Ponvert Nathaniel, Byrne Frank, Rivera Monique J, Rohula Timo, Olkowski Sandra, De Silva Weligodage Heshani, McRoberts Neil, Brown Judith K
School of Plant Sciences, The University of Arizona, Tucson, Arizona, United States of America.
Department of Entomology, The University of California, Riverside, Riverside, California, United States of America.
PLoS One. 2025 May 12;20(5):e0323908. doi: 10.1371/journal.pone.0323908. eCollection 2025.
Huanglongbing (citrus greening disease) is caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas) (Alphaproteobacteria) and is one of the most destructive bacterial-vector diseases affecting the citrus industry. The bacterium is transmitted by the Asian citrus psyllid (ACP; Diaphorina citri). Early detection in citrus trees is challenging due to uneven distribution of CLas throughout the tree and a long pre-symptomatic phase of the disease. Due to these limitations, ACP sampling has been suggested as a more reliable early detection strategy. The objective of this study was to develop and optimize approaches for detecting CLas in ACP adults and nymphs collected in citrus groves in California using real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). The goal was to establish the optimal number of ACP adults and nymphal instar life stages (stages 1-2, 3, or 4-5) that yielded the most reliable detection of CLas (Cq values ≤ 38). Results indicated that CLas detection correlated with psyllid developmental stage, with the 4th-5th instar nymphs (sample size of five to ten per tube) or adult ACP (sample size of three to ten per tube) providing the most consistent qPCR detection. While CLas detection rates increased with adult ACP age, nymphs were preferred for field sampling as adult ACP might have dispersed from non-infected trees, potentially misrepresenting the grove's CLas status. Detection by droplet digital PCR confirmed the presence and genome copies of CLas in a subset of ACP across life stages. In field populations, detection rates in nymphs were consistent or stable throughout the year, whereas CLas detection in adults exhibited seasonal variation, with CLas detection and genome load peaking in January. These targeted ACP sampling strategies and optimized laboratory processing methods will facilitate CLas detection in psyllids for streamlining citrus greening disease management.
黄龙病（柑橘黄龙病）由“亚洲韧皮杆菌”（CLas）（α-变形菌纲）引起，是影响柑橘产业最具破坏性的细菌媒介病害之一。该细菌通过亚洲柑橘木虱（ACP；柑橘木虱）传播。由于CLas在整棵树中分布不均以及该病有较长的无症状前期，因此在柑橘树中进行早期检测具有挑战性。由于这些限制，有人建议采用ACP采样作为一种更可靠的早期检测策略。本研究的目的是开发和优化检测方法，利用实时定量PCR（qPCR）和液滴数字PCR（ddPCR）检测加利福尼亚柑橘园中采集的ACP成虫和若虫体内的CLas。目标是确定能最可靠检测到CLas（定量PCR值≤38）的最佳ACP成虫数量和若虫龄期（1-2龄、3龄或4-5龄）。结果表明，CLas检测与木虱发育阶段相关，4-5龄若虫（每管样本量为5至10只）或成年ACP（每管样本量为3至10只）提供了最一致的qPCR检测结果。虽然随着成年ACP年龄的增长，CLas检测率会升高，但由于成年ACP可能已从未感染的树木中扩散，可能会误判果园的CLas状况，因此若虫更适合用于田间采样。液滴数字PCR检测证实了不同生命阶段的部分ACP中存在CLas及其基因组拷贝数。在田间种群中，若虫的检测率全年保持一致或稳定，而成虫的CLas检测呈现季节性变化，1月份CLas检测和基因组载量达到峰值。这些有针对性的ACP采样策略和优化的实验室处理方法将有助于在木虱中检测CLas，从而简化柑橘黄龙病的管理。
