Wide absence of yellows disease symptoms in elm in Germany
A total of 6,486 elm samples from 339 sites were collected. The plant samples comprised 2,630 Scots elm, 2,049 European white elm and 1,807 field elm samples (Table 1, Fig. 3A). The individuals’ ages ranged from one-year-old seedlings to trees of more than 400 years. The trunk diameter of the trees ranged from 0.5 cm to 3.5 m.
‘Ca. P. ulmi’-specific symptoms were rarely observed, considering the number of trees, the infection rate at some sites, their different ages and environment. However, in 2017, 2018 and 2019, more than 15 symptomatic Scots elms, approximately 2 to 3 m in height, were observed along roadsides in Müncheberg, Brandenburg (Fig. 1A, B). The trees showed numerous witches’ brooms clearly visible in winter time and during new shoot development in July/August. These plants showed an early bud break at the witches’ broom sites in March compared to non-symptomatic parts of the trees. The branches and leaves outside the witches’ brooms resembled those of healthy trees. One tree, about the same height and in the same area, showed all branches severely stunted, and the leaves small and brittle. In 2018, field elms displaying little leaf, yellowing and stunting symptoms were observed in Ingelheim am Rhein, Rhineland-Palatinate (Fig. 1C, D, E) and near Haßfurt, Bavaria. In many natural habitats of Scots elms and field elms, the assessment on the presence of elm yellows symptoms was severely compromised by the Dutch elm disease which caused wilting and dieback. These symptoms were aggravated during the 2018 summer drought in Germany. Despite the fungal infection, samples were included in the survey.
‘Ca. P. ulmi’-specific TaqMan assay
A universal quantitative real-time assay for the detection of phytoplasma presence and for control of the template quality (18Sr DNA plant) was routinely used [30]. A specific-assay for the detection of ‘Ca. P. ulmi’ was developed in this work. The universal and the specific assay have been applied within this study enabling the detection of phytoplasma presence and of ‘Ca. P. ulmi’ in particular, respectively. The selected forward and reverse primers and the TaqMan probe of the specific assay are located in the spacer region at positions 1,677 to 1,698, 1,700 to 1,721 and 1,754 to 1,772, respectively, relative to the first nucleotide of the ‘Ca. P. ulmi’ sequence deposited in GenBank under the accession number AF122911 and amplified a fragment of 96 bp. The ‘Ca. P. ulmi’ sequences showed the majority of the differences to the sequences of alder witches’ broom, "flavescence dorée", rubus stunt, ‘Ca. P. balanitae’ and ‘Ca. P. ziziphi’ in the region between the forward primer (fEY_spacer-rt) and the TaqMan probe (qEY_spacer-rt) annealing sites. The reverse primer (rEY_spacer-rt) differs only by a T or a G at the 3’ end relative to the sequences of alder witches’ broom, "flavescence dorée", rubus stunt and ‘Ca. P. ziziphi’, resepectively, or by an internal nucleotide difference relative to ‘Ca. P. balanitae’ (Fig. 2). Temperature gradient assays revealed an optimum temperature of 56°C, for both product yield and assay specificity. At this temperature, only the ‘Ca. P. ulmi’ strains ULW and EYC maintained in Catharanthus roseus and the ‘Ca. P. ulmi’-infected field samples were amplified (data not shown).
To assess the number of phytoplasmas in the phloem tissue of elm samples, DNA standards consisting of cloned 16S-23S ribosomal DNA from strain ULW with copy numbers from 1008 to 1001 per µl were PCR-amplified with the universal phytoplasma and ‘Ca. P. ulmi’-specific assays. One µl of DNA extract from a healthy elm tree was added to each reaction, to simulate the assay conditions with unknown samples. Both assays showed a dynamic range of amplification with Ct values of 18.2 to 37.6 for the lowest and highest dilution, respectively, with serial dilution steps differing by Ct values of 2 to 3 (Table 2).
Quantitative real-time PCR results highlight high incidence of infection in elm stands
The internal 18Sr DNA amplification control showed strong amplification (Ct values 9 to 22), due to the high content of plant DNA but also confirmed the template quality (data not shown). Samples with a Ct value > 22 were re-assayed, or the DNA extraction was repeated. With the universal phytoplasma assay, 1,803 of the 6,486 elm samples were rated phytoplasma-positive, representing an infection rate of 27.8% based on the total number of samples (Table 3). With the specific qPCR assay 1801 samples tested positive (Table 3). To estimate the number of phytoplasmas present in the positive samples, four Ct categories were established. With the universal qPCR assay the majority of the positive samples grouped in the Ct range > 22 ≤ 28 followed by the Ct range > 18 ≤ 22 representing 80% of all positive samples. Almost 7% of the positive samples revealed Ct values below 18. The specific qPCR assay had a different performance. Here, the majority of positive samples grouped in the Ct rage > 22 ≤ 28, followed by the Ct range > 28 ≤ 34. In comparison with the universal qPCR assay, 73% of positive samples grouped between Ct 18 and Ct 28. Only 5 samples showed Ct values of 18 or below. However, considering the set threshold level of Ct ≤ 34 both assays identified the same number of ‘Ca. P. ulmi’-positive samples.
