Citrus crop production is affected by biotic and abiotic challenges that have a significant impact worldwide. Citrus nematode (Tylenchulus semipenetrans) is one of the most common diseases of citrus. The use of some natural or synthetic substances in plant defense mechanism or to induce a source of resistance in plants is a desirable goal, and one of these is Trehalose. In this study, the role of Validamycin A and Trehalose in inducing plant resistance against T.semipenetrans of sour orange (Citrus aurantium L.) and Troyer citrange (Poncirus trifoliate (L) Raf. x Citrus sinensis (L) Osb,), rootstocks was investigated. Induced resistance (IR) of such treatments was assessed through cytological, biochemical, and molecular approaches. The results showed that the total number of nematodes was significantly inhibited by each of the tested treatments, with reproduction factor (RF) ranging from 0.43 to 0.73 in sour orange, and from 0.69 to 1.2 in Troyer citrange. Among the two treatments, Trehalose significantly (P≤0.05) suppressed total nematodes with RF = 0.49 and 0.88 with reduction % 47.4 and 35.3, respectively, for sour orange and Troyer citrange. In addition, the behavior of SA and PAL genes involved in plant defense responses was determined. Our findings strongly suggest that the use of Trehalose improved the defense responses of both sour orange and Troyer citrange rootstocks against T. semipenetrans.