Stem rot and wilt on Euonymus
Stammfäule und Welke an Euonymus
Journal für Kulturpflanzen, 67 (10). S. 329–336, 2015, ISSN 1867-0911, DOI: 10.5073/JfK.2015.10.01, Verlag Eugen Ulmer KG, Stuttgart
In August 2008, stem rot and wilt symptoms of unknown origin were observed on Euonymus japonica. From the symptomatic stem base a fungus belonging to the genus Calonectria (anamorph: Cylindrocladium) was isolated (isolate JKI 2140). The isolate was morphologically very similar to Calonectria colhounii as well as to Ca. fujianensis and Ca. pseudocolhounii, except for the larger conidia. Sequence analysis of genes (ITS, BT, TEF-1α, HIS3) showed high similarity to Ca. colhounii, Ca. eucalypti, Ca. fujianensis and Ca. pseudocolhounii. The taxonomic status of the fungal isolate from E. japonica is not yet clear. It belongs to the complex Ca. colhounii but a definitive allocation to or separation from a known Calonectria species is not possible on the basis of a single isolate. The fungus is provisionally named Calonectria colhounii compl.
The pathogenicity of the fungus was tested on E. japonica and E. fortunei. The disease symptoms originally observed on field plants of E. japonica were reproduced and the fungus was re-isolated. Thus the pathogenicity of isolate JKI 2140 on both Euonymus species is proved. Since the first occurrence of this infestation there were no further notifications of stem canker and wilt on Euonymus spp. in Germany. Therefore the importance of this pathogen on Euonymus is considered to be low.
Key words: Calonectria, Cylindrocladium, Euonymus, ornamental shrub, symptoms, morphology, sequence analysis
Im August 2008 trat an Euonymus japonica eine Stammfäule mit Welke unbekannter Ursache auf. Aus der Triebbasis wurde ein Pilz der Gattung Calonectria (Anamorphe: Cylindrocladium) isoliert (Isolat JKI 2140). Das Isolat wies morphologisch große Ähnlichkeit mit Ca. colhounii, Ca. fujianensis sowie Ca. pseudocolhounii auf, allerdings waren die Konidien im Durchschnitt größer als für diese drei Arten beschrieben. Die Sequenzanalysen ergaben hohe Übereinstimmungen mit Ca. colhounii, Ca. eucalypti, Ca. fujianensis und Ca. pseudocolhounii. Der taxonomische Status des Pilzisolates aus E. japonica ist noch nicht eindeutig geklärt. Es gehört zum Artenkomplex Ca. colhounii, eine sichere Zuordnung zu oder Abtrennung von einer der bekannten Arten aus diesem Komplex lässt sich aber anhand eines einzigen Isolates nicht treffen. Der Pilz wird vorläufig als Calonectria colhounii compl. bezeichnet.
Die Pathogenität des Pilzes wurde an E. japonica und an E. fortunei geprüft. Die ursprünglich an E. japonica beobachteten Symptome ließen sich reproduzieren, der Pilz reisolieren. Die Pathogenität von Isolat JKI 2140 an beiden Euonymus-Arten ist damit nachgewiesen. Seit dem Erstauftreten dieses Pilzes gab es keine weiteren Meldungen über eine Stammgrundfäule mit Welkeerscheinungen an Euonymus in Deutschland. Die Bedeutung dieses Erregers an Euonymus ist deshalb als gering einzustufen.
Stichwörter: Calonectria, Cylindrocladium, Euonymus, Ziergehölz, Symptome, Morphologie, Sequenzierung
Wilt symptoms were found on one-year-old plants of Euonymus japonica in a nursery in the German federal state Hesse (Fig. 1a). The nursery manager had purchased rods to establish a mother stock for propagation. Approx. 15% of the stock plants showed disease symptoms. Only the cultivar Microphyllus was affected. The typical symptom was wilting of single shoots, pale green leaves without gloss. The shoot base was necrotic and the cortex peeled from the woody part (Fig. 1b). The wilting seemed to be a consequence of this damage. From the lower stem part a fungus was isolated which was allocated to the genus Calonectria because of stipe extensions with vesicles on the conidiophores. The fungal isolate was morphologically very similar to Ca. colhounii Peerally.
