Towards on-site testing of Phytophthora species †

Rapid detection and accurate identi ﬁ cation of plant pathogens in the ﬁ eld is an ongoing challenge. In this study, we report for the ﬁ rst time on the development of a helicase-dependent isothermal ampli ﬁ cation (HDA) in combination with on-chip hybridization for the detection of selected Phytophthora species. The HDA approach allows e ﬃ cient ampli ﬁ cation of the yeast GTP-binding protein ( Ypt 1) target gene region at one constant temperature in a miniaturized heating device. The assay's speci ﬁ city was determined by on-chip DNA hybridization and subsequent silver nanoparticle deposition. The silver deposits serve as stable endpoint signals that enable the visual as well as the electrical readout. Our promising results point to the direction of a near future on-site application of the combined techniques for a reliable detection of Phytophthora species.


Introduction
Fungal-like organisms of the genus Phytophthora belong to some of the world's most devastating plant pathogens. 1 Phytophthora fragariae (P. fragariae), Phytophthora kernoviae (P. kernoviae), Phytophthora ramorum (P. ramorum) and Phytophthora rubi (P. rubi) are on the EPPO A2 list of the European and Mediterranean Plant Protection Organization (EPPO) (https:// www.eppo.int/QUARANTINE/listA2.htm). These species are sufficiently dangerous to recommend a regulation as quarantine organisms. The pathogen P. ramorum 2 for instance is the causal agent of Sudden Oak Death in the forests of the west coast of the United States (http://www.suddenoakdeath.org) and Larix decline in the United Kingdom. 3 A wide range of trees, shrubs and plants in natural and landscaped environments as well as in nursery industries can be affected by Phytophthora species. Therefore the prevention of the worldwide spread of these fungus-like plant pathogens due to an increasing trade between countries is of great importance. Thus, suitable detection methods are mandatory in order to facilitate effective screening to control and eradicate Phytophthora. In particular, rapid and reliable approaches which are inexpensive plus eld applicable are needed to signicantly minimize the delay between sampling and diagnosis.
Common techniques for routine diagnosis of Phytophthora on the species level rely upon molecular biological, immunological or microbiological approaches. [4][5][6] The last two methods are time consuming, laborious, and require extensive knowledge of classical taxonomy. An accurate discrimination between various Phytophthora species was successfully realized by the polymerase chain reaction (PCR). 5 Nevertheless, their application in the eld is hampered due to the need for thermal cycling instruments. [7][8][9][10][11][12] An important step towards on-site detection of regulated Phytophthora species is provided by isothermal nucleic acid amplication techniques. [7][8][9][10][11][12] Recently, several articles highlighted the loop-mediated amplication (LAMP) for a DNAbased Phytophthora specication. [13][14][15] Here the Bst DNA polymerase amplies the target gene region under conditions that omit the use of a thermal cycler. [16][17][18] Although LAMP allows a convenient usage and is highly sensitive, primer designing is arduous and requires dedicated soware. Moreover, an initial heat denaturation of the double-stranded template DNA prior to the isothermal amplication is oen mandatory. Thus, LAMP needs a two temperature prole and cannot be claimed as really isothermal.
A further improvement of isothermal amplication that mimics in vivo DNA replication is introduced by the helicasedependent amplication (HDA). [19][20][21][22] Similar to the common PCR, the target gene region, which is enclosed by two primers, is selectively amplied. In more detail, a DNA helicase separates the double-stranded DNA and the resulting strands are immediately coated by single-stranded binding proteins (SSBs). Two sequence-specic primers bind to the template and get extended by the DNA polymerase. The newly synthesized DNA strands serve as matrices for a new amplication cycle which allows an exponential amplication. 19 This approach possesses several advantages compared to other isothermal amplication methods. Firstly, a helicase unwinds and separates the doublestranded DNA; a prior heat denaturation step and subsequent thermal cycling are unnecessary. Thus, HDA can be referred to as a real isothermal technique with performance at one constant temperature for the entire process. Secondly, only one specic primer pair has to be designed. Last but not least kits are commercially available that enable a more convenient usage. 22,23 These depicted properties offer promising potential towards the development of on-site detection systems for plant pathogens. 19,[24][25][26] Optimized HDA protocols have already been adapted for the detection of several bacterial pathogens like Clostridium difficile, 27,28 Staphylococcus aureus, 23,29,30 Neisseria gonorrhoeae, 20,23,31,32 Mycobacterium tuberculosis; 33,34 as well as different viruses. [35][36][37][38][39] Within this context, we adapted the HDA approach for Phytophthora pathogen detection for the rst time. The DNA for this isothermal amplication was isolated from cultivated Phytophtora species or infected rhododendron leaves. A subsequent precise specication of the phylogenetically closely related Phytophthora species was realized by on-chip DNA hybridization.
DNA extraction was performed by using the InviMag Plant DNA Mini Kit according to the recommendation of the manufacturer (Invitek, Berlin, Germany).

