Pathogenicity and infection behaviour of Exserohilum rostratum on wheat and associated collateral hosts

Exserohilum leaf spot is a newly arising fungal disease that primarily affects monocots. The isolate collected from diseased wheat leaves produced typical dark brown lesions upon inoculation to healthy plants. Thirty-two plant species of 14 families were evaluated for susceptibility to Exserohilum rostratum as a potential pathogen. The artificial inoculation using detached leaf assay showed symptoms on major cereals like Triticum aestivum, Oryza sativa, Echinochloa esculenta, Panicum miliaceum and Eleusine coracana. The symptoms were reddish-brown in members belonging to Poaceae. Histopathological studies revealed that conidia produce the appressoria within 24 h and penetrate the host through stomata or epidermal cells after germination. Study shows that collateral hosts serve as an infection reservoir, allowing it to survive without its primary host. These secondary hosts aid the pathogen in continuing the infection cycle and spreading the disease. The infections on major cereals like wheat and rice indicate its importance as an emerging plant pathogen.


Introduction
Exserohilum K.J. Leonard & Suggs has been described as a pathogen in cereals and grasses (Kusai et al. 2016;Hernandez-Restrepo et al. 2018). Every year about 10-20% of losses due to diseases are reported in cereals (Figueroa et al. 2018). Major wheat diseases are rusts, spot blotch, smut, common root rot, Fusarium head blight and several bacterial and viral diseases (Gulyaeva et al. 2020). Helminthosporium Link is classified as one of the influential groups under anamorphic ascomycetes, including pathogens of plants and animals. Based on conidial ontogeny and morphology, this group is divided into three genera Exserohilum, Bipolaris Shoemaker, and Drechslera S. Ito (Alcorn 1988). Presently, less than 50 species are described as true Helmithosporium spp. Based on the conidial and protruding hilum, the Exserohilum was erected from Bipolaris by Leonard and Suggs (1974) with the sexual morph Setosphaeria rostrata K.J. Leonard. E. rostratum is a phytopathogen with more than 30 plant species in 28 genera as hosts. It is the causative agent of blight, leaf spot, and rots, including seedling damping-off and foot and root rot. Many economically important crops, including rice, wheat, sweet sorghum, sugarcane (Poaceae), tomato (Solanaceae), and banana (Musaceae), have been reported to be infected by this pathogen (Kusai et al. 2016;Hernandez-Restrepo et al. 2018). E. rostratum infects the host in a biotrophic mode, entering through a natural opening such as the stomata or directly through infection pegs. It eventually adopts a necrotrophic lifestyle after successful penetration, killing the cells by producing secondary metabolites and toxins. E. rostratum has been identified as a soil-borne saprophyte. The moist environment promotes abundant mycelium growth and pathogen spread via wind and rain on various collateral hosts, including monocotyledonous grasses (Sharma et al. 2014;Kusai et al. 2016). Exserohilum pathogenic species include E. pedicellatum (A.W. Henry) K.J. Leonard & Suggs, E. prolatum K.J. Leonard & Suggs producing 1 3 leaf spot on maize and wheat. However, E. rostratum K.J. Leonard & Suggs infect numerous hosts, including wheat, banana, maize and several grasses (Lin et al. 2011). Exserohilum leaf blight is characterized by leaf and stem lesions that spread to the entire leaf after a few days of infection (Kusai et al. 2016). A strong interaction between the pathogen and the environment is also reported (Kusai et al. 2016). Climate change has also brought behavioural changes in pathogens, and many pathogens of minor significance have become major (El-Sayed and Kamel 2020). E. rostratum is a potential emerging plant pathogen whose outbreak is expected due to climate change leading to increased temperatures. However, the biology and ecology of this pathogen are least known. Therefore, this study aimed to determine the host range and histological studies during infection.

Materials and methods
All the investigations were carried out during 2019-2021 at the laboratory and polyhouse facility, Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India (North Eastern Plain Zone, India, 25.26° N and 83.99°E).

