Pestalotiopsis jiangsuensis sp. nov. Causing Needle Blight on Pinus massoniana in China

Pinus massoniana Lamb. is an important, common afforestation and timber tree species in China. Species of Pestalotiopsis are well-known pathogens of needle blight. In this study, the five representative strains were isolated from needle blight from needles of Pi. massoniana in Nanjing, Jiangsu, China. Based on multi-locus phylogenetic analyses of the three genomic loci (ITS, TEF1, and TUB2), in conjunction with morphological characteristics, a new species, namely Pestalotiopsis jiangsuensis sp. nov., was described and reported. Pathogenicity tests revealed that the five representative strains of the species described above were pathogenic to Pi. massoniana. The study revealed the diversity of pathogenic species of needle blight on Pi. massoniana. This is the first report of needle blight caused by P. jiangsuensis on Pi. massoniana in China and worldwide. This provides useful information for future research on management strategies of this disease.


Introduction
Pinus massoniana Lamb. is the most widely distributed timber tree species with the largest afforestation area in China [1], which provides a large amount of timber, oleoresin [2], carbon storage [3], and ecological products [4], and also has potential biomedical properties [5].However, Pi. massoniana was found dead at the top of needles in plantations in Nanjing, Jiangsu Province with a high incidence, which seriously threatened the economic and ecological value.
Many pathogens have been reported to damage Pi. massoniana in the world; for example, its forestry and pine forests were threatened by outbreaks of pine wilt disease (PWD) caused by Bursaphelenchus xylophilus (pinewood nematode; PWN) [6].Damping-off and root rot disease caused by Fusarium oxysporum has been found in Pi. massoniana [7,8].Pseudofusicoccum kimberleyense and Pse.violaceum can cause dead branch disease of Pi. massoniana [9].Pestalotiopsis funerea affected the needles of young Pi.massoniana trees and caused them to gradually dry up and fall off [10].In addition, insect-parasitic entomopathogenic fungi such as Penicillium citrinum, Purpurecillium lilacinum, and Fusarium spp.were also confirmed to be pathogenic to Pi. massoniana [11].However, as an important economic tree species, the host-pathogen relationship of Pi. massoniana needs more studies, and additional pathogens may be found.
Pestalotiopsis species are widely distributed in the world as endophytes, plant pathogens, or saprobes [12][13][14][15][16][17], mainly in tropical and temperate regions and have a wide range of host plants [15,18,19].Initially, the characteristics of conidia, such as color, size, and appendages, are the key to the identification of Pestalotiopsis and related genera [20,21].Those taxonomic groups related to the genus Pestalotiopsis are also called pestalotioid fungi.
Needle blight caused by Pestalotiopsis is a common disease in young pine forests, and the disease is widely distributed and causes serious damage.For example, Pestalotiopsis funerea can infect Pinus tabulaeformis [27], Pi. taeda [28], Pi. massoniana [10], etc. and cause needle blight.Xu et al. (2017) [29] reported that the pathogen causing the needle blight of Pi. sylvestris was P. citrina.The disease began to occur in 1974 and became popular in 1980, and it has become the main coniferous disease of trees [30,31].Needle blight not only reduced the stock of trees but even led to the death of trees, which greatly threatened forestry production [32][33][34].
In March 2023, the needles of Pi. massoniana with the characteristics of needle blight were collected in Nanjing, Jiangsu Province.The earlier identification of Pi. massoniana needle blight in a previous study was in a different geographical area [10]; thus, the main purpose of this study was to determine the pathogen of Pi. massoniana needle blight and its pathogenicity by Koch's postulates.

Field Survey and Fungal Isolation
In March 2023, needle lesions were found on Pinus massoniana in Lishui District, Nanjing, Jiangsu, China.The entire planting area of the Pi.massoniana forest was about 1800 m 2 .The symptoms of trees were visually observed and the needles with the symptoms were collected.Five symptomatic Pi. massoniana trees were randomly sampled.After macroscopic and microscopic observation of the collected pine needles, the pine needle fragments at the intermediate area of the diseased and healthy portions were cut off, and the surface was disinfected in 70% ethanol for 30 s, in 1% NaClO for 90 s, and then washed in sterile water for 90 s three times.Pine needle fragments were dried on sterile filter paper and incubated on potato dextrose agar (PDA) in the dark at 25 • C for 3 days.The hyphal tips of fungi emerging from tissue pieces were transferred to new PDA to obtain pure cultures.The isolates were obtained from needle blight samples of Pi. massoniana.

