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. 2024 Mar 21;10(3):230. doi: 10.3390/jof10030230

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

Hui Li 1,2, Bing-Yao Peng 1,2, Jun-Ya Xie 1,2, Yu-Qing Bai 1,2, De-Wei Li 3, Li-Hua Zhu 1,2,*
Editor: Zonghua Wang
PMCID: PMC10970983  PMID: 38535238

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.

Keywords: multi-locus phylogeny, new species, pine

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).

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
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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).

Figure 1.

Figure 1

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Symptoms of needle blight on Pinus massoniana in the field (A–C).

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).

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
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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.

Figure 2.

Figure 2

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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.

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).

Figure 3.

Figure 3

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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.

3.3. Taxonomy

Pestalotiopsis jiangsuensis Li-Hua Zhu, Hui Li, and D.W. Li, sp. nov. Figure 4

Figure 4.

Figure 4

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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.

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.

Pestalotiopsis funerea has two to four apical appendages, and Pestalotiopsis lawsoniae has two apical appendages. They also have differences with P. jiangsuensis. In addition, P. funerea has a longer basal appendage than that of P. jiangsuensis (5–7) µm vs. (1.4–6.3) µm [43,44].

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.

Figure 5.

Figure 5

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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.

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.

Acknowledgments

The authors would like to thank those who provided assistance and advice for this study.

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.

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.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was supported by National Key R & D Program of China (2022YFD1401005), and the National Natural Science Foundation of China (grant number 31971659).

Footnotes

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