Fungal isolation and identification
Wheat leaves infected by E. rostratum were collected from Dharwad, Karnataka (15.3518° N,75.1291° E). Infected samples were air-dried and stored in a labelled paper bag. The pure culture of the fungus was obtained by single spore isolation. The spore suspension of fungal isolates was streaked on autoclaved 4% water agar (WA). The single spore was picked and cultured aseptically on potato dextrose agar (PDA) PH 7.0, and it was then incubated for 24 h at 28 ℃. Pure culture of the fungus culture was maintained on PDA (Chand et al. 2013). For spore measurements, a minimum of 30 spores were analyzed per culture using Adobe CS5 Software.
For molecular identification, DNA was isolated following Al-Samarrai and Schmid (2000). In brief, fungal colonies of age 3-4 weeks were harvested and washed in 0.1% W/V CsCl solution following centrifugation at 13000 rev/ min for 10 min. Freeze-dried mycelium was ground to a fine powder in liquid nitrogen and resuspended in 500 µl of lysis buffer (40 mmol/l Tris-acetate, 20 mmol/l sodium acetate, 1 mmol/l EDTA, 1% w/v SDS pH 7.8). RNAse A (2 µl of 10 mg/ml) was added, and the mixture was incubated for 5 min at 37 °C. To precipitate most polysaccharides, proteins, and cell debris, 165 µl of 5 mol/l NaCl solution was added, and components were mixed by inverting the tube several times. The suspension was centrifuged for 20 min at 4 °C at 13000 rev/min, and the supernatant was immediately transferred to a new tube, and chloroform (400 µl) and phenol ((400 µl) were added. Two volumes of 95% ethanol were used to precipitate the DNA in the aqueous supernatant. The precipitated DNA was washed three times with 70% ice-cold ethanol, dried, and dissolved in 50 ml TE buffer (10 mmol Tris-HCl, 0.1 mmol/l EDTA, pH 7.8) before being stored at 20 °C.
Fungal barcodes Internal Transcribed Spacer (ITS) region of the rRNA (White et al. 1990) and Translation elongation factor 1-alpha (Tef1α, Raja et al. 2017) were used for identification. In brief, for the ITS region, primers ITS1F (CTT GGT CAT TTA GAG GAA GTA A) and ITS4 (TCC TCC GCT TAT TGA TAT GC) were used in PCR protocol: 95 °C -5 min, 94 °C -30 s, 52 °C -30 s, 72 °C -1 min, repeat 35 cycles, 72 °C -8 min and 4 °C on hold. For the Tef1α region, primers EF1-983F (GCY CCY GGH CAY CGT GAY TTY AT) and EF1-2218R (AT GAC ACC RAC RGC RAC RGT YTG) were used in PCR protocol: 94 C -2 min, 66 °C -56 °C touchdown (9 cycles), 94 °C -30 s, 56 °C -1 min, 72 °C -1 min, repeat for 36 cycles, 72 °C -10 min and 4 °C on hold. PCR-purified products were commercially sequenced at Eurofins Pvt. Ltd., India, following Sanger sequencing. Sequences were checked and edited in BioEDIT (v 7.2) for good quality and further subjected to nBLAST for homology in the GenBank database (https:// www. ncbi. nlm. nih. gov/ genba nk/) to confirm the species further. For phylogenetic analysis, additional sequences were retrieved from GenBank (See Table S1 for sequence details). MEGA11 (Tamura et al. 2021) was used for the phylogenetic tree construction. The alignment of sequences was done using MUSCLE (Edgar 2004). During the sequence alignment, gaps and missing data were removed. The phylogenetic tree was based on the Neighbor-Joining method (Saitou and Nei 1987) with 1000 bootstrap repetitions to calculate the confidence levels for each branch.

