<8) MycoKeys MycoKeys 121: 291-310 (2025) DOI: 10.3897/mycokeys.121.155321 Research Article Two new species of Diaporthe (Diaporthaceae, Diaporthales) from Actinidia chinensis in Guizhou Province, China Chunguang Ren™®, Yu Liu™, Wenwen Su", Zhengcheng Han?, Weijie Li2® 1 Guizhou Institute of Mountain Resources, Guiyang, Guizhou 550001, China 2 GuiZhou Botanical Garden, Guiyang, Guizhou 550001, China Corresponding author: Weijie Li (13765071358@163.com) OPEN Qrccess Academic editor: Nattawut Boonyuen Received: 9 April 2025 Accepted: 1 August 2025 Published: 1 September 2025 Citation: Ren C, Liu Y, Su W, Han Z, Li W (2025) Two new species of Diaporthe (Diaporthaceae, Diaporthales) from Actinidia chinensis in Guizhou Province, China. Mycokeys 121: 291-310. https://doi. org/10.3897/mycokeys.121.155321 Copyright: © Chunguang Ren et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Diaporthe spp. are well known to be plant pathogens, endophytes, or saprophytes on a wide range of economically significant crops, ornamental plants, and forest trees. In the present study, we aimed to investigate the diversity of Diaporthe species, which cause kiwifruit soft rot in Guizhou Province. Five strains of fungi were isolated from kiwifruit in- fected with soft rot in Guizhou province. These strains were identified using morpholog- ical and multilocus sequences analysis of the rDNA internal transcribed spacer region (ITS), calmodulin (ca/), histone H3 (his3), translation elongation factor 1-alpha (tef7), and B-tubulin (tub2). The results confirmed two new species — D. shuichengensis sp. nov. and D. liupanshuiensis sp. nov. This study identifies two new soft-rot pathogens of kiwifruit and provides a reference for future disease-management studies. Key words: Diaporthaceae, DNA phylogeny, kiwifruit, morphology Introduction Kiwifruit (Actinidia chinensis Planch.) contains various essential amino acids, vi- tamin C, dietary fiber, and dietary minerals. It is favored by consumers because it has a high nutritional value and plays an important role in several biological functions, such as cosmetology and skin care (Wojdylo et al. 2017; Lian et al. 2019; Zhu et al. 2019; Wang et al. 2021). China is one of the four major kiwi- fruit-producing countries in the world, accounting for approximately half of the global kiwifruit production (Shan et al. 2021). The kiwifruit industry has experi- enced continuous growth in recent years; this has resulted in the expansion of planting areas and an increase in the incidence of diseases. During storage, kiwi- fruit is highly susceptible to soft rot, which is primarily caused by Botryosphaeria dothidea (Moug.) Ces. & De Not. and Diaporthe spp. (Zhou et al. 2015; Diaz et al. 2017; Pan et al. 2020). Botryosphaeria dothidea, Alternaria alternata (Fr.) Keissl., Plectosphaerella cucumerina (Lindf.) W. Gams, Neofusicoccum parvum (Penny- cook & Samuels) Crous, Slippers & A. J. L. Phillips, Diaporthe spp., and Fusarium oxysporum have been reported as pathogens of kiwifruit rot in Guizhou Province (Wang et al. 2022a), China. This constraint significantly hinders the development of China's kiwifruit industry. Therefore, the identification of pathogens of kiwifruit soft rot disease is of great significance for industrial development. * These authors contributed equally to this work. 291 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Diaporthe spp. are globally distributed plant pathogens; they cause various diseases, such as branch dieback, leaf spot disease, and wilt disease, there- by affecting plant growth, decreasing production/yield, and even causing plant death in severe cases (Mctavish et al. 2018; Thomidis et al. 2019; Ariyawansa et al. 2021; Nair et al. 2021). The genus Diaporthe Nitschke was established in 1870 by Nitschke (1870). Saccardo (1883) proposed that the genus Phomopsis Sacc. & Roum. is the ana- morphic stage of Diaporthe owing to its ability to produce two conidia types. With the implementation of the fungal nomenclature rule of “one fungus, one name, the genus Diaporthe gained nomenclatural precedence and was used as the genus name for any newly established species and recombined species (Huang et al. 2013; Rossman et al. 2015). Classification of this genus typically relies on morphological features, ordered as follows: colony characteristics, mycelium features (including hyphae), asexual structures (conidiomata, con- idiophores, conidia), and sexual structures (ascospores) (Santos et al. 2011; Gomes et al. 2013; Guarnaccia and Crous 2017; Yang et al. 2018b). Studies have shown that the morphological features of fungi from the genus Diaporthe exhibit variability and plasticity between and within species; additionally, ob- server subjectivity during observation and recording can affect