First report of Cryptosporiopsis tarraconensis causing leaf lesion of Corylus avellana in Central Europe (Poland)

Abstract

Key message

As a result of our research, we determined that Cryptosporiopsis tarraconensis—as a new species for Central Europe—is the causative agent of leaf lesions in natural populations of hazel (Corylus avellana). Until now, this species had not been described in a natural population of C. avellana or out of the temperate climate. This is the fifth notification of this rare fungus in the world and the first from Central Europe and the natural population of the host.

Cryptosporiopsis tarraconensis Gené and Guarro is a rare fungal species (Ascomycota, Helotiales, Dermateaceae), occurring on hazel (Corylus avellana L.) buds, leaves, and twigs (Tagliavento et al. 2021). It can cause leaf desiccation and the dry rot of buds, leading to their abortion (Gené et al. 1990; Roohvarzi et al. 2013; Tagilavento et al. 2021). Hitherto C. tarraconensis has been found in the world only 4 times, each in temperate regions: in Spain (Gené et al. 1990), in Iran (Roohvarzi et al. 2013) in Italy (Tagilavento et al. 2021) and in Türkiye (Altin and Gulcu 2023), causing considerable damage to hazel orchards. Until now, this species had not been described in a natural population of C. avellana or out of the temperate climate. This is the fifth notification of this rare fungus in the world and the first from Central Europe and the natural population of the host.

The leaf lesions of C. avellana were observed in September 2021 in 15 locations in Wigry National Park (NE Poland), located in an oak-hornbeam forest (Table 1). On the infected leaves two types of lesions were visible: one consisting of round, brown spots with a black point in the middle, and a second made of irregular, wet lesions, mostly occurring on the leave’s edges (Appendix Figure 2). Depending on the location, the lesions were observed on a single tree or up to 100% of the trees at each location, and occurring from single leaves to 45% of a tree’s leaves (Table 1).

Table 1 An assessment of leaf lesion infection on hazel trees in 15 studied plots

Symptomatic tissues were disinfected for 30 s in 0.5% NaClO and rinsed in sterile water. Then, the leaves were cut into 2–5 mm fragments, placed on a PDA medium (Biocorp, Poland), and incubated at 21 ± 2 °C in darkness. After 10 days 810 fungal colonies were obtained, from which 134 were C. tarraconensis strains. The species identification of two main morphotypes: CtC1-21Ca and CtC7-21Ca, was classified, based on morphology, as C. tarraconensis (Gené et al. 1990; Sutton 1980). After 14 days of inoculation, a white-brown, velvet mycelium of C. tarraconensis was obtained, reaching 2,5–3,5 cm diam on the PDA medium, raising in the center with age (Appendix Figure 3). On the reverse of the colony, mostly dark yellow coloration with concentric brown rings was observed. After circa 30 days of incubation, ochre to dark brown globular fruiting bodies developed (Appendix Figure 4). Under a light microscope acervuli 115–250 µm were observed, exuding whitish conidial masses (Appendix Figure 4). The conidia were solitary, unicellular, hyaline, smooth, and cylindrical with evident basal scarring, in size (9) 12–16 (19) × (6) 7–8 (8,5) µm. All the macroscopic and microscopic morphological traits so far described suggest the presence of the fungus C. tarraconensis.

To prove the morphological identification, an analysis of the sequences of the ITS (internal transcribed spacer) region was conducted. Mycelia were grown on potato dextrose broth and freeze-dried prior to genomic DNA extraction using the CTAB method. The ITS sequences of C. tarraconensis isolates analyzed in these studies were deposited in NCBI GenBank with acc no. OM642119 and OM642128.

Due to the ambiguous result of the comparative analysis of our sequences with sequences available in GenBank NCBI, additional analysis was carried out. To confirm molecular identification and dispel doubts we made a phylogenetic analysis based on the ITS sequences of CtC1-21Ca and CtC7-21Ca, 8 sequences showing an identity match with them at the level over 99% and 6 chosen sequences of Helotiales and Pleosporales representatives (Appendix Table 2). Our sequences were 100% identical for two (EU707431, MT012300) of the six sequences of C. terraconensis deposited in GenBank originated from C. avellana and 99,82% similar with the next four (KF225578, EU707430, OM567738, OM567657) with a difference of one nucleotide (Fig. 1). Blastn analysis also revealed their identity in the first set with the singular GenBank isolate of Piggotia coryli (Roberge ex Desm.) Sutton (basionym-Cheilaria coryli Roberge ex Desm. (MH859025)). However, a match of our isolates to this species was ruled out. The phylogenetic tree (Fig. 1) shows a separate grouping of Pezicula cinnamomea (DC.) Sacc., Botrytis cinerea Pers. and Sclerotinia sclerotiorum (Lib.) de Bary from the Helotiales order, which includes C. tarraconensis, and isolates from Pleosporales order, i.e., Epicoccum nigrum Link, Alternaria alternata (Fr.) Keissl. and Bipolaris sorokiniana Shoemaker, to which belongs P. coryli. Also, the level of identity between C. tarraconensis and the sequences of other species of both Pleosporales and Helotiales is below 93%, while the level of identity between C. tarraconensis is 100% or close to 100%. Therefore, this indicates that our isolates cannot belong to the species P. coryli and confirms the results of microscopic observations. The dendrogram was constructed in MEGA11 Toolbar (Tamura et al. 2021) using the Maximum Likelihood algorithm. The Kimura 2-parameter model was applied (Kimura 1980). Node support was calculated using 1000 bootstrap replicants.

Fig. 1

Phylogenetic relationship between C. tarraconensis isolates CtC1-21Ca (OM642119) and CtC7-21Ca (OM642128) as analyzed in these studies, and 8 sequences showing an identity match with them at a level over 99% with 6 chosen sequences of Helotiales and Pleosporales representatives

The pathogenicity test was conducted on fresh, 1-month-old leaves of C. avellana. To inoculate pathogens on the leaves, two 10 μl of dilutions of C. tarraconensis isolates (C1 and C7) with a concentration of 1 × 10⁶ conidia/ml each were prepared. After inoculation, the plants were kept under controlled conditions in a dew chamber (21 ± 2 ˚C at over 90% humidity) with controlled plants. After 7 days, the fungus was re-isolated from the inoculated plants. Morphologically identical to the original, C. tarraconensis isolate was reisolated from the infected tissues, thus fulfilling Koch’s postulates (Appendix Figure 5).

Appendix

Fig. 2

Disease symptoms on Corylus avellana leaves were observed at Wigry National Park in 2021. A Regular, round, brown spots with a dark point in the middle of the spot. B Regular spots spread to the top of the leaf, creating long necrosis, limited by the leave’s nerves, C and D wet, irregular lesions occurring on the borders of the leaves

Fig. 3

Culture features of Cryptosporiopsis tarraconensis on PDA plates, 14 days after inoculation (A, B upper view of the plate and C, D reverse view)

Fig. 4

Observation of acervuli and conidia of Cryptosporiopsis tarraconensis. An acervulus (scale bar = 0.5 mm) and conidia (scale bar = 50 μm) were recorded by optical microscope

Fig. 5

Symptoms of leaf lesions caused by Cryptosporiopsis tarraconensis on Corylus avellana after 7 days of reinoculation

Table 2 Details on isolates taken from GenBank NCBI for phylogenetic comparative analysis based on ITS sequences