Host range and inoculation studies
The identified culture was multiplied (Accession No. MTCC 13118 at MTCC, Chandigarh, India) on PDA after inoculating a bit of mycelium in the plate's centre and incubating at 25 ℃ for a week. The inoculum was prepared by washing the conidia from the cultures grown in Petri plates using sterile water. Spore suspensions were prepared and adjusted to 10 4 spores/ml with a drop of Tween 20 (0.05%) as a spreader. Spore suspensions were sprayed on the leaves at the Zadok's stage 45 (Zadoks et al. 1974). A host range test was conducted on locally available and reported weeds (Table 1). Under polyhouse conditions (plastic tunnel), seeds of different hosts were grown in pots (20 cm diameter) filled with sterilized mixtures with equal portions (v/v) of soil, sand and clay (5 seeds/pot). All the plants were inoculated when they were 35 days old. The spore suspension was sprayed on each plant with hand-held atomizers. For each isolate, a separate was used. The inoculated plants were covered with plastic bags for 48 h to maintain high humidity. Three replicates of each host were sprayed with the isolate (MTCC 13118). For negative control, a set of plants were sprayed with sterile water and covered with polythene for 48 h. Six days post-inoculation, plants were examined for the appearance of symptoms.
The method described above was followed in the detached leaf assay technique with three leaves as three replications and control in Petri plates for 31 hosts (Bhattarai et al. 2020). The incubation period (IP) was recorded from the inoculation to the appearance of the first visible symptoms described by Aquino et al. (1995). The total number of lesions was counted on each infected leaf one week after inoculation. The leaf was divided into four parts with the help of a marker pen, and the number of lesions was counted (LN) in each part (Smith and Campbell 1996). Lesion size (LS) was determined by measuring the length and width of the lesion produced by the pathogen one week after inoculation (Bashyal et al. 2011). The grey value was obtained by transforming the RGB image into an 8-bit greyscale and calculating the mean grey value of the pixels making up each lesion. The greyscale ranges from 0 (black) to 255 (white) and can be used to describe the degree of the colour of the lesion in E. rostratum (Lendenmann et al. 2014;Stewart et al. 2016).

Measurement of the lesion number, the colour of the lesion, mean grey value and lesion size of the infected leaves
Photography of individual infected leaves was done by the NIKON Camera D5200 (Nikon Japan) on red background with a white scale (Poudel et al. 2019). The photographs of symptoms were taken from randomly selected three tagged plants in each row for all the hosts after inoculation. These photographs were saved in jpg format. The lesions were analyzed for colour measurement on a grey scale by Adobe CS5 Software. Selected infected leaves were opened in the software and went to the analysis settings for modification; pixel length, logical length, and logical unit (https:// helpx. adobe. com/ photo shop/ using/ color-modes. html). The number of lesions was measured using the analysis menu's count tool. Grey values were displayed, extracted to the notepad, and finally recorded in the excel file.

Histopathology
The leaves of the different hosts were collected at 0, 24 and 48 hours after inoculation (hai). Histopathology was performed using a bleaching solution (Ethanol: Glacial acetic acid: Glycerol = 3:1:1 v/v). Cut leaf segments (1 cm × 1 cm) were poured in 5 ml bleaching solution into a test tube (10 ml) containing leaf segments in each test tube. The bleaching solution and leaf segments were placed in boiling water for 15-20 min (Sillero and Rubiales 2002). The transparent leaf segments were used for microscopic study after treating the leaf with Trypan blue solution (Trypan blue, 0.025 mg + ethanol, 50 ml + 50 ml, lactophenol + 50 ml distilled water) (Sillero and Rubiales 2002). Spore germination and infection processes were observed using the Nikon Eclipse E 200 microscope (Nikon Instruments Inc). The observations on the germination percentage (G%), germ tube length (LGT), total count formation of appressoria (NAF), total penetration count through a stoma (SP) and total count penetration (EP) through the epidermal cell were noted at 0, 24, 48 hai (Bashyal et al. 2011).