Morphological Identification
Colony morphology and pigment production on PDA was observed after 7 days at 25 • C with a 12/12 h light/dark cycle and inspected daily for fungal sporulation.Acervuli and conidial masses were observed under a Zeiss stereo microscope (SteRo Discovery v20, Oberkochen, Germany).The micromorphological characteristics of five isolates were observed by Zeiss Axio Imager A2m microscope (Carl Zeiss, Oberkochen, Germany), such as shape, color, septation, appendages, and size of conidia, conidiophores, and acervuli.

Genealogical Concordance Phylogenetic Species Recognition Analyses
The phylogenetically related ambiguous species were analyzed using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) to determine the recombination level in closely related species by performing a pairwise homoplasy index (PHI) test according to the method described by Quaedvlieg et al. (2014) [42].A PHI result below 0.05 (Φw < 0.05) indicated significant recombination in the dataset.The relationships between closely related species were visualized in splits graphs with the LogDet transformation and splits decomposition options.

Pathogenicity Test
In this study, 12 two-year-old healthy Pi.massoniana seedlings and the three isolates representing the highest isolation frequency of Pestalotiopsis species were selected to perform the pathogenicity tests: BM 1-1, BM 1-2, BM 1-3-Pestalotiopsis jiangsuensis sp.nov.The tested plants were taken from the GuDong Green Seedling Base in Hechi, Guangxi Province, China.Healthy needles of Pi. massoniana were injured with a sterile needle.One wound was made per pine needle and conidial suspension (10 6 conidia•mL −1 ) was sprayed on the wounds.Three plants were inoculated with each isolate, and the control was treated with sterile water.Inoculated seedlings and control seedlings were placed in a tent (1.5 × 1.2 × 1.5 m) with a humidifier (300 mL/h) to maintain RH 70%.The tent was placed in a greenhouse at 25 ± 2 • C and observed continuously for 10 days.All experiments were conducted three times.

Disease Symptoms and Fungal Isolation
In March 2023, the incidence of needle blight of Pi. massoniana found in Nanjing, Jiangsu Province was ca.60%, and the needle disease incidence of each Pi.massoniana was as high as 80%.The early symptom was a small yellow lesion at the needle tip, which extended from the needle tip downwards, and the lesion turned gray; a dark brown band encircled the needle at the junction with the healthy part (Figure 1A-C).Eventually the lesion area expanded until all the needles were necrotic.Ninety Pestalotiopsis strains were isolated and determined, based on the colony morphologies on PDA and ITS sequence blasting, with an isolation frequency of 90% (90/100).Five representative isolates (BM 1-1, BM 1-2, BM 1-3, BM 1-4, and BM 1-5) were selected for further study and deposited at the China Forestry Culture Collection Center (CFCC).

Disease Symptoms and Fungal Isolation
In March 2023, the incidence of needle blight of Pi. massoniana found in Nanjing, Jiangsu Province was ca.60%, and the needle disease incidence of each Pi.massoniana was as high as 80%.The early symptom was a small yellow lesion at the needle tip, which extended from the needle tip downwards, and the lesion turned gray; a dark brown band encircled the needle at the junction with the healthy part (Figure 1A-C).Eventually the lesion area expanded until all the needles were necrotic.Ninety Pestalotiopsis strains were isolated and determined, based on the colony morphologies on PDA and ITS sequence blasting, with an isolation frequency of 90% (90/100).Five representative isolates (BM 1-1, BM 1-2, BM 1-3, BM 1-4, and BM 1-5) were selected for further study and deposited at the China Forestry Culture Collection Center (CFCC).

Phylogenetic Analyses
The concatenated sequence dataset of ITS, TEF1, and TUB2 included the five representative isolates, 120 taxa, and one outgroup taxon (Neopestalotiopsis protearum CBS 114178) with a total of 1637 base pairs (1-554 for the TEF1, 555-1163 for ITS, and 1164-1637 for TUB2) including gaps were obtained.The hosts, locations, and GenBank accession numbers of Pestalotiopsis species used for phylogenetic analyses in this study were shown in Table 2.The tree topology of the phylogenetic tree of ML and BI systems was congruent, and the bootstrap support values of RA × ML greater than 70% and the Bayesian posterior probabilities greater than 0.90 were denoted at nodes.In the phylogenetic analyses, five isolates formed a separate clade (ML/BI = 100/1), which was clustered into a big branch with four ex-type strains with a significant support (ML/BI = 98/0.92:Pestalotiopsis foliicola CFCC 54440, P. pinicola KUMCC 19-0183, P. suae CGMCC 3.23546, and P. rosea MFLUCC 12-0258.Based on the three-locus phylogenetic analyses and morphology, five strains (BM 1-1, BM 1-2, BM 1-3, BM 1-4, and BM 1-5) were identified as a new species of Pestalotiopsis (Figure 2).

