SUMMARY The witches’ broom disease of lime (WBDL) suppos- edly caused by Candidatus Phytoplasma aurantifolia (Ca. P. aurantifolia) is the most devastating disease of lime in Oman and southern parts of Iran. The present study describes developing a quantum dot (QD)-based nano-biosensor for the highly sensitive detection of phy- toplasma in infected trees. The immunodominant mem- brane protein (IMP) expressed in the surface of phyto- plasma was selected as a target protein for construction of a specific binding antibody. The antibody (anti-IMP) was effectively conjugated to tioglicolic acid-modified cadmium-telluride quantum dots (CdTe-QDs) synthe- sized in an aqueous solution via electrostatic interaction. Dye (rhodamine) molecules were attached to the IMP, then, the donor-acceptor complexes (QDs-Ab-IMP- Rhodamine) were formed based on the antigen-anti- body interaction. The mutual affinity of the antigen and the antibody brought the CdTe QDs and rhodamine to- gether close enough to allow the fluorescence resonance energy transfer (FRET) to occur. The immunosensor constructed showed a high sensitivity and specificity of 100%, a detection limit of 5 ca. P. aurantifolia/μl, and acceptable stability, so it could be used for detection with consistent results. Key words: biosensing; detection, Candidatus Phyto- plasma aurantifolia, quantum dots (QDs), FRET, im- munodominant membrane protein (IMP). INTRODUCTION Over the past decade, there has been a growing at- tention towards the various applications of nanobio- thechnology. More specifically, nanoparticles due to Corresponding author: M. Tabatabaei; M.R. Safarnejad Fax: +98.2612704539; +98.2122403691 E-mail: meisam_tab@yahoo.com; safarnejad@iripp.ir their unique features including size-dependent, bright and extremely stable emissions have attracted a great deal of attention. One of the most promising nanomate- rials are quantum dots (QDs) which have been widely used in a broad range of bio-related applications, such as imaging, cell tracking/trafficking and multiplexed biosensors used for disease diagnosis (Zhang et al., 2010). In-time detection of pathogenic agents has al- ways posed serious challenges and as a result, it is be- coming increasingly difficult to ignore the necessity to apply novel tools such as QDs for rapid, accurate, and cost-effective detection purposes. Among the plant pathogens, Candidatus Phytoplasma aurantifolia (Ca. P. aurantifolia), the causal agent of the witches’ broom disease of lime trees (generally referred to as WBDL) (Zreik et al., 1995), is regarded highly criti- cal in some parts of the world. The disease was first ob- served in Oman followed by its catastrophic incidence in Iran (Bové, 1986; Bové et al., 2000). The symptoms in- clude yellowing, deformation, weakening and stunting of the plants which, eventually, lead to their death (Zamharir et al., 2011). The disease has already caused the eradication of thousands of hectares of WBDL-af- fected trees impinging on the livelihood of rural commu- nities in the affected areas. Currently, the detection methods used for Ca. P. au- rantifolia have some drawbacks limiting their straight- forward application. For instance, serological methods are not sensitive enough for the detection of low amounts of the pathogen at the initial stages of infection (Lee et al., 1998). On the other hand, DNA based- methods are not able to discriminate live from dead pathogens. DNA hybridization depends on radioactive and/or non-radioactive probes which may not be readily available, are costly and lengthen the detection time. To overcome these obstacles and provide more sensitive tools for establishing the presence of phytoplasmas in the plants, alternative approaches such as the use of bio- nanosensors should be considered. Since the introduction of biocompatible QDs, the number of biotechnological applications reported for these versatile materials has grown rapidly. Many appli- Journal of Plant Pathology (2012), 94 (3), 525-534 Edizioni ETS Pisa, 2012 525 DETECTION OF CANDIDATUS PHYTOPLASMA AURANTIFOLIA WITH A QUANTUM DOTS FRET-BASED BIOSENSOR F. Rad 1,3 , A. Mohsenifar 1,4 , M. Tabatabaei 1,2 , M.R. Safarnejad 1,5 , F. Shahryari 2,4 , H. Safarpour 1,3 , A. Foroutan 6 , M. Mardi 2 , D. Davoudi 2 and M. Fotokian 3 1 Nanosystems Research Team (NRT), Microbial Biotechnology and Biosafety, Agriculture Biotechnology Research Institute of Iran (ABRII), 31535-1897 Karaj, Iran 2 Agriculture Biotechnology Research Institute of Iran (ABRII), 31535-1897 Karaj, Iran 3 Shahed University, 3319118651 Tehran, Iran 4 Tarbiat Modares University (TMU), 14155-4838 Tehran, Iran 5 Iranian Research Institute of Plant Protection,19395-1454 Tehran, Iran 6 Mazandaran University, 47416-95447 Sari, Iran