DataSheet_1_‘Priming’ protects Piper nigrum L. from Phytophthora capsici through reinforcement of phenylpropanoid pathway and possible enhancement of Piperine biosynthesis.docx

Piper nigrum L. (black pepper), a woody perennial spice crop indigenous to India is positioned at the phylogenetically unique basal lineage of angiosperms. Cultivation of this major spice crop is constrained by rampant fungal and viral infections leading to a lack of disease-free planting material. The major disease that poses severe threat to P. nigrum plantations and nurseries is ‘quick wilt’ caused by the oomycete Phytophthora capsici, which affects the leaf, stem, spike, collar and root. In this paper, we report the consequence of priming in modulating Piper nigrum defense against Phytophthora capsici. Glycol Chitosan (GC) was used to infiltrate detached leaves of mature P. nigrum plants. It was observed that pre-treatment of GC for 24 hours resulted in significant reduction of disease symptoms in infected leaves, as evidenced by the marked decrease in the size of lesions, and also delayed the appearance of symptoms up to 72 hpi. Experiments repeated in P. nigrum seedlings under controlled growth conditions indicate that delayed disease symptoms of GC pre-treated leaves do not spread to healthy uninfiltrated leaves suggesting a priming-associated systemic defense response. An ROS-mediated manifestation of Hypersensitive Response (HR) induced by Chitosan was also evident in pre-treated leaves. A corresponding visual indication of increased lignification was observed, which correlated with an enhanced lignin content of GC-treated leaves. Enhanced callose deposition was also apparent in GC infiltrated leaves, establishing a stimulatory effect of GC in triggering HR through ROS production, enhanced lignification and callose deposition. Key genes of the core phenylpropanoid and isoprenoid pathways along with major defense signalling pathway genes of P. nigrum, including pathogenesis-related genes and hormone signalling genes showed significant transcript enrichment consequential to GC treatment. A significant quantitative enhancement in Piperine content was evident in GC-infiltrated leaves. The systemic nature of priming on disease protection was established through experiments conducted in rooted cuttings monitored for 30 days after disease infection. This is the first report that provides strong molecular evidence endorsing the twofold advantage of defense priming in P. nigrum by improving crop protection with a concomitant enhancement in Piperine biosynthesis.

黑胡椒（Piper nigrum L.）是原产于印度的木本多年生香辛料作物，在系统发育上隶属于独特的被子植物基部类群。该主栽香辛料作物的种植受猖獗的真菌与病毒病害制约，导致无病种植材料匮乏。对胡椒种植园与苗圃构成严重威胁的主要病害是由辣椒疫霉（Phytophthora capsici）引起的“快速萎蔫病”，该病害可侵染叶片、茎秆、花穗、根颈与根系。本研究探讨了抗病启动（priming）对胡椒抵御辣椒疫霉的调控效应。我们采用乙二醇壳聚糖（Glycol Chitosan，GC）处理成熟胡椒植株的离体叶片。实验发现，经GC预处理24小时后，侵染叶片的病害症状显著减轻：病斑面积明显缩小，且症状出现时间延迟至接种后72小时（hpi）。在可控生长条件下对胡椒幼苗开展的重复实验显示，GC预处理叶片的延迟发病症状并未扩散至未处理的健康叶片，表明存在与抗病启动相关的系统防御响应。预处理叶片中还观察到由壳聚糖诱导的、活性氧（Reactive Oxygen Species, ROS）介导的超敏反应（Hypersensitive Response, HR）表征。同时观察到木质化程度增强的直观表征，且该现象与GC处理叶片的木质素含量升高呈正相关。GC浸润的叶片还出现了胼胝质（callose）沉积增强的现象，证实GC可通过产生活性氧、促进木质化与胼胝质沉积来触发超敏反应，发挥刺激调控作用。GC处理后，胡椒核心苯丙烷途径（phenylpropanoid pathway）与类异戊二烯途径（isoprenoid pathway）的关键基因，以及包括病程相关基因（pathogenesis-related genes）、激素信号通路基因在内的主要防御信号通路基因，均出现了显著的转录富集。GC浸润的叶片中，胡椒碱（Piperine）含量出现了显著的定量提升。通过对生根插条开展病害接种后30天的监测实验，证实了抗病启动对病害防护的系统效应。本研究首次提供了强有力的分子证据，证实胡椒的抗病启动具有双重优势：既可提升作物病害防护能力，同时可促进胡椒碱的生物合成。