Caliciopsismoriondi, a new species for a fungus long confused with the pine pathogen C.pinea

Abstract The genus Caliciopsis (Eurotiomycetes, Coryneliales) includes saprobic and plant pathogenic species. Caliciopsis canker is caused by Caliciopsispinea Peck, a species first reported in the 19th century in North America. In recent years, increasing numbers of outbreaks of Caliciopsis canker have been reported on different Pinus spp. in the eastern USA. In Europe, the disease has only occasionally been reported causing cankers, mostly on Pinusradiata in stressed plantations. The aim of this study was to clarify the taxonomy of Caliciopsis specimens collected from infected Pinus spp. in Europe and North America using an integrative approach, combining morphology and phylogenetic analyses of three loci. The pathogenicity of the fungus was also considered. Two distinct groups were evident, based on morphology and multilocus phylogenetic analyses. These represent the known pathogen Caliciopsispinea that occurs in North America and a morphologically similar, but phylogenetically distinct, species described here as Caliciopsismoriondisp. nov., found in Europe and at least one location in eastern North America. Caliciopsismoriondi differs from C.pinea in various morphological features including the length of the ascomata, as well as their distribution on the stromata.

Caliciopsis canker has been reported as an emerging disease of Pinus in the eastern USA (Munck et al. 2015(Munck et al. , 2016Costanza et al. 2018;Haines et al. 2018;Schulz et al. 2018a, b;Whitney et al. 2018) and is caused by the fungus Caliciopsis pinea. The pathogen gives rise to cankers and abundant resin bleeding on branches and stems of young and mature Pinus strobus trees, which can lead to crown wilting and defoliation and, in some cases, killing significant portions of the tree crowns. In the USA, C. pinea has been known since at least 1920 (Fitzpatrick 1920). It was considered "not uncommon" in eastern North America on P. strobus and on various conifer species in western North America (Ray 1936). After the accidental introduction of white pine blister rust (Cronartium ribicola J.C. Fisch.) into the USA in the early 1900s, Caliciopsis canker was ignored.
Caliciopsis pinea is considered native to North America (Harrison 2009). In Europe, Lanier (1965) reported this species for the first time from France on P. halepensis, P. insignis, P. nigra and P. pinaster, although the pathogen had been known in the region since the late 1800s (Rehm 1896). The disease was also reported in Italy (Capretti 1978(Capretti , 1980 on several different Pinus species, both native and non-native. While all of these reports of Caliciopsis canker have been attributed to C. pinea, a recent study by Munck et al. (2015) suggested that a closely related, but distinct lineage of the fungus was present on P. nigra and P. radiata in Italy. Recently, an extensive survey of pine plantations in central-south Italy revealed several P. radiata stands showing crown yellowing, die-back and profuse resin production on the stems and shoots associated with depressed cankers. Fungal fruiting bodies resembling those of Caliciopsis in all stages of development were found on the cankered tissues (N. Luchi unpublished data).
No comprensive phylogenetic study has been undertaken on Caliciopsis spp. associated with cankers on Pinus spp. including both Europe and North America. Given the findings of Munck et al. (2015), it is possible that a distinct species of Caliciopsis is present in Europe. Furthermore, this fungus could have a host range, ecology and epidemiology different to those of its North American congener. The aim of this study was to compare pine-infecting Caliciopsis isolates from Europe and North America, based on morphological features and phylogenetic inference and to determine whether these represent a single or more than one species.

