Plant-Associated Novel Didymellaceous Taxa in the South China Botanical Garden (Guangzhou, China)

Abstract

The South China Botanical Garden (SCBG), one of the largest and oldest botanical gardens in China, conserves important plant germplasms of endangered species. Therefore, ensuring tree health and studying the associated mycobiome of the phyllosphere is essential to maintaining its visual aesthetics. During a survey of plant-associated microfungal species in SCBG, we collected several coelomycetous taxa. Phylogenetic relationships were evaluated based on the analyses of ITS, LSU, RPB2, and β-tubulin loci. The morphological features of the new collections were compared with those of existing species, emphasizing close phylogenetic affinities. Based on the morphological comparisons and multi-locus phylogeny, we introduce three new species. These are Ectophoma phoenicis sp. nov., Remotididymella fici-microcarpae sp. nov., and Stagonosporopsis pedicularis-striatae sp. nov. In addition, we describe a new host record for Allophoma tropica in the Didymellaceae. Detailed descriptions and illustrations are provided along with notes comparing allied species.

1. Introduction

The South China Botanical Garden (SCBG), Chinese Academy of Sciences, Guangzhou was established in 1929 [1]. This is the largest modern botanical garden in Guangzhou, Guangdong Province, and it extends over 2854 acres comprising around 2400 plant species including alpine, arctic, aquatic, Mediterranean, tropical, and desert examples [1]. SCBG is one of the top plant germplasm conservation institutions in China and it contains 710 of the verified Red List plant taxa [2]. There are more than 14,000 trees including Chinese endemic species in ex-situ conservation [3]. Tree health and disease management are rather important in SCBG.

Didymellaceae was established by de Gruyter et al. [4] to accommodate Phoma and other allied genera. Aveskamp et al. [5] delimited the boundaries of the Didymellaceae based on morphology and combined ITS, LSU, SSU, and βtubulin loci analyses. Subsequent revisions of the Didymellaceae resolved generic boundaries and revealed more natural evolutionary relationships [6,7,8,9,10,11]. Currently, the family encompasses more than 5400 species in 44 accepted genera [12,13,14] (Species Fungorum. http://www.speciesfungorum.org/Names/Names.asp, accessed on 14 August 2022). Species of the Didymellaceae are cosmopolitan and distributed in a wide range of hosts and habitats [5,15,16]. Some species are plant pathogens causing leaf and stem lesions and fruit rots [5,7,15,17], while some species such as Allophoma hayatii Babaahm. and Mehr-Koushk., Calophoma petasitis Tibpromma, Camporesi, and K.D. Hyde are saprobes on dead plant parts [18,19,20]. Few species such as Phoma herbarum Westend. and Juxtiphoma eupyrena (Sacc.) Valenz-Lopez, Crous, Stchigel, and Guarro and Cano have been reported as human and animal pathogens [21,22].

In a survey of fungal species associated with plants in botanical gardens, we collected several saprobic, hyaline-spored, and didymellaceae-like coelomycetous taxa from the SCBG. The main objectives of this study are to analyze the taxonomic placement of our didymellaceous collections and then to describe the taxonomic novelties using morphology and the multi-locus phylogeny of ITS, LSU, RPB2, and β-tubulin sequences. Our study revealed that three of our isolates are new to science. Herein, we introduce these isolates as novel species namely Ectophoma phoenicis sp. nov., Remotididymella fici-microcarpae sp. nov., and Stagonosporopsis pedicularis-striatae sp. nov. A new collection of Allophoma tropica (R. Schneid. and Boerema) Qian Chen and L. Cai is described herein as a new host record. Detailed descriptions, illustrations, and notes are provided.

2. Material and Methods

2.1. Sampling Sites, Specimens, and Isolates

Dead plant specimens were collected from the south China Botanical Garden, Guangzhou, Guangdong Province, China (Figure 1), between June to September 2021. Specimens were detached from the host using sterile blades and packed in sterilized paper bags. The collection site is characterized by a tropical climate with abundant sunshine and rainfall throughout the year. The average annual temperature is 22 °C with around 2125 mm of rainfall per year [23].

Figure 1.

Entrance of Southern China Botanical Garden.

The collected dead samples (petioles, sepals, and stems) were brought to the laboratory in sterilized paper bags and examined with a stereomicroscope (Carl Zeiss Discovery V8). Conidia were cultured following the method described by Senanayake et al. [24]. The germinated conidia were aseptically transferred into fresh potato dextrose agar (PDA) plates, incubated at 25 °C in the dark to obtain pure cultures, and later transferred to PDA slants and stored at 4 °C for further study. Colony characters were recorded from PDA cultures. Fungarium specimens are deposited at the Herbarium of Zhongkai University of Agriculture and Engineering (ZHKU), and all the ex-type and living cultures are deposited at the Culture Collection of Zhongkai University of Agriculture and Engineering (ZHKUCC). Index Fungorum numbers (https://www.indexfungorum.org, accessed on 1 June 2022) and Facesoffungi numbers [25] were registered for the new species.

2.2. Morphological Studies

Microscopic mounts of fruiting structures in sterilized tap water were examined and photographed using a stereomicroscope fitted with a camera (ZEISS Axiocam 208). The micromorphological characteristics such as the structure of the conidiomatal wall, conidiogenous cells, and conidia were studied and photographed using a compound microscope (Nikon Eclipse 80i) fitted with a digital camera (Canon 450D). All microscopic measurements were made with the Tarosoft image framework (v. 0.9.0.7).

2.3. DNA Extraction, PCR Amplification, and Sequencing

Fresh mycelia grown on PDA for two weeks at 25 °C in the dark were used for DNA extraction using a fungal genomic DNA extraction kit (Biospin DNA Extraction Kit, Bioer Technology, Co. Ltd., Hangzhou, China) following the manufacturer’s protocols. Polymerase chain reactions (PCR) and sequencing were carried out for the complete ITS region, part of the LSU ribosomal DNA, the RNA polymerase II subunit (RPB2) gene, and the β-tubulin gene. The PCR amplification reactions were carried out with the following protocols in Table 1. The total volume of the PCR reaction was 25 µL containing 1 µL of DNA template, 1 µL of each forward and reverse primer, 12.5 µL of 2 × PCR Master Mix, and 9.5 µL of double-distilled sterilized water (ddH₂O). All the PCR thermal cycles include a final hold at 4 °C.

Table 1.

