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Taxonomic Paper
Lasiodiplodia syzygii sp. nov. (Botryosphaeriaceae) causing post-harvest water-soaked brown lesions on Syzygium samarangense in Chiang Rai, Thailand
expand article infoChao-Rong Meng, Qian Zhang, Zai-Fu Yang, Kun Geng§, Xiang-Yu Zeng, K. W. Thilini Chethana|,, Yong Wang
‡ 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
¶ School of Science, Mae Fah Luang University, Chiang Rai, Thailand
Open Access

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.

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.

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 μl 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 LR0R and LR5 for 28S rRNA (LSU) region (Vilgalys and Hester 1990, Rehner and Samuels 1994). Two protein-coding gene fragments, the β-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

Isolate no.

GenBank no.

ITS

LSU

tef 1

tub2

Lasiodiplodia americana

CFCC50065T

KP217059

MF410052

KP217067

KP217075

L. avicenniae

CMW 414673T

KP860835

KP860680

KP860758

L. brasiliense

CMM 4015T

JX464063

JX464049

L. brasiliense

CMW 35884

KU887094

KU886972

KU887466

L. bruguierae

CMW 41470T

KP860833

KP860678

KP860756

L. caatinguensis

CMM 1325T

KT154760

KT008006

KT154767

L. caatinguensis

IBL 40

KT154762

KT154755

KT154769

L. chinensis

CGMCC3.18061T

KX499889

KX499927

KX500002

L. citricola

IRAN 1522CT

GU945354

GU945340

KU887505

L. crassispora

WAC12533T

DQ103550

DQ377901

EU673303

KU887506

L. euphorbicola

CMM 3609T

KF234543

KF226689

KF254926

L. exigua

CBS 137785T

KJ638317

KJ638336

KU887509

L. gilanensis

IRAN 1523CT

GU945351

GU945342

KU887511

L. gonubiensis

CMW 14077T

AY639595

DQ377902

DQ103566

DQ458860

L. gravistriata

CMM 4564T

KT250949

KT250950

L. hormozganensis

IRAN 1500CT

GU945355

GU945343

KU887515

L. hyalina

CGMCC3.17975T

KX499879

KX499917

KX499992

L. indica

IBP 01T

KM376151

L. iraniensis

IRAN 1520CT

GU945348

GU945336

KU887516

L. laeliocattleyae

CBS 167.28T

KU507487

DQ377892

KU507454

L. lignicola

CBS134112

JX646797

JX646814

KU887003

JX646845

L. macrospora

CMM 3833T

KF234557

KF226718

KF254941

L. mahajangana

CMW 27801T

FJ900595

FJ900641

FJ900630

L. margaritacea

CMW 26162T

EU144050

KX464354

EU144065

KU887520

L. mediterranea

CBS 137783T

KJ638312

KJ638331

KU887521

L. missouriana

UCD2193MOT

HQ288225

HQ288267

HQ288304

L. mitidjana

ALG111T

MN104115

MN159114

L. parva

CBS 456.78T

EF622083

KF766362

EF622063

KU887523

L. parva

CBS 494.78

EF622084

EU673258

EF622064

EU673114

L. plurivora

CBS 120832T

EF445362

KX464356

EF445395

KU887524

L. pontae

CMM 1277T

KT151794

KT151791

KT151797

L. pseudotheobromae

CBS 116459T

EF622077

EU673256

EF622057

EU673111

L. pyriformis

CMW 25414T

EU101307

EU101352

KU887527

L. rubropurpurea

WAC 12535T

DQ103553

DQ377903

DQ103571

EU673136

L. sterculiae

CBS 342.78T

KX464140

JX681073

KX464634

KX464908

L. subglobosa

CMM 3872T

KF234558

KF226721

KF254942

L. syzygii

MFLUCC 19-0219.1T

MT990531

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

MW081993

MW081990

MW087103

MW087106

L. thailandica

CPC 22795T

KJ193637

KJ193681

L. theobromae

CBS 164.96T

AY640255

EU673253

AY640258

KU887532

L. venezuelensis

WAC 12539T

DQ103547

DQ377904

DQ103568

KU887533

L. viticola

UCD 2553ART

HQ288227

HQ288269

HQ288306

L. vitis

CBS 124060T

KX464148

KX464367

KX464642

KX464917

Botryosphaeria dothidea

CMW 8000T

AY236949

AY928047

AY236898

AY236927

B. fabicerciana

CBS 127193T

HQ332197

MF410028

HQ332213

KF779068

Table 2.

