBS 141356	Citrus limon, leaf	Spain	–	KY855632	KY855687	KY855961	KY855745	KY855806	KY855904
	CPC 28128	Citrus limon, leaf	Spain	–	KY855633	KY855688	KY855962	KY855746	KY855807	KY855905
	CPC 28129	Citrus limon, leaf	Spain	–	KY855634	KY855689	KY855963	KY855747	KY855808	KY855906
P. paracitricarpa	CPC 27169 = CBS 141357	Citrus limon, leaf litter	Greece	–	KY855635	KY855690	KY855964	KY855748	KY855809	KY855907
	CPC 27170 = CBS 141358	Citrus limon, leaf litter	Greece	–	KY855636	KY855691	KY855965	KY855749	KY855810	KY855908
	CPC 27171 = CBS 141359	Citrus limon, leaf litter	Greece	–	KY855637	KY855692	KY855966	KY855750	KY855811	KY855909
	CPC 27172 = CBS 141360	Citrus limon, leaf litter	Greece	–	KY855638	KY855693	KY855967	KY855751	KY855812	KY855910
	CPC 31246	Citrus limon, leaf litter	Greece	–	–	–	–	–	–	–
	CPC 31247	Citrus limon, leaf litter	Greece	–	–	–	–	–	–	–
	CPC 31248	Citrus limon, leaf litter	Greece	–	–	–	–	–	–	–
	CPC 31249	Citrus limon, leaf litter	Greece	–	–	–	–	–	–	–
	ZJUCC200933	Citrus sinensis, fruit	China	–	JN791626	JN791544	JN791468	KY855752	KY855813	KY855911
	ZJUCC200937	Citrus sinensis, fruit	China	–	JN791627	JN791546	JN791470	KY855753	KY855814	KY855912
P. spinarum	CBS 292.90	Chamaecyparis pisifera	France	–	JF343585	JF343669	JF343606	JF343773	KF206301	KY855913

CPC: Culture collection of P.W. Crous, housed at CBS; CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; ZJUCC: Zhejiang University Culture Collection, China; MFLUCC: Mae Fah Luang University Culture Collection; CGMCC: China, General Microbiological Culture Collection, Beijing, China; VIC: Culture collection of Federal University of Viçosa, Viçosa, Brazil. Ex-type and ex-epitype cultures are indicated in bold.

ITS: internal transcribed spacers 1 and 2 together with 5.8S nrDNA; actA: partial actin gene; tef1: partial translation elongation factor 1-α gene; gapdh: partial glyceraldehyde-3-phosphate dehydrogenase gene; LSU: partial 28S (large subunit) nrDNA; rpb2: partial RNA polymerase II second largest subunit gene. Sequences generated in this study indicated in italics.

P. citricarpa isolates genotyped in this study.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted using a Wizard® Genomic DNA Purification Kit (Promega Corporation, WI, USA) following the manufacturer's instructions. Partial regions of six loci were amplified. The primers V9G (de Hoog & Gerrits van den Ende 1998) and ITS4 (White ) were used to amplify the internal transcribed spacer region (ITS) of the nuclear ribosomal RNA operon, including the 3′ end of the 18S rRNA, the first internal transcribed spacer region, the 5.8S rRNA gene; the second internal transcribed spacer region and the 5′ end of the 28S rRNA gene. The primers EF1-728F (Carbone & Kohn 1999) and EF2 (O’Donnell ) were used to amplify part of the translation elongation factor 1-α gene (tef1). The primers ACT-512F and ACT-783R (Carbone & Kohn 1999) were used to amplify part of the actin gene (actA). The 28S large subunit nrDNA (LSU) was amplified using primers LR0R (Moncalvo ) and LR5 (Vilgalys & Hester 1990). The RNA polymerase II second largest subunit (rpb2) was amplified with RPB2-5F2 (Sung ) and fRPB2-7cR (Liu ). Glyceraldehyde-3-phosphate dehydrogenase (gapdh) was amplified using primers Gpd1-LM and Gpd2-LM (Myllys ). For P. citricarpa isolates the alternative primers Gpd1 (Guerber ) and GPDHR2 (Glienke ) were used to amplify gapdh. The PCR amplification mixtures and cycling conditions for ITS, actA, tef1, LSU and gapdh were followed as described by Glienke . Due to the lack of available rpb2 gene sequences of Phyllosticta isolates, we generated these sequences for all the strains used for this study (except for P. citrimaxima CPC 20276 = CBS 136059, culture has been lost). The rpb2 PCR was performed in a total volume of 25 μL and the mixture consisted of 1 μL genomic DNA, 1× PCR Buffer (Bioline GmbH, Luckenwalde, Germany), 0.75 μM MgCl2, 1.85 μM of each dNTP, 0.45 μM of each primer and 0.5 μL BioTaq Taq DNA polymerase (Bioline GmbH, Luckenwalde, Germany). A touchdown PCR protocol was used for rpb2: initial denaturation (94 °C, 5 min), five amplification cycles (94 °C, 45 s; 60 °C, 45 s; 72 °C, 2 min), five amplification cycles (94 °C, 45 s; 58 °C, 45 s; 72 °C, 2 min), 30 amplification cycles (94 °C, 45 s; 54 °C, 45 s; 72 °C, 2 min) and a final extension step (72 °C, 8 min). The PCR products were sequenced in both directions using the BigDye® Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems Life Technologies, Carlsbad, CA, USA), after which amplicons were purified through Sephadex G-50 Fine columns (GE Healthcare, Freiburg, Germany) in MultiScreen HV plates (Millipore, Billerica, MA). Purified sequence reactions were analysed on an Applied Biosystems 3730xl DNA Analyzer (Life Technologies, Carlsbad, CA, USA). The DNA sequences generated were analysed and consensus sequences were computed using the program SeqMan Pro (DNASTAR, Madison, WI, USA).