Gene regions, respective primer pairs, and thermal cycler conditions used in this study.

Gene Region	Primer Pairs	Optimized Thermal Cycler Conditions
ITS	ITS1/ITS4	94 °C: 3 min; (94 °C: 45 s, 56 °C: 50 s) 35 cycles 72 °C: 10 min
LSU	LR0R/LR5	94 °C: 3 min; (94 °C: 45 s, 56 °C: 50 s) 35 cycles 72 °C: 10 min
RPB2	fRPB2-5F/fRPB2-7cR	95 °C: 5 min; (95 °C: 1 min, 52 °C: 50 s) 35 cycles 72 °C: 10 min
β -tubulin	bt2a/bt2b	95 °C: 5 min; (94 °C: 1 min, 55 °C: 90 s) 35 cycles 72 °C: 10 min

The PCR products were observed on a 1% agarose electrophoresis gel stained with ethidium bromide. Purification and sequencing of PCR products were carried out at Sunbiotech Company, Beijing, China. Sequence quality was checked, and sequences were condensed with DNASTAR Lasergene v. 7.1 [26]. Sequences derived in this study were deposited in GenBank and accession numbers were obtained (Table 2).

Table 2.

Taxa used in the present phylogenetic analyses and GenBank numbers of their sequences.

Species	Strain no	GenBank Accession Numbers
		ITS	LSU	RPB2	β-tubulin
Allophoma anatiae	CBS 124673	MN973472	MN943674	MT018048	MT005571
Allophoma cylindrispora	UTHSC DI16-233	LT592920	LN907376	LT593058	LT592989
Allophoma zantedeschiae	CBS 131.93	FJ427084	GU238159	KT389557	FJ427188
Allophoma hayatii	CBS 142859	KY684812	KY684814	MF095108	KY684816
Allophoma labilis	CBS 124.93	GU237765	GU238091	KT389552	GU237619
Allophoma piperis	CBS 268.93	GU237816	GU238129	KT389554	GU237644
Allophoma siamensis	MFLU 17-2422	MK347742	MK347959	MK434912	MK412867
Allophoma pterospermicola	LC12182	MK088570	MK088577	MK088584	MK088591
Allophoma pterospermicola	CGMCC 3.19245	MK088573	MK088580	MK088587	MK088594
Allophoma thunbergiae	GUCC2070.7	MW036298	MW040201	MW116819	MW116823
Allophoma minor	CBS 325.82	GU237831	GU238107	KT389553	GU237632
Allophoma alba	CBS 120422	MN973469	MN943671	MT018044	MT005568
Allophoma nicaraguensis	CBS 506.91	GU237876	GU238058	KT389551	GU237596
Allophoma tropica	CBS 436.75	GU237864	GU238149	KT389556	GU237663
Allophoma tropica	CBS 537.66	MH858877	MH870533	MT018043	N/A
Allophoma tropica	ZHKUCC 22-0169	OQ275206	OQ275192	OQ343373	OQ336257
Allophoma tropica	ZHKUCC 22-0170	OQ275207	OQ275193	OQ343374	OQ336258
Allophoma oligotrophica	LC 6245	KY742040	KY742194	KY742128	KY742282
Ectophoma insulana	CBS 140548	MN973482	MN943686	MT018071	MT005582
Ectophoma insulana	CBS 252.92	MN973481	MN943685	MT018070	MT005581
Ectophoma pomi	CBS 267.92	GU237814	GU238128	N/A	GU237643
Ectophoma iranica	SCUA-K1G1	MK519382	MK519389	N/A	MK519562
Ectophoma pomi	CBS 121.93	MN972933	N/A	MN983570	MN983948
Ectophoma multirostrata	CBS 110.79	FJ427030	GU238110	LT623264	FJ427140
Ectophoma multirostrata	CBS 274.60	FJ427031	GU238111	LT623265	FJ427141
Ectophoma sp.	URM 408	MH384820	MH370603	MH370607	N/A
Ectophoma phoenicis	ZHKUCC 22-0163	OQ275208	OQ275194	OQ343375	OQ336259
Ectophoma phoenicis	ZHKUCC 22-0164	OQ275209	OQ275195	OQ343376	OQ336260
Remotididymella ageratinae	G13388	MN864294	MN864298	MN871530	MN871533
Remotididymella anemophila	C74	MN864293	MN864296	MN871529	MN871532
Remotididymella anthropophila	CBS 142462	LT592936	N/A	LT593075	LT593005
Remotididymella bauhiniae	MFLU 18-2118	MK347737	MK347954	MK434914	MK412884
Remotididymella brunnea	CBS 993.95	NR_170781	N/A	MT018064	MT005576
Remotididymella capsici	CBS 679.77	MN973478	MN943681	MT018066	MT005578
Remotididymella destructiva	CBS 133.93	GU237779	GU238064	LT623257	GU237602
Remotididymella fici-microcarpae	ZHKUCC 22-0165	OQ275210	OQ275196	OQ343377	OQ336261
Remotididymella fici-microcarpae	ZHKUCC 22-0166	OQ275211	OQ275197	OQ343378	OQ336262
Remotididymella humicola	CBS 120117	NR_170782	MN943680	MT018065	MT005577
Stagonosporopsis actaeae	CBS 106.96	GU237734	GU238166	KT389672	GU237671
Stagonosporopsis actaeae	CBS 105.96	GU237733	GU238165	MT018018	GU237670
Stagonosporopsis ailanthicola	MFLUCC 16-1439	KY100872	KY100874	KY100876	KY100878
Stagonosporopsis ajacis	CBS 176.93	GU237790	GU238167	MT018035	GU237672
Stagonosporopsis andigena	CBS 269.80	GU237817	GU238170	MT018026	GU237675
Stagonosporopsis artemisiicola	CBS 102636	GU237728	GU238171	KT389674	GU237676
Stagonosporopsis astragali	CBS 178.25	GU237792	GU238172	MT018030	GU237677
Stagonosporopsis bomiensis	CGMCC 3.18366	KY742123	KY742277	KY742189	KY742365
Stagonosporopsis chrysanthemi	CBS 500.63	GU237871	GU238190	MT018012	GU237695
Stagonosporopsis chrysanthemi	CBS 137.96	GU237783	GU238191	MT018011	GU237696
Stagonosporopsis citrulli	FLAS-F-58996	KJ855546	N/A	N/A	KJ855602
Stagonosporopsis crystalliniformis	CBS 713.85	GU237903	GU238178	KT389675	GU237683
Stagonosporopsis cucumeris	CBS 386.65	MN973455	MN943657	MT018021	MT005554
Stagonosporopsis caricae	CBS 119735	MN973042	N/A	MN983680	MN984054
Stagonosporopsis caricae	CBS 102399	MN973041	N/A	MN983679	MN984053
Stagonosporopsis dennisii	CBS 110.96	MN973046	N/A	MN983685	MN984058
Stagonosporopsis dorenboschii	CBS 320.90	GU237830	GU238184	MT018039	GU237689
