MALDI-TOF MS as a method for rapid identification of Phytophthora de Bary, 1876

Pathogen isolation, cultivation, genetic identification, and preservation

Oomycetous pathogens were isolated from damaged plants of different ornamental, forest and fruit woody plant species and from water in 2008–2017. Woody plants of many species from different stands (nurseries, orchards, gardening centers and ornamental gardens, forest and riparian stands) with disease symptoms such as yellowing or wilting foliage, dieback, root and collar rot, or rot of feeding roots were identified and examined for the presence of damaged plant tissues. Tissues exhibiting characteristic lesions were aseptically sampled and transported to the laboratory. The samples were rinsed under tap water, and transition zones between necrotized and healthy plant tissues were identified. Segments (3 ×3 mm) from the edges of active lesions were excised, surface disinfected by immersion in 95% ethanol for 10 s, rinsed in deionized sterile water and dried with sterile filter paper. Then, the segments were placed on selective PARPNH agar medium (V8 juice 200 mL, ampicillin 200 ppm, rifampicin 10 ppm, quintozene 25 ppm, nystatin 50 ppm, hymexazol 50 ppm, agar 15 g, CaCO₃ 3 g, and deionized water 800 mL) in 9-cm Petri dishes and incubated at 20 °C in the dark. After two to five days, segments exhibiting characteristic oomycetous hyphae were transferred onto plates containing V8A medium (V8 juice 200 mL, agar 15 g, CaCO₃ 3 g, deionized water 800 mL). Pure isolates were transferred to oatmeal agar (HiMedia Ltd, India) slants in tubes and stored in refrigeration boxes at 12 °C in the Czech Collection of Phytopathogenic Oomycetes (CCPO https://www.vukoz.cz/index.php/en/collections/collection-of-phytopathogenic-oomycetes-of-rilog).

Samples of soil substrate from plants with damaged feeding roots were collected from three sites at depths of 10–20 cm in the root zone of the affected plants. A mixed sample of approximately 1,000–2,000 cm³ was then created from a single plant and transported to the laboratory. Soil samples were processed via a baiting method. Approximately 150 cm³ of soil substrate containing damaged roots was placed in a deep plastic bowl, and deionized water was added to cover the sample by 2–3 cm. The bait (young, healthy, rhododendron leaves of the susceptible Rhododendron yakushimanum cv. Silberwolke) was rinsed under tap water, placed on the water surface and incubated at room temperature under natural light in a standard day/night regime. When characteristic lesions appeared on the baits, the segments with necrotized tissues were excised and processed as described above. Floating oomycetous zoospores were trapped from water in situ using healthy leaves of R. yakushimanum cv. Silberwolke placed in a sterilized bag made from loosely woven fabric. Four bags containing leaf bait were allowed to float just under the surface of the investigated watercourses, and after one week of incubation, the samples were collected, transported to the laboratory and processed as leaf baits as described above.

Total genomic DNA of the isolates was extracted from pure cultures using a DNeasy UltraClean Microbial Kit (QIAGEN, DE) according to the manufacturer’s instructions. For molecular identification, the nuclear rDNA ITS region and/or the COI region were amplified by polymerase chain reaction (PCR). The primer pairs used to amplify the ITS region were ITS1/ITS4 or ITS4/ITS5 (White et al., 1990), and the COI region was amplified with OomCoxILevup/Fm85mod primers (Robideau et al., 2011). PCR was performed in a Mastercycler Nexus Gradient GSX 1 thermal cycler (Eppendorf, DE), and products were visualized via agarose gel electrophoresis in 1% TBE buffer using a 100-bp DNA ladder (New England Biolabs, USA) as a size marker. PCR products were purified and sequenced in both directions by Macrogen Europe B.V. (NL) using the same primers used for PCR amplification. The obtained sequences were edited and aligned in BIOEDIT (Hall, 1999) and compared with sequences in GenBank using a BLAST similarity search (http://www.ncbi.nlm.nih.gov). All sequences obtained in this study were deposited in the NCBI GenBank database (Table S1).

