Biodiversity Data Journal 9: e60604 OO) doi: 10.3897/BDJ.9.e60604 open access Taxonomic Paper Lasiodiplodia syzygii sp. nov. (Botryosphaeriaceae) causing post-harvest water-soaked brown lesions on Syzygium samarangense in Chiang Rai, Thailand Chao-Rong Meng?, Qian Zhang}, Zai-Fu Yang*, Kun Geng§, Xiang-Yu Zeng}, K. W. Thilini Chethana!T, Yong Wangt* + Department of Plant Pathology, Agricultural College, Guizhou University, Guiyang, China § Guiyang plant protection and inspection station, Guiyang, China | Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand 4] School of Science, Mae Fah Luang University, Chiang Rai, Thailand Corresponding author: Yong Wang (yongwangbis@aliyun.com) Academic editor: Renan Barbosa Received: 10 Nov 2020 | Accepted: 23 Dec 2020 | Published: 07 Jan 2021 Citation: Meng C-R, Zhang Q, Yang Z-F, Geng K, Zeng X-Y, Thilini Chethana KW, Wang Y (2021) Lasiodiplodia syzygii sp. nov. (Botryosphaeriaceae) causing post-harvest water-soaked brown lesions on Syzygium samarangense in Chiang Rai, Thailand. Biodiversity Data Journal 9: e60604. https://doi.org/10.3897/BDJ.9.e60604 Abstract Background Syzygium samarangense (Wax apple) is an important tropical fruit tree with high economic and nutrient value and is widely planted in the tropics or subtropics of Asia. Post-harvest water-soaked brown lesions were observed on mature fruits of ornamental wax apples in Chiang Rai Province, Thailand. A fungus with morphological characters, similar to Lasiodiplodia, was consistently isolated from symptomatic fruits. Phylogenetic analyses, based on ITS, LSU, TEF1-a and tub2, revealed that our isolates were closely related to, but phylogenetically distinct from, Lasiodiplodia rubropurpurea. © Meng C etal. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Meng C et al New information Morphological comparisons indicated that pycnidia and conidiogenous cells of our strains were significantly larger than L. rubropurpurea. Comparisons of base-pair differences in the four loci confirmed that the species from wax apple was distinct from L. rubropurpurea and a new species, L. syzygii sp. nov., is introduced to accommodate it. Pathogenicity tests confirmed the newly-introduced species as the pathogen of this post-harvest water-soaked brown lesion disease on wax apples. Keywords Botryosphaeriaceae, fruit disease, new pathogen, wax apple Introduction Wax apple [Syzygium samarangense (Blume) Merrill and Perry] belongs to the Myrtaceae and was naturalised in the Philippines thousands of years ago (Lim 2012, Shen et al. 2012). As a kind of juicy tropical fruit like watermelon with economic importance, it has been commonly and widely cultivated in many Asian countries (Nesa et al. 2014). Every part of S. samarangense also has potential medicinal values (Shen et al. 2012). Due to the fruit characteristics, such as thin peel and tender pulp with high respiratory intensity, wax apples are prone to damage by pathogens and cannot be stored for a long time (Yang et al. 2009). This causes a significant post-harvest loss. Many studies suggest that wax apple is mainly threatened by fungal diseases. For example, a new fruit rot of wax apple caused by Phytophthora palmivora was reported in southern Taiwan during the rainy periods in 1982 (Lin et al. 1984). Yang et al. (2009) and Che et al. (2015) reported Lasiodiplodia theobromae as the causal agent of black spot disease on harvested wax apple fruits. Pestalotiopsis samarangensis was isolated from the fruit rot in wax apples from markets in Thailand (Maharachchikumbura et al. 2013). Chrysoporthe deuterocubensis caused cankers on wax apple and branches in Taiwan (Fan et al. 2013). The present study reports a new post-harvest water-soaked brown lesion disease on wax apples caused by Lasiodiplodia sp. in Chiang Rai, Thailand. Morphological and multi-locus phylogenetic analyses revealed that our strain represented a novel species. A pathogenicity test on fruits confirmed the pathogenic relationship between L. syzygii and Syzygium samarangense. Lasiodiplodia syzygii sp. nov. (Botryosphaeriaceae) causing post-harvest ... 