Stagonosporopsis helianthi	CBS 200.87	KT389545	KT389761	KT389683	KT389848
Stagonosporopsis hortensis	CBS 572.85	GU237893	GU238199	KT389681	GU237704
Stagonosporopsis inoxydabilis	CBS 425.90	GU237861	GU238188	KT389682	GU237693
Stagonosporopsis loticola	CBS 562.81	GU237890	GU238192	KT389684	GU237697
Stagonosporopsis lupini	CBS 101494	GU237724	GU238194	KT389685	GU237699
Stagonosporopsis nemophilae	CBS 715.85	MN973460	MN943662	MT018031	MT005559
Stagonosporopsis oculo-hominis	CBS 634.92	GU237901	GU238196	KT389686	GU237701
Stagonosporopsis papillata	LC 8171	KY742127	KY742281	KY742193	KY742369
Stagonosporopsis pini	MFLUCC 18-1549	MK347800	MK348019	MK434860	MK412886
Stagonosporopsis rudbeckiae	CBS 109180	GU237745	GU238197	MT018015	GU237702
Stagonosporopsis sambucella	CBS 130003	MN973459	MN943661	MT018029	MT005558
Stagonosporopsis stuijvenbergii	CBS 144953	MN823449	MN823300	MN824475	MN824623
Stagonosporopsis tanaceti	CBS 131484	NR_111724	KP161044	MT018013	JQ897496
Stagonosporopsis pedicularis-striatae	ZHKUCC 22-0167	OQ275212	OQ275198	OQ343379	OQ336263
Stagonosporopsis pedicularis-striatae	ZHKUCC 22-0168	OQ275213	OQ275199	OQ343380	OQ336264
Stagonosporopsis trachelii	CBS 379.91	GU237850	GU238173	KT389687	GU237678
Stagonosporopsis valerianellae	CBS 273.92	GU237819	GU238200	MT018033	GU237705
Stagonosporopsis weymaniae	CBS 144959	MN823453	MN823304	MN824479	MN824627
Stagonosporopsis centaureae	MFLUCC 16-0787	KX611240	KX611238	N/A	N/A
Stagonosporopsis heliopsidis	CBS 109182	GU237747	GU238186	KT389679	GU237691
Stagonosporopsis pogostemonis	ZHKUCC 21-0001	MZ156571	MZ191532	MZ203135	MZ203132
Stagonosporopsis rhizophila	CGMCC 3.19852	MG833824	MG833789	MN422105	MN422099
Stagonosporopsis cucurbitacearum	CBS 133.96	GU237780	GU238181	KT389676	GU237686
Stagonosporopsis cucurbitacearum	CBS 109171	GU237922	GU238180	N/A	GU237685
Stagonosporopsis ailanthicola	CBS 140554	MN973462	MN943664	MT018036	MT005561
Epicoccum oryzae	CBS 173.34	MN973499	N/A	MT018098	MT005599
Epicoccum viticis	LC 5126	KY742118	N/A	KY742186	KY742360
Similiphoma crystallifera	CBS 193.82	GU237797	GU238060	LT623267	GU237598
Didymella heteroderae	CBS 109.92	FJ426983	GU238002	KT389601	FJ427098
Didymella suiyangensis	LC 7439	KY742089	KY742243	KY742168	KY742330
Cumuliphoma indica	CBS 65478	FJ427044	GU238123	LT623262	FJ427154
Cumuliphoma pneumoniae	UTHSC DI16-249	NR_158277	N/A	LT593063	LT592994
Paraboeremia selaginellae	CBS 122.93	GU237762	GU238142	MT018189	GU237656
Paraboeremia litseae	CGMCC 318109	KX829029	KX829037	KX829045	KX829053
Macroventuria anomochaeta	CBS 502.72	GU237873	GU237985	MT018193	GU237545
Macroventuria wentii	CBS 526.71	GU237884	GU237986	KT389642	GU237546
Juxtiphoma eupyrena	CBS 527.66	FJ427000	GU238073	LT623269	FJ427111
Juxtiphoma eupyrena	CBS 374.91	FJ426999	GU238072	LT623268	FJ427110
Vacuiphoma ferulae	CBS 353.71	MH860160	MH871928	MT018196	MT005655
Vacuiphoma oculihominis	FMR 138.01	LT592954	N/A	LT593093	LT593023
Nothophoma infossa	CBS 123395	FJ427025	FJ899743	KT389659	FJ427135
Nothophoma anigozanthi	CBS 381.91	GU237852	GU238039	KT389655	GU237580
Ascochyta medicaginicola	BRIP 45051	KY742044	KY742198	KY742132	KY742286
Ascochyta koolunga	CBS 373.84	KT389481	KT389698	KT389560	KT389775
Calophoma rosae	CGMCC 3.18347	KY742049	KY742203	KY742135	KY742291
Calophoma vodakii	CBS 173.53	KT389497	KT389714	MT018233	KT389791
Neomicrosphaeropsis novorossica	MFLUCC 14-0578	KX198709	KX198710	N/A	N/A
Neomicrosphaeropsis rossica	MFLUCC 14-0586	KU752192	KU729855	N/A	N/A
Phoma herbarum	CBS 127589	KT389539	MH876049	KT389664	KT389838
Phoma herbarum	CBS 134.96	KT389535	KT389753	KT389661	KT389834
Phomatodes nebulosa	CBS 740.96	KT389540	KT389758	KT389667	KT389839
Leptosphaerulina americana	CBS 213.55	GU237799	GU237981	KT389641	GU237539
Leptosphaerulina arachidicola	CBS 275.59	GU237820	GU237983	MT018278	GU237543
Pseudoascochyta novaezelandiea	CBS 141689	LT592892	LT592893	LT592895	LT592894
Pseudoascochyta pratensis	CBS 141688	LT223130	LT223131	LT223133	LT223132
Briansuttonomyces eucalypti	CBS 114879	KU728479	KU728519	MT018239	KU728595
Xenodidymella applanata	CBS 115577	KT389546	KT389762	KT389688	KT389850
Xenodidymella asphodeli	CBS 375.62	KT389549	KT389765	KT389689	N/A
Neodidymelliopsis cannabis	CBS 121.75	GU237761	GU237972	N/A	GU237535
Neodidymelliopsis achlydis	CBS 256.77	KT389531	KT389749	MT018293	KT389829
Neoascochyta europaea	CBS 819.84	KT389510	KT389728	KT389645	KT389808
Neoascochyta graminicola	CBS 102789	KT389518	KT389736	KT389649	KT389816
Leptosphaeria conoidea	CBS 616.75	JF740201	JF740279	KT389639	KT389804
Heterophoma poolensis	CBS 113.20	GU237751	GU238119	MT018056	GU237638
Boeremia diversispora	CBS 101194	GU237716	GU237929	KT389564	GU237491
Boeremia foveata	CBS 109176	GU237742	GU237946	KT389578	GU237508
Leptosphaeria doliolum	CBS 505.75	JF740205	GU301827	KT389640	JF740144
Heterophoma nobills	CBS 507.91	GU237877	GU238065	KT389638	GU237603

