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Fusarium wilt caused by Fusarium udum Butler (Fud) is a constant threat to pigeonpea productivity in several parts of the world. Understanding the molecular basis of pigeonpea-Fud interaction is necessary to improve resistance to Fud and thereby the productivity of pigeonpea. Temporal and spatial changes were studied in defense enzymes namely catalase, peroxidase (PO), polyphenol oxidase (PPO), Phenylalanine ammonia lyase (PAL), β-1,3-glucanase and chitinase at pre-initiation (S1), disease initiation (S2) and severe diseased (S3) stages in root, stem and leaf tissues of 2 susceptible and 2 resistant cultivars of pigeonpea inoculated with Fud. Comparison of various treatment combinations revealed that all the defence enzymes showed temporal and spatial variation in activity/expression pattern. PO increased in all the tissues of all the varieties from S1 to S2 and declined thereafter in S3 stage, and induction was higher in resistant cultivars. Catalase and PPO were induced highly in resistant cultivars inoculated with Fud, and the pattern was stage-specific and tissue-specific in nature. Catalase induction occurred at S2 in uninoculated plants whereas it started at S1 in Fud inoculated plants. PPO level diminished from S2 to S3 stage, and in susceptible varieties, it reached even below the control. PAL, β-1,3-glucanase and chitinase activity were induced at S1, and S2 and values at S3 decreased progressively. Induction of chitinase was significantly high in leaves whereas β-1,3-glucanase activity was high in stem tissues. Both control and challenged plants had a higher level of β-1,3-glucanase activity at later stages of disease development, but the proportionate increase was much higher in resistant cultivars. The activity/expression pattern of these defence enzymes reveals their use as biochemical markers for resistance and provides scope for manipulating their expression and development of wilt-resistant transgenic pigeonpea.