3 Materials and methods Sample collection, isolation and morphology Rotten wax apple fruits were occasionally collected from a food market near Mae Fah Luang University in Chiang Rai, Thailand. On the third day after the wax apple fruits were collected, it was observed that there were conidiomata bulges on the surface of the fruit, white hyphae and the fruit turned black, rotted and had cytoplasmic extravasation. Diseased samples were conserved in self-sealing bags and then taken back to the laboratory and photographed. Before isolation, diseased fruits were surface disinfected with 70% ethanol for 30 s, 1% sodium hypochlorite (NaClO) for 1 min and repeatedly twice rinsed in sterile distilled water for 30 s. Pure cultures were obtained by single-conidium isolation following a modified method outlined by Chomnunti et al. (2011) and Maharachchikumbura et al. (2013). The morphology of fungal colonies was recorded following the method of Hu et al. (2007). Fungal mycelium and spores were observed under a light microscope and photographed. The holotype specimen is deposited in the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). The ex-type and isotype cultures are deposited in the Culture Collection at the Department of Plant Pathology, Agriculture College, Guizhou University, P.R. China (GUCC) and the Mae Fah Luang University Culture Collection (MFLUCC) in Thailand. DNA extraction, PCR reaction and sequencing Fungal cultures were grown on PDA at 28°C. When colonies nearly covered the entire Petri dish (90 mm diam.), fresh mycelia were scraped from the agar surface with sterilised scalpels. Genomic DNA was extracted using a BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416) following the manufacturer’s protocol. DNA amplification was performed in a 25 ul reaction volume following Liang et al. (2018). Primers ITS1 and ITS4 (White et al. 1990) were used to amplify the internal transcribed spacer regions and intervening 5.8S rRNA region (ITS) and LROR and LR6d for 28S rRNA (LSU) region (Vilgalys and Hester 1990, Rehner and Samuels 1994). Two protein-coding gene fragments, the B-tubulin (tub2) and translation elongation factor 1-alpha (TEF1-a) were amplified with primer pairs BT2A/ BT2B (Glass and Donaldson 1995, O'Donnell and Cigelnik 1997) and EF1-688F/ EF1-986R, respectively (Carbone and Kohn 1999, Alves et al. 2008). Purification and sequencing of the PCR amplicons were done by SinoGenoMax, Beijing. The DNA sequences are deposited in the GenBank and their accession numbers are provided in Table 1. The DNA base differences of the four loci amongst our strains and ex-type or representative strains of relative taxa are shown (Table 2). Table 1. Table 1 GenBank accession numbers of isolates included in this study. Ex-type isolates are labelled with superscript T. Species Lasiodiplodia americana eS ete ee eS ren ee eee aor Le [ser ore ee ee i i et | ee ee (Es Le L. L. L. L. pseudotheobromae . avicenniae . brasiliense . brasiliense . bruguierae . caatinguensis . caatinguensis . chinensis . citricola . Crassispora . euphorbicola . exigua . gilanensis . gonubiensis . gravistriata . hormozganensis . hyalina . indica . iraniensis . laeliocattleyae . lignicola . Macrospora . mahajangana . Margaritacea . mediterranea . missouriana mitidjana parva parva plurivora pontae Isolate no. CFCC50065' CMW 414673" CMM 4015" CMW 35884 CMW 41470' CMM 1325° IBL 40 CGMCC3.18061" IRAN 1522CT WAC12533! CMM 3609! CBS 137785" IRAN 1523C' CMW 14077" CMM 4564' IRAN 1500C' CGMCC3.17975' IBP 017 IRAN 1520CT CBS 167.287 CBS134112 CMM 3833! CMW 278017 CMW 26162" CBS 137783! UCD2193MOT ALG1117 CBS 456.78" CBS 494.78 CBS 120832" CMM 1277! CBS 116459! Meng C etal GenBank no. ITS KP217059 KP860835 JX464063 KU887094 KP860833 KT154760 KT154762 KX499889 GU945354 DQ103550 KF 234543 KJ638317 GU945351 AY639595 KT250949 GU945355 KX499879 KM376151 GU945348 KU507487 JX646797 KF 234557 FJ900595 