Abbreviations: CBS, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CGMCC, China General Microbiological Culture Collection, Beijing, China; MFLUCC, Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; BMU: Department of Dermatology, Beijing Medical University, Beijing, China; UTHSC, Fungus Testing Laboratory at the University of Texas Health Science Center, San Antonio, Texas, USA; BRIP, Plant Pathology Herbarium, Department of Employment, Economic, Development and Innovation, Queensland, Australia; FMR, Facultat de Medicina, Universitat Rovira i Virgili, Reus, Spain; LC, Cai’s personal collection deposited in laboratory, housed at CAS, PR China; SCUA: the Collection of Fungal Cultures, Department of Plant Protection, Shahid Chamran University of Ahvaz, Iran; GZCC, Guizhou culture collection, Guizhou, China; ZHKUCC, University of Agriculture and Engineering Culture Collection (China). “N/A” denotes sequences that are not available. Newly generated sequences in this study are indicated in bold.

2.4. Phylogenetic Analyses

BLASTn searches were made using NCBI BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 18 March 2022). The newly generated sequences assist in taxon sampling for phylogenetic analyses. All the ex-type strains of species were included if available, and other authentic strains were selected when sequences from ex-type strains were unavailable. [27,28,29,30,31,32,33] were followed to obtain sequences from GenBank (Table 2). The concatenated ITS, LSU, RPB2, and β-tubulin sequence dataset for the family Didymellaceae comprised 123 strains with the outgroup taxon as Leptosphaeria conoidea (De Not.) Sacc. (CBS 616.75) and Leptosphaeria doliolum (Pers.) Ces. & De Not. (CBS 505.75). DNA sequences of all the single gene regions were aligned using the online version of MAFFT v. 7.0362 [34] with default settings and manually adjusted using BioEdit 7.1.3 [35] when necessary.

Maximum likelihood analysis was performed by RAxML [36] implemented in raxmlGUIv. 1.5 [37] using the ML + rapid bootstrap setting and the GTR + I + G model of nucleotide substitution with 1000 replicates. For the Bayesian inference (BI) analyses, the optimal substitution model for the combined datasets was determined to be GTR + I + G using the MrModeltest software v. 2.2 [38]. The BI analyses were computed in MrBayes v. 3.2.6 [39] with four simultaneous Markov chain Monte Carlo chains from random trees over 5 M generations (trees were sampled every 1000th generation). The distribution of log-likelihood scores was observed to check whether sampling was in the stationary phase and Tracer v1.5 was used to check if further runs were required to reach convergence [40]. The consensus tree and posterior probabilities were calculated after discarding the first 20% of the sampled trees as burn-in. The phylogram was visualized in FigTree v. 1.4 [41].

2.5. PHI Analyses

The PHI test was performed using SplitsTree4 v. 4.17.1 to determine the recombination level within phylogenetically closely related species. The concatenated four-locus dataset (ITS + LSU + RPB2 + β-tubulin) was used for the analyses. PHI test results (Φw) ≥0.05 indicated no significant recombination within the dataset. The relationships between closely related taxa were visualized in split graphs with both the Log-Det transformation and splits decomposition options.

3. Results

3.1. Phylogenetic Analyses

The alignment comprised 2308 nucleotide characters (488 of ITS, 893 of LSU, 595 of RPB2, 332 of β-tubulin). Maximum likelihood analysis yielded the best ML tree (Figure 2) with a likelihood value of −25,904.846543 and the following model parameters: estimated base frequencies A = 0.246664, C = 0.258093, G = 0.262080, and T = 0.233164; substitution rates AC = 1.535206, AG = 4.889606, AT = 1.722258, CG = 0.744297, CT = 10.883910, and GT = 1.0; proportion of invariable sites I = 0.587394; gamma distribution shape parameter: α = 0.536998. The alignment contained a total of 857 distinct alignment patterns and 9.44% of undetermined characters.

Figure 2.

Phylogram generated from a maximum likelihood analysis based on a combined ITS, LSU, RPB2, and β-tubulin sequence alignment. Maximum likelihood bootstrap support values greater than 70% and Bayesian posterior probabilities greater than 0.90 are given at the nodes. The tree is rooted with Leptosphaeria conoidea (CBS 616.75) and Leptosphaeria doliolum (CBS 505.75). Ex-type strains are given in bold and the newly generated sequences are indicated in red bold.

After discarding the first 20% of generations in the Bayesian analyses, 4000 trees remained from which the 50% consensus tree and Bayesian Interference (BI) posterior probabilities were calculated (Figure 2). All individual trees generated under different criteria from single gene datasets were similar in topology and not significantly different from the final trees generated from the concatenated datasets of the Didymellaceae. Topologies of the ML and Bayesian trees were similar to each other and there were no significant differences.