species iden- tification (Gomes et al. 2013; Udayanga et al. 2012). Moreover, researchers have found that some species can infect a variety of hosts, whereas different species can infect the same host (Thompson et al. 2011). Therefore, relying solely on morphological features and host specificity as the classification crite- ria for Diaporthe species can lead to ambiguity in the results (Elfar et al. 2013; Thompson et al. 2015). Since the dawn of the molecular analysis era, phyloge- netic analyses based on multigene sequencing has been widely used to clas- sify fungi from the Diaporthe genus. Gomes et al. (2013) constructed the first taxonomic system for Diaporthe by re-examining type specimens or strains of Diaporthe using five gene fragments (ITS-cal-tub2-tef1-his3); this system is still being used (Bai et al. 2023). Therefore, in the present study, we aimed to investigate the diversity of Dia- porthe species, which cause kiwifruit soft rot in Guizhou Province; we examined five isolates from kiwifruit soft rot symptomatic samples by combined mor- phological and phylogenetic analyses. These isolates were found to represent two new Diaporthe species, which are described and discussed in the present study. The discovery of these new Diaporthe species would help researchers to understand the diversity. Materials and methods Sampling, fungal isolation, and morphological observations. From 2022 to 2024, kiwifruit soft rot samples were collected from Liupanshui City (25°19'44'N, 104°18'24"E), Guizhou Province, China. The diseased tissue along the edge of the kiwifruit (5 x 5 mm) was cut using a dissecting knife, which was sterilized at a high temperature, immersed in 75% ethanol for 30 s for surface disinfection, and then rinsed thrice with sterile distilled water. After drying on a sterile filter paper, the samples were placed on a potato dextrose agar (PDA) culture medi- um in a 25 °C incubator for 2-4 days. Hyphae were selected from the periphery of the colonies and inoculated onto new PDA plates. MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 299 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Five-millimeter diameter mycelial plugs of purified strains were inoculated onto PDA medium (9 cm diameter Petri dishes). These cultures were incubat- ed in a BOXUN SPX-250B-Z Biochemical Incubator (Shanghai Boxun Medical Biological Instrument Corp., China, all the incubators mentioned in this paper belong to the same brand.) darkness at 25 °C, with three replicates per strain. The resulting colony growth on the medium was recorded. Mycelial plugs were inoculated onto a WA medium containing pine needles, fennel stems (Santos et al. 2010), and clover stems (Udayanga et al. 2014). The strains were cultured in an intelligent light incubator at 25 °C with a 12/12 (light/dark) cycle (until conidi- omata were produced. For microscopic examination, fungal structures mounted in clear lactic acid were observed using a Leica DM4 B compound microscope at x1000 magnification. At least 30 conidiomata and conidia were measured to calculate mean size/length. The holotypes were stored in the herbarium of the Institute of Mountain Resources, Guizhou Academy of Sciences, China. Ex-type living culture was deposited at the Culture Collection Management Center of the Institute of Mountain Resources, Guizhou Academy of Sciences, China. DNA extraction and amplification DNA extraction was performed using a fungal genomic DNA extraction kit (DP2033, BioTeke Corporation) according to Liang et al. (2011). Partial regions of the isolates rDNA-ITS region (ITS), B-tubulin (tub2), translation elongation factor 1-alpha (tef7), calmodulin (ca/), and histone H3 (his3) regions were am- plified using the primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1-728F/EF1-986R, cal-228F/cal-737R (Carbone and Kohn 1999), and CYLH3F/H3-1b (Crous et al. 2004), respectively. The PCR reaction mixture (25 pL) comprised 12.5 uL Tag Mix (Sangon, Shanghai, China), 1 uLDNA template, 1 uL of each forward and reverse primer (10 um) (Sangon, Shanghai, China), and 9.5 pL ddH,O (Sangon, Shanghai, China). The PCR program was as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of de- naturation at 95 °C for 30 s, annealing for 30 s at 55 °C for ITS, 60 °C for tub2, 52 °C for tef71, 54 °C for cal, 57 °C for his3, and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. The amplified PCR products were sent to Shanghai Sangon for sequencing. Phylogenetic analysis The obtained forward and reverse sequences were checked and assembled using SeqMan v. 7.0. The ITS, tub2, tef1, cal, and his3 sequences in Table 1 were downloaded from GenBank, based on Dissanayake et al. (2024). Multiple sequence alignments were performed using the online MAFFT tool (https:// www.ebi.ac.uk/Tools/msa/mafft/) (Katoh et al. 2019). Prior to conducting the Bayesian inference (BI) analyses, the best nucleotide substitution model for each gene was determined using jModelTest 2.0 (Posada 2008) based on the Akaike information criterion (AIC). The Bayesian posterior probabilities were es- timated using Markov Chain Monte Carlo sampling (MCMC) in MrBayes v3.2.7 (Ronquist et al. 2012). Six simultaneous Markov Chains were run for 1,000,000 generations, trees were sampled every 100" generation, and 25% of the aging samples were discarded. A maximum likelihood (ML) analysis was conducted MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 293 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Table 1. Isolates and GenBank accession numbers used in the phylogenetic analysis of Diaporthe. Newly sequenced material is indicated in bold. Strains marked with “*” are ex-type or ex-epitype strains. enecies GenBank Accession Number 19-0124 MycoKeys 121: 291-310 (2025 SS , DOI: 10.3897/mycokeys.121.155321 294 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) GenBank Accession Number Species Strain/Isolate CBS 101339* B3180 CBS 176.77 MFLUCC 10-0580a* CBS 143770* CBS 133182* COAD 2913 NFCCI 4385 KC343181 MT043790 KC343183 JQ619887 MG600223 KC343188 MT311197 MN061372 D. pseudomangiferae KC344149 KC343423 rad === D. pseudooculi KC344151 JX275441 MG600227 KC344156 KC343425 JX197433 MG600219 KC343430 MT313691 D. pseudophoenicicola D. pterocarpicola D. racemosae D. raonikayaporum D. rosiphthora D. salsuginosa MN431500 seemed a phylli phylli ops 101339* | kc34381 _Kesaai49 | KC343423 83180 | MT043790 | [Sie el 68817677 KC343183 _Kesaais1 | Kc343425 ___MFLUCC 10-0580" | JQ619887 | 275441 JX197433 ops 143770" | MG600223 | _MG600227__MG600219_ __eps131s2* | KC343188 _Ke34a156 | KC343430 CAD 2918 | MT311197 | = MT313691 —_NFccl4385 | MNO61872_ _MIN431500 |= MycoKeys 121: 291-310 (2025 ~S , DOI: 10.3897/mycokeys.121.155321 tub2 KC343604 MN224558 MZ504691 MF418342 PP567107 PP567108 PP567109 KC343619 KY435648 MT309439 KC343623 KC343624 ON081662 MF418344 MW022499 KC343627 KC343649 KC343648 KC343655 KY435654 KX999243 KX999246 KC343665 KC343667 MG600221 KC343672 295 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Species D. schini D. searlei D. sennae D. shuichengensis D. shuichengensis D. siamensis D. siamensis D. spinosa . taiwanensis . taoicola . tarchonanthi . terebinthifolii D D D D. tecomae D D. terebinthifolii D . viniferae Strain/Isolate GenBank Accession Number ITS cal his3 tef1 tub2 CBS 133181* KC343191 KC343917 | KC344159 KC343433 KC343675 BRIP 66528* MN708231 = MN696540 = = CFCC 51636* KY203724 KY228885 KY228891 KY228875 = SC-7* PP537966 | PP567095 § PP567100 | PP599035 PP567105 SC-8 PP537967 | PP567096 | PP567101 PP567110 PP567106 MFLUCC 10-0573a* JQ619879 JX275393 JX275429 JX197423 = MFLUCC 12-0300 KT459417 KT459451 KT459435 KT459467 = PSCG 279 MK626925 | MK654801 | MK691235 | MK691126 MK726155 NTUCC 18-105-1* MT241257 | MT251199 | MT251202 | MT251196 Zi MFLUCC 16-0117* KU557567 | KU557636 | KU557591 = = CBS 146073* MT223794 = MT223733 = MT223759 CBS 100547* KC343215 KC343941 KC344183 KC343457 KC343699 CBS 133180* KC343216 KC343942 | KC344184 KC343458 KC343700 LGMF907 KC343217 KC343943 KC344185 KC343459 KC343701 JZB320071* MK341550 | MK500107 | MK500112 | MK500119 z on the CIPRES web portal using RAxML-HPC BlackBox v.8.2.12 (Miller et al. 2010), with the GTR + GAMMAI substitution model and 1000 bootstrap repli- cations performed for testing. Phylogenetic trees were viewed in FigTree v1.4. The assembled sequences were submitted to the GenBank database to obtain accession numbers. Pathogenicity test Healthy “Guichang” kiwifruits were selected (n = 15), disinfected with 75% alco- hol, washed twice with sterile water, and then placed on an ultra-clean bench to dry naturally. After drying, three points were stabbed in the middle of each fruit with sterile needles. At the puncture site, 1 mL of the spore suspension (10°8 per/mL) was inoculated and covered with sterile cotton to ensure constant moisturization. Five kiwi fruits inoculated with sterile water was used as the control. Each treatment had 5 fruits, and the experiment was repeated 3 times. Fruits were cultured at a constant temperature of 25 °C under 85% relative hu- midity and a 12/12 h light/dark cycle for 5 d in an incubator. The incidence was observed and recorded every day. To confirm the fungi as the causative agents, Koch's postulates were fulfilled: the fungi were consistently detected in dis- eased hosts, isolated and cultured in vitro, then inoculated into healthy, suscep- tible hosts which subsequently developed the disease. Fungi re-isolated from lesions post-infection were confirmed as identical to the original inoculum. Results Symptoms of