Widespread occurrence of Candidatus Phytoplasma ulmi infecting elm species in Germany

Background: Candidatus Phytoplasma ulmi is the causative agent of elm yellows and has been categorised in the European Union as a quarantine pathogen in the past. For central and northern European countries, information on the occurrence and distribution of the pathogen and its impact on elms is scarce, so a survey of native elm trees has been conducted in Germany. Results: About 6,500 samples in total, from Ulmus minor , Ulmus laevis and Ulmus glabra , were collected nationwide. Phytoplasma detection was performed by applying a universal 16Sr DNA-based real-time PCR assay and a novel Ca . P. ulmi species-specific real-time PCR assay targeting the 16S-23S spacer region. Both assays revealed that 28% of the samples were infected by Ca . P. ulmi, but infection rates of the elm species and regional incidences differed. The infection of trees is not correlated to disease-specific symptoms. The survey identified a regional disparity of infection which was high in east, south and central Germany, whereas only a few infected sites were found in the western and northern parts of the country. No correlation was apparent between altitude level and the prevalence of infection. First insights into the monitoring of the seasonal titre of Ca . P. ulmi in an infected tree by real-time PCR revealed high colonisation in all parts of the tree throughout the year. Conclusions: Ca . P. ulmi-infection is widely present in elms in Germany. The rare occurrence of symptoms indicates either a high degree of tolerance in elm populations or a low virulence of pathogen strains enabling high infection rates in a long-living host.

However, all symptomatic elm trees tested phytoplasma-negative. The unexpected high presence of Ca. P. ulmi, and the absence of visual disease symptoms found in this study, prompted a nationwide survey of the pathogen's distribution and occurrence in the native elm species U. glabra, U. laevis and U. minor. The results of this survey, comprising a distribution map with incidence levels at sampling sites and a newly designed elm yellows-specific real-time PCR assay, are presented herein.

Results
Wide absence of disease symptoms of elms in Germany A total of 6,486 elm accessions from 339 sites were collected. The accessions 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.  To assess the number of phytoplasmas in the phloem tissue of infected elm samples, DNA standards with copy numbers from 10 08 to 10 01 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). based on the total number of samples (Table 3). 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 between 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 However, the fact that only 16SrV-group phytoplasmas were found in elm trees might indicate a certain host-pathogen group specificity.

Rare symptom formation indicates tolerance in a long-living host
Despite the high number of phytoplasmas in the sieve tubes, German elms seem to react in a tolerant way upon infection, which stands in striking contrast to the reactions of American and Asian elm species [12,16]. The few symptomatic U.
glabra and U. minor trees that were found, however, displayed typical Ca. P. ulmi symptoms with witches' brooms, stunting and leaf chlorosis. Mittempergher (2000) also concluded after extended observations in Italian elm breeding stations that Unequal distribution of Ca. P. ulmi might be linked to vectors Infection hotspots were located in the eastern, central and south-western parts of Germany, whereas infected sites became rare towards the north and north-western regions. The most plausible explanation is that an insect vector migrated from the southern-to-eastern side into the territory moving towards the north and west.
Hence, the distribution map given in Fig. 3B

Ca. P. ulmi infection is common in European elm stands
This survey demonstrated a nationwide distribution of Ca. P. ulmi in all three native elm species. The number of infected individuals is such that the eradication of this quarantine pathogen is impossible. In 2017, the EPPO realised the more general occurrence of Ca. P. ulmi in its member states and moved the pathogen from Annex I/A 1 to Annex I/A2, recognising its regional presence. However, even this categorisation does not seem to be justified in light of the general distribution demonstrated in this survey. The recent finding of Ca. P. ulmi in Belgium [23] demonstrates its presence also west of Germany, although the confirmed numbers    Overall infection rate of elm species at different altitude levels. Altitude, number of sites and