Phylogenetic Analyses
The concatenated sequence dataset of ITS, TEF1, and TUB2 included the five representative isolates, 120 taxa, and one outgroup taxon (Neopestalotiopsis protearum CBS 114178) with a total of 1637 base pairs (1-554 for the TEF1, 555-1163 for ITS, and 1164-1637 for TUB2) including gaps were obtained.The hosts, locations, and GenBank accession numbers of Pestalotiopsis species used for phylogenetic analyses in this study were shown in Table 2.The tree topology of the phylogenetic tree of ML and BI systems was congruent, and the bootstrap support values of RA × ML greater than 70% and the Bayesian posterior probabilities greater than 0.90 were denoted at nodes.In the phylogenetic analyses, five isolates formed a separate clade (ML/BI = 100/1), which was clustered into a big branch with four ex-type strains with a significant support (ML/BI = 98/0.92:Pestalotiopsis foliicola CFCC 54440, P. pinicola KUMCC 19-0183, P. suae CGMCC 3.23546, and P. rosea MFLUCC 12-0258.Based on the three-locus phylogenetic analyses and morphology, five strains (BM 1-1, BM 1-2, BM 1-3, BM 1-4, and BM 1-5) were identified as a new species of Pestalotiopsis (Figure 2).Importantly, the PHI test of new species shows that no significant recombination (Φw = 0.071) events were observed between Pestalotiopsis sp.(undescribed taxon) and phylogenetically related species P. foliicola CFCC 54440, P. pinicola KUMCC 19-0183, P. suae CGMCC 3.23546, and P. rosea MFLUCC 12-0258 (Figure 3).Importantly, the PHI test of new species shows that no significant recombination (Φw = 0.071) events were observed between Pestalotiopsis sp.(undescribed taxon) and phylogenetically related species P. foliicola CFCC 54440, P. pinicola KUMCC 19-0183, P. suae CGMCC 3.23546, and P. rosea MFLUCC 12-0258 (Figure 3).Notes: Pestalotiopsis jiangsuensis is a species often having one to four tubular apical appendages, which are phylogenetically and morphologically well distinguished from P. foliicola CFCC 54440, P. pinicola KUMCC 19-0183, P. suae CGMCC 3.23546, and P. rosea MFLUCC 12-0258.Although the five strains studied are a sister clade of P. foliicola CFCC 54440, P. pinicola KUMCC 19-0183, P. suae CGMCC 3.23546, and P. rosea MFLUCC 12-0258, the number of apical appendages is quite different.Pestalotiopsis folicola, P. pinicola and P. suae have two to three apical appendages; P. rosea has one to three tubular apical appendages, and some appendages are branched.The strains in this study have one to four apical appendages, and the appendages are unbranched.Notes: Pestalotiopsis jiangsuensis is a species often having one to four tubular apical appendages, which are phylogenetically and morphologically well distinguished from P. foliicola CFCC 54440, P. pinicola KUMCC 19-0183, P. suae CGMCC 3.23546, and P. rosea MFLUCC 12-0258.Although the five strains studied are a sister clade of P. foliicola CFCC 54440, P. pinicola KUMCC 19-0183, P. suae CGMCC 3.23546, and P. rosea MFLUCC 12-0258, the number of apical appendages is quite different.Pestalotiopsis folicola, P. pinicola and P. suae have two to three apical appendages; P. rosea has one to three tubular apical appendages, and some appendages are branched.The strains in this study have one to four apical appendages, and the appendages are unbranched.