MALDI-TOF MS biotyping

After genetic identification, cultures of 43 selected strains belonging to 26 taxa of Phytophthora and one species belonging to the genus Pythium as an outgroup were incubated in 0.5 L of Tryptone Soya Broth (TSB, Sigma Aldrich, USA) with shaking in the dark for 3–7 days (according to the intensity of growth of each species) to develop sufficient mycelial biomass. Samples from the liquid nutrient medium were transferred to 70% ethanol and stored in Eppendorf tubes at −20 °C until analysis. An overview of the species, including their collection numbers, is given in Table S1.

For protein extraction and subsequent MALDI-TOF MS, mycelium was harvested from the liquid nutrient medium samples by centrifugation in a 1.5-mL Eppendorf tube and divided into small, approximately equal-sized pieces of 5–10 mg using micro scissors. Distilled water (300 µL) was added to each piece of mycelium and mixed thoroughly. Then, 900 µL of ethanol was added, followed by thorough mixing. The sample was centrifuged at 20000 × g for 2 minutes (Rotanta 450 R, Hettich, DE), the supernatant was decanted, and the sample was briefly centrifuged again. The rest of the supernatant was carefully removed from the pellet by pipetting, and the sample was subsequently allowed to dry at room temperature. Next, 10–20 µL of 70% formic acid (Sigma-Aldrich, DE) was added to fully immerse the dry pellet, followed by thorough mixing and incubation for 3 min. Finally, an equal volume of ultrapure acetonitrile (Fluka, DE) was added, followed by thorough mixing and centrifugation at 20000 × g for 2 minutes. One microliter of the supernatant was immediately applied to a spot of an MTP 384 ground steel BC target plate for MALDI-TOF MS (Bruker, DE) and allowed to dry at room temperature. Immediately after drying, 1 µL of HCCA (α-cyano-4-hydroxycinnamic acid, Bruker, DE) matrix solution (acetonitrile 50%, water 47.5% and trifluoracetic acid 2.5%; 10 mg/mL HCCA) was applied to each spot and allowed to dry again at room temperature. Each sample was applied to 8 spots around the calibration spot. For calibration, 0.5 L of Bruker Bacterial Test Standard (BTS, Bruker, DE) was applied to the center spot, followed by the application of matrix solution as described for the supernatant. Each spot was measured three times by MALDI-TOF MS Autoflex Speed (Bruker, DE) and flexControl 3.4 (Build 135) with a standard MALDI Biotyper method (MBT_FC.par). Ion source 1 was set to 19.38 kV, ion source 2 was set to 18.18 kV, and detection was set from 2 to 20 kDa. All experiments were performed in independent biological triplicates; three spectra were measured for each sample, and each spectrum was collected as 2000 shots in 200 steps.

Data processing

For each measurement, the spectra were manually inspected by flexAnalysis 3.4 Compass 1.4 (Bruker Daltonics, DE), and then MSPs were processed by the standard MALDI Biotyper MSP creation method using MALDI Biotyper Version 3.1 (Build 66). MALDI Biotyper was used to compare the results based on the similarity of the spectra in the range of 2–20 kDa. For evaluation, the standard method of identification of MSPs was chosen; the frequency threshold for spectrum adjustment was 50, the frequency threshold for calculating the score was 5, the maximum primary spectrum mass error was 2000, the mass tolerance for the modified spectrum was 350, and the accepted mass tolerance of the peak was 600 par.