Fig. 1. Symptoms on E. japonica ‘Microphyllus‘, natural infestation
a – Green wilt
b – Rot on the stem base.
Calonectria-species are pathogenic to a wide range of plant species and may cause different symptoms. In Germany, important species on ornamental shrubs are Ca. morganii (Anamorph: Cylindrocladium scoparium) and Ca. pseudonaviculata (Anamorph: Cylindrocladium pseudonaviculatum), better known as Cylindrocladium buxicola. Cy. scoparium causes basal stem rot and wilt on Rhododendron simsii and Erica gracilis (Timonin and Self, 1955; Kelling, 1981; Neubauer and Zinkernagel, 1996). Cy. buxicola causes leaf and twig blight on Buxus sp. (Henricot and Culham, 2002; Brand, 2005). In 1973 Ca. colhounii (Anamorph: Cy. colhounii), the causal agent of a leaf spot disease on tea plants in Mauritius, was described by Peerally as a new species. According to Jeon et al. (2010) Ca. colhounii was established on hosts belonging to 14 genera. The fungus may not only cause leave blight but also basal stem rot. Ca. colhounii as a pathogen on blueberries is of particular importance. Reports about leaf spots on Vaccinium corymbosum are known from China (Luan et al., 2006); and in Korea basal stem rot was reported on blueberry seedlings originating from USA (Jeon et al., 2010). Sadowsky et al. (2011) attributed necrotic stems and leaves on V. corymbosum and V. angustifolium also to an infection by Ca. colhounii. In 2008, the disease was observed for the first time in the USA. Ca. colhounii was also isolated from Gaultheria with leaf spots (El-Goll et al., 1997); however, in this case no pathogenicity test was performed.
The only report of Ca. colhounii in Europe comes from Belgium where Ca. colhounii was identified as the causal agent of leaf spots on Rhododendron (Inghelbrecht et al., 2011). Current knowledge suggests that Ca. colhounii is a species complex (L. Lombard, pers. comm. 15.11.2013). Two species isolated recently from Eucalyptus infested by Cylindrocladium leaf blight (CLB) (Chen et al., 2011), Ca. fujianensis and Ca. pseudocolhounii, can also be allocated to this complex.
The identification of the fungal isolate from E. japonica was done using morphological analysis according to Crous (2002). Agar plugs of a single-spore culture of the isolate JKI 2140 were transferred to 2% malt extract agar (MEA) and incubated at 25°C in the dark. The anamorph was studied by light microscopy after incubation for seven days on a special nutrient-poor agar (SNA) (Nirenberg, 1976) at 25°C under NUV-light (12 h). In two separate approaches with respectively 40 conidiospores, conidia were measured at x 1000 magnification and the 95% confidence levels were determined. The minimum and maximum ranges are given in parentheses.
For perithecia induction the isolate was cultivated at 25°C on MEA under NUV-light. Mature perithecia were studied using the Kulzer Histo-Technique ISO 7100 based on hydroxyethyl methacrylate (HEMA) according to Gerritts (1985). From the embedded perithecia thin sections each 10 μm thick were prepared with a rotary microtom.
The cardinal temperatures were determined by assessment of the radial growth on MEA after incubation for 6 days in the dark.
Four loci were amplified and sequenced: parts of the big and small subunit (LSU/SSU) as well as the 5.8S rDNA and the embedded internal transcribed spacer (ITS) regions 1 and 2, the ß-tubulin gene (BT), the translation-elongation-factor TEF-1α as well as the histone H3 (HIS3). The DNA was isolated by means of the Invisorb® Spin Plant Mini Kits (STRATEC Molecular GmbH, Berlin) from mycelium of a MEA cultivated single-spore culture of the isolate JKI 2140. The following primers were used: for the amplification of the ITS-region the ITS1- and ITS4-Primer of White et al. (1990), for the fragment of the β-tubulin genes the primers Bt2a and Bt2b of Glaas and Donaldson (1995), for the fragment of the histone H3 genes the primers H3–1a and H3–1b (Glaas and Donaldson, 1995) as well as a gene section of the TEF-1 α by means of the primers EF1–526F and EF1–1567R (Rehner, 2001). Successful amplification of the gene sections were cleaned (MSB® Spin PCRapace Kit, STRATEC Molecular GmbH, Berlin) and sent to the company LGC Genomics (Berlin) for sequencing in both directions. Subsequently the sequences were assembled (CLC Maim Worknech, Qiagen Aarhus) and blasted (BLAST, Altschul et al., 1997) against previously gene-bank-stored sequences (NCBI, http://www.ncbi.nlm.nih.gov).
