Leaf spot disease caused by Alternaria alternata severely affects the growth and yield of a high repute medicinal plant, Withania somnifera(L.) Dunal. Thus, to substantiate the growing demand of this plant, it is critical to develop suitable strategies to combat the effects of leaf spot disease. Reactive oxygen species (ROS) modulation is one of the key mechanisms for achieving biotic stress tolerance as it evokes multiple stress adaptive pathways in plants. Hence, in the present investigation, ROS generating and scavenging systems have been evaluated together in leaf spot disease resistant and susceptible accessions of W. somnifera. The levels of various types of ROS have been detected using different staining methods and biochemical assays have been performed to estimate the enzymatic activities of ROS generating and scavenging enzymes. The results of enzymatic activities have been consolidated at the transcript level using real-time PCR. Results show that leaf spot resistant accession has higher basal levels of hydrogen peroxide due to the increased enzymatic activities of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase and superoxide dismutase (SOD) enzymes. Hydrogen peroxide is well known as a signaling molecule that regulates a plethora of biotic stress-responsive pathways. The threshold levels of this molecule are maintained by the action of a series of antioxidant enzymes which also showed increased levels in the present investigation. Different oxidative stress markers including proline and malondialdehyde (MDA) have also been analyzed. Our study demonstrates that ROS dynamics in leaf spot resistant accession of W. somnifera due to the coordinate action of ROS generating and scavenging system could be one of the reasons for resistance against A. alternata. Hence, modulation of ROS machinery could a feasible approach for achieving leaf spot disease resistance in W. somnifera.

由链格孢菌引起的叶斑病严重影响着名望很高的药用植物Withania somnifera（L.）Dunal的生长和产量。因此，为了证实该植物不断增长的需求，开发合适的策略来对抗叶斑病的影响至关重要。活性氧（ROS）调节是实现生物逆境耐受性的关键机制之一，因为它唤起了植物中的多种逆境适应途径。因此，在本研究中，已经在叶斑病抗性和易感W. somnifera品种中一起评估了ROS产生和清除系统。。已经使用不同的染色方法检测了各种类型的ROS的水平，并且已经进行了生化测定以估计ROS产生和清除酶的酶活性。酶促活性的结果已使用实时PCR在转录水平上进行了合并。结果表明，由于烟酰胺腺嘌呤二核苷酸磷酸氢（NADPH）氧化酶和超氧化物歧化酶（SOD）酶的酶促活性，抗叶斑病的种质具有较高的基础水平的过氧化氢。过氧化氢是众所周知的信号分子，可调节多种生物应激反应途径。该分子的阈值水平通过一系列抗氧化剂酶的作用得以维持，该抗氧化剂在本研究中也显示出增加的水平。还分析了包括脯氨酸和丙二醛（MDA）在内的各种氧化应激指标。我们的研究表明，ROS动态在叶片抗斑病中的应用。由于ROS产生和清除系统的协同作用，W。somnifera可能是抗交链孢菌的原因之一。因此，调节ROS机械可能是实现W. somnifera叶斑病抗性的可行方法。