The phylogenetic analyses of this study (Figure 2) showed that the Didymellaceae comprises a total of 27 well-supported genera. We included 32 sequences from four new collections representing eight isolates in this analysis. Newly generated sequences from two isolates (ZHKUCC 22-0167, ZHKUCC 22-0168) grouped with Stagonosporopsis astragali (Cooke and Harkn.) Aveskamp, and Gruyter and Verkley (CBS 178.25) with strong statistical support (89% in ML, 0.90 in BI). Furthermore, another two isolates (ZHKUCC 22-0169, ZHKUCC 22-0170) grouped with the type strain (CBS 537.66) and another strain (CBS 436.75) of Allophoma tropica with a strong support value (99% in ML, 0.99 in BI). Other two isolates (ZHKUCC 22-0163, ZHKUCC 22-0164) form a distinct basal clade to Ectophoma multirostrata (P.N. Mathur, S.K. Menon and Thirum.) Valenz.-Lopez, Cano, Crous, Guarro and Stchigel-E. Iranica M. Mehrabi, Larki and Farokhinejad subclade with strong statistical support (98% in ML, 0.99 in BI). Moreover, two new isolates (ZHKUCC 22-0165, ZHKUCC 22-0166) form a sister clade to Remotididymella ageratinae H.B. Zhang, A.L. Yang and L. Chen (G1338) with strong support value (100% in ML, 1.00 in BI).

3.2. Taxonomy

Allophoma Q. Chen & L. Cai, Stud. Mycol. 82: 162 (2015).

Type species: Allophoma tropica (R. Schneid. and Boerema) Q. Chen and L. Cai. Chen et al.

Notes: Allophoma was introduced to accommodate phoma-like taxa with various-shaped conidia [7]. Currently, 14 species are accepted in the genus [14] (Species Fungorum. http://www.speciesfungorum.org/Names/Names.asp, accessed on 14 August 2022). Allophoma species are phytopathogens or saprobes on leaves, pods, and stems and some are human and animal pathogens [33,42,43]. Allophoma labilis (Sacc.) Qian Chen and L. Cai often causes leaf necrosis, canker, and stem lesions, or stem rot in various plants [18,44,45,46,47,48], and A. tropica can cause necrosis in a wide variety of ornamental plants [42]. In addition, A. cylindrispora Valenz-Lopez, Stchigel, Guarro and Cano was isolated from a lesion of the human eye [43] and A. oligotrophica Qian Chen, Crous and L. Cai is an air borne fungus [49].

Allophoma tropica (R. Schneid. and Boerema) Q. Chen and L. Cai, Stud. Mycol. 82: 164 (2015).

Basionym: Phoma tropica R. Schneid. and Boerema, Phytopath. Z. 83 (4): 361 (1975)

Index Fungorum number: IF814071; Facesoffungi number: FoF 13240, Figure 3.

Figure 3.

Allophoma tropica (ZHKU 22-0099) (a) Examined material. (b) Conidiomata on the substrate. (c) Vertical section of a conidioma. (d,e) Conidiogenous cells attached to conidia. (j) Conidia. (f,g) Chlamydospores in cultures. (h,i) Colonies on PDA (i from the bottom). Scale bars: e = 200 μm, f–j = 10 μm.

Saprobic on the sepals of Canna sp. Sexual morph: Undetermined. Asexual morph: Coelomycetous. Appearing as black spots. Hyphae 4–6 μm wide, initially hyaline, become olivaceous to brown, septate. Conidiomata 80–250 μm high, 100–300 μm diam. (x = 210 × 280 μm, n = 20), pycnidial, mostly solitary, rarely aggregated, superficial to erumpent, uniloculate, subglobose to irregular, dark brown, glabrous, conspicuous dark ostiole. Ostiole single, dark, distinct, non-papillate or sometimes slightly papillate. Pycnidial wall 5–10 μm thick (x = 8 μm, n = 10), 1–3-layered, composed of pale brown cells of textura angularis. Conidiophores are reduced to conidiogenous cells. Conidiogenous cells 5–8 × 3–4 μm (x = 7 × 3.6 μm, n = 10), phialidic, ampulliform to doliiform, hyaline, smooth. Conidia 3–5 × 1–2.5 μm (x = 4 × 2 μm, n = 20), ellipsoidal, smooth and thin-walled, hyaline, aseptate, with small guttules. Conidial exudates not observed. Chlamydospores globose 10–25 μm diam. (x = 20 μm, n = 20) to irregular 10–15 × 6–15 μm (x = 13 × 10 μm, n =20), unicellular or multicellular, intercalary or terminal, solitary or in chains, smooth, verruculose or incidentally tuberculate, subhyaline to pale brown.

Culture characters: Colonies on PDA reaching 5 cm after 7 days at 25 °C in the dark, white to off-white, aerial mycelium less, circular, flat, smooth to slightly waved margin; reverse off-white. Cultures were not sporulating and no pigments were produced.

Material examined: China, Guangdong Province, Guangzhou City, South China Botanical Garden (23°11′12″ N 113°21′51″ E), on sepals of Canna sp. (Cannaceae), 17 June 2021, N.D. Kularathnage, NDK 71-3 (ZHKU 22-0099), living cultures ZHKUCC 22-0169, ZHKUCC 22-0170.

Hosts and distribution: on Aphelandra sp. from the Netherlands, on Saintpaulia ionantha from Germany, on Gossypium sp. from Bolivia [42], on Lactuca sativa from Italy [50], on Syzygium cumini from India [46], and on sepals of Canna sp. from China (the present study).

Notes: The multi-locus phylogeny of ITS, LSU, RPB2, and β-tubulin showed that our isolates (ZHKUCC 22-0169 and ZHKUCC 22-0170) are affiliated with Allophoma tropica (CBS 537.66, CBS 436.75) with high statistical support (ML/BI 99/0.99) (Figure 2). A single gene comparison of ITS, LSU, RPB2, and β-tubulin of our isolates (ZHKUCC 22-0169, ZHKUCC 22-0170) with the type strain of Allophoma tropica (CBS 436.75) revealed base pair differences of 2/488, 1/893, 0/595, and 2/332, respectively. Our specimen fits well with the type collection of Allophoma tropica (DSM 63365) [51] in all morphological aspects [52].

No species of Allophoma had been reported from Canna sp., and our collection is the first Allophoma species reported from this host genus. Two species, A. pterospermicola Qian Chen and L. Cai and A. thunbergiae Jun Yuan and Yong Wang, have been described from Guangxi and Guizhou Provinces in China [49,53], and A. tropica is the third species reported in the country.

Ectophoma Valenz-Lopez, Cano, Crous, Guarro and Stchigel, Stud. Mycol. 90: 34 (2017)

Type species: Ectophoma multirostrata (P.N. Mathur, S.K. Menon and Thirum.) Valenz-Lopez, J.F. Cano, Crous, Guarro, and Stchigel.

