Introduction

Pulsatilla koreana is a perennial herb of the Ranunculaceae, which is widely distributed in northeast China, Korea and Japan. It is highly valued as a traditional Chinese medicine whose extracts contain antimicrobial cinnamic acid derivatives (Lee et al. 2001), anemonin, the cytotoxic hederagenin, etc. (Mimaki et al. 1999; Bang et al. 2005); it is used to treat amoebic dysentery, malaria, and internal hemorrhoids (Lin et al. 2011). In addition, P. koreana shows excellent activity against plant pathogens, including Fusarium graminearum on wheat and Xanthomonas oryzae pv. Oryzae on rice (Zheng et al. 1999).

Sclerotinia spp. are well known as destructive and cosmopolitan pathogens on a wide range of host plants worldwide (Njambere et al. 2008). Three pathogenic species of Sclerotinia are commonly reported, viz. S. sclerotiorum (Lib.) de Bary, S. minor Jagger and S. trifoliorum Erikss. (Kohn et al. 1988). S. sclerotiorum is the most ubiquitous, with a host range of over 400 plant species (Boland and Hall 1994), S. minor causes diseases on a range of crops in at least 53 plant genera, and S. trifoliorum is reported to cause diseases in 21 genera, and they all cause significant economic losses (Kohn 1979). A new Sclerotinia from Japan was described as Sclerotinia nivalis I. Saito, distinguished morphologically in the sclerotial anamorph and the teleomorph produced in culture (Saito 1997). S. nivalis has been found in many plants including Lactuca sativa (Li et al. 2000), Sedum sarmentosum (Fan et al. 2012), Atractylodes japonica (Zhou et al. 2012), Aralia elata (Lee et al. 2010), and others. It is distinguished from S. sclerotiorum, S. minor and S. trifoliorum by cultural characteristics, number and size of sclerotia, pathogenicity, the molecular mass of major proteins bands on SDS-PAGE, and the patterns of esterase isozymes in sclerotial extracts (Saito 1997; Li et al. 1998).

During early spring of 2012, typical symptoms of Sclerotinia rot were observed on P. koreana in many production fields in Liaoning province, China. The plants generally showed cottony white mycelium, black sclerotia and rotting of the basal stems. Affected plants were distributed in patches throughout the rows, the disease incidence ranging from 8.6 to 25.8 % in 2012. The disease has become increasingly serious. In 2013, its incidence was highest reaching up to 60.4 % and imposing a direct threat to sustainable plant production. Therefore, this study was undertaken to identify and characterize the pathogen morphologically in cultural characteristics, by analysis of the ITS region of rDNA and comparison with 11 isolates of S. nivalis and S. sclerotiorum, and by carrying out pathogenicity tests.

Materials and methods

Field survey and collection of diseased samples

Surveys of disease development on P. koreana were conducted from April 2011 to June 2013 in three major production fields in Medicinal Herb Garden in Shenyang Agricultural University (SYAU) in Shenyang City, and in Fushun and Benxi Counties, Liaoning Province, China. Fushun County is rainy; the mean annual precipitation is up to 1000 mm, in Shenyang City it is 700–800 mm, and in Benxi County 900 mm. The severity of the disease was rated in terms of percentage of infected plants among 100 plants observed with five replicates at each production field. Diseased plants with typical symptoms were collected and photographed.

Isolation of the causal agent

Sclerotia were washed with water, surface-sterilized in 70 % ethanol for 2 s and then in 1 % sodium hypochlorite for 5 min, washed thoroughly with sterilized distilled water, and cut in half with a sterilized razor blade. Sclerotial pieces were placed on potato dextrose agar (PDA) containing 0.1 % lactic acid in 9-cm Petri dishes, with the cut face downwards. The plates were incubated at 20 °C in the dark. Isolates were purified by transferring hyphal tips to PDA slants. The slants were incubated at 20 °C for about 7 days, and then transferred to a refrigerator (4 °C) (Saito 1997).

In total 53 isolates (35 from Fushun County, 8 from Benxi City and 10 from Shenyang City) were obtained from sclerotia of diseased samples collected in the production fields of P. koreana. Based on morphological and cultural characteristics, five representative isolates were selected for assessing growth rates (diameter of colonies) and number and size of sclerotia, viz. PSnF-0, PSnF-09, PSnB-01, PSnS-02 and PSnS-R. PSnF-0 and PSnF-09 were obtained from the sclerotia in Fushun County, PSnB-01 in Benxi City, and PSnS-02 and PSnS-R in Shenyang City.

