Biodiversity Data Journal : Taxonomic Paper
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Taxonomic Paper
Allophoma species (Pleosporales: Didymellaceae) associated with Thunbergia grandiflora in Guangxi Province, China
expand article infoJun Yuan, Xiang-Yu Zeng, Kun Geng§, Nalin N. Wijayawardene|, Jayarama D. Bhat, Shi-Ping Wu#, Yong Wang, Zai-Fu Yang
‡ Department of Plant Pathology, Agricultural College, Guizhou University, Guiyang, China
§ Guiyang plant protection and inspection station, Guiyang, China
| Center for Yunnan Plateau Biological Resources Protection and Utilization, Qujing Normal University, Qujing, China
¶ No. 128/1-J, Azad Co-Op Housing Society, Curca P.O, Goa, India
# The Institute of Plant Protection, Guizhou Academy of Agricultural Sciences, Guiyang, China
Open Access

Abstract

Background

Thunbergia grandiflora belongs to the family Acanthaceae and is a widely distributed dicotyledonous plant in tropical and subtropical regions. Three isolates of Allophoma (Dothideomycetes, Pleosporales, Didymellaceae) were collected from leaves of T. grandiflora in Guangxi Province, China.

New information

Phylogenetic analyses of a combined ITS–LSU–rpb2tub2 dataset indicate that one of our three strains represents an undescribed species with close affinity to A. minor and the other two strains clustered amongst other isolates of A. pterospermicola. Evidence from morphology and sequence analysis indicates that GUCC 2070.7 is a new species that we introduce here as A. thunbergiae. This is the first report about taxa of Allophoma from this host plant.

Keywords

one new species, Didymellaceae, phylogeny, taxonomy

Introduction

Didymellaceae was established by De Gruyter et al. (2009) with Didymella as the type genus. It is the largest family in the Pleosporales and accommodates more than 5400 taxon names (Crous et al. 2004), including saprobic, endophytic and pathogenic species (Aveskamp et al. 2008, Aveskamp et al. 2010, Marin-Felix et al. 2017). A great part of Didymellaceae species are reported as plant pathogens, which cause severe economic losses to many crops (Aveskamp et al. 2008). Recently, Didymellaceae was revised, based on morphological and phylogenetic analyses of ex-type sequences of LSU, ITS, rpb2 and tub2 loci, resulting in 19 genera (Chen et al. 2015, Chen et al. 2017), Currently, 37 genera are accepted (Wijayawardene et al. 2017, Wijayawardene et al. 2020, Valenzuela-Lopez et al. 2018, Hou et al. 2020a, Hou et al. 2020b, Phukhamsakda et al. 2020).

Allophoma is presently accepted with 14 species (Hongsanan et al. 2020, Hou et al. 2020a, Hyde et al. 2020, Wijayawardene et al. 2020) and two of them were firstly obtained from Guizhou and Guangxi Provinces, China (Chen et al. 2017, Marin-Felix et al. 2019). The genus includes several important plant-pathogenic taxa, for example, Allophoma labilis (basionym: Phoma labilis), which often cause leaf necrosis, canker and stem lesions or stem rot, resulting in a negative effect on the health of plants (Zimowska 2011, Garibaldi et al. 2012, Nagarjun and Suryanarayana 2016, O'Neill and Mayne 2016, Babaahmadi et al. 2018,Jayasiri et al. 2019). Allophoma is characterised by superficial or immersed pycnidial conidiomata with ostioles, a 2−5-layered pseudo-parenchymatous wall, phialidic conidiogenous cells and aseptate variously-shaped, mostly guttulate conidia. The size of the pycnidia, conidiogenous cells and conidia are used to distinguish amongst different species in Allophoma (Chen et al. 2015).

In recent years, most species of fungi have been described from Asia, mostly China (Cheek et al. 2020). Our research group investigates the fungi on medicinal plants in south-western China, which has, thus far, resulted in the discovery of several new taxa (Long et al. 2019, Zhang et al. 2020a, Zhang et al. 2020b, An et al. 2021). Here, we studied diseased leaves of Thunbergia grandiflora collected from the Medicinal Botanical Garden in Nanning City, Guangxi Province, China. Following isolation, purification, morphological examination and phylogenetic analyses, a new species and one known species were discovered.

