Palmer Jade T T, Vink Jochem N A, Castro Leticia M, Craig Oliver J S, Davison Emily E, Gerth Monica L
School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
Microbiol Spectr. 2025 Apr 8;13(5):e0013525. doi: 10.1128/spectrum.00135-25.
species are eukaryotic microorganisms responsible for severe dieback and root rot in plants worldwide, impacting crops, forests, and other important ecosystems. In New Zealand, leads to fatal dieback in kauri (), long-lived endemic trees of significant cultural and ecological importance. A critical aspect of the lifecycle is the production of oospores-thick-walled spores essential for long-term survival in soil, dispersal, and disease inoculation. However, their heterogeneous distribution in soils, robust structure, and dormant state make them challenging to detect using soil baiting or DNA-based methods. Soil baiting is the basis of most current testing for , but baiting-based methods have low sensitivity, are slow, and require specialised facilities. To address these challenges, we developed and validated a PCR-based method for detecting oospores directly from soil. Our approach includes a technique for separating oospores from soil, improved oospore lysis and DNA extraction, and a primer pair that targets a repeat region of the genome with high sensitivity and specificity. The primers amplified the target product in all tested isolates without cross-reactivity against eight non-target species. The detection limit was 1 femtogram of DNA via endpoint PCR. Performance assessment against 65 soil samples from kauri forests revealed in 69% of samples compared to only 11% detected by existing methods. By eliminating the need for baiting, our assay enhances the speed, accuracy, and accessibility of testing, thereby facilitating more comprehensive monitoring and improved disease management.
species are notorious plant pathogens responsible for severe dieback and root rot diseases, significantly impacting crops, forests, and irreplaceable natural ecosystems. Rapid and accurate detection of these pathogens is essential for effective disease management. In New Zealand, threatens the country's endemic kauri forests. In this study, we developed and validated a PCR-based method for detecting oospores in soil. Oospores are long-lived, thick-walled spores that serve as key propagules for survival in soil and the spread of disease. Their robust structure and dormant state make them particularly challenging to detect using traditional soil baiting techniques or DNA-based methods. Our method is fast, accurate, and requires minimal equipment, enabling local testing and thereby empowering communities and enhancing surveillance efforts. Although developed for , this method could be adapted for other plant pathogens, potentially improving disease management across various agricultural and ecological contexts.
该物种是真核微生物，在全球范围内导致植物严重衰退和根腐病，影响农作物、森林及其他重要生态系统。在新西兰，它导致贝壳杉（一种具有重要文化和生态意义的长寿本土树种）致命衰退。该物种生命周期的一个关键方面是卵孢子的产生——卵孢子是厚壁孢子，对在土壤中长期存活、传播和疾病接种至关重要。然而，它们在土壤中的分布不均、结构坚固且处于休眠状态，使得使用土壤诱饵法或基于DNA的方法检测它们具有挑战性。土壤诱饵法是当前大多数该物种检测的基础，但基于诱饵的方法灵敏度低、速度慢且需要专门设施。为应对这些挑战，我们开发并验证了一种基于PCR的直接从土壤中检测该物种卵孢子的方法。我们的方法包括一种从土壤中分离卵孢子的技术、改进的卵孢子裂解和DNA提取方法，以及一对针对该物种基因组重复区域的引物，具有高灵敏度和特异性。这些引物在所有测试的该物种分离株中均扩增出目标产物，且与八个非目标该物种无交叉反应。通过终点PCR检测，检测限为1飞克该物种DNA。对来自贝壳杉森林的65个土壤样本进行性能评估发现，与现有方法仅检测到11%的样本相比，我们的方法在69%的样本中检测到了该物种。通过无需诱饵，我们的检测方法提高了检测速度、准确性和可及性，从而有助于更全面的监测和改善病害管理。
该物种是臭名昭著的植物病原体，导致严重的衰退和根腐病，对农作物、森林及不可替代的自然生态系统造成重大影响。快速准确地检测这些病原体对于有效的病害管理至关重要。在新西兰，该物种威胁着该国的本土贝壳杉森林。在本研究中，我们开发并验证了一种基于PCR的检测土壤中该物种卵孢子的方法。卵孢子是长寿的厚壁孢子，是在土壤中生存和疾病传播的关键繁殖体。它们坚固的结构和休眠状态使得使用传统土壤诱饵技术或基于DNA的方法检测它们特别具有挑战性。我们的方法快速、准确且所需设备最少，能够进行本地检测，从而增强社区能力并加强监测工作。尽管该方法是针对该物种开发的，但可适用于其他植物病原体，有可能改善各种农业和生态环境下的病害管理。
