Screening of QPM & OPV, sweet corn, baby corn and Pop Corn genotypes against Turcicum Leaf Blight disease of maize

Introduction

Maize (Zea mays L.) is one of the most adaptable, versatile, and economically important cereal crops, cultivated across a wide range of agro-climatic conditions. Often referred to as the "queen of cereals," maize possesses the highest genetic yield potential among cereal crops. In recent years, besides its primary use for grain production, maize has gained significance as a raw material for various industrial applications. Different types of maize include field corn, quality protein maize (QPM), popcorn, sweet corn, and baby corn. Among these, the speciality corns such as QPM, popcorn, sweet corn, and baby corn are valued for their enhanced nutritional profiles and health benefits. Due to their increasing consumer demand and premium market value, these speciality types are being widely promoted and cultivated across the globe, offering lucrative opportunities for farmers. However, all these maize types are susceptible to several foliar fungal diseases, among which Turcicum Leaf Blight (TLB), also known as Northern Corn Leaf Blight (NCLB), is of major concern. The disease not only affects grain yield but also reduces the quality of straw, which is an important cattle feed component (Ahangar et al., 2016) [2] . Early onset of epidemics causes premature death of leaves, thereby diminishing their fodder value (Hooda et al., 2017) [13] . TLB is caused by Exserohilum turcicum (Pass.) Leonard and Suggs [syn. Helminthosporium turcicum (Pass.) Leonard and Suggs; perfect stage: Setosphaeria turcica (Luttrell) Leonard and Suggs. The pathogen is identified primarily by its asexual stage (E. turcicum), as its sexual stage (Trichometasphaeria turcica Luttrell) is rarely observed under natural conditions (Luttrell, 1957) [18] . It exhibits a wide host range and considerable pathogenic variability (Muiru et al., 2010) [23] .

The disease was first reported in New Jersey, USA, in 1878, and is now prevalent in the northeastern United States, sub-Saharan Africa, China, Latin America, and India (Adipala et al., 1993; Dingerdissen et al., 1996) [1,11] . In India, TLB was first documented by Butler in 1907 and is now widespread across several states, including Karnataka, Maharashtra, Andhra Pradesh, Bihar, Uttarakhand, Uttar Pradesh, Odisha, Punjab, West Bengal, Himachal Pradesh, Jammu & Kashmir, Sikkim, and the northeastern states such as Manipur, Meghalaya, Mizoram, Nagaland, Tripura, and Assam (Butler, 1907; Mitra, 1981; Chenhulu and Hora, 1962; Payak and Renfro, 1968; Laxminarayana and Shankar Lingam, 1983; Kaul, 1997) [7,21,8,29,19,16] . In the northeastern region (NER) of India, maize is predominantly cultivated under rainfed hilly upland and jhum (shifting cultivation) systems (Ramkrushna et al., 2022) [30] . It serves as an essential kharif crop, ensuring food security and being used both for direct consumption and as livestock feed. In Manipur, maize is cultivated over an area of approximately 25.53 thousand hectares (Anonymous, 2020) [5] . The pathogen E. turcicum can infect maize plants at any growth stage, from seedlings to maturity. Although TLB occurs worldwide, its severity is greater in regions characterized by low temperatures, high humidity, and cloudy weather conditions (Jeevan et al., 2023) [15] . In Manipur, the disease incidence (DI) has been reported to range between 51-71% (Nongmaithem et al., 2022) [28] . Yield losses of up to 70% have been documented (Yeshitila, 2003) [36] , with reductions exceeding 50% when infection occurs before flowering (Raymundo et al., 1981; Tefferi et al., 1996) [31,34] . Severe infection can reduce photosynthetic rates by up to 91% when disease severity exceeds 50% (Pant et al., 2001) [24] . Similarly, yield losses may approach 50% when disease intensity is high two to three weeks after pollination (Shurtleff, 1980; Dey et al., 2017) [27,10] , and under epiphytotic conditions, losses can range from 28% to 91% (Ribeiro et al., 2016) [25] . Although chemical treatments and crop management practices can mitigate TLB, the most sustainable, ecofriendly, and cost-effective strategy for disease management is the deployment of resistant cultivars. Host-plant resistance, achieved through resistance breeding, remains the most practical approach to minimizing yield losses. Therefore, the present investigation was undertaken to screen QPM, open-pollinated varieties (OPVs), sweet corn, baby corn, and popcorn genotypes for resistance against Turcicum Leaf Blight disease of maize under field conditions.