Pathogenicity Test
In the experiment of Koch's postulates, the three representative isolates were pathogenic to Pi. massoniana needles.The development of disease symptoms was observed during a 10-day period.At 5 d, all the Pestalotiopsis jiangsuensis isolates developed gray to graybrown lesions on wounded needles of Pi. massoniana (Figure 5B-D).At 10 d, the lesion expanded, and in severe cases, the whole needle was necrotic (Figure 5F-H).No symptoms developed on the needles of the control (Figure 5A,E).In this study, the pathogenicity of Pestalotiopsis jiangsuensis is strong; for example, the lesions spread almost to the whole needle after 10 days.It may also relate to its high isolation rate.Pestalotiopsis jiangsuensis was successfully re-isolated from 100% of the inoculated plants and identified based on morphological features and phylogenetic analysis of ITS.Thus, Koch's postulates had been fulfilled.
In the experiment of Koch's postulates, the three representative isolates were pathogenic to Pi. massoniana needles.The development of disease symptoms was observed during a 10-day period.At 5 d, all the Pestalotiopsis jiangsuensis isolates developed gray to gray-brown lesions on wounded needles of Pi. massoniana (Figure 5B-D).At 10 d, the lesion expanded, and in severe cases, the whole needle was necrotic (Figure 5F-H).No symptoms developed on the needles of the control (Figure 5A,E).In this study, the pathogenicity of Pestalotiopsis jiangsuensis is strong; for example, the lesions spread almost to the whole needle after 10 days.It may also relate to its high isolation rate.Pestalotiopsis jiangsuensis was successfully re-isolated from 100% of the inoculated plants and identified based on morphological features and phylogenetic analysis of ITS.Thus, Koch's postulates had been fulfilled.

Discussion
Pestalotiopsis was established by Steyeart (1949) [45] and typified with Pestalotiopsis guepinii Steyaert.Pestalotiopsis sensu lato was classified based on conidia with five-celled, the middle three intermediate colored cells, and hyaline end cells.After that, its taxonomic characteristics gradually changed into conidia spindle-shaped, with five-celled, with colorless or nearly colorless cells at both ends, dark cells in the middle, and one or more branched or unbranched apical appendages arising from the apical cell, with or without basal stalk [20,21,46,47].The excessive overlap of conidia makes it difficult to identify Pestalotioid species only by morphological characteristics [19].Although some additional taxonomic features can also be used as the basis for the identification of Pestalotiopsis-such as the pigmentation of median cells, which is an important character to distinguish Pestalotiopsis funerea and P. triseta [23,48]-there are still great limitations [17,22,49].However, the application of molecular data in the identification of Pestalotiopsis species has greatly improved the accuracy and credibility [22,23,26,50,51].Pestalotiopsis sensu lato was