Additional previously identified isolates were also used, viz. GmS, BcS and HaS of S. sclerotiorum from Glycine max, Brassica napus and Helianthus annuus, respectively; in addition to SN110812 (Zhou et al. 2012), SsSn-24 (Fan et al. 2012) and Let-19 (Li et al. 2000) of S. nivalis from Atractylodes lancea, Sedum sarmentosum and Lactuca sativa, respectively.

Morphological and cultural characteristics

Mycelial plugs (5 mm diam.) taken from the growing edge of colonies were incubated at 20 °C in dark on PDA, oatmeal agar (OA), oatmeal-yeast extract agar (OA-YE), 5 % malt extract agar (MEA), malt extract-peptone-dextrose agar (MEA-peptone) and Czapek-Dox solution agar (Czapek) (Saito 1997). To determine the effect of temperature on radial growth and sclerotium production, PDA plates were inoculated with mycelial disks (5 mm diam) and incubated from 5 to 30 °C at 5 °C intervals. Diameters of growing colonies were measured daily. The number and pattern of sclerotial formation were assessed and measured using a vernier caliper (0–150 mm) after 14 days. Colonies of each isolate were photographed. The experiment was repeated twice with four replicates.

DNA extraction, PCR amplification and sequence analysis

The five isolates were cultured by shaking in liquid PD medium at 20 °C for 7 days. Mycelia were collected by centrifugation. Genomic DNA was extracted by using the TianGen DNA secure Plant kit according to the manufacturer’s protocol. The rDNA-ITS (ITS) region was amplified as previously described (Zhou et al. 2012) with primers ITS1/ITS4. A total reaction volume of 50 μl contained 25 μl 2× Taq MasterMix, 2 μl of each primer (ITS1 and ITS4), 1 μl DNA template, and 20 μl RNase-free water. The PCR cycling conditions were 94 °C for 5 min followed by 35 cycles of 94 °C for 1 min, 61 °C for 1 min, 72 °C for 1 min, and a final extension step of 72 °C for 10 min in a S1000TM thermal cycler (Bio-Rad Laboratories, Inc, USA). Gels were visualized on 1 % agarose gel electrophoresis and photographed under UV light.

Sequencing of the ITS region was done by Sangon Biotechnology Ltd (Shanghai, China). ITS sequences were aligned with previously published sequences from closely related Sclerotinia species maintained in GenBank. Phylogenetic analysis was performed using MEGA version 4.0 with neighbor-joining.

Pathogenicity tests

Roots of P. koreana (2 years old) were dug up from Medical Plants Gardens in Shenyang Agricultural University, Liaoning Province and gently washed. Each healthy root was inoculated with three 5 mm diam mycelial plugs from a 7-day-old culture of each isolate in three replicates, and incubated in a sealed plastic container on a layer of moist filter paper. Roots were either wounded at the point of inoculation with a fine needle to a depth of 1.0 and 0.25 mm diam or left unwounded. Sterile PDA was used to inoculate the control plants. The experiment was repeated twice.

Lesion development was assessed after 3 days, and the lesion diameter was recorded. Each root was rated for disease incidence and severity at 24 h intervals on a continuous scale of 0 to 6 in which 0 = no visible lesions, 1 = brown lesions up to 4 mm diam, 2 = lesions of 4–8 mm, 3 = lesions of 8–12 mm, 4 = lesions of 12–16 mm, 5 = lesions > 12 mm, coalesced with each other, and 6 = fully rotten roots. A disease severity index (DSI) was calculated using the formula:

$$ DSI=\frac{\left({\mathrm{X}}_0\times 0\right)+\left({X}_1\times 1\right)+\left({X}_2\times 2\right)+\left({X}_3\times 3\right)+\left({X}_4\times 4\right)+\left({X}_5\times 5\right)+\left({X}_6\times 6\right)}{\left({\mathrm{X}}_0+{\mathrm{X}}_1+{\mathrm{X}}_2+{\mathrm{X}}_3+{\mathrm{X}}_4+{\mathrm{X}}_5+{\mathrm{X}}_6\right)} $$

in which X0, X1, X2, X3, X4, X5, and X6 are the numbers of roots with rotting severity values of 0, 1, 2, 3, 4, 5, and 6, respectively (Rahman and Punja 2005). Data of disease severity in all three experiments were pooled together after testing the homogeneity of variance (P ≤ 0.05). The pathogen was re-isolated from the artificially inoculated plants after showing typical symptoms of the disease, satisfying Koch’s postulates.

Results

Disease incidence and symptoms

Surveys of disease development on P. koreana were conducted from April 2011 to June 2013 in three production fields. No disease was seen in 2011 and 2012, except for a disease incidence of 8.6–25.8 % in Fushun in 2012. In 2013 disease incidence reached up to 60.4 % and resulted in devastating loss in the 4th year of production in Fushun Country. The disease incidence ranged from 1.3–10.0 % in Benxi, and 0–3.0 % in Shenyang.

