Pestalotiopsis jiangsuensis sp. nov. Causing Needle Blight on Pinus massoniana in China
1
College of Forestry and Grassland, Nanjing Forestry University, Nanjing 210037, China
2
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
3
The Connecticut Agricultural Experiment Station Valley Laboratory, Windsor, CT 06095, USA
*
Author to whom correspondence should be addressed.
J. Fungi 2024, 10(3), 230; https://doi.org/10.3390/jof10030230
Submission received: 3 February 2024
/
Revised: 3 March 2024
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Accepted: 19 March 2024
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Published: 21 March 2024
Abstract
: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.
1. 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. Afterwards according to the relationship between conidial morphology and multi-locus phylogeny [14,19,22,23], Pestalotiopsis sensu lato was divided into three genera by Maharachchikumbura et al. (2014) [15]—Pestalotiopsis sensu stricto, Neopestalotiopsis, and Pseudopestalotiopsis. Three genera correspond to three types of conidia, conidia with light brown or olivaceous concolorous median cells (Pestalotiopsis sensu stricto), conidia with versicolorous median cells (Neopestalotiopsis), and conidia with dark-colored concolorous median cells (Pseudopestalotiopsis) [14,19,22,24]. Pestalotioid species identification remains a major challenge because of the conidia of overlap, and the classification is complex [22,25,26].
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.
2. Materials and Methods
2.1. 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 m2. 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.
2.2. 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.
2.3. Genomic DNA Extraction, PCR, and Sequencing
Fungal genomic DNA of fungi cultured on PDA for 5 days was extracted by the cetyltrimethylammonium bromide (CTAB) method, and three distinct DNA regions were amplified by polymerase chain reactions (PCR). Three genomic loci, including the internal transcribed spacer (ITS), the partial translation elongation factor 1-alpha (TEF1), and partial β-tubulin (TUB2), were amplified with primers ITS5/ITS4 [35], EF1-728F/EF1-986R [36], and T1/Bt-2b [37,38], respectively. The protocols for amplification are shown in Table 1. Each 50 μL PCR mixture consisted of 25 μL of Premix TaqTM (Takara Biomedical Technology Company Limited, Beijing, China), 19 μL of dd H2O, 2 μL of forwarding primer, 2 μL of reverse primer, and 2 μL of DNA template. PCR purification and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd. (Nanjing, China).
2.4. Phylogenetic Analyses
Sequences with similarity of the ITS sequences generated in the present study were searched with the BLAST program on GenBank (https://blast.ncbi.nlm.nih.gov/, accessed on 3 November 2023), and the reference sequences used in this study were obtained. Concatenated multi-locus data (ITS, TEF1, and TUB2) were used for phylogenetic analyses with maximum likelihood (ML) and Bayesian Inference (BI). Neopestalotiopsis protearum (CBS 114178) was designated as an outgroup. The DNA sequences were aligned with MAFFT ver. 7.313 [39] and adjusted with BioEdit ver. 7.0.9.0 [40]. Maximum likelihood (ML) analysis was conducted on the multi-locus alignments using IQtree ver. 1.6.8 [41] with the GTR + F + I + G4 replacement model and the bootstrap method with 1000 replications to assess clade stability. RA × ML bootstrap support values were set at ML ≥ 70. Bayesian inference was analyzed using MrBayes ver. 3.2.6 with the GTR + I + G + F model (2 parallel runs, 2,000,000 generations) according to Quaedvlieg et al. (2014) [42]. Bayesian posterior probability values were set at PP ≥ 0.90. The phylogenetic trees were created in Figtree ver. 1.4.4. (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 2 December 2023).
2.5. 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.
2.6. 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 (106 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.
3. Results
3.1. 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).
3.2. 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).
3.3. Taxonomy
Pestalotiopsis jiangsuensis Li-Hua Zhu, Hui Li, and D.W. Li, sp. nov. Figure 4
Index Fungorum No: IF 900494
Etymology: the epithet referring to the province where the holotype was collected.
