Morphological and molecular characterization of two graminicolous Exserohilum species associated with cultivated rice and early barnyard grass from Sri Lanka

The genus Exserohilum (Order Pleosporales, Class Dothideomycetes) comprises plant pathogenic hyphomycetous fungi, associated with poaceous hosts. Although numerous pathogenic species of Exserohilum are known globally, only E. turcicum and E. rostratum have been reported from Sri Lanka. In the present study, samples showing the symptoms of leaf blight of Oryza sativa (cultivated rice) and sheath blight of Echinochloa oryzoides (early barnyard grass) were collected and causal agents were primarily identified as Exserohilum spp. based on morphological characters. Molecular phylogenetic analyses based on three loci namely, nuclear ribosomal internal transcribed spacer (ITS), partial glyceraldehyde 3-phosphate dehydrogenase (GPDH) and translational elongation factor (TEF-1α) were used to infer evolutionary relationships and accurate identification. These isolates from O. sativa and Echinochloa oryzoides were identified as Exserohilum rostratum and E. oryzicola respectively. Both records are novel plant-fungal associations from Sri Lanka based on available data. This study suggests the need for morphological and molecular reassessments of emerging and poorly known species of fungi associated with cereals, their wild relatives and other economically important hosts in Sri Lanka.

Exserohilum species are encountered as pathogenic fungi of humans and plants and also frequently found as saprobic, endophytic and soil-borne fungi. Human pathogenic Exserohilum spp. are generally opportunistic fungi which may also cause life-threatening infections in immune-compromised humans. The most commonly reported human pathogenic species is E. rostratum, whereas some cases are attributed to E. longirostratum and E. macginnisii (McGinnis et al., 1986;De Hoog et al., 2000;Al-Attar et al., 2006). These pathogens have been reported on immune-compromised patients causing skin and corneal infection, invasive diseases, and allergic fungal sinusitis (Adler et al., 2006).
The plant family Poaceae comprises of important cereal crops such as rice, wheat, millet and corn which provide major dietary needs of the human population. Pleosporalean fungal pathogens, bearing brown asexual spores, are often associated with cereal crops, their wild relatives and weeds in the family Poaceae in different life styles including, epiphytes, endophytes, saprophytes or pathogens (Hernandez-Restrepo et al., 2018). Understanding the host associations and host ranges of fungi is important due to the possibilities of host shift of these species from weed hosts to important crops, as observed in many species. For example, E. fusiforme (syn. E. oryzicola) has originally been identified as pathogenic on the weed, Echinochloa crus-galli, causing numerous small leaf lesions and later known to cause small linear spots on, cultivated rice plants (Alcorn, 1991).
Majority of Exserohilum species are associated with grasses and important crops in the family Poaceae causing leaf blights of corn and millet, leaf spots and foot rots of wheat and damping-off of sugarcane seedlings (Sivanesan, 1987). The type species of the genus, Exserohilum turcicum, is the causative agent of northern leaf blight of corn which is a widespread foliar disease characterized by oblong, straw-colored to greyish necrotic lesions and causing significant death of foliar tissue. The reduction of effective photosynthetic area of leaves may lead to severe cases of grain yield losses of 20-25 % (Smith et al., 1988).
Although Exserohilum species are widely known emerging fungi on cereal hosts and weeds with worldwide distribution, only two species, E. turcicum and E. rostratum, have been recorded so far from Sri Lanka (Farr and Rossman, 2020). Information on the diversity and DNA sequence data of these common cereal pathogenic fungi is important to establish control measures for emerging fungal diseases (Udayanga, 2019). Therefore, the major aim of this study was to use molecular and morphological data to characterize freshly collected isolates of Exserohilum species associated with rice and associated grass species collected from two selected locations in Sri Lanka.

Sample collection, isolation and morphological studies
Samples were collected from field surveys carried out in Kegalle and Gampaha districts and all the specimen information (date of collection, collector, locality, host and symptomatology) were recorded and the samples were brought to the laboratory for further processing.
Fresh specimens were observed under stereomicroscope (Optika, LAB 30) and, instances where fungal structures were not visible, they were incubated for another 24 h in a moist chamber. Single spore isolation was done from the sporulating samples to isolate fungi (Chomnunti et al., 2011). Pure cultures were prepared on Potato Dextrose Agar (PDA) and stock cultures were maintained on Corn Meal Agar (CMA) slants. To determine colony morphology, cultures were triplicated on several media; PDA, CMA and Malt Extract Agar (MEA), and incubated at 25 °C for 12 h each in light and dark conditions. The color notations were recorded according to the standard color charts (Rayner, 1970). Micro-morphological characters were observed under compound light microscope (Optika, B 290) and measurements of structures were obtained under imaging facility. At least 30 length and width measurements were made from conidia of each isolate. Digital microscopic images were generated to illustrate the morphological characteristics. For all morphological measurements, statistical data (mean, minimum, maximum and standard deviation) were calculated and used in taxonomic descriptions. The specimens collected were dried and preserved as reference herbarium material at the herbarium, University of Sri Jayewardenepura (USJ) and the cultures are maintained at the fungal collection (USJCC) at the Department of Botany, Faculty of Applied Sciences, University of Sri Jayewardenepura, Sri Lanka.