EU144050 KJ638312 HQ288225 MN104115 EF622083 EF622084 EF445362 KT151794 EF622077 LSU MF410052 DQ377901 DQ377902 DQ377892 JX646814 KX464354 KF 766362 EU673258 KX464356 EU673256 tef 1 KP217067 KP860680 JX464049 KU886972 KP860678 KT008006 KT154755 KX499927 GU945340 EU673303 KF 226689 KJ638336 GU945342 DQ103566 KT250950 GU945343 KX499917 GU945336 KU507454 KU887003 KF226718 FJ900641 EU144065 KJ638331 HQ288267 MN159114 EF622063 EF622064 EF445395 KT151791 EF622057 tub2 KP217075 KP860758 KU887466 KP860756 KT154767 KT154769 KX500002 KU887505 KU887506 KF254926 KU887509 KU887511 DQ458860 KU887515 KX499992 KU887516 JX646845 KF254941 FJ900630 KU887520 KU887521 HQ288304 KU887523 EU673114 KU887524 KT151797 EU673111 Lasiodiplodia syzygii sp. nov. (Botryosphaeriaceae) causing post-harvest ... Species Isolate no. GenBank no. ITS LSU tef 1 tub2 L. pyriformis CMW 254147 EU101307 - EU101352 KU887527 L. rubropurpurea WAC 12535! DQ103553 DQ377903 DQi103571 EU673136 L. sterculiae CBS 342.78" KX464140 JX681073 KX464634 KX464908 L. subglobosa CMM 3872! KF234558 - KF226721 KF 254942 L. syzygii MFLUCC 19-0219.1' M1T990531 MT990548 MW016943 MW014331 L. syzygii GUCC 9719.2 MW081991 MW081988 | MW087101 MW087104 L. syzygii GUCC 9719.3 MW081992 MW081989 MW087102 MW087105 L. syzygii sp. nov. GUCC 9719.4 MVWV081993 MW081990 MW087103 MW087106 L. thailandica CPC 22795! KJ193637 - KJ193681 - L. theobromae CBS 164.96" AY640255 EU673253 AY640258 KU887532 L. venezuelensis WAC 12539! DQ103547 DQ377904 DQ103568 KU887533 L. viticola UCD 2553AR™ HQ288227 - HQ288269 HQ288306 L. vitis CBS 124060" KX464148 KX464367 KX464642 KX464917 Botryosphaeria dothidea CMW 8000! AY236949 AY928047 AY 236898 AY236927 B. fabicerciana CBS 127193" HQ332197 MF410028 HQ332213 KE779068 Table 2. DNA base pair differences between Lasiodiplodia syzygii and L. rubropurpurea in four separate loci. 5 oe = ex-type L. syzygiumae strains Lasiodiplodia rubropurpurea WAC 12535 ITS (1-530) — LSU (531-1421) TEF1-a(1422-1748) @-tubulin (1749-2177) MFLUCC 19-0257=GUCC 9719.1" 7 5 34 9 GUCC 9719.2 7 5 34 GUCC 9719.3 7 5 34 9 GUCC 9719.4 7 5 34 9 Total number of differences 55 Phylogenetic analyses Sequences of 45 Lasiodiplodia isolates, representing all species known from culture, were aligned using the online version of MAFFT v. 7.307 (Katoh and Standley 2016) and manually improved, where necessary, using MEGA v. 6.06 (Koichiro et al. 2013). Mesquite v. 2.75 (Maddison 2008) was used to concatenate the aligned sequences of the different loci. Ambiguous regions were excluded from analyses using AliView (Larsson 2014), gaps were treated as missing data and optimised manually with Botryosphaeria dothidea (CMW8000) and B. fabicerciana (CBS 127193) as the outgroups (Table 1). The alignment document has been deposited in TreeBASE (www.treebase.org) and the accession 6 Meng C etal number is 27461. Phylogenetic analyses were constructed by Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference methods. First, the ambiguous regions were excluded from the alignment and gaps were treated as missing data. The MP analysis was done with PAUP v. 4.0610 (Swofford 2002), using the heuristic search option with 1,000 random taxa addition and tree bisection and reconnection (TBR) as the branch swapping algorithm. Maxtrees was set to 5000. Tree length (TL), consistency index (Cl), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for each tree generated. The Maximum Likelihood (ML) analysis was performed using I|Q-tree (Nguyen et al. 2015, Chernomor et al. 2016). Nucleotide substitution models were selected under the Akaike Information Criterion (AIC) by jModelTest2 (Darriba et al. 2012) on XSEDE in the CIPRES web portal (Miller et al. 2010). For the ITS dataset, the TPM3uf+!l model was selected (-InL = 1316.7068), for LSU, the TrN+1 (-InL = 1643.7273), for TEF1-a, the HKY+I+G (-InL = 2399.0528) and for B-tubulin, the TIM3+G (-InL = 1161.0392). ML was inferred under partitioned models. Non-parametric bootstrap analysis was implemented with 1000 