Sampling and isolation of fungal strains
Isolates used in this study were obtained from a number of surveys of Caliciopsis canker on native and non-native Pinus spp. in plantations and naturally regenerated eastern white pine stands growing in different areas of Europe (EU) and North America (NA). Isolates from NA were obtained from Georgia, North Carolina, Tennessee and Virginia (this study) and Maine, Massachusetts, New Hampshire in the USA (Munck et al. 2015). Isolates from EU were obtained from France, Italy and Spain (Table 1).
Samples from Italy were obtained from pine trees with Caliciopsis canker symptoms from three different locations in Tuscany (Central Italy). Five shoots with Caliciopsis canker from five different trees were collected at each of the three Italian sites. Other isolates from Pinus and other host species used in this study included Caliciopsis pinea LSVN1233 (from France, supplied by Dr. R. Ioos), C. pinea SP 1 (from Spain, supplied by Dr. P. Capretti), C. pinea CBS 139.64 (from Canada), C. orientalis CBS 138.64 (from Canada) and C. pseudotsugae CBS 140.64 (from Canada). All isolates are maintained in the culture collections of the Institute for Sustainable Plant Protection -National Research Council (IPSP-CNR, Italy) and the Department of Bioagricultural Sciences, Colorado State University.
Samples were placed in paper bags and transferred to the laboratory for isolation. Pine twigs (5 cm long; 0.5 to 1 cm diameter) were surface disinfested with 75% ethanol (1 min) and 3% sodium hypochorite (NaOCl) (3 min), after which they were rinsed three times in sterile water. A sterile scalpel was used to remove the outer portions. Necrotic tissues were cut in small pieces and placed in 90-mm Petri dishes containing 1.5% Potato Dextrose Agar (PDA, DIFCO, Detroit, Michigan, USA), amended with streptomycin (0.050 g/l). All plates were incubated in the dark at 20 °C for 10-15 days. Fungal colonies with a morphology resembling C. pinea were transferred to fresh plates to obtain pure cultures.

Morphology and culture characteristics
Caliciopsis fruiting bodies on cankered bark of the Italian specimens were mounted on glass slides in 80% lactic acid, amended with bromothymol blue and examined using a  dissection microscope (SMZ800, Nikon, Japan). The length and width of 50 released ascospores and 30 fruiting bodies were measured under a light microscope (Axioskop 50 Zeiss, Germany) and images captured with a Nikon Digital Sight DS-5M camera (Nikon Instruments Software-Elements Basic Research). The means and range dimensions of fruiting bodies and ascospores were compared with those reported in literature ( Table 2). The morphology of fungal colonies was determined for cultures grown for four weeks on 1.5% Malt Extract Agar (MEA, DIFCO, Detroit, Michigan, USA) and 1.5% PDA.

Daily growth rate in culture
Six-mm diameter mycelial plugs were taken from the margins of actively growing seven-day-old colonies on PDA, using a flame-sterilised cork borer and were placed at the centres of 90-mm Petri dishes containing 1.5% PDA or 1.5% MEA. Five replicates were used for each of five selected strains (IT6, IT9, IT16, IT17, IT22) at each temperature. Dishes were incubated at 4 °C, 15 °C and 20 °C. Two measurements of colony diameter perpendicular to each other were made at 3, 7, 14, 21 and 28 days and daily growth rate was calculated as an average for each strain on each substrate. Data were analysed using a factorial ANOVA, considering temperature and substrate as the independent factor and daily growth rate as the dependent factor. For the phylogenetic analyses, partial regions of three loci were amplified. Amplification of the internal transcribed spacer ITS region (including spacers ITS1 and ITS2 and the 5.8S gene of the rDNA) was done using primers ITS1 and ITS4 (White et al. 1990), of β-tubulin 1 (Bt1) gene using primers Bt2a and Bt2b (Glass and Donaldson 1995) and of translation elongation factor 1-α (EF1-α) gene using primers EF1-983F and EF-gr (Rehner and Buckley 2005). PCR reaction mixtures (25 µl) contained 1 µl of genomic DNA, 2.5 µl of each 10 µM primer, 0.5 µl of 10 mM dNTPs (GeneSpin, Milan, Italy), 2.5 µl of 10X PCR Buffer (GeneSpin), 3 µl of 25 nM MgCl and 0.5 µl (5 U/µl) of Taq polymerase (TaqPol, GeneSpin). Amplifications were carried out in a Mastercycler (Eppendorf, Hamburg, Germany) using the following PCR conditions: for ITS and Bt1: initial denaturation at 95 °C for 5 min; followed by 35 cycles of denaturation at 94 °C for 90 sec, annealing at 56 °C for 1 min and extention at 72 °C for 2 min; and a final elongation step at 72 °C for 10 min. For EF1-α: initial denaturation at 95 °C for 8 min; followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec and extention at 72 °C for 1 min; and a final elongation step at 72 °C for 5 min (modified from Pepori et al. 2015). Amplification products were separated by electrophoresis on gels containing 1% (w/v) of agarose LE (GeneSpin). The approximate length (bp) of the amplification products was determined using the 100-bp DNA ladder Ready to Load (Genespin). PCR amplicons were purified with a miPCR Purification Kit (Metabion International, Planegg, Germany) and sequenced in both directions at Macrogen (Seoul, South Korea). The quality of amplified nucleotide sequences was checked with the software package Geneious version 10.0.9 (Biomatters, Auckland, New Zealand). Generated sequences were submitted to NCBI GenBank (Table 1).