Scanning electron microscope (SEM)
The infected leaf lesions were dissected into small 1 mm 2 pieces. The primary chemical fixation was carried out for 18 h at 4 °C in 0.1 M phosphate buffer (pH 7.2). After that, the samples were rinsed with 0.1 M phosphate buffer (pH 7.2) (Kiernan 2000). The sample conductivity was increased by post-fixing it with 2% osmium tetroxide for 2 h at room temperature. Specimens were initially dehydrated by immersing them in the following ethanol gradients: initially, 50% and 70% (two times for 10 min), then 95% (two times for 5 min), and finally 100% (two times for 1 min). The samples were then dehydrated with acetone twice for 30 s before being dried in liquid CO 2 using the critical point procedure (Riau et al. 2010). A sticky carbon disc or tape was used to mount the specimen to a metal stub to increase conductivity. The conductive material was sputtered for 6 min in a sputter coater, and the metal was coated in a controlled manner. The coating was designed to be thick enough to prevent charging (usually around 10 nm) but not so thick that it obscured the surface details of the specimen. The coated samples were examined with a ZEISS EM 10 SEM (Department of Geology, BHU) at 20 keV (Low-voltage electron microscope) at magnifications ranging from 100 to 5000-fold. Photoshop (Adobe Photoshop v. CS5) was used to combine the images.

Statistical analysis
The data were subjected to ANOVA (analysis of variance) and LSD (least significant differences) to determine the significant differences between means. The correlation coefficients between disease and histological components were calculated using R software (Version 4.2.1, R Core team 2022).

Results
Isolation and identification of the pathogen E. rostratum isolated from the infected leaf samples has been deposited at CSIR-Microbial Type Culture Collection (MTCC), Chandigarh, India, with accession number MTCC 13118. The conidial dimensions of the isolate ranged from 14-85 µm in length and were 7-17 µm wide. The conidia had protruding hilum, conidiophores slightly curved or erect, septate and geniculate, olivaceous-brown, conidiogenous cells with circular conidial scars. A dark septum was observed on both polar ends of each conidium. The conidial dimensions of the isolates ranged from 85-114 µm in length and were 7-17 µm wide. The conidia had protruding hilum, conidiophores slightly curved or erect, 5 to 15 distoseptate and geniculate, olivaceous-brown, conidiogenous cells with circular conidial scars (Fig. 1). A dark septum was observed on both polar ends of each conidium. Additionally, the identity of the isolate was confirmed based on the phylogenetic analysis of ITS and Tef-1α regions of E. rostratum (Fig. 2). The sequences are deposited at NCBI GenBank with accession MN599631 (ITS) and OM752309 (Tef1 α). The fungal strain was identified as E. rostratum based on the similarity with most central strains CBS 112815, type strain CBS 571.73, CBS 412.93, CBS 467.75, and CBS 325.87. Estimates of evolutionary divergence between sequences are presented as Table S1.

Symptomatology on different hosts and pathogenicity aspects
For Triticum aestivum, Echinochloa esculenta, Eleusine coracana, Panicum miliaceum and other hosts, small brown to dark brown lesions were formed, and later, these lesions coalesced, leading to blight appearance. Furthermore, in Sorghum bicolour, Saccharum spontaneum and Saccharum officinarum where lesions coalesce to produce reddish colour symptoms. (Figs. 3 and 4). Mangifera indica has not shown infection.
Incubation period: The incubation period for different hosts under the polyhouse and detached leaf technique differed significantly. In detached leaf assay, the incubation period was three days on Cynodon dactylon, Megathyrsus maximus, Panicum virgatum and Sorghum bicolour. The prolonged incubation period was five days on Oryza sativa, Saccharum officinarum, Eichhornia crassipes, Zea mays, Ficus religiosa and Ficus hispida (Table 2). Under polyhouse, the incubation period was 4.34 days on Zea mays and the shortest on Dactyloctenium aegyptium, Setaria   (Table 3).
Lesion Count / leaf: Pathogenicity using detached leaf assay is presented in Table 2. The maximum number (93.50) recorded Eichhornia crassipes, followed by Sorghum bicolor (66.0) and the minimum number (8.50) in Vigna radiata. In the greenhouse, the maximum lesion number (147) was in Panicum miliaceum, followed by (131) in Sorghum bicolor. In Triticum aestivum, the lesions were (38.5) ( Table 3).