Materials and Methods

Collection of Diseased Samples and Pathogen Isolation

Diseased maize leaf samples exhibiting typical symptoms of Turcicum Leaf Blight (TLB), characterized by elongated necrotic lesions, were collected in paper poly bags from different maize-growing areas of Manipur during the survey and transported to the laboratory for pathogen isolation. The causal pathogen, Exserohilum turcicum, was isolated from infected leaf tissues following the standard hyphal tip isolation technique. The obtained pure cultures were maintained on Potato Dextrose Agar (PDA) slants and stored at 5±1 °C in a refrigerator for subsequent laboratory and field investigations.

Isolation of the pathogen

Isolation of the fungus was done through the standard tissue isolation technique. The necrotized leaf lesions, along with the healthy portions, were surface sterilized in 1:1000 sodium hypochlorite solution for 30 sec and again washed thoroughly three times in sterile distilled water to remove the traces of sodium hypochlorite. The sterilized bits were then transferred aseptically to sterile Petri plates that contained PDA media. The inoculated petri dishes were incubated at room temperature (25±1 °C) for periodic observation of fungal growth.

Hyphal tip isolation

For the maintenance of a pure culture of the pathogen, the hyphal tip isolation method was used. From 15 days 15-dayold culture, the spore suspension was diluted in sterilised distilled water to get eight to ten spores per ml. From this, one ml of suspension was uniformly spread on two per cent solidified water agar plates and incubated at 27±1 °C for 12 hr. With a marker pen, a single spore was marked on the backside of the Petri plate with the help of a microscope. After periodically observing under the microscope, the hyphae coming from each cell of the single spore were then traced and marked. With the help of a cork borer, the tip of the hyphae was cut carefully and then transferred to PDA plates and incubated at 27±1 °C for 10 days. Again, the mycelial bits of the fungus from incubated plates were transferred to the petri plates containing PDA and further incubated at 27±1 °C for 10 days. Thus, the pure culture obtained was free from sectoring or saltation. For the identification of E. turcicum, spore morphology and colony characteristic studies were done on PDA.

Mass multiplication of the inoculum

Sterilized sorghum grains were used for the mass multiplication of E. turcicum (Joshi et al., 1969) [14] . About 40 to 45 g of sorghum grains were dispensed in a 500 ml conical flask, soaked in water for about 3-4 hrs and excess water was drained off. The sorghum grains were autoclaved twice at 15 psi for 1 hour, inoculated with the fungus under aseptic conditions, and incubated at 25-27 °C. Once in 2-3 days, the flasks were shaken for uniform growth of E. turcicum on grains. The above impregnated sorghum grains were dried by spreading uniformly on a clean paper sheet in the shade.

Field Screening of Maize Genotypes for TLB

A field experiment was conducted during kharif 2024 at the Andro Research Farm, College of Agriculture, Central Agricultural University, Imphal, Manipur (24.763793° N, 94.052882° E; 755.02±2.5 m above mean sea level) to evaluate maize genotypes for resistance to Exserohilum turcicum, the causal organism of Turcicum Leaf Blight (TLB). The experiment was laid out in an alpha lattice design with a plot length of 2 m. Each genotype was sown on 29 June 2024, maintaining a spacing of 20 × 60 cm and following the recommended agronomic practices, except for plant protection measures.

Inoculum Preparation and Inoculation Procedure

Pure cultures of E. turcicum were maintained on potato dextrose agar (PDA) medium and incubated at 25±1 °C for 20 days to promote sporulation. A spore suspension was prepared by flooding the culture plates with sterile distilled water and gently scraping the surface to dislodge the conidia. The resulting suspension was filtered through double-layered muslin cloth to remove mycelial fragments. The spore concentration was adjusted to 3×10⁵ spores ml⁻¹ using a haemocytometer. The suspension was sprayed uniformly on the maize plants at the three-to four-leaf stage twice at 30 and 40 DAS using a hand-held atomizer during the evening hours to facilitate infection. Immediately after inoculation, a fine water mist was applied to maintain adequate humidity and promote pathogen establishment.