Multi-locus phylogenetic analyses
BLAST searches of the generated sequences were done against the NCBI GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the most closely-related sequences. Sequences were compared to those of known Caliciopsis species and other Coryneliaceae obtained from GenBank: ITS sequences of Lagenulopsis bispora (KP881709); EF1-α sequence of Caliciopsis pinea (DQ677937). Lagenulopsis bispora (KP881709) was selected as outgroup in the ITS dataset, whereas C. orientalis (CBS 138.64) and C. pseudotsugae (CBS 140.64) were selected as outgroup taxa in the EF1-α and Bt1 datasets. The software package Geneious (Auckland, New Zealand) was used for manual optimisation and alignment (ClustalW) of the sequences. Gaps were treated as missing data.
Phylogenetic analyses of all obtained sequences were performed using MEGA 7 (Kumar et al. 2016), Maximum Parsimony (MP) was performed using the heuristic research option with random stepwise addition with 1000 replicates, tree bisection and reconnection (TBR) as branch swapping algorithm and random taxon addition of sequences for the construction of MP trees. All characters were weighted equally and character state transitions were treated as unordered. Parameters measured for parsimony included tree length (TL), consistency index (CI), rescaled consistency index (RC), retention index (RI) and homoplasy index (HI). Bootstrap support values were evaluated using 5000 bootstrap replicates (Hills and Bull 1993).
Datasets were also analysed by Bayesian Inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003), with a General Time Reversible (GTR) model and gamma distributed rate variation across sites. Six Markov Chain Monte Carlo (MCMC) chains (Larget and Simon 1999) were run for 3 million generations, starting from a random tree and using the default temperature. Every 100 th tree was sampled and the first 100,000 generations were discarded as burn-in. The burn-in period was determined after testing for stationarity of likelihood values, that is, by plotting numbers of generation versus the log probability and checking for the convergent diagnostic PSRF, which approached 1 (Ronquist et al. 2005). The consensus tree was calculated in MrBayes with the command sumt (Ronquist et al. 2005). The resulting phylogenetic tree was visualised using TreeView (Page 1996) and edited in TreeGraph2 (Stöver and Müller 2010).

Inoculation tests
An inoculation experiment was carried out at the IPSP-CNR nursery facilities, located at Antella, Province of Florence, Italy (43°43'N, 11°22'E; 170 m a.s.l.). Three-yearold seedlings of Pinus halepensis, P. pinaster and P. pinea, with 36 plants per species, were planted in a randomised block design. The plants were maintained in rows 1 m apart and grown in a substrate comprised of commercially-produced loam and drip irrigated. The site had been completely cleared and ploughed prior to planting and was weeded each month.
Inoculations were performed in June 2014. A 6-mm diameter cork borer was used to remove the bark and expose the cambium on each plant. A plug of mycelium of the test fungus that had been grown in Petri dishes on 1.5% PDA for 20 days at 25 °C in the dark was inserted, with the mycelium side placed downwards into each wound. For inoculations, four different Italian Caliciopsis isolates (IT5, IT7, IT20 and IT22), recovered from infected Pinus sp. in the field, were used (Table 1). The inoculation trial was performed using eight trees per isolate on each of the Pinus spp. Four plants for each Pinus sp. were mock-inoculated using sterile PDA as controls.
Pathogenicity was assessed, based on the length of lesions (mm) after six months. Statistical analyses were performed by using Statistica 10.0 (StatSoft Inc. 1984-2011. To fulfil Kock's postulates, re-isolations were carried out from the lesions on all the inoculated and control plants.

Morphology and culture characteristics
Fruiting bodies on bark taken from infected trees were black ascomata assembled in tufts with ascigerous swellings at the apices containing ascospores. The Italian specimens had different morphological characteristics from those reported in literature for Caliciopsis pinea (Table 2, Figures 1, 2). Colonies of the Italian strains grown on MEA were white, appressed to the agar surface, turning to brown with time.