Thermophilic helicase-dependent amplication
We decided to exploit an asymmetric thermophilic HDA (tHDA) approach that amplies target DNA efficiently at 65 C and requires less protein components than the ambient temperature platform. 22,35 This second-generation HDA approach led to higher specicity and sensitivity. 20 For the adaption of the Phytophthora target DNA amplication 41 to the HDA system we used the commercially available IsoAmp II tHDA kit from Bio-Helix. The IsoAmp® II Universal tHDA kit (BioHelix, Beverly, MA, USA) was utilized according to the manufacturer's recommendations. The reaction mix contained 1x annealing buffer II, 4 mM MgSO 4 , 40 mM NaCl, 1 mM BSA, 3.5 ml dNTPs, 75 nM biotin-labeled reverse primer, 25 nM forward primer and 3.5 ml of IsoAmp enzyme mix in a nal volume of 50 ml. The primers and capture probes (Table 1; Fig. 1) were designed within the yeast GTP-binding protein (Ypt1) target region, 42,43 using the program Sequencher 5.1. Primers fulll the following criteria: (i) a length of 29-34 bp, (ii) an optimized melting temperature of 64 C (AE2 C) and a G/C base content between 45 and 55%. Capture probes were constructed to achieve (i) the highest discrimination of target sequences in relation to non-corresponding sequences, (ii) a length of 30-35 bp and (iii) a melting temperature between 62 and 65 C. A low tendency for sequence secondary structure formation is expressed in delta G values 44 between À1 and 1.5. The primer positions are close to the capture probes because the complete amplicon should not extend more than 110 bp to ensure amplication by the Bst polymerase. The tHDA reaction was conducted asymmetrically whereas the ratio between the forward and biotin-labeled reverse primer was 1 : 4.
The HDA reaction mixture was incubated for 90 min at 65 C (thermophilic) in a miniaturized heating module allowing simple temperature management (Fig. 2). A Peltier heat pump element covered with a copper plate and a heat sink at the other side was used to create isothermal temperature conditions. A polycarbonate plate (thickness 4 mm) with drill-holes (diameter 4 mm, reaction volume 50 ml) was placed between these elements and sealed with a thin foil for incubating the HDA reaction mixture. The size of the heating element is 15 Â 15 mm with an electric power of 8.5 Watt. The temperature was measured with a PT1000 platinum resistor thermometer pasted within the copper plate. An electronic controller used this temperature signal to generate a pulse-width modulation (PWM) signal to switch the Peltier element for holding a constant temperature of 65 C. The operating points for this controller were set by USB connection from a PC.

Agarose gel electrophoresis
Successful DNA amplication was veried on a 2% (w/v) agarose gel. For visualization the DNA was stained with GelRed (VWR International GmbH, Darmstadt, Germany) according to the recommendations of the manufacturer. The molecular weight marker 'GeneRuler 100 bp DNA Ladder' was purchased from Thermo Scientic.

On-chip DNA hybridization
The preparation of the chips was performed as previously described. 45,46 The Ypt1 region was chosen to design the species-specic capture probes 41 (Fig. 1). These capture probes (Euro-ns MWG Operon, Ebersberg, Germany; Table 1) were dissolved in spotting buffer (160 mM Na 2 SO 4 , 130 mM Na 2 HPO 4 ) to a nal concentration of 20 mM and spotted within the electrode gaps of the chip platform (Nanoplotter 2.1 GeSim, Germany; spotting layout see ESI †). A biotin-labeled non-complementary probe was immobilized as a positive control to verify successful enzyme binding via biotin-streptavidin interaction and subsequent silver deposition. Aer UV-linking at 254 nm for 5 min the chips were washed with 0.1 Â saline-sodium citrate (SSC)/ 0.5% sodium dodecyl sulphate (SDS).
The specic detection of Phytophthora species was performed in a microuidic device as previously described. 41,46,47 20 ml of the HDA products were dissolved in 50 ml buffer (5 Â SSC/ 0.1% SDS) and applied on the chips for 15 min at 58 C using an interval ow and further processed.