Description: Sporadic black and gregarious conidiomata produced on PDA after 7 days under light at 25 °C, globose, semi-immersed, dark brown to black, up to 400 μm diam (Figure 4B); conidiophores indistinct and reduced to conidiogenous cells. Conidiogenous cells (4.5-) 7.0–12.8 (−15.3) × (2.4-) 3.3 –5.6 (−6.5) µm (11.4 ± 2.5 × 4.4 ± 0.9 µm, n = 30), hyaline, ampulliform or cylindrical, and sometimes slightly wide at the base (Figure 4C). Conidia phragmospores, (20.3-) 22.1–25.5 (−27.3) × (6.2-) 6.7–8.2 (−8.7) µm (23.4 ± 1.8 × 7.5 ± 0.5 µm, n = 30), fusoid, ellipsoid, straight to slightly curved, 4-septate (Figure 4D); basal cell hyaline, obconic, thin-walled, 3.5–5.9 μm long; three median cells (12.7-) 13.7–15.5 (−16.5) × (6.2-) 6.7–7.4 (−7.9) µm (14.2 ± 1.0 × 7.2 ± 0.5 µm, n = 30), doliiform, wall rugose, concolorous, brown, septa darker than the rest of the cell (second cell from the base 4.2–5.9 μm long; third cell 4.8–5.7 μm long; fourth cell 4.0–5.4 μm long); apical cell hyaline, smooth-walled, conic or trapezoid, tapering toward the apex, 2.6–4.4 μm long, with 1–4 tubular apical appendages (mostly 2 and very few 4), arising from the apical crest, unbranched, filiform, 8.7–23.4 μm long; basal appendage single, tubular, unbranched, centric, 1.4–6.3 μm long.
Culture characteristics: Colonies on PDA flat with sparse aerial mycelia on the surface after 7 d at 25 °C, edge undulate, pale honey-colored, and reverse pale brown in the center and pale luteous margin (Figure 4A).
Holotype: China, Jiangsu province, Nanjing city, Lishui district, Baima National Agricultural Science and Technology Park, 119°10′44″ N, 31°36′28″ E (DMS), isolated from needles of Pinus massoniana, 1 March 2023, Hui Li, holotype CFCC 59538. Holotype is a living specimen being maintained via lyophilization at the China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing, China, and ex-type BM 1-1 is stored at Forest Pathology Laboratory, Nanjing Forestry University.
Habitat and host: On needles of Pinus massoniana with needle blight.
Known distribution: Nanjing, Jiangsu Province, China.
Additional specimens examined: China, Jiangsu province, Nanjing city, Lishui district, Baima National Agricultural Science and Technology Park, 119°10′44″ N, 31°36′28″ E (DMS), isolated from needles of Pinus massoniana, 1 March 2023, Hui Li, cultures: CFCC 59539 (=BM 1-2), CFCC 59540 (=BM 1-3), CFCC 59541 (=BM 1-4), and CFCC 59542 (=BM 1-5).
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.
3.4. 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 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.
4. 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 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.
5. 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.
Author Contributions
Conceptualization, L.-H.Z.; methodology, H.L., J.-Y.X. and Y.-Q.B. software, H.L.; validation, H.L.; formal analysis, H.L.; investigation, H.L., J.-Y.X. and Y.-Q.B.; resources, L.-H.Z.; data curation, H.L.; writing—original draft preparation, H.L.; writing—review and editing, D.-W.L.; visualization, H.L. and B.-Y.P.; supervision, D.-W.L.; project administration, L.-H.Z.; funding acquisition, L.-H.Z. All authors have read and agreed to the published version of the manuscript.
Funding
This work was supported by National Key R & D Program of China (2022YFD1401005), and the National Natural Science Foundation of China (grant number 31971659).
Institutional Review Board Statement
Not applicable for studies not involving humans or animals.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data are contained within the article.
Acknowledgments
The authors would like to thank those who provided assistance and advice for this study.
Conflicts of Interest
The authors declare no conflicts of interest.
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Figure 1.
Symptoms of needle blight on Pinus massoniana in the field (A–C).
Figure 2.
Phylogenetic relationship of Pestalotiopsis jiangsuensis isolates: BM 1-1, BM 1-2, BM 1-3, BM 1-4, and BM 1-5, based on concatenated sequences of ITS, TEF1, and TUB2 genes/region. RA × ML bootstrap support values (ML ≥ 70) and Bayesian posterior probability values (PP ≥ 0.90) were shown at the nodes (ML/PP). Neopestalotiopsis protearum (CBS 114178) is used as an outgroup. Bar = 0.04 substitution per nucleotide position. The sequences from this study are in red. The ex-type strains are in bold.
Figure 2.