DNA sequence analysis
The final combined ITS-EF1-α -Bt1 data matrix of Caliciopsis included 53 ingroup and 2 outgroup sequences (length = 137, CI = 0.9444, RI = 0.99633, RC = 0.98178, HI = 0.940979) (Figure 3), comprising 1611 alignment characters, including gaps. Of these, 1434 characters were constant and 112 characters were parsimony informative ( Figure 3). Single ITS, EF1-α and Bt1 datasets included, respectively, 458, 762 and 373 characters (Figure 4, Suppl. material 1: Figure S1 and Suppl. material 2: Figure S2) Phylogenetic analysis, resulting in the most parsimonious tree from the concatenated dataset, showed that isolates, previously identified as Caliciopsis pinea, based on morphology, grouped in two different clades. One of these clades (Clade I) included most of the US strains and the C. pinea isolate CBS 139.64. The other clade (Clade II) included all EU isolates and three US strains from P. resinosa from a single location in New Hampshire (US64, US65, US66). Maximum Parsimony and Bayesian Inference produced nearly identical topologies for all single locus datasets: ITS, which included different species of Caliciopsis and other Coryneliaceae (Corynelia africana, C. fructigena, C. uberata  Figure S1 and Suppl. material 2: Figure S2).
Across the three loci sequenced, there were 31 fixed polymorphisms separating Clade I from Clade II. Of these, 12 were in the ITS region, 11 in EF1-α and 7 in Bt1 ( Figure 5). The USA isolates US64, US65 and US66 shared the same fixed polymorphisms present in Clade II samples. However, samples US64 together with isolates SP1, LSVN1233 and IT17 did not have the insertion in position 459 of ITS, which was one of the fixed polymorphisms in Clade II samples. Ten fixed polymorphisms were specific to Clade I. Of these, two were in the ITS, two in the EF1-α and six in the Bt1, where three in position 105, 122 and 128 were in common with isolates in Clade II. Fixed, unique polymorphisms were identified in all three loci, which produced congruent trees from the individual loci that separated Clade I (C. pinea) from Clade II isolates, suggesting that the latter isolates represent a novel species different from C. pinea. Description. Stromata developing beneath the surface of host periderm as small, more or less circular structures, giving little external evidence of their presence at early stages. Continued growth causing the bark to break and the minute cushion-shaped  stromata, developing a lobed appearance and increasing in diameter and thickness, in black short-stalked columnar ascomata. Ascomata mostly frequent protruding at the margin of cankers, single or double, rarely triple, stalks not branched, (0.45) 0.84 ± 0.02 (1.2) mm high and (51) 79 ± 2 (135) µm width. Ascigerous swelling, terminal, (106) 281 ± 8 (406) µm high and (81) 142 ± 5 (268) µm diameter, forming a brownish pulverulent mass of extruded ascospores. Asci bitunicate, clavate, 8-spored, slightly curved, pedicellate, (26) 37 ± 6 (53) µm long; pedicel 1-3 µm diameter; sporiferous part (12) 13 ± 0.4 (14.2) µm long and (5.3) 6.3 ± 0.4 (7.4) µm wide. Ascospores yellow-green colour, sub-globose to ellipsoidal and often aggregated in small masses, (3) 4.4 ± 0.07 (6.2) µm long and (1.8) 2.5 ± 0.04 (3.5) µm wide, brown when mature. Spermogonia sub-globose, papillate, sessile, aggregated below ascomatal tubes. Conidia unicellular, hyaline, smooth, slightly fusiform.
Culture characteristics. Cultures incubated on 2% PDA, showed optimal temperature for growth at 20 °C, with slow-growth rate (1.4 mm/day). Colonies white appressed to the agar, circular to irregular, becoming fawn-colored with age, areas towards margin floccose; mycelium velutinous with funicolose areas or strongly funicolose in the inner and older parts of the mycelium. Reverse colony brownish, with brown diffusion zone in old cultures; branching septate hyphae, with frequent anastomoses and tips with dendroid branching.