Notes: Ectophoma was established to accommodate two phoma-like taxa, Phoma multirostrata (P.N. Mathur, S.K. Menon and Thirum.) Dorenb. and Boerema and P. pereupyrena Gruyter, Noordel. and Boerema which formed a distinct lineage in the Didymellaceae [28]. Currently, there are four accepted species (Species Fungorum. http://www.speciesfungorum.org/Names/Names.asp, accessed on 14 August 2022). This genus is characterized by pycnidial conidiomata with one or more short necks, phialidic conidiogenous cells, and aseptate conidia [28]. Ectophoma species have been isolated from different woody and herbaceous plants as opportunistic plant pathogens or from soil inhabitants [51,54]. Ectophoma species have been reported from Greece, India, Iran, South Africa, and The Netherlands.

Ectophoma phoenicis Kular. sp. nov.

Index Fungorum number: IF900158; Facesoffungi number: FoF 13241; Figure 4.

Figure 4.

Ectophoma phoenicis (ZHKU 22-0093, holotype). (a) Host. (b,c) Conidiomata on the substrate. (d) Vertical section of a conidioma. (e–g) Conidiogenous cells attached to conidia. (j) Conidia. (h,i) Colonies on PDA (i) from the bottom. Scale bars: d = 100 μm, e–h = 15 μm.

Etymology: in reference to the host genus, Phoenix.

Saprobic on dead petioles of Phoenix roebelenii O’Brien. Sexual morph: Undetermined. Asexual morph: Coelomycetous, Conidiomata 80–110 μm high, 100–130 μm wide (x = 100 × 120 μm, n = 10), pycnidial, solitary or rarely aggregated, immersed in substrate, conical to subglobose, brown to blackish brown, coriaceous, ostiolate, apapillate. Pycnidial wall comprises 4–7 layered, brown cells of textura angularis. Conidiophores are reduced to conidiogenous cells. Conidiogenous cells 15–20 μm high, 3–8 μm wide (x = 18 × 5 μm, n = 10), phialidic, cylindrical to doliiform, discrete, hyaline to grey, smooth. Conidia 6–8 × 2–4 μm (x = 7 × 3 μm, n = 20), oblong to ellipsoidal, aseptate, with polar guttules, hyaline, smooth, thin-walled, straight.

Culture characters: Colonies on PDA reaching 4 cm diam. after 7 days of incubation at 25 °C, circular, flat, floccose, filiform, pale brown to greyish brown, darker in the central area, then paler ring and darker ring around the center; reverse brown. Hyphae pale brown, branched, septate. No pigments or chlamydospores observed.

Material examined: China, Guangdong Province, Guangzhou City, South China Botanical Garden (23°11′12″ N 113°21′51″ E) on dead petioles of Phoenix roebelenii (Arecaceae), 17 June 2021, N.D. Kularathnage, NDK 78-1 (ZHKU 22-0093, holotype), ex-type culture ZHKUCC 22-0163; ibid. NDK 78-2 (ZHKU 22-0094, isotype), ex-type culture ZHKUCC 22-0164.

Notes: The multi-locus analysis of ITS, LSU, RPB2, and β-tubulin showed that our isolates (ZHKUCC 22-0163, ZHKUCC 22-0164) clustered within Ectophoma and formed a sister clade to E. iranica and E. multirostrata with 98% in ML and 0.99 in BI support values (Figure 2). A single gene comparison of ITS, RPB2, and β-tubulin locus of our isolates (ZHKUCC 22-0163, ZHKUCC 22-0164) with the type strain of E.iranica (CBS 144681) and E. multirostrata (CBS 274.60) revealed the base pair differences of 7/488, N/A, 3/332 and 6/488, 6/595, 2/332, respectively. The LSU sequences of all Ectophoma species are identical. The genetic distinctness and phylogenetic stability of our isolates were further confirmed by PHI analysis of the Ectophoma clade. The result showed that Φw = 0.99 and this means there was no significant genetic recombination (Φw ≥ 0.05) between these novel isolates with existing Ectophoma species (Figure 5).

Figure 5.

The results of a PHI test of Ectophoma clade using both LogDet transformation and splits decomposition. PHI test results Φw = 0.99.

Morphologically, our collection differs from E. iranica by its floccose, grey colonies, apapillate pycnidia, cylindrical to doliiform, hyaline to grey conidiogenous cells, and ellipsoidal, straight conidia. In contrast, E. iranica has pale brown to greyish brown colonies, conidiomata with 1–2(3)-narrowed necks, and hyaline to pale brown, oblong to ellipsoidal, straight or sometimes very slightly curved conidia [29]. Ectophoma iranica is a phytopathogen that forms leaf spots on Dracaena compacta and Catharanthus roseus [29] while our collections are saprobes. Furthermore, our collections differ from E. multirostrata by its bi-guttulated conidia, and conidiomata with single ostiole while E. multirostrata is characterized by eguttulate conidia or sometimes with 2–3, polar guttules and necks with several ostioles. Ectophoma multirostrata is a plurivorous opportunistic plant pathogen isolated from soil and also from some plant samples [51] and our species is a saprobe.

Remotididymella Valenz-Lopez, Crous, Cano, Guarro and Stchigel, Stud. Mycol. 90: 35 (2017).

Type species: Remotididymella destructiva (Plowr.) ValenzuelaLopez, Cano, Crous, Guarro and Stchigel.

Notes:Remotididymella is characterized by aseptate, hyaline, smooth- and thin-walled, allantoid or cylindrical, guttulate conidia. Currently, there are eight species listed under this genus (https://www.indexfungorum.org, accessed on 1 June 2022) and the sexual morph has been reported only for R. bauhiniae Jayasiri, E.B.G. Jones and K.D. Hyde [48]. Species of Remotididymella have been reported from Ageratina adenophora, Bauhinia sp., Capsicum annuum, Lycopersicon sp., and Solanum sp. as saprobes or pathogens. However, some species have been isolated from air, soil in tropical forests, and human respiratory tract [12,28,32,48]. Remotididymella species have been reported from China, Guadeloupe, Papua New Guinea, Thailand, The Republic of Fiji, and the United States.

Remotididymella fici-microcarpae Kular. sp. nov.

Index Fungorum number: IF900161; Facesoffungi number: FoF 13242; Figure 6.

Figure 6.