DNA extraction, PCR amplification and sequencing
Genomic DNA were extracted from the morphologically identified Exserohilum fungal isolates following the modified Sodium Dodecyl Sulphate (SDS) method as described in Arnold and Lutzoni (2007). The PCR amplifications were carried out in the BIORAD T 100 Thermal cycler according to the protocols described in Manamgoda et al. (2012) with the primer pairs for ITS region with ITS1 and ITS4 (White et al., 1990), GPDH with gpd1 and gpd2 (Berbee et al., 1999) and TEF1-α with EF1-983F and EF1-2218R (Rehner and Buckley, 2005). The PCR products were visualized on 2 % agarose gel electrophoresis. PCR product purification and Sanger sequencing of the successfully amplified samples were carried out in Macrogen Inc, Korea.

Sequence alignment, phylogenetic analyses and species recognition
Raw sequences were assembled on BioEdit v7.0.5 programme for windows. Initial alignments of assembled DNA sequences were accomplished using BioEdit v7.0.5, optimized with MAFFT v. 7 using default settings (http:// mafft.cbrc.jp/alignment/server/) (Katoh and Standley, 2013). Preliminary identification of the isolates was carried out using newly generated ITS, GPDH and TEF1-α sequences with all available ex-type sequences from the GenBank as listed in Table 1.
Phylogenetic analyses were performed in two different criteria; Maximum Parsimony (MP) and Maximum Likelihood (ML) in order to infer evolutionary relationships among closely related species. Sequence data generated in this study were deposited in GenBank (Table 1).
Maximum Parsimony was performed with PAUP v. 4.0b10 (Swofford, 2003). Trees were inferred using the heuristic search option with 1000 random sequence additions. Descriptive tree statistics for parsimony [Tree length (TL), Consistency Index (CI), Retention Index (RI), Rescaled Consistency Index (RC) and Homoplasy Index (HI)] were calculated for trees generated in the parsimony analysis. Maximum likelihood trees were constructed using the RAxML v.7.4.2 Black Box (Stamatakis et al., 2008) in the CIPRES Science Gateway platform (Miller et al., 2010). For the combined dataset all free model parameters were obtained using RAxML with ML estimate of 25 per site rate categories. Phylogenetic trees generated were visualized by FigTree v. 1.4 (Rambaut and Drummond, 2008).

Molecular Phylogeny
In the present study, two different Exserohilum species from rice and early barnyard grass were accurately identified. The updated backbone phylogenetic tree for the genus Exserohilum presented in Figure 1 includes  According to the phylogram generated, two isolates clustered in distinct clades within the genus representing two distinct species. The strain USJCC-0010 isolated from the host Echinochloa oryzoides, grouped as more closely related with Exserohilum fusiforme. Recent phylogenetic assessments of the genus Exserohilum (Hernández-Restrepo et al., 2018) have shown that E. fusiforme is conspecific with closely related E. oryzicola. Therefore the isolate USJCC-0010 was identified as E. oryzicola. Similarly, Hernández-Restrepo et al. (2018) revealed that E. antillanum, E. gedarefense, E. longirostratum, E. macginnisii and E. prolatum are conspecific with E. rostratum. Therefore, the isolate USJCC-0011, which clustered in the broadly classified "rostratum clade" was hereby determined as E. rostratum. Although the aforementioned five species are determined to be one species, E. rostratum, by Hernández-Restrepo et al. (2018) in the phylogeny, sequence variabilities are observed within all three gene loci. Therefore, these species may be segregated in to different taxa if the sampling and gene regions are increased in future studies.
Colony characteristics: Colonies on PDA dark greenish center and olivaceous green to the periphery, flat, entire margin slightly undulated, sparse aerial mycelia, reaching 4.4 cm diam. after 7-d of incubation. Colonies on MEA dark green and olivaceous green concentric rings, flat, attaining approximately 5.8 cm diam.; on CMA brownish aerial mycelia, flat colony, approximately 7.9 cm in 7-d.  (Farr and Rossman, 2020).
Based on the available sources and databases, we confirm that the two species Exserohilum oryzicola on Echinochloa oryzoides and Exserohilum rostratum on Oryza sativa are novel plant-fungal association records. This study highlights the potential occurrence of Exserohilum associated with rice and associated weeds in Sri Lanka. Further studies in combination with phytopathological surveys incorporated with molecular data could reveal many unknown fungi and fungal-hosts associations, significance in agriculture and biosecurity. Therefore, this study urges the need for molecular identification and taxonomic studies in Sri Lanka for the control of emerging plant and human pathogens and also to update quarantine measures, pathogen lists for cereal and fiber crops and weeds.

ACKNOWLEDGMENT
University of Sri Jayewardenepura is acknowledged for the Research grant ASP/01/RE/SCI/2018/036 to work on the taxonomy and molecular phylogeny of graminicolous hyphomycetes in Sri Lanka. The study is also partially funded by Emory Simmons research award to DSM by Mycological Society of America in 2018.