Screening of short and medium-duration genotypes against Turcicum leaf blight disease of maize

Introduction

Maize (Zea mays L.) is one of the most important staple food crops, originating from Central America and Mexico, and it is often referred to as the "queen of cereals". It is cultivated in almost 170 countries worldwide, occupying an area of 193.7 million hectares and a total production of 1,147.7 million tons having average productivity of 5.75 tons per hectare (Anonymous, 2020). Globally, the United States of America ranks first in terms of production and productivity (361.09 million tons and 10.73 tonnes/ha, respectively), followed by China (215.81 million tons and 5.80 tonnes/ha, respectively). India ranks sixth in total maize production (23.67 million tons), area of 9.25 million hectares and productivity is 2.55 tonnes/ha (FAOSTAT, 2014) [8] . It faces various biotic and abiotic stresses throughout its growth period, foliar diseases being one of the most important diseases causing significant yield losses. One major foliar disease of maize is the Turcicum leaf blight (TLB), also called as northern corn leaf blight (NCLB) in the United States of America, caused by the ascomycete fungus, Setosphaeria turcica and its conidial stage Exserohilum turcicum (Pass.) Leonard and Suggs. (syn. Heliminthosporium Turcicum Pass). It is a heterothallic facultative parasitic fungus (Luttrell, 1957) [11] , and it reproduces both asexually and sexually. It was reported first by Passerine (1876) in Perma, Italy and was followed by a serious outbreak of TLB in Connecticut, New England, in 1889, which was reported by Drechsler in 1923. It can lead to severe yield losses of 25% to 90% in various regions of India, which depend on the disease severity of TLB outbreaks (Chenulu and Hora, 1962) [6] . Yield losses have reached 50% when the disease has reached severe levels at 2-3 weeks after pollination (Shurtleff, 1980) [16] . The disease is mostly favoured by high humidity and mild temperature (Ullstrup, 1970). It affects the crop from the seedling stage till maturity, and the symptom of the disease is shown by having greyish green or brown leaf lesions with a long elliptical shape, which first appear on the lower leaves and gradually extend throughout the foliage. Premature death of blighted leaves occurred if the disease started during the early stage of the crop. The application of chemical treatments and different crop management practices can manage TLB disease, but the most eco-friendly and cost-efficient strategy is to adopt host-plant resistance. Among different practices, resistance breeding is the most practical, cost-effective means to manage the disease. Hence, the present investigation was undertaken to screen short and mediumduration entries against Turcicum Leaf Blight 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) [10] . 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 28 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) [1] . 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).

Results and Discussion

Screening of Medium-and Short-Duration Maize Hybrids for Resistance to Turcicum Leaf Blight Medium-Duration Hybrids

A total of thirty-three medium-duration maize hybrids were evaluated for their reaction to Turcicum Leaf Blight (TLB) under field conditions. Among them, fourteen genotypes, 2). 3). The present findings are in agreement with the earlier studies of Harlapur et al. (1999) [9] , who screened thirtyseven maize inbreds under artificially inoculated conditions and reported that CI- [12] screened 135 genotypes and reported that none were completely resistant, while 34 genotypes were moderately resistant, 73 moderately susceptible, and 29 susceptible to TLB. Bantu et al. (2021) [4] evaluated seventy medium-maturing maize inbreds under artificial epiphytotic conditions and observed that the per cent disease index (PDI) ranged from 13.3 to 80.0, and the area under the disease progress curve (AUDPC) varied between 300.0 and 1591.7. Babita and Mani (2011) [3] screened temperate maize lines against northern corn leaf blight and identified five inbreds showing resistant reactions. Singh et al. (2014) [15] evaluated 118 maize genotypes under artificial epiphytotic conditions and reported 26 resistant, 56 moderately resistant, 26 susceptible, and 10 highly susceptible genotypes. Likewise, Manu et al. (2017) [13] screened 115 maize inbred lines against TLB and found 21 resistant (disease score 1), 51 moderately resistant, and the remaining lines ranged from moderately susceptible to highly susceptible. Chandrashekara et al. (2012) [5] evaluated thirty-five inbred lines under artificial epiphytotic conditions at Hawalbagh, Almora, India, and reported 10 resistant, 18 moderately resistant, 3 susceptible, and 4 highly susceptible entries. The inbred lines V373, V398, V407, V418, VQL2, and CM 145 exhibited a high degree of resistance to TLB.

