Fighting Plant Pathogens Worldwide with the Maxwell® RSC PureFood GMO and Authentication Kit

Among the one trillion or so species that share space on our planet, complex relationships have emerged over time. Such relationships, in which two or more species closely interact, are collectively termed symbiosis. Although it’s commonly assumed that symbiotic relationships are mutually beneficial, this example constitutes only one type of symbiosis (known as mutualism). The traditional predator-prey relationship, clearly a one-sided arrangement, is also an example of symbiosis.

Olive trees in Italy are being affected by the plant pathogen Xylella fastidiosa

The sheer diversity of microbial species has led to the development of many well-characterized relationships with plants and animals. Perhaps the best-known example of mutualism in this context is the process of nitrogen fixation. In this process, various types of bacteria that live in water, soil or root nodules convert atmospheric nitrogen into forms that are readily used by plants. On the other hand, some types of bacteria-plant relationships are parasitic: the bacteria rely on the plant for survival but end up damaging their host. Parasitic relationships can have devastating ecological and economic consequences when they affect food crops.

Plant Pathogens: Know Your Enemy

Xylella fastidiosa is a parasitic bacterial species that infects a variety of plants. Its host range extends across more than 500 plant species, including crops such as grapevines, almonds, citrus and olives (1). Although the first case of a disease caused by X. fastidiosa was reported in 1892 in Anaheim, California, the plant pathogen was assumed to be a virus because no causative bacteria could be cultured from infected plants (2). It was not until 1973 that the true nature of the pathogen was discovered by electron microscopy, and the bacteria were cultured successfully in 1978.

Four subspecies of X. fastidiosa are known, and they infect different types of plants. They have similar life cycles, and all of them are transmitted through a variety of insects that feed on the xylem of plants. At low bacterial levels, the infected plants may not display any symptoms. At higher levels, the bacteria form biofilms within the xylem tissue, interfering with transport of water through the plant. This problem leads to leaf scorching, and stunted growth of leaves and fruit (2).

In 2013, for the first time the plant pathogen, X. fastidiosa subsp. pauca was detected in the European Union (EU), affecting olive and almond crops, as well as oleander trees (3). Despite the implementation of control measures, the pathogen remains a serious problem in several EU countries, including Italy, France, Spain and Portugal (3). As part of the response to this pathogen, a multidisciplinary, four-year research project, XF-ACTORS, was launched in November 2016. The research objectives were to develop further understanding of the X. fastidiosa strains found in the EU, as well as “implementation of tools for pest risk assessment, for prevention and reduction of the impact of the Xylella-induced diseases.”

Validating Maxwell® Solutions for Plant Pathogen Detection

Recently, the XF-ACTORS group published a report that examined methods to detect the pathogen in plants and insects (4). It included two parts:

  1. A test performance study (TPS) to compare the performance of an automated DNA extraction protocol using the Maxwell® RSC PureFood GMO and Authentication Kit to previously validated methods: cetyl trimethylammonium bromide (CTAB) extraction and a modified DNeasy® mericon® Food Kit (QIAGEN).
  2. A laboratory proficiency test (PT) to assess the efficiency of different laboratories performing molecular detection of X. fastidiosa.

The study was developed and organized by the Institute for Sustainable Plant Protection, National Research Council of Italy (CNR). Participating laboratories included national reference laboratories and expert laboratories in several EU countries.

The laboratories processed crude leaf sap from randomized Xylella-free plants and sap spiked with heat-inactivated X. fastidiosa subsp. pauca bacterial suspensions at varying concentrations from 106 to 10 cfu/ml. In addition, samples were prepared from insect homogenates and spiked at the same concentration range as for the sap preparations. For plant samples, DNA was extracted using the Maxwell® RSC PureFood GMO and Authentication Kit on the Maxwell® RSC Instrument, CTAB, and the mericon® kit with either manual extraction or on the Qiacube® instrument. Insect samples were processed with the Maxwell® Kit and compared to the CTAB protocol. The extracted DNA was then used for qPCR with Xyllela TaqMan® probes developed previously (5). None of the plant or insect samples at the lowest bacterial concentration (10 cfu/ml) showed consistent qPCR results, so they were excluded from the analysis.

For plant samples, all three extraction procedures showed average diagnostic sensitivity >98%, diagnostic specificity close to 100%, and accuracy ~99%. However, the CTAB-extracted samples showed lower Cq values compared to the Maxwell® and mericon® samples.

For insect samples, the diagnostic sensitivity of the qPCR assays was >98% for Maxwell® DNA vs. 95% for the CTAB DNA, and the diagnostic specificity was 98% vs. 89%, respectively.  The Maxwell® DNA samples also showed greater interlaboratory reproducibility in qPCR, compared to CTAB-extracted DNA.

The authors concluded that, although CTAB produced higher DNA yields, the commercial DNA extraction kits resulted in better qPCR performance. Further, the Maxwell® Kit, combined with automated extraction on the Maxwell® RSC Instrument, was more efficient for processing plant and insect samples compared to CTAB.

For more information on purification of X. fastidiosa DNA from infected plant tissue, download Scientific Applications Note PA143.

References

  1. EFSA, (2018) Update of the Xylella spp. host plant database. EFSA J. 16, e05408.
  2. Rapicavoli, J. et al. (2018) Xylella fastidiosa: an examination of a re-emerging plant pathogen. Mol. Plant Pathol. 19(4), 786–800.
  3. Schneider, K. et al. (2020) Impact of Xylella fastidiosa subspecies pauca in European olives. Proc. Natl. Acad. Sci. USA 117(17), 9250–9259.
  4. Saponari, M. and Loconsole, G. (2021) Evaluation of molecular methods for the detection of Xylella fastidiosa. Interlaboratory Comparison EU-XF-IC-2020-03.
  5. Harper, S.J. et al. (2010) Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications. Phytopathol. 100, 1282–1288.

Looking for more information about plant DNA purification? This blog might help.

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Ken is a science writer at Promega Corporation. Although his PhD is in molecular biology, he enjoys researching and writing about everything from M-theory to graptolites. When he's not spending time with family or serving his canine and feline overlords, Ken engages in a quest for a mythical creature known as "spare time". If he succeeds, he hopes to return to writing fiction so he can keep his brain in balance.

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