Disease Development and Assessment

Following inoculation, the field was periodically irrigated to maintain moderate humidity conducive to disease development. Disease observations were recorded at regular intervals, and disease severity was assessed using the standard 1-9 rating scale (Table 1) recommended by the Indian Institute of Maize Research (IIMR), Ludhiana (Anonymous, 2014) [14] . Observations began 45 days after sowing when typical Turcicum Leaf Blight (TLB) symptoms were well expressed, and assessments were continued at weekly intervals for six consecutive weeks. Based on the mean disease scores, the genotypes were categorized as follows: The genotypes showing disease scores between 1.0-3.0 were considered as resistant (R), 3.1-5.0 as moderately resistant (MR), 5.1-7.0 as moderately susceptible (MS), 7.1-9.0 as susceptible (S). [9] ; Mitiku et al. (2014) [22] Rating 3). The present findings are in accordance with studies of Kachapur et al. (2014) [17] which reported that among the fifty new germplasm lines screening against TLB, they found that GPM-378, GPM-408, GPM-496, GPM-524 and GPM-537 showed resistant reaction and GPM-375, GPM-440, GPM-540 and GPM-569 showed susceptible reaction against TLB. Mir et al. (2015) [20] reported that among the screening of 10 inbred lines, three were found to be moderately resistant, five lines were found to be moderately susceptible, and two were found to be susceptible to TLB. Similarly, Ahanger et al. (2016) [37] reported that field trial against sixty isolates of maize against E. turcicum under artificially inoculated field conditions, twenty-six genotypes were found to be resistant and moderately resistant. Later, screening of these twenty-six genotypes against twelve isolates of E. turcicum under artificially epiphytotic conditions revealed that eight genotypes were found to be resistant and eight as moderately resistant. Shikari and Zafar (2009) [26] also reported that inbred NAI-147 and composite Girija have shown resistance against Turcicum leaf blight. Yousuf et al. (2018) [37] also reported that among the seventy landraces, forty-three lines were reported as resistant, eighteen were found to be moderately resistant, five as moderately susceptible and Tral 3 was found to have the highest per cent disease index (PDI) as 78.91 per cent and was rated to be susceptible.

Conclusion

The present investigation demonstrated considerable variability among maize genotypes in their response to Exserohilum turcicum, the causal agent of Turcicum Leaf Blight (TLB). Several short-and medium-duration hybrids exhibited resistance or moderate resistance, highlighting their potential as valuable genetic resources for resistance breeding. Identification of such resistant sources is crucial for developing durable and sustainable TLB resistance, particularly in regions prone to frequent disease outbreaks. The incorporation of resistance genes from these sources into susceptible but agronomically superior cultivars can greatly enhance yield stability and resilience. Furthermore, the use of these resistant genotypes in population improvement and hybrid development programs will significantly enhance maize germplasm and lead to the development of high-yielding, TLB-resistant maize cultivars for sustainable maize production.

