RESUMO
Maize is a widely grown cereal crop in India and ranks third to wheat and rice in production (https://iimr.icar.gov.in). During a field survey in Kharif season in 2018, foliar chlorosis at the base and middle of leaves, and twisted top symptoms were observed in 40-50 days old maize plants in Belagavi district, Karnataka, India. Again during Kharif season in 2021, similar symptoms were observed on commercial maize hybrids and sugarcane at Agricultural Research Station, Sankeshwar Karnataka. The symptoms resembled Pokkah boeng disease of sugarcane (Vishwakarma et al. 2013). Symptomatic sugarcane and maize leaves were sampled, surface sterilized with 1.0% sodium hypochlorite, and 70% ethanol, and transferred on Potato dextrose agar, incubated for 10 days at 27±1°C. Fungal growth initiated with white mycelium later turned to pinkish-white with hyaline spores. The morphological features and sporulation patterns of maize and sugarcane samples were similar (e-Xtra 1). Microconidia were formed in long chains and clusters with oval to club-shaped, 0-septate, monophialide-borne microspores. DNA from representative pure culture isolates was extracted using the CTAB protocol (Doyle and Doyle, 1990). The ITS region of r-DNA was amplified with ITS1/ITS4 primers and sequenced. BLAST analyses of sequences of maize and sugarcane culture isolates at NCBI database revealed 100% homology with Fusarium verticillioides MK264336 (Lin et al., 2016). PCR amplification with Fusarium verticilliodes specific primers VER1/VER2 (Mule et al., 2004) confirmed the organism. CBS-KNAW Fungal Biodiversity Centre's Fusarium MLST database also revealed over 98.89% homology with Fusarium verticilliodes (NRRL 46612). The fungal isolates were named Fusarium verticilliodes maize isolate SNK 01 (ON110289) and Fusarium verticilliodes sugarcane isolate SNK 01 (ON564879), and their sequences were deposited in the GenBank. To test pathogenicity, artificial inoculation using maize isolate SNK 01 and cross-inoculation of sugarcane isolate SNK 01 were done on ten maize plants by spraying a conidial suspension (2×106 conidia ml-1) on nonwounded leaves. The plants sprayed with sterile water were used as control. After ten days, typical Pokkah boeng symptoms were observed in the plants inoculated with both maize and sugarcane isolates. Diseased leaves turned pale yellowish-green with small brown spots and a chlorotic appearance, further, these developed into stripes (e-Xtra 2). Wrinkling of leaves was noticed followed by splitting and rotting. No symptoms were noticed in the water-treated control. The pathogens re-isolated from diseased plants inoculated with maize and sugarcane isolates were similar morphologically and identical to the original isolates, fulfilling Koch's postulates. Hitherto, Fusarium verticilliodes was known to cause post-flowering stalk rot in maize. However, this is the first report of Pokkah boeng disease on maize in India caused by F. verticillioides. Considering the economic value of the maize crop, this identification can help develop appropriate disease management strategies to control the disease. References Lee, S. B., et al. 1988. A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genet. Newsl. 35:23-24. Lin, Z., et al. 2016. Deciphering transcriptomic response of Fusarium verticillioides in relation to nitrogen availability and the development of sugarcane Pokkah boeng disease. Sci. Rep. 6, 29692. Mule, G., et al. 2004. A Species-Specific PCR assay based on the Calmodulin partial gene for identification of Fusarium verticillioides, F. proliferatum and F. subglutinans. European J. Plant Path. 110:495-502 Vishwakarma, S.K., et al. 2013. Pokkah Boeng: an emerging disease of sugarcane. J. Plant Pathol. Microb. 4(3):170. https://iimr.icar.gov.in. Director's desk, ICAR-Indian Institute of Maize Research. (Accessed September 8, 2022).
RESUMO
A rapid and efficient sap inoculation method for tobacco streak virus (TSV) was developed in sunflower. Sap from TSV-infected sunflower plants was freshly extracted in phosphate buffer and diluted serially from 10(-1) to 10(-8). Two-day old seedlings of sunflower were injured at the meristem and immersed in the sap for 10 min, maintained at 20 °C for 2-3 days and shifted to greenhouse. The surviving seedlings in the respective sap dilution were scored for symptoms of sunflower necrosis disease (SND). SND symptoms were seen in 80 % of the seedlings inoculated with a sap dilution of 10(-5). ELISA and RT-PCR analysis of coat protein and movement protein of TSV confirmed SND symptoms. The methodology was also found to be reproducible when the sap from the infected plants was inoculated onto healthy plants. The main aim of the study was to develop a primary screening strategy for the selection of transgenics developed for SND resistance. This methodology can also be extended for the analysis of resistance against other viruses.