Phylogenetic analyses

Novel sequences generated in this study were queried against the NCBI's GenBank nucleotide database to determine the closest relatives for a taxonomic framework of the studied isolates. Alignments of different gene regions, including sequences obtained from this study and sequences downloaded from GenBank, were initially performed by using the MAFFT v. 7 online server (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh & Standley 2013), and then manually adjusted in MEGA v. 6.06 (Tamura ). Additional reference sequences were selected based on recent studies on Phyllosticta species (Glienke et al., 2011, Wang et al., 2012, Wikee et al., 2013a).

Phylogenetic analyses were based on both Bayesian Inference (BI) and Maximum Parsimony (MP) analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analysis. MrBayes v. 3.2.5 (Ronquist ) was used to generate phylogenetic trees under optimal criteria per partition. The Markov Chain Monte Carlo (MCMC) analysis used four chains and started from a random tree topology. The heating parameter was set to 0.2 and trees were sampled every 100 generations. Analyses stopped once the average standard deviation of split frequencies was below 0.01. The MP analysis was done using PAUP (Phylogenetic Analysis Using Parsimony, v. 4.0b10; Swofford 2003). Phylogenetic relationships were estimated by heuristic searches with 100 random addition sequences. Tree bisection-reconnection was used, with the branch swapping option set on “best trees” only with all characters weighted equally and alignment gaps treated as fifth state. Tree length (TL), consistency index (CI), retention index (RI) and rescaled consistence index (RC) were calculated for parsimony and bootstrap analysis (Hillis & Bull 1993) was based on 1 000 replications. Sequences generated in this study were deposited in GenBank (Table 2) and alignments and phylogenetic trees in TreeBASE (www.treebase.org). Nomenclatural novelties were deposited in MycoBank (Crous ).

Taxonomy

A subset of isolates of the four Phyllosticta species collected in this study was morphologically characterised. After 14 d of incubation in the dark at 27 °C, the morphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at ×1 000 magnification were determined for each isolate using a Zeiss Axioscope 2 microscope with interference contrast (DIC) optics. Colony colour and growth rate were established on MEA, potato dextrose agar (PDA) and OA according to Crous . Sporulation was induced on pine needle agar (PNA) (Smith ) and synthetic nutrient-poor agar (SNA) under near UV-light. Colony colour was determined on MEA, OA and PDA using the colour charts of Rayner (1970). Colony growth rates were assessed on MEA, OA and PDA in 90 mm Petri plates at 9–39 °C at 3 °C intervals. Three plates were used for each culture/media and two measurements of colony diameter perpendicular to each were made after 3, 6, 9 and 12 d of incubation in the dark, after which averages were computed. For each species × growth medium × incubation time combination, data were normalised to the maximum growth observed for that combination. The combined dataset with relative growth values (0 = no growth, 1 = maximum growth) was subjected to non-linear regression using the BETE function: Y = (a × ((X − Tmin)/(Tmax − Tminx)) ˆ b × (1−((X − Tmin)/(Tmax − Tminx)) ˆ c (Analytis, 1977, Leggieri et al., 2017). Goodness of fit was determined through linear regression of the predicted against actual relative growth values.

Mating type identification

The mating types of P. citricarpa strains were determined based on PCR amplification of a diagnostic region from each mating type idiomorph by using four primers, MAT111F3 (5′-GCAATGTGGCAGCGCAATCC-3′) and MAT111R3 (5′-TCTGGACCATCGGACTCATC-3′) for MAT1-1-1, and MAT121F6 (5′-GATCGTGGCAGGAGGCTTTG-3′) and MAT121R6 (5′-AACGACCAGCGATCGGTAAG-3′) for MAT1-2-1 (Amorim ). The same reaction mixtures were used for the amplification of both primers sets. A total volume of 12.5 μL containing 1 μL genomic DNA, 1× PCR Buffer (Bioline GmbH, Luckenwalde, Germany), 0.63 μM MgCl2, 0.7 μM of each dNTP, 0.25 μM of each primer and 0.5 μL BioTaq Taq DNA polymerase (Bioline GmbH, Luckenwalde, Germany), was used.

The PCR programme for the primers MAT111F3–MAT111R3 consisted of initial denaturation (94 °C, 3 min), 25 amplification cycles (94 °C, 30 s; 60 °C, 30 s; 72 °C, 1 min), and a final extension step (72 °C, 10 min). For the primers MAT121F6–MAT121R6, 30 amplification cycles (94 °C, 30 s; 55 °C, 30 s; 72 °C, 1 min) were used. The amplified fragments were separated by electrophoresis at 100 V for 25 min on a 1 % (w/v) agarose gel stained with GelRed™ (Biotium, Hayward, CA, USA), and viewed under ultra-violet light. Sizes of amplicons were determined against a HyperLadder™ I molecular marker (Bioline).

Genotyping of P. citricarpa isolates

Fifteen published polymorphic SSR markers (Wang et al., 2016, Carstens et al., 2017) were used to compare the genotypes of the P. citricarpa isolates found in this study with populations from Australia, Brazil, China, South Africa and the USA (Carstens ). The primer labelling as well as the PCR reactions and cycling conditions were as previously described in Carstens . The SSR alleles were scored using Genemapper software v. 4 (Life Technologies). To determine the within-population genetic diversity the following were calculated in GenAlEx v. 6.5 (Peakall & Smouse, 2012): number of alleles (Na), number of effective alleles, number of private alleles, number of polymorphic loci and Nei's measure of gene diversity (Nei 1973). A zero value for Nei's gene diversity is an indication that there is no genetic diversity within the population. Isolates with identical alleles across all the loci were considered clones or multilocus genotypes (MLGs). For the allele-based genetic analyses, a per population clone-corrected dataset was used. To assess the genetic variation between the European populations and those from other continents, an analysis of molecular variance (AMOVA) was conducted. The statistical significance was tested using 999 permutations. In order to perform this analysis, the 12 P. citricarpa populations from Carstens were included in the dataset. The AMOVA was performed in GenAlEx v. 6.5 (Peakall & Smouse 2012).