Remotididymella fici-microcarpae (ZHKU 22-0095, holotype). (a,b) Conidiomata on the substrate. (c) Vertical section of a conidioma. (d–f) Conidiogenous cells attached to conidia. (g) Conidia. (h) Surface view of colony on PDA. (i) Reverse view of colony on PDA. Scale bars: c = 100 μm, d–g = 15 μm.

Etymology: in reference to the host name Ficus microcarpa.

Saprobic on dead stems of Ficus microcarpa L.f Sexual morph: Undetermined. Asexual morph: Conidiomata 180–230 × 130–180 μm, (x = 200 × 150 μm, n = 20), pycnidial, dark brown, mostly solitary, rarely aggregated, immersed, glabrous, conical to irregularly-shaped, with a single papillate ostiolar neck. Pycnidial wall 15–25 μm thick (x = 19 μm, n = 10), 6–8-layered, composed of brown, flattened cells of textura angularis. Conidiogenous cells 12–15 × 8–11 μm (x = 14 × 9 μm, n = 20), phialidic, ampulliform, hyaline, smooth-walled. Conidia 5–8 × 3.5–4.5 μm (x = 7 × 4 μm, n = 20), mostly fusiform to allantoid, aseptate, hyaline, smooth, thin-walled, indistinct guttules.

Culture characters: Colonies on PDA reached 6 cm diam. after 7 days, circular, flat, filiform margin, center pale, margin darker, margin comprised of filiform hyphal tips, aerial mycelia less, brown, reverse pale brown. Cultures not sporulating and no pigments were produced.

Material examined: China, Guangdong Province, Guangzhou City, South China Botanical Garden (23°11′12″ N 113°21′51″ E), on stems of Ficus microcarpa (Moraceae), 17 June 2021, Senanayake I.C, S2 3-1 (ZHKU 22-0095, holotype), ex-type culture ZHKUCC 22-0165, ibid. S2 3-1 A (ZHKU 22-0096, isotype), ex-type living culture ZHKUCC 22-0166.

Notes: The combined gene analysis of ITS, LSU, RPB2, and β-tubulin (Figure 2) showed that our isolates (ZHKUCC 22-0165, ZHKUCC 22-0166) grouped with the type strain of Remotididymella ageratinae (CGMCC 3.19991) with a strong support value (100% in ML, 1.00 in BI). Result of PHI analysis of Remotididymella species showed a value of Φw = 0.26 and there was no significant genetic recombination (Φw ≥ 0.05) between these novel species of Remotididymella and other species in this genus (Figure 7). A comparison of the DNA sequences of ITS, LSU, RPB2, and β-tubulin locus of our isolates (ZHKUCC 22-0165, ZHKUCC 22-0166) with the type strain of R. ageratinae revealed base pair differences of 6/488, 8/893, 9/595, 3/332, respectively.

Figure 7.

The results of a PHI test of closely related taxa (Remotididymella clade) using both LogDet transformation and splits decomposition. PHI test results. Φw = 0.26.

Morphologically, our collections differ from R. ageratinae by having small conidiomata (180–230 × 130–180 μm), large conidiogenous cells (12–15 × 8–11 μm), and fusiform to allantoid conidia with indistinct guttules, whereas R. ageratinae has large conidiomata (107–409 × 121–503 µm), small conidiogenous cells (8–9 × 10 µm), and oblong to cylindrical, obovoid, sometimes slightly curved to reniform conidia with distinct guttules. Therefore, we introduce our collection as Remotididymella fici-microcarpae sp. nov. based on morphology and phylogeny.

Stagonosporopsis Died., Annls mycol. 10 (2): 142 (1912).

Type species: Stagonosporopsis hortensis (Sacc. and Malbr.) Petr.

Notes: There are 52 accepted species in this genus (Species Fungorum. http://www.speciesfungorum.org/Names/Names.asp, accessed on 14 August 2022). Members of the Stagonosporopsis are saprobes on dead plant materials, and some species have been reported from house dust and garden soil [12]. Some species of Stagonosporopsis can cause devastating diseases on a wide range of economically important plants, including those found in farmlands, forests, grasslands, and other natural ecosystems [5]. Stagonosporopsis species have been reported as severe pathogens of some crops and ornamentals in several countries, including Australia China, France, India, Italy, Turkey, and the United States [55,56,57,58,59].

Stagonosporopsis pedicularis-striatae Kular. sp. nov.

Index Fungorum number: IF900160; Facesoffungi number: FoF 13243, Figure 8.

Figure 8.

Stagonosporopsis pedicularis-striatae (ZHKU 22-0097, holotype). (a) Host. (b) Material examined. (c) Vertical section of a conidioma. (d) Papilla. (e,f) Conidiogenous cells attached to conidia. (g,h) Conidiogenous cells. (i) Conidia. (j) Colony on PDA (k) from the bottom. Scale bars: c = 100 μm, d = 40 μm, e–i = 10 μm.

Etymology: in reference to the host name Pedicularis striata.

Saprobic on dead stems of Pedicularis striata Pallas. Sexual morph: Undetermined. Asexual morph: Coelomycetous. Conidiomata 150–200 × 300–400 μm (x = 180 × 350 μm, n = 10), pycnidial, solitary or aggregated, scattered, subglobose, coriaceous, brown to dark brown, thin-walled, glabrous, ostiolate. Ostiole single, slightly papillate. Pycnidial wall 10–15 μm thick, pseudoparenchymatous, 4–5-layered, composed with brown, angularis to globosa cells. Conidiophores arereduced to conidiogenous cells. Conidiogenous cells 4–6 × 4–5 μm (x = 5.5 × 4.5 μm, n = 20), phialidic, subglobose, hyaline, smooth. Conidia 7–9 × 3–5 μm (x = 8 × 4 μm, n = 20), oblong to ellipsoid, with ends rounded, smooth and thin-walled, aseptate, small guttulate.

Culture characters: Colonies on PDA reached 7 cm diam. after 7 days, circular, flat, smooth, entire margin, aerial mycelia concentrated at the margin, brown, center dark, margin pale; reverse pale brown, center pale, margin darker. Cultures not sporulating and no pigments are produced.

Material examined: China, Guangdong Province, Guangzhou City, South China Botanical Garden (23°11′12″ N 113°21′51″ E) on dead petioles of Pedicularis striata (Orobanchaceae), 17 June 2021, Kularathnage N.D., S1 1010 (ZHKU 22-0097, holotype), ex-type culture ZHKUCC 22-0167; ibid. S1-1002 (ZHKU 22-0098, isotype), ex-type culture ZHKUCC 22-0168.