Phylogenetic relationship of Pestalotiopsis jiangsuensis isolates: BM 1-1, BM 1-2, BM 1-3, BM 1-4, and BM 1-5, based on concatenated sequences of ITS, TEF1, and TUB2 genes/region. RA × ML bootstrap support values (ML ≥ 70) and Bayesian posterior probability values (PP ≥ 0.90) were shown at the nodes (ML/PP). Neopestalotiopsis protearum (CBS 114178) is used as an outgroup. Bar = 0.04 substitution per nucleotide position. The sequences from this study are in red. The ex-type strains are in bold.
Figure 3.
Pairwise homoplasy index (PHI) test of Pestalotiopsis isolates: BM 1-1, BM 1-2, BM 1-3, BM 1-4, and BM 1-5 and closely related P. foliicola, P. pinicola, P. suae, and P. rosea using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the data set.
Figure 3.
Pairwise homoplasy index (PHI) test of Pestalotiopsis isolates: BM 1-1, BM 1-2, BM 1-3, BM 1-4, and BM 1-5 and closely related P. foliicola, P. pinicola, P. suae, and P. rosea using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the data set.
Figure 4.
Morphological characteristics of Pestalotiopsis jiangsuensis sp. nov. BM 1-1. (A) Fungal colony on PDA, 5 d growth from above (L) and below (R). (B) Conidiomata and conidial masses. (C) Conidiophores, conidiogenous cells, and conidia. (D) Conidia. Scale bars: (B) = 500 μm, (C,D) = 20 μm.
Figure 4.
Morphological characteristics of Pestalotiopsis jiangsuensis sp. nov. BM 1-1. (A) Fungal colony on PDA, 5 d growth from above (L) and below (R). (B) Conidiomata and conidial masses. (C) Conidiophores, conidiogenous cells, and conidia. (D) Conidia. Scale bars: (B) = 500 μm, (C,D) = 20 μm.
Figure 5.
Pathogenicity of representative isolates of Pestalotiopsis jiangsuensis sp. nov. (BM 1-1, BM 1-2, and BM 1-3) on Pinus massoniana. (A) No symptoms were observed on control pine needles treated with sterile water after 5 days. (B–D) Symptoms on pine needles inoculated with conidial suspensions of BM 1-1, BM 1-2, and BM 1-3 after 5 days, respectively. (E) No symptoms observed on control pine needles treated with sterile water after 10 days. (F–H) Symptoms on pine needles inoculated with conidial suspensions of BM 1-1, BM 1-2, and BM 1-3 after 10 days.
Figure 5.
Pathogenicity of representative isolates of Pestalotiopsis jiangsuensis sp. nov. (BM 1-1, BM 1-2, and BM 1-3) on Pinus massoniana. (A) No symptoms were observed on control pine needles treated with sterile water after 5 days. (B–D) Symptoms on pine needles inoculated with conidial suspensions of BM 1-1, BM 1-2, and BM 1-3 after 5 days, respectively. (E) No symptoms observed on control pine needles treated with sterile water after 10 days. (F–H) Symptoms on pine needles inoculated with conidial suspensions of BM 1-1, BM 1-2, and BM 1-3 after 10 days.
Table 1.
Reaction conditions used in PCR amplification and sequencing.
| Locus | PCR Primers (Forward/Reverse) | PCR: Thermal Cycles: (Annealing Temperature in Bold) |
|---|---|---|
| ITS | ITS5/ITS4 | 94 °C: 3 min, (94 °C: 45 s, 55 °C: 45 s, 72 °C: 1 min) ×35 cycles, 72 °C: 10 min |
| TEF1 | EF1-728F/EF1-986R | 94 °C: 3 min, (94 °C: 45 s, 55 °C: 45 s, 72 °C: 1 min) ×35 cycles, 72 °C: 10 min |
| TUB2 | T1/Bt-2b | 94 °C: 3 min, (94 °C: 45 s, 56 °C: 60 s, 72 °C: 1 min) ×35 cycles, 72 °C: 10 min |
Table 2.
Host, Origin, and GenBank accession numbers of strains of Pestalotiopsis species used for phylogenetic analyses.
Table 2.