kiwifruit after picking Under natural conditions, blisters appear on fruit surfaces when diseased. The flesh inside the fruit is light yellow and in severe cases, it undergoes perforated decay and produces an odour (Fig. 1). Mycokeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 296 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Figure 1. Kiwifruit soft rot symptoms. Phylogenetic analysis Two parallel phylogenetic analyses were performed to optimally resolve the positions of our novel strains. Analysis 1 (Fig. 2) examined 57 taxa within the D. arecae species complex framework (Dissanayake et al. 2024), using D. salsugi- nosa as outgroup. Analysis 2 (Fig. 3) included 47 taxa representing a distinct clade near D. arezzoensis (outgroup), which preliminary BLAST searches sug- gested as the closest known relatives of our strains SC-7 and SC-8. Analysis 1: The “RAxML-HPC BlackBox” software was utilized for conducting ML analysis and the “GTRGAMMA + |” model was employed to estimate the proportion of invariant sites. The final value of the highest scoring tree was -—15906.15, which was obtained from an ML analysis of the dataset (ITS + tef7 + cal + his3 + tub2). The parameters of the rate heterogeneity model used to an- alyze the dataset were estimated using the following frequencies: A = 0.2223, C = 0.3167, G = 0.2361, T = 0.2247; substitution rates AC = 1.1093, AG = 3.0045, AT = 1.1820, CG = 0.7598, CT = 3.5346 and GT = 1.00, as well as the gamma distribution shape parameter a = 0.953082. For BI analysis, the “MrBayes on XSEDE” application was utilized along with the “GTR” model. Similar tree to- pologies were obtained by ML and BI methods, and the best scoring ML tree is shown in Fig. 2. Three strains in group 1 forming independent branches. Three new strains clustered into an independent clade with close relationships to D. podocarpi-macrophylli Y.H. Gao & L. Cai (strains GCMCC3.18281 and LC6144). Analysis 2: The final value of the highest scoring tree was —23691.68, which was obtained from the ML analysis of the dataset (ITS + tef7 + cal + his3 + tub2). MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 997 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) 98/1 - D. arecae (D. CERT Gn SES UGTA) BRIP 54781 -/0.99 D. arecae (D. musigena) 29519 -/0.98 D. arecae (D. lite scola) RIP 54900 D. arecae (D. arengae) CBS 114979 -/0.98 J D. arecae (D. sennae) CFCC 51636 bel D. arecae (D. taoicola) MFLUCC 16-0117 D. arecae ee anhuiensis) CNUCC 201901 D. camelliaeoleiferae HNZZ027 D. arecae (D. oculi) HHUF 30565 D. arecae oe endocitricola) ZHKUCC20-0012 . arecae (D. ARES EASES) CNUCC201903 D. arecae (D. eugeniae) CBS 444.82 D. arecae BBPPCA257 D. arecae (D. pascoei) BPPCA147 1 D. arecae Ce viniferae) JZB320071 D. arecae (D. pandanicola) MFLUCC 17-0607 D. arecae (D. averrhoae) SCHM 3605 D. arecae (D. pseudomangiferae) CBS 101339 74/0.99 1410.95 D. arecae (D. limonicola) CBS 142549 ; D. arecae (D. melitensis) CBS 142551 D. arecae (D. taiwanensis) NTUCC 18-105-1 D. arecae KKUC21243 D. arecae (D. krabiensis) MFLUCC 17-2481 91/0.98_1 D. arecae (D. phyllanthicola) RS 129 . arecae (D. phyllanthicola 74) lite D D. phyllanthicola) SCHM 3680 7 D. arecae (D. delonicis) MFLU 16-1059 D. arecae (D. POEUG COE MEIC g2) CBS 176.77 84/0.99 100/1- D. arecae PPB 345 D. arecae PPBMR340 D. arecae CCBS 161.64 D. arecae (D. pterocarpicola) MFLUCC 10-0580a D. arecae (D. ceratozamiae) HCH260 D. arecae (D. pescicola) MFLUCC 16-0105 D. caricae-papayae NIBM ABIJP . arecae (D. ceratozamiae fi D D jae) CBS 131306 93/1 eh D. arecae (D. fulvicolor) PSCG051 D. arecae (D. hunanensis) HNZZ023 )() D. arecae (D. acuta) CGMCC3.19600 D. arecae (D.acuta) PSCG046 D. arecae (D. spinosa) PSCG 279 D. arecae (D. chrysa EAs SAUCC1 94.35 D. arecae (D. pica aris) SC 3621 . arecae CCGMCC3.24296 D. arecae (D. nelumbonis) A-SER3 D. arecae (D. OTS: JZB320091 D. arecae (D. cercidis) CFCC 5256 D. arecae (D. searlei ) BRIP 66528 D. arecae (D. drenthii ) BRIP 66524 D. arecae (D. meliae ) CFCC 53089 73/-- D. liupanshuiensis SC-20 100/. LD. liupanshuiensis SC-19 10.94 D. liupanshuiensis SC-18 eg a RO D. arecae (D. podocarpi-macrophylli) LC6229 D. arecae (D. podocarpi-macrophylli) CGMCC3.18281 D. arecae (D. poenacocut) B3180 D. osmanthi GUCC9165 D. salsuginosa NFCCI 4385 0.03 Figure 2. Phylogenetic tree generated from maximum likelihood analysis based on combined ITS, tef7, tub2, cal and his3 sequence data for the Diaporthe arecae species complex and related taxa, rooted to D. salsuginosa (NFCCI 4385). The ML and BI bootstrap support values above 70% and 0.90 BYPP are shown at the first and second positions, respectively. The codes referring to the strains from the current study are indicated in red. MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 298 