Pathogenicity

Two isolates of each of the four Phyllosticta species isolated from specimens collected in Europe (P. capitalensis: CPC 27825, CPC 27917; P. paracapitalensis: CPC 26517, CPC 26700; P. citricarpa: CPC 27909, CPC 27913; P. paracitricarpa: CPC 27169, CPC 27170), were inoculated into mature, untreated fruits of sweet orange (Citrus sinensis Osbeck), cultivar ‘Valencia’ (from Spain), following the method described by Perryman to obtain indicative results about pathogenicity. Three fruits per replicate for each isolate were inoculated and were arranged in a randomised complete block design. Fruits were washed and surface disinfected by immersion for 10 min in 70 % ethanol, and rinsed twice in autoclaved water. A suspension of conidia (1.0 × 105 conidia/mL) was obtained from cultures grown on PDA for 15 d at 27 °C, and was injected, 100 mL at a time, into 12 inoculation points on the surface of oranges. The suspension was inoculated by inserting a hypodermic sterile needle into the albedo (the white pith area just below the peel), approx. 2 mm deep. Control fruits were inoculated with sterile water. The inoculation points on each fruit were labelled with a dot made with a permanent marker. The inoculated oranges were incubated in sterile plastic boxes at 20 °C, with 100 % relative humidity, under a lighting rig providing a 12 h photoperiod. Lesion development was evaluated 5, 10 and 25 d after inoculation. The inoculated fungi were re-isolated from any tissue showing lesions and the identity of the re-isolated fungi was confirmed by sequencing loci tef1 and LSU.

Results

A total of 64 monosporic isolates resembling those of the genus Phyllosticta were collected. The Phyllosticta isolates were recovered from five species of Citrus at 11 different sites. Among them, 32 isolates were obtained from fresh leaves, 28 were associated with leaf litter and four with leaf spot symptoms (Table 2). During the surveys performed no CBS symptoms were observed.

The combined species phylogeny of Phyllosticta consisted of 100 sequences, including the outgroup sequences of Neofusicoccum mediterraneum (culture CBS 121718). A total of 3 142 characters were included in the phylogenetic analyses; 693 characters were parsimony-informative, 315 were variable and parsimony-uninformative and 2 134 characters were constant. The maximum of 1 000 equally most parsimonious trees were saved (Tree length = 1 829, CI = 0.750, RI = 0.972 and RC = 0.729). Bootstrap support values from the parsimony analysis were plotted on the Bayesian phylogeny presented in Fig. 1. For the Bayesian analysis, MrModeltest suggested that the ITS partition should be analysed with a fixed state frequency distribution and all other loci with Dirichlet state frequency distributions. The following models were used in the Bayesian analysis: SYM+I+G (ITS), HKY+I (actA), GTR+G (tef1, gapdh, rpb2) and GTR+I (LSU).

Fig. 1

Consensus phylogram resulting from a Bayesian analysis of the combined ITS, actA, tef1, gapdh, LSU and rpb2 sequence alignments. Bootstrap support values and Bayesian posterior probability values are indicated at the nodes. Substrate and country of origin, where known, are indicated next to the strain numbers. The tree was rooted to Neofusicoccum mediterraneum (CBS 121718).

In the Bayesian analysis, the ITS partition had 189 unique site patterns, the actA partition had 116 unique site patterns, the tef1 partition had 158 unique site patterns, the gapdh partition had 105 unique site patterns, the LSU partition had 76 unique site patterns, the rpb2 partition had 245 unique site patterns and the analysis ran for 1 900 000 generations, resulting in 38 002 trees of which 28 502 trees were used to calculate the posterior probabilities (Fig. 1). The main difference between the Bayesian and MP trees was the position of P. bifrenariae; in the Bayesian tree this species clustered basal to P. citricarpa whereas it was basal to the broader lineage containing the species clades of P. citricarpa to P. citribraziliensis in the parsimony analysis (data not shown). All other species clades were identical between the two analyses. The tree resolved 15 Phyllosticta species, two of which (P. paracapitalensis and P. paracitricarpa) are described as new in the Results – Taxonomy section.

Nucleotide variations were observed in 49 base positions within the alignment of P. capitalensis isolates and those of the new species, P. paracapitalensis, included in this study (Table 3), and in 14 positions for P. citricarpa and the new species P. paracitricarpa (Table 4). Between the P. capitalensis and P. paracapitalensis clades, differences were present in all regions sequenced except for ITS. Specifically, 20 fixed nucleotide changes were observed over 3 142 nucleotides (one for actA, four for tef1, one for gapdh and 14 for rpb2). Moreover, seven fixed nucleotide changes were observed between P. citricarpa and P. paracitricarpa clades (five for tef1 and two for LSU). ITS, LSU and tef1 were sequenced to identify a further eight isolates of P. citricarpa (CPC 31171, CPC 31172, CPC 31173, CPC 31174, from Malta and CPC 31179, CPC 31180, CPC 31181, CPC 31182 from Portugal) and four isolates of P. paracitricarpa (CPC 31246, CPC 31247, CPC 31248, CPC 31249 from Greece) (data not shown).

Table 3

Nucleotide differences observed among P. paracapitalensis and P. capitalensis isolates used in this study. Base positions include spaces caused by alignment gaps and refer to the position in the alignment deposited in TreeBASE. Base positions representing fixed nucleotide differences between the two species are in bold.

Table 4

Nucleotide differences observed among P. paracitricarpa and P. citricarpa isolates used in this study. Base positions include spaces caused by alignment gaps and refer to the position in the alignment deposited in TreeBASE. Base positions representing fixed nucleotide differences between the two species are in bold.

