Diseases of Cymbopogon citratus (Poaceae) in China: Curvularia nanningensis sp. nov.

Qian Zhang; Zai-Fu Yang; Wei Cheng; Nalin N. Wijayawardene; Kevin D. Hyde; Zhuo Chen; Yong Wang

doi:10.3897/mycokeys.63.49264

Research Article

Diseases of Cymbopogon citratus (Poaceae) in China: Curvularia nanningensis sp. nov.

Qian Zhang^‡, Zai-Fu Yang^‡, Wei Cheng^‡, Nalin N. Wijayawardene^§, Kevin D. Hyde^|, Zhuo Chen^‡, Yong Wang^‡

‡ Guizhou University, Guiyang, China

§ Qujing Normal University, Qujing, China

| Mae Fah Luang University, Chiang Rai, Thailand

Corresponding author: Zhuo Chen ( gychenzhuo@aliyun.com )

Corresponding author: Yong Wang ( yongwangbis@aliyun.com )

Academic editor: Huzefa Raja

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.

Citation: Zhang Q, Yang Z-F, Cheng W, Wijayawardene NN, Hyde KD, Chen Z, Wang Y (2020) Diseases of Cymbopogon citratus (Poaceae) in China: Curvularia nanningensis sp. nov. MycoKeys 63: 49-67. https://doi.org/10.3897/mycokeys.63.49264

Abstract

Five Curvularia strains isolated from diseased leaves of lemongrass (Cymbopogon citratus) in Guangxi Province, China, were examined. NCBI-Blast searches of ITS sequences suggested a high degree of similarity (99–100%) to Curvularia akaii, C. akaiiensis, C. bothriochloae, C. heteropogonis and C. sichuanensis. To accurately identify these strains, we further analysed their morphology and phylogenetic relationships based on combinations of ITS, GAPDH, and tef1 gene sequences. Morphological observations indicated that the key character differing from similar species was conidial size, whereas phylogenetic analyses indicated that the five strains represent one species that is also distinct from C. akaii, C. akaiiensis and C. bothriochloae by conidial size and conidiophore length. Thus, the strains examined are found to represent a new species described herein as Curvularia nanningensis. The pathogenicity test on the host and detached leaves confirmed the new species to be pathogenic on Cymbopogon citratus leaves. Standardised requirements for reliable identification of Curvularia pathogens are also proposed.

Keywords

Cymbopogon, phylogeny, plant disease, Pleosporaceae, taxonomy

Introduction

Cymbopogon citratus Stapf (lemongrass), believed to be a native of Malaysia, is now widely distributed in all continents and particularly in America, China, Guatemala and Southeast Asia. Essential oil from lemongrass is often used in aromatherapy (Williamson et al. 1996; Noel et al. 2002; Yang and Lei 2005; Shah et al. 2011). As a traditional Chinese medicine, lemongrass is known to provide relief from a variety of ailments including eczema, cold, headache and stomach-ache (Zhou et al. 2011). Guatemala is known to be the main exporter of lemongrass with about 250 tons per year. China produces 80 to 100 tons of lemongrass annually and the USA and Russia each imports about 70 tons per year (DAFF 2012). Depending on climatic conditions, lemongrass can be severely infected with a rust disease caused by Puccinia nakanishikii Dietel in Hawaii and California (Gardner 1985; Koike and Molinar 1999). In Brazil, a rust on lemongrass caused by another Puccinia species named P. cymbopogonis Massee has been reported (Vida et al. 2006). Joy et al. (2006) summarised the various disease symptoms and their causal agents of lemongrass.

Curvularia spp. infect many herbaceous plants including Cymbopogon Spreng. (Smith et al. 1989). Helminthosporium cymbopogi C.W. Dodge (≡ Curvularia cymbopogonis (C.W. Dodge) J.W.Groves & Skolko) is responsible for a severe disease of lemongrass in the lowlands of Guatemala (Dodge 1942). Barua and Bordoloi (1983) discovered C. verruciformis causing disease on Cymbopogon flexuosus Stapf. Curvularia andropogonis (Zimm.) Boedijn led to foliage blight of Cymbopogon nardus (L.) Rendle in the Philippines (Sato and Ohkubo 1990). Thakur (1994) reported C. lunata (Wakker) Boedijn as the causal agent of a new blight disease of Cymbopogon martini (Roxb.) Wats. var. motia Burk. Chutia et al. (2006) discovered that a leaf blight of Cymbopogon winterianus Jowitt is caused by Curvularia spp., resulting in a dramatic change in oil yield and its constituents. Recently, Santos et al. (2018) characterised the morphological and molecular diversity of the isolates of C. lunata, associated with Andropogon Linn. seeds.

