First report of Curvularia dactylocteniicola causing leaf spots on Zea mays and Sorghum in Sri Lanka

: The genus Curvularia includes pathogens, endophytes and saprobes associated with a diverse range of plants belonging to the family Poaceae. These species are responsible for diseases on cereal crops such as rice, maize, wheat, millet, sorghum and poaceous weedy hosts. During a field survey of cereal pathogens conducted in Galle, Hambanthota, and Matale districts of Sri Lanka, diseased leaf samples were collected randomly from Zea mays and an indigenous cereal landrace of Sorghum sp. traditionally known as ‘Swayanjatha’. Morphology and molecular phylogeny based on combined nuclear ribosomal internal transcribed spacers 1 and 2 with 5.8S region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-α (TEF1) revealed that the causative agent of the leaf spots is Curvularia dactylocteniicola . Results of the pathogenicity tests confirmed cause of the disease on Zea mays and Sorghum sp. To the best of our knowledge, this is the first record of C. dactylocteniicola causing leaf spots on Zea mays and Sorghum sp. in Sri Lanka.


INTRODUCTION
Cereal crops and their crop wild relatives are the most common hosts for taxa in the genus Curvularia (Pleosporales, Dothideomycetes).The species of Curvularia have reported as saprobes, endophytes and pathogens with worldwide distribution (Manamgoda et al., 2012;Marin-Felix et al., 2017, 2020;Tan et al., 2014Tan et al., , 2018)).Pathogenic species of Curvularia have caused significant damage specifically on cereal crops (Kusai et al., 2016;Manamgoda et al., 2015;Qostal et al., 2019).However, prior to the molecular era the taxonomy of this genus was confusing and it was solely based on morphological characters.The current species recognition criteria of the genus has established based on multi-locus phylogeny.The most commonly used loci to construct multi-locus phylogenies of the genus are nuclear ribosomal internal transcribed spacers 1 and 2 with 5.8S region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-α (TEF1) (Manamgoda et al., 2012;2015;Marin-Felix et al., 2017;2020;Tan et al., 2014;2018).

Curvularia dactylocteniicola was first reported on
Dactyloctenium aegyptium in Thailand causing oval or fusiform, brown to brown-red leaf spots often surrounded by yellow halo and was first described by Marin-Felix et al. (2017).However, there were no available reports of this species from Zea mays and Sorghum from Sri Lanka.The aims of the present study were; i) to isolate the causal agents associated with the leaf spot disease of cereal crops cultivated in Sri Lanka; ii) to test the pathogenicity of isolates by fulfilling Koch's postulates; and iii) to characterize the isolated fungi using morphological, molecular and phylogenetic analyses.

Sample collection, Isolation and Morphological studies
During the period of 2018-2021, a noteworthy leaf spot disease was encountered on Zea mays and Sorghum sp.cultivated in Sri Lanka.Samples were collected during the field surveys conducted in Galle, Hambanthota and Matale districts.Four (04) fungal isolates were obtained following the single spore isolation method (Chomnunti et al., 2011) and cultures were maintained on potato dextrose agar (PDA, HiMedia-India) at 25 °C, with a light regime of 12 h in light and 12 h in dark.In order to determine colony characters, cultures were in triplicate on three different media: PDA, corn meal agar (CMA, HiMedia-India) and malt extract agar (MEA, Criterion-USA), at 25 °C, with a light regime of 12 h in light and 12 h in dark.Standard color chart by Rayner (1970) was used to record colony colors.Digital images of fungal structures were captured using a Carl Zeiss compound light microscope equipped with an AxioCam digital camera and ZEN lite software (Carl Zeiss Microscopy, Thornwood, NY, USA).The statistical data (mean, minimum, maximum, and standard deviation) for each micro-morphological measurement utilized in the morphological descriptions were recorded.
The specimens collected were deposited in USJ-H (University of Sri Jayewardenepura Herbarium) and the living fungal cultures are maintained in USJCC (University of Sri Jayewardenepura Culture Collection).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fungal isolates according to the protocol mentioned in Ferdinandez et al. (2021).Amplification of fungal ITS region and TEF loci were carried out according to the conditions and primers described in Manamgoda et al. (2012).Glyceraldehyde-3phosphate dehydrogenase (GAPDH) locus was amplified using the PCR conditions mentioned in Ferdinandez et al. (2021).DNA sequences generated in this study were deposited in NCBI GenBank (Table 1).

Sequence alignment, phylogenetic analyses and species recognition
Raw sequences generated for fresh isolates were assembled with BioEdit v7.0.5 for Windows.To confirm the identity, phylogenetic analyses were performed using both Maximum Parsimony (MP) and Maximum Likelihood (ML) criteria (Ferdinandez et al., 2020) along with ex-type and reference DNA sequences of the genus Curvularia (Ferdinandez et al., 2021).