Host, Origin, and GenBank accession numbers of strains of Pestalotiopsis species used for phylogenetic analyses.
| Species a | Strain Number b | Host | Origin | GenBank Accession Number c | ||
|---|---|---|---|---|---|---|
| ITS | TUB2 | TEF1 | ||||
| Pestalotiopsis abietis | CFCC 53011 T | Abies fargesii | China | MK397013 | MK622280 | MK622277 |
| P. adusta | ICMP 6088 T | Prunus cerasus | Fiji | JX399006 | JX399037 | JX399070 |
| P. aggestorum | LC6301 T | Camellia sinensis | China | KX895015 | KX895348 | KX895234 |
| P. anacardiacearum | IFRDCC 2397 T | Mangifera indica | China | KC247154 | KC247155 | KC247156 |
| P. anhuiensis | CFCC 54791 T | Cyclobalanopsis glauca | China | ON007028 | ON005056 | ON005045 |
| P. appendiculata | CGMCC 3.23550 T | Rhododendron decorum | China | OP082431 | OP185516 | OP185509 |
| P. arengae | CBS 331.92 T | Arenga undulatifolia | Singapore | KM199340 | KM199426 | KM199515 |
| P. arceuthobii | CBS 434.65 T | Arceuthobium campylopodum | USA | KM199341 | KM199427 | KM199516 |
| P. australasiae | CBS 114126 T | Knightia sp. | New Zealand | KM199297 | KM199409 | KM199499 |
| P. australis | CBS 114193 T | Grevillea sp. | Australia | KM199332 | KM199383 | KM199475 |
| P. biciliata | CBS 124463 T | Platanus × hispanica | Slovakia | KM199308 | KM199399 | KM199505 |
| P. brachiata | CGMCC 3.18151 T | Rhizophora apiculata | Thailand | MK764274 | MK764340 | MK764318 |
| P. brassicae | CBS 170.26 T | Brassica napus | New Zealand | KM199379 | - | KM199558 |
| P. camelliae | MFLUCC 12-0277 T | Camellia japonica | China | JX399010 | JX399041 | JX399074 |
| P. camelliae-oleiferae | CSUFTCC 08 T | Camellia oleifera | China | OK493593 | OK562368 | OK507963 |
| P. cangshanensisi | CGMCC 3.23544 T | Rhododendron delavayi | China | OP082426 | OP185517 | OP185510 |
| P. castanopsidis | CFCC 54430 T | Castanopsis lamontii | China | OK339732 | OK358508 | OK358493 |
| P. chamaeropis | CBS 186.71 T | Chamaerops humilis | Italy | KM199326 | KM199391 | KM199473 |
| P. changjiangensis | CFCC 54314 T | Castanopsis tonkinensis | China | OK339739 | OK358515 | OK358500 |
| P. changjiangensis | CFCC 54433 | Castanopsis tonkinensis | China | OK339740 | OK358516 | OK358501 |
| P. chiaroscuro | BRIP 72970 T | Sporobolus natalensis | Australia | OK422510 | - | - |
| P. chinensis | MFLUCC 12-0273 T | Taxus sp. | China | JX398995 | - | - |
| P. clavata | MFLUCC 12-0268 T | Buxus sp. | China | JX398990 | JX399025 | JX399056 |
| P. colombiensis | CBS 118553 T | Eucalyptus urograndis | Colombia | KM199307 | KM199421 | KM199488 |
| P. cyclobalanopsidis | CFCC 54328 T | Cyclobalanopsis glauca | China | OK339735 | OK358511 | OK358496 |
| P. daliensis | CGMCC 3.23548 T | Rhododendron decorum | China | OP082429 | OP185511 | OP185518 |
| P. dianellae | CBS 143421 T | Dianella sp. | Australia | MG386051 | MG386164 | - |
| P. digitalis | MFLU 14-0208 T | Digitalis purpurea | New Zealand | KP781879 | KP781883 | - |
| P. diploclisiae | CBS 115587 T | Diploclisia glaucescens | China | KM199320 | KM199419 | KM199486 |
| P. disseminata | CBS 143904 | Persea americana | New Zealand | MH554152 | MH554825 | MH554587 |
| P. distincta | LC3232 T | Camellia sinensis | China | KX894961 | KX895293 | KX895178 |
| P. diversiseta | MFLUCC12-0287 T | Rhododendron sp. | China | JX399009 | JX399040 | JX399073 |
| P. dracaenae | HGUP 4037 T | Dracaena fragrans | China | MT596515 | MT598645 | MT598644 |