Starting in 2010, there have been outbreak reports of pathogenic Curvularia in Asian countries, especially India and Pakistan (Pandey et al. 2014; Avasthi et al. 2015; Majeed et al. 2015). As China is a neighbouring country, we felt obligated to evaluate the potential threat of Curvularia to our crops. A severe Curvularia leaf blight disease was observed in three farms of Curcuma aromatica Salisb. in Hainan Province during 2010 (Chen et al. 2013). Gao et al. (2012) reported a new rice black sheath spot disease caused by C. fallax Boedijn in Hunan Province. Our research group is also conducting a disease survey on the occurrence of Curvularia diseases in Southwest China since 2017. Two new pathogens (C. asianensis Manamgoda, L. Cai & K.D. Hyde and C. microspora Y. Liang, K.D. Hyde, J. Bhat & Yong Wang bis), which affected Epipremnum pinnatum (L.) Engl. and Hippeastrum rutilum Herb. (Liang et al. 2018; Wang et al. 2018), respectively, were found.

Meanwhile, a severe leaf blast disease on lemongrass was found in Guangxi Province, China, that first appeared on the tips of leaves. As the infection progressed, more than 30% of leaves showed different degrees of abnormalities, while in the later stages more than 50% of the upper leaves appeared diseased and disease incidence reached 80% or above in the lower leaf blades. We provide a detailed morphological description and phylogenetic analyses of the pathogen confirming it as a new Curvularia species. Koch’s postulates (see later text) have been carried out to confirm its pathogenicity. Our study provides a further understanding of Curvularia disease on lemongrass in China.

Materials and methods

Isolation

Leaves of Cymbopogon citratus showing leaf blast symptoms were collected from Guangxi Medicinal Botanical Garden in Nanning, China, during 2017. Diseased leaf pieces were surface disinfected with 70% ethanol for 30 s, 1% NaClO for 1 min and repeatedly rinsed in sterile distilled water for 30 s. For isolation of Curvularia, conidia were removed from the diseased tissue surface using a sterilised needle and placed in a drop of sterilised water followed by microscopic examination. The spore suspension was drawn with a Pasteur pipette and transferred to a Petri dish with 2% water agar (WA) or 2% malt extract agar (MEA) and 100 mg/l streptomycin to inhibit the growth of bacteria. The plates were incubated for 24 h in an incubator (25°C) and examined for single spore germination under a dissecting microscope. Germinating conidia were transferred separately to new 2% MEA plates (Chomnunti et al. 2014).

Morphological studies

Single germinated spores were transferred to PDA or MEA and incubated at 28°C in a light incubator with 12 h light/12 h darkness. Ten days later, the colony and morphological characters were recorded according to Manamgoda et al. (2011, 2012). Colony diameters on PDA and MEA were measured at 1, 3, 5 and 7 days post-inoculation and average growth rates were calculated. Conidia and conidiophores were examined using a compound microscope fitted with a digital camera (Olympus BX53). The holotype specimen is deposited in the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). An ex-type culture is deposited in the Culture Collection of the Department of Plant Pathology, Agriculture College, Guizhou University, China (GUCC) and Mae Fah Luang University Culture Collection (MFLUCC) in Thailand (Table 1).

Table 1.

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Sequences used for phylogenetic analysis.

