Pestalotiopsis pini sp. nov., an Emerging Pathogen on Stone Pine ( Pinus pinea L.)

: Research Highlights : Pestalotiopsis pini sp. nov. is an emerging pathogen on stone pine, Pinus pinea L., in Portugal. Background and Objectives : Stone pine is one of the most important forest tree species in Portugal and in the whole Mediterranean basin. Pestalotiopsis species are common endophytes, saprobes or pathogens in a variety of hosts and environments. The objective of the present study was to identify the Pestalotiopsis species associated with the symptomatic stone pine trees. Materials and Methods : Samples of stone pine trees showing shoot blight and stem necrosis were obtained from stone pine orchards and urban areas in Portugal, and the isolated Pestalotiopsis species were identiﬁed based on morphology and combined ITS, TEF and TUB DNA sequence data. Artiﬁcial inoculations on one-year-old stone pine seedlings were performed with the two species most frequently found in association with shoot blight disease. Results : Five Pestalotiopsis spp. were isolated. A taxonomic novelty, Pestalotiopsis pini is described, representing a new pathogen for stone pine. Conclusions: Pestalotiopsis species may represent a threat to the health of pine forests in the Mediterranean basin. Future research should be done in order to increase our knowledge about the potential impact of pestalotioid species in stone pine, in order to develop management strategies against these pathogens.


Introduction
Stone pine, Pinus pinea L., is one of the most important forestry species in Portugal and the Mediterranean basin. Stone pine forests play an important role in the economy of the areas where they are planted, especially due to the high value of edible pine nuts, which are the main resource of this industry [1]. Pinus pinea is broadly considered a robust species. In recent years, pine nut production has been decreasing due to several factors, including pests and diseases [1,2].
Pestalotiopsis is a widely distributed genus of appendage-bearing conidia belonging to the family Sporocadaceae [3]. Fungi within this genus are normally considered secondary pathogens that can be responsible for a variety of plant diseases, including cankers, dieback, leaf spots, needle blight, tip blight,

Fungal Isolation
Isolates were obtained from samples of Pinus pinea showing shoot blight, trunk necrosis, needle blight and pine cone decay. A sample of Pinus pinaster Aiton with shoot blight was also analysed. After macro-and microscopic observation of the sampled material, small pieces from the leading edge of the lesions were surface sterilized for 1 min in 1% NaClO and plated onto potato dextrose agar (PDA) amended with 0.5 mg/mL of streptomycin sulphate in order to avoid bacterial growth. Materials were incubated for seven days with a 12 h light period at 23 ± 2 • C. The hyphal tips of fungi emerging from tissue pieces were transferred to PDA, and single-spore cultures were subsequently established. Fungal isolates were deposited in the culture collection of INIAV Institute (Micoteca da Estação Agronómica Nacional (MEAN)) ( Table 1). Table 1. Details of Pestalotiopsis isolates obtained in this study (bold) and of strains representing species of Pestalotiopsis and related genera retrieved from GenBank and used in phylogenetic analyses.

Species
Collection No. 1

Morphology
Colony morphology was observed after 7 days of cultivation on PDA at 23 ± 2 • C at 12 h daylight. Conidiomatal development was observed on Synthetic Nutrient-poor Agar (SNA) by cultivating the isolates on autoclaved pine needles placed on the surface of SNA. Colony colour was determined on PDA using the colour charts of Rayner [19]. Conidia and conidiogenous cells were mounted in distilled water, and at least 30 measurements per structure were recorded at 400× magnification under a compound light microscope (Olympus BX51, Olympus Corporation, Tokyo, Japan) using the program Olympus DP-Soft, or under a Nikon Eclipse 80i compound microscope with differential interference contrast (DIC) illumination, equipped with a Nikon DS-Ri2 high definition colour digital camera.