Materials and methods

Isolation and morphological study

The samples were collected in 2017 at the Medicinal Botanical Garden, Nanning, Guangxi, China. Single spore isolates were obtained on oatmeal agar (OA), malt extract agar (MEA) and potato dextrose agar (PDA), followed by incubation at 25 °C. Colony diameters were measured after 1 week (Boerema et al. 2004). The colour of colonies of inoculated Petri dishes was determined following Rayner (1970). Morphological structures were examined and photographed using a Nikon Eclipse 80i microscope. Micro-morphological descriptions and measurements of mature conidiomata, conidia and conidiogenous cells on OA or MEA and PDA cultures were based on Aveskamp et al. (2010). The holotype specimen is deposited at the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). An ex-type culture of the new taxon is deposited at the Culture Collection of the Department of Plant Pathology, Agriculture College, Guizhou University (GUCC) (Table 1).

Table 1.

Sequences that were used for phylogenetic analysis. The accession numbers in bold are those generated in this study. Ex-type strains are marked by an asterisk (*).

Species

Strain number

GenBank accession numbers

LSU

ITS

rpb2

tub2

Allophoma alba

CBS 120422

MN943671

MN973469

MT018044

MT005568

A. anatii

CBS 124673

MN943674

MN973472

MT018048

MT005571

A. cylindrispora

CBS 142453*

LN907376

LT592920

LT593058

LT592989

A. hayatii

CBS 142859

KY684814

KY684812

MF095108

KY684816

A. hayatii

CBS 142860

KY684815

KY684813

MF095109

KY684817

A. labilis

CBS 124.93

GU238091

GU237765

KT389552

GU237619

A. minor

CBS 325.82*

GU238107

GU237831

KT389553

GU237632

A. nicaraguensis

CBS 506.91*

GU238058

GU237876

KT389551

GU237596

A. oligotrophica

CBS 497.91

GU238059

GU237870

GU237597

LT623247

A. oligotrophica

CGMCC 3.18114*

KY742194

KY742040

KY742128

KY742282

A. oligotrophica

CGMCC 3.18115

KY742195

KY742041

KY742129

KY742283

A. oligotrophica

CGMCC 3.18116

KY742196

KY742042

KY742130

KY742284

A. piperis

CBS 268.93*

GU238129

GU237816

KT389554

GU237644

A. piperis

CBS 108.93

GU238130

GU237921

KT389555

GU237645

A. pterospermicola

CGMCC 3.19245*

MK088580

MK088573

MK088587

MK088594

A. pterospermicola

LC12181

MK088576

MK088569

MK088583

MK088590

A. pterospermicola

LC12182

MK088577

MK088570

MK088584

MK088591

A. pterospermicola

LC12183

MK088578

MK088571

MK088585

MK088592

A. pterospermicola

LC12184

MK088579

MK088572

MK088586

MK088593

A. pterospermicola

GUCC2070.6

MW040200

MW036297

MW116818

MW116822

A. pterospermicola

GUCC2070.3

MW040199

MW036296

MW116817

MW116821

A. siamensis

MFLU 17-2281

MK347959

MK347742

MK434912

MK412867

A. thunbergiae

GUCC2070.7

MW040201

MW036298

MW116819

MW116823

A. tropica

CBS 436.75*

GU238149

GU237864

KT389556

GU237663

A. zantedeschiae

CBS 131.93

GU238159

FJ427084

KT389557

FJ427188

A. zantedeschiae

CBS 229.32

KT389690

KT389473

KT389558

KT389767

A. zantedeschiae

ICMP 16850

KY742197

KY742043

KY742131

KY742285

Stagonosporopsis loticola

CBS 562.81*

GU238192

GU237890

KT389684

GU237697

DNA isolation, PCR and sequencing

Fungal mycelia were scraped off the surface of the pure culture plate with a sterile scalpel. Total genomic DNA was extracted using the A BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416, BIOMIGA, San Diego, California, USA). Four loci of each fungal strains were amplified, including the internal transcribed spacer (ITS) region with primers V9G (De Hoog and Van den Ended 1998) and ITS4 (White et al. 1990); the large subunit (LSU) of the ribosomal RNA gene with primers LR0R (Hopple 1994), LR5 and LR7 (Vilgalys and Hester 1990); the second-largest subunit of the RNA polymerase II (rpb2) wih primers RPB2-5F2 (Sung