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 resistant or moderately resistant reactions, 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 utilization of these resistant genotypes in population improvement and hybrid development programs will contribute significantly to the improvement of maize germplasm and the development of high-yielding, TLBresistant maize cultivars for sustainable maize production.

References

1. Anonymous. Indian Institute of Maize Research. Annexure. 2014;1:1001-1010.
2. Anonymous. Area, production and productivity of maize. 2020. Retrieved from: https://www.fao.org/faostat/en/#data/QCL
3. Babita C, Mani VP. Screening for resistance against northern corn leaf blight (Exserohilum turcicum (Pass.) KJ Leonard & Suggs) in temperate maize lines. Indian Journal of Plant Genetic Resources. 2011;24(2):343345.
4. Bantu R, Devlash R, Rana SK, Guleria SK. Evaluation of medium maturing maize inbred lines for resistance to Turcicum leaf blight caused by Exserohilum turcicum. Himachal Journal of Agricultural Research. 2021;47(1):120-124.
5. Chandrashekara C, Jha SK, Agrawal PK, Singh NK, Bhatt JC. Screening of extra early maize inbred under artificial epiphytotic conditions for the north-western Himalayan region of India. Maize Genetics Cooperation Newsletter. 2012;86:1-4.
6. Chenulu VV, Hora TS. Studies on losses due to Helminthosporium blight of maize. Indian Phytopathology. 1962;15:235-237.
7. Chung CL, Jamann T, Longfellow J, Nelson R. Characterization and fine-mapping of a resistance locus for northern leaf blight in maize. Theoretical and Applied Genetics. 2010;121(2):205-227.
8. Harlapur SI, Wali MC, Krishna SM, Prashanth M. Resistant sources of Turcicum leaf blight of maize caused by Exserohilum turcicum. Karnataka Journal of Agricultural Sciences. 1999;12:212-213.
9. Joshi LM, Goel LB, Renfro BL. Multiplication of inoculum of Helminthosporium Turcicum on sorghum seeds. Indian Phytopathology. 1969;22:48.
10. Luttrell ES. Leptosphaeria (Metasphaeria) perfect stages for Helminthosporium turcicum and H. rostratum. Phytopathology. 1957;47:313.
11. Mallikarjuna N, Puttaramanaik N, Kumar K, Raveendra HR, Kumar VBS. Evaluation of maize germplasm for resistance to Turcicum leaf blight. International Journal of Pure and Applied Bioscience. 2018;6(2):1601-1605.
12. Manu TG, Gangadhara NB, Murali R, Nagaraja H. Identification of sources of resistance against Turcicum leaf blight of maize. International Journal of Chemical Studies. 2017;5(5):1664-1668.
13. Mitiku M, Eshte Y, Shiferaw W. Evaluation of maize variety for northern leaf blight (Trichometasphaeria turcica) in the South Omo zone. World Journal of Agricultural Research. 2014;2(5):237-239.
14. Singh R, Srivastava RP, Mani VP, Khandelwal RS, Ram L. Screening of maize genotypes against northern corn leaf blight. The Bioscan. 2014;9(4):1689-1693.
15. Shurtleff MC. Compendium of corn diseases. 2nd ed. St. Paul, MN: The American Phytopathological Society; 1980. p. 105.
16. Ullstrup AJ. A comparison of monogenic and polygenic resistance to Helminthosporium turcicum in corn. Phytopathology. 1970;60:1597-1599.