Pathogenicity tests
To confirm the pathogenicity of the fresh isolates, healthy plants of Zea mays and Sorghum sp. were inoculated with conidial suspension of 1 x 10 6 conidia/mL of respective pathogen isolates (Kaspary et al., 2019).A similar amount of distilled water droplets was applied to the control plants.Both inoculated and non-inoculated plants were then covered to maintain high humidity and left in a plant house at 25 °C under a 12 h photoperiod.The same fungus was recovered from inoculated plants after five days of inoculation confirming Koch's postulates.

RESULTS AND DISCUSSION
The combined multi-locus phylogeny consisted of a total of 46 taxa and the alignment statistics for the parsimony analysis revealed 2246 total characters, with 1649 constant, 392 parsimony-informative, and 205 variable characters were parsimony uninformative.The most parsimonious tree resulting from the MP analysis of the combined dataset is presented here.Resulted phylogram revealed that the three isolates from Zea mays and a single isolate from Sorghum sp. were similar to C. dactylocteniicola which forms a monophyletic group with the ex-type isolate (CPC 28810) of the same species (Figure 1).Therefore, we provide a modern description based on the fresh collection of the species below with full illustrations, notes on habitats and recorded hosts and geographic distribution.Leaf spots (0.5-2 cm), oval or fusiform, brown to brown-red often surrounded by yellow halo which gradually increased up to 4-5 cm long as dark brown patches.Asexual morph: On CMA Hyphae 3-4 μm, hyaline, septate, branched.Conidiophores up to 316 µm long, simple to branched, septate, straight or flexuous, pale brown to brown.Conidiogenous cells hyaline to pale brown, smooth-walled, terminal or intercalary, monotretic to polytretic.Conidia (16)19-24(26) × 7-9(10) μm (av.= 21, SD = 3, n = 50; av.= 8, SD =1, n = 50), hyaline to pale brown, usually straight, sometimes curved, smoothwalled, ellipsoid, usually 3-distoseptate; hila 1-2 μm flat or slightly protruding, darkened.Chlamydospores formation is observed.Sexual morph: Not observed.
Culture characteristics: Colonies on PDA reaching 50 mm diameter after 7 d at 25 °C, colonies from above: convex, cottony appearance with moderate aerial mycelia, margin entire, mouse grey centre, olivaceous grey at the margins, reverse: dark brown at the margin, black in the centre.Colonies on CMA reaching 67 mm diameter after 7 d at 25 °C, colonies from above: flat, hairy appearance, circular, margin entire, olivaceous grey at the margin, pale brown in the centre, concentric ring growth; reverse: pale brown centre to periphery.Colonies on MEA reaching 67 mm diameter after 7 d at 25 °C, colonies from above: convex, circular, velvety appearance, margin entire, olivaceous green at the margin, grey in the centre; reverse: black from centre to margin (Figure 2 b, c, d).

Pathogenicity tests
Characteristic leaf spots developed after five days of inoculation on test plants while non-inoculated control plants remained asymptomatic (Figure 3).In order to fulfill Koch's postulates, the pathogen was re-isolated

CONCLUSION
According to the morphological and molecular data obtained, the fungal isolates recovered from diseased leaves were identified as Curvularia dactylocteniicola.To our knowledge, this is the first report of C. dactylocteniicola on Z. mays and Sorghum sp.(traditional).Thus, the current study contributes the records of host-fungal association and emphasizes the potential risk posed to commonly consumed cereals in Sri Lanka.
Following the first report from Thailand, Curvularia dactylocteniicola has been recorded as a pathogen on Saccharum officinarum in China (Raza et al., 2019) and on Sorghum bicolor in Indonesia (Hidayat and Ramadhani, 2019).Zea mays and Sorghum sp. have reported to be infected with several species of Curvularia including, C. australiensis (Chang et al., 2016), C. geniculata (Manzar et al., 2021), C. lunata (Akram et al., 2014;Garcia-Aroca et al., 2018).Nevertheless, C. dactylocteniicola has not been recorded as a pathogen on Zea mays before.Defining the species limits of the genus Curvularia is confusing as nomenclatural changes and refinements have occurred frequent within last three decades.There is no clear morphological boundary between the close associated genera Bipolaris and Curvularia (Manamgoda et al., 2012).Thus, establishment of generic boundaries from molecular phylogenetic assessments have given away the problems in species identification within the genus Curvularia with several refinements as well (Manamgoda et al., 2012;Marin-Felix et al., 2017;Tan et al., 2014Tan et al., , 2018)).Therefore, accurate identification of the causative agent of the leaf spot disease observed during this study was accomplished with the aid of morphological data and DNA sequence data incorporated multi-locus phylogenetic analyses.

Figure 1 :
Figure 1: Maximum Parsimony phylogram for Curvularia spp.based on the combined ITS, GAPDH, and TEF1 alignment.MP and ML bootstrap support values above 70% are shown at the nodes respectively.Ex-type cultures are marked with an asterisk.New records from the current study are indicated in bold red.The tree is rooted with Bipolaris maydis CBS13727/C5.

Table 1 :
Curvularia dactylocteniicola isolates identified and the DNA sequences generated in this study.