DNA Extraction, PCR Amplification and Sequencing
Genomic DNA was extracted using the "DNA, RNA and Protein Purification-NucleoSpin Plant II" (Macherey-Nagel-MN) following the manufacturer's instructions. Fresh mycelium was disrupted by vortexing with approximately 200 µL glass beads (450-600 µm diameter) added to the extraction buffer [20].
All PCR reactions were performed in a 25 µL reaction containing DNA template (diluted 10×), 10× PCR reaction buffer, 3 mM MgCL 2 , 0.5 mM of each deoxyribonucleotide triphosphate, 1 U of Taq DNA Polymerase, (BioTaqTM DNA Polymerase-Bioline, London, UK) and 2 µM of each primer, for ITS and TUB amplification, or 6 µM of each primer, for TEF amplification.
PCR reactions were performed in a Biometra TGradient thermo cycler (Biometra, Göttingen, Germany) with the following thermal cycling conditions, for ITS: initial denaturation at 94 • C for 3 min, followed by 30 cycles consisting of denaturation at 94 • C for 30 s, annealing at 55 • C for 30 s and extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min; for TEF: initial denaturation at 94 • C for 8 min, followed by 35 cycles consisting of denaturation at 94 • C for 15 s, annealing at 55 • C for 20 s and extension at 72 • C for 1 min, and a final extension at 72 • C for 5 min; and for TUB: initial denaturation at 94 • C for 1 min, followed by 30 cycles consisting of denaturation at 94 • C for 1 min, annealing at 55 • C for 1 min and extension at 72 • C for 1 min, and a final extension at 72 • C for 5 min.
PCR products were sequenced in both directions at STABVida Sequencing Laboratory (Caparica, Portugal) on an ABI PRISM 3730xl DNA analyser (Applied Bio systems) using the same primers as those used for the amplification reactions. The resulting nucleotide sequences were edited using the programs FinchTV version 1.4.0 (Geospisa Inc.) and BioEdit version 7.2.6 [25], and a consensus sequence was made from the forward and reverse sequences. Sequences obtained in this study were deposited in GenBank (see Table 1).

Phylogenetic Analyses
A BLAST engine search was used for sequence similarity searching on GenBank (NCBI-National Centre for Biotechnology Information). Based on blast search results and the literature, additional sequences were selected from GenBank and incorporated in the analyses (Table 1). Sequence alignments of the three individual loci (ITS, TEF, TUB) were made using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/ server/index.html), and were then manually edited using BioEdit version 7.2.6. Single gene datasets were combined using SequenceMatrix [26].
Phylogenetic analyses of the combined three-locus sequence dataset comprised Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI).
MP analysis was performed using Phylogenetic Analysis Using Parsimony (PAUP) v. 4.0b10 [29]. Gaps were treated as missing data. Trees were inferred using heuristic search with random stepwise addition and tree-bisection reconnection (TBR). Maxtrees were set to 10,000 and branches of zero length were collapsed. Bootstrap support values with 1000 replications [30] were calculated for tree branches. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for trees generated under different optimality criteria.
BI was performed by using the Markov Chain Monte Carlo method (MCMC) with MrBayes v. 3.2.6 [31]. JModelTest2 on XSEDE [32], implemented via the CIPRES portal, was used to determine the best-fit nucleotide substitution model for each partition using the Akaike Information Criterion (AIC) [33]. The GTR + I + G model was selected as the most suitable for ITS and TUB data partitions, and the GTR + G model was selected for TEF data partition. Four MCMC chains were run simultaneously, starting from random trees for 1,000,000 generations. Trees were sampled every 100 generations for a total of 10,000 trees. The burn-in fraction was set to 0.25, after which posterior probabilities were determined from a majority-rule consensus tree [34].