The disease initially appeared on plants in early April when daily temperatures reached −5–10 °C and snow melted. The typical symptoms were distributed in patches throughout the rows (Fig. 1a). The lower mature leaves of infected plants first became yellow and wilted, basal stem areas and roots showed a black-brown water-soaked rot at the same time under conditions of high humidity (Fig. 1b). Over time, the rot gradually enlarged, coalesced, and stems and roots were covered partly or wholly with white mycelium, and the internal root tissue was softened or decayed, producing cavities generally filled with white cottony mycelium (Fig. 1c). Black, irregular sclerotia (average 0.8–3.5 × 0.8–6.9 mm) were observed within the pith cavity of split stems and rotted roots (Fig. 1c, d). Ultimately, the basal stem and roots rotted and the plants wilted and died quickly.

Fig. 1
figure 1

Symptoms of Sclerotinia rot of Pulsatilla koreana a Symptoms in the field; b Comparision of highly diseased (l), little diseased (m) and healthy plants (r), with symptoms on stalks and petioles; c, d Sclerotia in the pith cavity of rotted roots (c) and split stems (d)

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Morphological and cultural characteristics

Colonies produced masses of white aerial mycelium on PDA at 20 °C (Fig. 2a), with small white tufts distributed among sclerotia. Some mycelium in the colony centre, especially where submerged in the medium, was light brown (Fig. 2a, b). After 2 weeks, sclerotia 0.5–4.5 mm diam were spherical, elongated, some sclerotia were aggregated to form irregular shapes, and some were produced near the colony margin, arranged in concentric rings (Fig. 2b), firmly attached to the agar surface visible through the bottom of the Petri dish.

Fig. 2
figure 2

Morphological characteristics of isolates GmS, BcS, HaS (S. sclerotiorum), PSnF-0, PSnF-09, PSnB-01, PSnS-02, PSnS-R, SN110812, SsSn-24 and Let-19 (S. nivalis) on potato dextrose agar after incubation at 20 °C for 7 days (a), and 15 days (b)

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The optimum temperatures for mycelial growth and sclerotium formation of the 5 representative isolates PSnF-0, PSnF-09, PSnB-01 PSnS-02 and PSnS-R were 20 and 15 °C, respectively. The growth rate was 10.5–12.7 mm/d (average 11.9 mm/d) and no growth at 30 °C, but some at 5 °C (Fig. 3a). More sclerotia were formed at 15 °C than at 20 °C (Fig. 3b). The optimum temperature for mycelial growth, colony morphology and sclerotial numbers and distribution pattern of the five isolates is similar to that of isolates SN110812, SsSn-24 and Let-19 of S. nivalis. Isolates Gm-S, Bc-S and Ha-S of S. sclerotiorum grew between 5 and 30 °C, faster than S. nivalis under the same conditions. The optimum temperature for S. sclerotiorum was 25 °C, with a daily growth rate of 23.1–23.7 mm (average 23.3 mm/d) (Fig. 3a). Sclerotia were formed at the edge of the colonies, fewer but larger than in S. nivalis.

Fig. 3
figure 3

Cultural characteristics of 11 isolates PSnF-0, PSnF-09, PSnB-01, PSnS-02, PSnS-R, let-19, SsSn-24, SN110812, GmS, BcS and Ha-S. a Average daily diametral growth rates at different temperatures; b Numbers of sclerotia at different temperatures; c Average growth rates on PDA, OA, OA-YE, MEA, MEA- peptone and Czapek; d Numbers of sclerotia on PDA, OA, OA-YE, MEA, MEA-peptone and Czapek

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Eleven tested isolates grew well on PDA, OA, OA-YE, MEA and MEA-peptone, but poorly on Czapek (Fig. 3c). More sclerotia were formed by the five isolates from P. koreana and three others of S. nivalis on MEA-peptone and MEA than on PDA (Fig. 3d), but they were smaller, mostly remained immature, and were more or less flat in shape and deep grayish-olive or dark olive-gray in color, and colonies were olive-buff or light grayish-olive. The colony appearance on OA was basically similar to that on PDA except that the aerial mycelium was produced more profusely than on the latter substrate, especially in the center of the colony at an early stage prior to sclerotium formation. No sclerotia were produced on Czapek.

Based on colony characteristics and sclerotia, the five isolates from P. koreana matched of Sclerotinia nivalis (Zhou et al. 2012; Saito 1997; Li et al. 2000). The morphological identification is further confirmed by ITS sequence analysis.