| P. dracaenicola | MFLUCC 18-0913 T | Dracaena sp. | Thailand | MN962731 | MN962733 | MN962732 |
| P. dracontomelon | MFLUCC 10-0149 T | Dracontomelon dao | Thailand | KP781877 | - | KP781880 |
| P. eleutherococci | HMJAU 60190 | Eleutherococcus brachypus | China | OL996127 | OL898722 | - |
| P. endophytica | MFLUCC 18-0932 T | Magnolia garrettii | Thailand | MW263946 | - | MW417119 |
| P. ericacearum | IFRDCC 2439 T | Rhododendron delavayi | China | KC537807 | KC537821 | KC537814 |
| P. etonensis | BRIP 66615 T | Sporobolus jacquemontii | Australia | MK966339 | MK977634 | MK977635 |
| P. ficicola | SAUCC230046 T | Ficus microcarpa | China | OQ691974 | OQ718749 | OQ718691 |
| P. foliicola | CFCC 54440 T | Castanopsis faberi | China | ON007029 | ON005057 | ON005046 |
| P. formosana | NTUCC 17-009 T | Neolitsea villosa | China | MH809381 | MH809385 | MH809389 |
| P. furcata | MFLUCC 12-0054 T | Camellia sinensis | Thailand | JQ683724 | JQ683708 | JQ683740 |
| P. fusoidea | CGMCC 3.23545 T | Rhododendron delavayi | China | OP082427 | OP185519 | OP185512 |
| P. gaultheriae | IFRD 411-014 T | Gaultheria forrestii | China | KC537805 | KC537819 | KC537812 |
| P. gibbosa | NOF 3175 T | Gaultheria shallon | Canada | LC311589 | LC311590 | LC311591 |
| P. grandis-urophylla | E72-04 | Eucalyptus grandis | Brazil | KU926710 | KU926718 | KU926714 |
| P. grevilleae | CBS 114127 T | Grevillea sp. | Australia | KM199300 | KM199407 | KM199504 |
| P. guangxiensis | CFCC 54308 T | Quercus griffithii | China | OK339737 | OK358513 | OK358498 |
| P. guizhouensis | CFCC 57364 T | Cyclobalanopsis glauca | China | ON007035 | ON005063 | ON005052 |
| P. hawaiiensis | CBS 114491 T | Leucospermum sp. | USA | KM199339 | KM199428 | KM199514 |
| P. hispanica | CBS 115391 | Eucalyptus globulus | Portugal | MW794107 | MW802840 | MW805399 |
| P. hollandica | CBS 265.33 T | Sciadopitys verticillata | Netherlands | KM199328 | KM199388 | KM199481 |
| P. humus | CBS 336.97 T | Soil | Papua New Guinea | KM199317 | KM199420 | KM199484 |
| P. hydei | MFLUCC 20-0135 T | Litsea petiolata | Thailand | MW266063 | MW251112 | MW251113 |
| P. iberica | CAA 1004 T | Pinus radiata | Spain | MW732248 | MW759035 | MW759038 |
| P. inflexa | MFLUCC 12-0270 T | Unidentified tree | China | JX399008 | JX399039 | JX399072 |
| P. intermedia | MFLUCC 12-0259 T | Unidentified tree | China | JX398993 | JX399028 | JX399059 |
| P. italiana | MFLUCC 12-0657 T | Cupressus glabra | Italy | KP781878 | KP781882 | KP781881 |
| P. jiangsuensis | CFCC 59538 | Pinus massoniana | China | OR533577 | OR539191 | OR539186 |
| CFCC 59539 | OR533578 | OR539192 | OR539187 | |||
| CFCC 59540 | OR533579 | OR539193 | OR539188 | |||
| CFCC 59541 | OR533580 | OR539194 | OR539189 | |||
| CFCC 59542 | OR533581 | OR539195 | OR539190 | |||
| P. jiangxiensis | LC4399 T | Camellia sp. | China | KX895009 | KX895341 | KX895227 |
| P. jinchanghensis | LC6636 T | Camellia sinensis | China | KX895028 | KX895361 | KX895247 |
| P. kaki | KNU-PT-1804 T | Diospyros kaki | Korea | LC552953 | LC552954 | LC553555 |
| P. kandelicola | NCYUCC 19-0355 T | Kandelia candel | China | MT560723 | MT563100 | MT563102 |
| P. kenyana | CBS 442.67 T | Coffea sp. | Kenya | KM199302 | KM199395 | KM199502 |
| P. knightiae | CBS 114138 T | Knightia sp. | New Zealand | KM199310 | KM199408 | KM199497 |