replicates. Bayesian Inference (BI) analyses was conducted in MrBayes 3.2 (Ronquist et al. 2012). MrModeltest v.2.3 (Nylander 2004) was used to estimate the best evolutionary models under the Akaike Information Criterion (AIC). HKY+I was selected as the best model for ITS, for LSU, HKY+I+G, for TEF1-a, HKY+I+G and for B-tubulin, GTR+G was selected as the best model. Six Markov Chain Monte Carlo runs were launched with random starting trees for 1,000,000 generations and sampling every 1,000 generations. The first 25% resulting trees were discarded as burn-in. Pathogenicity tests One isolate of the new Lasiodiplodia species (GUCC 9719.1) was grown on PDA and when the cultures covered the entire surface of the Petri dish, mycelia were scraped off with a sterilised blade. Conidiomata were crushed with a glass rod to prepare a spore suspension of 1x 10° spores/ml. Pathogenicity testing was carried out on five healthy fruits of wax apple bought from the market. Inoculations were carried out in April 2020. The surface of the fruits was wiped with 70% ethanol and allowed to air-dry. Three fruits were slightly wounded by pin-pricking and 3 ml of spores suspension was sprayed on to the wound. The other two wounded fruits were maintained as control and inoculated with 2 ml of sterile deionised water. All inoculated fruits were placed in plastic bags, labelled and a high level of humidity was maintained for seven days by the addition of wet sterile cotton wool in each bag in an illuminated incubator at 28 + 3°C. Daily observations were made on the development of disease symptoms. When fruits developed the symptoms, they were removed from the bags. Two isolates obtained from the diseased tissue were grown on PDA and then sequenced with primer pairs of the above four DNA markers to confirm the identity. Lasiodiplodia syzygii sp. nov. (Botryosphaeriaceae) causing post-harvest ... t Taxon treatment Lasiodiplodia syzygii C.R. Meng, Qian Zhang & Yong Wang bis, sp. nov. MycoBank 837701 Materials Holotype: a. scientificName: Lasiodiplodia syzygil; kingdom: Fungi; class: Dothideomycetes; order: Botryosphaeriales; family: Botryosphaeriaceae; genus: Lasiodiplodia; country: Thailand; stateProvince: Chiang Rai; catalogNumber: HGUP 9719; recordedBy: Wang Yong; identifiedBy: Chao-Rong Meng; dateldentified: 2020; type: ex-type living culture GUCC 9719.1; MFLU 19-0565, isotype, isotype living culture MFLUCC 19-0257. Other material: a. scientificName: Lasiodiplodia syzygil; kingdom: Fungi; class: Dothideomycetes; order: Botryosphaeriales; family: Botryosphaeriaceae; genus: Lasiodiplodia; country: China; stateProvince: Guiyang; catalogNumber: HGUP 9720 and HGUP 9721; recordedBy: Wang Yong; identifiedBy: Chao-Rong Meng; dateldentified: 2020; type: living cultures GUCC 9719.2, GUCC 9719.3 and GUCC 9719.4 Description Pathogenic on Syzygium samarangense. Sexual morph: Undetermined. Asexual morph (Fig. 2): Conidiomata up to 2 mm diam., pycnidial, covered with hyphae, black, globose, ostiolate, solitary, separate, uniloculate, immersed to semi-immersed. Conidiomatal wall composed of thick-walled, dark brown cells of ftextura angularis, becoming thin-walled and hyaline towards the inner region. Paraphyses cylindrical, aseptate, hyaline. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 10-14.5 x 3.5-4.5 um (average = 11 x 3.7 um, n = 20), hyaline, smooth, holoblastic forming conidia at their tips. Conidia thick-walled, wal/ up to 1 um wide, ovoid with both ends rounded, hyaline and remaining so for a long time, becoming pale brown with obsolete striations and occasionally with 1-septate after discharging from the conidioma, (27—)30—32(—36) x (13—)15—17(—20) um (average = 31.3 Lasiodiplodia citricola |RAN 1522C T Lasiodiplodia mitidjana ALG111 T 89/79/0.96 _ | | asiodiplodia parva CBS 456.78 T Lasiodiplodia parva (cont).CBS 494.78 Lasiodiplodia euphorbicola CMM 3609 T Lasiodiplodia hormozganensis IRAN 1500C T Lasiodiplodia laeliocattleyae CBS 167.28 T Lasiodiplodia vitis CBS 124060 