Pathogenicity Tests
Two isolates representing the most common Pestalotiopsis species isolated from stone pine trees with shoot blight disease in this study were selected to perform the pathogenicity tests: MEAN1095-Pestalotiopsis pini sp. nov. and MEAN1096-Pestalotiopsis australis Maharachch., K.D. Hyde & Crous.
To carry out the pathogenicity tests, 93 one-year-old stone pine seedlings were sourced from a nursery, where they were cultivated from seeds of a certified orchard. For each isolate and for the control treatment, 31 seedlings were randomly chosen and distributed along a plastic cell pack (6 × 11 plastic cells container). Each plastic cell pack with plants was randomly located in the greenhouse test area. The plants were then acclimatized during one month under greenhouse conditions, with temperatures varying from 18 to 28 • C, watered as needed (circa 2 L per plastic cell pack container, twice a week).
Spore suspensions of each isolate were prepared from cultures on PDA, grown at 25 ± 1 • C for 14 days (four plates/isolate). Sterile deionized water was added to the cultures and spores were dislodged by a sterile glass rod. The spore suspensions were resuspended in sterile deionized water and concentration adjusted to 1 × 10 5 conidia mL −1 with a haemocytometer.
The inoculations were performed by two combined methods. First, the stems were damaged by gently piercing them with a dissection needle that was previously dipped into the spore solution, while, in the control, the stems were pierced with a sterile needle. Five to six wounds were made per plant, approximately 3 cm apart from each other, in the upper third of the stem. Secondly, based on Talgø et al. [35], some needles were removed from plants, and the injured area subsequently brushed with the spore suspension. Sterile water was used in the control. Each container was covered with a plastic bag and maintained for one week to enhance fungal development.
The seedlings were kept in the greenhouse for four months (18 July to 17 November 2017). At the end of the experiment, the number of affected plants was noted, and in order to attest Koch's postulates, re-isolations of fungi were carried out from the disease margins of three symptomatic seedlings, following the methodology described in Section 2.1.

Fungal Isolation and Identification
Among other fungi, a total of 18 pestalotiopsis-like colonies were observed. After morphological observation and ITS sequence analyses, five isolates were identified as belonging to Heterotruncatella and 13 to Pestalotiopsis. Further molecular studies were performed to identify the Pestalotiopsis species isolated.

Phylogenetic Analyses of Combined ITS, TEF and TUB Sequences
To determine the phylogenetic position of the Pestalotiopsis isolates, phylogenetic analyses were performed based on the combined ITS, TEF, and TUB sequence data. The combined alignment contained sequences from 104 strains (including two outgroups) with 1427 characters (including alignment gaps), divided in three partitions with 494 (ITS), 491 (TEF) and 442 (TUB) characters; 417 of these were parsimony-informative, 151 were variable and parsimony-uninformative, and 859 were constant. The combined Pestalotiopsis dataset was analysed using ML, MP and BI (Figure 1). The phylograms from the three analyses showed similar results in topology, and hence the best scoring tree resulting from ML analyses, with a final likelihood value of −10,646.254559, is shown in Figure 1 Etymology: Named after the host genus from which it was isolated, Pinus. Host/Distribution: On needles, shoots and trunks of Pinus pinea and on Pinus pinaster in Portugal (this study). Seen on Pinus radiata in Chile and on Pinus sp. in the USA also [3].

Pathogenicity
Two isolates, representing the most common Pestalotiopsis species isolated from pine trees with shoot blight disease in the present study, were submitted to pathogenicity tests by artificial inoculation on stone pine seedlings: MEAN1095-Pestalotiopsis pini sp. nov. and MEAN1096-Pestalotiopsis australis.
The development of disease symptoms was observed during a four-month period. Initial symptoms started after four weeks on seedlings inoculated with the Pe. pini isolate. Seedlings started to show yellowish and wilted needles in the apical third of the trunk. By the end of the experiment, symptomatic plants exhibited a dried apex in the inoculated branch/trunk (Figure 3). In total, 19.4% (6/31) of the plants inoculated with Pe. pini isolate MEAN 1095 were symptomatic. No symptoms were observed on the control treatment, nor in plants inoculated with Pe. australis isolate MEAN 1096. Pestalotiopsis pini was successfully re-isolated from the three symptomatic plants sampled, thus fulfilling Koch's postulates and confirming its pathogenicity to stone pine.  [12,36]. They are clearly separated in the phylogram based on combined ITS, TEF, and TUB sequence data, Pe. pini isolates formed a separate clade with strong support values on the three analyses performed (ML, MP and BI), (see Figure 1).

