Working Report: Plant Health Initiative Work Package 2 Post-Flowering Stalk Rot (PFSR) of Maize: A disease of complex etiology resurging in South Asia Sudha k. Nair, Zerka Rashid International Maize and Wheat Improvement Center (CIMMYT), ICRISAT Campus, Hyderabad, India Background and Objective: Post-flowering stalk rots (PFSR) are reported from all major maize growing ecologies and are caused by at least six genera of fungal pathogens, which generally occur as a complex, along with secondary colonizers (Afolabi et al., 2008). PFSR are expected to be exacerbated by the changing climates in Latin America, Asia and Sub-Saharan Africa, especially increases in temperature. The level of impact is determined by a number of factors, including the weather during the growing season, the amount of stress on the plants, hybrid genetics and the populations of the stalk rot pathogens in the field. In Asia, PFSR is reported from many countries, including Nepal, Cambodia, China, India, Indonesia, Laos, Pakistan, Philippines, Thailand and Vietnam (Lal and Singh, 1984; Yang et al., 2010; Subedi et al., 2016). Among the different stalk rots in the complex, Fusarium stalk rot (FSR), Charcoal rot (CR) and Late wilt, are more prevalent and destructive in the Asian tropics (Khokhar et al., 2014). Due to the complex nature of the stalk rot complex, with multiple primary and secondary infections, we still do not have comprehensive understanding of the prevalence and spread of the stalk rot pathogens in different maize agro-ecologies in Asia. This limits the capacity to plan defensive strategies, including development and deployment of resistant varieties. As these diseases affect the crop, at a much later stage in the life cycle, the economic impact of the disease is high, and hence is one of the biggest challenges faced by small and marginal farmers. Hence, characterizing the pathogen spectrum that causes this disease is important towards planning of IPDM strategies including resistance breeding. Hence this study is being undertaken in maize growing areas in India and Nepal as part of Work Package 2 of PHI contributing to Output-6 (WP2-OP6: Knowledge on P&D shifts and virulence variation with strategies for augmenting IPDM and resistance breeding). Materials and Methodology: The study in 2022-2024 involved collection of PFSR affected maize plant stalks from different maize growing agro-ecologies across India and Nepal, isolation of the pathogens and morphological and molecular characterization using pathogen specific primers. Two hundred and thirty-four (234) Infected samples were collected from seven maize growing states across India and 72 samples were collected from 20 locations in Nepal (Table 1, Fig. 1). A total of 199 and 42 pure cultures were isolated from infected samples from India and Nepal respectively. The colony and microscopic morphology were studied for initial identification of the pathogen genera. Genomic DNA was extracted from the pure cultures of Fusarium spp. identified and translation elongation factor (TEF-1) region of the fungi was amplified using published primer sequences (O'Donnell et al., 1998). Figure 1: Mapping of PFSR samples collected from India and Nepal (The different color place holders indicate different seasons/years) Table 1. List of the locations surveyed for collection of post flowering stalk rot samples during 2022 to 2024 Region Season Year Bihar, India Dry 2022, 2023 Punjab, India Spring, Rainy 2022, 2023, 2024 Rajasthan, India Rainy 2022 Telangana, India Dry, Rainy 2022, 2023 Andhra Pradesh, India Dry, Rainy 2022, 2023 Karnataka, India Dry, Rainy 2022, 2023 Maharashtra, India Dry, Rainy 2022, 2023, 2024 Nepal Rainy 2023, 2024 Amplified product was used to obtain the raw sequence from SeqStudio Genetic Analyzer at National Fungal Culture Collection of India (NFCCI), Pune, India. BLASTN analysis was done with the sequences deposited at CBS-KNAW Fungal Biodiversity Centre’s Fusarium MLST website (https://fusarium.mycobank.org/) for Fusarium species identification. Results and Inference: Ten pathogen species/species complex causing PFSR disease were identified from the samples collected from India and five from Nepal (Figure 2; Figure 3). Eight of the pathogen species detected belonged to the Fusarium genus. Apart from Fusarium Spp., Macrophomina phaseolina and Sarocladium Zeae were detected, of which the former represented ~24% of the diseased sample isolates from India. Eight different species/species complex of Fusarium were identified, where