Morphological observations, supported by phylogenetic inference, were used to distinguish two known species (P. capitalensis and P. citricarpa) from two novel species. Culture characteristics were noted as dissimilar. The colour of upper and lower surfaces of Petri dishes were determined (Fig. 2). The BETE function fitted the relative growth data very well (R2 values 0.81 to 0.87) and predicted cardinal and optimal temperatures of 12.5–27.2–34.0 °C for P. citricarpa, 10.7–26.4–33.2 °C for P. paracitricarpa, 9.4–27.3–33.3 °C for P. capitalensis, and 11.8–28.6–33.3 °C for P. paracapitalensis (Fig. 3). After 9 d of incubation at 27 °C, P. capitalensis and P. paracapitalensis grew significantly faster (8.6–8.7 mm/d) on PDA and OA than P. citricarpa (4.8 and 6.6 mm/d, respectively) and P. paracitricarpa (4.0 and 5.4 mm/d, respectively), while growth of these species were significantly slower on MEA (5.7, 4.4, 4.5 and 3.3 mm/d, respectively). The isolates also differed morphologically from the other Phyllosticta species associated with citrus worldwide (Table 5). Based on the results of both the phylogenetic and morphological analyses, the two new species are described below.

Fig. 2

Cultural characteristics of Phyllosticta species collected from citrus in Europe after 7 d at 27 °C on MEA, OA and PDA (respectively in 1st, 2nd and 3rd column). A–C.P. paracapitalensis. D–F.P. capitalensis. G–I.P. paracitricarpa. J–L.P. citricarpa.

Fig. 3

Relative growth (0 to 1 scale) values on MEA, OA and PDA of Phyllosticta species collected in this study as influenced by incubation temperatures of 9–39 °C as fitted to a BETE function [Y = (a × ((X − Tmin)/(Tmax − Tminx)) ˆ b × (1−((X − Tmin)/(Tmax − Tminx)) ˆ c] with parameter values of a, Tmin, Tmax, b, c, and goodness of fit for P. capitalensis (8.942, 9.357, 33.261, 2.988, 0.665, R2 = 0.835), P. paracapitalensis (9.715, 11.820, 33.310, 3.551, 0.408, R2 = 0.806), P. citricarpa (6.932, 12.541, 33.962, 2.179, 0.749, R2 = 0.866) and P. paracitricarpa (6.281, 10.687, 33.247, 2.283, 0.471, R2 = 0.873).

Table 5

Morphological characteristics of Phyllosticta spp. associated with citrus.

Species	Ascomata		Asci		Ascospores		Conidiomata		Conidiogenous cells		Conidia		Spermatia		Reference
	Size (μm)	Shape	Size (μm)	Shape	Size (μm)	Shape	Size (μm)	Shape	Size (μm)	Shape	Size (μm)	Shape	Size (μm)	Shape
P. capitalensis	250	globose to pyriform	58–80 × 11–15	clavate	15–17 × 5–6	limoniform	300 × 250	globose to ampulliform	7–10 × 3–5	subcylindrical to ampulliform to doliiform	(10–)11–12(–14) × (5–)6–7	ellipsoid to obovoid	–	–	Hennings (1908)
P. citriasiana	–	–	–	–	–	–	120–240 × 125–225	globose, subglobose to ellipsoidal	7–17 × 3–5	subcylindrical to ampulliform or doliiform	(10–)12–14(–16) × (5–)6–7(–8)	ellipsoid to obovoid	3–5 × 1–2	bacilliform to ellipsoid	Wulandari et al. (2009)
P. citribraziliensis	–	–	–	–	–	–	250	globose	7–20 × 3–4	subcylindrical to doliiform	10–12 × 6–7	ellipsoid to obovoid	–	–	Glienke et al. (2011)
P. citricarpa	–	–	–	–	–	–	250	globose to ampulliform	7–12 × 3–4	subcylindrical to doliiform	(10–)11–12(–14) × ( –)7(–8)	ellipsoid to obovoid	–	–	Van der Aa (1973)
P. citrichinaensis	100–300 × 100–200	subglobose to pyriform	42–81 × 10–14	subclavate to cylindrical	14–20 × 7–8	fusiform to ellipsoidal	100–200 × 100–200	globose or subglobose	6–12 × 2–5	lageniform	(7–)8–12(−13) × 6–9	ellipsoid to obovoid	7–9 × 1–2	bacilliform	Wang et al. (2012)
P. citrimaxima	–	–	–	–	–	–	150–160 × 120–130	globose	3–5 × 1–2	cylindrical	5(–8) × (3–)4(–7)	ellipsoid	–	–	Wikee et al., 2013a, Wikee et al., 2013b
P. paracapitalensis	up to 300	globose	40–75 × 10–12	subcylindrical to clavate	16–17 × 6 (–7)	limoniform	up to 250	globose	7–15 × 3–4	subcylindrical	(9–)12–13(–14) × (6–)7	ellipsoid to obovoid	–	–	This study
P. paracitricarpa	–	–	–	–	–	–	250	globose	12–17 × 3–4	subcylindrical	(9–)11–13(–15) × 7–8(–9)	ellipsoid to obovoid	–	–	This study

Guarnaccia & Crous, sp. nov. MycoBank MB817204; Fig. 4.

Fig. 4

Phyllosticta paracapitalensis (CBS 141353). A. Ascomata forming on PNA. B. Asci with ascospores. C. Ascospores. D. Conidiomata forming on SNA. E. Conidiogenous cells giving rise to conidia. F. Conidia with mucoid sheaths and apical appendages. Scale bars = 10 μm.

Etymology: Named after its close morphological resemblance and phylogenetic relationship to P. capitalensis.