Discussion
Pestalotiopsis was established by Steyeart (1949) [45] and typified with Pestalotiopsis guepinii Steyaert.Pestalotiopsis sensu lato was classified based on conidia with five-celled, the middle three intermediate colored cells, and hyaline end cells.After that, its taxonomic characteristics gradually changed into conidia spindle-shaped, with five-celled, with colorless or nearly colorless cells at both ends, dark cells in the middle, and one or more branched or unbranched apical appendages arising from the apical cell, with or without basal stalk [20,21,46,47].The excessive overlap of conidia makes it difficult to identify Pestalotioid species only by morphological characteristics [19].Although some additional taxonomic features can also be used as the basis for the identification of Pestalotiopsissuch as the pigmentation of median cells, which is an important character to distinguish Pestalotiopsis funerea and P. triseta [23,48]-there are still great limitations [17,22,49].However, the application of molecular data in the identification of Pestalotiopsis species has greatly improved the accuracy and credibility [22,23,26,50,51].Pestalotiopsis sensu lato was segregated into three genera by Maharachchikumbura et al. (2014) [15] as Pestalotiopsis sensu stricto, Neopestalotiopsis, and Pseudopestalotiopsis, based on both morphological characteristics and phylogenetic analyses.Gu et al. (2022) [17] identified six new Pestalotiopsis species from Rhododendron, based on phylogenetic analyses of combined ITS, TEF1, and TUB2 genes/region along with morphological characteristics.Maharachchikumbura et al. (2012) [14] identified 23 species of Pestalotiopsis from different host plants in China, including 14 new species, based on phylogenetic analysis of ITS, TEF1, and TUB2 genes/region and morphology.More importantly, concatenating ITS, TUB2, and TEF1 sequences can provide better identification information for Pestalotiopsis [14,52].
The Global Biodiversity Information Facility (https://www.gbif.org/,accessed on 24 November 2023) displays 9320 records of Pestalotiopsis from all over the world, including years and coordinates [53].The data show that most of them are distributed in Australia, Brazil, China, and the United States.Pestalotiopsis as a plant pathogen has a wide range of symptoms on the hosts, such as withering or chlorosis of leaves, dead shoots or tips, and canker [15].In Pinus spp., it may be characterized by shoot blight, trunk necrosis, needle blight, and pinecone decay [54].It is not uncommon that a species of Pestalotiopsis was successfully isolated from needles of Pinus species [34].For example, Pestalotiopsis neglecta and P. citrina isolated from Pi. sylvestris can cause the needles to turn yellow partially or completely and even cause death of the trees [29,34].Pestalotiopsis bessey isolated from Pi. halenpesis can cause the entire needles to turn dark gray-brown and eventually cause the death of the trees [55,56].Pestalotiopsis pini isolated from Pi. Pinea can cause the needles and branches to wither, trunk necrosis, and pinecone rot [54].Pestalotiopsis is also an endophytic fungus of some Pinus spp., such as P. funerea, and it was isolated from the healthy needles of Pi. pinaster [57].
Interestingly, the pathogen of Pi. massoniana needle blight isolated in a previous study was P. funerea [58], but the pathogen obtained in this study was Pestalotiopsis jiangsuensis, which indicated that the pathogens of the same genus on the same host were diverse.Silva et al. [54] isolated P. disseminata and P. pini from Pi. Pinea, and their results also confirmed this view.Similarly, the same species of Pestalotiopsis can be found on different plant hosts, such as P. funereal, which was isolated from Pi. tabulaeformis, Pi. taeda, and Pi.massoniana [10,27,28].Pestalotiopsis chamaeropis was isolated from Quercus sp., Castanopsis sp., and Camellia sp.[15,49,59].However, in the current study the samples were only collected from one site.In future research, the investigation areas should be expanded to study fungal diversity on Pinus spp.and related ecological functions.

Conclusions
In this study, we examined five strains, all of which were pathogenic to Pi. massoniana.Combined with morphology, multi-locus phylogenetic analyses, and GCPSR principle, these five strains were identified to be a new species to science, Pestalotiopsis jiangsuensis.This is the first report of needle blight caused by P. jiangsuensis on Pi. massoniana in China and worldwide, and it will provide useful information for future studies on all the phytopathological perspectives of this fungus and the management strategies of this newly emerged disease.

Figure 1 .
Figure 1.Symptoms of needle blight on Pinus massoniana in the field (A-C).

Figure 1 .
Figure 1.Symptoms of needle blight on Pinus massoniana in the field (A-C).

Table 1 .
Reaction conditions used in PCR amplification and sequencing.

Table 2 .
Host, Origin, and GenBank accession numbers of strains of Pestalotiopsis species used for phylogenetic analyses.

Table 2 .
Cont.Strains isolated from the current study are given in bold.T = ex-type culture.b CFCC = China Forestry Culture Collection Center, China; ICMP = International Collection of Microorganisms from Plants, Auckland, New Zealand; LC = working collection of Lei Cai, housed at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; IFRDCC = International Fungal Research and Development Culture Collection, Kunming, Yunnan China; CGMCC = China General Microbiological Culture Collection Center, Beijing, China; CBS = culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; MFLUCC = Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; CSUFTCC = Central South University of Forestry and Technology Culture Collection, Hunan, China; BRIP = Plant Pathology Herbarium, Department of Employment, a Economic, Development and Innovation, Queensland, Australia; MFLU = Mae Fah Luang University Herbarium, Thailand; HGUP = Plant Pathology Herbarium of Guizhou University, Guizhou, China; HMJAU = Herbarium of Mycology of Jilin Agricultural University, Jilin, China; SAUCC = Shandong Agricultural University Culture Collection, Taian, Shandong, China; NTUCC = The Department of Plant Pathology and Microbiology, National Taiwan University Culture Collection, Taipei, Taiwan (ROC); NOF = The Fungus Culture Collection of the Northern Forestry Centre, Alberta, Canada; E = The "Coleção de culturas de fungos fitopatogênicos Prof. Maria Menezes", Universidade Federal Rural de Pernambuco, Recife, Brazil; CAA = culture collection of Artur Alves, housed at Department of Biology, University of Aveiro, Aveiro, Portugal; KNU = Kyungpook National University, Daegu, South Korea;