The sequences of the single gene sections of the isolate JKI 2140 were compared phylogenetically with the Calonectria-species listed in Chen et al. (2011). The sequences were aligned using the program Bioedit 7.2.5. (Hall, 1999) (CLUSTALW multiple alignment) and the nucleotide differences were analyzed. The model with the highest Bayesian information criterion (BIC) and thus the statistically most stable model for the phylogenetical analysis of the Calonectria-species was the time reversible algorism according to Tamura-Nei (1993) which was used for further analyses by means of the maximum-likelihood (ML) method. These calculations were done with the software MEGA 6 (Tamura et al., 2013). Phylogenetic relationships were estimated by heuristic searches based on 1000 random repeats. The analyses included 57 partial gene sequences per gene (BT, HIS3, TEF-1α), representing 28 Calonectria species to calculate an unrooted maximum likelihood tree (Chen et al., 2011).
One-year-old Euonymus japonica ‘Microphylla‘ and E. fortunei ‘Emerald’n Gold‘ were inoculated with a single-spore culture of Calonectria JKI 2140 using four different methods:
1. Spraying of the plants with a watery conidia suspension of 105/ml until run-off (4.5 ml per plant). |
2. Excision of the two leaves at a nodium and application of an agar plug colonized by the fungus onto the fresh wound, inoculation of three shoots per plant. |
3. Wounding of the base of the main shoot by roughening with fine sandpaper type P150, subsequently application of an agar plug colonized by the fungus |
4. Mixing of a homogenized Calonectria culture from one Petri dish with 1 L of growing medium. |
The inoculation points of method 2 and 3 were wrapped with wet cellulose and parafilm for three weeks. Control plants were treated in the same way but without the fungal isolate. Twenty plants were used for each variant. The plants were incubated in a climate chamber at 21°C/16°C (day/night) with 90–100% relative humidity and a photoperiod of 12 hours. Within the first three weeks the plants were kept under a plastic tunnel and were regularly irrigated overhead. The assessment of symptoms was done three and ten weeks after the inoculation. For re-isolation samples from the edge of the lesions on the stem were surface disinfected and placed on potato-dextrose-agar (PDA). The agar plates were incubated at 20°C in the dark.
The isolate JKI 2140 developed white aerial mycelium on MEA in the dark, partly with irregular colony margins. The colony reverse turned orange (Fig. 2) due to the production of chlamydospores. The chlamydospores formed chocolate brown microsclerotia.
Fig. 2. Colony morphology on MEA
a – 7 days old culture
b – 14 days old culture.
The conidiophores were arranged penicillately (Fig. 3a). Their stipe extensions were very long, narrow and septate, and ended in a claviform vesicle (Fig. 3b). The macroconidia were straight, cylindrical, rounded on both ends, 3-septate and were held together by hyaline slime in parallel cylindrical clusters. The average size of the conidia was 79 × 7 μm (Tab. 1).
Fig. 3. Morphological characteristics of isolate JKI 2140
a – Penicillate conidiophores
b – Stipe extensions with vesicles
c – Perithecium on MEA
d – Section of perithecium with asci and ascospores.
