First report of Alternaria alternata causing leaf spot on Oat (Avena sativa) in India.

Oat (Avena sativa L.) is an important cereal crop grown worldwide primarily for food and animal feed. In November 2019, a leaf spot disease was observed on the oat plants at Mandya (12.5218° N, 76.8951° E), Karnataka, India. The disease incidence on plants was ranged between 43 to 57 percent. Initially, the symptoms appeared on leaves as small dark-brown spots surrounded by a yellow halo later turned to irregular necrotic spots with a yellow halo. A total of thirty leaves showing typical symptoms were collected from ten plants (three leaves per plant), cut into an area of 4-5 mm pieces at the junction of infected and healthy tissues. Cut tissues were soaked in 75% ethanol for 45 seconds, followed by 1% sodium hypochlorite solution for 1 min, rinsed five times in sterile distilled water, air dried, and placed on PDA and incubated at 25 ± 1 ℃. After 7 days of incubation, greyish fungal colonies appeared on PDA. Single-spore isolation method was employed to recover the pure cultures for five isolates. The colonies initially produced light-greyish aerial mycelia, then turned to dark-greyish upon maturity. Conidia were obclavate to pyriform and measured 17.34 to 46.97 µm long, 5.38 to 14.31 µm wide with 0 to 3 longitudinal, and 1 to 6 transverse septa with short beak (2.73 to 10.17µm) (n = 50.) Based on the morpho-cultural characteristics, the isolates were identified as Alternaria spp., and PCR assay using species-specific primers (AAF2/AAR3; Konstantinova, et al. 2002) confirmed the taxonomic identity of all five isolates as A. alternata. To further confirm the identity, the internal transcribed spacer (ITS), small subunit (SSU), glyceraldehyde-3-phosphate dehydrogenase (gapdh), RNA polymerase second largest subunit (rpb2), Alternaria major allergen (Alt a1), endopolygalacturonase (endoPG), an anonymous gene region OPA10-2, KOG1058 and translation elongation factor 1-alpha (tef1) of two isolates MAAS-1 and MAAS-2 were PCR amplified using the primers described previously (Woudenberg et al. 2015; Praveen et al. 2020) and the resultant PCR products were sequenced and deposited in NCBI GenBank (ITS: MW487388, MW741962, SSU: MW506220, MW752854, gapdh: MW506221, MW752855, rpb2: MW506222, MW752856, Alt a1: MW506223, MW752857, endoPG: MW506224, MW752858, OPA10-2: MW506225, MW752859, KOG1058: MW506226, MW752860, and tef1: MW506227, MW752861) which showed (99.62%, 99.81%), (100%, 100%), (100%, 99.66%), (100%, 100%), (99.58%, 99.15%), (99.55%, 99.32%), (99.53%, 99.68%), (99.23%, 99.56%) and (99.17%, 99.58%) identity with ITS (AF347031), SSU (KC584507), gapdh (AY278808), rpb2 (KC584375), Alt a1 (AY563301), endoPG (JQ811978), OPA10-2 (KP124632), KOG1058 (KP125233) and tef1 (KC584634) genes/genomic regions of type strain CBS916.96 of A. alternata respectively, confirming the identity of MAAS-1 as A. alternata. For pathogenicity assay, the conidial suspension (2 × 106 conidia/ml) of MAAS-1 isolate was artificially sprayed until runoff on ten healthy oat plants (cv. Kent, 35 days old) and ten plants sprayed with sterile water served as control. All plants were covered with polyethylene covers and kept under the greenhouse at 28 ± 1 ℃. The pathogenicity assay was repeated three times. After six days post-inoculation, small dark-brown spots with light-yellow halo appeared on leaves of MAAS-1inoculated plants. In comparison, no symptoms were observed on control plants. The fungal pathogen was re-isolated from the artificially infected plants and confirmed as A. alternata based on morpho-cultural characteristics and PCR assays. The leaf spot disease of Oat caused by A. alternata has already been reported from China (Chen et al. 2020); to our knowledge, it is the first report of A. alternata causing leaf spot on Oat in India. The leaf spot disease is an emerging threat to oat cultivation, and it reduces the grain yield and leaf quality; therefore, its management is essential for increasing productivity.

First Confirmed Report of Leaf Blight on Browntop Millet Caused by Bipolaris setariae in Southern Peninsular India.