DNA base pair differences between Lasiodiplodia syzygii and L. rubropurpurea in four separate loci. T = ex-type

L. syzygiumae strains

Lasiodiplodia rubropurpurea WAC 12535T

ITS (1–530)

LSU (531–1421)

TEF1-a(1422–1748)

β-tubulin (1749–2177)

MFLUCC 19-0257=GUCC 9719.1T

7

5

34

9

GUCC 9719.2

7

5

34

9

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 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. 4.0b10 (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 (CI), 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 IQ-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+I model was selected (-lnL = 1316.7068), for LSU, the TrN+I (-lnL = 1643.7273), for TEF1-a, the HKY+I+G (-lnL = 2399.0528) and for β-tubulin, the TIM3+G (-lnL = 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 β-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 1× 105 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.

Taxon treatment

Lasiodiplodia syzygii C.R. Meng, Qian Zhang & Yong Wang bis, sp. nov.

Materials    Download as CSV 
Holotype:
  1. scientificName:
    Lasiodiplodia syzygii
    ; 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
    ; dateIdentified:
    2020
    ; type:
    ex-type living culture GUCC 9719.1; MFLU 19-0565, isotype, isotype living culture MFLUCC 19-0257.
Other material:
  1. scientificName:
    Lasiodiplodia syzygii
    ; 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
    ; dateIdentified:
    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 textura angularis, becoming thin-walled and hyaline towards the inner region. Paraphyses cylindrical, aseptate, hyaline. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 10–14.5 × 3.5–4.5 μm (average = 11 × 3.7 μm, n = 20), hyaline, smooth, holoblastic forming conidia at their tips. Conidia thick-walled, wall up to 1 μm 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) × (13–)15–17(–20) μm (average = 31.3 × 16.4 μm, n = 50), L/W = 1.9.

Figure 1.  

One of 850 most parsimonious trees obtained from a combined analyses of the ITS, LSU, TEF1-a and β-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 B. dothidea (CMW 8000). The branch of the new Lasidiodiplodia species is highlighted with pink.

Figure 2.  

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 μm, c = 140 μm, d = 50 μm, e = 20 μm, f–m = 10 μm.

Culture characteristics: Conidia germinate on PDA within 24 hours at room temperature (25–30°C) with germ tubes produced from both ends of the conidia. Colonies with white fluffy mycelium on PDA, after 7 days become olivaceous-grey at the centre, white at the edge, raised, fluffy, dense filamentous.

Notes: 

Lasiodiplodia syzygii strains are closely related to L. rubropurpurea, but formed a distinct, well-supported clade in the phylogenetic analyses. Base-pairs comparisons between L. syzygii ex-type strain (GUCC 9719.1) and ex-type strain of L. rubropurpurea (WAC 12535) found seven base differences (1.3%) in ITS region and five differences (0.6%) on LSU, but nine differences (2.1%) in tub2 and 34 in TEF1-a (10.4%) (Table 2). Lasiodiplodia syzygii produced larger pycnidia (up to 2 mm) and larger conidiogenous cells (10–14.5 × 3.5–4.5 μm) than L. rubropurpurea (0.5–1.5 mm and 7–13 × 3–5 μm) (Burgess et al. 2006).

Etymology

In reference to the host from which the fungus was first isolated.

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 β-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, CI = 0.64, RI = 0.81, RC = 0.52 and HI = 0.36) and the first 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.  

Symptoms developing on Syzygium samarangense fruits inoculated with Lasiodiplodia syzygii. a. Symptom at 3rd day; b. Symptom at 5th day; c. Symptom at 7th day; d. Control.

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).

References