Table 1. Size of conidiospores and ascospores of five Calonectria-species belonging to the complex Ca. colhounii
Size [μm] | Ca. colhounii compl. | Ca. colhounii | Ca. pseudocolhounii sp.nov. | Ca. fujianensis sp. nov. | Ca. eucalypti |
(JKI 2140) | |||||
Macroconidiospores | |||||
Length | (66–)67–82(–84)* | (30–)50–65(–80) | (49–)55–65(–74) | (48–)50–55(–60) | (66–)69–75(–80) |
Width | (5–)6–7.5(–8) | (4–)5–6(–7) | (3,5–)4–5(–5.5) | (2,5–)3.5–4.5(–5) | (5–)6 |
Average L × W | 79 × 7 | 65 × 5 | 60 × 4.5 | 52.5 × 4.5 | 72 × 6 |
Ascospores | |||||
Length | (41–)40–69(–67) | (30–)50–65(–75) | (44–)50–62(–74) | (38–)49–62(–72) x | (25–)30–36(–56) |
Width | (5–)6–7(–9) | (4–)5–6(–8) | (5–)6–7(–8) | (5–)6–7.5(–8) | (3–)5–6(–8) |
Average L × W | 54 × 7 | 55 × 6 | 56 × 6.5 | 55.5 × 6.8 | 33 × 6 |
* 95% confidence interval, minimum and maximum in parenthesis | |||||
After four to six weeks perithecia developed on MEA. Mature perithecia were dark yellow to light orange or orange-brown (Fig. 3c). They contained numerous asci with four ascospores each (Fig. 3d). Data on perithecia and ascospore size are listed in Tab. 1.
Cardinal temperatures for the mycelium growth: Minimum > 5°C, Maximum < 35°C, Optimum 25°C.
Based on the morphological characteristics the fungal isolate from E. japonica JKI 2140 was allocated to the species complex Calonectria colhounii.
The ITS-sequence of the isolate JKI 2140 (550 Bp) shared 100% identity with the sequence of Ca. colhounii isolate PDIC 660–1L (access. no. JF742647) and 99.6% with the isolate CBS 293.79 (access. no. GQ280565.1). No ITS-sequences of Ca. pseudocolhounii and Ca. fujianensis are in the GeneBank. For the other gene fragments (HIS3, BT, TEF-1α) the sequence similarity was between 97% and 99%. (Tab. 2). Ca. fujianensis had the lowest differences in the number of nucleotides (SNPs) within the three genes. Phylogenetical analysis placed the isolate JKI 2140 into the species complex Ca. colhounii (Chen et al., 2011) with a very close relationship to Ca. fujianensis (Fig. 4).
Table 2. Sequence comparison ofCa. colhounii compl. JKI 2140 with closely relatedCalonectria-species
Sequence identity of the examined gene sections (%) and number of single nucleotide polymorphisms (SNPs)
Species | Ca. colhounii compl. JKI 2140 | ||||||
isolate no. | HIS3 | 453 bp | BT | 360 bp | TEF-1α | 493 bp | |
% | SNP | % | SNP | % | SNP | ||
Ca. colhounii | CBS 293.79 | 98.7 | 6 | 99.6 | 2 | 97.1 | 13 |
CBS 114704 | 98.7 | 6 | 98.9 | 5 | 96.9 | 14 | |
Ca. eucalypti | CBS 125273 | 97.6 | 11 | 98.5 | 7 | 99.6 | 2 |
CBS 126275 | 97.6 | 11 | 98.5 | 7 | 99.6 | 2 | |
Ca. pseudocolhounii | CMW 27213 | 98.5 | 7 | 99.1 | 4 | 99.8 | 1 |
CMW 27209 | 98.5 | 7 | 99.1 | 4 | 99.8 | 1 | |
Ca. fujianensis | CMW 27257 | 99.3 | 3 | 99.8 | 1 | 99.3 | 3 |
CMW 27263 | 99.3 | 3 | 99.8 | 1 | 99.3 | 3 | |
Fig. 4. Unrooted maximum likelihood-tree with Calonectria-isolate JKI 2140 and gene-bank reference-sequences of known Calonectria-species, based on the loci HIS3, BT and TEF-1α (Chen et al., 2011). The distances were calculated using the Tamura-Nei model TN93 + G + I. The bootstrap test was done with 1000 repeats. Only values above 50% are shown. A total 174 partial sequences of 28 Calonectria-species were used for phylogenetic tree construction. The analysis was done using MEGA6.