Species name	Strain number	GenBank Accession numbers
Species name	Strain number	ITS	GAPDH	tef1
Curvularia aeria	CBS 294.61^T	HE861850	HF565450	–
C. affinis	CBS 154.34^T	KJ909780	KM230401	KM196566
C. ahvazensis	CBS 144673^T	KX139029	MG428693	MG428686
C. akaii	CBS 317.86	KJ909782	KM230402	KM196569
C. akaiiensis	BRIP 16080^T	KJ415539	KJ415407	KJ415453
C. alcornii	MFLUCC 10-0703^T	JX256420	JX276433	JX266589
C. americana	UTHSC 08-3414^T	HE861833	HF565488	–
C. asiatica	MFLUCC 10-0711^T	JX256424	JX276436	JX266593
C. australiensis	BRIP 12044^T	KJ415540	KJ415406	KJ415452
C. australis	BRIP 12521^T	KJ415541	KJ415405	KJ415451
C. bannonii	BRIP 16732^T	KJ415542	KJ415404	KJ415450
C. beasleyi	BRIP 10972^T	MH414892	MH433638	MH433654
C. beerburrumensis	BRIP 12942^T	MH414894	MH433634	MH433657
C. boeremae	IMI 164633^T	MH414911	MH433641	–
C. borreriae	CBS 859.73	HE861848	HF565455	–
C. borreriae	MFLUCC 11-0422	KP400638	KP419987	KM196571
C. bothriochloae	BRIP 12522^T	KJ415543	KJ415403	KJ415449
C. brachyspora	CBS 186.50	KJ922372	KM061784	KM230405
C. buchloes	CBS 246.49^T	KJ909765	KM061789	KM196588
C. carica-papayae	CBS 135941^T	HG778984	HG779146	–
C. chiangmaiensis	CPC 28829^T	MF490814	MF490836	MF490857
C. chlamydospora	UTHSC 07-2764^T	HG779021	HG779151	–
C. clavata	BRIP 61680b	KU552205	KU552167	KU552159
C. coatesiae	BRIP 24261^T	MH414897	MH433636	MH433659
C. coicis	CBS 192.29^T	JN192373	JN600962	JN601006
C. colbranii	BRIP 13066^T	MH414898	MH433642	MH433660
C. crustacea	BRIP 13524^T	KJ415544	KJ415402	KJ415448
C. cymbopogonis	CBS 419.78	HG778985	HG779129	–
C. dactyloctenicola	CPC 28810^T	MF490815	MF490837	MF490858
C. dactyloctenii	BRIP 12846^T	KJ415545	KJ415401	KJ415447
C. deightonii	CBS 537.70	LT631356	LT715839	–
C. ellisii	CBS 193.62^T	JN192375	JN600963	JN601007
C. eragrosticola	BRIP 12538^T	MH414899	MH433643	MH433661
C. eragrostidis	CBS 189.48	HG778986	HG779154	–
C. geniculata	CBS 187.50^T	KJ909781	KM083609	KM230410
C. gladioli	CBS 210.79	HG778987	HG779123
C. graminicola	BRIP 23186^T	JN192376	JN600964	JN601008
C. gudauskasii	DAOM 165085	AF071338	–	–
C. harveyi	BRIP 57412^T	KJ415546	KJ415400	KJ415446
C. hawaiiensis	BRIP 11987^T	KJ415547	KJ415399	KJ415445
C. heteropogonicola	BRIP 14579^T	KJ415548	KJ415398	KJ415444
C. heteropogonis	CBS 284.91^T	JN192379	JN600969	JN601013
C. hominis	CBS 136985^T	HG779011	HG779106	–
C. homomorpha	CBS 156.60^T	JN192380	JN600970	JN601014
C. inaequalis	CBS 102.42^T	KJ922375	KM061787	KM196574
C. intermedia	CBS 334.64	HG778991	HG779155	–
C. ischaemi	CBS 630.82^T	JX256428	JX276440	–
C. kenpeggii	BRIP 14530^T	MH414900	MH433644	MH433662
C. kusanoi	CBS 137.29^T	JN192381	–	JN601016
C. lamingtonensis	BRIP 12259^T	MH414901	MH433645	MH433663
C. lunata	CBS 730.96^T	JX256429	JX276441	JX266596
C. malina	CBS 131274^T	JF812154	KP153179	KR493095
C. mebaldsii	BRIP 12900^T	MH414902	MH433647	MH433664
C. micropus	CBS 127235^T	HE792934	LT715859	–
C. microspora	GUCC 6272^T	MF139088	MF139106	MF139115
C. miyakei	CBS 197.29^T	KJ909770	KM083611	KM196568
