First detection of Phytophthora chrysanthemi on Chrysanthemum indicum in Germany

Chrysanthemum is a commercially important plant in Germany cultivated in both field and greenhouse production. In 2015, approximately 200 potted Chrysanthemum indicum hybrids, mostly cultivar 'Palisade', in a nursery in Hessen, Germany showed wilting symptoms (Fig. 1). Oospores were observed microscopically in infected roots (Fig. 2). From the roots of diseased plants a Phytophthora sp. (isolate JKI-050-15-8-01-2-0) was recovered on carrot piece agar (CPA), malt extract and SNA agar.

Chlamydospores and very low numbers of sporangia and oospores were observed on CPA or V8 agar amended with calcium carbonate (V8A). Higher numbers of healthy oospores developed on chrysanthemum agar (CA). Low numbers of sporangia were produced on CPA and V8A or after flooding with pure water, pea extract, or Petri solution. Sporangia released zoospores immediately after rinsing and showed nested and internal proliferation (Fig. 3). Cardinal temperatures for vegetative growth on CPA and size and shape of oogonia (mean 35.4 µm; n=50) and oospores (mean 28.4 µm; n=50) grown on CA were similar to those described for Phytophthora chrysanthemi (Naher et al., 2011). Chlamydospores produced on CPA were slightly larger (mean 44.0 µm; n=50) than reported.

To confirm morphological identification, ITS, 28S rDNA, β-tubulin, TEF1 alpha and COXI loci of JKI-050-15-8-01-2-0 were sequenced with the primers listed in Table 1. The ITS sequence showed 100% identity to a P. chrysanthemi isolate from Croatia (GenBank Accession No. KJ508824) and 99-100% to those from Japan and USA (AB437135, AB437136, AB511826, AB511827, AB688343, EU596361). The 28S rDNA sequence showed 99-100% identity to isolates from Japan (AB465508, AB465349, AB511313, AB511314, AB688485) and USA (FJ868725, EU596366) and for the β-tubulin sequence (Japan: AB511995 - AB511998; USA: EU596363, FJ868721). The COXI sequence showed 99% identity to a sequence of a Japanese isolate (AB688212) of P. chrysanthemi. However, the TEF1 alpha sequence had 98-99% identity to P. chrysanthemi isolates from the USA (EU596364, FJ868722) but only 96% identity to the Japanese reference isolates (AB511925, AB511927 - AB511929).

To fulfill Koch’s postulates, ten rooted cuttings of C. indicum 'Palisade White' and 'Palisade Yellow' were inoculated by drenching and draining the soil with 40 ml of a mycelium suspension in sterile tap water of isolate JKI-050-15-8-01-2-0 grown on V8A. A homogenate of sterile V8A was applied to ten rooted cuttings of each cultivar as a negative control. The plants were incubated in a growth chamber with a 14 h photoperiod in a 25/20°C day/night regime. Two days after inoculation some inoculated plants started to wilt. After two weeks all inoculated plants showed severe wilting. The roots on these plants were brown and necrosis had spread to the stem base. Oospores were observed in the infected roots and P. chrysanthemi was re-isolated from infected plants after surface disinfection. Negative controls remained asymptomatic and the pathogen was not isolated.

Phytophthora chrysanthemi was first described as a new species on Chrysanthemum in Japan (Naher et al., 2011). In 2015, it was reported from Croatia (Tomiċ & Iviċ, 2015) and in 2016 from the USA (Randall-Schadel, 2016). According to our knowledge, this is the first report of P. chrysanthemi in Germany.