Optical and electrical signal readout
The amount of silver deposits was measured optically and electrically. The optical readout was realized by scanning with a reecta ProScan 7200 slide scanner (reecta GmbH, Rottenburg, Germany) with a 8 bit grey value and a resolution of 3600 dpi and the subsequent analysis of grey values was performed with ImageJ soware (National Institutes of Health, USA). The grey value is calculated by mean grey value calculation, subtracting the measured background value from the sample values and setting the positive control to 100%. The mean grey value of the internal hybridization control (negative control I) of all experiments was used to set the threshold which is three times the standard deviation (6.41% AE 6.46%).
For conductance measurement, the DC resistance is computed using an in-house developed portable chip-reader 48 and converted to electrical conductance.

Results and discussion
Optimization of helicase-dependent amplication for Phytophthora species Within the present study we explored for the rst time HDA as an attractive alternative amplication method for plant pathogens. The HDA reaction was established and optimized for selected Phytophthora species (Table 1) by using a robust, miniaturized heating module combined with an existent detection platform.
First, specic primers and capture probes were designed. The capture probes for P. fragariae, P. ramorum and P. kernoviae were recently published by Schwenkbier et al. 41 The positions of primers and capture probes were set to amplify a region within the yeast GTP-binding protein 1 (Ypt1) gene (Fig. 1). Established isothermal LAMP-based Phytophthora detection systems used   capture probes that hybridize with the internal transcribed spacer 1 region (ITS1). Due to the fact that by targeting the Ypt1 instead of the ITS1 region a higher specicity is achievable concomitant with easier adaptability to other regulated Phytophthora species, we addressed this molecular target with our chip-based amplication and detection assay. Extensive studies with the Ypt1 region from Phytophthora species showed that it is the best region to get species-specic base pairs within a length of 30-40 bp. 49 Isolated genomic DNA from various Phytophthora cultures was used as a template for the amplication of specic Ypt1 target gene regions via the tHDA approach (Fig. 1). The primer pair HDA_frag. allowed the amplication of P. fragariae and P. rubi Ypt1 DNA parts. These two species differ in only one single base. And as both P. fragariae and P. rubi are on the EPPO A2 list, it is not stringently required to discriminate between those plant pathogens. The amplication of P. ramorum was realizable with the primers HDA_ram.F/R. Lastly, the primer set HDA_ker. was used to amplify a fragment of P. kernoviae within the Ypt1 gene region. A further HDA approach was conducted with genomic DNA isolated from rhododendron leaves infected with P. ramorum/kernoviae. Additionally, an asymmetric amplication strategy was chosen to generate ssDNA, which facilitates the subsequent hybridization.
The isothermal amplication was performed in a miniaturized HDA reaction module consisting of a polycarbonate plate providing reaction cavities of 50 ml and a heating device to ensure a constant temperature of 65 C (Fig. 2). It offers several advantages including an accurate temperature control and a small size that ensure its portability. As there is a heater for both, bottom and top plus another cooling from the bottom, we achieve efficient heat conduction. Furthermore the system can be easily adapted to various chip formats and reaction volumes since the height is changeable.
The resulting HDA products were analyzed by agarose gel electrophoresis (Fig. 3). In general the asymmetric tHDA approach led to two distinct bands in the analytical gel. The faster migrating DNA emerged single-stranded and the higher molecular weight band represented double-stranded DNA. The asymmetric tHDA reaction was successfully realized with genomic DNA isolated from cultures (Fig. 3a) as well as from infected rhododendron leaves (Fig. 3b). Thus, the newly designed HDA primer pairs allow for the successful amplication of Ypt1 target gene regions of regulated Phytophthora species by asymmetric isothermal HDA.