Conidiomata (on PNA) pycnidial, solitary, black, erumpent, globose, exuding colourless conidial masses; pycnidia up to 250 μm diam, elongated in culture on PNA; pycnidial wall of several layers of textura angularis, to 30 μm thick; inner wall of hyaline textura angularis. Ostiole central, to 20 μm diam. Conidiophores subcylindrical to ampulliform, reduced to conidiogenous cells, or with 1–2 supporting cell, that can be branched at the base, 7–20 × 4–6 μm. Conidiogenous cells terminal, subcylindrical, hyaline, smooth, coated in a mucoid layer, 7–15 × 3–4 μm; proliferating several times percurrently near apex. Conidia (9–)12–13(–14) × (6–)7 μm, solitary, hyaline, aseptate, thin and smooth-walled, granular, or with a single large central guttule, fusoid-ellipsoid, tapering towards a narrow truncate base, 3–4 μm diam, enclosed in a persistent mucoid sheath, 2–3 μm thick, and bearing a hyaline, apical mucoid appendage, (4–)5–7(–8) × 1.5(–2) μm, flexible, unbranched, tapering towards an acutely rounded tip. Ascomata solitary or in clusters of 2–3, erumpent, globose, up to 300 μm diam, with elongated neck to 500 μm long, with central ostiole; wall of 3–6 layers of brown textura angularis. Asci bitunicate, 8-spored, stipitate, with small pedicel and well developed apical chamber, hyaline, subcylindrical to clavate, 40–75 × 10–12 μm. Ascospores bi- to multiseriate, hyaline, smooth, granular with large central guttule, aseptate, straight, rarely curved, widest in the middle, limoniform with mucoid caps at obtuse ends, (15–)16–17(–18) × 6(–7) μm.

Culture characteristics: On MEA, colonies appear woolly, flat, irregular, initially white with abundant mycelium, gradually becoming greenish to dark green after 2–3 d with white hyphae on the undulate margin; reverse dark green to black. On OA, colonies appear flat with a regular margin, initially hyaline with abundant mycelium, gradually becoming dark greenish after 3–4 d; reverse dark green to black. On PDA, colonies appear irregular, woolly, initially white, gradually becoming greenish to dark green after 2–3 d with white hyphae on the undulate margin; reverse black. After 12 d in the dark at 27 °C, mycelium reached the edge of the Petri dish. The optimum growth rate was observed at 27 °C. No growth was observed at 12 °C and 39 °C.

Specimen examined: Italy, Sicily, on living leaf of Citrus × floridana, 4 Mar. 2015, V. Guarnaccia (holotype CBS H-22663, culture ex-type CPC 26517 = CBS 141353).

Notes: Phyllosticta paracapitalensis was isolated from leaves of Citrus limon and C. ×floridana as an endophyte. This species is similar to P. capitalensis, its sister species, but represents a distinct taxon, supported by molecular and morphological differences. Phyllosticta paracapitalensis differs from P. capitalensis in having longer ascomatal necks, narrower asci, and slightly larger ascospores. The asexual morph presents solitary and globose conidiomata that differ from those of P. capitalensis (aggregated and globose to ampuliform). Furthermore, the ostioles are larger and the conidiogenous cells are longer than P. paracapitalensis.

Guarnaccia & Crous, sp. nov. MycoBank MB817205. Fig. 5.

Fig. 5

Phyllosticta paracitricarpa (CBS 141357). A, B. Conidiomata forming on PNA. C, D. Conidiogenous cells giving rise to conidia. E, F. Conidia with mucoid sheaths and apical appendages. Scale bars = 10 μm.

Etymology: Named after its close morphological resemblance and phylogenetic relationship to P. citricarpa.

Conidiomata (on PNA) pycnidial, solitary, black, erumpent, globose, exuding colourless conidial masses; pycnidia up to 250 μm diam, elongated in culture on PNA; pycnidial wall of several layers of textura angularis, 20–30 μm thick; inner wall of hyaline textura angularis. Ostiole central, up to 10 μm diam. Conidiophores subcylindrical to ampulliform, reduced to conidiogenous cells, or with 1–2 supporting cell, that can be branched at the base, 15–25 × 4–5 μm. Conidiogenous cells terminal, subcylindrical, hyaline, smooth, coated in a mucoid layer, 12–17 × 3–4 μm; proliferating several times percurrently near apex. Conidia (9–)11–13(–15) × 7–8(–9) μm, solitary, hyaline, aseptate, thin and smooth-walled, granular, or with a single large central guttule, ellipsoid to obovoid, tapering towards a narrow truncate base, 3–4 μm diam, enclosed in a thin persistent mucoid sheath, 1–1.5 μm thick, and bearing a hyaline, apical mucoid appendage, (8–)10–12(–15) × 1.5(–2) μm, flexible, unbranched, tapering towards an acutely rounded tip.

Culture characteristics: Colonies on MEA flat, with irregular edge; surface initially yellow becoming leaden grey in the centre, yellow at margin, and leaden grey underneath. On PDA colonies were flat, rather regular and slow growing, initially white-grey mycelium, gradually becoming greenish to dark green, with white hyphae at the margin; reverse black. On OA flat, spreading, olivaceous grey, becoming pale dark grey towards the margin, with sparse to moderate aerial mycelium; surrounded by a diffuse yellow pigment in the agar medium. After 12 d in the dark the optimum growth was observed at 27 °C on MEA, OA and PDA (33, 53 and 41 mm). No growth was observed at 9 °C and 39 °C.

Specimen examined: Greece, Mastro, on leaf litter of Citrus limon, 6 May 2015, V. Guarnaccia (holotype CBS H-22664, culture ex-type CPC 27169 = CBS 141357).