Browntop millet (Brachiaria ramosa (L.) Stapf), which is native to the United States, was recently introduced into India as one of the small millet crop. In September 2018, leaf blight symptoms were observed on cv. Dundu Korale on the adaxial side of the leaves in a field at Bangalore, India (13.0784oN, 77.5793oE). Initial lesions were brown with small yellow halo that ranged from 1 to 5 mm and eventually enlarged exhibiting light brown centers. Afterwards, spots coalesced and leaves were blighted. About 75% of the plants were infected in the field of 0.5 ha. Samples of symptomatic and asymptomatic leaves were collected, and nine isolates were recovered from culture on potato dextrose agar (PDA). Single conidial isolation was performed. Colonies were grey to olive green with regular margins at 7 days when cultured on PDA at 27 ± 1oC and 16 h light and 8 h dark cycles. Conidiophores were single or in clusters from 4.92 to 6.04 µm in width. Conidia were fusoid, cylindrical to slightly curved ranging from 38.50 to 130 µm in length and from 8.30 to 17 µm in width, with 4 to 10 distosepta (n =100). Hilum was flat to inconspicuous or slightly protruded. Based on the morphology, the pathogen was identified as a Bipolaris species according to the genus standard descriptions of Helminthosporium (Alcorn 1988). Misra and Prakash (1972) reported that Helminthosporium setariae causing leaf spot on browntop millet in India, but they did not provide any morphological or molecular evidence of identification. For molecular identification, the genomic DNA of isolate BTMH3 was extracted and the internal transcribed spacer region (ITS) and glyceraldehyde-3-phosphate dehydrogenase gene (GPD) were amplified using the pairs of primers ITS1/ITS4 (White et al. 1990) and GPD1/GPD2 (Berbee et al. 1999), respectively. BLAST analysis of ITS (MT750301; 562 matching base pairs) and GPD (MT896702; 562 matching base pairs) sequences showed 99.82% of identity with sequences of Bipolaris setariae (Sawada) Shoemaker type strain CBS 141.31 (ITS: EF452444 and GPD: EF513206). Pathogenicity was proved by spraying 10 healthy 20-day-old browntop millet plants with conidial suspension (106 conidia/ml). Control plants were sprayed with distilled water. Plants were covered with transparent polythene bags in a greenhouse at 28 ± 2oC and high relative humidity of 90%. Symptoms were observed at five days post inoculation. The pathogen was re-isolated from infected areas using the same protocol as before, whereas the control plants were symptomless. The re-isolated pathogen was confirmed as B. setariae based on morphological characters and PCR assay. To the best of our knowledge, this is the first report of leaf blight on browntop millet caused by B. setariae in the southern peninsular region of India. Disease specimen was deposited in Herbarium Cryptogamae Indiae Orientalis (HCIO), New Delhi, India with accession number 52209. Grain yield losses caused by leaf blight on browntop millet remain to be determined, however our findings indicate that cultivar cv. Dundu Korale is susceptible to B. setariae.

First report of Alternaria alternata causing leaf blight on little millet (Panicum sumatrense) in India.

Little millet (LM) is a minor cereal crop grown in the Indian sub-continent. During October 2018, dark brown, circular to oval necrotic spots surrounded by concentric rings were observed on the upper leaf surface of the LM (cv. VS-13) grown in the fields of the University of Agricultural Sciences, Bengaluru, India (13.0784oN, 77.5793oE). As the disease progressed, infected leaves became blighted. Disease incidence up to 53% was recorded in 3 fields of 0.4-hectare area each. Thirty symptomatic leaves were collected to isolate the associated causal organism. The margins of diseased tissue were cut into 5 × 5-mm pieces, surface-sterilized in 75% ethanol for 45 seconds followed by 1% sodium hypochlorite for 1 min, finally rinsed in sterile distilled water five times and placed on PDA. After 7 days of incubation at 25°C, greyish fungal colonies appeared on PDA. Single-spore isolations were performed to obtain ten isolates. Pure cultures of the fungus initially produced light gray aerial mycelia that later turned to dark grey. All isolates formed obclavate to pyriform conidia measured 22.66-48.97µm long and 6.55-13.79µm wide with 1-3 longitudinal and 2-7 transverse septa with a short beak (2.55-13.26µm) (n=50). Based on the conidial morphology, the fungus was identified as Alternaria sp. Further, the taxonomic identity of all ten isolates was confirmed as A. alternata using species-specific primers (AAF2/AAR3, Konstantinova et al. 2002) in a PCR assay. Later, one of the isolate UASB1 was selected, and its internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (gapdh), major allergen Alt a 1 (Alt a 1), major endo-polygalacturonase (endoPG), OPA10-2, and KOG1058 genes were amplified in PCR (White et al. 1990; Berbee et al. 1999; Woudenberg et al. 2015), and the resultant products were sequenced and deposited in the NCBI GenBank (ITS, MN919390; gapdh, MT637185; Alt a 1, MT882339; endoPG, MT882340; OPA10-2, MT882341; KOG1058, MT882342). Blastn analysis of ITS, gapdh, Alt a 1, endoPG, OPA10-2, KOG1058 gene sequences showed 99.62% (with AF347031), 97.36% (with AY278808), 99.58% (with AY563301), 99.10% (with JQ811978), 99.05% (with KP124632) and 99.23% (with KP125233) respectively, identity with reference strain CBS916.96 of A. alternata, confirming UASB1 isolate to be A. alternata. For pathogenicity assay, conidial suspension of UASB1 isolate was spray inoculated to ten healthy LM (cv. VS-13) plants (45 days old) maintained under protected conditions. The spore suspension was sprayed until runoff on healthy leaves, and ten healthy plants sprayed with sterile water served as controls. Later, all inoculated and control plants were covered with transparent polyethylene bags and were maintained in a greenhouse at 28±2 ◦C and 90% RH. The pathogenicity test was repeated three times. After 8 days post-inoculation, inoculated plants showed leaf blight symptoms as observed in the field, whereas no disease symptoms were observed on non-inoculated plants. Re-isolations were performed from inoculated plants, and the re-isolated pathogen was confirmed as A. alternata based on morphological and PCR assay (Konstantinova et al. 2002). No pathogens were isolated from control plants. There is an increasing acreage of LM crop in India, and this first report indicates the need for further studies on leaf blight management and the disease impacts on crop yields.