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) 100/) D. kyushuensis ch-D-1 10/1 D. kyushuensis STE-U2675 Pt D. chiangraiensis MFLUCC 17-1670 100/0.972 > oe D. chiangraiensis MFLUCC 17-1669 ie Cl D. eleutherrhenae 02 100/0.97{ D. eleutherrhenae 01 96/0.99/~ _- D. siamensis MFLUCC 12-0300 D. siamensis MFLUCC 10-0573a D. cinnamomi CFCC 52569 95/1 {| 200 D. foliorum CMRP 1330 ; D. foliorum CMRP 1321 D. raonikayaporum CBS 133182 D. mayteni CBS 133185 100/1, D. terebinthifolii LGMF907 100/) | D. terebinthifolii CBS 133180 100/1 D. tecomae CBS 100547 100/I+~_- D. schini CBS 133181 S66 D. rosiphthora COAD 2913 D. racemosae CBS 143770 96/0.99 D. hordei CBS 481.92 98/0.99) D. helianthi CBS 344.94 D. megalospora CBS 143.27 95/1 100/) D. ambiguaCBS 114015 D. ambiguaCBS 117167 D. goulteri BRIP 55657a 98/0.90~1] | D. longispora CBS 194.36 D. cyatheaeY MJ 1364 100/ . Shuichengensis SC-7 75/- D. shuichengensis SC-8 100/1- D. passiforae DJY 16A1-5 L00/IN|, D. passiforae A aL en GUCC 420.9 91/1004 ' D. passiforae (D. eucommiigena) GUCC 420.19 100/ D. passiforae (D. malorum) CAA734 100/1] JL DD. passiforae (D. malorum) CAA953 D. passiforae CBS 132527 D. minusculata CGMCC3.20098 a 100/1) D. brasiliensisCBS 133183 93/1 100/1} ' D. brasiliensisLGMF926 81/0.99 fl 100/I~L, D. caatingaensisURM7485 100/1 Ur D. caatingaensisURM7484 SS N 100/1r D. paranensis LMICRO417 D. paranensis CBS 133184 D. tarchonanthi CBS 146073 100/Ir D. oxe CBS 133187 D. oxe CBS 133186 D. arezzoensisMFLU 19-2880 0.05 Figure 3. Phylogram of Diaporthe spp. constructed using the ITS, tub2, tef1, cal and his3 gene sequences. The ML and BI bootstrap support values above 70% and 0.90 BYPP are shown at the first and second positions, respectively. The codes referring to strains from the current study are indicated in red. MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 The parameters of the GTR model used to analyze the dataset were estimated based on the following frequencies: A = 0.216529, C = 0.322542, G = 0.239361, T = 0.221568; substitution rates AC = 1.141925, AG = 3.613388, AT = 1.476437, CG = 1.061871, CT = 5.063639 and GT = 1.0000, as well as the gamma distribution 299 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) shape parameter a = 0.783643. For BI analysis, the “MrBayes on XSEDE” applica- tion was utilized along with the “GTR” model. Similar tree topologies were obtained by ML and BI methods, and the best scoring ML tree is shown in Fig. 3. Two new strains clustered into an independent clade with close relationships to D. passiflo- rae Crous & L. Lombard (strain DJY16A1-5). Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analysis Afive-locus concatenated dataset (ITS, cal, tub2, tef7, his3) was used to deter-mine the recombination level within D. podocarpi-macrophylli (CGMCC3.18281), D. podocarpi-macrophylli (LC6229), D. pseudooculi (B3180) and SC18 (Fig. 4), whereas a three-locus concatenated dataset (ITS, tub2, tef1) was used to de- termine the recombination level within D. eucommiigena (GUCC 420.9), D. malo- rum (CAA734), D. passiforae DJY16A1-5, and strains SC8 (Fig. 5). Chaiwan et al. (2022) noted that if the PHI is below the 0.05 threshold (Ow< 0.05), it indicates that there is significant recombination in the dataset. This means that related species in a group and recombination levels are not different. If the PHI is above [10.01 D. podocarpi-macrophylli CGMCC3.18281 D. pseudooculi B3180 SC18 D. podocarpi-macrophylli LC6229 Figure 4. Results of the pairwise homoplasy index (PHI) test of the new Diaporthe strains and its closely-related species using both LogDet transformation and splits decomposi- tion. PHI test results (Ow) < 0.05 indicate significant recombination within the dataset. The new strains are in bold type. —— 0.001 D. malorum CAA734 _ SD. eutcommiigena GUCC 420.9 D. passiforae DJY 16A1-5 Figure 5. Results of the pairwise homoplasy index (PHI) test of the new Diaporthe strains and its closely-related species using both LogDet transformation and splits decomposi- tion. PHI test results (Ow) < 0.05 indicate significant recombination within the dataset. The new strains are in bold type. MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 300 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) the 0.05 threshold (®w > 0.05), it indicates that it is not significant, which means that the related species in a group level are different. The result of the pairwise homoplasyindex (PHI) test of D. podocarpi-macrophylli (CGMCC3.18281), D. podocarpi-macrophylli (LC6229), D. pseudooculi (B3180) and strains SC18,was 1.0 and revealed that those species and strains SC18 were different (Fig. 4). The result of the pairwise homoplasy index (PHI) test of D. eucommiigena (GUCC 420.9), D. malorum (CAA734), D. passiforae DJY16A1-5, and