Antifungal potential of zinc against leaf spot disease in chili pepper caused by Alternaria alternata

Abstract

The fungal pathogen, Alternaria alternata is responsible for causing leaf spot disease in many plants, including chili pepper. Zinc (Zn) an essential micronutrient for plant growth, also increases resistance in plants against diseases, and also acts as an antifungal agent. Here, in vitro effects of ZnSO₄ on the propagation of A. alternata were investigated, and also in vivo, the effect of foliar application of ZnSO₄ was investigated in chili pepper plants under disease stress. In vitro, ZnSO₄ inhibited fungal growth in a dose-dependent manner, with complete inhibition being observed at the concentration of 8.50 mM. Hyphae and conidial damage were observed along with abnormal activity of antioxidant enzymes, Fourier-transform infrared spectroscopy confirmed the major changes in the protein structure of the fungal biomass after Zn accumulation. In vivo, pathogen infection caused the highest leaf spot disease incidence, and cumulative disease index, which resulted in a significant reduction in the plant's growth (length and biomass), and physiochemical traits (photosynthetic pigment, activity of catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase). The heat map and principal component analysis based on disease, growth and, physico-chemical variables generated useful information regarding the best treatment useful for disease management. Foliar Zn (0.036 mM) acted as a resistance inducer in chili pepper plants that improved activities of antioxidants (CAT and POX), and defense compounds (PPO and PAL), while managing 77% of disease. The study indicated foliar ZnSO₄ as an effective and sustainable agriculture practice to manage Alternaria leaf spot disease in chili pepper plants.

Keywords: Biplot; Heat map; Leaf spot; Micronutrient; Physiology; Zinc.

Fig. 1

The Zn toxicity thresholds or EC50 (effective concentration) for Alternaria alternata on the basis of growth assays in nutrient agar (a) and nutrient broth (b) containing various zinc concentrations incubated at 25 °C for 10 days

Fig. 2

Effect of different concentrations (0.75–7.50 mM) of ZnSO₄ on activity of defense-related enzymes in Alternaria alternata. Error bars indicate standard errors of mean of three replicates. Values with different letters show significant difference (p ≤ 0.05) as determined by Tukey's HSD test

Fig. 3

Fourier-transform infrared spectra of Alternaria alternata biomass after 10 days of ZnSO₄ exposure

Fig. 4

Colony morphology and microscopic examination of Alternaria alternata at different concentrations of Zn after incubation at 25 °C for 10 days. (A) Growth in nutrient agar; (B, C) microscopic observations at ×40 and ×10 under compound microscope, respectively

Fig. 5

Effect of foliar ZnSO₄ on leaf spot disease in chili pepper caused by Alternaria alternata. ×1: 0.009 mM; ×2: 0.018 mM and ×3: 0.036 mM of ZnSO₄. Values with different letters show significant difference (p ≤ 0.05) as determined by Tukey's HSD test

Fig. 6

Effect of foliar ZnSO₄ on disease and growth in chili pepper caused by Alternaria alternata. T₁, negative control (uninoculated plants); T₂, T₃ and T₄, uninoculated plants received 0.009 (×1), 0.018 (×2) and 0.036 (×3) mM of ZnSO₄; T₅, positive control (pathogen inoculated plants); T₆, T₇ and T₈, pathogen inoculated plants received 0.009 (X1), 0.018 (X2) and 0.036 (X3) mM of ZnSO₄

Fig. 7

Growth responses and % of increase/decrease in growth attributes due to foliar application of ZnSO₄ in chili pepper affected by leaf spot disease caused by Alternaria alternata. Error bars indicate standard errors of mean of three replicates. Values with different letters show significant difference (p ≤ 0.05) as determined by Tukey's HSD test. ×1: 0.009 mM; ×2: 0.018 mM and ×3: 0.036 mM of ZnSO₄

Fig. 8

Effect of ZnSO₄ on defense-related enzymes in chili pepper leaf under stress of Alternaria alternata. Error bars indicate standard errors of mean of three replicates. Values with different letters show significant difference (p ≤ 0.05) as determined by Tukey's HSD test. ×1: 0.009 mM; ×2: 0.018 mM and ×3: 0.036 mM of ZnSO₄

Fig. 9

PCA biplot for phenotypic and genotypic traits of chili pepper plant due to effect of Alternaria alternata and foliar ZnSO₄ application in chili pepper plant. T₁, negative control (without pathogen or ZnSO₄); T₂, T₃ and T₄, uninoculated plants received 0.009, 0.018 and 0.036 mM of ZnSO₄, respectively; T₅, positive control (pathogen inoculated plants); T₆, T₇ and T₈, pathogen inoculated plants received 0.009, 0.018 and 0.036 mM of ZnSO₄, respectively; SL, shoot length; SFW, shoot fresh weight; SDW, shoot dry weight; RL, root length; RFW, root fresh weight; RDW, root dry weight; TCC, total chlorophyll content; CC, carotenoids; TPC, total protein content; CAT, catalase; POX, peroxidase; PPO, polyphenol oxidase; PAL, phenylalanine ammonia lyase

Fig. 10

Heat map visualization of variables and the treatments due to effect of Alternaria alternata and foliar ZnSO₄ application in chili pepper plant. T₁, negative control (without pathogen or ZnSO₄); T₂, T₃ and T₄, uninoculated plants received 0.009, 0.018 and 0.036 mM of ZnSO₄, respectively; T₅, positive control (pathogen inoculated plants); T₆, T₇ and T₈, pathogen inoculated plants received 0.009, 0.018 and 0.036 mM of ZnSO₄, respectively