NucleoƟdes variaƟons In Sb Sb Sb Sb Sb Sb Sb Sb Sb In In In In In Sb Sb
Sb Sb Sb Sb Sb Sb Sb Sb Sb Sb Sb Sb Sb Sb Sb Sb but all had profuse resin production at the inoculation points. Caliciopsis moriondi fruiting bodies were clearly visible on P. halepensis, while no fructifications were seen in any of the other inoculated Pinus species.
Hosts and distribution. Pathogen of pine trees P. nigra, P. radiata and P. resinosa, causing cankers and resin production in Europe (France, Italy, Spain) and North America (New Hampshire, USA).
Etymology. The name moriondi honours Prof. Francesco Moriondo (1926Moriondo ( -2014. Francesco Moriondo was the founder of forest pathology as a discipline distinct from plant pathology in Italy. In this respect, he preferred a more ecological view of the topic as opposed to the typical mechanistic approach. During his career, he encouraged many young students to consider the reasons for the appearance of symptoms on trees, rather than considering only the causative agents. He also emphasised the importance of minor pathogens in the ecosystem, of which Caliciopsis moriondi (then C. pinea) was one.
Notes. Caliciopsis moriondi is commonly associated with a canker disease on Pinus spp. It differs subtly from C. pinea, based on morphological traits, including shorter ascomata, protruding and isolated from the stroma, rarely in groups of two-three, but never in more numerous groups, such as is common for C. pinea (Table 2).

Discussion
This study included a large number of isolates previously believed to be Caliciopsis pinea. Analysis of DNA sequences of the ITS, Bt1 and EF1-α regions clearly showed that these isolates represented two distinct taxa. One of these represented C. pinea and the other an undescribed species, which we have formally described here as C. moriondi.
Caliciopsis moriondi can be distinguished from C. pinea based on various morphological features including the length of the ascomata, as well as by their distribution on the stromata. In the absence of sequence data, previous authors confused isolates obtained in Europe with C. pinea, which was originally described from North America by Fitzpatrick (1920). Caliciopsis moriondi as the fungus is now known, has been found in Italy, France and Spain, mainly on P. radiata trees and, on one occasion, on P. nigra. Based on the wide sampling in this study, it appears likely that C. pinea does not occur in Europe. Delatour (1969) described Caliciopsis pinea as a weak pathogen by basing his assessment on inoculations of P. pinaster in France. The results of the present study suggest that it is more likely that this author was working with C. moriondi. This view is supported by the illustrations of C. pinea by Lanier (1965) showing ascomata very similar to those of C. moriondi.
Caliciopsis moriondi was able to cause only mild symptoms when inoculated on Mediterranean Pinus spp. in pathogenicity trials. The symptoms were most noticeable on P. halepensis and less severe on P. pinaster and P. pinea, confirming the results of Delatour (1969). Caliciopsis moriondi was able to produce ascocarps when inoculated on P. halepensis, but not on P. pinaster and P. pinea.
Interestingly, the European isolates of Caliciopsis moriondi were mainly found on Pinus radiata. Our inoculation tests, as well as those of Delatour (1969), suggest that the non-native P. radiata is more susceptible than Mediterranean Pinus spp. Unfortunately, P. radiata and P. nigra plants were not available when this pathogenicity test was undertaken. A further inoculation experiment on these two Pinus spp., which are widely planted in Europe, will be necessary in order to assess their susceptibility to C. moriondi.
The results of this study suggest that Caliciopsis moriondi is native to Europe. This is supported by the fact that it caused only mild symptoms on artificially inoculated European Pinus spp. Yet on naturally infected non-native P. radiata, it gave rise to symptoms similar to those caused by the pitch canker pathogen Fusarium circinatum, which is an important quarantine pathogen in Europe and also commonly found on P. radiata (Capretti et al. 2013). Future studies will be necessary to determine whether infections on these trees are caused by F. circinatum or C. moriondi and the duplex real-time PCR assay developed and validated by Luchi et al. (2018) should be useful in this regard.
Caliciopsis moriondi and C. pinea are two vicariant species and it appears that the European C. moriondi has been accidentally introduced in North America. We hypothesise that this might have occurred at the end of the 1800s when European nurseries produced large volumes of Pinus spp. for the establishment of North American plantations (Maloy 1997). Caliciopsis moriondi could easily have moved on infected, but asymptomatic, seedlings at that stage.
The results of this study suggest that Caliciopsis pinea is not present in Europe. Its pathogenicity on European pines has never been assessed. Since the beginning of the present century, there has been a renewed interest in this species due to the damage it causes to the plantations of P. strobus in the north-eastern United States (Munck et al. 2015(Munck et al. , 2016). An accidental introduction of this species into Europe could pose a threat to European pine plantations and natural forests. Consequently, it will be important to assess the susceptibility of European Pinus spp. to this pathogen and to prepare an ad hoc pest risk assessment for it. This study has received funding from the project "Holistic management of emerging forest pests and diseases" (HOMED) a European Union's Horizon 2020 Programme for Research & Innovation under grant agreement No 771271.