Pathogenicity
Two isolates, representing the most common Pestalotiopsis species isolated from pine trees with shoot blight disease in the present study, were submitted to pathogenicity tests by artificial inoculation on stone pine seedlings: MEAN1095-Pestalotiopsis pini sp. nov. and MEAN1096-Pestalotiopsis australis.
The development of disease symptoms was observed during a four-month period. Initial symptoms started after four weeks on seedlings inoculated with the Pe. pini isolate. Seedlings started to show yellowish and wilted needles in the apical third of the trunk. By the end of the experiment, symptomatic plants exhibited a dried apex in the inoculated branch/trunk (Figure 3). In total, 19.4% (6/31) of the plants inoculated with Pe. pini isolate MEAN 1095 were symptomatic. No symptoms were observed on the control treatment, nor in plants inoculated with Pe. australis isolate MEAN 1096. Pestalotiopsis pini was successfully re-isolated from the three symptomatic plants sampled, thus fulfilling Koch's postulates and confirming its pathogenicity to stone pine.

Discussion
In the present study Pestalotiopsis pini is described as a new species causing shoot blight and stem necrosis on Pinus pinea. Based on the morphology and molecular phylogenetic analyses of combined ITS, TEF and TUB sequence data, this taxon proved distinct from other species known from pine, or from DNA sequence data. Four other species of Pestalotiopsis were identified in association with symptomatic stone pines, namely, Pe. australis, Pe. biciliata, Pe. disseminata and Pe. hollandica.
Pestalotiopsis pini isolates obtained in this study (MEAN 1095, MEAN 1092, MEAN 1094, MEAN 1167) were grouped along with two unclassified Pestalotiopsis sp. strains included in the revision of Sporocadaceae, performed by Liu et al. [3], namely CBS 110326 and CBS 127.80. In the latter study, the authors retained these two isolates as an "informal species" "Pestalotiopsis sp.7 FL-2019", due to the lack of more isolates and limited phylogenetic support. In our phylogenetic analyses (Figure 1), these two strains were grouped with the four isolates obtained in this study, forming a separate clade with strong support values in all the phylogenetic analyses performed (MLBS = 100%, MPBS = 99%, BIPP = 1.00).
In the present study, Pe. pini was isolated from blighted shoots of P. pinea and P. pinaster trees in pine plantations, and from the necrotic wood of a decayed stone pine trunk located in Monsanto Forest Park in Lisbon. Pathogenicity tests performed confirmed that Pe. pini is pathogenic to stone pine. Furthermore, in the Monsanto Forest Park, various stone pine trees exhibited the same symptoms, and no other potential pathogens were isolated along with Pe. pini, suggesting that this could be a primary pathogen for this host. Interestingly, despite Pestalotiopsis species generally not being regarded as host-specific and normally being found on a wide range of plants and substrates [9], the two Pe. pini strains included in the study of Liu et al. [3] were also isolated in pines-Pinus sp. in the USA (CBS 110326) and Pinus radiata D. Don. in Chile (CBS 127.80)-although no information about the health of these pine trees is available.
In this study, Pestalotiopsis australis was isolated from blighted stone pine shoots in P. pinea orchards. This is the first report of Pe. australis isolated from conifers and in Europe. Under the conditions of the trials, no symptom development occurred in any of the inoculated seedlings,