Discussion
Exserohilum leaf spot is a new emerging fungal disease in the present scenario. In this study, the fungus was isolated from diseased leaves of wheat exhibiting leaf blight. The fungus was identified based on conidia morphology and molecular characterization of ITS and Tef 1α sequences which showed the same homology (95-100%) as other isolates of E. rostratum in GenBank (Cardona and González 2007;Lin et al. 2011). Kalekar (1973) reported E. rostratum on wheat and paddy (Kusai et al. 2016). The primary symptoms appeared dark to purplish spots and became LGT), total count formation of appressoria (NAF), total penetration count through a stoma (SP) and total count penetration (EP) through the epidermal cells. Different colours indicated correlation coefficient among the disease components as more than medium size blue (P < 0.001) medium size blue colour (P < 0.01) followed by small size blue colour (P < 0.05), and non-significant difference (NS) indicated by very small size blue colour, light to dark brown colour chlorotic to necrotic areas. (Farag 2020;Brunings 2009). This pathogen produces lesions with reddish borders in bean plants seven days after inoculation (Cardona and González 2007). Lesions of E. rostratum on Thysanolaena latifolia were elliptical between leaf veins and sometimes appeared as yellow halos, ranging from 0.2 cm to 1.2 cm long (Brunings 2009). The higher densities of inoculums lead to disease progression, coalescing lesions resulting in widespread necrosis and death of leaves (Brunings 2009). Plant pathogens shift to new hosts for their survival and host expansion in nature ). E. rostratum was isolated from the diseased leaf of wheat and more infectious to the members of Poaceae than dicots. E. rostratum would establish a compatible interaction with the Poaceae hosts, resulting in divergence in the population. Using grey values, we obtained a degree of the lesion's colour based on the assumption that with a higher grey value, the lesion represents more sporulation and disease spread on different host leaves. The fungal, conidial germination takes place by specific stimuli from plant surfaces. (Ijadpanahsaravi et al. 2021). The pegs from the globose appressorium penetrated the leaf epidermal or stomata and colonized host cells. The chemicals such as calcium, potassium ions, sucrose, phenols, and plant extract influence the appressoria formation by a fungal pathogen (Lee and Bostock 2006). These chemicals stimulate or inhibit the number of appressoria on different hosts (Visai and Vanoli 1997;Podila et al. 1993). The bulbous appressorium development in tiger grass was reported by Lin et al. (2012), which is also reported in our experiment. The appressorium penetrates through stomata and epidermal cells and colonization of E. rostratum inside the mesophyll cells of rice, wild sugarcane, and maize (Lin et al. 2012;Sun et al. 2022). This investigation revealed similar results in wheat infected with E. rostratum. A significant positive correlation between disease and histological components was observed. Poudel et al. (2019) and Yusuf et al. (2016) in B. sorokiniana infections in wheat revealed a positive and significant correlation between lesion number and lesion area and sporulating lesions.
This study reveals that E. rostratum was mainly pathogenic to the members of Poaceae. Reddish colour lesions were evident in sugarcane, sorghum and wild sugarcane while remaining hosts exhibited light to dark brown lesions in both detached leaf and poly house assay. A maximum number of appressoria was observed at 24 hai and positively correlated with lesion size and number. Collateral hosts act as reservoirs for the pathogen to survive without their primary host. These collateral hosts help the pathogen to continue the infection chain and spread the disease. These hosts also support the pathogen's evolution by selecting the aggressive strain. However, there is a need to investigate the pathogen and adopt cultural, biological, and chemical management techniques to prevent future outbreaks of virulent strains.