F.verticillioides (~34%) was the major species identified. It has been reported as the major causal pathogen of PFSR in India (Khokhar et al., 2014), apart from M.phaseolina. In Nepal, the major pathogen species reported was F.verticillioides 0.00 10.00 20.00 30.00 40.00 Fusarium verticillioides F. incarnatum-equiseti species complex Macrophomina phaseolina Figure 2: Pathogens characterized from PFSR infected maize samples from India (53%), followed by F.solani (19%). The Fusarium species that is found to gain prominence is Fusarium incarnatum equiseti species complex, which was first reported in India in 2020 from Karnataka and Telengana (Swamy et al., 2020). In the present study, it was detected in ~27% of samples studied, which included the samples from Bihar, where in the Dry season of 2022, there has been severe incidence of PFSR in farmers’ fields (Arshad Anwar, BAU, Personal communication). This Fusarium species complex has not yet been reported as a major pathogen causing PFSR in India yet, and hence host resistance deployment efforts were also not targeted towards this pathogen. Similarly, in Nepal also, FIEC has never been reported, but the present study showed 15% samples infected with this pathogen. Initial studies on differential response of standard genotype set points to similar pattern of responses to F.verticillioides and FIEC, with higher levels of severity observed for the latter (Figure 4). Apart from this important finding, multiple pathogens were detected suggesting possible co-infection (Table 2), and in majority of such cases, the co-infection was observed between F. verticillioides and F. incarnatum equiseti complex or F. verticillioides and M. phaseolina. This also suggests the importance of resistance breeding for combined resistance to multiple pathogens. Table 2. Locations where multiple pathogen co-infected PFSR diseased stalks were collected 0.00 10.00 20.00 30.00 40.00 50.00 60.00 1 3 5 Fusarium fujikuroi Fusarium incarnatum-equiseti species complex Fusarium solani species complex Macrophomina phaseolina Fusarium oxysporum species complex Figure 3: Mapping of PFSR samples collected from India and Nepal 0.0 2.0 4.0 6.0 8.0 10.0 1 3 5 7 9 11 13 15 17 19 D is ea se S co re Germplasm FIEC FV Figure 4: Differential response of standard genotype set with known F. verticilliodes reaction Stalk rot pathogen Complex Locations Fusarium verticillioides-Macrophomina phaseolina Dharwad, Gadag, Behrampur, Simbly, Malavalli, Patancheru, Daulatabad, Karimnagar, Rahuri, Peddapuram, Ahmed nagar Fusarium verticillioides-Fusarium incarnatum-equiseti species complex Kuresaila, Shirga, Baldaour, Khageria, Banmauki, Rannuehach, Bihpur, Belgavi, Bhagalpur, Behrampur, Sidhwanbet, Sidhwanbet, Kang- Patrapurt, Mehatpur, Kapurthala, Langroya, Sadullapur, Patencheru, Kurnool, Yesgoan, Pimpari, Malegoan, Kaldhabasti Fusarium nygamai - Fusarium solani Baldaour- Bihar Fusarium verticillioides- Fusarium nygamai Purnea, Shirga-Bihar Fusarium verticillioides-Fusarium incarnatum-equiseti species complex- F.solani Garhshanker-Punjab Fusarium verticillioides-Fusarium proliferatum Tamatya, Udaipur, Rajasthan Fusarium incarnatum-equiseti species complex-Macrophomina phaseolina Gotiya-Rajasthan, Rannabanaur, Sherigheri-Karnataka Partners: ICAR-Indian Institute of Maize Research (ICAR-IIMR), Ludhiana, India National Maize Research Program-Nepal (NMRP), Rampur, Nepal Acharya N. G. Ranga Agricultural University (ANGRAU), Peddapuram, India Bihar Agricultural University (BAU), Sabour, India Maharana Pratap University of Agriculture and Technology (MPUAT), Udaipur, India Mahatma Phule Krishi Vidyapeeth (MPKV), Rahuri, India Professor Jayashankar Telangana State Agricultural University (PJTSAU), Hyderabad, India Punjab Agricultural University (PAU), Ludhiana, India Rallis India Limited, Maharashtra, India References: Afolabi CG, Ojiambo, PS, Ekpo EJA, Menkir A, Bandopadhyay R (2008). Novel sources of resistance to Fusarium stalk rot of maize in tropical Africa. Plant Dis. 92, 772 – 780. Khokhar MK, Hooda KS, Sharma SS, Singh V (2014) Post Flowering Stalk Rot Complex of Maize - Present Status and Future Prospects. Maydica 59: 226-242. Lal S and Singh IS (1984) Breeding for resistance to downy mildews and stalk rots in maize. Theor. Appl. Genet. 69: 111-19. 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Theor Appl Genet. 121(4):673-87. https://doi: 10.1007/s00122-010-1339-0 http://dx.doi.org/10.3126/jmrd.v2i1.16213 https://doi.org/10.1016/j.cropro.2019.105035