Discussion
In the present study Pestalotiopsis pini is described as a new species causing shoot blight and stem necrosis on Pinus pinea. Based on the morphology and molecular phylogenetic analyses of combined ITS, TEF and TUB sequence data, this taxon proved distinct from other species known from pine, or from DNA sequence data. Four other species of Pestalotiopsis were identified in association with symptomatic stone pines, namely, Pe. australis, Pe. biciliata, Pe. disseminata and Pe. hollandica.
Pestalotiopsis pini isolates obtained in this study (MEAN 1095, MEAN 1092, MEAN 1094, MEAN 1167) were grouped along with two unclassified Pestalotiopsis sp. strains included in the revision of Sporocadaceae, performed by Liu et al. [3], namely CBS 110326 and CBS 127.80. In the latter study, the authors retained these two isolates as an "informal species" "Pestalotiopsis sp.7 FL-2019", due to the lack of more isolates and limited phylogenetic support. In our phylogenetic analyses (Figure 1), these two strains were grouped with the four isolates obtained in this study, forming a separate clade with strong support values in all the phylogenetic analyses performed (MLBS = 100%, MPBS = 99%, BIPP = 1.00).
In the present study, Pe. pini was isolated from blighted shoots of P. pinea and P. pinaster trees in pine plantations, and from the necrotic wood of a decayed stone pine trunk located in Monsanto Forest Park in Lisbon. Pathogenicity tests performed confirmed that Pe. pini is pathogenic to stone pine. Furthermore, in the Monsanto Forest Park, various stone pine trees exhibited the same symptoms, and no other potential pathogens were isolated along with Pe. pini, suggesting that this could be a primary pathogen for this host. Interestingly, despite Pestalotiopsis species generally not being regarded as host-specific and normally being found on a wide range of plants and substrates [9], the two Pe. pini strains included in the study of Liu et al. [3] were also isolated in pines-Pinus sp. in the USA (CBS 110326) and Pinus radiata D. Don. in Chile (CBS 127.80)-although no information about the health of these pine trees is available.
In this study, Pestalotiopsis australis was isolated from blighted stone pine shoots in P. pinea orchards. This is the first report of Pe. australis isolated from conifers and in Europe. Under the conditions of the trials, no symptom development occurred in any of the inoculated seedlings, suggesting that Pe. australis may behave as an endophyte on stone pine. Pestalotiopsis australis has been reported from Proteaceae hosts, it was isolated from Grevillea sp. in Australia and South Africa, and from Protea neriifolia × susannae cv. 'Pink Ice' and dead leaves of Brabejum stellatifolium L. in South Africa [3,9].
Pestalotiopsis hollandica was isolated from the blighted shoots of stone pine trees in stone pine orchards. Pestalotiopsis hollandica was first described from Sciadopityaceae (Sciadopitys verticillata (Thunb.) Siebold & Zucc.) in the Netherlands [9] and it has already been isolated from conifers in Spain, namely from Cupressus sempervirens L. (Cupressaceae) [37]. Isolate MEAN 1091 was closely related to the reference strain of Pe. hollandica. However, Pe. hollandica was not well resolved from Hyde, suggesting that these isolates may represent a single species, as suggested by Liu et al. [3]. Some of those species' names have also been associated with conifers in the past [9,38].
Pestalotiopsis biciliata was isolated from a dry conelet (1st year) from a stone pine orchard. This species was first described by Maharachchikumbura et al. [9], isolated from dry needles of Taxus baccata L. in the Netherlands, from Paeonia sp. in Italy and from Platanus × hispanica in Slovakia. Pe. biciliata was also isolated from dry needles of Taxus baccata in the UK [3]. The fungus was referred to as the causal agent of fruit rot on withered grapes in Italy [8], and is associated with grapevine trunk diseases in France [10]. Recently Pe. biciliata was also reported as a foliar pathogen of Eucalyptus spp. [11].