et al. 2007) and fRPB2-7cR (Liu et al. 1999); and β-tubulin (tub2) with primers Btub2Fd and Btub4Rd (Woudenberg et al. 2009). DNA amplifications were performed in 25-μl reaction volumes, containing 2.5 μl 10 × PCR buffer, 1 μl of each primer (10 μM), 1 μl template DNA, 0.25 μlTaq DNA polymerase (Promega, Madison, WI, USA) and 18.5 μl ddH2O. The PCR cycling conditions for ITS were as follows: initial denaturation at 95°C for 5 min; then 35 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 45 s and extension at 72°C for 90 s; and final extension at 72°C for 10 min. For LSU: initial denaturation at 98°C for 3 min; then 35 cycles of denaturation at 98°C for 30 s, annealing at 45°C for 27 s and extension at 72°C for 30 s; and final extension at 72°C for 10 min. For rpb2: initial denaturation at 95°C for 5 min; then 40 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 2 min and extension at 72°C for 90 s; and final extension at 72°C for 10 min. For tub2: initial denaturation at 94°C for 3 min; then 35 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 45 s and extension at 72°C for 1 min; and final extension at 72°C for 10 min. The amplification products were sent to SinoGenoMax (Beijing) for sequencing. The newly-generated DNA sequences were submitted to GenBank (accession numbers in Table 1). The DNA base differences on four loci amongst our strains and ex-type or representative strains of relative Allophoma taxa are shown in Table 2.

Table 2.

DNA base differences amongst our strains and related species in four gene regions.

Species

Strain number

ITS (1-494bp)

rpb2 (495-1090bp)

tub2 (1091-1424bp)

LSU (1425-2729bp)

Allophoma thunbergiae

GUCC 2070.7

0

0

0

0

A. piperis

CBS 268.93 *

21

39

26

0

A. minor

CBS 325.82*

6

1

9

0

A. pterospermicola

GUCC 2070.3

0

0

0

0

A. pterospermicola

GUCC 2070.6

5

0

1

4

A. pterospermicola

CGMCC 3.19245*

1

0

1

3

A. siamensis

MFLU 17-2281

4

55

1

6

Sequence alignment and phylogenetic analyses

The related DNA sequences for phylogenetic analyses in this study were downloaded from GenBank (Table 1). Amongst them, Stagonosporopsis loticola (CBS 562.81) is regarded as outgroup taxon. Alignments for four individual loci were constructed (ITS, rpb2, tub2 and LSU) in MAFFT v7.307 online version (Katoh and Standley 2016) and were manually edited in MEGA v. 6.0 when necessary (Tamura et al. 2013). The concatenated aligned dataset and each locus were analyzed separately using Maximum Likelihood (ML), Bayesian Inference (BI) and Maximum Parsimony (MP).The best fit substitution model for each gene was tested from eleven substitution schemes by using ‘jModelTest2 on XSEDE’ tool (Darriba et al. 2012) at the CIPRES web portal (Miller et al. 2010), and determined by the Bayesian information criterion (BIC).ML analysis was performed using RAxML-HPC2 v. 8.2.12 (Stamatakis 2014) as implemented on the CIPRES Science Gateway, with the GTR+G+I model and 1,000 rapid bootstrap (BS) replicates for four genes. For BI analysis, the best substitution model for each partition was determined with the program MrModeltest 2.2 (Nylander 2004) to be GTR+G+I. BI analysis was performed using MrBayes v.3.2.6 (Ronquist et al. 2012) as implemented on the Cipres portal (Miller et al. 2010). Parameters and tree samples were summarized with a burn-in fraction of 0.25, which were checked against the log likelihood by sampled generation plot. MP analysis was performed in PAUP v. 4.0b10 (Swofford 2002) using the heuristic search option with 1,000 random taxa additions and tree bisection and reconnection (TBR) as the branch-swapping algorithm. The maxtrees were set as 5000 to build up the phylogenetic tree. The Tree Length (TL), Consistency Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were calculated for each tree generated.

