Propidium monoazide combined with qPCR to differentiate live and dead conidia of Neofabraea actinidiae

Q3 Agricultural and Biological Sciences

New Zealand Plant Protection Pub Date : 2018-07-30 DOI:10.30843/NZPP.2018.71.218

Lucia R. Ramos, I. Pushparajah, M. Kabir, B. E. Parry, K. Everett

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引用次数: 0

Abstract

Neofabraea actinidiae can occasionally cause post-harvest rot in kiwifruit. Quantitative polymerase chain reaction (qPCR) analysis represents a feasible and accurate option for identifying and quantifying this rot but is limited because qPCR results do not differentiate live and dead conidia. Propidium monoazide (PMA) is a photoreactive dye that penetrates into the damaged cell-wall membranes of dead conidia binding to the DNA and thus suppressing its amplification by qPCR. A commercial kit containing PMA was trialled for differentiating between live and dead N. actinidiae conidia. The most suitable conditions were 1 Î¼M PMA with 10 min light emitting diode (LED) exposure, and could clearly distinguish high concentrations of live from similar concentrations of dead conidia when tested separately and as a mixture. Low concentrations of live N. actinidiae conidia could be distinguished from dead ones when tested separately, but not as a mixture. Additional work is needed to optimise the effectiveness of the PMA binding and apply this concept in the orchard.

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单叠氮丙啶联合qPCR技术鉴别猕猴桃活孢子和死孢子

猕猴桃新芽偶尔会引起猕猴桃采后腐烂。定量聚合酶链反应(qPCR)分析是鉴定和定量这种腐烂的一种可行和准确的选择，但由于qPCR结果不能区分活的和死的分生孢子，因此存在局限性。单叠氮丙啶(PMA)是一种光反应性染料，可穿透死亡分生孢子的受损细胞壁膜，与DNA结合，从而抑制其qPCR扩增。一个含有PMA的商业试剂盒被用于区分活的和死的N. actinidiae分生孢子。最合适的条件是1 Î¼M PMA和10 min发光二极管(LED)暴露，并且可以清楚地区分高浓度的活分生孢子和类似浓度的死分生孢子，无论是单独测试还是混合测试。低浓度活的猕猴桃分生孢子在单独试验时可与死的分生孢子区分开来，而在混合试验时则不能区分。需要进一步的工作来优化PMA结合的有效性，并在果园中应用这一概念。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

New Zealand Plant Protection Agricultural and Biological Sciences-Insect Science

CiteScore

1.10

自引率

0.00%

发文量

期刊介绍： New Zealand Plant Protection is the journal of the New Zealand Plant Protection Society. It publishes original research papers on all aspects of biology, ecology and control of weeds, vertebrate and invertebrate pests, and pathogens and beneficial micro-organisms in agriculture, horticulture, forestry and natural ecosystems of relevance to New Zealand.