Phytophthora specication by on-chip hybridization
A subsequent on-chip hybridization step for proper discrimination of selected Phytophthora species concerning their Ypt1 gene region was performed. The resulting hybridization signals were detectable by the naked eye due to the formed silver deposits in the case of matching the capture and target probe. We used HDA-generated DNA from P. fragariae, P. kernoviae, P. ramorum (template DNA isolated from cultures) and P. ramorum or P. kernoviae-infected plant samples (template DNA isolated from infected rhododendron leaves) to verify the functionality of on-chip hybridization. The results of ve independent experiments for each of the Phytophthora isolates are displayed in Fig. 4 (le panel: diagrams with grey values for the spotted capture probes; right panel: chips with silver deposits). Specic signals were obtained for tHDA amplicons of P. fragariae, which indicated that a sufficient amount of ssDNA specically bound to the matching capture probes (Fig. 4a). Besides the specic signals for P. fragariae, signals for P. rubi can also be detected. This can be explained by the fact that the amplied sequence of both Phytophthora species only differs in one single base. In the case of P. kernoviae the tHDA amplicons also yielded high and specic hybridization signals (Fig. 4b). In contrast, a slightly lower but still very distinct hybridization signal was detectable for P. ramorum (Fig. 4c). These experiments were repeated independently for at least ve times per species to ensure the reproducibility. Due to the necessity of short fragments for the HDA, the hybridization efficiency is great and the signals are still specic. The target DNA binds selectively to the capture probes without showing any false-positive signals. Thus, the specicity of the capture probes is illustrated.
In addition to the grey value analysis an electrical detection was performed. A matching hybridization and subsequent enzyme binding result in the deposition of silver between the electrode gaps of the chip. The metallic silver enables the closure of the gap and the electric resistance on each individual spot can be measured. The conductivity signals of P. fragariae, P. kernoviae and P. ramorum reect the results obtained by grey value analysis (Fig. 4).
The results for the infected plant samples are illustrated in Fig. 5 (le panel: diagrams with grey values for the spotted capture probes; right panel: chips with silver deposits). The hybridization signals of amplied target DNA, which was isolated from P. ramorum or P. kernoviae infected rhododendron leaves appear signicantly. Thus, also the combined technology of tHDA and on-chip hybridization allowed a discrimination of Phytophthora species in real plant samples.

Conclusions
Within this contribution, the identication of some EPPO-listed Phytophthora species by combining isothermal asymmetric tHDA with on-chip hybridization is introduced for the rst time.
The optical as well as electrical detection provided valid results for the analysis of the regulated plant pathogens. Isothermal amplication techniques were developed as an alternative to PCR for target gene amplication omitting the use of a thermocycler. In particular, HDA enables truly isothermal amplication without the need for prior heat denaturation or elaborated primer design, which is mandatory for LAMP. To date, no report addresses the HDA technique for the amplication of plant pathogens, in particular Phytophthora. Our developed asymmetric tHDA approach was successfully applied to amplify isolated template DNA from Phytophthora cultures and infected plant material. For an effective discrimination of several regulated Phytophthora species the Ypt1 region was chosen to design species-specic capture probes. These probes are located within a 450 bp region of the Ypt1 gene. Current HDA protocols allow the amplication of DNA fragments with a maximum length of 120 bp, hence three different primer pairs had to be designed to cover all species investigated in this study. For the establishment of our HDA experiments we started with only one primer pair per reaction.
HDA combined with chip-based detection of regulated Phytophthora species offers great potential for on-site detection. Signicant improvements can allow the use of portable testing devices directly in the eld or at the location, where a suspicious plant has to be investigated. This can concentrate sampling, detection as well as intervention and, thereby, reduce the delay between taking a sample of infected plants and obtaining a valid result. In order to realize a putative eld application, isothermal nucleic acid amplication was optimized to substitute PCR, which requires a cost-intensive thermocycler. We demonstrated that the tHDA-based amplication as well as onchip detection can be conducted in miniaturized and portable devices that enable on-site operating performance. The tHDA performance omits the need for thermal cycling and laborious technical requirements. Additionally, the development of disposable, low-cost chips can facilitate the near future availability of portable devices for chip-based DNA analytics. Further, the generated silver spots on the chips represent robust and long-lasting endpoint signals, which are already detectable by the naked eye. In contrast to a recently reported study based on HDA and uorescence detection of bacterial pathogens, our colorimetric approach eliminated signal loss due to fading or expensive detection equipment. Also a conductance measurement is realizable via metallic silver, bridging the electrode gaps in the case of a matching DNA hybridization. The resulting decreased electrical resistance can be readout with our proprietary portable chip reader. Taken together, the presented results concerning an isothermal amplication and subsequent onchip detection of Phytophthora pathogens in plant samples, realized in simple, modular, miniaturized devices, display great potential for upcoming on-site applications.