The acquired spectrum of the sample was transformed into a peak pattern utilizing dedicated spectral analysis tools. The peak pattern was then compared to reference peak lists of organisms in the reference library, and a log(score) value between 0.00 and 3.00 was calculated. In brief, identification is based on average peak intensity, peak position and peak occurrence frequency, and each factor is assigned a value between 0 and 1. The three values are then summed, and the result is normalized to 1000. The decimal logarithm of the result is taken as the calculated score, such that the maximum achievable score is 3 (= log 1000). The values are then classified. The calculated score determines the highest match at the top of the list. The higher the log(score) value, the higher the degree of similarity to a given organism in the reference library. Values ≥ 2 indicate high-confidence identifications. Log (score) values between 1.70 and 1.99 need to be confirmed by additional methods. Values below 1.70 indicate that no organism identification is possible (MALDI Biotyper 3.0 User Manual Revision 2, 2011). Data correlations were visualized as score-oriented dendrograms generated from spectra or MSPs in MALDI Biotyper Version 3.1 (Build 66) using the centroid linkage method.

MSP Library

Over 3500 mass spectra were processed and manually reviewed for quality control to obtain a total of 144 MSPs from 43 different samples; these MSPs were added to a custom MSP library ( http://doi.org/10.5281/zenodo.4271724). The produced MSPs were compared with the available MBT Library BDAL 9.0 (Bruker Daltonics, DE), which includes 8,468 MSPs, and the MBT Filamentous Fungi Library V1.0 (365 MSPs). After validation and verification of the method, the samples were compared with respect to species and strain classification.

Spectra of different Phytophthora strains were searched against the reference Bruker Library, which contains 2,969 species and 8,468 MSPs of various microorganisms, mainly bacteria and fungi, but no Phytophthora strains. None of the sample mass spectra matched the reference organisms, as the obtained identification scores were less than 1.70. The MSPs of the Phytophthora strains were then added to the Bruker MALDI Biotyper Library.

Library validation and verification

The same extraction method using ethanol and formic acid was used for validation. Intraday variability was determined based on successful measurement of 8 spots of the same sample in three replicates, and interday variability was based on the similarity of the spectra of samples measured on different days. Each sample was measured at least 3 times. The procedure and conditions were always identical. Figure 1 shows a comparison of the spectra of a sample of Phytophthora lacustris with collection number 295.09. The log scores of identical samples measured on different days are very similar (score > 2.6).

Blank analysis was performed to further validate the method. One of the 43 samples was randomly selected and measured in 3 spots by MALDI Biotyper Realtime Classification (Bruker, DE). The sample was correctly identified as P. lacustris in accordance with the previous DNA classification. The spectra of the blank sample matched the MSP in our database as shown in Table 1.

From genus to strain using the custom library

In total, 42 samples within the genus Phytophthora and 1 sample belonging to the related genus Pythium, which was included in the collection for quality control purposes, were compared. The MSP spectra are compared in the form of log score values in Table S2. All 42 samples matched their own MSPs (biological repeats) measured on another day with log scores greater than 2.3. For one sample (P. gonapodyides 419.10), the highest match category had a score of 2.3 with only one biological replicate and a second score of 2.2, which is satisfactory for probable species identification. The results of the reliability of identification of all species and strains are summarized in Table 2. Two limit values were determined according to the manufacturer’s recommendations. At a limit value of 2, the confidence of strain identification was 78.3%; at a lower limit value of 1.7, the success rate was 90.7%.

Comparison within a species

The spectra of different strains of P. lacustris analyzed in flexAnalysis (Fig. 2) showed protein ion peaks in the 6,500 and 14,500 m/z regions for all samples, but the intensities differed among the samples. All spectra were smoothed and baseline aligned prior to evaluation. For P. lacustris, reliable identification of strains from different locations and hosts was also achieved within the species using the log score as shown in Table 3. The MSP dendrogram based on comparison of the MALDI-TOF mass spectra is shown in Fig. 3.

The MSP spectra of P. alni subsp. uniformis 239.08 were very similar to those of P. cambivora, P. alni subsp. alni and P. rubi. The log scores ranged from 2.4 to 2, indicating some association among these species. However, the dendrogram placed these species in different clusters (Fig. 4). There was also a match of log scores at the species level between P. cactorum 862.17 and P. hedraiandra. For the genus Pythium, only consistency among biological replicates was observed. The log score compared to Phytophthora was less than 1.6 (Table 4)