Taxon treatments

Allophoma thunbergiae Jun Yuan & Yong Wang bis, sp. nov.

Material    Download as CSV 
Holotype:
  1. scientificName:
    Allophoma thunbergiae
    ; order:
    Pleosporales
    ; family:
    Didymellaceae
    ; genus:
    Allophoma
    ; country:
    China
    ; stateProvince:
    GuangXi
    ; locality:
    Nanning City, Guangxi Medicinal Botanical Garden
    ; verbatimCoordinates:
    22°51’N, 108°19’E
    ; recordedBy:
    Jun Yuan
    ; identifiedBy:
    Jun Yuan
    ; dateIdentified:
    2020
    ; collectionID:
    HGUP 2070.7
    ; occurrenceID:
    GUCC 2070.7

Description

Pathogenic on the leaf spot of Thunbergia grandiflora. Lesions initially on the upper leaf surface, scattered, distinct, irregular, the maximum length of the spot more than 10-15 mm, the edge of the spots yellow, the centre of necrotic section brown, on the lower leaf surface similar. Sexual morph: Undetermined. Asexual morph (Fig. 1): Coelomycetous. Conidiomata pycnidial, mostly solitary or aggregated, subglobose to irregular, dark brown, glabrous, covered with some hyphal outgrowths, produced on the agar surface or (semi-)immersed, ostiolate, (39−)44−200 × (48−)49−230 μm (x̄ = 108.9 × 138.9 μm, n = 20). Ostioles 1−3, with a short neck, slightly papillate or sometimes non-papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 3−4 layered, 14−32 μm thick (x̄ = 20.8 μm, n = 10). Conidiogenous cells phialidic, hyaline, smooth, ampulliform to doliiform, 4.5−7 × 4−5 μm (x̄ = 4.9 × 4.6 μm, n = 10), with a distinct periclinal thickening. Conidia oblong to cylindrical, slightly obovoid, smooth and thin-walled, hyaline, aseptate, 3−5 × 1.5−2.5 μm (x̄ = 3.6 × 2.2 μm, n = 20), with two minutes guttules. Conidial exudates not recorded.

Figure 1.  

Allophoma thunbergiae (GUCC2070.7) a. Leaf symptoms on the host; b, c. Pycnidia forming on PDA; d, e. Colony on PDA (front and reverse); f, g. Colony on MEA (front and reverse); h, i. Colony on OA (front and reverse); j, k. Section of pycnidium; l. Section of pycnidial wall; m, n. Conidiogenous cells; o. Conidia. Scale bars: b, c = 500 μm; j = 100 μm; k = 50 μm; l = 20 μm; m−o = 5 μm.

Culture characteristics: Colonies on PDA, 46−57 mm diameter after 1 week, irregular at margin, aerial mycelia floccose, grey with a white margin, brown near the centre; reverse pale brown, with a white margin. Colonies on MEA 44−47 mm diameter after 1 week, regular at margin, covered by brown, dense aerial mycelia, yellow near the centre; reverse greyish-brown. Colonies on OA, 41−46 mm diameter after 1 week, irregular at margin, covered by white aerial mycelia sparse, brownish, reverse buff to yellowish-olivaceous.

Etymology

In reference to the host (Thunbergia grandiflora), from which the fungus was isolated.

Allophoma pterospermicola Qian Chen & L. Cai, Stud. Mycol. 94: 4 (2019)

Material    Download as CSV 
  1. scientificName:
    Allophoma pterospermicola
    ; order:
    Pleosporales
    ; family:
    Didymellaceae
    ; genus:
    Allophoma
    ; country:
    China
    ; stateProvince:
    GuangXi
    ; locality:
    Nanning City, Guangxi Medicinal Botanical Garden
    ; verbatimCoordinates:
    22°51’N, 108°19’E
    ; recordedBy:
    Jun Yuan
    ; identifiedBy:
    Jun Yuan
    ; dateIdentified:
    2020
    ; collectionID:
    HGUP 2070.3 and HGUP 2070.6
    ; occurrenceID:
    GUCC 2070.3 and GUCC 2070.6