C. mosaddeghii	IRAN 3131C^T	MG846737	MH392155	MH392152
C. muehlenbeckiae	CBS 144.63^T	HG779002	HG779108	–
C. neergaardii	BRIP 12919^T	KJ415550	KJ415397	KJ415443
C. nanningensis sp. nov.	GUCC 11000	MH885316	MH980000	MH980006
	GUCC 11001	MH885317	MH980001	MH980007
	GUCC 11002	MH885318	MH980002	MH980008
	GUCC 11003	MH885319	MH980003	MH980009
	GUCC 11005^T	MH885321	MH980005	MH980011
C. neoindica	BRIP 17439	AF081449	AF081406	–
C. nicotiae	CBS 655.74^T = BRIP 11983	KJ415551	KJ415396	KJ415442
C. nodosa	CPC 28800^T	MF490816	MF490838	MF490859
	CPC 28801	MF490817	MF490839	MF490860
	CPC 28812	MF490818	MF490840	MF490861
C. nodulosa	CBS 160.58	JN601033	JN600975	JN601019
C. oryzae	CBS 169.53^T	KP400650	KP645344	KM196590
C. ovariicola	CBS 470.90^T	JN192384	JN600976	JN601020
C. pallescens	CBS 156.35^T	KJ922380	KM083606	KM196570
C. palmicola	MFLUCC 14-0404	MF621582	–	–
C. papendorfii	CBS 308.67^T	KJ909774	KM083617	KM196594
C. perotidis	CBS 350.90^T	JN192385	KJ415394	JN601021
C. petersonii	BRIP 14642^T	MH414905	MH433650	MH433668
C. pisi	CBS 190.48^T	KY905678	KY905690	KY905697
C. platzii	BRIP 27703b^T	MH414906	MH433651	MH433669
C. portulacae	CBS 239.48^T = BRIP 14541	KJ415553	KJ415393	KJ415440
C. prasadii	CBS 143.64^T	KJ922373	KM061785	KM230408
C. protuberata	CBS 376.65^T	KJ922376	KM083605	KM196576
C. pseudobrachyspora	CPC 28808^T	MF490819	MF490841	MF490862
C. pseudolunata	UTHSC 09-2092^T	HE861842	HF565459	–
C. pseudorobusta	UTHSC 08-3458	HE861838	HF565476	–
C. ravenelii	BRIP 13165^T	JN192386	JN600978	JN601024
C. reesii	BRIP 4358^T	MH414907	MH433637	MH433670
C. richardiae	BRIP 4371^T	KJ415555	KJ415391	KJ415438
C. robusta	CBS 624.68^T	KJ909783	KM083613	KM196577
C. rouhanii	CBS 144674^T	KX139030	MG428694	MG428687
C. ryleyi	BRIP 12554^T	KJ415556	KJ415390	KJ415437
C. senegalensis	CBS 149.71	HG779001	HG779128	–
C. sesuvii	Bp-Zj 01^T	EF175940	–	–
C. shahidchamranensis	IRAN 3133C^T	MH550084	MH550083	–
C. soli	CBS 222.96^T	KY905679	KY905691	KY905698
C. sorghina	BRIP 15900^T	KJ415558	KJ415388	KJ415435
C. spicifera	CBS 274.52	JN192387	JN600979	JN601023
C. sporobolicola	BRIP 23040b^T	MH414908	MH433652	MH433671
C. subpapendorfii	CBS 656.74^T	KJ909777	KM061791	KM196585
C. trifolii	CBS 173.55	HG779023	HG779124	–
C. tripogonis	BRIP 12375^T	JN192388	JN600980	JN601025
C. tropicalis	BRIP 14834^T	KJ415559	KJ415387	KJ415434
C. tsudae	ATCC 44764^T	KC424596	KC747745	KC503940
C. tuberculata	CBS 146.63^T	JX256433	JX276445	JX266599
C. uncinata	CBS 221.52^T	HG779024	HG779134	–
C. variabilis	CPC 28813	MF490820	MF490842	MF490863
	CPC 28814	MF490821	MF490843	MF490864
	CPC 28815^T	MF490822	MF490844	MF490865
	CPC 28816	MF490823	MF490845	MF490866
C. verruciformis	CBS 537.75	HG779026	HG779133	–
C. verruculosa	CBS 150.63	KP400652	KP645346	KP735695
	CPC 28792	MF490825	MF490847	MF490868
	CPC 28809	MF490824	MF490846	MF490867
C. warraberensis	BRIP 14817^T	MH414909	MH433653	MH433672
Bipolaris drechsleri	MUS0028	KF500532	KF500535	KM093761
B. maydis	CBS 136.29^T	AF071325	KM034846	KM093794