| P. krabiensis | MFLUCC 16-0260 T | Pandanus sp. | Thailand | MH388360 | MH412722 | MH388395 |
| P. lespedezae | SY16E | Pinus armandii | China | EF055205 | - | EF055242 |
| P. leucadendri | CBS 121417 T | Leucadendron sp. | South Africa | MH553987 | MH554654 | MH554412 |
| P. licualacola | HGUP4057 T | Licuala grandis | China | KC492509 | KC481683 | KC481684 |
| P. linearis | MFLUCC 12-0271 T | Trachelospermum sp. | China | JX398992 | JX399027 | JX399058 |
| P. linguae | ZHKUCC 22-0159 | Pyrrosia lingua | China | OP094104 | OP186108 | OP186110 |
| P. lithocarpi | CFCC 55100 T | Lithocarpus chiungchungensis | China | OK339742 | OK358518 | OK358503 |
| P. lushanensis | LC4344 T | Camelia sp. | China | KX895005 | KX895337 | KX895223 |
| P. macadamiae | BRIP 63738b T | Macadamia integrifolia | Australia | KX186588 | KX186680 | KX186621 |
| P. malayana | CBS 102220 T | Macaranga triloba | Malaysia | KM199306 | KM199411 | KM199482 |
| P. menhaiensis | CGMCC 3.18250 T | Camellia sinensis | China | KU252272 | KU252488 | KU252401 |
| P. microspora | SS1-033I | Cornus canadensis | Canada | MT644300 | - | - |
| P. monochaeta | CBS 144.97 T | Quercus robur | Netherlands | KM199327 | KM199386 | KM199479 |
| P. montellica | MFLUCC12-0279 T | Fagraea bodeni | China | JX399012 | JX399043 | JX399076 |
| P. nanjingensis | CSUFTCC 16 T | Camellia oleifera | China | OK493602 | OK562377 | OK507972 |
| P. nanningensis | CSUFTCC 10 T | Camellia oleifera | China | OK493596 | OK562371 | OK507966 |
| P. neglecta | TAP1100 T | Quercus myrsinaefolia | Japan | AB482220 | LC311599 | LC311600 |
| P. neolitseae | NTUCC 17-011 T | Neolitsea villosa | China | MH809383 | MH809387 | MH809391 |
| P. novae-hollandiae | CBS 130973 T | Banksia grandis | Australia | KM199337 | KM199425 | KM199511 |
| P. olivacea | SY17A | Pinus armandii | China | EF055215 | EF055251 | - |
| P. oryzae | CBS 353.69 T | Oryza sativa | Denmark | KM199299 | KM199398 | KM199496 |
| P. pallidotheae | MAFF 240993 T | Pieris japonica | Japan | AB482220 | LC311584 | LC311585 |
| P. pandanicola | MFLUCC 16-0255 T | Pandanus sp. | Thailand | MH388361 | MH412723 | MH388396 |
| P. papuana | CBS 331.96 T | Coastal soil Papua | New Guinea | KM199321 | KM199413 | KM199491 |
| P. parva | CBS 278.35 | Leucothoe fontanesiana | Thailand | KM199313 | KM199405 | KM199509 |
| P. phoebes | SAUCC230093 T | Phoebe zhenna | China | OQ692028 | OQ718803 | OQ718745 |
| P. photinicola | YB28-2 | Mango | China | MK228997 | MK360938 | MK512491 |
| P. pini | MEAN 1092 T | Pinus pinea | Portugal | MT374680 | MT374705 | MT374693 |
| P. pinicola | KUMCC 19-0183 T | Pinus armandii | China | MN412636 | MN417507 | MN417509 |
| P. portugallica | CBS 393.48 T | - | Portugal | KM199335 | KM199422 | KM199510 |
| P. rhizophorae | MFLUCC 17-0416 T | Rhizophora apiculata | Thailand | MK764283 | MK764349 | MK764327 |
| P. rhododendri | IFRDCC 2399 T | Rhododendron sinogrande | China | KC537804 | KC537818 | KC537811 |
| P. rhodomyrtus | CFCC 55052 | Cyclobalanopsis augustinii | China | OM746311 | OM839984 | OM840083 |
| P. rosarioides | CGMCC 3.23549 T | Rhododendron decorum | China | OP082430 | OP185513 | OP185520 |
| P. rosea | MFLUCC 12-0258 T | Pinus sp. | China | JX399005 | JX399036 | JX399069 |
| P. scoparia | CBS 176.25 T | Chamaecyparis sp. | China | KM199330 | KM199393 | KM199478 |
| P. sequoiae | MFLUCC 13-0399 T | Sequoia sempervirens | Italy | KX572339 | - | - |