Description

Pathogenic on the leaf spot of Thunbergia grandiflora. Lesions initially on the upper leaf surface, scattered, distinct, irregular, the maximum length of the spot more than 10-13 mm, the edge of the spots yellow, the necrotic section brown at the later stage connected to form the dead leaves, on the lower leaf surface similar. Sexual morph: Undetermined. Asexual morph (Fig. 2): Coelomycetous. Conidiomata pycnidial, mostly aggregated and those aggregates are solitary, scattered, globose, subglobose or sometimes irregular, dark brown, glabrous, covered with some hyphal outgrowths, produced on the toothpick surface, ostiolate, (42−)52−208 × (25−)63−147 μm (x̄ = 108.1 × 99.3 μm, n = 20). Ostiole single, with a short neck, slightly papillate. Pycnidial wall pseudoparenchymatous, composed of oblong to isodiametric cells, 3−6 layers, 18−36 μm thick (x̄ = 23.6 μm, n = 10). Conidiogenous cells phialidic, hyaline, smooth, ampulliform to doliiform, 3.5−6 × 3.5−4 μm (x̄ = 3.8 × 4.3 μm, n = 10). Conidia ellipsoidal to oblong, incidentally slightly obovoid, smooth and thin-walled, hyaline, aseptate, 2.5−4 × 1.5−2.5 μm (x̄ = 3.5 × 2.5 μm, n = 20), with 2 distinct polar guttules. Conidial exudates not recorded.

Figure 2.  

Allophoma pterospermicola (GUCC2070.3) a, b. Colony on PDA (front and reverse); c, d. Colony on MEA (front and reverse); e, f. Colony on OA (front and reverse); g. Leaf symptoms on the host; h-j. Pycnidia forming on the toothpick; k. Pycnidium; l. section of pycnidium; m. Section of pycnidial wall; n, o. Conidiogenous cells; p. Conidia. Scale bars: h, i = 500 μm; j = 100 μm; k = 50 μm; l, m = 20 μm; n−p = 5 μm.

Culture characteristics: Colonies on PDA, 46−50 mm diameter after 1 week, regular at margin, densely covered by floccose aerial mycelia, grey, with a white concentric ring near the margin; reverse pale black, with a white concentric ring near the margin. Colonies on MEA, 52−58 mm diameter after 1 week, regular at margin, dull green, aerial mycelia floccose, aerial mycelia sparsely, grey near the centre; reverse changing towards margin from the centre greyish-brown to brown. Colonies on OA 34−47 mm diameter after 1 week, irregular at margin, covered by floccose aerial mycelia, mycelia sparse in some furrowed zone, reverse buff to pale olivaceous.

Analysis

Phylogenetic analyses (Fig. 3

Figure 3.  

Phylogenetic tree inferred from a Maximum Parsimonious analysis, based on a concatenated alignment of ITS, rpb2, tub2 and LSU sequences. ML bootstrap support values (MLBS) ≥ 70, BI posterior probabilities (BIPP) ≥ 0.90, and MP boostrap support values (MPBS) ≥ 70 are given at the nodes. The tree was rooted to Stagonosporopsis loticola (CBS 562.81). Newly-generated isolates are in bold. Ex-type strains are marked by an asterisk (*).

Discussion

Phoma sensu lato was previously a large genus with phoma-like species (De Gruyter et al. 2012), but was recently characterised using molecular data, resulting in many species that were transferred to new genera, such as Allophoma (Chen et al. 2015). In this study, our isolates from Thunbergia (GUCC 2070.3, GUCC 2070.6 and GUCC 2070.7) represent species of Allophoma (Didymellaceae). One of these isolates, GUCC 2070.7, was retrieved close to A. minor in our phylogenetic tree (Fig. 3). In Table 3, we provide a comprehensive comparison of pycnidia, conidiogenous cells and conidia, which indicates that strain GUCC 2070.7 has smaller pycnidia (39−200 × 48−230 μm vs. 150−280 × 150−220 μm) and larger conidiogenous cells (4.5−7 × 4−5 μm vs. 4−5.5 × 3−4.5 μm) than A. minor. The phylogenetic analyses and comparison of DNA base pairs confirm that strain GUCC 2070.7 is different from A. minor sensu Jeewon and Hyde (2016). In summary, strain GUCC 2070.7 represented an undescribed species, A. thunbergiae, whereas strains GUCC 2070.3 and GUCC 2070.6 are A. pterospermicola, based on phylogenetic analyses and morphological studies (Chen et al. 2017, Fig. 3, Table 2). Thunbergia grandiflora, native to China, is here reported as a host for Allophoma species for the first time.