DNA Extraction and Sequencing

Fungal cultures were grown on PDA at 28°C until the entire Petri dish (90 mm) was colonised. Fresh fungal mycelia were scraped off the surface of the PDA using a sterilised scalpel. A BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416, BIOMIGA, Inc., San Diego, California, USA) was used to extract the genomic DNA. DNA amplification was performed in a 25 μl reaction volume which contained 2.5 μl 10 × PCR buffer, 1 μl of each primer (10 μM), 1 μl template DNA, 0.25 μl Taq DNA polymerase (Promega, Madison, WI, USA) and 18.5 μl ddH₂O. Primers used and thermal cycling programme for PCR amplification of the ITS (ITS4/ITS5), GAPDH (gpd1/gpd2) and tef1 (EF-526F/1567R) genes were followed as described previously (White et al. 1990; Berbee et al. 1999; Schoch et al. 2009; Liang et al. 2018).

Phylogenetic analyses

DNA sequences originated from five strains (GUCC 11000, GUCC 11001, GUCC 11002, GUCC 11003 and GUCC 11005) and reference sequences of ex-type or representative sequences of Curvularia species were downloaded from GenBank database (Table 1) with strains of Bipolaris maydis (Y. Nisik. & C. Miyake) Shoemaker (CBS 136.29) and B. drechsleri Manamgoda & Minnis (MUS0028) as outgroup taxa. Alignments for each locus were performed in MAFFT v7.307 online version (Katoh and Standley 2016) and manually verified in MEGA 6.06 (Tamura et al. 2013). Phylogenetic analyses were performed by Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian methods. Sequences were optimised manually to allow maximum alignment and maximum sequence similarity as detailed in Manamgoda et al. (2012). MP analyses were performed in PAUP v. 4.0b10 (Swofford 2003) using the heuristic search option with 1,000 random taxa additions and tree bisection and reconnection (TBR) as the branch-swapping algorithm. Five thousand maxtrees were set to build up the phylogenetic tree. The characters in the alignment matrix were ordered according to ITS+GAPDH+tef1 with equal weight, and gaps were treated as missing data. The MP phylogenetic analysis of Curvularia ITS sequences included pathogens from China, India and Pakistan and the wrong sequence (KN879930), actually belonging to Alternaria alternata (taxon:5599), was selected as the outgroup. The Tree Length (TL), Consistency Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were calculated for each tree generated. The resulting PHYLIP file was used to generate the ML tree on the CIPRES Science Gateway (https://www.phylo.org/portal2/login.action) using the RAxML-HPC2 black box with 1000 bootstrap replicates and GTRGAMMA as the nucleotide substitution model. For Bayesian inference analysis, the best model of evolution (GTR+I+G) was determined using MrModeltest v2 (Nylander 2004). Bayesian inference analysis was done using MrBayes v 3.2.6 (Ronquist et al. 2012). Bayesian analyses were launched with random starting trees for 2 000 000 generations and Markov chains were sampled every 1000 generations. The first 25% resulting trees were discarded as burn-in. Alignment matrices are available in TreeBASE under the study ID 25080.

Koch’s Postulate test

To confirm the pathogenicity of the fungus, five healthy plants of Cymbopogon citratus were inoculated with 5 mm diameter mycelial plugs of the five isolates (GUCC 11000, GUCC 11001, GUCC 11002, GUCC 11003 and GUCC 11005) cut from the margins of 10-day-old actively growing cultures; the control was treated with sterile agar plugs. The plants were kept for two days in an illuminating incubator at 28° ± 3°C. Additionally, two plants were sprayed with distilled water and kept as control under the same conditions. Both inoculated (host and detached leaves) and control plants were kept for two days in an illuminating incubator at 28 ± 3°C. After four days of incubation, the inoculated plants and leaves were observed for the development of symptoms (Zhang et al. 2018). Infected leaves were collected and the fungus was re-isolated using PDA medium and the ITS sequence was compared with original strains.

Results

Phylogenetic analyses

First, we compared the DNA sequence identity of ITS, GAPDH and tef1 gene regions (Table 2). Among our five strains, there was only one base difference. In the ITS gene region, for C. akaiiensis, the base sequence was identical to our strains; only 1 difference for C. bothriochloae; base differences were 8 for C. akaii, 9 for C. deightonii and 5 for C. sichuanensis. Only C. heteropogonis had noticeable (25) base differences with our strains. In the GAPDH and tef1 gene regions, the mutation rate of DNA bases was apparently faster than the ITS region. There were between 9 to 19 base differences in GAPDH and 3 to 8 in tef1. This means that in Curvularia, GAPDH has a faster evolutionary rate than ITS and tef1 and therefore some mycologists have suggested the use of ITS+GAPDH for phylogenetic analysis and GAPDH as a secondary barcode marker for accurate identification.