Notes: Phyllosticta paracitricarpa was isolated from Citrus limon leaf litter in Europe (this study) and from lesions on C. sinensis fruits in China (Wang ). This species is similar to P. citricarpa, its sister species, but represents a distinct taxon, based on phylogenetic analyses and morphological differences. Phyllosticta paracitricarpa differs from P. citricarpa in having longer and slightly narrower conidiophores, larger conidiogenous cells and conidia. Phyllosticta paracitricarpa colonies on MEA appear yellow becoming leaden-grey in the centre, and yellow at the margin, different from P. citricarpa colonies that are olivaceous grey.

Mating type identification of P. citricarpa

The Phyllosticta mating type primer sets were successful in amplifying the respective portions of the MAT1-1-1 or the MAT1-2-1 idiomorphs of the 21 P. citricarpa isolates tested (Table 2). The primer pair MAT111F3–MAT111R3 amplified a fragment of approximately 606 bp in eight isolates, and the primer pair MAT121F6–MAT121R6 amplified 692-bp-fragments in the remaining 13 isolates.

The 20 P. citricarpa isolates from four localities in three countries (Malta, Italy and Portugal) were regarded as four “putative” populations (due to the low number of isolates obtained and the sampling strategy employed) and were genotyped with the 15 SSR markers. Among the 20 isolates that were analysed, only two MLGs were identified. The two populations from Malta and the population from Italy shared a single MLG; the other MLG was identified in the population from Portugal. None of the 15 SSR markers were polymorphic in the populations from Italy, Malta and Portugal and therefore indicated very low gene diversity in the populations (0.000; results not shown). The population from Portugal shared its single MLG with all three populations from Australia, while the populations from Italy and Malta shared one MLG, which was not shared with any of the populations from Australia, Brazil, China, Portugal, South Africa or the USA. For the AMOVA analyses, the data from the three populations from Italy and Malta were combined as one population (Italy+Malta) as these three populations shared one MLG. Pairwise PhiPT values (Table 6) indicated that the Portugal population was genetically significantly (P ≤ 0.05) differentiated from the China (PhiPT = 0.634; P = 0.001), Italy+Malta (PhiPT = 1.000; P = 0.001), South Africa (PhiPT = 0.311; P = 0.002), and the USA (PhiPT = 1.000; P = 0.001) populations. The Portugal population was not significantly differentiated from the Australia population (PhiPT = 0.000; P = 0.418), and also not from the Brazil population (PhiPT = 0.322; P = 0.155). The Italy+Malta population was significantly (P ≤ 0.05) differentiated from the Australia (PhiPT = 0.258; P = 0.001), China (PhiPT = 0.651; P = 0.002), South Africa (PhiPT = 0.365; P = 0.002), Brazil (PhiPT = 0.483; P = 0.043), the USA (PhiPT = 1.000; P = 0.001) and Portugal (PhiPT = 1.000; P = 0.001) populations.

Table 6

Pairwise PhiPT values (below the diagonal) averaged over 15 microsatellite loci of Phyllosticta citricarpa populations from Australia, Brazil, China, Italy+Malta, Portugal, South Africa and the United States. Significance P-values are indicated above the diagonal.

	Australia	Brazil	China	Italy + Malta	Portugal	South Africa	USA
Australia	–	0.011	0.001	0.001	0.418	0.001	0.422
Brazil	0.097	–	0.001	0.043	0.155	0.313	0.342
China	0.649	0.659	–	0.002	0.001	0.001	0.001
Italy + Malta	0.258	0.483	0.651	–	0.001	0.002	0.001
Portugal	0.000	0.322	0.634	1.000	–	0.002	0.001
South Africa	0.165	0.013	0.700	0.365	0.311	–	0.452
USA	0.000	0.013	0.674	1.000	1.000	0.000	–

After 25 d, some inoculation points (approx. 75 %) showed atypical lesions. The lesions developed only on fruits inoculated with P. citricarpa (CPC 27909, CPC 27913) and P. paracitricarpa isolates (CPC 27169, CPC 27170). No lesions were observed on fruits inoculated with P. capitalensis (CPC 27825, CPC 27917), P. paracapitalensis (CPC 26517, CPC 26700) (Fig. 6), or on control fruits (not shown). The lesions caused by P. citricarpa and P. paracitricarpa were similar (Fig. 6). The latter species were consistently re-isolated from the fruit lesions, albeit from lesions atypical of the CBS disease, and identified by sequencing and comparing the loci tef1 and LSU.

Fig. 6

Fruit of Citrus sinensis (‘Valencia’) artificially inoculated with Phyllosticta spp. A. Lesions caused by P. citricarpa. B. Lesions caused by P. paracitricarpa. C, D. No symptoms were observed on fruits inoculated with P. capitalensis and P. paracapitalensis.

Discussion

Phylogenetic studies published on the genus Phyllosticta in recent years have substantially reshaped its taxonomy (Glienke et al., 2011, Wang et al., 2012, Wikee et al., 2013a). The present study represents the first results of fresh collections of several Phyllosticta isolates and species associated with citrus in Europe, and the first DNA sequence analyses of strains from almost all continents.

Phyllosticta capitalensis has been recorded worldwide as a common endophyte of diverse host plants (Baayen ). Phyllosticta citricarpa is confined to Citrus species on which it causes CBS in summer rainfall citrus growing areas in several countries. Despite the fact that these two species are morphologically distinct, their identification has often been confused (Everett & Rees-George 2006). Conidia of P. citricarpa (11–12 × 7 μm) are similar to those of P. capitalensis (11–12 × 6–7 μm), but have a thinner mucoid sheath. Moreover, P. citricarpa strains produce a distinct yellow pigment on OA, and are slower growing than P. capitalensis. The most recent studies focussing on the taxonomy of Phyllosticta species showed the occurrence of additional species associated with Citrus. Glienke described P. citribraziliensis from healthy leaves. An additional three species were reported as Citrus pathogens in Asia: P. citriasiana and P. citrimaxima cause Citrus Tan Spot on pomelo fruits (Wulandari et al., 2009, Wikee et al., 2013a) and P. citrichinaensis causes a brown spot and red-brown protuberant freckle on citrus leaves and fruits (Wang ).