T Lasiodiplodia macrospora CMM 3833 T Lasiodiplodia subglobosa CMM 3872 T Lasiodiplodia gravistriata CMM 4564 T Lasiodiplodia avicenniae CMW 414673 T Lasiodiplodia pseudotheobromae CBS 116459 T Lasiodiplodia mediterranea CBS 137783 T 78I5S/-1| | | asiodiplodia sterculiae CBS 342.78 T Lasiodiplodia chinensis CGMCC3 18061 T Lasiodiplodia lignicola CBS134112 T 95/85/1_| | Lasiodiplodia americana CFCC50065 T Lasiodiplodia exigua CBS 137785 T Lasiodiplodia mahajangana CMW 27801 T 98/93/1_| || Lasiodiplodia caatinguensis CMM 1325 T Lasiodiplodia caatinguensis (cont).IBL 40 Lasiodiplodia viticola UCD 2553AR T 91/71/0.9 Lasiodiplodia brasiliense CMM 4015 T Lasiodiplodia brasiliense CMW 35884 T Lasiodiplodia theobromae CBS 164.96 T Lasiodiplodia indica IBP 01 T Lasiodiplodia bruguierae CMW 41470 T 90/98/1_ | 67/-/0.99_ Tr 82/89/0.99 99/95/0.98 TORRE 99/99/1 * Lasiodiplodia hyalina CGMCC3 17975 T 97/89/ Lasiodiplodia thailandica CPC 22795 T Lasiodiplodia iraniensis |RAN 1520C T Lasiodiplodia pontae CMM 1277 T Lasiodiplodia gilanensis |RAN 1523C T |_ Lasiodiplodia missouriana UCD2193MO T Lasiodiplodia plurivora CBS 120832 T Lasiodiplodia gonubiensis CMW 14077 T 82/84/0.99 Lasiodiplodia rubropurpurea WAC 12535 T Lasiodiplodia venezuelensis WAC 12539 T Lasiodiplodia crassispora WAC12533 T Lasiodiplodia pyriformis CMW 25414 T Lasiodiplodia margaritacea CMW 26162 T 400/100/1 ; Botryosphaeria dothidea CMW 8000 T Botryosphaeria fabicerciana CBS 127193 T 100/100/1 0.03 Figure 1. EES] One of 850 most parsimonious trees obtained from a combined analyses of the ITS, LSU, TEF1-a and B-tubulin sequence dataset. Bootstrap values > 50% and BPP values > 0.90 are provided at the nodes and separated by “/’. Bootstrap values < 50% and Bayesian posterior probability (BPP) values < 0.90 were labelled with “-”. The tree was rooted with Botryosphaeria fabicerciana (CBS 127193) and 8B. dothidea (CMW 8000). The branch of the new Lasidiodiplodia species is highlighted with pink. Lasiodiplodia syzygii sp. nov. (Botryosphaeriaceae) causing post-harvest ... 9 Figure 2. EES] Lasiodiplodia syzygii (MFLUCC 19-0257). a. infected fruit; b, c. Conidiomata on the host; d. Section through a conidioma; e. Conidia developing amongst paraphyses; f-h. Conidia formed on conidiogenous cells; i-m. Immature conidia; n-o. Colonies on PDA culture; n. From above; o. From below. Scale bars: b = 300 um, c = 140 um, d = 50 um, e = 20 um, f—m = 10 um. Analysis Phylogenetic analyses Four Lasiodiplodia strains isolated from Syzygium samarangense were sequenced. The final alignment of ITS, LSU, TEF1-a and tub2 comprised of 2177 characters, viz. ITS: 1— 530, LSU: 533-1423, TEF1-a: 1426-1752 and £-tubulin: 1755-2183. Of these, 1843 characters were constant and 73 were parsimony-uninformative. Maximum parsimony analysis of the remaining 261 parsimony-informative characters resulted in 850 most parsimonious trees (TL = 676, Cl = 0.64, RI = 0.81, RC = 0.52 and HI = 0.36) and the first 10 Meng C et al one is shown as Fig. 1. The ML and Bayesian analyses resulted in trees with similar topologies. Strains GUCC 9719.1, GUCC 9719.2, GUCC 9719.3 and GUCC 9719.4 formed an independent well-supported clade sister to Lasiodiplodia rubropurpurea (MP: 100%, ML: 100% and Bayesian posterior probability: 1) Comparison of the DNA base-pair differences between our strains and L. rubropurpurea species in four gene regions (Table 2) confirmed the presence of two species; therefore, a new species is introduced for those isolates from wax apple. Pathogenicity test on the fruits of wax apple At the third day after inoculation, water-soaked areas with a few white hyphae began to appear on all inoculated fruits similar to the naturally-infected wax apples (Fig. 2a and Fig. 3a). The water-soaked symptom of diffusion with abundant hyphae producing mycelium further appeared on inoculated Syzygium samarangense fruits after five days (Fig. 3b). At the 7th day after inoculation, the symptoms spread throughout the fruit (Fig. 3c), together with many white mycelia and more hyphae accompanied by cytoplasmic exosmosis. The control fruits (Fig. 3d) did not show any symptom. The fungi were re-isolated from the lesions of inoculated wax apple fruits and the re-identified (GUCC 9719.3 and GUCC 9719.4) sequencing four gene regions. Figure 3. EE Symptoms developing on Syzygium samarangense fruits inoculated with Lasiodiplodia syzygii. a. Symptom at 3 day; b. Symptom at 5" day; c. Symptom at 7" day; d. Control. Lasiodiplodia syzygii sp. nov. (Botryosphaeriaceae) causing post-harvest ... 