Table 2.

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DNA sequence differences between Curvularia nanningensis and related species in three gene regions.

Species	Strain number	ITS (1–547 bp)	GAPDH (550–1031bp)	tef1 (1034–1899 bp)
C. nanningensis	GUCC11000	0	1	0
	GUCC11001	0	0	0
	GUCC11002	0	1	0
	GUCC11003	0	1	0
	GUCC11005^T	0	0	0
C. akaii	CBS 317.86	8	9	4
C. akaiiensis	BRIP 16080 ^T	0	10	5
C. bothriochloae	BRIP 12522 ^T	1	19	8
C. deightonii	CBS 537.70	9	13	–
C. heteropogonis	CBS 284.91 ^T	25	12	3
C. sichuanensis	HSAUP II.2650-1 ^T	5	–	–

The alignment of Curvularia combining three gene fragments (ITS, GAPDH and tef1) comprised 116 strains belonging to 104 taxa. In order to accurately identify our strains, phylogenetic analysis included all ex-type and published strains of all Curvularia spp. described recently (Hyde et al. 2017; Marin-Felix et al. 2017; Dehdari et al. 2018; Heidari et al. 2018; Hernández-Restrepo et al. 2018; Mehrabi-Koushki et al. 2018; Tan et al. 2018; Jayawardena et al. 2019) which are listed in Table 1. The final alignment comprised 2032 characters (each gene fragment was separated with 2 “N”) including gaps (ITS: 1−600, GAPDH: 603−1162 and tef1: 1165−2032). Among these characters, 2032 are constant, 125 variable characters are parsimony-uninformative and 503 are parsimony-informative. The parameters of the phylogenetic trees are TL = 2590, CI = 0.38, RI = 0.72 and HI = 0.62. In the Curvularia phylogenetic tree (Figure 1), all isolates grouped together with 100% (MP and ML) bootstrap support. Our strains (GUCC 11000, 11001, 11002, 11003 and 11005) formed a strongly supported group (MP: 100%; ML: 100%; BPP: 1.00) with a close relationship to C. akaii, C. akaiiensis, C. bothriochloae, C. deightonii and C. heteropogonis with high bootstrap support (MP: 94%; ML: 97%; BPP: 1.00). In this group, the five examined strains were closer to C. akaii, C. akaiiensis and C. bothriochloae and also showed high bootstrap support (MP: 82% and ML: 94%; BPP: 0.98).

Figure 1.

Maximum Parsimony (MP) topology of Curvularia generated from a combination of ITS, GAPDH and tef1 sequences. Bipolaris maydis (CBS 136.29) and B. drechsleri (MUS0028) were used as outgroup taxa. MP and ML above 50% and BPP values above 0.90 were placed close to topological nodes and separated by “/”. The bootstrap values below 50% and BPP values below 0.90 were labelled with “-”. Our main research clade was labelled with green colour.

The phylogenetic analysis of the ITS gene region evaluated all new Curvularia pathogens recently described from China, India and Pakistan. The aligned matrix consisted of fifty-four ITS sequences and included ex-type sequences of 13 Curvularia species (Supplementary Table 1). The phylogenetic tree (Figure 2) indicated that ITS BLAST searches only provided limited value for pathogenic identification. In Curvularia lunata, only one sequence WCCL (MG063428) showed a very close relationship with the ex-type strain sequence of C. lunata CBS 730.96 (MG722981). The other eight sequences were grouped into two branches, e.g. taxon:5503 (LN879926) which might belong to C. aeria, while the other seven formed an independent lineage. ITS sequences did not separate Curvularia affinis, C. asianensis and C. fallax and some of their sequences even clustered with C. australiensis HNWB9-1 (KT719300). After multi-gene analysis, the phylogenetic distance was shown to be unreliable and may suggest whether they belong perhaps to different species.

Figure 2.

Maximum Parsimony (MP) analysis of Curvularia pathogens in China, India and Pakistan based on ITS sequences. Alternaria alternata (taxon:5599) was used as outgroup taxon. Bootstrap values (≥ 50%) of the MP method are shown near the nodes.

Taxonomy

Curvularia nanningensis Qian Zhang, K.D. Hyde & Yong Wang bis, sp. nov.

MycoBank No: 829056

Facesoffungi number: FoF 05596

Figure 3A–I

Diagnosis

Characterised by the size of conidia.