Scots elm trees showed in general a higher phytoplasma titre compared to the other elm species. However, there was no correlation between the phytoplasma titre and ‘Ca. P. ulmi’-symptoms (data not shown). The amplification curves in assays with the DNA of a non-infected elm tree and a no-template control always ranged below the threshold line.
Comparison of qPCRs revealed rare occurrence of other phytoplasmas
The ‘Ca. P. ulmi’-specific assay detected two positive samples less than the universal phytoplasma assay (Table 3). The difference in the two cases was due to trees infected by other phytoplasma strains. Partial sequence analysis of two P1/P7 PCR fragments revealed that one phytoplasma sequence (1,631 bp) was identical to the sequence of "flavescence dorée" phytoplasma strain FD70 (acc. no. AF176319) from France, whilst the other sequence (1,631 bp) was identical to phytoplasmas found in Artemisia vulgaris (acc. no. MK440304) and Alnus glutinosa (acc. no. MK440303) in Poland. An alignment of both sequences to the ‘Ca. P. ulmi’ sequence AF122911 revealed six nucleotide exchanges within the 16S gene and mispairing at the binding sites of primers and probe of the specific assay. However, the 16Sr RNA gene of both phytoplasmas was identical to "flavescence dorée" phytoplasma strain FD70 which is a member of the elm yellows group 16SrV-C. The sequences have been deposited in GenBank under the accession numbers MN394841 and MN394842.
Distribution map of ‘Ca. P. ulmi’ in Germany highlights the presence of hot spots
Elm samples were collected at 339 sites in Germany. The elm species were not homogeneously distributed across the territory (Fig. 3A). The approximate species frequency in the federal states is reflected by the numbers given in Table 1. The detected presence of ‘Ca. P. ulmi’ was likewise not homogeneous (Fig. 3B). Regions with sites showing an infection rate of more than 66.7% were clustered in Saxony, Saxony-Anhalt and Brandenburg. Other hotspots occurred along the upper Rhine valley, and some were present in Bavaria and Hesse. Sites showing a lower infection rate were mostly found in the vicinity of these hotspots. The infection rate decreased towards the west and north, and only five sites were found above a virtual line drawn from Trier to Rostock (Fig. 3B). At one of these sites in North Rhine-Westphalia, U. laevis showed an infection rate of 30% (six out of 20 samples). The four sites in Schleswig-Holstein showed infection rates of 2.5% (one out of 40 samples) and 20% (four out of 20 samples) for U. glabra, and 5% (one out of 20 samples) and 15% (six out of 40 samples) for U. laevis and U. minor, respectively.
Infection rate correlated to altitude or tree age
The majority of sampling sites were located in the German lowlands at altitudes ≤ 100 m above the average mean sea level (AMSL). However, quite a few sites were also located in the low mountain range, from 300 to 1,100 m AMSL. About the same number of sites ranged in between (Fig. 4). The proportion of sites free of phytoplasmas, and those with a low (up to 1/3 of individuals), high (up to 2/3 of individuals) and extreme infection rate (up to 100%), were almost identical among the zones, thereby indicating a broad habitat for potential insect vectors.
Except for monumental trees and seedlings, the ages of trees were unknown and calculated on the basis of the trunk diameter. For trees up to 5 cm, 10 cm, 20 cm and 50 cm in diameter, age was calculated at 10, 20, 39 and 98 years, respectively. The oldest tree was a European white elm in Gülitz (Brandenburg), estimated to be 400 to 700 years old and with a diameter of 3.5 m. The number of infected individuals was determined in relation to trunk diameter (Fig. 5). The graph revealed different disease progressions for the three species. While U. glabra showed a steady increase of infection with age, U. laevis showed a strong increase of infected individuals in trees up to 20 years of age, reaching a plateau thereafter. A different situation was observed for U. minor. An infection rate of 20% was determined in the youngest age group, and this number did not change much in the other age categories. The graph also shows the diminishing number of older trees for Scots elms and field elms, due to the mortal effects of Dutch elm disease on their population. Of particular interest was the situation of old and monumental trees of more than 100 cm in trunk diameter. 111 trees with an estimated age of 195 years and older were included in this survey. Thirty-eight individuals were infected by ‘Ca. P. ulmi’, comprising 29 U. laevis-, eight U. glabra- and one U. minor tree
All-season colonisation of elm
To assess the seasonal fluctuation of pathogen numbers, a monthly screening of different plant parts from an infected Scots elm tree was performed with the universal qPCR assay. The mean monthly Ct values of all samples within the examined period ranged from 24.2 to 28.6. The June to December Ct means were slightly lower than the January to May means. The Ct means between trunk samples and root samples of the same month never differed by more than 3. The Ct values of the individual monthly trunk samples were close and never apart by more than four cycles. The lowest phytoplasma titre was recorded from January to March in buds, with Ct means ranging from 28.4 to 31.5. Considering the lowest average Ct (24.2) value, a phytoplasma number of 1008 per gram of phloem tissue was calculated. The highest Ct average (Ct 31.5) found in bud material represents a phytoplasma number two orders of magnitude lower.