Citrus Black Spot and symptoms similar to that caused by P. citricarpa, P. citriasiana, P. citrimaxima and P. citrichinaensis have never been reported in citrus-producing European countries (European Union, 1998, Kotzé, 2000). Climatic conditions play a primary role in the ability of P. citricarpa to establish and to cause CBS disease, most notably warm summer rainfall conditions that would allow spore production, dissemination and infection during periods of fruit susceptibility (Kiely, 1948a, Kiely, 1948b, Kotzé, 1963, Kotzé, 1981, McOnie, 1967, McOnie, 1964, Huang and Chang, 1972, Lee and Huang, 1973, Noronha, 2002, Fourie et al., 2013, Yonow et al., 2013, Magarey et al., 2015).

Given the long history of trade in citrus propagation material between Europe and Asia, where CBS is endemic and also regarded as the centre of origin of citrus, (Ramón-Laca, 2003, Mabberley, 2004, Nicolosi, 2007), and the potential for illegal movement of plant propagating material, the likely coincidental spread of citrus-specific Phyllosticta species to Europe could reasonably be expected. To investigate this possibility, several surveys were carried out during this study, resulting in the collection of 64 Phyllosticta isolates. A subset of 52 European isolates were compared to several reference isolates using partial gene sequences of six different loci, as well as morphological characteristics. Based on a comparison with sequences retrieved from GenBank of an additional 43 strains (Glienke et al., 2011, Wang et al., 2012, Wikee et al., 2013a), four distinct Phyllosticta species, including two new species, were delineated from several Citrus species growing in five European countries.

The distribution of the Phyllosticta species isolated in this study varied in terms of host and tissue type from which they were recovered. Phyllosticta capitalensis was recovered in all countries sampled and P. paracapitalensis in Italy and Spain only. Both species were isolated from asymptomatic leaves. Phyllosticta citricarpa and P. paracitricarpa were isolated from leaf litter only. Phyllosticta citricarpa was found in Italy, Malta and Portugal, whereas P. paracitricarpa was isolated only from samples collected in Greece. Phyllosticta capitalensis was associated with P. paracapitalensis in the same specimens collected in Spain, but in this survey P. citricarpa and P. paracitricarpa were not found associated with P. capitalensis.

Wang reported two sub-clades (I and II) of P. citricarpa associated with Citrus spp. in China by comparison of ITS, actA and tef1 sequences data. In this study, we used partial regions of an additional three loci, and fixed nucleotide differences were observed within the tef1 and LSU regions, supporting the splitting of the “P. citricarpa” clade in two taxa: P. citricarpa s.str. and the new species P. paracitricarpa. Moreover, this study establishes the presence of P. paracitricarpa only in Asia and Europe and represents the first report of P. citricarpa in Europe. Phyllosticta paracitricarpa was isolated from fruit lesions in China and caused lesions on citrus fruit in the pathogenicity test performed in this study. Further surveys and research is required to determine the importance of P. paracitricarpa as a citrus pathogen.

The origin of P. citricarpa in Europe is not clear at present. On a genotypic level, the P. citricarpa populations from Italy+Malta and Portugal represented two respective clones, differing from each other in both their MLGs and mating types. These populations further differed from one another in their degree of connectivity and differentiation from the other populations from Australia, Brazil, China, South Africa and the USA. Analysis of molecular variance showed that populations from Portugal and Australia are more strongly connected to each other than to other populations. Interestingly, “Lisbon” lemon was introduced into Australia from Portugal in 1824 (Morton 1987), while CBS was first described in Australia in 1895 (Benson 1895). Very little connectivity was evident between the Portuguese population and those from the other continents, including the population from Italy+Malta. Also, the Italy+Malta population seemed to be distinct from the other populations. These findings suggest two separate introductions into Europe. However, in order to determine whether there were other introductions of P. citricarpa into Europe and to infer the origin of these introductions, additional populations from Europe, Asia and the Oceania countries need to be studied. The description of P. paracitricarpa from Greece and China suggests connectivity in this species with Asia.

No evidence of CBS disease in European citrus trees was observed in this study. The P. citricarpa isolates were found in leaf litter of old C. limon and C. sinensis trees (20 to 60 years old) that were situated in gardens, and not found in any of the commercial orchards or nurseries surveyed. Fruit is not considered a pathway for spread (USDA APHIS 2010) and evidence that might suggest a fruit pathway (such as nearby compost heap, waste disposal or processing plants; Baker ) was not observed. Movement of infected plant material is regarded as the most likely means of long-distance spread of P. citricarpa (Kiely, 1948b, Kotzé, 1981). Whilst import of citrus plants for planting is presently not permitted, unless it is plant propagation material that is handled through appropriate quarantine procedures, the introduction of P. citricarpa found in Portugal, Malta and Italy therefore most likely occurred via the introduction of plants many years ago or via illegal movement of such plants.

Phyllosticta citricarpa was found at very low frequency only in a few of the sites investigated, while P. paracitricarpa was found only at one site in Greece. CBS disease symptoms were never observed. Our results indicate that the presence of P. citricarpa and P. paracitricarpa is not associated with disease under European climatic conditions.

Twenty-three P. capitalensis strains were isolated as endophyte from leaves of four Citrus species collected. This taxon can occur in fruit or leaf lesions caused by other fungi or insects (Wikee ). Indeed, in this study, P. capitalensis was found associated with leaf lesions (caused by insects) of the ornamental C. medica var. sarcodactylis. Wikee indicated that the phylogeny of Phyllosticta derived from the ITS and actA genomic loci is sufficiently robust to differentiate most taxa, except those closely related to P. capitalensis. In our study, sequences of a partial region of rpb2 of Phyllosticta spp. helped to resolve differences in nucleotides within P. capitalensis. Moreover, fixed nucleotide differences were observed in tef1, demonstrating the separation of the new species P. paracapitalensis with highly supported independent lineages in the phylogenetic tree. Phyllosticta paracapitalensis was isolated as endophyte from commercial orchards of C. limon in Spain and from C. floridana cultivated in ornamental plant nurseries in Italy. One strain (CBS 173.77) isolated from C. aurantiifolia in New Zealand during February 1974, previously identified as P. capitalensis, grouped with the European isolates of P. paracapitalensis in the present phylogenetic analyses. Further studies must be conducted on a wider global selection of strains to clarify its host association and distribution.