| P. shaanxiensis | CFCC 54958 T | Quercus variabilis | China | ON007026 | ON005054 | ON005043 |
| P. shorea | MFLUCC 12-0314 T | Shorea obtusa | Thailand | KJ503811 | KJ503814 | KJ503817 |
| P. sichuangensis | CGMCC 3.18244 T | Camellia sinensis | China | KX146689 | KX146807 | KX146748 |
| P. silvicola | CFCC 55296 T | Cyclobalanopsis kerrii | China | ON007032 | ON005060 | ON005049 |
| P. spatholobi | SAUCC231201 T | Spatholobus suberectus | China | OQ692023 | OQ718798 | OQ718740 |
| P. spathulata | CBS 356.86 T | Gevuina avellana | Chile | KM199338 | KM199423 | KM199513 |
| P. spathuliappendiculata | CBS 144035 T | Phoenix canariensis | Australia | MH554172 | MH554845 | MH554607 |
| P. suae | CGMCC3.23546 T | Rhododendron delavayi | China | OP082428 | OP185521 | OP185514 |
| P. telopeae | CBS 114161 T | Telopea sp. | Australia | KM199296 | KM199403 | KM199500 |
| P. terricola | CBS 141.69 T | Soil | Pacific Islands | MH554004 | MH554680 | MH554438 |
| P. thailandica | MFLUCC 17-1616 T | Rhizophora apiculata | Thailand | MK764285 | MK764351 | MK764329 |
| P. trachycarpicola | IFRDCC 2240 T | Trachycarpus fortunei | China | JQ845947 | JQ845945 | JQ845946 |
| P. tumida | CFCC 55158 T | Rosa chinensis | China | OK560610 | OL814524 | OM158174 |
| P. unicolor | MFLUCC 12-0276 T | Rhododendron sp. | China | JX398999 | JX399030 | - |
| P. verruculosa | MFLUCC 12-0274 T | Rhododendron sp. | China | JX398996 | - | JX399061 |
| P. vismiae | HHL-DG | Rhizophora stylosa | China | HM535704 | HM573246 | - |
| P. yanglingensis | LC4553 T | Camellia sinensis | China | KX895012 | KX895345 | KX895231 |
| P. yunnanensis | HMAS 96359 T | Podocarpus macrophyllus | China | AY373375 | - | - |
| Neopestalotiopsis protearum | CBS 114178 T | Leucospermum cuneiforme | Zimbabwe | JN712498 | KM199463 | LT853201 |
a 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, 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; NCYUCC = The National Chiayi University Culture Collection, Jiayi, Taiwan; ZHKUCC = the culture collection of Zhongkai University of Agriculture and Engineering, Guangzhou City, Guangdong, China; TAP = Tamagawa University, Tokyo, Japan; MAFF = Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, Japan; MEAN = Instituto Nacional de Investigação Agrária e Veterinária I. P.; KUMCC = Kunming Institute of Botany Culture Collection, Yunnan, China; HMAS = Mycological Herbarium, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. c ITS = internal transcribed spacer; TUB2 = b-tubulin; TEF1 = translation elongation factor1-α.
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MDPI and ACS Style
Li, H.; Peng, B.-Y.; Xie, J.-Y.; Bai, Y.-Q.; Li, D.-W.; Zhu, L.-H. Pestalotiopsis jiangsuensis sp. nov. Causing Needle Blight on Pinus massoniana in China. J. Fungi 2024, 10, 230. https://doi.org/10.3390/jof10030230
AMA Style
Li H, Peng B-Y, Xie J-Y, Bai Y-Q, Li D-W, Zhu L-H. Pestalotiopsis jiangsuensis sp. nov. Causing Needle Blight on Pinus massoniana in China. Journal of Fungi. 2024; 10(3):230. https://doi.org/10.3390/jof10030230
Chicago/Turabian StyleLi, Hui, Bing-Yao Peng, Jun-Ya Xie, Yu-Qing Bai, De-Wei Li, and Li-Hua Zhu. 2024. "Pestalotiopsis jiangsuensis sp. nov. Causing Needle Blight on Pinus massoniana in China" Journal of Fungi 10, no. 3: 230. https://doi.org/10.3390/jof10030230
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