strains SC8 was 1.0 and revealed that those species and strains SC8 were different (Fig. 5). Taxonomy Diaporthe liupanshuiensis C. G. Ren, sp. nov. Index Fungorum: IF901897 Fig. 6 Diagnosis. Distinguished from the phylogenetically closely related species D. podocarpi-macrophylli by its shorter alpha conidia. Etymology. Referring to the locality of the holotype, Liupanshui City, Guizhou Province, China. Figure 6. Diaporthe liupanshuiensis sp. nov. (SC-18). A. Upper view of the colony; B. Reverse view of the colony; C. Con- idiomata; D-F. Conidiogenous cells; G. Alpha conidia. Scale bars: 10 um (D-F); 5 um (G). MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 301 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Table 2. Morphological comparison of the new species with other Diaporthe species. Taxon conidiogenous Layer Alpha conidia Beta-conidia References D. arecae not observed 7.2-9.6 x 2.4 um 14.4-24 x 1.2 um Pereira et al. 2023 D. pseudooculi Conidiophores 5-12 x 2-5 um, Conid- | 6-9 x 2-3.5 um (ay, 7.3 21.5-33.9 © t= Yang et al. iogenous cells, 12-18 x 2 um x 2.8 um, n = 50) 1.7 um (av.27.0 x 2021 1.4 um, n = 30) D. podocarpi-macrophylli Alpha conidiophores 6-18 x 1.5-3um | 3.5-8.5x1-3um(x= | 8.5-31.5x0.5-2 um Gao etal. (x =12.3+2.6x2.1+0.3,n=30).Beta) 6.3+1.7x2.1+0.7, X= TOS ETE. 2017 conidiophores 10.5-27 x 1.5-2.5 um n= 50) 0.4, n = 30), (x = 15.3+ 4.3 x 2.1 +0.3,n = 30). SC-18 Conidiophores reduce to conidiog- 2.5-6.9 x 1.1-2.7 um not observed. This study enous cells. Conidiogenous cells: (mean = 5.4 x 2.2,n = 50) 18.1-40.4 x 1.2-2.5 um (mean = 30 x 1.8, n = 30). Description. Conidiomata: pycnidial, spherical or conical, black, and scat- tered and secrete irregular yellow conidial horns at the top when mature. Conid- iophores reduce to conidiogenous cells. Conidiogenous cells: colorless, trans- parent, upright, elongate cylindrical; size, 18.1-40.4 x 1.2-2.5 um (mean = 30 x 1.8, n = 30). Alpha conidia: transparent, smooth, undivided, cylindrical to fusi- form, sharp at both ends or round at one end, and slightly sharp at one end; size, 2.5-6.9 x 1.1-2.7 um (mean = 5.4 x 2.2, n = 50). Beta conidia: not observed. Culture characteristics. After 15 days of culture on PDA in the dark at 25 °C, the surface of the colony was white and the opposite side was light brown, with one or more concentric rings. Holotype. CHINA * The Guizhou Province: Liupanshui City (26°27'18.35'N, 105°02'45.60"E), from kiwifruit soft rot, October 11, 2023, Chunguang Ren (ho- lotype GZMHT SC-18.; ex-type living SC-18; living culture: SC-19 and SC-20). Notes. The three strains of D. liupanshuiensis sp. nov. were clustered into an independent clade with a close relationship with D. podocarpi-macrophyl- li and D. pseudooculi with high bootstrap value (0.94 BI). Compared with the typical characteristics of the known species (Table 2), D. liupanshuiensis sp. nov. differs from D. podocarpi-macrophylli and D. pseudooculi in that it possess- es smaller alpha conidia (2.5-6.9 x 1.1-2.7 um vs.3.5—-8.5 x 3 um and 6-9 x 2-3.5 um). Thus, the morphological characteristics and molecular phylogenet- ic results support D. liupanshuiensis as a new species. Diaporthe shuichengensis C.G. Ren sp. nov. Index Fungorum: IF901898 Fig:-7 Diagnosis. Diaporthe shuichengensis can be distinguished from the closely relat- ed species D. passiflorae and D. malorum based on the ITS, tef7, tub2, his3, and cal loci. Diaporthe shuichengensis differs from D. passiflorae in that it possesses longer alpha conidia and from D. malorum in that it possesses wider beta conidia. Etymology. Referring to the locality of the holotype, Shuicheng City, Guizhou Province, China. MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 302 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Figure 7. Diaporthe shuichengensis sp. nov. (SC-7). A. Upper view of the colony; B. Reverse view of the colony; C. Conidi- omata; D-E. Conidiogenous cells; F—H. Alpha- and beta-conidia. Scale bars: 10 um (D-E); 5 um (F-H). Description. Conidiomata: pycnidial, globose or conical, growing on the surface of pine needles, gray to black, with white villous hyphae on the sur- face. Conidiophores reduce to conidiogenous cells. Conidiogenous cells: col- orless, transparent, smooth, and without branches, and acuminate apex; size, 14.8-30.9 x 1.3-2.6 um (mean = 22.5 x 1.9, n = 30). Alpha conidia: transpar- ent, elliptic, obtuse at both ends, with 2 oil droplets, no septum; size, 5.2-8.1 x 1.3-2.8 um (mean = 6.6 x 1.9, n = 30); Beta conidia: unicellular, septate, and linear; one of their ends was straight and the other was slightly curved; size, 17.3-29.7 x 1.3-2.7 um (mean = 23.3 x 2.0, n = 30). Culture characteristics. After 15 days of culture on PDA at 25 °C under dark conditions, the colonies were white to