Type

China, Guangxi Province, Nanning City, Guangxi Medicinal Botanical Garden, 22°51’N, 108°19’E, on blighted leaves of Cymbopogon citratus, 30 September 2017, Q. Zhang, ZQ0091 (HGUP 11005, holotype, MFLU19-1227, isotype), GUCC 11005 and MFLUCC 19-0092, ex-type.

Description

Pathogenic on Cymbopogon citratus. Fungus initially producing white to grey lesions with dark borders on all parts of the shoot, later enlarging and coalescing over entire leaf.

Colonies on PDA irregularly circular, with mycelial growth rate = 1.0 cm/day, vegetative hyphae septate, branched, subhyaline to brown, smooth to verruculose, 2–3 µm, anastomosing. Aerial mycelium dense, felted, initially pale grey, becoming darkened and greyish-green at maturity, producing black extracellular pigments. On MEA, the colony morphology similar to PDA, with growth rate = 1.35 cm/day. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Conidiophores macronematous, arising singly, simple or branched, flexuous, 8–10 septate, geniculate, pale brown to dark brown, paler towards apex, 120–200 × 2–3 µm (av. = 170 × 2.5 µm, n = 30). Conidiogenous cells polytretic, sympodial, terminal, sometimes intercalary, cicatrised, with thickened and darkened conidiogenous loci up to 1.0–1.2 µm diam., smooth. Mature conidia 3 to rarely 4 septa, acropleurogenous, obovoid, usually straight to curved at the slightly wider, smooth-walled, larger third cell from the base, 24.5–36.0 × 14.0–20.5 µm (av. = 29.5 × 17.5 µm, n = 50), sub-hyaline to pale brown end cells, pale brown to dark brown at intermediate cells, with conspicuous or sometimes slightly protuberant hilum. Germination of conidia bipolar.

Distribution

China, Guangxi Province, Nanning City.

Other material examined

China, Guangxi Province, Nanning city, Guangxi Medicinal Botanical Garden, on blight leaves of C. citratus, 30 September 2017, Q. Zhang, ZQ0087 (HGUP 11000); ZQ0088 (HGUP 11001); ZQ0089 (HGUP 11002); ZQ0090, (HGUP 11003).

Etymology

With reference to the location, Nanning City where the fungus was isolated.

Figure 3.

Curvularia nanningensis (GUCC11005, holotype) A, B diseased symptom C colony on PDA from above D colony on PDA from below E−G conidia and conidiophores H−I conidia. Scale bars: 50 μm (E), 20 μm (F), 10 μm (G−I).

Pathogenicity test

Four days after inoculation, blast symptoms appeared on all inoculated plants, which were similar to symptoms of plants in the field (Figures 3A, B, 4A, B). Non-treated control plants remained healthy without any symptoms (Figure 4C). Curvularia nanningensis was re-isolated from the lesions of inoculated plants and the identity of the fungus was confirmed by sequencing the ITS region. Meanwhile, a detached leaf-experiment was also conducted in an illuminated incubator at 28 ± 3°C, where similar symptoms appeared on healthy inoculated leaves of Cymbopogon citratus after four days (Figure 4 D right), while the control leaf (Figure 4 D left) did not show symptoms.

Figure 4.

Pathogen inoculation and symptom (4 days). A Cymbopogon citratus inoculated and disease symptom B inoculation point and disease symptom C control D detached experiment. Left. Control. Right. Inoculation point and disease symptoms.

Discussion

Phylogenetic analysis based on combined DNA sequences of ITS, GAPDH and tef1 showed that our strains were related to three Curvularia species named C. akaii (Tsuda & Ueyama) Sivan., C. akaiiensis Sivan. and C. bothriochloae Sivan., Alcorn & R.G. Shivas. The main morphological characters that discriminate our strains from related species are the size-range of conidia and length of conidiophores. Curvularia bothriochloae produced conidia measuring 30–47 × 15–25 µm (Sivanesan et al. 2003) while C. akaiiensis produced the smallest conidia (22.5–27.5 × 7.5–15.5 µm). Conidial length of C. nanningensis was very close to C. akaii (24–34 µm) (Tsuda and Ueyama 1985) but the conidia of our species were broader than those of C. akaii (8.7–13.8 µm). Conidiophores of C. nanningensis were shorter than those of C. bothriochloae (360–425 µm) (Alcorn 1990). In the case of C. sichuanensis Meng Zhang & T.Y. Zhang, only one ITS sequence AB453881 was available in GenBank for analysis. While examining our sequences, only 4–5 bp differences were revealed in 499 bp characters between C. nanningensis and C. sichuanensis, thus indicating a close relationship between the two strains based on ITS sequence data and likely between the two species. However, according to Zhang et al. (2007), the conidial width of C. sichuanensis (10–15 µm) is smaller than C. nanningensis (14–20.5 µm) on PDA. For C. sichuanensis, the conidial wall of the median cell is deepened and thickened while C. nanningensis obviously does not have these characters. Meanwhile, the hilum of conidia in C. sichuanensis is obviously protuberant while C. nanningensis lacked this character.