11 Discussion This study revealed a new species of Lasiodiplodia, L. syzygi from rotting fruits of Syzygium samarangense. Phylogenetic analyses, based on ITS, LSU, TEF1-a and tub2, showed that it is phylogenetically closer to L. rubropurpurea. Comparisons of DNA base- pair differences in the four loci, as well as morphological differences, confirmed the novelty of this species. The fungus was proved to be pathogenic and, therefore, it is the causal agent of the post-harvest water-soaked brown lesions on wax apple. Wax apple (Syzygium samarangense) is known to be affected by many fungal pathogens that often cause economic losses. These include Colletotrichum gloeosporioides (Udayanga et al. 2013) and Lasiodiplodia theobromae which was the causal agent of black spot disease (Che et al. 2015), Pestalotiopsis spp. and Phytophthora spp. The fruit disease of the current study did not show any typical symptoms of black spot caused by L. theobromae. Furthermore, the pink or orange spore masses, typical of anthracnose caused by C. gloeosporioides or epidermal to superficial, acervular conidiomata reported by Maharachchikumbura et al. (2013) for Pestalotiopsis, were not seen in the current study. The fruit rot caused by Phytophthora spp. spread more rapidly (only 2 or 3 days up to a whole fruit) and results in a sour taste on fruits. However, the L. syzygii needed about seven days to completely rot the fruit and did not cause any sour taste in the fruits. Thus, the study reports a new disease on wax apple. Lasiodiplodia resides in Botryosphaeriaceae, Botryosphaeriales (Hongsanan et al. 2020) and comprises several species known to cause important or potentially important diseases on woody hosts, mostly in the tropics or sub-tropics (Phillips et al. 2019). Very few species of this family appear to be host-specific (Dissanayake et al. 2016). In south-western China and adjoining areas, agriculture and forestry play an important role in the local economy, which might facilitate the spread of this wax apple disease. Thus, research needs to focus on the occurrence of this newly-discovered pathogen in other economically-important plants and in other locations, as well as how to manage it by biological or chemical control approaches. It is also remarkable to find a new disease on such an important commercial fruit indicating that there are numerous new taxa to be discovered in Thailand (Hyde et al. 2018) and Botryosphaeriaceae (Hyde et al. 2020). Acknowledgements This research is supported by the following projects: National Natural Science Foundation of China (No. 31972222, 31560489), Program of Introducing Talents of Discipline to Universities of China (111 Program, D20023), Talent Project of Guizhou Science and Technology Cooperation Platform ({[2017]5788-5 and [2019]5641), Guizhou Science, Technology Department of International Cooperation Base project ([2018]5806) and Guizhou Science and Technology Innovation Talent Team Project ([2020]5001) and Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (RDg6130001). 12 Meng C etal References ° Alves A, Crous PW, Correia A, Phillips AJL (2008) Morphological and molecular data reveal cryptic speciation in Lasiodiplodia theobromae . Fungal Diversity 28: 1-13. ° Burgess Tl, Barber PA, Mohali S, Pegg G, de Beer W, Wingfield MJ (2006) Three new Lasiodiplodia spp. from the tropics, recognized based on DNA sequence comparisons and morphology. 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