light green in color, with the back appear- ing white to purple. Holotype. CHINA * The Guizhou Province: Shuicheng City (26°25'8.65'N, 104°57'33.67"E), from kiwifruit soft rot, October 11, 2023, Chunguang Ren; (ho- lotype GZMHT SC-7.; ex-type living culture: SC-7; living culture: SC-8). Notes. The two strains of D. shuichengensis sp. nov. formed a distinct clade with high bootstrap value (100% ML, 1 Bl); they were closely related to D. pas- siflorae, D. malorum and D. eucommiigena. Compared with the typical charac- MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 303 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Table 3. Morphological comparison of the new species with other Diaporthe species. Taxon conidiogenous Layer Alpha conidia Beta-conidia Gamma conidia ee D. malorum 4 on pine needles (5.0)—6.3-(7.5) x | on fennel twigs (17.4)— | not observed. | Santos (1.5)-2.2-(3.2) ym (mean +S.D.= | 21.5-(26.6) x (0.8)- et al. 6.3 +0.5x2.2+0.3um,n=100), | 1.3-(2.0) um (mean + 2017 on fennel twigs (5.6)-7.0-(8.7) x | S.D.=21.542.1x1.3+4 2.2-3.4 um (mean + S.D. = 7.0 + 0.3 um, n = 50). 0.6 x 2.8 + 0.3 um, n = 100). D. passiflorae Conidiophores hyaline, §.5=)6-7(48)* 2=)2 5=3(- (14-)16-18(-20) x 10-12 x Crous et 20-30 x 2.5-4 um. 3.5) ym. 1.5(-2) pm. 2-2.5ym. al. 2012 Conidiogenous cells, 7-15 x 1.5-2.5 pm D. eucommiigena | Conidiogenous cells 5.5-8 x 1.5-3 um (x = 7x 2.3 um; | 27-37 x 1-2 um (x = f > Ox Wang 12-27.5% 1.5-3 1m n = 30). 32x 1.3 um;n=10). | 1.5-2.5 um (x=) etal. (x = 19 x 2.2 um; n = 20) 8.6x2.1 um; | 2022b n= 20). sce-7 Conidiogenous cell 5.2-8.1 x 1.3-2.8 pm (mean = VAS 2 Te 3 not observed. This 14.8-30.9 x 1.3- 6.6 x 1.9,n = 30) 2.7 um (mean = 23.3 x study 2.6 um (mean = 22.5 x 2.0, n = 30) 1.9,n = 30) MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 teristics of the known species (Table 3). D. shuichengensis sp. nov. differs from D. passiflorae and D. malorum in that it possesses larger beta conidia 17.3- 29.7 x 1.3-2.7 um vs.(14—)16-18(-20) x 1.5(—2) um. and (17.4)-21.5-(26.6) x (0.8)—1.3-(2.0) um). D. shuichengensis was distinguished from eucommiigena by its shorter beta conidia (17.3-29.7 x 1.3-2.7 um vs. 27-37 x 1-2 um). Thus, the morphological characteristics and molecular phylogenetic results support D. shuichengensis as a new species. Pathogenicity test results The SC-7 and SC-18 strains were inoculated into healthy “Guichang” kiwifruits, which were then cultured at 25 °C and 85% humidity for 5-7 d. After 5 d of inoculation, liquid discharge was noted at the inoculation sites. After peeling, noticeable soft rot lesions were observed on the fruit surface; they were round or oval, and the flesh was softened. The cross-cut fruit displayed lesions that extended to the core, as well as rotten flesh and a bad odor (Fig. 8). No symp- toms were observed in the fruits of the control group (CK). Five days after inoc- ulation, isolates were obtained from the diseased fruits and cultured again. The morphological characteristics and cultural traits were consistent with those observed before inoculation; the strains were identified as pathogenic fungi. Discussion Kiwifruit soft rot poses a globally significant threat to postharvest quality. While Botryosphaeria dothidea and Diaporthe spp. are established primary pathogens (Kim et al. 2015; Zhou et al. 2015; Li et al. 2016; Nazerian et al. 2019), pathogen dominance varies regionally: B. dothidea prevails in South Korea, New Zealand, and Chinese provinces including Shaanxi, Jiangxi, Guizhou, Beijing, Zhejiang, and Anhui (Zhou et al. 2015), whereas Diaporthe species dominate in Turkey, Chile, and Chinese provinces such as Sichuan, Hunan, and Fujian (Diaz et al. 2017; Liu 304 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Figure 8. Lesions on kiwifruits inoculated with Diaporthe shuichengensis sp. nov. SC-7 and Diaporthe liupanshuiensis sp. nov. SC-18 strains. CK: Control group (inoculated with sterile water). et al. 2020). Our study reveals two novel Diaporthe species associated with this disease in Guizhou, China. Critically, these findings must be interpreted within the framework of the major taxonomic revision of Diaporthe proposed by Dissanayake et al. (2024), which consolidates numerous species into refined complexes using multi-locus phylogenetics (ITS, tef7, tub2, cal, his3) and challenges historical over- reliance on host association for species delimitation. The integration of morphological and molecular approaches has advanced the systematics of Diaporthe, with ITS, tub2, cal, tef1, and his3 loci proving effec- tive for species discrimination (Gao et al. 2016; Yang et al. 2018b, 2020, 2021). Although Index Fungorum records approximately 1,201 species in this genus, Dissanayake et al. (2024) note that traditional taxonomy based on