DNA extraction, PCR amplification and phylogeny

PCR fragments of the ITS region were approximately 500 bp in size. The GenBank accession numbers of isolates PSnF-0, PSnF-09, PSnB-01, PSnS-02, PSnS-R were KM211696, KM211697, KM211698, KM211699 and KM265189. The sequences were identical to those of S. nivalis (isolates SN110812 (Acc. No. JX294862), SsSn-24 (Acc. No. JN415129) and Let-19 (Acc. No. JN415131)). The phylogenetic tree shows that the isolates from P. koreana in China form a unique and well-supported clade that groups with the S. nivalis reference isolates (Fig. 4). These results indicate that the isolates of S. nivalis are conspecific and different from S. sclerotiorum.

Fig. 4
figure 4

Phylogenetic tree for S. nivalis and related species based on NJ analysis of ITS sequence. The figures at the nodes indicate the bootstrap support calculated for 1000 repetitions. The scale bar indicates 0.005 substitutions per nucleotide position

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Pathogenicity test

In the pathogenicity test the five isolates from P. koreana infected the roots at 20 °C under humid conditions. The same fungi were reisolated from the lesions, satisfying Koch’s postulates. Lesions appeared on wounded roots as early as 3 days after inoculation, but after 4–6 days on unwounded roots (Fig. 5a, b). A similar trend was observed in the DSI on unwounded roots (DSI = 0.7–3.0, average 2.0, after 7 days) and wounded roots (DSI = 2.3–3.2, average 2.9, after 7 days) (Fig. 5b).

Fig. 5
figure 5

Development of Sclerotinia rot following inoculation of 2-year-old roots (a wounded; b unwounded) of P. koreana with isolates PSnF-0, PSnF-09, PSnB-01, PSnS-02 and PSnS-R, and isolates SN110812, SsSn-24 and Let-19 of S. nivalis and isolates Gm-S and Bc-S and Ha-S of S. sclerotiorum

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The other six reference isolates also infected the roots of P. koreana. With S. sclerotiorum lesions appeared almost simultaneously on wounded and unwounded roots, and expanded more widely than with S. nivalis. Isolates SN110812, SsSn-24 and Let-19 of S. nivalis had similar effects as the other 5 isolates from P. koreana.

Discussion

P. koreana is recently widely cultivated as a Chinese traditional herb in northeast China. In the production field, rust (Su et al. 2012a), root rot (Su and Fu 2013), smut (Fu et al. 2010) and leaf spot (Su et al. 2011, 2012b, c) are the main diseases on P. koreana. Our study reveals that Sclerotinia rot caused by S. nivalis was also present on P. koreana in Liaoning Province.

Sclerotinia nivalis was first reported by Saito from Japan, based on the morphology of the sclerotial anamorph and the teleomorph produced in culture (Saito 1997). S. nivalis can infect many plant species, including Aralia elata (Araliaceae) (Lee et al. 2010), Daucus carota and Angelica acutiloba (Umbelliferae), Plantago lanceolata (Plantaginaceae), Ajuga reptans (Labiatae), Ambrosia elatior, Arctium lappa, Chrysanthemum morifolium, Lactuca sativa (Saito 1997; Li et al. 2000), Atractylodes japonica (Compositae) (Zhou et al. 2012), and Sedum sarmentosum (Crassulaceae) (Fan et al. 2012). In the pathogenicity tests, isolates of S. nivalis from P. koreana, Atractylodes lancea, Sedum sarmentosum and Lactuca sativa, and isolates of S. sclerotiorum from Glycine max, Brassica napus and Helianthus annuus all infected the roots of P. koreana. Isolates of S. sclerotiorum gave the highest disease severity index. According to Purdy (1979), S. sclerotiorum also infect Ranunculaceae. In view of its pathogenicity and wide distribution, S. sclerotiorum may also become an important pathogen of P. koreana .

The optimum temperature for mycelial growth and sclerotium formation of S. nivalis in culture is about 20 °C (Saito 1997) and 15 °C (Zhou et al. 2012), respectively. The disease caused by S. nivalis occurs at low temperatures (Saito 1997). S. nivalis has not only been found in north-western areas in Hubei Province (Fan et al. 2012; Li et al. 1995) but also in some mountainous area in Liaoning Province of China (Zhou et al. 2012; Fu et al. 2012), where the temperatures in winter and early spring are low, ranging from −5 to 10 °C (Li et al. 2000). The most relevant environmental factors leading to disease were low temperature and high humidity (Zhang et al. 2012; Huang and Kozub 1991). Further research is required to gain information relating to dissemination mechanism, potential control measures and cultural practices to reduce the disease.