References

1. Adipala E, Lipps PE, Madden LV. Reaction of maize cultivars from Uganda to Exserohilum turcicum. Phytopathology. 1993;83:217-223.
2. Ahangar MA, Bhat ZA, Sheikh FA, Dar ZA, Lone AA, Hooda KS, Reyaz M. Pathogenic variability in Exserohilum turcicum and identification of resistant International Journal of Advanced Biochemistry Research https://www.biochemjournal.com sources to turcicum leaf blight of maize (Zea mays L.). J Appl Nat Sci. 2016;8(3):1523-1529.
3. Ali F, Yan J. Disease resistance in maize and the role of molecular breeding in defending against global threats. J Integr Plant Biol. 2012;54(3):134-151.
4. Anonymous. Indian Institute of Maize Research. Annexure. 2014;1:1001-1010.
5. Anonymous. Area, production and productivity of maize. 2020. Available from: https://www.fao.org/faostat/en/#data/QCL.
6. Anonymous. Department of Agriculture, Government of Manipur. Area, production and yield for the year 2020-2021. Available from: http://www.agrimanipur.gov.in/area-production-andyield-for-the-year-2020-2021/. Accessed 2023 Mar 23.
7. Butler EJ. Some diseases of cereals caused by Sclerospora graminicola. Memoirs of the Department of Agriculture in India, Botanical Series. 1907;2:1-24.
8. Chenulu VV, Hora TS. Studies on losses due to Helminthosporium blight of maize. Indian Phytopathol. 1962;15:235-237.
9. Chung CL, Jamann T, Longfellow J, Nelson R. Characterization and fine-mapping of a resistance locus for northern leaf blight in maize. Theor Appl Genet. 2010;121(2):205-227.
10. Dey U, Harlapur SI, Dhutraj DN, Das A. Screening of inbred lines and hybrids/composites against common rust of maize. Int J Bioresour Stress Manag. 2017;8(4):548-552.
11. Dingerdissen AL, Geiger HH, Lee M, Schechert A, Welz HG. Interval mapping of genes for quantitative resistance of maize to Setosphaeria turcica. Mol Breed. 1996;2:143-156.
12. Hooda KS, Khokhar MK, Shekhar M, Karjagi CG, Kumar B, Mallikarjuna N, et al. Turcicum leaf blightsustainable management of a re-emerging maize disease. J Plant Dis Prot. 2017;124:101-113.
13. Joshi LM, Goel LB, Renfro BL. Multiplication of inoculum of Helminthosporium turcicum on sorghum seeds. Indian Phytopathol. 1969;22:48.
14. Jeevan B, Harlapur SI, Nongmaithem N. Turcicum leaf blight. In: Compendium of maize diseases. Indian Phytopathological Society. 2023;11-15.
15. Kaul TN. Food and Agriculture Organization. Plant Prot Bull. 1957;5:93-96.
16. Kachapur RM, Kulkarni MS, Wali MC, Kalappanavar IK, Timmanna O. Genetic evaluation of maize germplasm lines against turcicum leaf blight (Exserohilum turcicum). Natl Symp Plant Dis. 2014;1112:25.
17. Luttrell ES. Leptosphaeria (Metasphaeria) perfect stages for Helminthosporium turcicum and H. rostratum. Phytopathology. 1957;47:313.
18. Laxminarayana C, Shankarlingam S. Turcicum leaf blight of maize: techniques of scoring for resistance. In: Proceedings of AICMIP. IARI; 1983. p. 16-24.
19. Mir SD, Ahmad M, Parray GA, Razvi SM, Zaffar G. Screening of maize inbred lines under artificial epiphytotic conditions for turcicum leaf blight (Exserohilum turcicum). Afr J Microbiol Res. 2015;9(7):481-483.
20. Mitra M. A comparative study of species and strains of Helminthosporium on certain Indian crops. Trans Br Mycol Soc. 1981;15:254-293.
21. Mitiku M, Eshte Y, Shiferaw W. Evaluation of maize variety for northern leaf blight (Trichometasphaeria turcica) in South Omo. World J Agric Res. 2014;2(5):237-239.
22. Muiru WM, Koopmann B, Tiedemann AV, Mutitu EW, Kimenju JW. Race typing and aggressiveness of Exserohilum turcicum isolates of Kenyan, German and Austrian origin. 2010.
23. Pant S, Kumar P, Chauhan VS. Effect of turcicum leaf blight on photosynthesis in maize. Indian Phytopathol. 2001;54(2):251-252.
24. Ribeiro RM, Amaral ATD Jr, Pena GF, Vivas M, Kurosawa RN, Goncalves LSA. History of northern corn leaf blight disease in recurrent selection cycles of popcorn. Acta Sci Agron. 2016;38:447-455.
25. Shikari AB, Zaffar G. Evaluation and identification of maize for Turcicum leaf blight resistance under cold temperate conditions. Maize Genet Coop Newsl. 2009;83.
26. Shurtleff MC. Compendium of corn diseases. 2nd ed. APS Press; 1980. p. 105.
27. Nongmaithem N, Sanjenbam D, Konsam J, Singh LNK, Devi TR. Survey and surveillance of maize diseases in Manipur. Pharma Innov J. 2022;11(5):557-560.
28. Payak MM, Renfro BL. Combating maize disease. Indian Farmer Dis. 1968;1:53-58.
29. Ramkrushna GI, Layek J, Das A, Verma BC, Das S, Mohapatra KP, Ngachan SV. Nutrient management in maize (Zea mays) under shifting cultivation. Indian J Agron. 2022;67(4):386-391.
30. Raymundo AD, Parkins JM, Hooker AL. Effect of HtN gene on northern corn leaf blight. Plant Dis. 1981;65(4):327-333.
31. Shurtleff MC. Compendium of corn diseases. 2nd ed. APS Press; 1980. p. 105.
32. Sethy SK, Nongmaithem N, Sinha B, Singh KI, Singh NO, Priyadarshini M. Morphological and cultural variability among Exserohilum turcicum isolates from Manipur. Int J Bioresour Stress Manag. 2023;14(7):1052-1060.
33. Tefferi A, Hulluka M, Welz HG. Assessment of yield loss in maize caused by northern leaf blight. J Plant Dis 1996;103(4):353-363.
34. Ullstrup AJ. A comparison of monogenic and polygenic resistance to Helminthosporium turcicum in corn. Phytopathology. 1970;60:1597-1599.
35. Yeshitila D. Cloning and characterization of xylanase genes from phytopathogenic fungi with reference to Helminthosporium turcicum. PhD thesis. Univ. of Helsinki; 2003.
36. Yousuf N, Dar SA, Lone AA, Ahanger MA, Dar ZA, Bhat MA, et al. Field screening of maize landraces for resistance to turcicum leaf blight. Int J Chem Stud. 2018;6(1):333-337.