Three weeks after inoculation with Calonectria-isolate JKI 2140 all four inoculation methods resulted in disease symptoms on E. japonica and E. fortunei. The most severe damage was observed after inoculation of the wounded nodes and the wounded stem base. After inoculation of the nodes lesions on the shoots developed acropetally, followed by wilting and dieback of the shoots. After inoculation at the stem base initial symptoms were pale green leaves followed by a wilting of the plants (Fig. 5a). The stem base of these plants showed chocolate brown lesions. Plants that were sprayed with a conidia suspension also developed symptoms starting with chocolate brown lesions on shoots. In addition some plants showed leaf spots (Fig. 5b). Compared to inoculation method 1 and 2 symptoms appeared slightly later. Using inoculation methods 1, 2 and 3, 100% of the plants developed symptoms on E. fortunei (Fig. 6) and E. japonica. Planting in contaminated substrate resulted in only one symptomatic plant of each Eunoymus species. The non-inoculated plants from the negative control did not develop disease symptoms.
Fig. 5. Disease symptoms on E. japonica after inoculation with isolate JKI 2140
a – Agar plug on stem base
b – Spraying with conidia suspension.
Fig. 6. Disease symptoms on E. fortunei 3 weeks after inoculation with isolate JKI 2140 on the stem base.
The fungus was re-isolated from the inoculated E. fortunei and E. japonica plants. The characteristics of the re-isolates corresponded with those of the original isolate.
A Calonectria was isolated from symptomatic E. japonica. A distinct allocation of the isolate JKI 2140 to a known species was not possible. It shows high morphological similarity to Ca. colhounii (Crous, 2002), but also to Ca. fujianensis (Chen et al., 2011) and Ca. pseudocolhounii except for its larger conidia (Tab. 1). The results of the sequence analysis suggest a close relationship of isolate JKI 2140 to Ca. fujianensis.
Ca. fujianensis, causal agent of leaf blight on Eucalyptus, was defined as a new species within the Ca. colhounii- complex by Chen et al. (2011). The differentiation to Ca. colhounii and Ca. pseudocolhounii is based on analysis of the gene regions ß-Tubulin, histone H3 and translation elongation factor-1 alpha (TEF-1α). The TEF-1 α gene of the isolate JKI 2140 shows a higher similarity to Ca. pseudocolhounii, Ca. eucalypti and Ca. fujianensis (> 99%) than to Ca. colhounii. The results indicate that the causal agent of the wilt on Euonymus is a new species within the Ca. colhounii-complex. As long as no further identical isolates are detected we suggest to call the isolate JKI-2140 Calonectria colhounii compl..
Due to cardinal temperatures min. below 5°C, max. 35°C and opt. 25°C, Ca. colhounii compl. is an eurythermical fungus. That indicates that the fungus is adopted to a large temperature range with a risk of infection from spring to autumn in Central Europe.
Inoculation at the stem base resulted in symptoms identical to those observed on the originally infected Euonymus. When the plants were sprayed with a conidia suspension stem lesions and leaf spots developed. These symptoms were similar to those on blueberry inoculated with Ca. colhounii (Sadowsky et al., 2011). The results of the pathogenicity tests indicate that Ca. colhounii compl. JKI 2140 is highly virulent. In three out of four inoculation variants severe disease symptoms developed, including death of the plants. Wounding is not needed for infection. Thus Ca. colhounii compl. differs from Cylindrocladiella parva. Cy. parva is described as a weak pathogen on E. fortunei that develops lesions all around the stem on the upper parts of the stem and extends acropetally (Brielmaier-Liebetanz et al., 2013, 2014). Ca. colhounii compl. seems to be only a minor pathogen of Euonymus spp. because no reports on stem rot and wilt on Euonymus were published since 2008. Nevertheless, careful visual monitoring of Euonymus-stocks for disease symptoms is recommended, especially on young plants. In case of suspicious symptoms plant samples should be diagnosed in a laboratory to avoid confusion with symptoms caused by Cy. parva and also Phytophthora sp., which was reported to cause dieback on E. japonica in the USA (Keim et al., 1981).
We would like to thank Dagmar Trautmann and Kerstin Rogge for excellent technical assistance, and Stefan König for generating the phylogram. We also thank P. Crous and L. Lombard (CBS Utrecht) for their helpful comments and Wolfgang Schweigkofler for reviewing the manuscript.
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