Notes: Our isolates (ZHKUCC 22-0167, ZHKUCC 22-0168) grouped with Stagonosporopsis astragali (CBS 178.25), forming a well-supported (89% in ML, 0.90 in BI) distinct clade in the combined gene analysis of ITS, LSU, RPB2 and β-tubulin (Figure 2). The result of PHI analysis of Stagonosporopsis species in this study shows Φw = 0.18 and there was no significant genetic recombination between these novel isolates and other Stagonosporopsis species (Figure 9). Comparison of the DNA sequences of ITS, LSU, RPB2, and β-tubulin locus of our isolates (ZHKUCC 22-0167, ZHKUCC 22-0168) with the type strain of S. astragali reveals the base pair differences of 5/488, 9/893, 7/595, 15/332 respectively.

Figure 9.

The results of PHI test of closely related taxa (Stagonosporopsis sub clade) using both LogDet transformation and splits decomposition. PHI test results. Φw = 0.18.

Morphologically, our collections are different from Stagonosporopsis astragali by short papillate, larger conidiomata (150–200 × 300–400 μm), and oblong to ellipsoid conidia without polar guttules. In contrast, S. astragali is an opportunistic pathogen producing smaller conidiomata (80–180 μm diam.) with 0–3 papillate ostioles, and cylindrical to allantoid conidia with numerous polar guttules. Therefore, we introduce this collection as Stagonosporopsis pedicularis-striatae sp. nov. This fungus has often recorded in North America (United States and Canada) on stems of various species of Astragalus [51].

4. Discussion and Conclusions

In a survey of the diversity and species richness of plant-associated fungi in the South China Botanical Garden, we collected several saprobic, hyaline-spored asexual species in the Didymellaceae. The Didymellaceae has recently undergone extensive revision based on its phylogenetic relationships and morphological characteristics [5,7,60]. In this study, we identified and introduced three new species in Didymellaceae (Ectophoma phoenicis, Remotididymella fici-microcarpae, and Stagonosporopsis pedicularis-striatae) along with a new host and locality record of Allophoma tropica based on polyphasic approaches according to the procedure of [61,62,63]. The guidelines for introducing novel species have been discussed in [64,65] were followed.

We described and illustrated a new locality report of Allophoma tropica. Allophoma tropica was recorded for the first time as a saprobe on leaves of Streptocarpus ionanthus and this study is the first record of Allophoma species on Canna in China. Furthermore, this study reported morphological characters of chlamydospores from cultures of Allophoma for the first time. The type collection of Allophoma tropica obtained from Streptocarpus ionanthus (H. Wendl.) Christenh. as a pathogen and morphological characters were obtained from pycnidia in culture while morphological characters of our collection were obtained from conidiomata on the substrate. Therefore, there are negligible differences in the morphological characteristics of fungi on a substrate and in media. There are four Ectophoma species listed in (Species Fungorum. http://www.speciesfungorum.org/Names/Names.asp, accessed on 14 August 2022). The type species of Ectophoma, E. multirostrata has been isolated from poultry farm soil and the live stem of Cucumis sativus in a greenhouse [28]. Ectophoma iranica and E. pomi are plant pathogens isolated from leaf spots of Dracaena compacta, Catharanthus roseus [29], and Coffea arabica [28]. Ectophoma insulana was isolated from the fruit of Olea europaea and from house dust [12]. Morphologically, our collections differ from E. iranica, and E. multirostrata and also our collection of Ectophoma phoenicis isolated from the dead petioles of Phoenix roebelenii are saprobic fungi. As such, this is the first record of saprobic behavior of Ectophoma species. Our collection of Ectophoma phoenicis was identified and introduced as a new species belonging to the genus Ectophoma.

Remotididymella fici-microcarpae is the first species in this genus reported from China and also from Ficus microcarpa. This was collected from dead stems as saprobes. However, Remotididymella species show a wide variation of life modes including plant and human pathogens and saprobes [12,32,48]. Morphologically, our collections differ from R. ageratinae by having immersed, brown, small conidiomata, large conidiogenous cells, and fusiform to allantoid conidia with indistinct guttules. Our collection of Remotididymella fici-microcarpae was identified and introduced as a new species belonging to the genus Remotididymella.

There are several Stagonosporopsis species that have been reported from China, and most of them are plant pathogens [31]. Stagonosporopsis vannaccii Baroncelli, Cafà, Castro, Boufleur and Massola was reported from leaf spots on Crassocephalum crepidioides in Guangxi [66] while Stagonosporopsis cucurbitacearum (Fr.) Aveskamp, Gruyter and Verkley are the cause of pumpkin gummy stem blight, which is one of the most devastating pumpkin crop diseases in North-East China [67]. Stagonosporopsis pogostemonis M. Luo, Y.H. Huang and Manawas. was reported from leaf spots and as a stem blight on Pogostemon cablin in South China [31]. However, Stagonosporopsis pedicularis-striatae was collected from dead stems as a saprobe. Morphologically, our collections differ from Stagonosporopsis astragali by their subglobose, slightly papillate, comparatively larger conidiomata (150–200 × 300–400 μm), and oblong to ellipsoid conidia. In terms of phylogenetic and morphological differences, we identified and introduced Stagonosporopsis pedicularis-striatae as a new species belonging to the genus Stagonosporopsis.

Author Contributions

Conceptualization, N.D.K.; Methodology, N.D.K.; formal analysis, N.D.K.; resources, N.D.K. and I.C.S.; data curation, N.D.K.; writing—original draft preparation, N.D.K.; writing—review and editing, N.D.K., I.C.S., D.N.W., M.D., S.L.S., J.S., B.X. and W.D.; supervision, W.D.; project administration, W.D.; funding acquisition, M.D., J.S. and W.D. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This research study is supported by the National Natural Science Foundation of China (Grant no. 32200015) and the Talent Program of Zhongkai University of Agricultural and Engineering (KA22016B787). National Natural Science Foundation of China (31860008; U1803232), and the foundations of Guangdong Provincial Department of Education (2022KCXTD015; 2022ZDJS020). National Natural Science Foundation of China (Nos. 32100012) and Talent Program of Zhongkai University of Agricultural and Engineering (KA200540852). Talent Program of Zhongkai University of Agricultural and Engineering (KA22016B746) and the Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China, Guangdong (KA21031C502).