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

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.


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.
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;, 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.

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. (2011Manamgoda et al. ( , 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).

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, HG779108 - Ex-type isolates were labeled with " T ".

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 (  (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 . Infected leaves were collected and the fungus was re-isolated using PDA medium and the ITS sequence was compared with original strains.

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 Table 2. DNA sequence differences between Curvularia nanningensis and related species in three gene regions.   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.
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.  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.

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

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.