Table 3.

The pycnidia, conidiogenous cells, and conidia morphology of the new species compared to known species of Allophoma.

Species

Pycnidia

Conidiogenous cells

Conidia

References

Shape

Size (μm)

Size (μm)

Shape

Size (μm)

Allophoma alba

(sub-)globose to ellipsoidal, whitish at onset

205−635 × 195−510

3.5−6.5 × 4.5−9

oblong, with both ends rounded, hyaline, smooth and thin-walled, aseptate

3−4.5 × 1.5−2.3

Hou et al. (2020b)

A. anatii

(sub-)globose to ellipsoidal

130−400 × 120−370

5−7 × 5.5−9

oblong with both ends rounded or ovoid, smooth and thin-walled, hyaline, aseptate

3.5−5.5 × 2−3

Hou et al. (2020b)

A. cylindrispora

glabrous, ovoid

120−210 × 90−140

3.5−4 × 4.5−5

aseptate, hyaline, smooth and thin-walled, cylindrical

3−4 × 2

Valenzuela-Lopez et al. (2018)

A. hayatii

(sub-)globose with 1−2 narrow and long necks

125 × 102

-

oblong to ellipsoidal

3.3−8 × 2.2−3.3

Babaahmadi et al. (2018)

A. labilis

globose

250 × 70

5−7 × 4−8

oblong to ellipsoidal

4−6.5 × 2–3

De Gruyter and Noordeloos (1992) Boerema et al. 2004

A. minor

globose to broadly ellipsoidal

150−280 × 150−220

4−5.5 × 3−4.5

ellipsoidal to ovoid or slightly allantoid

3.5−4.5 × 1.8−2.5

Aveskamp et al. (2010)

A. nicaraguensis

globose to flask-shaped

30−150 × 28−120

3−4.5 × 3.5−4.5

ellipsoidal to oblong

2.5−4 × 1.5−2.5

Chen et al. (2015)

A. oligotrophica

globose to subglobose

50−440 × 145−420

4.5−7 × 3.5−6.5

oblong to cylindrical

3−4.5 × 1.5−2.5

Chen et al. (2017)

A. piperis

subglobose

115−245 × 85−230

2.5−3.5 × 2−3

ellipsoidal to ovoid or slightly allantoid

3.5−5.5 × 1.5−2.5

Chen et al. (2015)

A. pterospermicola

globose to subglobose, brown, glabrous

60−330 × 67−280

6−10 × 3−6

oval to oblong, occasionally bacilliform

3−5.5 × 1.5−2

Marin-Felix et al. (2019)

A. siamensis

glabrous, ovoid

70−90 × 68−85

3−6 × 4−5

hyaline, cylindrical, aseptate

3−4 × 2−3

Jayasiri et al. (2019)

A. thunbergiae

subglobose to irregular, dark brown

39−200 × 48−230

4.5−7 × 4−5

oblong to cylindrical, incidentally slightly obovoid

3−5 × 1.5−2.5

this study

A. tropica

subglobose

100−300

2−6 × 3−6

ellipsoidal

3−4 × 1−2

Boerema et al. (2004)

A. zantedeschiae

subglobose or depressed

90−180

-

oval or ellipsoidal

4−7 × 2.5−3.5

Boerema (1993)

Acknowledgements

We would like to thank Dr. Danny Haelewaters and Ms. Subodini N. Wijesinghe for their meticulous reviews. This research is supported by the following funding sources: National Natural Science Foundation of China (nos. 31972222, 31660011), 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, [2019]5641, [2019]13), Guizhou Science, Technology Department of International Cooperation Base Project ([2018]5806), the Project of Guizhou Provincial Education Department ([2020]001) and Guizhou Science and Technology Innovation Talent Team Project ([2020]5001). Nalin N. Wijayawardene acknowledges the National Natural Science Foundation of China (No: 31950410558) and Guizhou Medical University (grant number FAMP201906K).

References