Morphological characteristics of isolates grown on several media were consistent with those already reported in literature (Baayen et al., 2002, Glienke et al., 2011, Wikee et al., 2013a). Optimal temperatures for P. citricarpa (27.2 °C) and P. capitalensis (27.3 °C) predicted from the BETE function fitted to the relative growth data were similar to those reported by previous studies (Kotzé, 1981, Er et al., 2014), but cardinal temperatures were more contracted with Tmin of (12.5 and 9.4 °C, respectively). Optimal temperatures for P. paracitricarpa and P. paracapitalensis were lower (26.4 °C) and higher (28.6 °C), respectively. Growth rates of P. capitalensis and P. paracapitalensis were similar and significantly faster than those of P. citricarpa and P. paracitricarpa.

Results of this study showed that two (P. citricarpa and P. paracitricarpa) of the four species isolated from specimens collected in Europe induced atypical lesions (necrosis) in artificially inoculated mature sweet orange fruit and could be re-isolated from these lesions, while P. capitalensis and P. paracapitalensis induced no lesions. From this assay, it appears that P. paracapitalensis is similar to P. capitalensis, demonstrating them to have similar ecologies, occurring as asymptomatic endophytes in citrus tissue. Considering that mature citrus fruit are resistant to P. citricarpa infection under field conditions (Kiely, 1948b, Schutte et al., 2003, Schutte et al., 2012, Miles et al., 2004), and since the harsh artificial inoculation technique used in the pathogenicity assay did not resemble natural field infection (i.e. direct penetration of unwounded tissue following long wetness periods; Kotzé, 1963, McOnie, 1967, Noronha, 2002) these findings should be regarded as preliminary. Phyllosticta paracitricarpa caused similar lesions to those caused by P. citricarpa in this assay and appears to be pathogenic, which is supported by its isolation from lesions on fruit in China, but further surveys are required to elucidate.

Including the two taxa newly described in this study, eight Phyllosticta species are now associated with citrus: P. citricarpa and P. capitalensis are present on all continents where citrus is cultivated, P. paracapitalensis is reported in Europe and New Zealand, while P. paracitricarpa is present in Asia and Europe. As previously published by several authors, the pathogenic P. citrichinaensis, P. citriasiana and P. citrimaxima are present only in Asia, and the endophyte P. citribraziliensis has been isolated only in South America (Wulandari et al., 2009, Glienke et al., 2011, Wang et al., 2012, Wikee et al., 2013a). The presence in Europe of both P. citricarpa and P. paracitricarpa was not associated with any visible signs of infection; indeed, neither CBS or Citrus Tan Spot have ever been reported or observed during the extensive surveys performed in the present study.

Recent studies performed in Florida, USA (Zhang et al., 2015, Wang et al., 2016), supported the heterotallism of P. citricarpa, finding only MAT1-2-1 isolates present in Florida (based on 113 isolates) while 26 strains from Australia displayed an equal ratio of the two mating types. Amorim recently showed that in Brazil the two idiomorphs occur in a 1:1 ratio. Furthermore, Tran reported for the first time the successful mating in vitro of P. citricarpa, confirming that this species is heterothallic and requires isolates of different MAT idiomorphs to be in direct physical contact for mating and production of sexual fruiting bodies. We found both MAT1-1-1 and MAT1-2-1 isolates present in Europe, but both mating types were not recovered together in the same country, indicating separate introductions that have not spread and remained isolated. A broader sampling is required, however, to determine whether this holds up when a larger population per area is sampled.

This study contributed significantly towards our understanding of the genotypic variation in P. capitalensis and P. citricarpa, splitting both groups into different taxa, and clearly showing that a multi-locus approach works well for distinguishing these species. The use of a three-gene analysis (ITS, actA, tef1) performed in a previous study (Wang ) showed two poorly supported subclades within P. citricarpa. We used a further three genomic loci (gapdh, LSU and rpb2) to confirm that the two subclades actually represent two distinct species.

In this study we established the presence of P. citricarpa and the similar new species, P. paracitricarpa, for the first time in Europe. In spite of the occurrence of these species, neither was associated with disease symptoms, evidently because of unfavourable climatic conditions (Yonow et al., 2013, Magarey et al., 2015). Whilst it appears that these fungi were introduced with plant material many years ago, they apparently persist under these unfavourable conditions, most likely endophytically, and possibly through asexual reproduction. The latter hypothesis is supported by the finding that only one mating type was found per locality, and that some P. citricarpa pycnidiospore infection events were predicted to occur in these regions (Magarey ). The number of suitable infection periods was, however, markedly fewer than those for regions where P. citricarpa causes CBS disease. Magarey doubted the ability of P. citricarpa to persist and cause disease at a location where there is a low frequency of suitable seasons. Likewise, the climate suitability modelling conducted by Paul and Yonow , indicated climatic unsuitability across the EU, with the exception of a few isolated areas around the Mediterranean Sea, where marginally suitable climatic conditions can be found. All these climate modelling studies were calibrated for climate suitability according to the presence, absence, distribution and severity of CBS disease, and not the potential presence of the fungus in the absence of disease. The findings from our study indicate that P. citricarpa was able to persist but did not induce CBS symptoms or spread, considering that it was found in only a few of the sites investigated and at very low frequency.