Pestalotiopsis disseminata was isolated from blighted shoots of stone pine trees in a stone pine orchard. Pe. disseminata was first described from Eucalyptus botryoides Sm. in Portugal [39], and has already been isolated from a wide range of hosts and locations worldwide [3,15,18,40], including the genus Pinus [15,16,18]. It was isolated as an endophyte from Pinus armandii in China, along with 18 other pestalotioid species [16]; from Pinus parviflora Siebold & Zucc. var. pentaphylla (Mayr) in Japan [18] and from seeds of P. pinea in Turkey, Pinus elliottii Engel., Pinus patula Schltdl & Cham, P. radiata, Pinus taeda L. in the USA and P. pinaster in Portugal [15].
Isolates identified in this study were associated with symptomatic stone pine trees with shoot blight, trunk necrosis and pinecone decay in Portugal. At least one of the five identified species, Pestalotiopsis pini sp. nov., is pathogenic to stone pine. In recent years, various species of Pestalotiopsis have been described [3,4,7,9,10], with many being associated with plant diseases and shown to be pathogenic to their host under certain biotic and abiotic conditions [4,5,8,11,41,42].
The symptoms observed in stone pine orchards in Portugal, in particular shoot blight disease, might be of special concern to the forest industry, since dry shoots in the tree canopy could lead to a decrease in pinecone development and pine nut production, which is the most profitable resource of this industry [1,2].
Shoot blight disease on stone pine and other pine species is normally associated with Diplodia sapinea (Fr.) Fuckel [43,44], and has recently also been associated with Sydowia polyspora (Bref. & Tavel) E. Müller [45]. In the present study, various Pestalotiopsis species were isolated from stone pine samples with similar symptoms, moreover, Pe. pini proved to be pathogenic on stone pine, causing dry shoots on artificially inoculated seedlings, thus suggesting that Pe. pini should also have an active role in the expression of shoot blight disease on stone pine. The fact that in the pathogenicity tests, Pe. pini only caused disease symptoms in approximately 20% of the inoculated seedlings may indicate relative host resistance due to genetic differences among the seedlings. Alternatively, the development of shoot blight disease is due to more than one factor, biotic or abiotic. In fact, D. sapinea, S. polyspora and other fungi were also present in some of the sampled symptomatic material (data not shown). Diverse authors also report more than one species involved in dieback and blight diseases, including pestalotioid species and other fungi [8,[45][46][47] and observed that some abiotic factors also have a major role in disease development, namely water stress and air temperature [41,42,47]. In this case, a synergic effect among Pe. pini and other pathogenic or endophytic fungi found in stone pine shoots may also trigger the development of shoot blight disease symptoms. Future research should be performed to evaluate shoot blight disease prevalence on P. pinea orchards in Portugal and other Mediterranean areas and the diverse biotic and abiotic agents that can be involved in disease development.
The present study represents a preliminary contribution of the Pestalotiopsis species diversity associated with shoot blight disease of stone pine in Portugal. Knowledge of Pestalotiopsis species associated with shoot blight and other pine diseases will provide a basis to better understand disease development and help to develop management strategies against these pathogens.

Conclusions
A novel fungal species, Pestalotiopsis pini was described. This study proves that Pe. pini is an emerging pathogen causing shoot blight and trunk necrosis on Pinus pinea in the Mediterranean area.
To our knowledge, this is also the first report of Pe. australis on conifers and in Europe, and of Pe. hollandica and Pe. biciliata on Pinus spp. and in Portugal. Information about Pestalotiopsis species associated with shoot blight and other diseases on pine species will help to provide a basis for a better understanding of disease development, and the development of management strategies against these pathogens. Funding: This research was funded by Instituto Nacional de Investigação Agrária e Veterinária, I.P. and by the Portuguese program PDR2020 (Programa de Desenvolvimento Rural 2014-2020) financed by the European Agricultural Fund for Rural Development (EAFRD), under the project "Grupo Operacional + Pinhão-Gestão integrada de agentes bióticos associados à perda de produção de pinhão" (PDR2020-101-031187). Article Processing Charges were supported by Laboratório de Patologia Vegetal Veríssimo de Almeida, Instituto Superior de Agronomia.