Abstract
Bacterial leaf scorch, caused by Xylella fastidiosa, is a major threat to blueberry production in the southeastern United States. Management of this devastating disease is challenging and often requires early detection of the pathogen to reduce major loss. There are several different molecular and serological detection methods available to identify the pathogen. Knowing the efficiency and suitability of these detection techniques for application in both field and laboratory conditions is important when selecting the appropriate detection tool. Here, we compared the efficiency and the functionality of four different molecular detection techniques (PCR, real-time PCR, LAMP and AmplifyRP® Acceler8™) and one serological detection technique (DAS-ELISA). The most sensitive method was found to be real-time PCR with the detection limit of 25 fg of DNA molecules per reaction (≈9 genome copies), followed by LAMP at 250 fg per reaction (≈90 copies), AmplifyRP® Acceler8™ at 1 pg per reaction (≈350 copies), conventional PCR with nearly 1.25 pg per reaction (≈ 440 copies) and DAS-ELISA with 1x105 cfu/mL of Xylella fastidiosa. Validation between assays with 10 experimental samples gave consistent results beyond the variation of the detection limit. Considering robustness, portability, and cost, LAMP and AmplifyRP® Acceler8™ were not only the fastest methods but also portable to the field and didn’t require any skilled labor to carry out. Among those two, AmplifyRP® Acceler8™ was faster but more expensive and less sensitive than LAMP. On the other hand, real-time PCR was the most sensitive assay and required comparatively lesser time than C-PCR and DAS-ELISA, which were the least sensitive assays in this study, but all three assays are not portable and needed skilled labor to proceed. These findings should enable growers, agents, and diagnosticians to make informed decisions regarding the selection of an appropriate diagnostic tool for X. fastidiosa on blueberry.
Highlights
Xylella fastidiosa is a xylem-limited, gram-negative, fastidious bacterium that causes economically important diseases in many plants including citrus, grapevine, almond, peach, and pear [1]
The detection limit for C-polymerase chain reaction (PCR) for RST 31/33 (Table 1) primers was found to be 1.25 pg/μl of DNA ( 440 copies) per reaction (Fig 1A), which was less sensitive than the other molecular assays tested in this study
Compared to all other C-PCR primers, HL5 and HL6 showed to be the most sensitive and could detect a concentration as low as 1 pg/ μl of DNA ( 350 copies, S3 Fig) which supports the findings by Francis et al [34] as the HL5 and HL6 primers were seen to be superior to RST 31/33 primers in detecting X. fastidiosa
Summary
Xylella fastidiosa is a xylem-limited, gram-negative, fastidious bacterium that causes economically important diseases in many plants including citrus, grapevine, almond, peach, and pear [1]. In the U.S, the bacterium was first reported to cause disease in grapes (Vitis vinifera L.) in Southern California in 1892 [2] and was isolated from grapevines with Pierce’s disease (PD) [3] It was later reported in several parts of California and other states including Texas, Florida, and Georgia [4,5,6,7,8,9]. Management of bacterial leaf scorch is challenging and only a few control options are available for this pathogen Among these options, the prompt removal of infected plants is one of the key strategies, as diseases caused by Xylella spp. can spread from ~8,000 ha to ~23,000 ha within just a few months [11]. Detection of this pathogen can aid in decreasing major crop loss and can further prevent the spread of the disease [13]
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