The pathogenicity test based on natural inoculation and detached leaves (Figure 3) confirmed that Curvularia nanningensis is a pathogen of Cymbopogon citratus blast disease. We previously named our strains as C. cymbopogonis following a previous report of the species by Groves and Skolko (1945) as a seed-borne pathogen of Cymbopogon nardus. Curvularia cymbopogonis is a common pathogen which also causes diseases of sugar-cane, rice, seedlings of itchgrass, Agrostis palustris Huds. and Dactylis glomerata L. (Santamaria et al. 1971; Walker and White 1979; Olufolaji 1996; Yi et al. 2002). A single strain named C. cymbopogonis (CBS 419.78) included in our analyses grouped distant from C. nanningensis but its reliability seems questionable and apparently belongs to a different species (Fig. 1). We further checked the original description of this species (Groves and Skolko 1945) and found that differences in conidial shape mainly resulted from conidial width (C. cymbopogonis: 11–13 µm vs C. nanningensis: 14–20.5 µm). Additionally, Groves and Skolko (1945), Hall and Sivanesan (1972) and Yi et al. (2002) reported that C. cymbopogonis produced 4 to 5-septate conidia, whereas conidia of C. nanningensis only had 3-septa. Curvularia spp. are important pathogens of lemongrass. Morphological studies together with phylogenetic analyses provided evidence that C. nanningensis is a new pathogen distinct from all hitherto reported diseases on lemongrass. Our findings expanded the documented diversity of Cymbopogon pathogens within the genus Curvularia and further clarified the taxonomy of this novel pathogen, Curvularia nanningensis.

Moreover, 29 first reports of Curvularia diseases on different plants in China, India and Pakistan were found in the literature from 2010 to the present. It is evident that in this vast geographical area, Curvularia spp. have maintained a close association with plant diversity and thereby possess a rich fungal diversity that is affected by crops distribution. Among them, six reports only provided morphological data and more than half (16) only referred to ITS sequence data and morphological description (Suppl. Table 1). For unknown reasons, Iftikhar et al. (2016) misidentified the Curvularia pathogen with an Alternaria sequence (LN879930.1). Our phylogenetic tree, based on 54 reported ITS sequence data of Curvularia diseases in these countries (Figure 2), also indicated that this approach is not effective for identifying these pathogens, especially in the case of C. lunata as a prevalent species. However, identification of Curvularia isolates by multi-gene phylogenetic analyses has withstood scrutiny (Liang et al. 2018; Wang et al. 2018; Zhang et al. 2018). Additionally, nearly all reports, even for severe diseases, are based on a single isolate, which preclude an objective evaluation. We, therefore, propose the following standardised steps as required for the reliable identification of Curvularia diseases: 1) collect several isolates from diseased samples, 2) obtain sequences of the ITS, GAPDH and tef1 or at least ITS+GAPDH for phylogenetic analysis, 3) perform BLAST searches with sequences originated from ex-type or representative strains in GenBank, and 4) combine morphological comparison and phylogenetic analysis for accurate identification.

Acknowledgments

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), Science and Technology basic work of MOST [2014FY120100], National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2014BAD23B03/03), Talent project of Guizhou Science and Technology Cooperation Platform ([2017]5788-5 and [2019]5641) and Guizhou Science, Technology Department International Cooperation Base project ([2018]5806). Nalin Wijayawardene thanks National Natural Science Foundation of China (No. NSFC 31950410558). We thank Mr Mike Skinner for linguistic editing.

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Supplementary material

Supplementary material 1

Table S1

Qian Zhang, Zai-Fu Yang, Wei Cheng, Nalin N. Wijayawardene, Kevin D. Hyde, Zhuo Chen, Yong Wang

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