morpholo- gy, host association, and multi-gene phylogenies may lead to overestimation or underestimation of species diversity. Their study delineates several phylogeneti- cally distinct sections within the genus, emphasizing that future research should focus on species within relevant sections for accurate phylogenetic placement. Our phylogenetic approach explicitly aligns with Dissanayake et al.’s (2024) framework. Strains SC-18, SC-19, and SC-20 (D. liupanshuiensis) formed a dis- tinct, well-supported lineage within the redefined D. arecae species complex (Fig. 2), exhibiting close affinity yet clear separation from D. podocarpi-mac- rophylli and D. pseudooculi (ML/BI support: 94%/1.00). Crucially, the Pairwise MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 305 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Homoplasy Index (PHI) test (Fig. 4, ®_w = 1.0, p > 0.05) detected no significant evidence of recombination, rejecting recombination between these taxa and supporting D. liupanshuiensis as an independent evolutionary lineage under the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) princi- ple. Morphologically, D. liupanshuiensis is distinguished by significantly smaller alpha conidia (2.5-6.9 x 1.1-2.7 um, Table 2). Similarly, strains SC-7 and SC-8 (D. shuichengensis) clustered within a clade adjacent to D. passiflorae and D. malorum (Fig. 3) with maximal support (100% ML/1.00 BI). The PHI test (Fig. 5) confirmed their genetic distinctiveness, while morphologically, D. shuichen- gensis possesses larger beta conidia than D. passiflorae and D. malorum but shorter than those of D. eucommiigena (Table 3). This integration of robust phy- logenetic isolation within the consolidated taxonomic framework, significant PHI values, and consistent morphological differences provides compelling evi- dence for the novelty of both D. liupanshuiensis and D. shuichengensis. We emphasize that Dissanayake et al.’s (2024) revision highlights the dynamic nature of species boundaries in Diaporthe. Expanded sampling—particularly in- cluding ex-type strains across diverse hosts and geographies—coupled with ge- nomic analyses, may reveal greater intraspecific variation within complexes rele- vant to our isolates (e.g., the D. arecae complex for SC-18). Should future studies adhering to this framework demonstrate that our isolates represent distinct lin- eages within redefined complexes (e.g., D. podocarpi-macrophylli), this would pri- marily expand the known host range and pathogenic potential of those consolidat- ed species, rather than negate their role as causal agents. This underscores the critical importance of depositing type cultures, sequences, and metadata (as im- plemented herein) to facilitate reevaluation within evolving taxonomic paradigms. This study identifies two novel Diaporthe species through integrated molecu- lar and morphological characterization, enriching our understanding of soft rot pathogens affecting ‘Guichang’ kiwifruit during storage and providing a founda- tion for disease management. Future research priorities include:(1) Elucidating the epidemiology and environmental triggers for these novel pathogens. (2) Assessing fungicide sensitivity profiles. (3) Investigating pathogenic molecular mechanisms. (4) Developing targeted control strategies. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of Al No use of Al was reported. Funding This work was supported by the National key research and development plan, integra- tion and demonstration of key technologies for improving quality and efficiency of kiwi- fruit industry in aquatic area (no. 2022YFD1601710) and Nanyong kiwi new variety (se- ries) introduction test demonstration (2021YFD1100300 Project 10 post-grant project). MycoKeys 121: 291-310 (2025), DOI: 10.3897/mycokeys.121.155321 306 Chunguang Ren et al.: Two new species of Diaporthe (Diaporthaceae, Diaporthales) Author contributions Each author played an indispensable role in this study. Ren Chunguang was mainly re- sponsible for experimental research and manuscript writing, Liu Yu and Su Wenwen provided experimental assistance, Han Zhengcheng was responsible for data analysis, and Professor Li Weijie was responsible for review and editing. Author ORCIDs Chunguang Ren @ https://orcid.org/0000-0003-2819-1489 Weijie Li © https://orcid.org/0000-0003-21 58-2356 Data availability All nucleotide sequences generated in this study have been deposited in GenBank under the following accession numbers: Diaporthe liupanshuiensis strain SC-18: ITS = PP537969, tefl = PP567111, tub2 PP567107, his3 = PP567102, cal = PP567097; Diaporthe shuichengensis strain SC-7: ITS = PP537966, tef1 = PP599035, tub2 PP567105, his3 = PP567100, cal = PP567095. 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